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.

Diabetic wound keratinocytes induce macrophage JMJD3-mediated Nlrp3 expression via IL-1R signaling.

Macrophage (Mφ) plasticity is critical for normal wound repair; however, in type 2 diabetic wounds, Mφs persist in a low-grade inflammatory state that prevents the resolution of wound inflammation. Increased NLRP3 inflammasome activity has been shown in diabetic wound Mφs; however, the molecular mechanisms regulating NLRP3 expression and activity are unclear. Here, we identified that diabetic wound keratinocytes induce Nlrp3 gene expression in wound Mφs through IL-1 receptor-mediated signaling, resulting in enhanced inflammasome activation in the presence of PAMPs and DAMPs. We found that IL-1 alpha is increased in human and murine wound diabetic keratinocytes compared to non-diabetic controls and directly induces Mφ Nlrp3 expression through IL-1 receptor signaling. Mechanistically, we report that the histone demethylase, JMJD3, is increased in wound Mφs late post-injury and is induced by IL-1 alpha from diabetic wound keratinocytes, resulting in Nlrp3 transcriptional activation through an H3K27me3-mediated mechanism. Using genetically engineered mice deficient in JMJD3 in myeloid cells (Jmjd3fl/fllyz2cre+), we demonstrate that JMJD3 controls Mφ-mediated Nlrp3 expression during diabetic wound healing. Thus, our data suggest a role for keratinocyte-mediated IL-1 alpha/IL-1R signaling in driving enhanced NLRP3 inflammasome activity in wound Mφs. These data also highlight the importance of cell crosstalk in wound tissues and identify JMJD3 and the ILR signaling cascade as important upstream therapeutic targets for Mφ NLRP3 inflammasome hyperactivity in nonhealing diabetic wounds.

Corneal transplantation: A walk to vision.

Corneal diseases are a major cause of blindness in the world. Corneal transplantation has been a cornerstone in the management of several of these advanced pathologies. This article discusses the evolution of corneal transplantation over a century, its indications, complications and briefly the various surgical techniques. Such tremendous technical improvisations from total corneal transplantation to lamellar keratoplasties have generated significant interest in the ophthalmic world and garnered momentum to the fight against blindness. Armed Forces Medical Services are also in vogue more than ever in this forward surge.

The Histone Methyltransferase SETDB2 Modulates Tissue Inhibitors of Metalloproteinase-Matrix Metalloproteinase Activity During Abdominal Aortic Aneurysm Development.

OBJECTIVE: To determine macrophage-specific alterations in epigenetic enzyme function contributing to the development of abdominal aortic aneurysms (AAAs). BACKGROUND: AAA is a life-threatening disease, characterized by pathologic vascular remodeling driven by an imbalance of matrix metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). Identifying mechanisms regulating macrophage-mediated extracellular matrix degradation is of critical importance to developing novel therapies. METHODS: The role of SET Domain Bifurcated Histone Lysine Methyltransferase 2 (SETDB2) in AAA formation was examined in human aortic tissue samples by single-cell RNA sequencing and in a myeloid-specific SETDB2 deficient murine model induced by challenging mice with a combination of a high-fat diet and angiotensin II. RESULTS: Single-cell RNA sequencing of human AAA tissues identified SETDB2 was upregulated in aortic monocyte/macrophages and murine AAA models compared with controls. Mechanistically, interferon-ß regulates SETDB2 expression through Janus kinase/signal transducer and activator of transcription signaling, which trimethylates histone 3 lysine 9 on the TIMP1-3 gene promoters thereby suppressing TIMP1-3 transcription and leading to unregulated matrix metalloproteinase activity. Macrophage-specific knockout of SETDB2 ( Setdb2f/fLyz2Cre+ ) protected mice from AAA formation with suppression of vascular inflammation, macrophage infiltration, and elastin fragmentation. Genetic depletion of SETDB2 prevented AAA development due to the removal of the repressive histone 3 lysine 9 trimethylation mark on the TIMP1-3 gene promoter resulting in increased TIMP expression, decreased protease activity, and preserved aortic architecture. Lastly, inhibition of the Janus kinase/signal transducer and activator of the transcription pathway with an FDA-approved inhibitor, Tofacitinib, limited SETDB2 expression in aortic macrophages. CONCLUSIONS: These findings identify SETDB2 as a critical regulator of macrophage-mediated protease activity in AAAs and identify SETDB2 as a mechanistic target for the management of AAAs.

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.

Efficacy and safety of brolucizumab versus aflibercept in patients with neovascular age-related macular degeneration: a randomized trial in Indian patients.

BACKGROUND: The current standard treatment for neovascular age-related macular degeneration (nAMD) involves intravitreal injections of anti-vascular endothelial growth factor (anti-VEGF) agents. The aim of the present study was to compare the effectiveness and safety of two anti-VEGF drugs: brolucizumab and aflibercept, in treatment-naïve nAMD Indian patients over a period of 48 weeks. METHODS: A prospective, randomized, single-centre, single-blinded, two-arm comparative study was conducted between March 2021 and February 2022. Of the 114 patients, 56 received intravitreal injections of brolucizumab (6 mg/50 µL) while 58 received aflibercept (2 mg/50 µL). The patients received 03 initial loading doses at 4-week intervals of both the agents and then respective therapies were given as individualized pro re nata (PRN) regimen based on the signs of active macular neovascularization. The functional and anatomical outcomes measured were mean change in best-corrected visual acuity (BCVA, logMAR), central macular thickness (CMT, µm), presence of intraretinal fluid, subretinal fluid or subretinal hyper-reflective material. Furthermore, the average number of additional injections required after the loading doses, the injection-free interval and safety of both the drugs were also assessed. RESULTS: Brolucizumab was found to be non-inferior to aflibercept in terms of mean change in BCVA (-0.13 ± 0.21 logMAR vs. -0.10 ± 0.15 logMAR) and reduction in CMT (-112.59 ± 81.23 µm vs. -86.38 ± 71.82 µm). The percentage of eyes with IRF and SHRM was comparable between both the groups while fewer eyes treated with brolucizumab indicated SRF presence than aflibercept after the loading doses. These beneficial effects of brolucizumab were observed with significant (p < 0.0001) lesser number of injections (1.8 ± 1.1 vs. 3.8 ± 1.5) from week 12 to week 48. Moreover, the probability of no injections after the loading doses was significantly higher with brolucizumab compared to aflibercept indicating prolonged injection-free intervals. The average ocular side effects were comparable in the two groups. One adverse event of severe vitritis requiring treatment with oral steroids occurred in Brolucizumab group, while no such event occurred in Aflibercept group. CONCLUSION: The results of the present study suggest non-inferiority of brolucizumab PRN regimen to aflibercept PRN regimen in treatment naïve nAMD Indian patients while achieving longer inter-injection intervals. Trial registration Clinical Trial Registration of India (CTRI/2021/06/034415). Registered 03 March, 2021, http://ctri.nic.in/Clinicaltrials/pmaindet2.php?trialid=54328&EncHid=&userName = .

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.

Dextran-Mimetic Quantum Dots for Multimodal Macrophage Imaging In Vivo, Ex Vivo, and In Situ.

Macrophages are white blood cells with diverse functions contributing to a healthy immune response as well as the pathogenesis of cancer, osteoarthritis, atherosclerosis, and obesity. Due to their pleiotropic and dynamic nature, tools for imaging and tracking these cells at scales spanning the whole body down to microns could help to understand their role in disease states. Here we report fluorescent and radioisotopic quantum dots (QDs) for multimodal imaging of macrophage cells in vivo, ex vivo, and in situ. Macrophage specificity is imparted by click-conjugation to dextran, a biocompatible polysaccharide that natively targets these cell types. The emission spectral band of the crystalline semiconductor core was tuned to the near-infrared for optical imaging deep in tissue, and probes were covalently conjugated to radioactive iodine for nuclear imaging. The performance of these probes was compared with all-organic dextran probe analogues in terms of their capacity to target macrophages in visceral adipose tissue using in vivo positron emission tomography/computed tomography (PET/CT) imaging, in vivo fluorescence imaging, ex vivo fluorescence, post-mortem isotopic analyses, and optical microscopy. All probe classes exhibited equivalent physicochemical characteristics in aqueous solution and similar in vivo targeting specificity. However, dextran-mimetic QDs provided enhanced signal-to-noise ratio for improved optical quantification, long-term photostability, and resistance to chemical fixation. In addition, the vascular circulation time for the QD-based probes was extended 9-fold compared with dextran, likely due to differences in conformational flexibility. The enhanced photophysical and photochemical properties of dextran-mimetic QDs may accelerate applications in macrophage targeting, tracking, and imaging across broad resolution scales, particularly advancing capabilities in single-cell and single-molecule imaging and quantification.

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.

Facilitator lessons from online psychoeducational group for relational well-being in India during COVID-19 pandemic.

The COVID-19 pandemic has affected the mental health of individuals, along with their couple and familial relationships, necessitating an effective response. Teletherapy offers an option to address these relationship concerns amidst pandemic-specific mobility restriction. Against this setting, Sukoon, a project of Tata Institute of Social Sciences, India, initiated a five-session online psychoeducational group series on relational wellbeing. This paper explores facilitator's reflections and learnings based on session documentation and facilitator notes. Preparing well and selecting participants carefully for online psychoeducational groups was critical to success. Effectiveness was enhanced by flexibly adapting the therapy process (didactic and interactive elements) to fit online delivery and the cultural context. Identifying the potential of online psychoeducational groups for relational wellbeing could make it a valuable addition to the COVID-19 pandemic mental health response toolkit. PRACTITIONER POINTS: Effective preparation and careful selection of group members is key to the success of therapist facilitated online psychoeducational groups.Psychoeducational groups comprising didactic and interactive elements are more suitable for effective online group processes.Use of co-facilitators managing various channels of communication (audio, chat) is important. Group facilitators need to be cognisant of challenges of online medium and address them in an ongoing manner.

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.

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.

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.

The Histone Methyltransferase Setdb2 Modulates Macrophage Phenotype and Uric Acid Production in Diabetic Wound Repair.

Macrophage plasticity is critical for normal tissue repair to ensure transition from the inflammatory to the proliferative phase of healing. We examined macrophages isolated from wounds of patients afflicted with diabetes and of healthy controls and found differential expression of the methyltransferase Setdb2. Myeloid-specific deletion of Setdb2 impaired the transition of macrophages from an inflammatory phenotype to a reparative one in normal wound healing. Mechanistically, Setdb2 trimethylated histone 3 at NF-κB binding sites on inflammatory cytokine gene promoters to suppress transcription. Setdb2 expression in wound macrophages was regulated by interferon (IFN) ß, and under diabetic conditions, this IFNß-Setdb2 axis was impaired, leading to a persistent inflammatory macrophage phenotype in diabetic wounds. Setdb2 regulated the expression of xanthine oxidase and thereby the uric acid (UA) pathway of purine catabolism in macrophages, and pharmacologic targeting of Setdb2 or the UA pathway improved healing. Thus, Setdb2 regulates macrophage plasticity during normal and pathologic wound repair and is a target for therapeutic manipulation.

SIRT3 Regulates Macrophage-Mediated Inflammation in Diabetic Wound Repair.

Control of inflammation is critical for the treatment of nonhealing wounds, but a delicate balance exists between early inflammation that is essential for normal tissue repair and the pathologic inflammation that can occur later in the repair process. This necessitates the development of novel therapies that can target inflammation at the appropriate time during repair. Here, we found that SIRT3 is essential for normal healing and regulates inflammation in wound macrophages after injury. Under prediabetic conditions, SIRT3 was decreased in wound macrophages and resulted in dysregulated inflammation. In addition, we found that FABP4 regulates SIRT3 in human blood monocytes, and inhibition of FABP4 in wound macrophages decreases inflammatory cytokine expression, making FABP4 a viable target for the regulation of excess inflammation and wound repair in diabetes. Using a series of ex vivo and in vivo studies with genetically engineered mouse models and diabetic human monocytes, we showed that FABP4 expression is epigenetically upregulated in diabetic wound macrophages and, in turn, diminishes SIRT3 expression, thereby promoting inflammation. These findings have significant implications for controlling inflammation and promoting tissue repair in diabetic wounds.

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.