Histone demethylase JARID1C/KDM5C regulates Th17 cells by increasing IL-6 expression in diabetic plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) are first responders to tissue injury, where they prime naive T cells. The role of pDCs in physiologic wound repair has been examined, but little is known about pDCs in diabetic wound tissue and their interactions with naive CD4+ T cells. Diabetic wounds are characterized by increased levels of inflammatory IL-17A cytokine, partly due to increased Th17 CD4+ cells. This increased IL-17A cytokine, in excess, impairs tissue repair. Here, using human tissue and murine wound healing models, we found that diabetic wound pDCs produced excess IL-6 and TGF-ß and that these cytokines skewed naive CD4+ T cells toward a Th17 inflammatory phenotype following cutaneous injury. Further, we identified that increased IL-6 cytokine production by diabetic wound pDCs is regulated by a histone demethylase, Jumonji AT-rich interactive domain 1C histone demethylase (JARID1C). Decreased JARID1C increased IL-6 transcription in diabetic pDCs, and this process was regulated upstream by an IFN-I/TYK2/JAK1,3 signaling pathway. When inhibited in nondiabetic wound pDCs, JARID1C skewed naive CD4+ T cells toward a Th17 phenotype and increased IL-17A production. Together, this suggests that diabetic wound pDCs are epigenetically altered to increase IL-6 expression that then affects T cell phenotype. These findings identify a therapeutically manipulable pathway in diabetic wounds.

Macrophage-specific inhibition of the histone demethylase JMJD3 decreases STING and pathologic inflammation in diabetic wound repair.

Macrophage plasticity is critical for normal tissue repair following injury. In pathologic states such as diabetes, macrophage plasticity is impaired, and macrophages remain in a persistent proinflammatory state; however, the reasons for this are unknown. Here, using single-cell RNA sequencing of human diabetic wounds, we identified increased JMJD3 in diabetic wound macrophages, resulting in increased inflammatory gene expression. Mechanistically, we report that in wound healing, JMJD3 directs early macrophage-mediated inflammation via JAK1,3/STAT3 signaling. However, in the diabetic state, we found that IL-6, a cytokine increased in diabetic wound tissue at later time points post-injury, regulates JMJD3 expression in diabetic wound macrophages via the JAK1,3/STAT3 pathway and that this late increase in JMJD3 induces NFκB-mediated inflammatory gene transcription in wound macrophages via an H3K27me3 mechanism. Interestingly, RNA sequencing of wound macrophages isolated from mice with JMJD3-deficient myeloid cells (Jmjd3f/fLyz2Cre+) identified that the STING gene (Tmem173) is regulated by JMJD3 in wound macrophages. STING limits inflammatory cytokine production by wound macrophages during healing. However, in diabetic mice, its role changes to limit wound repair and enhance inflammation. This finding is important since STING is associated with chronic inflammation, and we found STING to be elevated in human and murine diabetic wound macrophages at late time points. Finally, we demonstrate that macrophage-specific, nanoparticle inhibition of JMJD3 in diabetic wounds significantly improves diabetic wound repair by decreasing inflammatory cytokines and STING. Taken together, this work highlights the central role of JMJD3 in tissue repair and identifies cell-specific targeting as a viable therapeutic strategy for nonhealing diabetic wounds.

Single-cell Transcriptomics Reveals Dynamic Role of Smooth Muscle Cells and Enrichment of Immune Cell Subsets in Human Abdominal Aortic Aneurysms.

OBJECTIVE: To determine cell-specific gene expression profiles that contribute to development of abdominal aortic aneurysms (AAAs). BACKGROUND: AAAs represent the most common pathological aortic dilation leading to the fatal consequence of aortic rupture. Both immune and structural cells contribute to aortic degeneration, however, gene specific alterations in these cellular subsets are poorly understood. METHODS: We performed single-cell RNA sequencing (scRNA-seq) analysis of AAAs and control tissues. AAA-related changes were examined by comparing gene expression profiles as well as detailed receptor-ligand interactions. An integrative analysis of scRNA-seq data with large genome-wide association study data was conducted to identify genes critical for AAA development. RESULTS: Using scRNA-seq we provide the first comprehensive characterization of the cellular landscape in human AAA tissues. Unbiased clustering analysis of transcriptional profiles identified seventeen clusters representing 8 cell lineages. For immune cells, clustering analysis identified 4 T-cell and 5 monocyte/macrophage subpopulations, with distinct transcriptional profiles in AAAs compared to controls. Gene enrichment analysis on immune subsets identified multiple pathways only expressed in AAA tissue, including those involved in mitochondrial dysfunction, proliferation, and cytokine secretion. Moreover, receptor-ligand analysis defined robust interactions between vascular smooth muscle cells and myeloid populations in AAA tissues. Lastly, integrated analysis of scRNA-seq data with genome-wide association study studies determined that vascular smooth muscle cell expression of SORT1 is critical for maintaining normal aortic wall function. CONCLUSIONS: Here we provide the first comprehensive evaluation of single-cell composition of the abdominal aortic wall and reveal how the gene expression landscape is altered in human AAAs.

IFN-κ is critical for normal wound repair and is decreased in diabetic wounds.

Wound repair following acute injury requires a coordinated inflammatory response. Type I IFN signaling is important for regulating the inflammatory response after skin injury. IFN-κ, a type I IFN, has recently been found to drive skin inflammation in lupus and psoriasis; however, the role of IFN-κ in the context of normal or dysregulated wound healing is unclear. Here, we show that Ifnk expression is upregulated in keratinocytes early after injury and is essential for normal tissue repair. Under diabetic conditions, IFN-κ was decreased in wound keratinocytes, and early inflammation was impaired. Furthermore, we found that the histone methyltransferase mixed-lineage leukemia 1 (MLL1) is upregulated early following injury and regulates Ifnk expression in diabetic wound keratinocytes via an H3K4me3-mediated mechanism. Using a series of in vivo studies with a geneticall y engineered mouse model (Mll1fl/fl K14cre-) and human wound tissues from patients with T2D, we demonstrate that MLL1 controls wound keratinocyte-mediated Ifnk expression and that Mll1 expression is decreased in T2D keratinocytes. Importantly, we found the administration of IFN-κ early following injury improves diabetic tissue repair through increasing early inflammation, collagen deposition, and reepithelialization. These findings have significant implications for understanding the complex role type I IFNs play in keratinocytes in normal and diabetic wound healing. Additionally, they suggest that IFN may be a viable therapeutic target to improve diabetic wound repair.

Coronavirus induces diabetic macrophage-mediated inflammation via SETDB2.

COVID-19 induces a robust, extended inflammatory "cytokine storm" that contributes to an increased morbidity and mortality, particularly in patients with type 2 diabetes (T2D). Macrophages are a key innate immune cell population responsible for the cytokine storm that has been shown, in T2D, to promote excess inflammation in response to infection. Using peripheral monocytes and sera from human patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and a murine hepatitis coronavirus (MHV-A59) (an established murine model of SARS), we identified that coronavirus induces an increased Mφ-mediated inflammatory response due to a coronavirus-induced decrease in the histone methyltransferase, SETDB2. This decrease in SETDB2 upon coronavirus infection results in a decrease of the repressive trimethylation of histone 3 lysine 9 (H3K9me3) at NFkB binding sites on inflammatory gene promoters, effectively increasing inflammation. Mφs isolated from mice with a myeloid-specific deletion of SETDB2 displayed increased pathologic inflammation following coronavirus infection. Further, IFNß directly regulates SETDB2 in Mφs via JaK1/STAT3 signaling, as blockade of this pathway altered SETDB2 and the inflammatory response to coronavirus infection. Importantly, we also found that loss of SETDB2 mediates an increased inflammatory response in diabetic Mϕs in response to coronavirus infection. Treatment of coronavirus-infected diabetic Mφs with IFNß reversed the inflammatory cytokine production via up-regulation of SETDB2/H3K9me3 on inflammatory gene promoters. Together, these results describe a potential mechanism for the increased Mφ-mediated cytokine storm in patients with T2D in response to COVID-19 and suggest that therapeutic targeting of the IFNß/SETDB2 axis in T2D patients may decrease pathologic inflammation associated with COVID-19.

Inhibition of macrophage histone demethylase JMJD3 protects against abdominal aortic aneurysms.

Abdominal aortic aneurysms (AAAs) are a life-threatening disease for which there is a lack of effective therapy preventing aortic rupture. During AAA formation, pathological vascular remodeling is driven by macrophage infiltration, and the mechanisms regulating macrophage-mediated inflammation remain undefined. Recent evidence suggests that an epigenetic enzyme, JMJD3, plays a critical role in establishing macrophage phenotype. Using single-cell RNA sequencing of human AAA tissues, we identified increased JMJD3 in aortic monocyte/macrophages resulting in up-regulation of an inflammatory immune response. Mechanistically, we report that interferon-ß regulates Jmjd3 expression via JAK/STAT and that JMJD3 induces NF-κB-mediated inflammatory gene transcription in infiltrating aortic macrophages. In vivo targeted inhibition of JMJD3 with myeloid-specific genetic depletion (JMJD3f/fLyz2Cre+) or pharmacological inhibition in the elastase or angiotensin II-induced AAA model preserved the repressive H3K27me3 on inflammatory gene promoters and markedly reduced AAA expansion and attenuated macrophage-mediated inflammation. Together, our findings suggest that cell-specific pharmacologic therapy targeting JMJD3 may be an effective intervention for AAA expansion.

Dysregulation of intercellular signaling by MOF deletion leads to liver injury.

Epigenetic mechanisms that alter heritable gene expression and chromatin structure play an essential role in many biological processes, including liver function. Human MOF (males absent on the first) is a histone acetyltransferase that is globally downregulated in human steatohepatitis. However, the function of MOF in the liver remains unclear. Here, we report that MOF plays an essential role in adult liver. Genetic deletion of Mof by Mx1-Cre in the liver leads to acute liver injury, with increase of lipid deposition and fibrosis akin to human steatohepatitis. Surprisingly, hepatocyte-specific Mof deletion had no overt liver abnormality. Using the in vitro coculturing experiment, we show that Mof deletion-induced liver injury requires coordinated changes and reciprocal signaling between hepatocytes and Kupffer cells, which enables feedforward regulation to augment inflammation and apoptotic responses. At the molecular level, Mof deletion induced characteristic changes in metabolic gene programs, which bore noticeable similarity to the molecular signature of human steatohepatitis. Simultaneous deletion of Mof in both hepatocytes and macrophages results in enhanced expression of inflammatory genes and NO signaling in vitro. These changes, in turn, lead to apoptosis of hepatocytes and lipotoxicity. Our work highlights the importance of histone acetyltransferase MOF in maintaining metabolic liver homeostasis and sheds light on the epigenetic dysregulation in liver pathogenesis.

Epigenetic regulation of the PGE2 pathway modulates macrophage phenotype in normal and pathologic wound repair.

Macrophages are a primary immune cell involved in inflammation, and their cell plasticity allows for transition from an inflammatory to a reparative phenotype and is critical for normal tissue repair following injury. Evidence suggests that epigenetic alterations play a critical role in establishing macrophage phenotype and function during normal and pathologic wound repair. Here, we find in human and murine wound macrophages that cyclooxygenase 2/prostaglandin E2 (COX-2/PGE2) is elevated in diabetes and regulates downstream macrophage-mediated inflammation and host defense. Using single-cell RNA sequencing of human wound tissue, we identify increased NF-κB-mediated inflammation in diabetic wounds and show increased COX-2/PGE2 in diabetic macrophages. Further, we identify that COX-2/PGE2 production in wound macrophages requires epigenetic regulation of 2 key enzymes in the cytosolic phospholipase A2/COX-2/PGE2 (cPLA2/COX-2/PGE2) pathway. We demonstrate that TGF-ß-induced miRNA29b increases COX-2/PGE2 production via inhibition of DNA methyltransferase 3b-mediated hypermethylation of the Cox-2 promoter. Further, we find mixed-lineage leukemia 1 (MLL1) upregulates cPLA2 expression and drives COX-2/PGE2. Inhibition of the COX-2/PGE2 pathway genetically (Cox2fl/fl Lyz2Cre+) or with a macrophage-specific nanotherapy targeting COX-2 in tissue macrophages reverses the inflammatory macrophage phenotype and improves diabetic tissue repair. Our results indicate the epigenetically regulated PGE2 pathway controls wound macrophage function, and cell-targeted manipulation of this pathway is feasible to improve diabetic wound repair.

Palmitate-TLR4 signaling regulates the histone demethylase, JMJD3, in macrophages and impairs diabetic wound healing.

Chronic macrophage inflammation is a hallmark of type 2 diabetes (T2D) and linked to the development of secondary diabetic complications. T2D is characterized by excess concentrations of saturated fatty acids (SFA) that activate innate immune inflammatory responses, however, mechanism(s) by which SFAs control inflammation is unknown. Using monocyte-macrophages isolated from human blood and murine models, we demonstrate that palmitate (C16:0), the most abundant circulating SFA in T2D, increases expression of the histone demethylase, Jmjd3. Upregulation of Jmjd3 results in removal of the repressive histone methylation (H3K27me3) mark on NFκB-mediated inflammatory gene promoters driving macrophage-mediated inflammation. We identify that the effects of palmitate are fatty acid specific, as laurate (C12:0) does not regulate Jmjd3 and the associated inflammatory profile. Further, palmitate-induced Jmjd3 expression is controlled via TLR4/MyD88-dependent signaling mechanism, where genetic depletion of TLR4 (Tlr4-/- ) or MyD88 (MyD88-/- ) negated the palmitate-induced changes in Jmjd3 and downstream NFκB-induced inflammation. Pharmacological inhibition of Jmjd3 using a small molecule inhibitor (GSK-J4) reduced macrophage inflammation and improved diabetic wound healing. Together, we conclude that palmitate contributes to the chronic Jmjd3-mediated activation of macrophages in diabetic peripheral tissue and a histone demethylase inhibitor-based therapy may represent a novel treatment for nonhealing diabetic wounds.

Epigenetic Regulation of TLR4 in Diabetic Macrophages Modulates Immunometabolism and Wound Repair.

Macrophages are critical for the initiation and resolution of the inflammatory phase of wound healing. In diabetes, macrophages display a prolonged inflammatory phenotype preventing tissue repair. TLRs, particularly TLR4, have been shown to regulate myeloid-mediated inflammation in wounds. We examined macrophages isolated from wounds of patients afflicted with diabetes and healthy controls as well as a murine diabetic model demonstrating dynamic expression of TLR4 results in altered metabolic pathways in diabetic macrophages. Further, using a myeloid-specific mixed-lineage leukemia 1 (MLL1) knockout (Mll1f/fLyz2Cre+ ), we determined that MLL1 drives Tlr4 expression in diabetic macrophages by regulating levels of histone H3 lysine 4 trimethylation on the Tlr4 promoter. Mechanistically, MLL1-mediated epigenetic alterations influence diabetic macrophage responsiveness to TLR4 stimulation and inhibit tissue repair. Pharmacological inhibition of the TLR4 pathway using a small molecule inhibitor (TAK-242) as well as genetic depletion of either Tlr4 (Tlr4-/- ) or myeloid-specific Tlr4 (Tlr4f/fLyz2Cre+) resulted in improved diabetic wound healing. These results define an important role for MLL1-mediated epigenetic regulation of TLR4 in pathologic diabetic wound repair and suggest a target for therapeutic manipulation.

TNF-α regulates diabetic macrophage function through the histone acetyltransferase MOF.

A critical component of wound healing is the transition from the inflammatory phase to the proliferation phase to initiate healing and remodeling of the wound. Macrophages are critical for the initiation and resolution of the inflammatory phase during wound repair. In diabetes, macrophages display a sustained inflammatory phenotype in late wound healing characterized by elevated production of inflammatory cytokines, such as TNF-α. Previous studies have shown that an altered epigenetic program directs diabetic macrophages toward a proinflammatory phenotype, contributing to a sustained inflammatory phase. Males absent on the first (MOF) is a histone acetyltransferase (HAT) that has been shown be a coactivator of TNF-α signaling and promote NF-κB-mediated gene transcription in prostate cancer cell lines. Based on MOF's role in TNF-α/NF-κB-mediated gene expression, we hypothesized that MOF influences macrophage-mediated inflammation during wound repair. We used myeloid-specific Mof-knockout (Lyz2Cre Moffl/fl) and diet-induced obese (DIO) mice to determine the function of MOF in diabetic wound healing. MOF-deficient mice exhibited reduced inflammatory cytokine gene expression. Furthermore, we found that wound macrophages from DIO mice had elevated MOF levels and higher levels of acetylated histone H4K16, MOF's primary substrate of HAT activity, on the promoters of inflammatory genes. We further identified that MOF expression could be stimulated by TNF-α and that treatment with etanercept, an FDA-approved TNF-α inhibitor, reduced MOF levels and improved wound healing in DIO mice. This report is the first to our knowledge to define an important role for MOF in regulating macrophage-mediated inflammation in wound repair and identifies TNF-α inhibition as a potential therapy for the treatment of chronic inflammation in diabetic wounds.

Pneumococcal conjugate vaccine modulates macrophage-mediated innate immunity in pneumonia caused by Streptococcus pneumoniae following influenza.

Pneumococcal conjugate vaccination (PCV) may prevent influenza-related pneumonia, including Streptococcus pneumoniae pneumonia. To investigate PCV efficacy against secondary pneumococcal pneumonia following influenza, PCV was administered intramuscularly 2 and 5 weeks before S. pneumoniae serotype-3 colonization of murine nasopharynges followed by intranasal challenge with a sublethal dose of influenza A virus. Bacterial and viral loads, including innate immune responses were compared across conditions. PCV vaccination improved the survival of mice with secondary pneumococcal pneumonia and significantly reduced the pulmonary bacterial burden. Increased monocyte/macrophage influx into the lungs, alleviated loss of alveolar macrophages and decreased neutrophil influx into the lungs occurred in PCV-treated mice irrespective of pneumococcal colonization. Higher monocyte chemoattractant protein 1 levels and lower levels of CXCL1, interferon-γ, interleukin-17A, and IL-10, were detected in PCV-treated mice. Additionally, PCV treatment activated the macrophage intracellular killing of S. pneumoniae. Collectively, PCV potentially modulates the host's innate immunity and specific antibodies induction. Macrophage-related innate immunity should be further explored to elucidate the efficacy and mechanisms of PCV versus influenza-related life-threatening diseases.

Ly6CLo Monocyte/Macrophages are Essential for Thrombus Resolution in a Murine Model of Venous Thrombosis.

Venous thrombosis (VT) resolution is a complex process, resembling sterile wound healing. Infiltrating blood-derived monocyte/macrophages (Mo/MΦs) are essential for the regulation of inflammation in tissue repair. These cells differentiate into inflammatory (CD11b+Ly6CHi) or proreparative (CD11b+Ly6CLo) subtypes. Previous studies have shown that infiltrating Mo/MΦs are important for VT resolution, but the precise roles of different Mo/MΦs subsets are not well understood. Utilizing murine models of stasis and stenosis inferior vena cava thrombosis in concert with a Mo/MΦ depletion model (CD11b-diphtheria toxin receptor [DTR]-expressing mice), we examined the effect of Mo/MΦ depletion on thrombogenesis and VT resolution. In the setting of an 80 to 90% reduction in circulating CD11b+Mo/MΦs, we demonstrated that Mo/MΦs are not essential for thrombogenesis, with no difference in thrombus size, neutrophil recruitment, or neutrophil extracellular traps found. Conversely, CD11b+Mo/MΦ are essential for VT resolution. Diphtheria toxoid (DTx)-mediated depletion after thrombus creation depleted primarily CD11b+Ly6CLo Mo/MΦs and resulted in larger thrombi. DTx-mediated depletion did not alter CD11b+Ly6CHi Mo/MΦ recruitment, suggesting a protective effect of CD11b+Ly6CLo Mo/MΦs in VT resolution. Confirmatory Mo/MΦ depletion with clodronate lysosomes showed a similar phenotype, with failure to resolve VT. Adoptive transfer of CD11b+Ly6CLo Mo/MΦs into Mo/MΦ-depleted mice reversed the phenotype, restoring normal thrombus resolution. These findings suggest that CD11b+Ly6CLo Mo/MΦs are essential for normal VT resolution, consistent with the known proreparative function of this subset, and that further study of Mo/MΦ subsets may identify targets for immunomodulation to accelerate and improve thrombosis resolution.

Inhibition of the stem cell factor 248 isoform attenuates the development of pulmonary remodeling disease.

Stem cell factor (SCF) and its receptor c-kit have been implicated in inflammation, tissue remodeling, and fibrosis. Ingenuity Integrated Pathway Analysis of gene expression array data sets showed an upregulation of SCF transcripts in idiopathic pulmonary fibrosis (IPF) lung biopsies compared with tissue from nonfibrotic lungs that are further increased in rapid progressive disease. SCF248, a cleavable isoform of SCF, was abundantly and preferentially expressed in human lung fibroblasts and fibrotic mouse lungs relative to the SCF220 isoform. In fibroblast-mast cell coculture studies, blockade of SCF248 using a novel isoform-specific anti-SCF248 monoclonal antibody (anti-SCF248), attenuated the expression of COL1A1, COL3A1, and FN1 transcripts in cocultured IPF but not normal lung fibroblasts. Administration of anti-SCF248 on days 8 and 12 after bleomycin instillation in mice significantly reduced fibrotic lung remodeling and col1al, fn1, acta2, tgfb, and ccl2 transcript expression. In addition, bleomycin increased numbers of c-kit+ mast cells, eosinophils, and ILC2 in lungs of mice, whereas they were not significantly increased in anti-SCF248-treated animals. Finally, mesenchymal cell-specific deletion of SCF significantly attenuated bleomycin-mediated lung fibrosis and associated fibrotic gene expression. Collectively, these data demonstrate that SCF is upregulated in diseased IPF lungs and blocking SCF248 isoform significantly ameliorates fibrotic lung remodeling in vivo suggesting that it may be a therapeutic target for fibrotic lung diseases.

Sepsis Induces Prolonged Epigenetic Modifications in Bone Marrow and Peripheral Macrophages Impairing Inflammation and Wound Healing.

OBJECTIVE: Sepsis represents an acute life-threatening disorder resulting from a dysregulated host response. For patients who survive sepsis, there remains long-term consequences, including impaired inflammation, as a result of profound immunosuppression. The mechanisms involved in this long-lasting deficient immune response are poorly defined. Approach and Results: Sepsis was induced using the murine model of cecal ligation and puncture. Following a full recovery period from sepsis physiology, mice were subjected to our wound healing model and wound macrophages (CD11b+, CD3-, CD19-, Ly6G-) were sorted. Post-sepsis mice demonstrated impaired wound healing and decreased reepithelization in comparison to controls. Further, post-sepsis bone marrow-derived macrophages and wound macrophages exhibited decreased expression of inflammatory cytokines vital for wound repair (IL [interleukin]-1ß, IL-12, and IL-23). To evaluate if decreased inflammatory gene expression was secondary to epigenetic modification, we conducted chromatin immunoprecipitation on post-sepsis bone marrow-derived macrophages and wound macrophages. This demonstrated decreased expression of Mll1, an epigenetic enzyme, and impaired histone 3 lysine 4 trimethylation (activation mark) at NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells)-binding sites on inflammatory gene promoters in bone marrow-derived macrophages and wound macrophages from postcecal ligation and puncture mice. Bone marrow transplantation studies demonstrated epigenetic modifications initiate in bone marrow progenitor/stem cells following sepsis resulting in lasting impairment in peripheral macrophage function. Importantly, human peripheral blood leukocytes from post-septic patients demonstrate a significant reduction in MLL1 compared with nonseptic controls. CONCLUSIONS: These data demonstrate that severe sepsis induces stable mixed-lineage leukemia 1-mediated epigenetic modifications in the bone marrow, which are passed to peripheral macrophages resulting in impaired macrophage function and deficient wound healing persisting long after sepsis recovery.

Histone Methylation Directs Myeloid TLR4 Expression and Regulates Wound Healing following Cutaneous Tissue Injury.

Myeloid cells are critical for orchestrating regulated inflammation during wound healing. TLRs, particularly TLR4, and its downstream-signaling MyD88 pathway play an important role in regulating myeloid-mediated inflammation. Because an initial inflammatory phase is vital for tissue repair, we investigated the role of TLR4-regulated, myeloid-mediated inflammation in wound healing. In a cutaneous tissue injury murine model, we found that TLR4 expression is dynamic in wound myeloid cells during the course of normal wound healing. We identified that changes in myeloid TLR4 during tissue repair correlated with increased expression of the histone methyltransferase, mixed-lineage leukemia 1 (MLL1), which specifically trimethylates the histone 3 lysine 4 (H3K4me3) position of the TLR4 promoter. Furthermore, we used a myeloid-specific Mll1 knockout (Mll1f/fLyz2Cre+ ) to determine MLL1 drives Tlr4 expression during wound healing. To understand the critical role of myeloid-specific TLR4 signaling, we used mice deficient in Tlr4 (Tlr4-/- ), Myd88 (Myd88 -/-), and myeloid-specific Tlr4 (Tlr4f/fLyz2Cre+) to demonstrate delayed wound healing at early time points postinjury. Furthermore, in vivo wound myeloid cells isolated from Tlr4-/- and Myd88 -/- wounds demonstrated decreased inflammatory cytokine production. Importantly, adoptive transfer of monocyte/macrophages from wild-type mice trafficked to wounds with restoration of normal healing and myeloid cell function in Tlr4-deficient mice. These results define a role for myeloid-specific, MyD88-dependent TLR4 signaling in the inflammatory response following cutaneous tissue injury and suggest that MLL1 regulates TLR4 expression in wound myeloid cells.

Role of Chemokine Receptor CCR4 and Regulatory T Cells in Wound Healing of Diabetic Mice.

Wound healing is a well-coordinated process that involves inflammatory mediators and cellular responses; however, if any disturbances are present during this process, tissue repair is impaired. Chronic wounds are one of the serious long-term complications associated with diabetes mellitus. The chemokine receptor CCR4 and its respective ligands, CCL17 and CCL22, are involved in regulatory T cell recruitment and activation in inflamed skin; however, the role of regulatory T cells in wounds is still not clear. Our aim was to investigate the role of CCR4 and regulatory T cells in cutaneous wound healing in diabetic mice. Alloxan-induced diabetic wild- type mice (diabetic) developed wounds that were difficult to heal, differently from CCR4-/- diabetic mice (CCR4-/- diabetic), and also from anti-CCL17/22 or anti-CD25-injected diabetic mice that presented with accelerated wound healing and fewer regulatory T cells in the wound bed. Consequently, CCR4-/- diabetic mice also presented with alteration on T cells population in the wound and draining lymph nodes; on day 14, these mice also displayed an increase of collagen fiber deposition. Still, cytokine levels were decreased in the wounds of CCR4-/- diabetic mice on day 2. Our data suggest that the receptor CCR4 and regulatory T cells negatively affect wound healing in diabetic mice.

GM-CSF Administration Improves Defects in Innate Immunity and Sepsis Survival in Obese Diabetic Mice.

Sepsis is the leading cause of death in the intensive care unit with an overall mortality rate of 20%. Individuals who are obese and have type 2 diabetes have increased recurrent, chronic, nosocomial infections that worsen the long-term morbidity and mortality from sepsis. Additionally, animal models of sepsis have shown that obese, diabetic mice have lower survival rates compared with nondiabetic mice. Neutrophils are essential for eradication of bacteria, prevention of infectious complications, and sepsis survival. In diabetic states, there is a reduction in neutrophil chemotaxis, phagocytosis, and reactive oxygen species (ROS) generation; however, few studies have investigated the extent to which these deficits compromise infection eradication and mortality. Using a cecal ligation and puncture model of sepsis in lean and in diet-induced obese mice, we demonstrate that obese diabetic mice have decreased "emergency hematopoiesis" after an acute infection. Additionally, both neutrophils and monocytes in obese, diabetic mice have functional defects, with decreased phagocytic ability and a decreased capacity to generate ROS. Neutrophils isolated from obese diabetic mice have decreased transcripts of Axl and Mertk, which partially explains the phagocytic dysfunction. Furthermore, we found that exogenous GM-CSF administration improves sepsis survival through enhanced neutrophil and monocytes phagocytosis and ROS generation abilities in obese, diabetic mice with sepsis.

Spred2 Deficiency Exacerbates D-Galactosamine/Lipopolysaccharide -induced Acute Liver Injury in Mice via Increased Production of TNFα.

Acute liver injury (ALI) is characterized by hepatocyte damage and inflammation. In the present study, we examined whether the absence of Sprouty-related EVH1-domain-containing protein 2 (Spred2), a negative regulator of the Ras/Raf/ERK/MAPK pathway, influences ALI induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS). Compared to wild-type mice, Spred2-/- mice developed exacerbated liver injury represented by enhanced hepatocyte damage and inflammation. Enhanced ERK activation was observed in Spred2-/--livers, and the MEK/ERK inhibitor U0126 ameliorated ALI. Hepatic tumour necrosis factor α (TNFα) and interleukin (IL)-1ß levels were increased in Spred-2-/--livers, and the neutralization of TNFα dramatically ameliorated ALI, which was associated with decreased levels of endogenous TNFα and IL-1ß. When mice were challenged with D-GalN and TNFα, much severer ALI was observed in Spred2-/- mice with significant increases in endogenous TNFα and IL-1ß in the livers. Immunohistochemically, Kupffer cells were found to produce TNFα, and isolated Kupffer cells from Spred2-/- mice produced significantly higher levels of TNFα than those from wild-type mice after LPS stimulation, which was significantly decreased by U0126. These results suggest that Spred2 negatively regulates D-GalN/LPS-induced ALI under the control of TNFα in Kupffer cells. Spred2 may present a therapeutic target for the treatment of ALI.