March 22, 2021
Combining 3D Printing and Biomaterials: A Translational Fix to Tracheal Defect ReconstructioN
Biofunctionalization of 3D-printed silicone implants with immunomodulatory hydrogels for controlling the innate immune response: An in vivo model of tracheal defect repair
By: Stacey Berger
With recent advances in biomaterials and manufacturing technology, there has been a surge in 3D-printed silicone implants for various medical applications. Improved 3D printing technology allows for highly anatomically correct models, while silicone provides nondegradable, biocompatible properties that are suitable for implantation. Such implants are used for tracheal defects following cancer surgeries or traumas that require tracheal regeneration. However, major challenges stemming from the immune response to these implants have blocked their widespread clinical use. Barthes et al. offered a solution in the form of immunomodulatory hydrogel coatings capable of controlling the innate immune response.
Barthes et al. aimed to optimize a hydrogel coating for a silicone implant that would effectively suppress an inflammatory response. To accomplish this, they created a protein cocktail, dubbed M2Ct2, composed of known pro-healing/anti-inflammatory cytokines IL-10 and prostaglandin E2 (PGE-2) that was loaded into a gelatin hydrogel. Stability of the hydrogel coating on silicone implants was tested in vitro in cell culture media using dyes and fluorescent labeling. Both labels showed the same trend of an initial loss of coating in the first week due to degradation, and then a constant, stable coating for the following week.
The authors performed an in vitro release study to quantify the loading and release of PGE-2 from the immunomodulatory hydrogel coating. The results showed 28% release of PGE-2 in the first 24 hours, followed by an additional 2% release over the following two weeks. This study shows that approximately 70% of initially loaded molecules remained in the hydrogel after two weeks of release, supporting the concept that hydrogels can act as reservoirs for proteins. Finally, an in vivo tracheal patch implantation in mice was performed to test the M2Ct2-loaded hydrogel coating compared to controls with no coating or with hydrogel containing no cytokines. After one week, the M2Ct2-loaded hydrogel group caused decreased expression of four pro-inflammatory cytokines (CXCL1/KV, CCL2/MCP-1, TNF-α and IFN-gamma) compared to the other groups, as measured using flow cytometry. The same trend was observed at the three-week time point.
These results showed promising outcomes of controlling the immune response with loaded hydrogels, and a framework for future studies that leverage such technologies. Moving forward with clinical translation of this technology will require testing the immunomodulatory coating in larger animal models.
Barthes, J., Lagarrigue, P., Riabov, V., Lutzweiler, G., Kirsch, J., Muller, C., . . . Dupret-Bories, A. (2021). Biofunctionalization of 3D-printed silicone implants with immunomodulatory hydrogels for controlling the innate immune response: An in vivo model of tracheal defect repair. Biomaterials, 268, 120549. doi:10.1016/j.biomaterials.2020.120549
By: Stacey Berger
With recent advances in biomaterials and manufacturing technology, there has been a surge in 3D-printed silicone implants for various medical applications. Improved 3D printing technology allows for highly anatomically correct models, while silicone provides nondegradable, biocompatible properties that are suitable for implantation. Such implants are used for tracheal defects following cancer surgeries or traumas that require tracheal regeneration. However, major challenges stemming from the immune response to these implants have blocked their widespread clinical use. Barthes et al. offered a solution in the form of immunomodulatory hydrogel coatings capable of controlling the innate immune response.
Barthes et al. aimed to optimize a hydrogel coating for a silicone implant that would effectively suppress an inflammatory response. To accomplish this, they created a protein cocktail, dubbed M2Ct2, composed of known pro-healing/anti-inflammatory cytokines IL-10 and prostaglandin E2 (PGE-2) that was loaded into a gelatin hydrogel. Stability of the hydrogel coating on silicone implants was tested in vitro in cell culture media using dyes and fluorescent labeling. Both labels showed the same trend of an initial loss of coating in the first week due to degradation, and then a constant, stable coating for the following week.
The authors performed an in vitro release study to quantify the loading and release of PGE-2 from the immunomodulatory hydrogel coating. The results showed 28% release of PGE-2 in the first 24 hours, followed by an additional 2% release over the following two weeks. This study shows that approximately 70% of initially loaded molecules remained in the hydrogel after two weeks of release, supporting the concept that hydrogels can act as reservoirs for proteins. Finally, an in vivo tracheal patch implantation in mice was performed to test the M2Ct2-loaded hydrogel coating compared to controls with no coating or with hydrogel containing no cytokines. After one week, the M2Ct2-loaded hydrogel group caused decreased expression of four pro-inflammatory cytokines (CXCL1/KV, CCL2/MCP-1, TNF-α and IFN-gamma) compared to the other groups, as measured using flow cytometry. The same trend was observed at the three-week time point.
These results showed promising outcomes of controlling the immune response with loaded hydrogels, and a framework for future studies that leverage such technologies. Moving forward with clinical translation of this technology will require testing the immunomodulatory coating in larger animal models.
Barthes, J., Lagarrigue, P., Riabov, V., Lutzweiler, G., Kirsch, J., Muller, C., . . . Dupret-Bories, A. (2021). Biofunctionalization of 3D-printed silicone implants with immunomodulatory hydrogels for controlling the innate immune response: An in vivo model of tracheal defect repair. Biomaterials, 268, 120549. doi:10.1016/j.biomaterials.2020.120549
June 12, 2020
Using the Dying to Improve the Living...Cells, that is
Efferocytosis-inspired membrane-coated nanoparticles mitigate inflammatory responses in macrophages.
By: Emily Valentine
Many nanoparticle-driven immunotherapies to date have shown great progress in attenuating inflammatory diseases. However, these therapies are limited by a high degree of off target, negative side-effects on other cells. By leveraging the highly specific recognition of apoptotic cells by macrophages, Kraynak et al. designed nanoparticles that mimicked this process to improve immunomodulation without adverse side effects.
Apoptotic cells (ACs) display PS (phosphatidylserine, a phospholipid typically hidden just beneath the membrane) on their cell surface, which declares, “Eat me!” to macrophages. Macrophages then bind, engulf, break down, and recycle the parts of the dying cell- a key process in homeostasis as well as tissue repair. This process also causes macrophages to convert from pro-inflammatory to an inflammation-resolving phenotype, as defined by changes in cytokine production. In this study, PLGA nanoparticles were coated with a liposomal formulation of plasma membrane from 3T3 fibroblasts, which contains PS, through a multi-step extrusion protocol, resulting in apoptotic body inspired PS/membrane-coated nanoparticles (PS-MNPs).
When murine macrophages were incubated with these nanoparticles in the presence of pro-inflammatory stimuli, they downregulated secretion of the pro-inflammatory cytokine TNFα as well as expression of pro-inflammatory genes like IL-1B, IL-6 and iNOS. Nanoparticle uptake was dose-dependent, as observed via flow cytometry, plateauing at 750-1000 μg/mL of PS-MNPs. Pro-resolving cytokine markers, Mrc1/CD206, were significantly upregulated compared to pro-inflammatory macrophage controls, although they did not reach the level of unactivated macrophages. These changes indicate that the macrophages switched phenotype from proinflammatory to pro-resolving in response to the novel nanoparticles.
To further evaluate the way PS-MNPs accomplish these results, the transcription factor NFkB was fluorescently marked and monitored for its role in initiating inflammatory phenotype macrophages. Following treatment with the inflammatory stimulus LPS, the level of NFkB fluorescence in PS-MNP treated groups was similar in magnitude to unstimulated macrophages, suggesting PS-MNPs inhibit NFkB activation. This effect was maintained whether the PS-MNPs were administered at the same time or 5 hours before treatment with LPS, although co-treatment had a stronger effect, suggesting important effects of timing. Finally, the authors measured uptake of the nanoparticles by macrophages compared to 3T3 fibroblasts, with macrophages outcompeting fibroblasts due to a reduction in fibroblast uptake. These results indicate specificity of these nanoparticles in targeting macrophages.
PS, an integral molecule in efferocytosis, has proven to be a promising molecule for the modification of nanoparticles for the attenuation of inflammatory diseases by effectively attracting macrophages, downregulating pro-inflammatory activity, and further promoting a pro-resolving phenotype in vitro. However, the results still need to be confirmed in vivo. In addition, the long-term effects of this nanocarrier will need to be evaluated in vitro and in vivo. These studies will allow the use of PS-MNPs for furthering understanding of the role of macrophages in perpetuating inflammation while working towards a novel anti-inflammatory therapeutic.
Kraynak, C. A., Yan, D. J., & Suggs, L. J. (2020). Modulating inflammatory macrophages with an apoptotic body-inspired nanoparticle. Acta Biomaterialia, 108, 250-260. doi:10.1016/j.actbio.2020.03.041
By: Emily Valentine
Many nanoparticle-driven immunotherapies to date have shown great progress in attenuating inflammatory diseases. However, these therapies are limited by a high degree of off target, negative side-effects on other cells. By leveraging the highly specific recognition of apoptotic cells by macrophages, Kraynak et al. designed nanoparticles that mimicked this process to improve immunomodulation without adverse side effects.
Apoptotic cells (ACs) display PS (phosphatidylserine, a phospholipid typically hidden just beneath the membrane) on their cell surface, which declares, “Eat me!” to macrophages. Macrophages then bind, engulf, break down, and recycle the parts of the dying cell- a key process in homeostasis as well as tissue repair. This process also causes macrophages to convert from pro-inflammatory to an inflammation-resolving phenotype, as defined by changes in cytokine production. In this study, PLGA nanoparticles were coated with a liposomal formulation of plasma membrane from 3T3 fibroblasts, which contains PS, through a multi-step extrusion protocol, resulting in apoptotic body inspired PS/membrane-coated nanoparticles (PS-MNPs).
When murine macrophages were incubated with these nanoparticles in the presence of pro-inflammatory stimuli, they downregulated secretion of the pro-inflammatory cytokine TNFα as well as expression of pro-inflammatory genes like IL-1B, IL-6 and iNOS. Nanoparticle uptake was dose-dependent, as observed via flow cytometry, plateauing at 750-1000 μg/mL of PS-MNPs. Pro-resolving cytokine markers, Mrc1/CD206, were significantly upregulated compared to pro-inflammatory macrophage controls, although they did not reach the level of unactivated macrophages. These changes indicate that the macrophages switched phenotype from proinflammatory to pro-resolving in response to the novel nanoparticles.
To further evaluate the way PS-MNPs accomplish these results, the transcription factor NFkB was fluorescently marked and monitored for its role in initiating inflammatory phenotype macrophages. Following treatment with the inflammatory stimulus LPS, the level of NFkB fluorescence in PS-MNP treated groups was similar in magnitude to unstimulated macrophages, suggesting PS-MNPs inhibit NFkB activation. This effect was maintained whether the PS-MNPs were administered at the same time or 5 hours before treatment with LPS, although co-treatment had a stronger effect, suggesting important effects of timing. Finally, the authors measured uptake of the nanoparticles by macrophages compared to 3T3 fibroblasts, with macrophages outcompeting fibroblasts due to a reduction in fibroblast uptake. These results indicate specificity of these nanoparticles in targeting macrophages.
PS, an integral molecule in efferocytosis, has proven to be a promising molecule for the modification of nanoparticles for the attenuation of inflammatory diseases by effectively attracting macrophages, downregulating pro-inflammatory activity, and further promoting a pro-resolving phenotype in vitro. However, the results still need to be confirmed in vivo. In addition, the long-term effects of this nanocarrier will need to be evaluated in vitro and in vivo. These studies will allow the use of PS-MNPs for furthering understanding of the role of macrophages in perpetuating inflammation while working towards a novel anti-inflammatory therapeutic.
Kraynak, C. A., Yan, D. J., & Suggs, L. J. (2020). Modulating inflammatory macrophages with an apoptotic body-inspired nanoparticle. Acta Biomaterialia, 108, 250-260. doi:10.1016/j.actbio.2020.03.041
May 21, 2020
Reprogramming macrophages via regulation of metabolic pathways
Knockdown of four metabolic transcriptional factors successfully switched M2 behavior to M1-like behavior.
By: Jessica Eager
Conversion of macrophage phenotypes from pro-inflammatory (M1) to immunosuppressive and/or wound healing (M2) has been observed in various disease states including tumors and sepsis. Treatments aiming to polarize in vivo macrophages to an M1-like phenotype have been suggested as methods to stimulate or reactivate the immune response. To better understand the regulation of M1 and M2 polarization, Horhold et al. evaluated the differences in gene regulatory and metabolic networks of each phenotype.
Gene set enrichment analysis of in vitro polarized M1 macrophages revealed upregulation of proinflammatory pathways, as expected, but also catabolic and glycolytic metabolic processes. Conversely, M2 macrophages upregulated biosynthesis of inosin monophosphate (IMP). To validate these differences, a computational model was created based upon the well-established flux balance analysis from Orth et al, which is based on the stoichiometric coefficients of each metabolic reaction. These results supported the observed metabolic shift between glycolysis and purine biosynthesis between phenotypes.
The authors then set out to identify transcriptional factors (TFs) that regulate this metabolic switch. DEGs within KEGG pathways for glycolysis, citrate cycle, pentose phosphate pathway, fatty acid metabolism, IMP synthesis, and arginine biosynthesis were selected. A literature review was also conducted to select genes known to distinguish between M1 and M2 macrophages. The result was a list of 112 TFs of interest. With the goal of minimizing the difference between predicted and observed expression, linear regression models were employed. Optimization and transcriptional activity parameters were obtained from ChIP databases. To determine TFs most likely to reprogram M1 macrophages into M2, the values of expression of each phenotype in the mathematical model were swapped. Five TFs were found to be likely to enable reprogramming: CTCF, E2F1, MYC, PPAR-y, and STAT6.
Four of the five predicted TFs were then functionally validated by knocking down their expression in M2 macrophages (iM1). Gene expression analysis of M1 and M2 genes showed a switch in behavior of iM1s at 24 and 48 hours after knockdown treatment. Cytokine secretion assays revealed that iM1s enhanced secretion of 6 of 7 pro-inflammatory factors investigated compared to mock-treated macrophages.
In summary, Horhold et al. demonstrated in vitro reprogramming of M2 macrophages into M1-like macrophages by targeting the TFs that regulate metabolic pathways. These data suggest that immunotherapies may be able to shift cells from an immunosuppressive to an immunostimulatory state by targeting these TFs, though future studies in vivo are required.
Hörhold F, Eisel D, Oswald M, Kolte A, Röll D, et al. (2020) Reprogramming of macrophages employing gene regulatory and metabolic network models. PLOS Computational Biology 16(2): e1007657.
By: Jessica Eager
Conversion of macrophage phenotypes from pro-inflammatory (M1) to immunosuppressive and/or wound healing (M2) has been observed in various disease states including tumors and sepsis. Treatments aiming to polarize in vivo macrophages to an M1-like phenotype have been suggested as methods to stimulate or reactivate the immune response. To better understand the regulation of M1 and M2 polarization, Horhold et al. evaluated the differences in gene regulatory and metabolic networks of each phenotype.
Gene set enrichment analysis of in vitro polarized M1 macrophages revealed upregulation of proinflammatory pathways, as expected, but also catabolic and glycolytic metabolic processes. Conversely, M2 macrophages upregulated biosynthesis of inosin monophosphate (IMP). To validate these differences, a computational model was created based upon the well-established flux balance analysis from Orth et al, which is based on the stoichiometric coefficients of each metabolic reaction. These results supported the observed metabolic shift between glycolysis and purine biosynthesis between phenotypes.
The authors then set out to identify transcriptional factors (TFs) that regulate this metabolic switch. DEGs within KEGG pathways for glycolysis, citrate cycle, pentose phosphate pathway, fatty acid metabolism, IMP synthesis, and arginine biosynthesis were selected. A literature review was also conducted to select genes known to distinguish between M1 and M2 macrophages. The result was a list of 112 TFs of interest. With the goal of minimizing the difference between predicted and observed expression, linear regression models were employed. Optimization and transcriptional activity parameters were obtained from ChIP databases. To determine TFs most likely to reprogram M1 macrophages into M2, the values of expression of each phenotype in the mathematical model were swapped. Five TFs were found to be likely to enable reprogramming: CTCF, E2F1, MYC, PPAR-y, and STAT6.
Four of the five predicted TFs were then functionally validated by knocking down their expression in M2 macrophages (iM1). Gene expression analysis of M1 and M2 genes showed a switch in behavior of iM1s at 24 and 48 hours after knockdown treatment. Cytokine secretion assays revealed that iM1s enhanced secretion of 6 of 7 pro-inflammatory factors investigated compared to mock-treated macrophages.
In summary, Horhold et al. demonstrated in vitro reprogramming of M2 macrophages into M1-like macrophages by targeting the TFs that regulate metabolic pathways. These data suggest that immunotherapies may be able to shift cells from an immunosuppressive to an immunostimulatory state by targeting these TFs, though future studies in vivo are required.
Hörhold F, Eisel D, Oswald M, Kolte A, Röll D, et al. (2020) Reprogramming of macrophages employing gene regulatory and metabolic network models. PLOS Computational Biology 16(2): e1007657.
March 3, 2020
Nanoparticles Alarming the Immune System to Wake Up and Smell the TumoR
Development of a dual pH sensitive nanoparticle to prevent tumor immune evasion via NF-κB and PD1 Inhibition in vitro and in vivo models.
By: Gina Cusimano
The immune system naturally monitors and targets tumor cells for destruction through a process called immune surveillance. Tumors, however, have developed evasion mechanisms to bypass immune surveillance. Immune checkpoint blockades, such as anti-PD1 (α-PD1), target one such evasion mechanism and have shown to be effective cancer immunotherapy. Still, more than half of patients do not respond to α-PD1, indicating a need for additional strategies that prevent immune evasion.
Tumors evade immune surveillance by secreting anti-inflammatory cytokines and coaxing tumor immune cells like regulatory T cells and macrophages to do the same. Studies have shown that expression of anti-inflammatory cytokines such as TGF-β and IL-10 are controlled via the NF-κB signaling pathway. In the hopes of synergizing NF-κB inhibition with α-PD1, Xiao et al. designed a nanoparticle that sequentially delivered α-PD1 into the tumor microenvironment followed by curcumin, an NF-κB inhibitor, into cancer cells and macrophages, for a dual strategy that targeted two different immune evasion mechanisms. Sequential delivery was achieved via changes in pH. The overall design strategy was that circulating PD1-expressing T cells would bind to the α-PD1 coated nanoparticles and traffic to the tumor, where they would release the α-PD1 in the slightly acidic tumor microenvironment (pH 6.5) due to cleavage of the pH-sensitive linkage. Following α-PD1 release, macrophages and cancer cells in the tumor would take up the nanoparticles into endocytic vesicles that fuse with the acidic lysosome (pH 5.5), thus resulting in intracellular release of curcumin due to protonation.
With this goal in mind, Xiao et al. conducted a series of in vitro and in vivo studies to test the efficacy of the system. In vitro studies using melanoma and macrophage cell lines showed that the nanoparticle system inhibited gene expression of anti-inflammatory cytokines, phospho-P65 (a target of NF-κB), and PDL1, the ligand for PD1. In vivo studies using a murine melanoma model showed successful T cell binding to the nanoparticles and trafficking to the tumor, decreased protein levels of anti-inflammatory cytokines, decreased numbers of regulatory T cells, and increased numbers of cytotoxic T cells in the tumor. Lastly, nanoparticle treatment resulted in increased expression of cytotoxic T cell activation markers, decreased tumor size, and increased survival.
Collectively, these data suggest that inhibition of NF-κB signaling pathway and PD1-PDL1 interactions work synergistically in combating cancer. A limitation of this study is curcumin’s potential to inhibit pro-inflammatory cytokine expression mediated by NF-κB, which was not evaluated in this study. Nonetheless, Xiao et al. showed the promise of increasing immune check point blockade effectiveness by targeting other tumor immune evasion mechanisms.
Xiao, Z., Su, Z., Han, S., Huang, J., Lin, L., & Shuai, X. (2020). Dual pH-sensitive nanodrug blocks PD-1 immune checkpoint and uses T cells to deliver NF-κB inhibitor for antitumor immunotherapy. Science Advances, 6(6), eaay7785. doi:10.1126/sciadv.aay7785
By: Gina Cusimano
The immune system naturally monitors and targets tumor cells for destruction through a process called immune surveillance. Tumors, however, have developed evasion mechanisms to bypass immune surveillance. Immune checkpoint blockades, such as anti-PD1 (α-PD1), target one such evasion mechanism and have shown to be effective cancer immunotherapy. Still, more than half of patients do not respond to α-PD1, indicating a need for additional strategies that prevent immune evasion.
Tumors evade immune surveillance by secreting anti-inflammatory cytokines and coaxing tumor immune cells like regulatory T cells and macrophages to do the same. Studies have shown that expression of anti-inflammatory cytokines such as TGF-β and IL-10 are controlled via the NF-κB signaling pathway. In the hopes of synergizing NF-κB inhibition with α-PD1, Xiao et al. designed a nanoparticle that sequentially delivered α-PD1 into the tumor microenvironment followed by curcumin, an NF-κB inhibitor, into cancer cells and macrophages, for a dual strategy that targeted two different immune evasion mechanisms. Sequential delivery was achieved via changes in pH. The overall design strategy was that circulating PD1-expressing T cells would bind to the α-PD1 coated nanoparticles and traffic to the tumor, where they would release the α-PD1 in the slightly acidic tumor microenvironment (pH 6.5) due to cleavage of the pH-sensitive linkage. Following α-PD1 release, macrophages and cancer cells in the tumor would take up the nanoparticles into endocytic vesicles that fuse with the acidic lysosome (pH 5.5), thus resulting in intracellular release of curcumin due to protonation.
With this goal in mind, Xiao et al. conducted a series of in vitro and in vivo studies to test the efficacy of the system. In vitro studies using melanoma and macrophage cell lines showed that the nanoparticle system inhibited gene expression of anti-inflammatory cytokines, phospho-P65 (a target of NF-κB), and PDL1, the ligand for PD1. In vivo studies using a murine melanoma model showed successful T cell binding to the nanoparticles and trafficking to the tumor, decreased protein levels of anti-inflammatory cytokines, decreased numbers of regulatory T cells, and increased numbers of cytotoxic T cells in the tumor. Lastly, nanoparticle treatment resulted in increased expression of cytotoxic T cell activation markers, decreased tumor size, and increased survival.
Collectively, these data suggest that inhibition of NF-κB signaling pathway and PD1-PDL1 interactions work synergistically in combating cancer. A limitation of this study is curcumin’s potential to inhibit pro-inflammatory cytokine expression mediated by NF-κB, which was not evaluated in this study. Nonetheless, Xiao et al. showed the promise of increasing immune check point blockade effectiveness by targeting other tumor immune evasion mechanisms.
Xiao, Z., Su, Z., Han, S., Huang, J., Lin, L., & Shuai, X. (2020). Dual pH-sensitive nanodrug blocks PD-1 immune checkpoint and uses T cells to deliver NF-κB inhibitor for antitumor immunotherapy. Science Advances, 6(6), eaay7785. doi:10.1126/sciadv.aay7785
February 13, 2020
Novel Cell Therapy strategy Eradicates Drug-Resistant Bacteria
Macrophage cell therapy using vitamin-derived lipid nanoparticles carrying mRNA and cathepsin B treats sepsis in a mouse model
By: Victoria Nash
The leading cause of death in hospitalized patients worldwide is sepsis. With more drug-resistant strains of bacteria than ever before, care providers urgently need new treatments. Hou et al. created a novel cell therapy to treat sepsis by harnessing macrophages carrying vitamin C lipid nanoparticles (VC-LNPs) that contain an mRNA (AMP-CatB) encoding for three products, a clinically approved antimicrobial peptide (AMP-IB367), cathepsin B (CatB), and a CatB-sensitive linker, to fight strains of multi-drug resistant bacteria.
Macrophage phagocytose and degrade sepsis-causing bacteria when lysosomes release degradation enzymes within the phagolysosome. AMP-CatB boosted the antimicrobial power of macrophages’ lysosomes by using CatB protein to translocate AMP-CatB proteins into lysosomes, which activates AMP-IB367 when CatB cleaves it from the protein via the CatB-sensitive linker. Delivering mRNA via vitamin-derived lipid nanoparticles (V-LNPs) utilized macrophages’ inherent phagocytic activity to ensure high delivery efficiencies of AMP-CatB mRNA.
VC-LNPs significantly out-performed other V-LNPs screened for mRNA delivery efficiency and expression. Cell line-derived and primary murine bone-marrow derived macrophages were loaded with an optimized formulation of VC-LNPs and challenged in vitro with multi-drug resistant Staphylococcus aureus (MDRSA) and Escherichia coli (E. coli). The enriched antimicrobial power of the macrophages’ lysosomes significantly reduced both bacterial populations.
In two different sepsis models, male C57BL/6 mice received an immunosuppressant drug, cyclophosphamide, to model an immunocompromised septic patient. One mouse model received MDRSA intraperitoneally, while the other received both MDRSA and E. coli intraperitoneally. Administering the cell therapy both intraperitoneally and intravenously significantly improved survival over either method individually. A second dose cleared all detectable bacteria. After one month, body weight, white blood cells, and lymphocytes recovered to normal levels in all mice.
This novel macrophage cell therapy with nanoparticles containing antimicrobial mRNA and cathepsin B treated sepsis-causing bacteria in immunosuppressed, septic mouse models. However, it is important to remember that the chemotherapeutically induced models of sepsis may not accurately represent the clinical situation, so testing in more sophisticated sepsis models is required to confirm their findings. Nonetheless, this is the first step towards a novel strategy to treat multi-drug resistant bacteria-induced sepsis in immunocompromised patients.
Hou, Xucheng, et al. “Vitamin Lipid Nanoparticles Enable Adoptive Macrophage Transfer for the Treatment of Multidrug-Resistant Bacterial Sepsis.” Nature Nanotechnology, vol. 15, no. 1, 6 Jan. 2020, pp. 41–46., doi:10.1038/s41565-019-0600-1.
By: Victoria Nash
The leading cause of death in hospitalized patients worldwide is sepsis. With more drug-resistant strains of bacteria than ever before, care providers urgently need new treatments. Hou et al. created a novel cell therapy to treat sepsis by harnessing macrophages carrying vitamin C lipid nanoparticles (VC-LNPs) that contain an mRNA (AMP-CatB) encoding for three products, a clinically approved antimicrobial peptide (AMP-IB367), cathepsin B (CatB), and a CatB-sensitive linker, to fight strains of multi-drug resistant bacteria.
Macrophage phagocytose and degrade sepsis-causing bacteria when lysosomes release degradation enzymes within the phagolysosome. AMP-CatB boosted the antimicrobial power of macrophages’ lysosomes by using CatB protein to translocate AMP-CatB proteins into lysosomes, which activates AMP-IB367 when CatB cleaves it from the protein via the CatB-sensitive linker. Delivering mRNA via vitamin-derived lipid nanoparticles (V-LNPs) utilized macrophages’ inherent phagocytic activity to ensure high delivery efficiencies of AMP-CatB mRNA.
VC-LNPs significantly out-performed other V-LNPs screened for mRNA delivery efficiency and expression. Cell line-derived and primary murine bone-marrow derived macrophages were loaded with an optimized formulation of VC-LNPs and challenged in vitro with multi-drug resistant Staphylococcus aureus (MDRSA) and Escherichia coli (E. coli). The enriched antimicrobial power of the macrophages’ lysosomes significantly reduced both bacterial populations.
In two different sepsis models, male C57BL/6 mice received an immunosuppressant drug, cyclophosphamide, to model an immunocompromised septic patient. One mouse model received MDRSA intraperitoneally, while the other received both MDRSA and E. coli intraperitoneally. Administering the cell therapy both intraperitoneally and intravenously significantly improved survival over either method individually. A second dose cleared all detectable bacteria. After one month, body weight, white blood cells, and lymphocytes recovered to normal levels in all mice.
This novel macrophage cell therapy with nanoparticles containing antimicrobial mRNA and cathepsin B treated sepsis-causing bacteria in immunosuppressed, septic mouse models. However, it is important to remember that the chemotherapeutically induced models of sepsis may not accurately represent the clinical situation, so testing in more sophisticated sepsis models is required to confirm their findings. Nonetheless, this is the first step towards a novel strategy to treat multi-drug resistant bacteria-induced sepsis in immunocompromised patients.
Hou, Xucheng, et al. “Vitamin Lipid Nanoparticles Enable Adoptive Macrophage Transfer for the Treatment of Multidrug-Resistant Bacterial Sepsis.” Nature Nanotechnology, vol. 15, no. 1, 6 Jan. 2020, pp. 41–46., doi:10.1038/s41565-019-0600-1.
December 3, 2019
Cadherin 11 inhibition as potential approach to treat Myocardial Infarction
Myeloid-cell derived cell-cell adhesion protein is implicated in impaired recovery following myocardial infarction
By: Ricardo Checchia Whitaker
Myocardial Infarction (MI) occurs when a coronary vessel is blocked, causing ischemia of the surrounding tissue. The process of cell recruitment and tissue remodeling in such injuries is still not fully understood and could be an essential target in an eventual treatment. Cadherin 11 (CDH11), a cell-cell adhesion protein, plays an important role in the fibrotic formation and migration of cells to damaged areas in other injuries. Therefore, Schroer et al. hypothesized that CDH11 may contribute to the pro-inflammatory and fibrotic environment following MI, hindering healing of damaged tissue.
Murine models of MI were employed to investigate the impact of CDH11 expression on cellular recruitment and cardiac function. Using flow cytometry, the authors found that most of the non-cardiomyocyte cells in the MI tissue were recruited from the bone marrow, and that these cells expressed much higher levels of CDH11 compared to cardiomyocytes, and to a non-injured control. Genetic depletion of CDH11 led to improved cardiac function, as measured by increased ejection fraction, decreased left ventricular mass, and decreased mortality.
Based on these results, the authors next investigated pharmacologic inhibition of CDH11 as a therapeutic strategy to improve recovery from MI. The SYN0012 antibody (a CDH11 inhibitor) or IgG2a isotype control were injected intraperitoneally into mice 24 hours after MI. Animals treated with the SYN002 antibody preserved baseline values of both ejection fraction and left ventricular volume, resulting in significantly improved outcomes compared to IgG2a treated animals. Finally, SYN0012-treated groups contained fewer myeloid cells, which expressed lower levels of markers of pro-inflammatory behavior and increased levels of markers of pro-healing and pro-angiogenic behavior.
The study demonstrated positive outcomes regarding repair of MI damaged tissue by inhibiting CDH11 expression. While the results suggest an effect driven by myeloid cells, the CDH11 blockade was not specific to this cell type so future studies will need to definitively determine the mechanism. In addition, for this therapy to be translated to humans, potential differences in cardiac function and cell recruitment mechanisms will need to be taken into consideration.
Alison K. Schroer, Hind Lal, W. David Merryman et al (2019) JCI Insight: Cadherin-11 blockade reduces inflammation-driven fibrotic remodeling and improves outcomes after myocardial infarction. 4(18):e131545
By: Ricardo Checchia Whitaker
Myocardial Infarction (MI) occurs when a coronary vessel is blocked, causing ischemia of the surrounding tissue. The process of cell recruitment and tissue remodeling in such injuries is still not fully understood and could be an essential target in an eventual treatment. Cadherin 11 (CDH11), a cell-cell adhesion protein, plays an important role in the fibrotic formation and migration of cells to damaged areas in other injuries. Therefore, Schroer et al. hypothesized that CDH11 may contribute to the pro-inflammatory and fibrotic environment following MI, hindering healing of damaged tissue.
Murine models of MI were employed to investigate the impact of CDH11 expression on cellular recruitment and cardiac function. Using flow cytometry, the authors found that most of the non-cardiomyocyte cells in the MI tissue were recruited from the bone marrow, and that these cells expressed much higher levels of CDH11 compared to cardiomyocytes, and to a non-injured control. Genetic depletion of CDH11 led to improved cardiac function, as measured by increased ejection fraction, decreased left ventricular mass, and decreased mortality.
Based on these results, the authors next investigated pharmacologic inhibition of CDH11 as a therapeutic strategy to improve recovery from MI. The SYN0012 antibody (a CDH11 inhibitor) or IgG2a isotype control were injected intraperitoneally into mice 24 hours after MI. Animals treated with the SYN002 antibody preserved baseline values of both ejection fraction and left ventricular volume, resulting in significantly improved outcomes compared to IgG2a treated animals. Finally, SYN0012-treated groups contained fewer myeloid cells, which expressed lower levels of markers of pro-inflammatory behavior and increased levels of markers of pro-healing and pro-angiogenic behavior.
The study demonstrated positive outcomes regarding repair of MI damaged tissue by inhibiting CDH11 expression. While the results suggest an effect driven by myeloid cells, the CDH11 blockade was not specific to this cell type so future studies will need to definitively determine the mechanism. In addition, for this therapy to be translated to humans, potential differences in cardiac function and cell recruitment mechanisms will need to be taken into consideration.
Alison K. Schroer, Hind Lal, W. David Merryman et al (2019) JCI Insight: Cadherin-11 blockade reduces inflammation-driven fibrotic remodeling and improves outcomes after myocardial infarction. 4(18):e131545
June 15, 2018
A novel approach for uncovering key transcriptional regulators
REGGAE prioritizes transcriptional regulators and shows potential to detect pathogenic mechanisms in a variety of diseases
By: Sarah
Transcriptional regulators control the transcription of DNA to RNA and as such, they play critical roles in nearly all biological processes. Recently, they have been considered as candidates for drug targets due to their role in a variety of diseases and their potential to regulate many target genes. While there are several approaches aimed at elucidating the influence of key transcriptional regulators on target genes, only one prioritizes these regulators. Prioritization means that every regulatory path with genes of interest will be investigated and analyzed. Indeed, without this prioritization, certain key regulatory pathways might be neglected. Therefore, T. Kehl et al. present an algorithm, REGGAE (REGulator-Gene Association Enrichment), designed to prioritize the influence of considered transcriptional regulators through a Kolmogorov-Smirnov-like test statistic which combines correlation coefficients with enrichment analysis. Specifically, their algorithm identifies the influential regulators involved in the differential expression of a set of target genes between two experimental groups and requires the following as inputs: (1) a gene expression matrix containing two samples and (2) a predefined collection of regulator-target gene interactions (RTIs).
To assess the capability of their approach to properly identify and prioritize influential transcriptional regulators, the authors compared its performance to seven related algorithms in two different application scenarios. In the first scenario, they tested for key regulators responsible for the phenotypic differences between breast cancer cell lines that do and do not contain receptors for estrogen (ER+ and ER-, respectively). In the second scenario, the objective was to determine the perturbed regulators in (a) mouse lymphomas with artificially induced overexpression of the MYC proto-oncogene and (b) human embryonic stem cells with knock-outs of fundamental regulators (NANOG, POUF1, and SOX2). Indeed, perturbed regulators are those that have differential activity resulting in gene expression alterations (e.g. overexpression or deletions).
When comparing ER+ and ER- breast cancer cell lines, REGGAE and another algorithm called TFRank performed best at ranking the key regulators of ER+ cells. When identifying perturbed regulators, REGGAE and TFRank were (in most cases) the only methods to produce results, with REGGAE slightly outperforming TFRank. In the knock-out experiments, REGGAE outperformed the alternative methods regarding upregulated target genes. Collectively, REGGAE demonstrated proficiency and prioritization in identifying the most influential regulators.
A notable limitation for methods such as these that analyze the effect of key transcriptional regulators is the lack in quality and quantity of available datasets of RTIs. To account for the missing information from certain cell types, the authors integrated the binding information for each regulator to include multiple cell types for a given species. While this strategy could lead to false positive and negative interactions, few faulty interactions are assumed to have only moderate effects on the results produced by REGGAE. Nonetheless, REGGAE outperformed almost all alternative methods and unlike other methods, provides information that makes interpreting the results simpler. Further, it shows potential to detect novel biomarkers as well as elucidate complex regulatory and pathogenic mechanisms in a variety of diseases.
Kehl T, Schneider L, Kattler K, et al (2018) REGGAE: a novel approach for the identification of key transcriptional regulators. Bioinformatics. doi: 10.1093/bioinformatics/bty372
By: Sarah
Transcriptional regulators control the transcription of DNA to RNA and as such, they play critical roles in nearly all biological processes. Recently, they have been considered as candidates for drug targets due to their role in a variety of diseases and their potential to regulate many target genes. While there are several approaches aimed at elucidating the influence of key transcriptional regulators on target genes, only one prioritizes these regulators. Prioritization means that every regulatory path with genes of interest will be investigated and analyzed. Indeed, without this prioritization, certain key regulatory pathways might be neglected. Therefore, T. Kehl et al. present an algorithm, REGGAE (REGulator-Gene Association Enrichment), designed to prioritize the influence of considered transcriptional regulators through a Kolmogorov-Smirnov-like test statistic which combines correlation coefficients with enrichment analysis. Specifically, their algorithm identifies the influential regulators involved in the differential expression of a set of target genes between two experimental groups and requires the following as inputs: (1) a gene expression matrix containing two samples and (2) a predefined collection of regulator-target gene interactions (RTIs).
To assess the capability of their approach to properly identify and prioritize influential transcriptional regulators, the authors compared its performance to seven related algorithms in two different application scenarios. In the first scenario, they tested for key regulators responsible for the phenotypic differences between breast cancer cell lines that do and do not contain receptors for estrogen (ER+ and ER-, respectively). In the second scenario, the objective was to determine the perturbed regulators in (a) mouse lymphomas with artificially induced overexpression of the MYC proto-oncogene and (b) human embryonic stem cells with knock-outs of fundamental regulators (NANOG, POUF1, and SOX2). Indeed, perturbed regulators are those that have differential activity resulting in gene expression alterations (e.g. overexpression or deletions).
When comparing ER+ and ER- breast cancer cell lines, REGGAE and another algorithm called TFRank performed best at ranking the key regulators of ER+ cells. When identifying perturbed regulators, REGGAE and TFRank were (in most cases) the only methods to produce results, with REGGAE slightly outperforming TFRank. In the knock-out experiments, REGGAE outperformed the alternative methods regarding upregulated target genes. Collectively, REGGAE demonstrated proficiency and prioritization in identifying the most influential regulators.
A notable limitation for methods such as these that analyze the effect of key transcriptional regulators is the lack in quality and quantity of available datasets of RTIs. To account for the missing information from certain cell types, the authors integrated the binding information for each regulator to include multiple cell types for a given species. While this strategy could lead to false positive and negative interactions, few faulty interactions are assumed to have only moderate effects on the results produced by REGGAE. Nonetheless, REGGAE outperformed almost all alternative methods and unlike other methods, provides information that makes interpreting the results simpler. Further, it shows potential to detect novel biomarkers as well as elucidate complex regulatory and pathogenic mechanisms in a variety of diseases.
Kehl T, Schneider L, Kattler K, et al (2018) REGGAE: a novel approach for the identification of key transcriptional regulators. Bioinformatics. doi: 10.1093/bioinformatics/bty372
April 20, 2018
A novel technique for classification of partially labeled data
A combination of linear discriminant analysis and semi-supervised machine learning techniques improves binary classification of complex datasets.
By: Jess
Classification techniques are powerful, predictive tools that utilize a part of a data set to obtain a rule that provides information about new observations. In many biological experiments, especially those involving clinical samples, data are often highly dimensional, missing key pieces of information, or derived from groups with very small sample sizes. Machine learning algorithms often prove to be inaccurate in these high-dimensional, low sample-size (HDLSS) situations. To address this challenge, Lu et al. set out to enrich the potential of a common technique, linear discriminant analysis (LDA), for binary classification problems, such as identification of lung carcinoma subtypes. Their strategy was to combine an optimized function for linear classification that incorporates a machine learning technique to account for sparse or missing values.
The team’s approach to combining these two disparate techniques involved two loss functions, one for misclassification of labeled data (L) and one for unlabeled data (U), as well as a penalty term (p) to control model complexity. Because they chose to account for both labeled and unlabeled data, this technique falls into the semi-supervised class of machine learning. After evaluating multiple types of L functions, the standard method of squared error loss was chosen for its previously determined efficacy in sparsity analyses. The U function was created by modifying an established function, known as the hinge loss function, to assign a large loss when the classification boundary goes through a densely populated area. The combination of these two functions enhanced discrimination between classes by simultaneously encouraging avoidance of high density areas while also promoting a large margin between clusters. The penalty term had 2 components: the normal penalty for sparsity and an additional penalty to prevent L from being forced to zero.
Four randomly generated data sets were used to test the accuracy of the developed function, called the semi-supervised sparse linear discriminant analysis function (S3LDA). One data set consisted of low-dimensional and completely labeled data, another contained partially labeled data, and two data sets were HDLSS. While S3LDA performed relatively similarly to previously established techniques for the completely labeled and partially labeled data sets in terms of overall accuracy, it showed great improvement in rates of false negative classification of HDLSS datasets for up to 500 dimensions.
Finally, the team investigated the utility of this approach for classifying the subtype of human lung carcinoma using microarray data. S3LDA outperformed the other classifiers only for labeled data, showing that there is still room for improvement. Application to scanned images of hand written digits also did not yield greater accuracy for labeled or mixed data. Because S3LDA generally performed on par with other techniques, it lends itself to applications with partially labeled, HDLSS data. Future work, however, is needed to improve S3LDA’s capacity for partially labeled data before it can be employed as adequately as currently established techniques.
Lu Q, Qiao X. Sparse Fisher’s linear discriminant analysis for partially labeled data. Stat Anal Data Min: The ASA Data Sci Journal, 2018;11:17–31.
By: Jess
Classification techniques are powerful, predictive tools that utilize a part of a data set to obtain a rule that provides information about new observations. In many biological experiments, especially those involving clinical samples, data are often highly dimensional, missing key pieces of information, or derived from groups with very small sample sizes. Machine learning algorithms often prove to be inaccurate in these high-dimensional, low sample-size (HDLSS) situations. To address this challenge, Lu et al. set out to enrich the potential of a common technique, linear discriminant analysis (LDA), for binary classification problems, such as identification of lung carcinoma subtypes. Their strategy was to combine an optimized function for linear classification that incorporates a machine learning technique to account for sparse or missing values.
The team’s approach to combining these two disparate techniques involved two loss functions, one for misclassification of labeled data (L) and one for unlabeled data (U), as well as a penalty term (p) to control model complexity. Because they chose to account for both labeled and unlabeled data, this technique falls into the semi-supervised class of machine learning. After evaluating multiple types of L functions, the standard method of squared error loss was chosen for its previously determined efficacy in sparsity analyses. The U function was created by modifying an established function, known as the hinge loss function, to assign a large loss when the classification boundary goes through a densely populated area. The combination of these two functions enhanced discrimination between classes by simultaneously encouraging avoidance of high density areas while also promoting a large margin between clusters. The penalty term had 2 components: the normal penalty for sparsity and an additional penalty to prevent L from being forced to zero.
Four randomly generated data sets were used to test the accuracy of the developed function, called the semi-supervised sparse linear discriminant analysis function (S3LDA). One data set consisted of low-dimensional and completely labeled data, another contained partially labeled data, and two data sets were HDLSS. While S3LDA performed relatively similarly to previously established techniques for the completely labeled and partially labeled data sets in terms of overall accuracy, it showed great improvement in rates of false negative classification of HDLSS datasets for up to 500 dimensions.
Finally, the team investigated the utility of this approach for classifying the subtype of human lung carcinoma using microarray data. S3LDA outperformed the other classifiers only for labeled data, showing that there is still room for improvement. Application to scanned images of hand written digits also did not yield greater accuracy for labeled or mixed data. Because S3LDA generally performed on par with other techniques, it lends itself to applications with partially labeled, HDLSS data. Future work, however, is needed to improve S3LDA’s capacity for partially labeled data before it can be employed as adequately as currently established techniques.
Lu Q, Qiao X. Sparse Fisher’s linear discriminant analysis for partially labeled data. Stat Anal Data Min: The ASA Data Sci Journal, 2018;11:17–31.
February 21, 2018
crystals that modulate Inflammation in Cutaneous Wounds
MSN-Ceria nanocomposite functions as an ROS-scavenging adhesive to accelerate wound healing
Cutaneous scarring of surgical wounds is a significant problem that can limit movement and result in loss of function. Though commonly used to close wounds, staples and sutures are traumatic and can aggravate inflammation, which can impair healing through the elevated production of reactive oxygen species (ROS). Therefore, Wu et al. sought to synthesize an ROS-reducing tissue adhesive that can accelerate healing without scarring.
Mesoporous silica nanoparticles (MSN) function as an adhesive when in solution by absorbing to the edges of a wound and forming connections with each other to close it. Ceria nanocrystals, which scavenge ROS through the oxidation of Ce3+ to Ce4+, were conjugated to the MSN via the addition of amine groups to the MSN that could bond with 2-Bromo-2-methylpropionic acid groups on the Ceria.
To measure MSN-Ceria’s antioxidant abilities, particles were incubated with a ROS-generating combination of xanthine and xanthine oxidase, and a water-soluble tetrazolium salt that changes color when reduced. MSN or Ceria alone acted as controls. Superoxide anions were measured spectrophotometrically, revealing that immobilization on MSN actually enhanced the ROS-scavenging capacity of Ceria, an effect that was attributed to improved dispersion of the Ceria nanocrystals.
The authors tested MSN-Ceria’s adhesion efficacy on cutaneous wounds on Sprague-Dawley rats by topically applying the dispersion, then holding the wound closed for 30 seconds. MSN alone and a saline control were also tested for comparison. Both MSN and MSN-Ceria demonstrated rapid closing of the wounds, while saline did not.
Finally, the regenerative capacity of MSN-Ceria on cutaneous wounds in rats was compared to that of MSN, Ceria, Silica Ludox (an adhesive), and a vehicle control. By day 5 post-treatment, there were fewer CD68-positive macrophages in the wounds of MSC-Ceria treated rats compared to those treated with MSN alone, suggesting reduced inflammation. On day 8, wounds treated with MSN-Ceria had almost healed, and exhibited a smoother appearance than all other groups. On day 12, staining with Masson’s trichrome revealed complete functional tissue structures in the MSN-Ceria group only, although there was no significant increase in blood vessel formation compared to all other groups. Importantly, the epidermal thickness was lowest in the MSN-Ceria group, suggesting decreased scarring.
MSN-Ceria demonstrated significant adhesive and antioxidant abilities and promoted faster wound healing without scarring. However, because blood vessel formation was not increased, it is uncertain whether its applications extend beyond healing superficial wounds.
Wu, Haibin, et al. “Ceria Nanocrystals Decorated Mesoporous Silica Nanoparticle Based ROS-Scavenging Tissue Adhesive for Highly Efficient Regenerative Wound Healing.” Biomaterials 151 (2018): 66-77.
Cutaneous scarring of surgical wounds is a significant problem that can limit movement and result in loss of function. Though commonly used to close wounds, staples and sutures are traumatic and can aggravate inflammation, which can impair healing through the elevated production of reactive oxygen species (ROS). Therefore, Wu et al. sought to synthesize an ROS-reducing tissue adhesive that can accelerate healing without scarring.
Mesoporous silica nanoparticles (MSN) function as an adhesive when in solution by absorbing to the edges of a wound and forming connections with each other to close it. Ceria nanocrystals, which scavenge ROS through the oxidation of Ce3+ to Ce4+, were conjugated to the MSN via the addition of amine groups to the MSN that could bond with 2-Bromo-2-methylpropionic acid groups on the Ceria.
To measure MSN-Ceria’s antioxidant abilities, particles were incubated with a ROS-generating combination of xanthine and xanthine oxidase, and a water-soluble tetrazolium salt that changes color when reduced. MSN or Ceria alone acted as controls. Superoxide anions were measured spectrophotometrically, revealing that immobilization on MSN actually enhanced the ROS-scavenging capacity of Ceria, an effect that was attributed to improved dispersion of the Ceria nanocrystals.
The authors tested MSN-Ceria’s adhesion efficacy on cutaneous wounds on Sprague-Dawley rats by topically applying the dispersion, then holding the wound closed for 30 seconds. MSN alone and a saline control were also tested for comparison. Both MSN and MSN-Ceria demonstrated rapid closing of the wounds, while saline did not.
Finally, the regenerative capacity of MSN-Ceria on cutaneous wounds in rats was compared to that of MSN, Ceria, Silica Ludox (an adhesive), and a vehicle control. By day 5 post-treatment, there were fewer CD68-positive macrophages in the wounds of MSC-Ceria treated rats compared to those treated with MSN alone, suggesting reduced inflammation. On day 8, wounds treated with MSN-Ceria had almost healed, and exhibited a smoother appearance than all other groups. On day 12, staining with Masson’s trichrome revealed complete functional tissue structures in the MSN-Ceria group only, although there was no significant increase in blood vessel formation compared to all other groups. Importantly, the epidermal thickness was lowest in the MSN-Ceria group, suggesting decreased scarring.
MSN-Ceria demonstrated significant adhesive and antioxidant abilities and promoted faster wound healing without scarring. However, because blood vessel formation was not increased, it is uncertain whether its applications extend beyond healing superficial wounds.
Wu, Haibin, et al. “Ceria Nanocrystals Decorated Mesoporous Silica Nanoparticle Based ROS-Scavenging Tissue Adhesive for Highly Efficient Regenerative Wound Healing.” Biomaterials 151 (2018): 66-77.
August 29, 2017
Protein-protein interaction networks to identify functional gene clusters
Using a macrophage cell line, gene expression analysis following nanoparticle exposure reveals 3 clusters, each with distinct functional significance.
By: Jess
The rise in use of nanomaterials calls into question the potential side effects of nanoparticle accumulation in the lungs. Recent studies have implicated nanoscale silica dust in the development of pulmonary inflammation, interstitial fibrosis, and lung cancer. Although the pathogenesis of lung injury following exposure has been studied, the underlying immunological mechanisms require further investigation.
To address the limitations in knowledge regarding interactions between nanomaterials and macrophages, the primary cell type of the inflammatory response, Zhang, et al. utilized publicly available microarray data from the Gene Expression Omnibus database. This dataset contains 6 groups of murine macrophages (n=3 samples per group) cultured in serum-free medium with different doses of nano-silica dust for 2 hours. Because preliminary analysis showed highly conserved gene expression across the groups, the treatments were pooled for comparison to the control.
The gene expression to background noise ratio was chosen as the test statistic for identifying differentially expressed genes (DEGs). Gene ontology functional annotation showed a wide distribution of genes related to the whole cell and extracellular regions, with the term “membrane” linked to 42.2% of DEGs. KEGG pathway analysis showed that the most significant action modes were cytokine-cytokine receptor interactions. The pathway with the greatest number of associated DEGs was PI3K-Akt signaling pathway, with 63 of the total 1972 mapped to it.
Protein-protein interactions were retrieved from a database of known and predicted interactions, known as STRING. The Molecular Complex Detection (MCODE) functionality of the open source software platform Cytoscape was used for remodeling of the initial STRING results. MCODE identified 3 modules (M1, M2, and M3), which were used in follow-up KEGG analysis and top 10 hub gene characterization. M1 was shown to play an import role in cytokine-cytokine receptor interaction and chemokine signaling pathway. M2 was mainly involved in biosynthesis of proteasome and steroids. M3 had close relationships with cell adhesion and the P13K-Akt signaling pathway.
Overall, this process provides a method for visualization of gene relatedness within the environment of interest, as well as insight into the biological mechanisms of these relationships. This method of visualization is important because the roles of molecular function hub genes in specific pathogeneses are seldom reported. The next step for this research is to further detail each module’s associated pathways in exploration of mechanisms, biomarkers, and therapeutic targets.
Zhang, L. (2017), et al. Bioinformatics methods for identifying differentially expressed genes and signaling pathways in nano-silica stimulated macrophages. Tumor Biology, 1–9.
By: Jess
The rise in use of nanomaterials calls into question the potential side effects of nanoparticle accumulation in the lungs. Recent studies have implicated nanoscale silica dust in the development of pulmonary inflammation, interstitial fibrosis, and lung cancer. Although the pathogenesis of lung injury following exposure has been studied, the underlying immunological mechanisms require further investigation.
To address the limitations in knowledge regarding interactions between nanomaterials and macrophages, the primary cell type of the inflammatory response, Zhang, et al. utilized publicly available microarray data from the Gene Expression Omnibus database. This dataset contains 6 groups of murine macrophages (n=3 samples per group) cultured in serum-free medium with different doses of nano-silica dust for 2 hours. Because preliminary analysis showed highly conserved gene expression across the groups, the treatments were pooled for comparison to the control.
The gene expression to background noise ratio was chosen as the test statistic for identifying differentially expressed genes (DEGs). Gene ontology functional annotation showed a wide distribution of genes related to the whole cell and extracellular regions, with the term “membrane” linked to 42.2% of DEGs. KEGG pathway analysis showed that the most significant action modes were cytokine-cytokine receptor interactions. The pathway with the greatest number of associated DEGs was PI3K-Akt signaling pathway, with 63 of the total 1972 mapped to it.
Protein-protein interactions were retrieved from a database of known and predicted interactions, known as STRING. The Molecular Complex Detection (MCODE) functionality of the open source software platform Cytoscape was used for remodeling of the initial STRING results. MCODE identified 3 modules (M1, M2, and M3), which were used in follow-up KEGG analysis and top 10 hub gene characterization. M1 was shown to play an import role in cytokine-cytokine receptor interaction and chemokine signaling pathway. M2 was mainly involved in biosynthesis of proteasome and steroids. M3 had close relationships with cell adhesion and the P13K-Akt signaling pathway.
Overall, this process provides a method for visualization of gene relatedness within the environment of interest, as well as insight into the biological mechanisms of these relationships. This method of visualization is important because the roles of molecular function hub genes in specific pathogeneses are seldom reported. The next step for this research is to further detail each module’s associated pathways in exploration of mechanisms, biomarkers, and therapeutic targets.
Zhang, L. (2017), et al. Bioinformatics methods for identifying differentially expressed genes and signaling pathways in nano-silica stimulated macrophages. Tumor Biology, 1–9.
August 8, 2017
Re-educating macrophages in cancer: utilizing nanoparticles to eliminate pathologY
Cancer immunotherapy is enhanced by delivering cytokines in pH-sensitive nanoparticles to tissue associated macrophages
By Carly and Kate
Immunotherapy is a promising frontier of cancer research in which treatments redirect endogenous immune cells to attack cancer inside the body. However, immunotherapy is not consistently successful when delivered to solid tumors. To bolster immunotherapy, Wang et al. developed a nanoparticle carrier system capable of delivering the cytokine interleukin-12 (IL-12) to tumors through the enhanced permeability and retention effect, in which blood-born particles tend to accumulate in tumors due to their leaky vasculature. The researchers chose IL-12 because it shifts tumor-associated macrophages from a tumor-supportive to a tumor-suppressive phenotype.
Noting the slightly acidic tumor microenvironment, researchers developed a polymeric nanoparticle (IL-12cP) via double emulsion fabrication utilizing the pH-sensitive polymer 3-(diethylamino)-1-propylamine (DEPA). Using dynamic light scattering and transmission electron microscopy, the researchers found that spherical IL-12cPs were hydrodynamically stable and capable of retaining IL-12 in neutral environments, but the particles expanded in diameter and released IL-12 upon entry into acidic tumor environments. Interestingly, flow cytometry analysis of the marker CD197 (aka CCR7) indicated that IL-12cP particles induced tumor-supportive macrophages to transition into tumor suppressive macrophages in vitro.
To test their system in vivo, the researchers completed two biodistribution studies by intravenously administering indocyanine green (ICG) or gold loaded particles into xenograft mouse models of melanoma, to investigate biodistribution at early and late time points, respectively. Both studies indicated large accumulation of particles in the tumor and liver but minimal accumulation in other off-target organs. Intravenous and intratumoral administration of IL-12cP were found to prevent tumor growth in vivo. Finally, the authors quantified iNOS and Arg-1 protein concentrations in the tumor using western blotting and found increased iNOS and decreased Arg-1 expression in IL-12cP treatment groups suggestive of a switch from a tumor-supportive phenotype to a tumor-suppressive phenotype.
In summary, Wang et al. showed that nanoparticles can utilize the tumor environment as an asset for targeted cancer treatments. Additionally, these data establish the utility of nanoparticles as re-education instruments for tumor associated macrophages and immunotherapeutic strategies. Further investigation of the mechanisms of nanoparticle distribution through intravenous delivery in addition to delivery efficiency and dosage could be used to optimize this system to reduce side effects further.
Wang, Yi, et al. "Polymeric nanoparticles promote macrophage reversal from M2 to M1 phenotypes in the tumor microenvironment." Biomaterials 112 (2017): 153-163.
By Carly and Kate
Immunotherapy is a promising frontier of cancer research in which treatments redirect endogenous immune cells to attack cancer inside the body. However, immunotherapy is not consistently successful when delivered to solid tumors. To bolster immunotherapy, Wang et al. developed a nanoparticle carrier system capable of delivering the cytokine interleukin-12 (IL-12) to tumors through the enhanced permeability and retention effect, in which blood-born particles tend to accumulate in tumors due to their leaky vasculature. The researchers chose IL-12 because it shifts tumor-associated macrophages from a tumor-supportive to a tumor-suppressive phenotype.
Noting the slightly acidic tumor microenvironment, researchers developed a polymeric nanoparticle (IL-12cP) via double emulsion fabrication utilizing the pH-sensitive polymer 3-(diethylamino)-1-propylamine (DEPA). Using dynamic light scattering and transmission electron microscopy, the researchers found that spherical IL-12cPs were hydrodynamically stable and capable of retaining IL-12 in neutral environments, but the particles expanded in diameter and released IL-12 upon entry into acidic tumor environments. Interestingly, flow cytometry analysis of the marker CD197 (aka CCR7) indicated that IL-12cP particles induced tumor-supportive macrophages to transition into tumor suppressive macrophages in vitro.
To test their system in vivo, the researchers completed two biodistribution studies by intravenously administering indocyanine green (ICG) or gold loaded particles into xenograft mouse models of melanoma, to investigate biodistribution at early and late time points, respectively. Both studies indicated large accumulation of particles in the tumor and liver but minimal accumulation in other off-target organs. Intravenous and intratumoral administration of IL-12cP were found to prevent tumor growth in vivo. Finally, the authors quantified iNOS and Arg-1 protein concentrations in the tumor using western blotting and found increased iNOS and decreased Arg-1 expression in IL-12cP treatment groups suggestive of a switch from a tumor-supportive phenotype to a tumor-suppressive phenotype.
In summary, Wang et al. showed that nanoparticles can utilize the tumor environment as an asset for targeted cancer treatments. Additionally, these data establish the utility of nanoparticles as re-education instruments for tumor associated macrophages and immunotherapeutic strategies. Further investigation of the mechanisms of nanoparticle distribution through intravenous delivery in addition to delivery efficiency and dosage could be used to optimize this system to reduce side effects further.
Wang, Yi, et al. "Polymeric nanoparticles promote macrophage reversal from M2 to M1 phenotypes in the tumor microenvironment." Biomaterials 112 (2017): 153-163.
June 8, 2017
Dual Growth Factor Deliver
Dual delivery of growth factors with coacervate-coated poly(lactic-co-glycolic acid) nanofiber improves neovascularization in a mouse skin flap model
By Greg
Promoting angiogenesis and vascularization in newly grown tissue is vital for acceptance of new tissue. In this study, Lee et al. wished to enhance acceptance of skin flaps to reduce distal necrotic effects that currently bar the path for full integration of skin flaps to treat injured skin. Lee et al. used a nanofiber scaffold loaded with two growth factors (GF), VEGF and TGF-β3, to achieve increased vascularization. VEGF was chosen for its ability to promote cell growth and migration as well as vascularization whereas TGF-β3 was chosen because it can reduce scar formation and encourage angiogenesis. To attach the GFs to the scaffold, Lee et al. first combined VEGF and TGF-β3 with negatively charged heparin in distilled water. A positively charged PLGA nanofiber scaffold was then submersed in the solution and subsequently dried. Next, the scaffold was rehydrated with distilled water, causing a 3-D coacervate, a GF-rich viscous liquid phase, to form on the fibers. To examine the scaffold’s effectiveness, Lee et al. conducted three sets of experiments, in vitro and in vivo, to characterize GF release profiles from the coacervate, proliferation of cells and neovascularization.
After ensuring that the GF/heparin was evenly distributed on the nanofiber scaffold, Lee et al. found that the release of VEGF was more sustained than that of TGF-β3 over 21 days. TGF-β3 was released rapidly, and they hypothesized that this was due to the more negative charge of the TGF-β3 at physiological pH, resulting in a weaker bond. Lee et al. next examined the proliferation of human dermal fibroblasts (hDFBs) and tubule formation of human umbilical vein endothelial cells (HUVECs) as an indicator of bioactivity. They discovered that the hDFB proliferation, as well as HUVEC tubule length and network formation, in vitro, were higher on the dual GF coated scaffold than control scaffolds that had only one GF attached with the other freely added or no GF at all. For the in vivo mouse model, they placed the scaffold underneath the skin flap. Blood perfusion, as measured by laser doppler perfusion, increased in dual GF implantations compared to all other controls. Next, they measured neovascularization by immunofluorescent staining of blood vessel markers CD31 and SM α-actin and discovered that the level was much higher in the dual GF method than any of the controls.
This work highlights the advantage of using both VEGF and TGF-β3 bound to a scaffold via heparin coacervate to help integrate skin flaps and promote better healing when compared to a scaffold with one or no GFs. The next step for this research would involve testing the effects on a skin graft, instead of a skin flap that has an intact blood supply at its base.
Min Suk Lee et al. “Dual delivery of growth factors with coacervate-coated poly(lactic-co-glycolic acid) nanofiber improves neovascularization in a mouse skin flap model”, Biomaterials 124 (2017): 65-77
By Greg
Promoting angiogenesis and vascularization in newly grown tissue is vital for acceptance of new tissue. In this study, Lee et al. wished to enhance acceptance of skin flaps to reduce distal necrotic effects that currently bar the path for full integration of skin flaps to treat injured skin. Lee et al. used a nanofiber scaffold loaded with two growth factors (GF), VEGF and TGF-β3, to achieve increased vascularization. VEGF was chosen for its ability to promote cell growth and migration as well as vascularization whereas TGF-β3 was chosen because it can reduce scar formation and encourage angiogenesis. To attach the GFs to the scaffold, Lee et al. first combined VEGF and TGF-β3 with negatively charged heparin in distilled water. A positively charged PLGA nanofiber scaffold was then submersed in the solution and subsequently dried. Next, the scaffold was rehydrated with distilled water, causing a 3-D coacervate, a GF-rich viscous liquid phase, to form on the fibers. To examine the scaffold’s effectiveness, Lee et al. conducted three sets of experiments, in vitro and in vivo, to characterize GF release profiles from the coacervate, proliferation of cells and neovascularization.
After ensuring that the GF/heparin was evenly distributed on the nanofiber scaffold, Lee et al. found that the release of VEGF was more sustained than that of TGF-β3 over 21 days. TGF-β3 was released rapidly, and they hypothesized that this was due to the more negative charge of the TGF-β3 at physiological pH, resulting in a weaker bond. Lee et al. next examined the proliferation of human dermal fibroblasts (hDFBs) and tubule formation of human umbilical vein endothelial cells (HUVECs) as an indicator of bioactivity. They discovered that the hDFB proliferation, as well as HUVEC tubule length and network formation, in vitro, were higher on the dual GF coated scaffold than control scaffolds that had only one GF attached with the other freely added or no GF at all. For the in vivo mouse model, they placed the scaffold underneath the skin flap. Blood perfusion, as measured by laser doppler perfusion, increased in dual GF implantations compared to all other controls. Next, they measured neovascularization by immunofluorescent staining of blood vessel markers CD31 and SM α-actin and discovered that the level was much higher in the dual GF method than any of the controls.
This work highlights the advantage of using both VEGF and TGF-β3 bound to a scaffold via heparin coacervate to help integrate skin flaps and promote better healing when compared to a scaffold with one or no GFs. The next step for this research would involve testing the effects on a skin graft, instead of a skin flap that has an intact blood supply at its base.
Min Suk Lee et al. “Dual delivery of growth factors with coacervate-coated poly(lactic-co-glycolic acid) nanofiber improves neovascularization in a mouse skin flap model”, Biomaterials 124 (2017): 65-77
March 23, 2017
Debris removal is linked to resolution of inflammation
MerTK receptor cleavage impaired efferocytosis in atherosclerosis.
By Anamika and Nathan
Atherothrombotic vascular disease is the leading cause of mortality in developed nations. Efferocytosis is the phenomenon in which macrophages engulf cellular debris and secrete anti-inflammatory factors in normal repair process. Atherosclerotic lesions are characterized by dysfunctional efferocytosis, which leads to impaired resolution of inflammation and further contributes to formation of plaque and a fibrous cap (layer of fibrous tissue containing macrophages and smooth muscle cells), thinning and thrombosis. However, the mechanism underlying the dysfunctional efferocytosis and how is it linked to impaired inflammation is poorly understood. Earlier studies have shown that macrophages near the necrotic core of human atheromas express higher levels of ADAM17 (also called tumor necrosis factor-α-converting enzyme) and lower levels of the efferocytosis receptor c-Mer tyrosine kinase (MerTK) than peripheral macrophages. Also, it has been shown that ADAM17 can cleave the MerTK receptor. In this study, Cai et al. tested the hypothesis that the cleavage of MerTK directly impairs efferocytosis and resolution of inflammation in atherosclerosis.
The authors first analyzed human carotid artery endarterectomy specimens and found that lesional soluble-Mer (a marker of MerTK cleavage) was positively correlated with necrosis. The authors confirmed these results in LDL receptor–deficient (Ldlr–/–) mice, a model of atherosclerosis. To further investigate whether MerTK cleavage directly promotes plaque necrosis, the authors used genetically engineered cleavage-resistant MerTK (MertkCR) mice. Excitingly, these mice did not show evidence of suppressed efferocytosis by macrophages following treatment with the atherogenic oxidized lipoprotein (oxLDL). Then, the authors transplanted myeloid cells expressing MerTKCR into mice with advanced atherosclerosis. Strikingly, compared to mice receiving myeloid cells from wild type mice, atherosclerotic lesions showed no change in total lesional area but they did have less plaque necrosis, less aortic sol-Mer, and increased uptake of apoptotic cells by macrophages, suggesting improved health via enhanced efferocytosis. To investigate the mechanism, the authors performed liquid chromatography-tandem mass spectrometry (LC-MS) to analyze the wide range of specialized pro-resolving mediators (SPMs) and pro-inflammatory lipid mediators in aortic extracts from MertkCR myeloid cell-treated mice. They found that the global content of SPMs, including resolving D2, D5, and E3 and cytokines TGF-β1 and IL-10, was significantly increased in MertkCR myeloid cell-treated mice compared to controls. Interestingly, treatment with resolvin D1 (RvD1), which is known to increase efferocytosis, blocked MerTK cleavage in macrophages in vitro and promoted MerTK expression in atherosclerotic mice, suggesting that MerTK is involved in inflammation resolution.
Taken together, these findings provide clear evidence for the central role of MerTK and its cleavage as one of the extensive dysregulated pathways in the development of atherothrombosis. However, the specific molecules that induce MerTK cleavage or that bridge apoptotic cells to MerTK receptors on macrophages remain largely unexplored in this study.
Bishuang Cai et al. 2017 “MerTK receptor cleavage promotes plaque necrosis and defective resolution in atherosclerosis” J Clin Invest. 2017;127(2):564–568
By Anamika and Nathan
Atherothrombotic vascular disease is the leading cause of mortality in developed nations. Efferocytosis is the phenomenon in which macrophages engulf cellular debris and secrete anti-inflammatory factors in normal repair process. Atherosclerotic lesions are characterized by dysfunctional efferocytosis, which leads to impaired resolution of inflammation and further contributes to formation of plaque and a fibrous cap (layer of fibrous tissue containing macrophages and smooth muscle cells), thinning and thrombosis. However, the mechanism underlying the dysfunctional efferocytosis and how is it linked to impaired inflammation is poorly understood. Earlier studies have shown that macrophages near the necrotic core of human atheromas express higher levels of ADAM17 (also called tumor necrosis factor-α-converting enzyme) and lower levels of the efferocytosis receptor c-Mer tyrosine kinase (MerTK) than peripheral macrophages. Also, it has been shown that ADAM17 can cleave the MerTK receptor. In this study, Cai et al. tested the hypothesis that the cleavage of MerTK directly impairs efferocytosis and resolution of inflammation in atherosclerosis.
The authors first analyzed human carotid artery endarterectomy specimens and found that lesional soluble-Mer (a marker of MerTK cleavage) was positively correlated with necrosis. The authors confirmed these results in LDL receptor–deficient (Ldlr–/–) mice, a model of atherosclerosis. To further investigate whether MerTK cleavage directly promotes plaque necrosis, the authors used genetically engineered cleavage-resistant MerTK (MertkCR) mice. Excitingly, these mice did not show evidence of suppressed efferocytosis by macrophages following treatment with the atherogenic oxidized lipoprotein (oxLDL). Then, the authors transplanted myeloid cells expressing MerTKCR into mice with advanced atherosclerosis. Strikingly, compared to mice receiving myeloid cells from wild type mice, atherosclerotic lesions showed no change in total lesional area but they did have less plaque necrosis, less aortic sol-Mer, and increased uptake of apoptotic cells by macrophages, suggesting improved health via enhanced efferocytosis. To investigate the mechanism, the authors performed liquid chromatography-tandem mass spectrometry (LC-MS) to analyze the wide range of specialized pro-resolving mediators (SPMs) and pro-inflammatory lipid mediators in aortic extracts from MertkCR myeloid cell-treated mice. They found that the global content of SPMs, including resolving D2, D5, and E3 and cytokines TGF-β1 and IL-10, was significantly increased in MertkCR myeloid cell-treated mice compared to controls. Interestingly, treatment with resolvin D1 (RvD1), which is known to increase efferocytosis, blocked MerTK cleavage in macrophages in vitro and promoted MerTK expression in atherosclerotic mice, suggesting that MerTK is involved in inflammation resolution.
Taken together, these findings provide clear evidence for the central role of MerTK and its cleavage as one of the extensive dysregulated pathways in the development of atherothrombosis. However, the specific molecules that induce MerTK cleavage or that bridge apoptotic cells to MerTK receptors on macrophages remain largely unexplored in this study.
Bishuang Cai et al. 2017 “MerTK receptor cleavage promotes plaque necrosis and defective resolution in atherosclerosis” J Clin Invest. 2017;127(2):564–568
January 26, 2017
Vexed about ineffective islet cell engraftment? Use Dex!
Local delivery of the anti-inflammatory agent dexamethasone from polydimethylsiloxane scaffolds improved transplantation of encapsulated islet cells in vivo.
By Emily Lurier and Stan de Vries
Type I diabetes is an autoimmune disease in which the body mistakenly attacks and kills insulin-producing beta cells. Without insulin, the body is incapable of regulating blood glucose levels, which can lead to tissue damage and even death. Researchers have attempted to graft donor islet cells into patients, but the ensuing inflammatory reaction causes significant early islet cell death. Therefore, Jiang et al. hypothesized that protecting the islet cells via encapsulation in an anti-inflammatory drug-loaded gel would increase graft efficacy and longevity by inhibiting the inflammatory response in vivo.
The authors developed a polydimethylsiloxane (PDMS) scaffold loaded with the anti-inflammatory therapeutic dexamethasone (Dex). The gels were then incubated in fibronectin to increase efficiency of cell attachment, and finally loaded with healthy murine-derived islet cells. To test the local release rate and optimal dose of Dex, gels loaded with four different concentrations of Dex (1, 0.5, 0.25, 0.1%) were implanted into the epididymal fat pad of mice with pharmacologically induced diabetes (n=4 mice per group) for 5,7, and 14 days. The gels loaded with the two lower doses of Dex restored normal glucose levels in more than 90% of the animals, effects that were sustained for up to 40 days post-retrieval of the gels from the mice. These doses of Dex also elicited higher levels of cellular infiltration and vascularization, as well as reduced fibrosis, compared to the drug-free control gel and gels loaded with higher doses of Dex, which ultimately improved engraftment efficiency. To investigate the mechanism in which Dex increased tissue integration and graft efficacy, the authors extracted scaffolds at 5, 7, and 14 days post-implantation to evaluate presence of macrophages during early and late stages of the engraftment response. By examining the presence of pro-inflammatory M1 (CD274+/CX4CR1-) and M2 (CX4CR1+/CD274-) macrophages, the authors showed that Dex-loaded scaffolds gave rise to a shift from M1 to a Dex-induced M2 phenotype which lead to increased engraftment and improved outcomes.
Overall, the authors identified that controlled delivery of Dex from scaffolds encapsulating allogenic islet cells and subsequently promoting the anti-inflammatory M2 phenotype at the engraftment site prolongs the viability and behavior of islet cells in vivo. This study also highlighted important considerations with respect to dose and timing of anti-inflammatory drug delivery.
Jiang, Kaiyuan, et al. "Local release of dexamethasone from macroporous scaffolds accelerates islet transplant engraftment by promotion of anti-inflammatory M2 macrophages." Biomaterials 114 (2017): 71-81.
By Emily Lurier and Stan de Vries
Type I diabetes is an autoimmune disease in which the body mistakenly attacks and kills insulin-producing beta cells. Without insulin, the body is incapable of regulating blood glucose levels, which can lead to tissue damage and even death. Researchers have attempted to graft donor islet cells into patients, but the ensuing inflammatory reaction causes significant early islet cell death. Therefore, Jiang et al. hypothesized that protecting the islet cells via encapsulation in an anti-inflammatory drug-loaded gel would increase graft efficacy and longevity by inhibiting the inflammatory response in vivo.
The authors developed a polydimethylsiloxane (PDMS) scaffold loaded with the anti-inflammatory therapeutic dexamethasone (Dex). The gels were then incubated in fibronectin to increase efficiency of cell attachment, and finally loaded with healthy murine-derived islet cells. To test the local release rate and optimal dose of Dex, gels loaded with four different concentrations of Dex (1, 0.5, 0.25, 0.1%) were implanted into the epididymal fat pad of mice with pharmacologically induced diabetes (n=4 mice per group) for 5,7, and 14 days. The gels loaded with the two lower doses of Dex restored normal glucose levels in more than 90% of the animals, effects that were sustained for up to 40 days post-retrieval of the gels from the mice. These doses of Dex also elicited higher levels of cellular infiltration and vascularization, as well as reduced fibrosis, compared to the drug-free control gel and gels loaded with higher doses of Dex, which ultimately improved engraftment efficiency. To investigate the mechanism in which Dex increased tissue integration and graft efficacy, the authors extracted scaffolds at 5, 7, and 14 days post-implantation to evaluate presence of macrophages during early and late stages of the engraftment response. By examining the presence of pro-inflammatory M1 (CD274+/CX4CR1-) and M2 (CX4CR1+/CD274-) macrophages, the authors showed that Dex-loaded scaffolds gave rise to a shift from M1 to a Dex-induced M2 phenotype which lead to increased engraftment and improved outcomes.
Overall, the authors identified that controlled delivery of Dex from scaffolds encapsulating allogenic islet cells and subsequently promoting the anti-inflammatory M2 phenotype at the engraftment site prolongs the viability and behavior of islet cells in vivo. This study also highlighted important considerations with respect to dose and timing of anti-inflammatory drug delivery.
Jiang, Kaiyuan, et al. "Local release of dexamethasone from macroporous scaffolds accelerates islet transplant engraftment by promotion of anti-inflammatory M2 macrophages." Biomaterials 114 (2017): 71-81.
January 5, 2017
It’s a vessel! It’s perfusable! It’s…a macrophage?
The unrecognized role of macrophages in vascular mimicry
By: Pam and Anamika
Macrophages, primary regulators of the inflammatory response, have been shown to play a supportive, albeit elusive, role in the growth of new blood vessels from pre-existing vasculature, a process referred to as angiogenesis. Based on a growing body of evidence implicating macrophages in both healthy and pathological angiogenesis, and the potential of macrophages to differentiate into endothelial-like cells, Barnett et al. hypothesized that macrophages may also participate in “vascular mimicry” – the development of functional vessels that lack endothelial cells.
To test this hypothesis, the authors subcutaneously implanted Matrigel, an extracellular matrix-based hydrogel that supports angiogenesis, into transgenic mice in which the CX3CR1 gene (expressed by monocytes, macrophages, and other white blood cells) was modified with green fluorescent protein (GFP) to enable cell tracking via confocal microscopy. The Matrigel plugs were explanted 10 days post-injection and analyzed via immunohistochemistry for the presence of macrophage marker F4/80. Remarkably, GFP+ F4/80+ macrophages assembled into an interconnected cellular network, suggesting that macrophages exhibit angiogenic behavior. Interestingly, Matrigel supplementation with either pro-angiogenic stimuli (vascular endothelial growth factor) or pro-inflammatory stimuli (interferon and/or granulocyte macrophage colony stimulating factor) induced similar macrophage networks, which may suggest that macrophages also respond to angiogenic stimuli. Time course analysis of the infiltration of GFP+ cells into the plug in vivo revealed tubular network formation and co-expression of CD31, prior to the appearance of endothelial blood vessels. Further immunohistochemical analysis revealed that vascular mimicry networks were comprised of many cells co-expressing macrophage and endothelial cell markers, including F4/80, CD163, CD206, CD11b, CD34, and TIE2, among others, though not all of the cells were GFP+. Notably, vascular mimicry networks were perfusable following intravenous injection of fluorescent dye, demonstrating that the networks connected to the systemic endothelial vasculature. Finally, inhibiting macrophage activity, either by pharmaceutically blocking macrophage differentiation from monocytes or by clodronate-induced apoptosis of macrophages, caused a significant reduction in the presence of F4/80+ CD31+ vascular mimicry cells, with a compensatory increase in F4/80- CD31+ cells, and significantly decreased the extent of plug perfusion. Using a murine knockout model of monocyte/macrophage-specific hypoxia-inducible factor 1-alpha (HIF-1a) the authors then demonstrated that hypoxia is a major driver of vascular mimicry network formation in both matrigel plugs and tumors, in which macrophages co-expressed CD11b, CD163, F4/80 and CD31.
Collectively, these data provide evidence that macrophages can form perfusable vascular networks in the absence of endothelial cells, and may contribute to angiogenesis in hypoxic environments, which has important implications for treating diseases characterized by abnormal vessel growth. However, the contributions of distinct macrophage populations in this process remain to be examined.
F.H. Barnett et al. 2016 “Macrophages form functional vascular mimicry channels in vivo.” Scientific reports 6, 36659.
By: Pam and Anamika
Macrophages, primary regulators of the inflammatory response, have been shown to play a supportive, albeit elusive, role in the growth of new blood vessels from pre-existing vasculature, a process referred to as angiogenesis. Based on a growing body of evidence implicating macrophages in both healthy and pathological angiogenesis, and the potential of macrophages to differentiate into endothelial-like cells, Barnett et al. hypothesized that macrophages may also participate in “vascular mimicry” – the development of functional vessels that lack endothelial cells.
To test this hypothesis, the authors subcutaneously implanted Matrigel, an extracellular matrix-based hydrogel that supports angiogenesis, into transgenic mice in which the CX3CR1 gene (expressed by monocytes, macrophages, and other white blood cells) was modified with green fluorescent protein (GFP) to enable cell tracking via confocal microscopy. The Matrigel plugs were explanted 10 days post-injection and analyzed via immunohistochemistry for the presence of macrophage marker F4/80. Remarkably, GFP+ F4/80+ macrophages assembled into an interconnected cellular network, suggesting that macrophages exhibit angiogenic behavior. Interestingly, Matrigel supplementation with either pro-angiogenic stimuli (vascular endothelial growth factor) or pro-inflammatory stimuli (interferon and/or granulocyte macrophage colony stimulating factor) induced similar macrophage networks, which may suggest that macrophages also respond to angiogenic stimuli. Time course analysis of the infiltration of GFP+ cells into the plug in vivo revealed tubular network formation and co-expression of CD31, prior to the appearance of endothelial blood vessels. Further immunohistochemical analysis revealed that vascular mimicry networks were comprised of many cells co-expressing macrophage and endothelial cell markers, including F4/80, CD163, CD206, CD11b, CD34, and TIE2, among others, though not all of the cells were GFP+. Notably, vascular mimicry networks were perfusable following intravenous injection of fluorescent dye, demonstrating that the networks connected to the systemic endothelial vasculature. Finally, inhibiting macrophage activity, either by pharmaceutically blocking macrophage differentiation from monocytes or by clodronate-induced apoptosis of macrophages, caused a significant reduction in the presence of F4/80+ CD31+ vascular mimicry cells, with a compensatory increase in F4/80- CD31+ cells, and significantly decreased the extent of plug perfusion. Using a murine knockout model of monocyte/macrophage-specific hypoxia-inducible factor 1-alpha (HIF-1a) the authors then demonstrated that hypoxia is a major driver of vascular mimicry network formation in both matrigel plugs and tumors, in which macrophages co-expressed CD11b, CD163, F4/80 and CD31.
Collectively, these data provide evidence that macrophages can form perfusable vascular networks in the absence of endothelial cells, and may contribute to angiogenesis in hypoxic environments, which has important implications for treating diseases characterized by abnormal vessel growth. However, the contributions of distinct macrophage populations in this process remain to be examined.
F.H. Barnett et al. 2016 “Macrophages form functional vascular mimicry channels in vivo.” Scientific reports 6, 36659.
November 15, 2016
Restoring efferocytosis to treat atherosclerosiS
Blocking the “anti-eat me” receptor CD47 restores efferocytosis, resulting in decreased atherosclerotic plaque formation.
By: Emily and Val
Efferocytosis, or programmed cell removal, of cellular debris and pathogens by professional phagocytes like macrophages, is essential to initiate wound healing resolution and to promote healthy tissue deposition following injury. Impaired efferocytosis has been linked to the development of a variety of pathologies including cancer, stroke and atherosclerotic plaque formation. The mechanism by which programmed cell removal becomes dysregulated is not well understood. Kojima et al. recently identified expression of CD47, a “don’t eat me” signal, in cancerous cells, which promoted tumor growth by inhibiting the phagocytic activity of macrophages. Therefore, the authors hypothesized that inhibiting CD47 expression in atherosclerosis may mitigate plaque formation by restoring the phagocytic capacity of macrophages.
To test this hypothesis, Kojima et al. analyzed CD47 expression in both diseased and healthy tissue and found that CD47 expression was upregulated in atherosclerotic plaques compared to healthy tissue. Based on these results, the authors treated an atheroprone (apoE-/-) mouse model with an inhibitory antibody against CD47 to determine the effects on atherosclerotic plaque formation. Histological analysis of explanted tissue showed that the treatment led to a significant decrease in atherosclerotic plaque formation in the aortic sinus and aorta. Then, an in vitro phagocytosis assay was used to determine that blocking CD47 in RAW 264.7 macrophages resulted in increased clearance of apoptotic and diseased cells. Assessment of the downstream signaling pathways of CD47 expression including phosphorylation of SHP1 via immunohistochemistry revealed that blocking CD47 reactivated efferocytosis without affecting apoptosis in the local environment. The authors also determined on the genetic and functional level that the pro-inflammatory molecule TNFa is a key regulator of CD47 expression. Nonetheless, macrophages primed with TNFa were still able to recover their efferocytotic capacity upon treatment with anti-CD47 antibodies.
In conclusion, Kojima et al. provided insights into the mechanistic nature of impaired efferocytosis in atherosclerotic plaque formation while strengthening the association between atherosclerosis and inflammation. Furthermore, administration of the anti-CD47 antibody in atherosclerosis represents a potential non-surgical method for treating and inhibiting plaque formation. However, the treatment was systemic, and so future studies should develop this treatment to be more targeted towards the pathological site.
Kojima, Y., Volkmer, J. P., McKenna, K., Civelek, M., Lusis, A. J., Miller, C. L., ... & Schadt, E. E. (2016). CD47-blocking antibodies restore phagocytosis and prevent atherosclerosis. Nature.
By: Emily and Val
Efferocytosis, or programmed cell removal, of cellular debris and pathogens by professional phagocytes like macrophages, is essential to initiate wound healing resolution and to promote healthy tissue deposition following injury. Impaired efferocytosis has been linked to the development of a variety of pathologies including cancer, stroke and atherosclerotic plaque formation. The mechanism by which programmed cell removal becomes dysregulated is not well understood. Kojima et al. recently identified expression of CD47, a “don’t eat me” signal, in cancerous cells, which promoted tumor growth by inhibiting the phagocytic activity of macrophages. Therefore, the authors hypothesized that inhibiting CD47 expression in atherosclerosis may mitigate plaque formation by restoring the phagocytic capacity of macrophages.
To test this hypothesis, Kojima et al. analyzed CD47 expression in both diseased and healthy tissue and found that CD47 expression was upregulated in atherosclerotic plaques compared to healthy tissue. Based on these results, the authors treated an atheroprone (apoE-/-) mouse model with an inhibitory antibody against CD47 to determine the effects on atherosclerotic plaque formation. Histological analysis of explanted tissue showed that the treatment led to a significant decrease in atherosclerotic plaque formation in the aortic sinus and aorta. Then, an in vitro phagocytosis assay was used to determine that blocking CD47 in RAW 264.7 macrophages resulted in increased clearance of apoptotic and diseased cells. Assessment of the downstream signaling pathways of CD47 expression including phosphorylation of SHP1 via immunohistochemistry revealed that blocking CD47 reactivated efferocytosis without affecting apoptosis in the local environment. The authors also determined on the genetic and functional level that the pro-inflammatory molecule TNFa is a key regulator of CD47 expression. Nonetheless, macrophages primed with TNFa were still able to recover their efferocytotic capacity upon treatment with anti-CD47 antibodies.
In conclusion, Kojima et al. provided insights into the mechanistic nature of impaired efferocytosis in atherosclerotic plaque formation while strengthening the association between atherosclerosis and inflammation. Furthermore, administration of the anti-CD47 antibody in atherosclerosis represents a potential non-surgical method for treating and inhibiting plaque formation. However, the treatment was systemic, and so future studies should develop this treatment to be more targeted towards the pathological site.
Kojima, Y., Volkmer, J. P., McKenna, K., Civelek, M., Lusis, A. J., Miller, C. L., ... & Schadt, E. E. (2016). CD47-blocking antibodies restore phagocytosis and prevent atherosclerosis. Nature.
November 3, 2016
MACROPHAGE RESPONSE TO BACTERIA REVEALS NOVEL GENES
Identification of four novel genes involved in macrophage response to gram-positive bacteria.
By: Carly and Brandon
Invasion of bacteria into the body through respiration or open wounds can lead to adverse health effects such as infection. The body’s first internal defense against the infiltration of bacteria is the innate immune system, and particularly macrophages. However, the mechanisms of how macrophages interact with bacteria are poorly understood. To address this challenge, Alper et al. explored the regulation of gene expression in macrophages when confronted with gram-positive bacteria.
The team cultured murine bone marrow-derived murine macrophages with the gram-positive bacterial cell wall component lipotechoic acid (LTA). The macrophages increased secretion of several pro-inflammatory cytokines, including IL-6, IL-12 and TNF-a. Then, whole genome association mapping analysis was utilized to identify 71 high-priority candidate genes that could be effectors of macrophage response to bacteria. Identification of these genes was constrained to genes having single nucleotide polymorphisms (SNPs) as well as other variants in DNA that associated with the genetic loci of IL-6, IL-12 and TNF-a. Functionality testing of these 71 genes was accomplished by inhibiting each identified gene using siRNAs in the RAW264.6 mouse macrophage cell line and correlating with downregulation of IL-6 secretion, resulting in the identification of Fbxo17, Bdkrb1, Blnk and Nkx6-1 as genes influencing the macrophage response to LTA. The same genes were then overexpressed using full-length cDNAs of each gene, leading to increased activation of the TNFa cascade, as measured by a reporter system for NF-kB-AP1. These results suggest that the genes Fbxo17, Bdkrb1, Blnk and Nkx6-1 influence macrophage response to gram-positive bacteria. To further verify results beyond the RAW264.6 cell line macrophage from mice that were genetically depleted of Bdkrb1 were exposed to three different strands of gram positive bacteria (LTA, HKSP, or HKSA) resulting in a downregulation of both IL-6 and TNFa production, further supporting that the identified gene Bdkrb1 is influential in the macrophage response to gram-positive bacteria.
Overall, Alper et al. identified four novel genes with strong evidence supporting their role in mediating the macrophage response to gram-positive bacteria. Better understanding of how the host immune system interacts with these bacteria will help in treating and possibly even preventing some of the resulting infections that can occur.
Alper, S., Warg, L. A., De Arras, L., Flatley, B. R., Davidson, E. J., Adams, J., . . . Yang, I. V. (2016). Novel innate immune genes regulating the macrophage response to gram positive bacteria. Genetics, 204(1), 327-327.
By: Carly and Brandon
Invasion of bacteria into the body through respiration or open wounds can lead to adverse health effects such as infection. The body’s first internal defense against the infiltration of bacteria is the innate immune system, and particularly macrophages. However, the mechanisms of how macrophages interact with bacteria are poorly understood. To address this challenge, Alper et al. explored the regulation of gene expression in macrophages when confronted with gram-positive bacteria.
The team cultured murine bone marrow-derived murine macrophages with the gram-positive bacterial cell wall component lipotechoic acid (LTA). The macrophages increased secretion of several pro-inflammatory cytokines, including IL-6, IL-12 and TNF-a. Then, whole genome association mapping analysis was utilized to identify 71 high-priority candidate genes that could be effectors of macrophage response to bacteria. Identification of these genes was constrained to genes having single nucleotide polymorphisms (SNPs) as well as other variants in DNA that associated with the genetic loci of IL-6, IL-12 and TNF-a. Functionality testing of these 71 genes was accomplished by inhibiting each identified gene using siRNAs in the RAW264.6 mouse macrophage cell line and correlating with downregulation of IL-6 secretion, resulting in the identification of Fbxo17, Bdkrb1, Blnk and Nkx6-1 as genes influencing the macrophage response to LTA. The same genes were then overexpressed using full-length cDNAs of each gene, leading to increased activation of the TNFa cascade, as measured by a reporter system for NF-kB-AP1. These results suggest that the genes Fbxo17, Bdkrb1, Blnk and Nkx6-1 influence macrophage response to gram-positive bacteria. To further verify results beyond the RAW264.6 cell line macrophage from mice that were genetically depleted of Bdkrb1 were exposed to three different strands of gram positive bacteria (LTA, HKSP, or HKSA) resulting in a downregulation of both IL-6 and TNFa production, further supporting that the identified gene Bdkrb1 is influential in the macrophage response to gram-positive bacteria.
Overall, Alper et al. identified four novel genes with strong evidence supporting their role in mediating the macrophage response to gram-positive bacteria. Better understanding of how the host immune system interacts with these bacteria will help in treating and possibly even preventing some of the resulting infections that can occur.
Alper, S., Warg, L. A., De Arras, L., Flatley, B. R., Davidson, E. J., Adams, J., . . . Yang, I. V. (2016). Novel innate immune genes regulating the macrophage response to gram positive bacteria. Genetics, 204(1), 327-327.
October 10, 2016
Hijacking macrophages to Target Tumors
Synthesizing and characterizing magnetically-controlled, drug-loaded macrophages for the treatment of cancer
By: Kate and Bhavani
Because current treatments for cancer tend to damage healthy cells and cause serious side effects, new methods must be created to attack tumors with precision. Macrophages, the primary innate immune cells of the body, have the unique capacity to home to and infiltrate cancerous tumors in the body. Furthermore, macrophages are especially phagocytic, making them natural targets to ingest drug-loaded nanoparticles and deliver them to tumors. However, two limitations of using macrophages as drug carriers are non-specific targeting and insufficient maintenance of drug concentrations at the intended site. Thus, Nguyen et al. designed “microrobots” – macrophages carrying chemotherapeutic drugs and magnetic particles – that would home to specific tumor sites and stay there through the external application of a magnetic field.
Iron oxide (Fe3O4) particles were synthesized using co-precipitation to make highly magnetic particles of 11 nm in diameter. Then, they were encapsulated together with the anti-cancer drug paclitaxel in multi-lamellar liposomes using a film hydration technique to prepare magnetic, drug-loaded particles (PTX-MLPs). These small (148 nm), magnetic (8 emu/g) liposomes were easily phagocytosed by murine macrophages in vitro with minimal cytotoxicity. In order to illustrate magnetic manipulation of macrophages after PTX-MLP phagocytosis, a three-dimensional magnetic field was used to control the movement of PTX-MLP-loaded macrophages in predetermined formations at an average velocity of 11μm/s in vitro. Using a transwell chemotaxis assay, untreated macrophages and macrophages with PTX-MLPs migrated in similar fashions when treated with cancer cell lysates or conditioned media– suggesting that phagocytosis would not interfere with endogenous tumor homing capabilities. The authors illustrated controlled release of paclitaxel over 24 hours from both PTX-MLP particles and macrophages containing PTX-MLP particles although the release rate was slightly reduced in the latter group. Finally, in vitro tumor cell cytotoxicity assays showed that both 4T1 and CT26 cancer cells were killed by conditioned media from macrophages that had taken up PTX-MLPs, but not by the particles alone, suggesting that the macrophages released paclitaxel and killed tumor cells through mitotic arrest – a process that may not harm homing macrophages.
Overall, these results support the idea that macrophage homing and subsequent killing of tumors could be enhanced through the use of magnetic, drug-loaded liposomes. Future studies characterizing these macrophage PTX-MLPs in vivo are essential to determine if these unique microrobots are capable of targeting cancerous cells in the body.
Nguyen VD, Han J, Go G, Zhen J, Zheng S, Le VH, Park JO, and Park S. Feasibility study of dual-targeting paclitaxel-loaded magnetic liposomes using electromagnetic actuation and macrophages. Sensors and Actuators B: Chemical. 2016; 240: 1226-1236.
By: Kate and Bhavani
Because current treatments for cancer tend to damage healthy cells and cause serious side effects, new methods must be created to attack tumors with precision. Macrophages, the primary innate immune cells of the body, have the unique capacity to home to and infiltrate cancerous tumors in the body. Furthermore, macrophages are especially phagocytic, making them natural targets to ingest drug-loaded nanoparticles and deliver them to tumors. However, two limitations of using macrophages as drug carriers are non-specific targeting and insufficient maintenance of drug concentrations at the intended site. Thus, Nguyen et al. designed “microrobots” – macrophages carrying chemotherapeutic drugs and magnetic particles – that would home to specific tumor sites and stay there through the external application of a magnetic field.
Iron oxide (Fe3O4) particles were synthesized using co-precipitation to make highly magnetic particles of 11 nm in diameter. Then, they were encapsulated together with the anti-cancer drug paclitaxel in multi-lamellar liposomes using a film hydration technique to prepare magnetic, drug-loaded particles (PTX-MLPs). These small (148 nm), magnetic (8 emu/g) liposomes were easily phagocytosed by murine macrophages in vitro with minimal cytotoxicity. In order to illustrate magnetic manipulation of macrophages after PTX-MLP phagocytosis, a three-dimensional magnetic field was used to control the movement of PTX-MLP-loaded macrophages in predetermined formations at an average velocity of 11μm/s in vitro. Using a transwell chemotaxis assay, untreated macrophages and macrophages with PTX-MLPs migrated in similar fashions when treated with cancer cell lysates or conditioned media– suggesting that phagocytosis would not interfere with endogenous tumor homing capabilities. The authors illustrated controlled release of paclitaxel over 24 hours from both PTX-MLP particles and macrophages containing PTX-MLP particles although the release rate was slightly reduced in the latter group. Finally, in vitro tumor cell cytotoxicity assays showed that both 4T1 and CT26 cancer cells were killed by conditioned media from macrophages that had taken up PTX-MLPs, but not by the particles alone, suggesting that the macrophages released paclitaxel and killed tumor cells through mitotic arrest – a process that may not harm homing macrophages.
Overall, these results support the idea that macrophage homing and subsequent killing of tumors could be enhanced through the use of magnetic, drug-loaded liposomes. Future studies characterizing these macrophage PTX-MLPs in vivo are essential to determine if these unique microrobots are capable of targeting cancerous cells in the body.
Nguyen VD, Han J, Go G, Zhen J, Zheng S, Le VH, Park JO, and Park S. Feasibility study of dual-targeting paclitaxel-loaded magnetic liposomes using electromagnetic actuation and macrophages. Sensors and Actuators B: Chemical. 2016; 240: 1226-1236.
May 26, 2016
Stubborn macrophages and poor IL10 in hyperglycemia
Chronic low-grade inflammation in type 2 diabetes causes hyporesponsiveness to interleukin-10
By: The Wounds team (Anamika, Brandon, Nicole and Donald)
Type 2 Diabetes (T2D) is characterized by chronic low grade inflammation and is associated with increased circulatory levels of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). Interleukin-10 (IL10) is an anti-inflammatory cytokine that helps to resolve inflammation and inhibit macrophage activation. Although inhibition of pro-inflammatory signaling in T2D has been described, the impact of T2D on signaling of anti-inflammatory molecules has not been evaluated.
Barry et al. explored the effects of high glucose levels on the ability of IL10 to inhibit inflammation in human T2D via macrophage models in vitro. The team first utilized whole blood from T2D and non-T2D patients to determine the difference in responsiveness to IL10 in high glycemic conditions after stimulation with pro-inflammatory bacterial lipopolysaccharide (LPS). They found that immune cells in the blood from healthy volunteers were more responsive to IL10 than those from T2D patients, as measured by a greater decrease in TNF-alpha secretion. Interestingly, flow cytometry revealed that expression of the IL10 receptor was not different between cells in the two groups, thus failing to explain the differences in their response to IL10. Then, the team examined the effect of hyperglycemic conditions on responsiveness to IL10. They cultured both cell line- and bone marrow-derived macrophages from mice in normal and high glucose conditions, and then stimulated them with LPS in the presence or absence of IL10. Interestingly, high glucose levels reduced the effectiveness of IL10 to inhibit LPS-induced TNF-alpha secretion. In cell line macrophages, high glucose levels reduced the ability of IL10 to induce STAT3 phosphorylation, thus decreasing downstream intracellular IL10 signal transduction. Finally, the team investigated a potential therapeutic to overcome IL10 resistance in hyperglycemia using AQX-MN100, which has anti-inflammatory actions that are similar to IL10 but through a STAT3-independent pathway involving SHIP1. Although the cells were resistant to IL10 in high glucose, AQX-MN100 effectively inhibited LPS-stimulated TNF–alpha secretion.
Collectively, these results indicate that macrophages from both T2D patients and hyperglycemic culture conditions have diminished IL10 function. Further investigations are warranted to identify key mechanisms involved in dysfunctional IL10 signaling pathway in T2D patients.
Barry JC, Shakibakho S, Durrer C, Simtchouk S, Jawanda KK, Cheung ST, Mui AL, and Little JP. Hyporesponsiveness to the anti-inflammatory action of interleukin-10 in type 2 diabetes. Scientific reports. 2016; 6:21244
By: The Wounds team (Anamika, Brandon, Nicole and Donald)
Type 2 Diabetes (T2D) is characterized by chronic low grade inflammation and is associated with increased circulatory levels of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). Interleukin-10 (IL10) is an anti-inflammatory cytokine that helps to resolve inflammation and inhibit macrophage activation. Although inhibition of pro-inflammatory signaling in T2D has been described, the impact of T2D on signaling of anti-inflammatory molecules has not been evaluated.
Barry et al. explored the effects of high glucose levels on the ability of IL10 to inhibit inflammation in human T2D via macrophage models in vitro. The team first utilized whole blood from T2D and non-T2D patients to determine the difference in responsiveness to IL10 in high glycemic conditions after stimulation with pro-inflammatory bacterial lipopolysaccharide (LPS). They found that immune cells in the blood from healthy volunteers were more responsive to IL10 than those from T2D patients, as measured by a greater decrease in TNF-alpha secretion. Interestingly, flow cytometry revealed that expression of the IL10 receptor was not different between cells in the two groups, thus failing to explain the differences in their response to IL10. Then, the team examined the effect of hyperglycemic conditions on responsiveness to IL10. They cultured both cell line- and bone marrow-derived macrophages from mice in normal and high glucose conditions, and then stimulated them with LPS in the presence or absence of IL10. Interestingly, high glucose levels reduced the effectiveness of IL10 to inhibit LPS-induced TNF-alpha secretion. In cell line macrophages, high glucose levels reduced the ability of IL10 to induce STAT3 phosphorylation, thus decreasing downstream intracellular IL10 signal transduction. Finally, the team investigated a potential therapeutic to overcome IL10 resistance in hyperglycemia using AQX-MN100, which has anti-inflammatory actions that are similar to IL10 but through a STAT3-independent pathway involving SHIP1. Although the cells were resistant to IL10 in high glucose, AQX-MN100 effectively inhibited LPS-stimulated TNF–alpha secretion.
Collectively, these results indicate that macrophages from both T2D patients and hyperglycemic culture conditions have diminished IL10 function. Further investigations are warranted to identify key mechanisms involved in dysfunctional IL10 signaling pathway in T2D patients.
Barry JC, Shakibakho S, Durrer C, Simtchouk S, Jawanda KK, Cheung ST, Mui AL, and Little JP. Hyporesponsiveness to the anti-inflammatory action of interleukin-10 in type 2 diabetes. Scientific reports. 2016; 6:21244
May 12, 2016
Making moves on miRNA-mediated macrophage modulation
Local delivery of miR-223 loaded nanoparticles reduces foreign body giant cell formation following biomaterial implantation
By: The Materials Team (Amanda, Emily, Kate, Claire, Nathan)
Implanted biomaterials are quickly recognized by macrophages as foreign contaminants in vivo via the foreign body response (FBR). During the FBR, macrophages fail to degrade and phagocytose the implant, causing macrophages to fuse together into multinucleated foreign body giant cells (FBGCs). Consequently, the biomaterial becomes surrounded by FBGCs and quarantined by a dense fibrous capsule that ultimately inhibits tissue-biomaterial integration and implant efficacy. While the hallmarks of the FBR are well defined, the mechanisms, regulation, and interplay of molecular mediators remain poorly understood. To begin to address this challenge, Moore et al. explored the role of a key micro RNA (miRNA), a small noncoding RNA molecule that regulates gene expression, in FBGC formation in vitro and in vivo.
In a previous study, Moore et al. created FGBCs by stimulating macrophages with interleukin-4 (IL-4), and identified miR-223 as a key inhibitor of macrophage fusion using a microarray. To extend this finding, macrophages from miR-223-deficient mice were cultured on non-degradable polydimethylsiloxane (PDMS) discs in vitro. The cells fused into FBGCs to a greater extent than macrophages from control, wild type mice. Subsequently, these results were further verified in vivo by intraperitoneal implantation of PDMS discs; a greater number of FBGCs were observed surrounding the discs in miR-223-deficient mice relative to wild type mice. Finally, in a proof-of-concept therapeutic study, the team implanted PDMS discs into the peritoneum of mice to recruit macrophages and subsequently injected poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) encapsulating a miR-223 mimic. NP treatment resulted in a significant decrease in FBGC formation relative to controls.
Together, these results support the potential of miR-223 as a therapeutic to reduce FBGC formation surrounding implanted biomaterials. Additional studies, including release kinetics of the miR-223 mimic from nanoparticles and longer term studies, are necessary to elucidate if the delivery of miR-223 is a viable strategy for improving biomaterial implant survival and efficacy.
Moore, Laura Beth, Andrew J. Sawyer, Jennifer Saucier-Sawyer, W. Mark Saltzman, and Themis R. Kyriakides. "Nanoparticle delivery of miR-223 to attenuate macrophage fusion." Biomaterials 89 (2016): 127-135.
By: The Materials Team (Amanda, Emily, Kate, Claire, Nathan)
Implanted biomaterials are quickly recognized by macrophages as foreign contaminants in vivo via the foreign body response (FBR). During the FBR, macrophages fail to degrade and phagocytose the implant, causing macrophages to fuse together into multinucleated foreign body giant cells (FBGCs). Consequently, the biomaterial becomes surrounded by FBGCs and quarantined by a dense fibrous capsule that ultimately inhibits tissue-biomaterial integration and implant efficacy. While the hallmarks of the FBR are well defined, the mechanisms, regulation, and interplay of molecular mediators remain poorly understood. To begin to address this challenge, Moore et al. explored the role of a key micro RNA (miRNA), a small noncoding RNA molecule that regulates gene expression, in FBGC formation in vitro and in vivo.
In a previous study, Moore et al. created FGBCs by stimulating macrophages with interleukin-4 (IL-4), and identified miR-223 as a key inhibitor of macrophage fusion using a microarray. To extend this finding, macrophages from miR-223-deficient mice were cultured on non-degradable polydimethylsiloxane (PDMS) discs in vitro. The cells fused into FBGCs to a greater extent than macrophages from control, wild type mice. Subsequently, these results were further verified in vivo by intraperitoneal implantation of PDMS discs; a greater number of FBGCs were observed surrounding the discs in miR-223-deficient mice relative to wild type mice. Finally, in a proof-of-concept therapeutic study, the team implanted PDMS discs into the peritoneum of mice to recruit macrophages and subsequently injected poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) encapsulating a miR-223 mimic. NP treatment resulted in a significant decrease in FBGC formation relative to controls.
Together, these results support the potential of miR-223 as a therapeutic to reduce FBGC formation surrounding implanted biomaterials. Additional studies, including release kinetics of the miR-223 mimic from nanoparticles and longer term studies, are necessary to elucidate if the delivery of miR-223 is a viable strategy for improving biomaterial implant survival and efficacy.
Moore, Laura Beth, Andrew J. Sawyer, Jennifer Saucier-Sawyer, W. Mark Saltzman, and Themis R. Kyriakides. "Nanoparticle delivery of miR-223 to attenuate macrophage fusion." Biomaterials 89 (2016): 127-135.
April 26, 2016
Mac attack on fibrin
Extravascular fibrin is cleared by CCR2-positive macrophages in a novel endocytic mediated pathway.
By: The Mechanisms Team (Reham, Pam, Claire, Kate, Val)
In response to tissue injury, fibrin is deposited into the extravascular space, which stems bleeding and creates a provisional matrix to support healing. The formation and clearance of a fibrin clot is imperative to a normal healing response, but failure to clear fibrin is linked to chronic inflammation and organ failure. While it is well established that fibrin is enzymatically degraded by plasmin, the cells and receptors responsible for its clearance remain elusive. Since previous work has demonstrated that leukocytes (white blood cells) are recruited to extravascular fibrin deposits, Motley et al. hypothesized that extravascular fibrin may be endocytosed by leukocytes and digested by intracellular enzyme-laden lysosomes.
To test their hypothesis, Motley et al. subcutaneously implanted fluorescently-labeled fibrin hydrogels in mice, along with markers for cellular nuclei and lysosomes. Using intravital imaging, they visualized the co-localization of degraded fibrin with lysosomes, suggesting that fibrin clearance is mediated by endocytosis and lysosomal activity. Next, using whole-mount staining and transgenic mice, they revealed that immune cells called macrophages were the primary cells endocytosing fibrin. To distinguish between subpopulations of macrophages involved, the authors implanted fluorescently labeled fibrin into different transgenic mice. Implanted fibrin was primarily removed by macrophages that stained positively for CCR2, suggesting they were of a pro-inflammatory phenotype. In contrast, macrophages staining positively for CX3CR1, indicating they were of an anti-inflammatory phenotype, were not involved in fibrin clearance. Importantly, selective depletion of CCR2+ macrophages mitigated fibrin clearance, further implicating pro-inflammatory macrophages in the fibrin uptake process. Conducting a series of inhibition experiments subsequently revealed that fibrin endocytosis by CCR2+ macrophages depends on activated plasminogen and is independent of fibrin receptors aMb2 and ICAM-1, mannose receptor (responsible for collagen endocytosis by macrophages), and the principal myeloid cell integrin binding site on fibrin.
Overall, this study identifies a novel pathway for extravascular fibrin clearance by macrophages. Ultimately, understanding the mechanisms behind fibrin clearance will enable the design of strategies to supplement ineffective clearance of pathological extravascular fibrin.
M.P. Motley et al. "A CCR2 macrophage endocytic pathway mediates extravascular fibrin clearance in vivo." Blood 2016; 127(9):1085-1096
By: The Mechanisms Team (Reham, Pam, Claire, Kate, Val)
In response to tissue injury, fibrin is deposited into the extravascular space, which stems bleeding and creates a provisional matrix to support healing. The formation and clearance of a fibrin clot is imperative to a normal healing response, but failure to clear fibrin is linked to chronic inflammation and organ failure. While it is well established that fibrin is enzymatically degraded by plasmin, the cells and receptors responsible for its clearance remain elusive. Since previous work has demonstrated that leukocytes (white blood cells) are recruited to extravascular fibrin deposits, Motley et al. hypothesized that extravascular fibrin may be endocytosed by leukocytes and digested by intracellular enzyme-laden lysosomes.
To test their hypothesis, Motley et al. subcutaneously implanted fluorescently-labeled fibrin hydrogels in mice, along with markers for cellular nuclei and lysosomes. Using intravital imaging, they visualized the co-localization of degraded fibrin with lysosomes, suggesting that fibrin clearance is mediated by endocytosis and lysosomal activity. Next, using whole-mount staining and transgenic mice, they revealed that immune cells called macrophages were the primary cells endocytosing fibrin. To distinguish between subpopulations of macrophages involved, the authors implanted fluorescently labeled fibrin into different transgenic mice. Implanted fibrin was primarily removed by macrophages that stained positively for CCR2, suggesting they were of a pro-inflammatory phenotype. In contrast, macrophages staining positively for CX3CR1, indicating they were of an anti-inflammatory phenotype, were not involved in fibrin clearance. Importantly, selective depletion of CCR2+ macrophages mitigated fibrin clearance, further implicating pro-inflammatory macrophages in the fibrin uptake process. Conducting a series of inhibition experiments subsequently revealed that fibrin endocytosis by CCR2+ macrophages depends on activated plasminogen and is independent of fibrin receptors aMb2 and ICAM-1, mannose receptor (responsible for collagen endocytosis by macrophages), and the principal myeloid cell integrin binding site on fibrin.
Overall, this study identifies a novel pathway for extravascular fibrin clearance by macrophages. Ultimately, understanding the mechanisms behind fibrin clearance will enable the design of strategies to supplement ineffective clearance of pathological extravascular fibrin.
M.P. Motley et al. "A CCR2 macrophage endocytic pathway mediates extravascular fibrin clearance in vivo." Blood 2016; 127(9):1085-1096
February 18, 2016
The Hydrogel Library: Developing an immunomodulatory encyclopedia
Triazole surface modified hydrogels successfully inhibit the foreign body response to implanted biomaterials.
By: The Materials Team (Amanda, Emily, Kate, Claire, Nathan)
The immune-mediated foreign body response plagues the field of biomaterials by promoting fibrosis around implants, which prevents tissue integration and limits molecular diffusion and device efficacy. Macrophages, the professional phagocytes and initiators of the foreign body response, amplify reactive biological cascades by fusing together to form multinucleated bodies that stimulate formation of the fibrous capsule. There is a critical need for biomaterials design strategies that overcome this response in order to extend biomaterials lifetime in vivo. To increase understanding of the foreign body response, Vegas et al. examined a diverse array of chemically-modified alginate hydrogels for their potential to inhibit the foreign body response in vivo.
The authors employed a combinatorial approach to modify sodium alginate hydrogel, a well-established and versatile biomaterial, with a variety of functional groups in order to develop a library of 774 polymer analogs. These analogs were evaluated in a murine subcutaneous implantation model for their ability to curb the foreign body response in a high-throughput assay. Using a fluorescent probe to detect immune cell activation in vivo, Vegas et al. identified the top 10 formulations. Three of the least immunogenic implants (Z2-Y12, Z1-Y15, and Z1-Y19), all of which contained triazole groups, elicited the least immune cell infiltration and fibrous capsule formation, as determined by live imaging and immunohistochemical staining.
To further evaluate their immunomodulatory performance, the three types of alginate microspheres were administered to the intraperitoneal space of non-human primates. Histological staining showed little fibrotic deposition on Z1-Y15 and Z1-Y19 spheres and no detectable fibrosis around Z2-Y12 spheres. Incredibly, Z1-Y12 sphere retrieval maintained little fibrotic formation as late as 6 months after implantation. In order to identify the properties responsible for the observed decrease in the immune and fibrotic response, the authors also studied the surface properties of the microcapsules, including topography. Despite a lack of topographical differences between the particles, there were no differences in the foreign body response, suggesting that the presence of the triazole groups may be primarily responsible for immunomodulatory behavior.
These results have major implications for the design of biomedical implants. The authors’ biomaterials design strategy and high-throughput in vivo model constitute a novel, systematic approach to the development of immunomodulatory biomaterials. However, further investigation is required to understand how triazole modifications prevent immune cell activation.
Vegas, A. J., Veiseh, O., Doloff, J. C., Ma, M., Tam, H. H., Bratlie, K., ... & Fenton, P. (2016). Combinatorial hydrogel library enables identification of materials that mitigate the foreign body response in primates. Nature biotechnology doi:10.1038/nbt.3462
By: The Materials Team (Amanda, Emily, Kate, Claire, Nathan)
The immune-mediated foreign body response plagues the field of biomaterials by promoting fibrosis around implants, which prevents tissue integration and limits molecular diffusion and device efficacy. Macrophages, the professional phagocytes and initiators of the foreign body response, amplify reactive biological cascades by fusing together to form multinucleated bodies that stimulate formation of the fibrous capsule. There is a critical need for biomaterials design strategies that overcome this response in order to extend biomaterials lifetime in vivo. To increase understanding of the foreign body response, Vegas et al. examined a diverse array of chemically-modified alginate hydrogels for their potential to inhibit the foreign body response in vivo.
The authors employed a combinatorial approach to modify sodium alginate hydrogel, a well-established and versatile biomaterial, with a variety of functional groups in order to develop a library of 774 polymer analogs. These analogs were evaluated in a murine subcutaneous implantation model for their ability to curb the foreign body response in a high-throughput assay. Using a fluorescent probe to detect immune cell activation in vivo, Vegas et al. identified the top 10 formulations. Three of the least immunogenic implants (Z2-Y12, Z1-Y15, and Z1-Y19), all of which contained triazole groups, elicited the least immune cell infiltration and fibrous capsule formation, as determined by live imaging and immunohistochemical staining.
To further evaluate their immunomodulatory performance, the three types of alginate microspheres were administered to the intraperitoneal space of non-human primates. Histological staining showed little fibrotic deposition on Z1-Y15 and Z1-Y19 spheres and no detectable fibrosis around Z2-Y12 spheres. Incredibly, Z1-Y12 sphere retrieval maintained little fibrotic formation as late as 6 months after implantation. In order to identify the properties responsible for the observed decrease in the immune and fibrotic response, the authors also studied the surface properties of the microcapsules, including topography. Despite a lack of topographical differences between the particles, there were no differences in the foreign body response, suggesting that the presence of the triazole groups may be primarily responsible for immunomodulatory behavior.
These results have major implications for the design of biomedical implants. The authors’ biomaterials design strategy and high-throughput in vivo model constitute a novel, systematic approach to the development of immunomodulatory biomaterials. However, further investigation is required to understand how triazole modifications prevent immune cell activation.
Vegas, A. J., Veiseh, O., Doloff, J. C., Ma, M., Tam, H. H., Bratlie, K., ... & Fenton, P. (2016). Combinatorial hydrogel library enables identification of materials that mitigate the foreign body response in primates. Nature biotechnology doi:10.1038/nbt.3462
February 17, 2016
The Pac-man of the immune system: ruffled multinucleated giant cells
Macrophage-derived multinucleated giant cells hold potential for immune-targeted therapeutics to clear large foreign particles.
By: The Mechanisms Team (Claire, Val, Reham, Kate)
Fusion of macrophages to generate multinucleated giant cells (MGCs) has been described in the presence of pathogenic contaminants, surgical implants, and chronic inflammatory conditions that are correlated with excessive scar formation and implant rejection. It is believed that MGCs form in a process called “frustrated phagocytosis” when macrophages band together to ramp up efforts to degrade foreign materials. How this process affects phagocytosis is not well understood.
To explore the phagocytic potential of MGCs compared to their unfused counterparts, Milde et al. stimulated murine bone marrow-derived macrophages with interleukin-4 to simultaneously generate a mixed population of MGCs and unfused, anti-inflammatory M2 macrophages. After separating the population by number of nuclei, the researchers found that MGCs were more effective at phagocytizing foreign complement-coated red blood cells and large debris (10-45um) compared to M2 macrophages. Inhibition of the complement component C3 via blocking antibodies reduced the phagocytic ability of MGCs relative to M2 macrophages, suggesting that C3 plays a paramount role in the regulation of MGC phagocytosis. Following activation with phorbol 12-myristate 13-acetate (PMA), which increases phagocytic activity of macrophages, phagocytic activity of C3-opsonized targets was significantly increased in M2 macrophages, but not with MGC compared to their respective controls. These results suggest that PMA activation induces changes in the CR3 receptor in M2 macrophages, but not in MGCs. In turn, MGC’s CR3 receptor may be pre-activated to facilitate enhanced phagocytosis. Additionally, MGC formation led to an increase in the surface area to volume ratio and the formation of CR3-dense ruffles, both of which were hypothesized to enhance the ability of MGCs to phagocytize larger particles. Finally, to confirm MGC’s phagocytic potential in vivo, authors employed a murine model of systemic amyloid amyloidosis, a disease that is characterized by the build-up of pathogenic, misfolded protein called amyloids in the spleen, liver and lungs. Injection of a complement-activating IgG into the amyloid tissue resulted in an influx of macrophages within 24 hours. Macrophage fusion into MGCs was noted 1-2 days post injection, and the MGC’s almost entirely eliminated pathogenic amyloids in the spleen and liver after 14 days.
Although this work remains to be validated in humans, this proof-of-concept study established that MGC-mediated pathogen clearance may be useful for treating chronic inflammatory diseases like systemic amyloid amyloidosis.
R. Milde et al. "Multinucleated Giant Cells Are Specialized for Complement-Mediated Phagocytosis and Large Target Destruction." Cell Reports 13 (2015) 1937-1948
By: The Mechanisms Team (Claire, Val, Reham, Kate)
Fusion of macrophages to generate multinucleated giant cells (MGCs) has been described in the presence of pathogenic contaminants, surgical implants, and chronic inflammatory conditions that are correlated with excessive scar formation and implant rejection. It is believed that MGCs form in a process called “frustrated phagocytosis” when macrophages band together to ramp up efforts to degrade foreign materials. How this process affects phagocytosis is not well understood.
To explore the phagocytic potential of MGCs compared to their unfused counterparts, Milde et al. stimulated murine bone marrow-derived macrophages with interleukin-4 to simultaneously generate a mixed population of MGCs and unfused, anti-inflammatory M2 macrophages. After separating the population by number of nuclei, the researchers found that MGCs were more effective at phagocytizing foreign complement-coated red blood cells and large debris (10-45um) compared to M2 macrophages. Inhibition of the complement component C3 via blocking antibodies reduced the phagocytic ability of MGCs relative to M2 macrophages, suggesting that C3 plays a paramount role in the regulation of MGC phagocytosis. Following activation with phorbol 12-myristate 13-acetate (PMA), which increases phagocytic activity of macrophages, phagocytic activity of C3-opsonized targets was significantly increased in M2 macrophages, but not with MGC compared to their respective controls. These results suggest that PMA activation induces changes in the CR3 receptor in M2 macrophages, but not in MGCs. In turn, MGC’s CR3 receptor may be pre-activated to facilitate enhanced phagocytosis. Additionally, MGC formation led to an increase in the surface area to volume ratio and the formation of CR3-dense ruffles, both of which were hypothesized to enhance the ability of MGCs to phagocytize larger particles. Finally, to confirm MGC’s phagocytic potential in vivo, authors employed a murine model of systemic amyloid amyloidosis, a disease that is characterized by the build-up of pathogenic, misfolded protein called amyloids in the spleen, liver and lungs. Injection of a complement-activating IgG into the amyloid tissue resulted in an influx of macrophages within 24 hours. Macrophage fusion into MGCs was noted 1-2 days post injection, and the MGC’s almost entirely eliminated pathogenic amyloids in the spleen and liver after 14 days.
Although this work remains to be validated in humans, this proof-of-concept study established that MGC-mediated pathogen clearance may be useful for treating chronic inflammatory diseases like systemic amyloid amyloidosis.
R. Milde et al. "Multinucleated Giant Cells Are Specialized for Complement-Mediated Phagocytosis and Large Target Destruction." Cell Reports 13 (2015) 1937-1948
December 14, 2015
Macrophages dragged into the ongoing war on salt: High salt attenuates alternative activation of macrophage
Diets high in dietary salt led to a decreased ability to activate anti-inflammatory macrophages.
By: The Wounds and Classification team (Brandon, Anamika, Sina, and Nicole)
Dietary salt (NaCl) intake is often listed as a contributor to a number of disease conditions such as cardiac disease, hypertension, and autoimmune disorders. Recently, Binger et al. shown that high salt has a proinflammatory effect that enhances the activation of LPS-induced macrophages (M1). Because M1 macrophage activation inhibits activation of M2 macrophages, which play very important role in resolution of inflammation, the authors examined the effects of high salt on anti-inflammatory (M2) activation of macrophages with interleukin-4 (IL-4) and IL-13.
Binger et al. isolated bone marrow derived macrophages from mice and stimulated them with IL-4 and IL-13 in the absence of salt and with 40mM NaCl (high salt) for 0 hours to 7 days. As early as 1 hour after the addition of stimuli there was a decrease in gene expression of M2 markers, with even greater blunting effects after one week. However, the addition of 40 mM NaCl did not have any effects on cell viability, cell size, or purity of macrophage cultures. Binger et al. then examined if this finding correlated with a shift to M1 (LPS activated) pro-inflammatory activation. Numerous different testing methods including flow cytometry, protein expression, and genome-wide transcriptome microarrays showed no M1 activation in the macrophages in the presence of high salt concentrations. Since M2 macrophages can suppress T cell proliferation, a process that can amplify inflammation in autoimmune disorders, T cells were co-cultured with pre-stimulated IL-4/IL-13 macrophages either in the presence of elevated salt concentrations or not. While T-cell proliferation was suppressed with control M2 macrophages, T cells proliferated freely in the presence of M2 macrophages stimulated in high salt conditions, suggesting that high salt reduces the immune-suppressive capacity of M2 macrophages. Finally, mice fed with high salt diets for 14 days exhibited delayed wound healing, though this delay did not occur until the later stages (3-7 days) of healing, when M2 macrophages are thought to play a primary role. Gene expression of M2 markers was decreased in wound samples from mice fed a high salt diet, confirming decreased M2 activation of macrophages in vivo.
Collectively, these results indicate that high salt attenuates the activation and function of M2 macrophages, both in vitro and in vivo. While further investigations are required to elucidate the underlying mechanism through which high salt suppresses the activation and function of M2 macrophages, this study provides enough fuel to ignite the war on salt for the years to come.
Binger, K. J., et al. (2015). "High salt reduces the activation of IL-4- and IL-13-stimulated macrophages." J Clin Invest 125(11): 4223-4238.
By: The Wounds and Classification team (Brandon, Anamika, Sina, and Nicole)
Dietary salt (NaCl) intake is often listed as a contributor to a number of disease conditions such as cardiac disease, hypertension, and autoimmune disorders. Recently, Binger et al. shown that high salt has a proinflammatory effect that enhances the activation of LPS-induced macrophages (M1). Because M1 macrophage activation inhibits activation of M2 macrophages, which play very important role in resolution of inflammation, the authors examined the effects of high salt on anti-inflammatory (M2) activation of macrophages with interleukin-4 (IL-4) and IL-13.
Binger et al. isolated bone marrow derived macrophages from mice and stimulated them with IL-4 and IL-13 in the absence of salt and with 40mM NaCl (high salt) for 0 hours to 7 days. As early as 1 hour after the addition of stimuli there was a decrease in gene expression of M2 markers, with even greater blunting effects after one week. However, the addition of 40 mM NaCl did not have any effects on cell viability, cell size, or purity of macrophage cultures. Binger et al. then examined if this finding correlated with a shift to M1 (LPS activated) pro-inflammatory activation. Numerous different testing methods including flow cytometry, protein expression, and genome-wide transcriptome microarrays showed no M1 activation in the macrophages in the presence of high salt concentrations. Since M2 macrophages can suppress T cell proliferation, a process that can amplify inflammation in autoimmune disorders, T cells were co-cultured with pre-stimulated IL-4/IL-13 macrophages either in the presence of elevated salt concentrations or not. While T-cell proliferation was suppressed with control M2 macrophages, T cells proliferated freely in the presence of M2 macrophages stimulated in high salt conditions, suggesting that high salt reduces the immune-suppressive capacity of M2 macrophages. Finally, mice fed with high salt diets for 14 days exhibited delayed wound healing, though this delay did not occur until the later stages (3-7 days) of healing, when M2 macrophages are thought to play a primary role. Gene expression of M2 markers was decreased in wound samples from mice fed a high salt diet, confirming decreased M2 activation of macrophages in vivo.
Collectively, these results indicate that high salt attenuates the activation and function of M2 macrophages, both in vitro and in vivo. While further investigations are required to elucidate the underlying mechanism through which high salt suppresses the activation and function of M2 macrophages, this study provides enough fuel to ignite the war on salt for the years to come.
Binger, K. J., et al. (2015). "High salt reduces the activation of IL-4- and IL-13-stimulated macrophages." J Clin Invest 125(11): 4223-4238.
Schooling immune cells: Preventing Type I Diabetes with a novel vaccine platforM
A novel, multi-component vaccine successfully prevents Type 1 diabetes onset in non-obese mice
By: The Materials Team (Emily, Amanda, Kate, Claire, and Nathan)
Vaccines are agents that introduce inactivated pathogen to the immune system to stimulate the production of specific antibodies to prevent future infection. Novel vaccines are currently being developed to prevent immune cells from mistakenly producing antibodies against normal molecules. For example, in Type I diabetes (T1D), adaptive immune cells mistakenly recognize insulin, the hormone that modulates glucose levels, as pathogenic. As a result, insulin-producing beta-cells in the pancreas are targeted and destroyed by the host’s immune cells. This phenomenon results in pancreatic degradation, hypoglycemia, and hypertension, among other potentially fatal symptoms. To combat this autoimmune activation, Yoon et al. developed a novel vaccine containing inactivated insulin, granulocyte macrophage colony stimulating factor (GM-CSF), which serves as a chemoattractant for immune cells, and CpG oligodeoxynucleotides (CpG), which safely activates the innate immune system. These innate immune cells suppress the adaptive immune response in the presence of CpG, and “educate” the adaptive immunity on how to respond to insulin.
The researchers loaded an injectable PuraMatrixTM peptide hydrogel containing CpG and GM-CSF with poly(lactide-co-clycolide) microparticles containing denatured insulin. 90% of GM-CSF and CpG were released from the hydrogel within 48 hours in vitro. Additionally, cell migration studies showed that GM-CSF successfully acted as a chemoattractant for bone marrow-derived immune cells from non-obese diabetic (NOD) mice in vitro. To confirm efficacy in vivo, NOD mice were then injected with the vaccine at 8, 10 and 12 weeks after they were born. All mice in the control group developed diabetes, while 40% of vaccine-treated mice remained diabetes-free after 28 weeks, implying at least partial efficacy of the vaccine. Interestingly, the vaccine injection sites developed inflamed tissue sites (granulomas), with evidence of a rare lymphatic endothelial marker (LYVE-1) characteristic of a tertiary lymphoid organ, which produce adaptive immune cells. These granulomas were resolved within 28 weeks and suggest that the vaccine promoted the formation of a temporary immune-educating microenvironment.
Because of only limited efficacy (<40%), further optimization will be critical for the future of this vaccine. Nonetheless, this study constitutes proof of concept that a biomaterial-based tolerance-inducing platform might be useful for the prevention of autoimmune diseases like T1D.
Yoon, Young Mee, et al. "A combination hydrogel microparticle-based vaccine prevents type 1 diabetes in non-obese diabetic mice." Scientific reports 5 (2015).
By: The Materials Team (Emily, Amanda, Kate, Claire, and Nathan)
Vaccines are agents that introduce inactivated pathogen to the immune system to stimulate the production of specific antibodies to prevent future infection. Novel vaccines are currently being developed to prevent immune cells from mistakenly producing antibodies against normal molecules. For example, in Type I diabetes (T1D), adaptive immune cells mistakenly recognize insulin, the hormone that modulates glucose levels, as pathogenic. As a result, insulin-producing beta-cells in the pancreas are targeted and destroyed by the host’s immune cells. This phenomenon results in pancreatic degradation, hypoglycemia, and hypertension, among other potentially fatal symptoms. To combat this autoimmune activation, Yoon et al. developed a novel vaccine containing inactivated insulin, granulocyte macrophage colony stimulating factor (GM-CSF), which serves as a chemoattractant for immune cells, and CpG oligodeoxynucleotides (CpG), which safely activates the innate immune system. These innate immune cells suppress the adaptive immune response in the presence of CpG, and “educate” the adaptive immunity on how to respond to insulin.
The researchers loaded an injectable PuraMatrixTM peptide hydrogel containing CpG and GM-CSF with poly(lactide-co-clycolide) microparticles containing denatured insulin. 90% of GM-CSF and CpG were released from the hydrogel within 48 hours in vitro. Additionally, cell migration studies showed that GM-CSF successfully acted as a chemoattractant for bone marrow-derived immune cells from non-obese diabetic (NOD) mice in vitro. To confirm efficacy in vivo, NOD mice were then injected with the vaccine at 8, 10 and 12 weeks after they were born. All mice in the control group developed diabetes, while 40% of vaccine-treated mice remained diabetes-free after 28 weeks, implying at least partial efficacy of the vaccine. Interestingly, the vaccine injection sites developed inflamed tissue sites (granulomas), with evidence of a rare lymphatic endothelial marker (LYVE-1) characteristic of a tertiary lymphoid organ, which produce adaptive immune cells. These granulomas were resolved within 28 weeks and suggest that the vaccine promoted the formation of a temporary immune-educating microenvironment.
Because of only limited efficacy (<40%), further optimization will be critical for the future of this vaccine. Nonetheless, this study constitutes proof of concept that a biomaterial-based tolerance-inducing platform might be useful for the prevention of autoimmune diseases like T1D.
Yoon, Young Mee, et al. "A combination hydrogel microparticle-based vaccine prevents type 1 diabetes in non-obese diabetic mice." Scientific reports 5 (2015).
November 5, 2015
Lumican: A new target in fibrosiS
Lumican, secreted by fibroblasts, plays a major role in differentiation of monocytes into fibrotic cells.
By: The Mechanisms Team (Claire, Pam, Val, Kate)
In the presence of tissue damage, fibroblasts and monocyte-derived, fibroblast-like cells called fibrocytes, communicate to form scar tissue, also called fibrosis. Fibrocytes are primarily found in fibrotic lesions and destructive pathologies such as cirrhosis of the liver or the stiffening of cardiac tissue that leads to congestive heart failure. While fibrocytes secrete cytokines, like TNFa, TGFb, and IL-13, that promote fibroblasts to produce extracellular matrix (ECM), tissue-resident fibroblasts also secrete factors that promote immune cell entry and retention during inflammation. Thus, Pilling et al. hypothesized that fibroblasts may also induce fibrocyte differentiation of immune cells in a positive feedback loop to potentiate fibrosis.
To test this hypothesis, researchers cultured fibroblasts with recombinant TNFa, an inflammatory cytokine that is produced by monocytes, macrophages, and fibrocytes. Chemical characterization of the conditioned media via mass spectroscopy identified several peaks, but only albumin, lumican, and Slit2 were known to be ECM proteins, which are the bridge between cells and tissues. The team had previously determined that albumin and Slit2 did not promote fibrocyte differentiation, but the role of lumican was unknown. Delivery of recombinant lumican to human peripheral blood mononuclear cells (PBMC) and monocytes promoted their differentiation into fibrocytes in vitro, while lumican depletion resulted in a significant reduction in fibrocyte differentiation. To determine lumican’s signaling in fibrocyte differentiation, the team performed blocking experiments and found that integrin receptors a2b1, aMb2, and aXb2 were essential to lumican’s efficacy in fibrocyte differentiation. Lastly, the authors assessed the ability of lumican to compete with inhibitors of fibrocyte differentiation, SAP and Slit2. Lumican reduced the inhibitory action of SAP on fibrocyte differentiation of PBMCs, suggesting that lumican and SAP compete to inhibit fibrocyte differentiation. In contrast, Slit2’s ability to inhibit fibrocyte differentiation of PBMCs was completely overshadowed by lumican, suggesting no competition.
Using a murine model of pulmonary fibrosis, the authors detected significantly more lumican in the alveolar walls compared to controls. Additionally, the team found increased lumican in patients with pulmonary fibrosis compared to patients with chronic obstructive pulmonary disease (COPD), a non-fibrotic inflammatory condition. Taken together, these results show that lumican is a major player in the crosstalk between fibroblasts and fibrocytes, suggesting that therapeutic approaches to regulate lumican signaling may aid in inhibiting fibrosis.
D. Pilling et al. "TNF-α–stimulated fibroblasts secrete lumican to promote fibrocyte differentiation." PNAS 112 (2015) 38: 11929-11934
By: The Mechanisms Team (Claire, Pam, Val, Kate)
In the presence of tissue damage, fibroblasts and monocyte-derived, fibroblast-like cells called fibrocytes, communicate to form scar tissue, also called fibrosis. Fibrocytes are primarily found in fibrotic lesions and destructive pathologies such as cirrhosis of the liver or the stiffening of cardiac tissue that leads to congestive heart failure. While fibrocytes secrete cytokines, like TNFa, TGFb, and IL-13, that promote fibroblasts to produce extracellular matrix (ECM), tissue-resident fibroblasts also secrete factors that promote immune cell entry and retention during inflammation. Thus, Pilling et al. hypothesized that fibroblasts may also induce fibrocyte differentiation of immune cells in a positive feedback loop to potentiate fibrosis.
To test this hypothesis, researchers cultured fibroblasts with recombinant TNFa, an inflammatory cytokine that is produced by monocytes, macrophages, and fibrocytes. Chemical characterization of the conditioned media via mass spectroscopy identified several peaks, but only albumin, lumican, and Slit2 were known to be ECM proteins, which are the bridge between cells and tissues. The team had previously determined that albumin and Slit2 did not promote fibrocyte differentiation, but the role of lumican was unknown. Delivery of recombinant lumican to human peripheral blood mononuclear cells (PBMC) and monocytes promoted their differentiation into fibrocytes in vitro, while lumican depletion resulted in a significant reduction in fibrocyte differentiation. To determine lumican’s signaling in fibrocyte differentiation, the team performed blocking experiments and found that integrin receptors a2b1, aMb2, and aXb2 were essential to lumican’s efficacy in fibrocyte differentiation. Lastly, the authors assessed the ability of lumican to compete with inhibitors of fibrocyte differentiation, SAP and Slit2. Lumican reduced the inhibitory action of SAP on fibrocyte differentiation of PBMCs, suggesting that lumican and SAP compete to inhibit fibrocyte differentiation. In contrast, Slit2’s ability to inhibit fibrocyte differentiation of PBMCs was completely overshadowed by lumican, suggesting no competition.
Using a murine model of pulmonary fibrosis, the authors detected significantly more lumican in the alveolar walls compared to controls. Additionally, the team found increased lumican in patients with pulmonary fibrosis compared to patients with chronic obstructive pulmonary disease (COPD), a non-fibrotic inflammatory condition. Taken together, these results show that lumican is a major player in the crosstalk between fibroblasts and fibrocytes, suggesting that therapeutic approaches to regulate lumican signaling may aid in inhibiting fibrosis.
D. Pilling et al. "TNF-α–stimulated fibroblasts secrete lumican to promote fibrocyte differentiation." PNAS 112 (2015) 38: 11929-11934
August 10, 2015
MVP of MMPs: MMP10
MMP10 influences the expression of collagenolytic MMPs in M2 macrophages to regulate scar formation.
By: the Wounds Team (Anamika, Sina, Nicole, Desiree, Claire, Tony, Val)
Scar is composed of fibrous tissue that replace normal skin after injury. Excessive scarring can lead to a variety of clinical problems, including fibrosis, loss of tissue function and restriction of movement. Matrix metalloproteinases (MMPs) are key enzyme that mediate remodeling of scar into healthy tissue. MMP10 is not typically considered the most important MMP for collagen degradation, but it is secreted by macrophages, which contribute to scar formation through the production of profibrotic cytokines. Thus, Rohani et al. speculated that macrophage-secreted MMP10 moderates scar formation by controlling expression of other, more potent collagen-degrading MMPs.
To test this hypothesis, the authors examined the role of MMP10 in the restoration of epithelial tissue following wounding. Although wound closure rates between healthy and transgenic animals that lack MMP10 were comparable, they did observe elevated collagen deposition and skin stiffness, and less collagen-degrading activity in MMP10-deficient wounds. Using a broad-spectrum MMP inhibitor as well as inhibitors for specific MMPs, the team found that MMP10 controlled secretion of other MMPs, especially MMP13. Depletion of macrophages diminished collagenolytic activity in healthy mice but not in MMP10-deficient mice, indicating that macrophage-derived MMP10 regulates collagenolytic activity. The specific ablation of a specific phenotype of macrophages, also known as M2 (involved in tissue remodeling), with M2pepKLA significantly decreased collagenolytic activity in healthy but not in MMP10-deficient samples, suggesting that MMP10 controls the expression of collagenolytic MMPs in M2 macrophages. Macrophages from healthy mice that were genetically engineered to have green fluorescent cells were transferred into MMP10-deficient mice. The influx of fluorescent macrophages normalized the increased collagen deposition in MMP10-deficient wounds, indicating that MMP-10 controls the collagenolytic activity of M2-biased resident macrophages in excisional skin wounds.
This study illustrated a critical role of macrophage-derived MMP10 that controls collagen resolution through the promotion of specific MMPs. However, whether MMP-10 has a similar role in moderating fibrosis in diseases such as chronic wounds has yet to be determined. Understanding these mechanisms can contribute to new therapies to reduce excessive scarring often seen following injuries.
Rohani MG et al., "MMP-10 Regulates Collagenolytic Activity of Alternatively Activated Resident Macrophages." J. of Investigative Dermatology, 2015. doi: 10.1038/jid.2015.167
By: the Wounds Team (Anamika, Sina, Nicole, Desiree, Claire, Tony, Val)
Scar is composed of fibrous tissue that replace normal skin after injury. Excessive scarring can lead to a variety of clinical problems, including fibrosis, loss of tissue function and restriction of movement. Matrix metalloproteinases (MMPs) are key enzyme that mediate remodeling of scar into healthy tissue. MMP10 is not typically considered the most important MMP for collagen degradation, but it is secreted by macrophages, which contribute to scar formation through the production of profibrotic cytokines. Thus, Rohani et al. speculated that macrophage-secreted MMP10 moderates scar formation by controlling expression of other, more potent collagen-degrading MMPs.
To test this hypothesis, the authors examined the role of MMP10 in the restoration of epithelial tissue following wounding. Although wound closure rates between healthy and transgenic animals that lack MMP10 were comparable, they did observe elevated collagen deposition and skin stiffness, and less collagen-degrading activity in MMP10-deficient wounds. Using a broad-spectrum MMP inhibitor as well as inhibitors for specific MMPs, the team found that MMP10 controlled secretion of other MMPs, especially MMP13. Depletion of macrophages diminished collagenolytic activity in healthy mice but not in MMP10-deficient mice, indicating that macrophage-derived MMP10 regulates collagenolytic activity. The specific ablation of a specific phenotype of macrophages, also known as M2 (involved in tissue remodeling), with M2pepKLA significantly decreased collagenolytic activity in healthy but not in MMP10-deficient samples, suggesting that MMP10 controls the expression of collagenolytic MMPs in M2 macrophages. Macrophages from healthy mice that were genetically engineered to have green fluorescent cells were transferred into MMP10-deficient mice. The influx of fluorescent macrophages normalized the increased collagen deposition in MMP10-deficient wounds, indicating that MMP-10 controls the collagenolytic activity of M2-biased resident macrophages in excisional skin wounds.
This study illustrated a critical role of macrophage-derived MMP10 that controls collagen resolution through the promotion of specific MMPs. However, whether MMP-10 has a similar role in moderating fibrosis in diseases such as chronic wounds has yet to be determined. Understanding these mechanisms can contribute to new therapies to reduce excessive scarring often seen following injuries.
Rohani MG et al., "MMP-10 Regulates Collagenolytic Activity of Alternatively Activated Resident Macrophages." J. of Investigative Dermatology, 2015. doi: 10.1038/jid.2015.167
June 22, 2015
Macrophages and Astrocytes in Spinal Cord Injuries: Friends or Foe?
M1 macrophages induce astrocyte proliferation and a reactive phenotype while M2 macrophages reduce the reactive phenotype and limit proliferation.
By: The Macrophage Characterization Team (Brandon, Sina, Val, Nicole, Emily)
Glial scar formation is the body’s response to spinal cord injury (SCI) in order to re-establish the local environment that exists prior to disruption of the blood-brain barrier (BBB). In the early response to SCI, resident central nervous system immune cells, or microglia, and peripheral blood derived macrophages, secrete pro-inflammatory cytokines. Pro-inflammatory macrophages (M1) remove damaged tissue around the SCI through phagocytosis, propagating neuronal cell death post-injury. Reactive astrocytes proliferate then deposit fibrous proteins that form the glial scar. At this time, anti-inflammatory macrophages (M2) may aid in remeylination of damaged axons, although their role in the process is poorly understood. Therefore, Haan et al. aimed to investigate how interactions between astrocytes and macrophages propagate glial scar formation.
To study glial scar formation in vivo, a mouse model was used to develop a moderate contusive SCI. Both the experimental and sham groups were allowed to recover for 7 days. The spinal cords were extracted and sectioned for immunohistochemistry, staining for the pan macrophage marker CD68, the M1 marker iNOS and the M2 marker Arg1. After injury, a larger population of CD68+/iNOS+ and CD68+/Arg1+ macrophages were present in the injury compared to the sham group, with a higher majority of Arg1+ M2-like cells compared to iNOS+ M1-like cells in the injured group. Cell metabolic assays were used in vitro to identify the role of murine primary macrophage-derived signals on spinal cord sections. Cells were stained to determine reactivity using the proliferative marker EdU. Conditioned media derived from cultured M1 macrophages promoted an increase in the metabolic activities, as measured by the MTT assay, of the spinal cord tissue compared to that from M2 macrophages. Murine primary astrocytes, derived from neonatal glial cells, cultured in M1 macrophage-derived conditioned media also increased their metabolic activity compared to astrocytes cultured in M2 macrophage-derived media. Interestingly, these results are in contrast to other studies of the role of M1 and M2 macrophages in promoting fibroblast proliferation outside of the central nervous system. Conditioned media derived from astrocytes cultured in the M2-conditioned media significantly reduced the amount of cells undergoing cell division in M1 and M2 macrophages. These results suggest that M1 macrophages induce a reactive phenotype of astrocytes, and that M2 macrophages counteract this effect by decreasing the reactive phenotype of astrocytes. In turn, astrocytes limit macrophage mitosis, suggesting a negative feedback loop. Taken together, these results suggest that M1 macrophages activate astrocytes to begin developing the glial scar.
Although catabolic assays shed light on the overall effects of the cells types influencing one another, future work can be done to better understand the exact factors mediating the feedback between macrophages and astrocytes. These findings illustrate the delicate balance between macrophage and astrocyte phenotypes, and suggest a need for more targeted therapies towards balancing their interactions.
To study glial scar formation in vivo, a mouse model was used to develop a moderate contusive SCI. Both the experimental and sham groups were allowed to recover for 7 days. The spinal cords were extracted and sectioned for immunohistochemistry, staining for the pan macrophage marker CD68, the M1 marker iNOS and the M2 marker Arg1. After injury, a larger population of CD68+/iNOS+ and CD68+/Arg1+ macrophages were present in the injury compared to the sham group, with a higher majority of Arg1+ M2-like cells compared to iNOS+ M1-like cells in the injured group. Cell metabolic assays were used in vitro to identify the role of murine primary macrophage-derived signals on spinal cord sections. Cells were stained to determine reactivity using the proliferative marker EdU. Conditioned media derived from cultured M1 macrophages promoted an increase in the metabolic activities, as measured by the MTT assay, of the spinal cord tissue compared to that from M2 macrophages. Murine primary astrocytes, derived from neonatal glial cells, cultured in M1 macrophage-derived conditioned media also increased their metabolic activity compared to astrocytes cultured in M2 macrophage-derived media. Interestingly, these results are in contrast to other studies of the role of M1 and M2 macrophages in promoting fibroblast proliferation outside of the central nervous system. Conditioned media derived from astrocytes cultured in the M2-conditioned media significantly reduced the amount of cells undergoing cell division in M1 and M2 macrophages. These results suggest that M1 macrophages induce a reactive phenotype of astrocytes, and that M2 macrophages counteract this effect by decreasing the reactive phenotype of astrocytes. In turn, astrocytes limit macrophage mitosis, suggesting a negative feedback loop. Taken together, these results suggest that M1 macrophages activate astrocytes to begin developing the glial scar.
Although catabolic assays shed light on the overall effects of the cells types influencing one another, future work can be done to better understand the exact factors mediating the feedback between macrophages and astrocytes. These findings illustrate the delicate balance between macrophage and astrocyte phenotypes, and suggest a need for more targeted therapies towards balancing their interactions.
June 10, 2015
The molecular clock of immunity: to sleep or not to sleep?
miR-155 regulates the inflammatory response by targeting the BMAL1 molecular clock in macrophages.
By: the Materials Team (Sina, Emily, Nathan, Amanda, Claire)
The circadian rhythm of cells greatly affects many systems in the body, including the immune system. Evolution has molded the inflammatory response to become strongest when the body is most likely to be infected by pathogens. Any disruption to the cellular circadian clock alters the animal’s immune response, although the mechanism is poorly understood. Previously, circadian oscillations of Toll-like receptors (TLRs) - a class of proteins that play a key role in the innate immune system - and effector genes have been identified. BMAL1, a transcription factor that is involved in controlling the molecular clock of some TLRs, is also responsible for the recruitment of classically activated, pro-inflammatory monocytes (those labeled Ly6hi) into wounded tissue. Based on the key role of BMAL1 as a molecular clock, Curtis et al. hypothesized that temporal oscillations in the inflammatory response in macrophages might be regulated through suppression of BMAL1 via a small non-coding RNA known as miR-155.
Mice are nocturnal, so to study the circadian rhythm of murine cells, Curtis et al. utilized two strategies. First, an exogenous time signal - also known as zeitgeber time - was used to induce consecutive 12-hour phases of rest and activity. Second, they knocked out members of the clock machinery of the immune cells, namely BMAL1 and miR-155 to gain better understanding of their role in regulating the circadian inflammatory response. Comparing the survival of myeloid cells in LPS-induced sepsis in BMAL1 knockout mice revealed increases in LPS lethality as well as in the induction of miR-155 compared to the control group. Bone marrow-derived macrophages from the knockout vs. control also showed higher expression of pro-inflammatory cytokines and decreased production of IL-10, a known anti-inflammatory cytokine. Using miR-155 knockout mice, the authors showed higher basal levels of BMAL1 mRNA at ZT0, suggesting that miR-155 inhibits BMAL1 expression. Additionally, knockout of miR-155 altered the molecular clock of the mice depicted by shorter free-running periods under constant darkness or light, followed by decreasing production of TNF-alpha. These results show that miR-155 regulates the inflammatory response by targeting the BMAL1 molecular clock in macrophages.
These findings provide a direct link between the circadian clock (BMAL1), a microRNA (miR-155) and the immune response in macrophages, furthering our understanding of the circadian mechanism of the inflammatory response. These results may lead to immunotherapies synchronized with natural circadian rhythms. Future work is needed to further investigate how alterations in circadian rhythm cycles, such as those induced by sleep deprivation, jet-lag, etc., may disrupt these processes.
Mice are nocturnal, so to study the circadian rhythm of murine cells, Curtis et al. utilized two strategies. First, an exogenous time signal - also known as zeitgeber time - was used to induce consecutive 12-hour phases of rest and activity. Second, they knocked out members of the clock machinery of the immune cells, namely BMAL1 and miR-155 to gain better understanding of their role in regulating the circadian inflammatory response. Comparing the survival of myeloid cells in LPS-induced sepsis in BMAL1 knockout mice revealed increases in LPS lethality as well as in the induction of miR-155 compared to the control group. Bone marrow-derived macrophages from the knockout vs. control also showed higher expression of pro-inflammatory cytokines and decreased production of IL-10, a known anti-inflammatory cytokine. Using miR-155 knockout mice, the authors showed higher basal levels of BMAL1 mRNA at ZT0, suggesting that miR-155 inhibits BMAL1 expression. Additionally, knockout of miR-155 altered the molecular clock of the mice depicted by shorter free-running periods under constant darkness or light, followed by decreasing production of TNF-alpha. These results show that miR-155 regulates the inflammatory response by targeting the BMAL1 molecular clock in macrophages.
These findings provide a direct link between the circadian clock (BMAL1), a microRNA (miR-155) and the immune response in macrophages, furthering our understanding of the circadian mechanism of the inflammatory response. These results may lead to immunotherapies synchronized with natural circadian rhythms. Future work is needed to further investigate how alterations in circadian rhythm cycles, such as those induced by sleep deprivation, jet-lag, etc., may disrupt these processes.
May 27, 2015
The Cells They are a-changing
Evidence that monocytes reprogram their behavior in response to local stimuli.
By: the FBR + Angiogenesis Team (Emily, Pam, Tony, Mark, Anamika, Max)
The inflammatory response serves as the body’s first line of defense against harmful stimuli, such as pathogens or injury. Dal-Secco et al. reasoned that understanding the inflammatory response to injury would facilitate our ability to resolve disease-driven inflammatory states, such as those in rheumatoid arthritis. The complexity of monocyte behavior stems from their ability to exist as both pro-inflammatory and anti-inflammatory cells. Pro-inflammatory monocytes express high levels of CCR2 and low levels of CX3CR1 (CCR2hiCX3CR1low), which are receptors involved in immune cell recruitment and adhesion. Alternatively, monocytes also exist as CCR2lowCX3CR1hi. The recruitment of these inflammatory cells to the site of injury is not well understood. In order to elucidate the dynamic role of monocytes in inflammation, Dal-Secco et al. tracked the migration of these distinct monocyte populations in mice, in response to inflammation induced by thermal injury to the liver.
Using spinning-disk fluorescent confocal intravital microscopy to visualize cells within living mice, the authors found that CCR2hiCX3CR1low cells accumulated around the injured area, formed a ring like structure, and remained at the injury site over the first 72 hours after injury. In contrast, CX3CR1hiCCR2low cells were not prevalent until 48 hours post-injury, but remained at the injury site with identical localization to the CCR2hiCX3CR1low population. Most notably, the authors observed that CX3CR1hiCCR2low cells did not migrate into the ring from surrounding tissue, suggesting that these cells were derived directly from the CCR2hiCX3CR1low population. To confirm that this phenotypic transition occurred, the authors characterized monocytes in a piece of liver tissue maintained in the laboratory, so that the migration of new cells to the tissue was prevented. The monocytes clearly transitioned from CCR2hi to CX3CR1hi over time. Moreover, accumulation of both CCR2hi and CX3CR1hi cells was inhibited in CCR2-deficient mice. Interestingly, local inhibition of IL-4 and IL-10 delayed the transition from CCR2hi to CX3CR1hi and impaired healing.
These findings provide direct evidence that stimuli in the local environment can regulate a phenotypic switch in monocytes. Understanding and controlling the differential behavior of monocytes may provide new avenues for therapeutic targets in wound healing and inflammatory disease.
Using spinning-disk fluorescent confocal intravital microscopy to visualize cells within living mice, the authors found that CCR2hiCX3CR1low cells accumulated around the injured area, formed a ring like structure, and remained at the injury site over the first 72 hours after injury. In contrast, CX3CR1hiCCR2low cells were not prevalent until 48 hours post-injury, but remained at the injury site with identical localization to the CCR2hiCX3CR1low population. Most notably, the authors observed that CX3CR1hiCCR2low cells did not migrate into the ring from surrounding tissue, suggesting that these cells were derived directly from the CCR2hiCX3CR1low population. To confirm that this phenotypic transition occurred, the authors characterized monocytes in a piece of liver tissue maintained in the laboratory, so that the migration of new cells to the tissue was prevented. The monocytes clearly transitioned from CCR2hi to CX3CR1hi over time. Moreover, accumulation of both CCR2hi and CX3CR1hi cells was inhibited in CCR2-deficient mice. Interestingly, local inhibition of IL-4 and IL-10 delayed the transition from CCR2hi to CX3CR1hi and impaired healing.
These findings provide direct evidence that stimuli in the local environment can regulate a phenotypic switch in monocytes. Understanding and controlling the differential behavior of monocytes may provide new avenues for therapeutic targets in wound healing and inflammatory disease.
May 15, 2015
Governor of cell migration in wound healing: Fidgetin-like 2
Fidgetin-like 2 protein impairs cell migration in wound healing via direct disruption of the microtubule cytoskeleton.
By: the Wounds team (Anamika, Sina, Nicole, Desiree, Claire, Tony, Reham, Val)
Wound healing is a complex process that is largely dependent on the migration of cells from the wound edge into the wound bed. Microtubules are highly dynamic, plastic polar tubules within cells that coordinate parameters of cell movement, assembly and organization of focal adhesion complexes (cell-matrix adhesions), and distribution and delivery of Golgi-derived and endosomal vesicles to the leading edges of wounds. Given that microtubule disruption has been shown in the past to suppress cell migration in vitro, Charafeddine et al. speculated that a previously uncharacterized microtubule-severing enzyme contributes to dysfunctional wound healing by impeding cell migration.
To test this hypothesis, the authors first conducted a siRNA-based screen of all known or putative microtubule severing or depolymerizing enzymes. Fidgetin-like 2 (FL2) protein produced the most pronounced effect on cellular migration of human osteosarcoma U2OS cells in vitro in a scratch closure assay. In fact, FL2 siRNA-treated cells had ~2.5-fold increase in migration rate compared to the control. This finding was also confirmed in human keratinocyte and mouse fibroblast cell lines. To elucidate the mechanism under which FL2 impairs cell migration, the team examined the subcellular distribution of FL2 and found cortical localization of FL2, suggesting a direct interaction between FL2 and microtubule cytoskeleton. Additionally, FL2 knockdown caused formation of dense microtubule bundles and increased size and maturity of focal adhesions – a structural link between cells and their surrounding environment that is important in cell migration. Most importantly, the authors encapsulated FL2 siRNA in nanoparticles as a means of locally mitigating FL2 activity at the wound site. Wounds treated with the FL2 siRNA showed faster re-epithelialization and collagen deposition and decreased size compared to control wounds in both excisional and burn murine wounds. In addition, FL2 siRNA-treated wounds were less inflamed and more organized wound bed than controls.
Taken together, these findings suggest that FL2 interferes with cell migratory machinery by directly influencing microtubule cytoskeleton and focal adhesions. Treatment strategies that target FL2 may have application in promoting wound closure and regeneration in vivo.
Charafeddine RA, et al., "Fidgetin-Like 2: A Microtubule-Based Regulator of Wound Healing." J. of Inv. Derm. 2015. doi:10.1038/jid.2015.94
By: the Wounds team (Anamika, Sina, Nicole, Desiree, Claire, Tony, Reham, Val)
Wound healing is a complex process that is largely dependent on the migration of cells from the wound edge into the wound bed. Microtubules are highly dynamic, plastic polar tubules within cells that coordinate parameters of cell movement, assembly and organization of focal adhesion complexes (cell-matrix adhesions), and distribution and delivery of Golgi-derived and endosomal vesicles to the leading edges of wounds. Given that microtubule disruption has been shown in the past to suppress cell migration in vitro, Charafeddine et al. speculated that a previously uncharacterized microtubule-severing enzyme contributes to dysfunctional wound healing by impeding cell migration.
To test this hypothesis, the authors first conducted a siRNA-based screen of all known or putative microtubule severing or depolymerizing enzymes. Fidgetin-like 2 (FL2) protein produced the most pronounced effect on cellular migration of human osteosarcoma U2OS cells in vitro in a scratch closure assay. In fact, FL2 siRNA-treated cells had ~2.5-fold increase in migration rate compared to the control. This finding was also confirmed in human keratinocyte and mouse fibroblast cell lines. To elucidate the mechanism under which FL2 impairs cell migration, the team examined the subcellular distribution of FL2 and found cortical localization of FL2, suggesting a direct interaction between FL2 and microtubule cytoskeleton. Additionally, FL2 knockdown caused formation of dense microtubule bundles and increased size and maturity of focal adhesions – a structural link between cells and their surrounding environment that is important in cell migration. Most importantly, the authors encapsulated FL2 siRNA in nanoparticles as a means of locally mitigating FL2 activity at the wound site. Wounds treated with the FL2 siRNA showed faster re-epithelialization and collagen deposition and decreased size compared to control wounds in both excisional and burn murine wounds. In addition, FL2 siRNA-treated wounds were less inflamed and more organized wound bed than controls.
Taken together, these findings suggest that FL2 interferes with cell migratory machinery by directly influencing microtubule cytoskeleton and focal adhesions. Treatment strategies that target FL2 may have application in promoting wound closure and regeneration in vivo.
Charafeddine RA, et al., "Fidgetin-Like 2: A Microtubule-Based Regulator of Wound Healing." J. of Inv. Derm. 2015. doi:10.1038/jid.2015.94
May 11, 2015
Radical particles behind a once-radical idea
Reactive oxygen species may provide an answer about the mechanisms behind dysfunctional immune responses in neurodegenerative disease.
By: the M1-to-M2 team (Pam, Claire, Desiree, Reham, Sina, Nathan, Tony)
Microglia, the inflammatory cells in the central nervous system, are functionally diverse cells that can change their behavior from pro-inflammatory (M1) to inflammation-resolving (M2) in response to environmental stimuli. While the ability of microglia to exhibit both M1 and M2 behavior is key for effective response to injury, several studies have shown that dysregulated M1/M2 activation is implicated in neurodegenerative diseases, like Parkinson’s. Although some evidence points to a role for reactive oxygen species (ROS), chemically reactive molecules containing oxygen that are typically secreted by cells during inflammation, the mechanisms that drive microglia into a pathologic, pro-inflammatory state are poorly understood. Based on the known roles of NF-kB p50 transcription factor – a protein involved in regulating transcription of NF-kB from DNA to messenger RNA – in macrophage M1/M2 signaling, and its sensitivity to oxygen, Taetzsch et al. reasoned that NF-kB p50 oxidation contributes to dysfunctional microglia activation and chronic neuroinflammation.
To this end, by stabilizing and identifying free radicals – atoms or groups of atoms with an unpaired number of electrons – Taetzsch et al. demonstrated for the first time that ROS generate an NF-kB p50 radical in mouse microglia. While the authors showed that microglia of NF-kB p50 knockout animals exhibit an enhanced M1 behavior in response to inflammatory stimulus, loss of NF-kB p50 function characterized by the presence of NF-kB p50 radicals was also found to augment the upregulation of inflammatory mediators. Based on these findings, Taetzsch et al. speculated that NF-kB p50 plays a role in the ability of microglia to respond to M2-promoting stimuli. Taetzsch et al. further showed that prolonged administration of ROS to microglia in vitro impaired NF-kB p50 protein-protein interactions and led to decreased NF-kB p50 DNA binding while increasing expression of the proinflammatory mediator TNFa at later time points. Treatment with a free radical scavenger failed to mitigate this inflammatory response in the brain of NF-kB p50 knockout mice, further implicating a role for NF-kB p50 in regulating microglial M2 response.
Taken together, these results suggest that NF-kB p50 is a key redox signaling mechanism driving the balance of M1/M2 activation in microglia. Importantly, while this study only identified one type of free radical involved in regulating microglial function, this research offers new insight into the mechanisms of dysfunctional activation. Understanding the factors that lead to this deleterious imbalance in neurodegenerative disease will provide therapeutic targets for achieving inflammatory resolution.
By: the M1-to-M2 team (Pam, Claire, Desiree, Reham, Sina, Nathan, Tony)
Microglia, the inflammatory cells in the central nervous system, are functionally diverse cells that can change their behavior from pro-inflammatory (M1) to inflammation-resolving (M2) in response to environmental stimuli. While the ability of microglia to exhibit both M1 and M2 behavior is key for effective response to injury, several studies have shown that dysregulated M1/M2 activation is implicated in neurodegenerative diseases, like Parkinson’s. Although some evidence points to a role for reactive oxygen species (ROS), chemically reactive molecules containing oxygen that are typically secreted by cells during inflammation, the mechanisms that drive microglia into a pathologic, pro-inflammatory state are poorly understood. Based on the known roles of NF-kB p50 transcription factor – a protein involved in regulating transcription of NF-kB from DNA to messenger RNA – in macrophage M1/M2 signaling, and its sensitivity to oxygen, Taetzsch et al. reasoned that NF-kB p50 oxidation contributes to dysfunctional microglia activation and chronic neuroinflammation.
To this end, by stabilizing and identifying free radicals – atoms or groups of atoms with an unpaired number of electrons – Taetzsch et al. demonstrated for the first time that ROS generate an NF-kB p50 radical in mouse microglia. While the authors showed that microglia of NF-kB p50 knockout animals exhibit an enhanced M1 behavior in response to inflammatory stimulus, loss of NF-kB p50 function characterized by the presence of NF-kB p50 radicals was also found to augment the upregulation of inflammatory mediators. Based on these findings, Taetzsch et al. speculated that NF-kB p50 plays a role in the ability of microglia to respond to M2-promoting stimuli. Taetzsch et al. further showed that prolonged administration of ROS to microglia in vitro impaired NF-kB p50 protein-protein interactions and led to decreased NF-kB p50 DNA binding while increasing expression of the proinflammatory mediator TNFa at later time points. Treatment with a free radical scavenger failed to mitigate this inflammatory response in the brain of NF-kB p50 knockout mice, further implicating a role for NF-kB p50 in regulating microglial M2 response.
Taken together, these results suggest that NF-kB p50 is a key redox signaling mechanism driving the balance of M1/M2 activation in microglia. Importantly, while this study only identified one type of free radical involved in regulating microglial function, this research offers new insight into the mechanisms of dysfunctional activation. Understanding the factors that lead to this deleterious imbalance in neurodegenerative disease will provide therapeutic targets for achieving inflammatory resolution.
