<p>MΦ that were differentiated in M-CSF (A) and GM-CSF (B) were further polarized using IFN... more <p>MΦ that were differentiated in M-CSF (A) and GM-CSF (B) were further polarized using IFN-γ or IL-4+IL-13. Characteristic features of M-CSF vs. GM-CSF MΦ are depicted in the upper part. Additional changes found in this study, induced by the polarization process are shown in the lower section with characteristic genes, surface markers and epigenetic changes for M-CSF and GM-CSF M1and M2 MΦ. </p
Despite many recent advances in the field, the underlying mechanisms governing pulmonary fibrosis... more Despite many recent advances in the field, the underlying mechanisms governing pulmonary fibrosis remain poorly understood. We have used an experimental system of fibrotic granuloma development via the embolization of Schistosoma mansoni eggs to the lungs. Egg embolization results in the release of highly antigenic glycoproteins (Schistosoma egg antigen,SEA) that promote a dominant Th2 immune response. We previously demonstrated that in comparison to wild-type mice, TLR9-/- mice showed increased granuloma size and augmented collagen deposition. In the present study, we investigated the mechanisms underlying the fibrotic change of this Th2-driven pulmonary granuloma system, and the contribution of TLR9 signals to this phenomenon. The chemokine receptor/ligand pair CXCR4/CXCL12 has been shown to play an important role in the homing of fibrocytes; these cells express CXCR4 and migrate in response to CXCL12 in a model of bleomycin-induced pulmonary fibrosis. Levels of CXCL12 were higher in the lungs of mice treated with S. mansoni eggs, but there were no difference between wild-type and TLR9-/- mice. Flow cytometry data indicated that TLR9-/- mice showed increased number of CD45+Col I+CXCR4+ fibrocytes in the lungs of TLR9-/- mice treated with S. mansoni eggs in comparison to wild-type mice. Thus, our results suggested that increased fibrocytes may contribute to the increased levels of fibrosis observed in TLR9-/- mice.
Macrophages are critical for the initiation and resolution of the inflammatory phase of wound rep... more Macrophages are critical for the initiation and resolution of the inflammatory phase of wound repair. In diabetes, macrophages display a prolonged inflammatory phenotype in late wound healing. Recently, Mixed-Lineage Leukemia-1 (MLL1) has been shown to direct gene expression by regulating NFκB-mediated inflammatory gene transcription. Thus, we hypothesized that MLL1 influences macrophage-mediated inflammation in wound repair. We utilized a myeloid-specific Mll1 knockout(Mll1(f/f)Lyz2(Cre+) ) to determine the function of MLL1 in wound healing. Mll1(f/f)Lyz2(Cre+) mice display delayed wound healing and decreased wound macrophage inflammatory cytokine production compared to controls. Further, wound macrophages from Mll1(f/f)Lyz2(Cre+) mice demonstrated decreased Histone-3 Lysine-4 trimethylation (H3K4me3; activation mark) at NFκB-binding sites on inflammatory gene promoters. Interestingly, early wound macrophages from 'pre-diabetic' mice displayed similarly decreased MLL1, H3K4...
The Notch ligand delta-like 4 (DLL4) is known to fine-tune the CD4(+) T cell cytokine response. D... more The Notch ligand delta-like 4 (DLL4) is known to fine-tune the CD4(+) T cell cytokine response. DLL4 is expressed on the surface of antigen-presenting cells (APCs) in a MyD88-dependent manner. We found that DLL4 expression was upregulated on bone marrow progenitor cells and APCs in mice infected with BCG Mycobacterium. Transfer of DLL4(+) progenitor cells from infected hosts resulted in an increase DLL4(+) myeloid cells in the spleen, indicating that expression of the dll4 gene is propagated throughout hematopoiesis. We also found an increase in DLL4(+) monocytes from individuals who were infected with Mycobacterium tuberculosis. In latent individuals, DLL4 expression correlated with increased cytokine production from T cells in response to PPD stimulation. Finally, antibody blockade of DLL4 reduced T cell cytokine production from naïve T cells stimulated with antigen. These results demonstrate that the Notch ligand DLL4 can influence T cell cytokine production in both humans and mi...
Influenza A virus (IAV) is an airborne pathogen that causes significant morbidity and mortality e... more Influenza A virus (IAV) is an airborne pathogen that causes significant morbidity and mortality each year. Macrophages (Mϕ) are the first immune population to encounter IAV virions in the lungs and are required to control infection. In the present study, we explored the mechanism by which cytokine signaling regulates the phenotype and function of Mϕ via epigenetic modification of chromatin. We have found that type I interferon (IFN-I) potently upregulates the lysine methyltransferase Setdb2 in murine and human Mϕ, and in turn Setdb2 regulates Mϕ-mediated immunity in response to IAV. The induction of Setdb2 by IFN-I was significantly impaired upon inhibition of the JAK-STAT signaling cascade, and chromatin immunoprecipitation revealed that both STAT1 and interferon regulatory factor 7 bind upstream of the transcription start site to induce expression. The generation of Setdb2LacZ reporter mice revealed that IAV infection results in systemic upregulation of Setdb2 in myeloid cells. In...
Impaired wound healing in Type 2 diabetes (T2D) is associated with a chronic inflammatory respons... more Impaired wound healing in Type 2 diabetes (T2D) is associated with a chronic inflammatory response mediated by accumulation of macrophages producing pro-inflammatory cytokines. While recent work has shown that epigenetic regulation plays a key role in influencing macrophage function, the specific alterations that occur in T2D macrophages and their effects on gene transcription have not been elucidated. Work in our laboratory has shown that Histone H3 Lys9 methylation that results in transcriptional silencing is mediated by Setdb2 (SET domain, bifurcated2) and plays a critical role in lung inflammation. Thus, we examined the role of Setdb2 in wound healing and macrophage activation in a murine model of T2D. Superficial wounding resulted in increased transcript levels of Setdb2 in wound macrophages of control mice compared with diabetic mice. Specific deletion of Setdb2 in macrophages (Setdb2lyz2) using cre recombinase resulted in delayed wound healing. Furthermore, bone marrow derived macrophages from Setdb2lyz2 cre+ mice show reduced expression of key regulatory mediators such as IFN-β. In summary, our data indicate that Setdb2 may play a protective role in wound healing by regulating inflammation.
Journal of Clinical Tuberculosis and Other Mycobacterial Diseases, Aug 1, 2021
Objective There is a clear need for improved biomarkers to diagnose HIV/TB coinfection. Although ... more Objective There is a clear need for improved biomarkers to diagnose HIV/TB coinfection. Although numerous tests can identify the existence of both of these microbes within the host, a parallel assessment of the host response to HIV/TB coinfection may prove as useful confirmation in cases where microbiological tests are inconclusive. To this end we assessed the levels of Notch ligands found in serum samples of patients with TB, HIV or HIV/TB coinfection. The Notch system is involved in almost every stage of development, including the maturation of the immune response. Upon exposure to a pathogen, the innate immune system will increase expression of Notch ligands Delta-like 1 and Delta-like 4. Previous research has demonstrated that Notch ligand expression is increased on monocytes from patients diagnosed with tuberculosis. We hypothesized that if Notch ligands were present in the peripheral blood of individuals diagnosed with TB, they may serve as a novel marker for infection. Design: Serum samples from patients with HIV, TB or HIV/TB coinfection were compared to serum from uninfected individuals to determine levels of DLL1 and DLL4 in a case controlled study. Methods DLL1 and DLL4 were measured by ELISA. Linear regression with post tests were used to determine if levels of DLL1 and DLL4 were increased in individuals with HIV/TB coinfection as compared to individuals infected with either HIV or TB or healthy controls. Results Delta-like 1 and Delta-like 4 were significantly increased in the serum of patients with HIV and HIV/ M. tuberculosis coinfection compared to other groups. Conclusions Assessment of Notch ligands in peripheral blood may enhance the diagnosis of individuals with active TB that are co-infected with HIV. The study will also need to be validated in in a larger cohort.
Classically-activated (M1) macrophages are known to play a role in the development of chronic inf... more Classically-activated (M1) macrophages are known to play a role in the development of chronic inflammation associated with impaired wound healing in Type 2 diabetes (T2D); however, the mechanism responsible for the dominant pro-inflammatory (M1) macrophage phenotype in T2D wounds is unknown. Since epigenetic enzymes can direct macrophage phenotypes, we assessed the role of histone methylation in bone marrow (BM) stem/progenitor cells in the programming of macrophages towards a pro-inflammatory phenotype. We have found that a repressive histone methylation mark, H3K27me3, is decreased at the promoter of the IL12 gene in BM progenitors and this epigenetic signature is passed down to wound macrophages in a murine model of glucose intolerance (diet-induced obese [DIO]). These epigenetically ‘pre-programmed’ macrophages result in poised macrophages in peripheral tissue and negatively impact wound repair. We found that in diabetic conditions, the H3K27 demethylase, Jmjd3, drives IL12 prod...
Macrophages can be divided into a classically (M1) or alternatively activated (M2) phenotype depe... more Macrophages can be divided into a classically (M1) or alternatively activated (M2) phenotype depending on the cytokine environment. Commonly, IFNg±LPS and IL-4±IL-13 drive the M1 and M2 phenotype, respectively. In humans however, understanding of the M1/M2 paradigm is still evolving. Recent findings also suggest that the growth factors GM-CSF and M-CSF influence human macrophage development towards a M1- or M2-like phenotype. We analyzed macrophages grown from human blood monocytes under GM-CSF or M-CSF culture conditions that were treated with either IFNg ± LPS or IL-4/IL-13. Cells were analyzed for the expression of genes associated with the M1 and M2 phenotype. To further explore mechanisms defining the M1/M2 cells, superarray was performed to evaluate mRNA differences in the expression of chromatin modifying enzymes. In both GM-CSF and M-CSF cultured cells IFNg/LPS increased the expression of genes associated with M1 macrophages, e.g. CXCL10 and CCR7; while treatment with IL-4/I...
Notch ligands present during interactions between T cells and dendritic cells (DCs) dictate cell ... more Notch ligands present during interactions between T cells and dendritic cells (DCs) dictate cell phenotype through a myriad of effects including the induction of T cell regulation, survival, and cytokine response. The presence of Notch ligands on DCs varies with the context of the inflammatory response; Jagged-1 is constitutively expressed, whereas Delta-like 1 and Delta-like 4 are induced in response to pathogen exposure. Although Delta-like and Jagged ligands send different signals through the same Notch receptor, the role of these two ligands in peripheral T cell immunity is not clear. The goal of our studies was to determine the role of Jagged-1 in the pathogen-free inflammation induced by OVA during allergic airway disease in mice. Our studies show that a deletion in DC-expressed Jagged-1 causes a significant increase in cytokine production, resulting in increased mucus production and increased eosinophilia in the lungs of mice sensitized and challenged with OVA. We also observ...
<p>MΦ that were differentiated in M-CSF (A) and GM-CSF (B) were further polarized using IFN... more <p>MΦ that were differentiated in M-CSF (A) and GM-CSF (B) were further polarized using IFN-γ or IL-4+IL-13. Characteristic features of M-CSF vs. GM-CSF MΦ are depicted in the upper part. Additional changes found in this study, induced by the polarization process are shown in the lower section with characteristic genes, surface markers and epigenetic changes for M-CSF and GM-CSF M1and M2 MΦ. </p
Despite many recent advances in the field, the underlying mechanisms governing pulmonary fibrosis... more Despite many recent advances in the field, the underlying mechanisms governing pulmonary fibrosis remain poorly understood. We have used an experimental system of fibrotic granuloma development via the embolization of Schistosoma mansoni eggs to the lungs. Egg embolization results in the release of highly antigenic glycoproteins (Schistosoma egg antigen,SEA) that promote a dominant Th2 immune response. We previously demonstrated that in comparison to wild-type mice, TLR9-/- mice showed increased granuloma size and augmented collagen deposition. In the present study, we investigated the mechanisms underlying the fibrotic change of this Th2-driven pulmonary granuloma system, and the contribution of TLR9 signals to this phenomenon. The chemokine receptor/ligand pair CXCR4/CXCL12 has been shown to play an important role in the homing of fibrocytes; these cells express CXCR4 and migrate in response to CXCL12 in a model of bleomycin-induced pulmonary fibrosis. Levels of CXCL12 were higher in the lungs of mice treated with S. mansoni eggs, but there were no difference between wild-type and TLR9-/- mice. Flow cytometry data indicated that TLR9-/- mice showed increased number of CD45+Col I+CXCR4+ fibrocytes in the lungs of TLR9-/- mice treated with S. mansoni eggs in comparison to wild-type mice. Thus, our results suggested that increased fibrocytes may contribute to the increased levels of fibrosis observed in TLR9-/- mice.
Macrophages are critical for the initiation and resolution of the inflammatory phase of wound rep... more Macrophages are critical for the initiation and resolution of the inflammatory phase of wound repair. In diabetes, macrophages display a prolonged inflammatory phenotype in late wound healing. Recently, Mixed-Lineage Leukemia-1 (MLL1) has been shown to direct gene expression by regulating NFκB-mediated inflammatory gene transcription. Thus, we hypothesized that MLL1 influences macrophage-mediated inflammation in wound repair. We utilized a myeloid-specific Mll1 knockout(Mll1(f/f)Lyz2(Cre+) ) to determine the function of MLL1 in wound healing. Mll1(f/f)Lyz2(Cre+) mice display delayed wound healing and decreased wound macrophage inflammatory cytokine production compared to controls. Further, wound macrophages from Mll1(f/f)Lyz2(Cre+) mice demonstrated decreased Histone-3 Lysine-4 trimethylation (H3K4me3; activation mark) at NFκB-binding sites on inflammatory gene promoters. Interestingly, early wound macrophages from 'pre-diabetic' mice displayed similarly decreased MLL1, H3K4...
The Notch ligand delta-like 4 (DLL4) is known to fine-tune the CD4(+) T cell cytokine response. D... more The Notch ligand delta-like 4 (DLL4) is known to fine-tune the CD4(+) T cell cytokine response. DLL4 is expressed on the surface of antigen-presenting cells (APCs) in a MyD88-dependent manner. We found that DLL4 expression was upregulated on bone marrow progenitor cells and APCs in mice infected with BCG Mycobacterium. Transfer of DLL4(+) progenitor cells from infected hosts resulted in an increase DLL4(+) myeloid cells in the spleen, indicating that expression of the dll4 gene is propagated throughout hematopoiesis. We also found an increase in DLL4(+) monocytes from individuals who were infected with Mycobacterium tuberculosis. In latent individuals, DLL4 expression correlated with increased cytokine production from T cells in response to PPD stimulation. Finally, antibody blockade of DLL4 reduced T cell cytokine production from naïve T cells stimulated with antigen. These results demonstrate that the Notch ligand DLL4 can influence T cell cytokine production in both humans and mi...
Influenza A virus (IAV) is an airborne pathogen that causes significant morbidity and mortality e... more Influenza A virus (IAV) is an airborne pathogen that causes significant morbidity and mortality each year. Macrophages (Mϕ) are the first immune population to encounter IAV virions in the lungs and are required to control infection. In the present study, we explored the mechanism by which cytokine signaling regulates the phenotype and function of Mϕ via epigenetic modification of chromatin. We have found that type I interferon (IFN-I) potently upregulates the lysine methyltransferase Setdb2 in murine and human Mϕ, and in turn Setdb2 regulates Mϕ-mediated immunity in response to IAV. The induction of Setdb2 by IFN-I was significantly impaired upon inhibition of the JAK-STAT signaling cascade, and chromatin immunoprecipitation revealed that both STAT1 and interferon regulatory factor 7 bind upstream of the transcription start site to induce expression. The generation of Setdb2LacZ reporter mice revealed that IAV infection results in systemic upregulation of Setdb2 in myeloid cells. In...
Impaired wound healing in Type 2 diabetes (T2D) is associated with a chronic inflammatory respons... more Impaired wound healing in Type 2 diabetes (T2D) is associated with a chronic inflammatory response mediated by accumulation of macrophages producing pro-inflammatory cytokines. While recent work has shown that epigenetic regulation plays a key role in influencing macrophage function, the specific alterations that occur in T2D macrophages and their effects on gene transcription have not been elucidated. Work in our laboratory has shown that Histone H3 Lys9 methylation that results in transcriptional silencing is mediated by Setdb2 (SET domain, bifurcated2) and plays a critical role in lung inflammation. Thus, we examined the role of Setdb2 in wound healing and macrophage activation in a murine model of T2D. Superficial wounding resulted in increased transcript levels of Setdb2 in wound macrophages of control mice compared with diabetic mice. Specific deletion of Setdb2 in macrophages (Setdb2lyz2) using cre recombinase resulted in delayed wound healing. Furthermore, bone marrow derived macrophages from Setdb2lyz2 cre+ mice show reduced expression of key regulatory mediators such as IFN-β. In summary, our data indicate that Setdb2 may play a protective role in wound healing by regulating inflammation.
Journal of Clinical Tuberculosis and Other Mycobacterial Diseases, Aug 1, 2021
Objective There is a clear need for improved biomarkers to diagnose HIV/TB coinfection. Although ... more Objective There is a clear need for improved biomarkers to diagnose HIV/TB coinfection. Although numerous tests can identify the existence of both of these microbes within the host, a parallel assessment of the host response to HIV/TB coinfection may prove as useful confirmation in cases where microbiological tests are inconclusive. To this end we assessed the levels of Notch ligands found in serum samples of patients with TB, HIV or HIV/TB coinfection. The Notch system is involved in almost every stage of development, including the maturation of the immune response. Upon exposure to a pathogen, the innate immune system will increase expression of Notch ligands Delta-like 1 and Delta-like 4. Previous research has demonstrated that Notch ligand expression is increased on monocytes from patients diagnosed with tuberculosis. We hypothesized that if Notch ligands were present in the peripheral blood of individuals diagnosed with TB, they may serve as a novel marker for infection. Design: Serum samples from patients with HIV, TB or HIV/TB coinfection were compared to serum from uninfected individuals to determine levels of DLL1 and DLL4 in a case controlled study. Methods DLL1 and DLL4 were measured by ELISA. Linear regression with post tests were used to determine if levels of DLL1 and DLL4 were increased in individuals with HIV/TB coinfection as compared to individuals infected with either HIV or TB or healthy controls. Results Delta-like 1 and Delta-like 4 were significantly increased in the serum of patients with HIV and HIV/ M. tuberculosis coinfection compared to other groups. Conclusions Assessment of Notch ligands in peripheral blood may enhance the diagnosis of individuals with active TB that are co-infected with HIV. The study will also need to be validated in in a larger cohort.
Classically-activated (M1) macrophages are known to play a role in the development of chronic inf... more Classically-activated (M1) macrophages are known to play a role in the development of chronic inflammation associated with impaired wound healing in Type 2 diabetes (T2D); however, the mechanism responsible for the dominant pro-inflammatory (M1) macrophage phenotype in T2D wounds is unknown. Since epigenetic enzymes can direct macrophage phenotypes, we assessed the role of histone methylation in bone marrow (BM) stem/progenitor cells in the programming of macrophages towards a pro-inflammatory phenotype. We have found that a repressive histone methylation mark, H3K27me3, is decreased at the promoter of the IL12 gene in BM progenitors and this epigenetic signature is passed down to wound macrophages in a murine model of glucose intolerance (diet-induced obese [DIO]). These epigenetically ‘pre-programmed’ macrophages result in poised macrophages in peripheral tissue and negatively impact wound repair. We found that in diabetic conditions, the H3K27 demethylase, Jmjd3, drives IL12 prod...
Macrophages can be divided into a classically (M1) or alternatively activated (M2) phenotype depe... more Macrophages can be divided into a classically (M1) or alternatively activated (M2) phenotype depending on the cytokine environment. Commonly, IFNg±LPS and IL-4±IL-13 drive the M1 and M2 phenotype, respectively. In humans however, understanding of the M1/M2 paradigm is still evolving. Recent findings also suggest that the growth factors GM-CSF and M-CSF influence human macrophage development towards a M1- or M2-like phenotype. We analyzed macrophages grown from human blood monocytes under GM-CSF or M-CSF culture conditions that were treated with either IFNg ± LPS or IL-4/IL-13. Cells were analyzed for the expression of genes associated with the M1 and M2 phenotype. To further explore mechanisms defining the M1/M2 cells, superarray was performed to evaluate mRNA differences in the expression of chromatin modifying enzymes. In both GM-CSF and M-CSF cultured cells IFNg/LPS increased the expression of genes associated with M1 macrophages, e.g. CXCL10 and CCR7; while treatment with IL-4/I...
Notch ligands present during interactions between T cells and dendritic cells (DCs) dictate cell ... more Notch ligands present during interactions between T cells and dendritic cells (DCs) dictate cell phenotype through a myriad of effects including the induction of T cell regulation, survival, and cytokine response. The presence of Notch ligands on DCs varies with the context of the inflammatory response; Jagged-1 is constitutively expressed, whereas Delta-like 1 and Delta-like 4 are induced in response to pathogen exposure. Although Delta-like and Jagged ligands send different signals through the same Notch receptor, the role of these two ligands in peripheral T cell immunity is not clear. The goal of our studies was to determine the role of Jagged-1 in the pathogen-free inflammation induced by OVA during allergic airway disease in mice. Our studies show that a deletion in DC-expressed Jagged-1 causes a significant increase in cytokine production, resulting in increased mucus production and increased eosinophilia in the lungs of mice sensitized and challenged with OVA. We also observ...
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Papers by Ronald Allen