Steve Kunkel

Two Mammalian MOF Complexes Regulate Transcription Activation by Distinct Mechanisms

In mammals, MYST family histone acetyltransferase MOF plays important roles in transcription activation by acetylating histone H4 on K16, a prevalent mark associated with chromatin decondensation, and transcription factor p53 on K120, which is important for activation of proapoptotic genes. However, little is known about MOF regulation in higher eukaryotes. Here, we report that the acetyltransferase activity of MOF is tightly regulated in two different but evolutionarily conserved complexes, MSL and MOF-MSL1v1. Importantly, we demonstrate that while the two MOF complexes have indistinguishable activity on histone H4 K16, they differ dramatically in acetylating nonhistone substrate p53. We further demonstrate that MOF-MSL1v1 is specifically required for optimal transcription activation of p53 target genes both in vitro and in vivo. Our results support a model that these two MOF complexes regulate distinct stages of transcription activation in cooperation with other histone modifying activities.

Inhibition of delta-like ligand 4 decreases Th1/Th17 response in a mouse model of multiple sclerosis

Notch is a family of receptors involved in the differentiation of several tissues, including the central nervous system and the immune system. One of the Notch ligands, delta-like 4 (Dll4), has been implicated in the differentiation of Th1 cells and the development of Th17 responses, which are involved in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis. Our results show that a single administration of an anti-Dll4 antibody is not enough to avoid the development of EAE or to ameliorate the already established clinical signs, despite the treatment reduces the proliferative T cell responses and decreases Th1/Th17 immune responses.

Notch regulates macrophage-mediated inflammation in diabetic wound healing

Macrophages are essential immune cells necessary for regulated inflammation during wound healing. Recent studies have identified that Notch plays a role in macrophage-mediated inflammation. Thus, we investigated the role of Notch signaling on wound macrophage phenotype and function during normal and diabetic wound healing. We found that Notch receptor and ligand expression are dynamic in wound macrophages during normal healing. Mice with a myeloid-specific Notch signaling defect (DNMAMLfloxedLyz2Cre+) demonstrated delayed early healing (days 1–3) and wound macrophages had decreased inflammatory gene expression. In our physiologic murine model of type 2 diabetes (T2D), Notch receptor expression was significantly increased in wound macrophages on day 6, following the initial inflammatory phase of wound healing, corresponding to increased inflammatory cytokine expression. This increase in Notch1 and Notch2 was also observed in human monocytes from patients with T2D. Further, in prediabetic mice with a genetic Notch signaling defect (DNMAMLfloxedLyz2Cre+ on a high-fat diet), improved wound healing was seen at late time points (days 6–7). These findings suggest that Notch is critical for the early inflammatory phase of wound healing and directs production of macrophage-dependent inflammatory mediators. These results identify that canonical Notch signaling is important in directing macrophage function in wound repair and define a translational target for the treatment of non-healing diabetic wounds.

The Histone Methyltransferase, MLL1, Directs Macrophage-Mediated Inflammation in Wound Healing and is Altered in a Murine Model of Obesity and Type 2 Diabetes

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. Mixed-lineage leukemia-1 (MLL1) has been shown to direct gene expression by regulating nuclear factor-κB (NF-κB)–mediated inflammatory gene transcription. Thus, we hypothesized that MLL1 influences macrophage-mediated inflammation in wound repair. We used a myeloid-specific Mll1 knockout (Mll1f/fLyz2Cre+) to determine the function of MLL1 in wound healing. Mll1f/fLyz2Cre+ mice display delayed wound healing and decreased wound macrophage inflammatory cytokine production compared with control animals. Furthermore, wound macrophages from Mll1f/fLyz2Cre+ mice demonstrated decreased histone H3 lysine 4 trimethylation (H3K4me3) (activation mark) at NF-κB binding sites on inflammatory gene promoters. Of note, early wound macrophages from prediabetic mice displayed similarly decreased MLL1, H3K4me3 at inflammatory gene promoters, and inflammatory cytokines compared with controls. Late wound macrophages from prediabetic mice demonstrated an increase in MLL1, H3K4me3 at inflammatory gene promoters, and inflammatory cytokines. Prediabetic macrophages treated with an MLL1 inhibitor demonstrated reduced inflammation. Finally, monocytes from patients with type 2 diabetes had increased Mll1 compared with control subjects without diabetes. These results define an important role for MLL1 in regulating macrophage-mediated inflammation in wound repair and identify a potential target for the treatment of chronic inflammation in diabetic wounds.

Ly6CHi Blood Monocyte/Macrophage Drive Chronic Inflammation and Impair Wound Healing in Diabetes Mellitus

Objective— Wound monocyte-derived macrophage plasticity controls the initiation and resolution of inflammation that is critical for proper healing, however, in diabetes mellitus, the resolution of inflammation fails to occur. In diabetic wounds, the kinetics of blood monocyte recruitment and the mechanisms that control in vivo monocyte/macrophage differentiation remain unknown. Approach and Results— Here, we characterized the kinetics and function of Ly6CHi [Lin− (CD3−CD19−NK1.1−Ter-119−) Ly6G−CD11b+] and Ly6CLo [Lin− (CD3−CD19−NK1.1−Ter-119−) Ly6G−CD11b+] monocyte/macrophage subsets in normal and diabetic wounds. Using flow-sorted tdTomato-labeled Ly6CHi monocyte/macrophages, we show Ly6CHi cells transition to a Ly6CLo phenotype in normal wounds, whereas in diabetic wounds, there is a late, second influx of Ly6CHi cells that fail transition to Ly6CLo. The second wave of Ly6CHi cells in diabetic wounds corresponded to a spike in MCP-1 (monocyte chemoattractant protein-1) and selective administration of anti-MCP-1 reversed the second Ly6CHi influx and improved wound healing. To examine the in vivo phenotype of wound monocyte/macrophages, RNA-seq–based transcriptome profiling was performed on flow-sorted Ly6CHi [Lin−Ly6G−CD11b+] and Ly6CLo [Lin−Ly6G−CD11b+] cells from normal and diabetic wounds. Gene transcriptome profiling of diabetic wound Ly6CHi cells demonstrated differences in proinflammatory and profibrotic genes compared with controls. Conclusions— Collectively, these data identify kinetic and functional differences in diabetic wound monocyte/macrophages and demonstrate that selective targeting of CD11b+Ly6CHi monocyte/macrophages is a viable therapeutic strategy for inflammation in diabetic wounds.

Targeting epigenetic mechanisms in diabetic wound healing

&NA; Impaired wound healing is a major secondary complication of type 2 diabetes that often results in limb loss and disability. Normal tissue repair progresses through discrete phases including hemostasis, inflammation, proliferation, and remodeling. In diabetes, normal progression through these phases is impaired resulting in a sustained inflammatory state and dysfunctional epithelialization in the wound. Due to their plasticity, macrophages play a critical role in the transition from the inflammation phase to the proliferation phase. Diabetes disrupts macrophage function by impairing monocyte recruitment to the wound, reducing phagocytosis, and prohibiting the transition of inflammatory macrophages to an anti‐inflammatory state. Diabetes also impedes keratinocyte and fibroblast function during the later phases resulting in impaired epithelialization of the wound. Several recent studies suggest that altered epigenetic regulation of both immune and structural cells in wounds may influence cell phenotypes and healing, particularly in pathologic states, such as diabetes. Specifically, it has been shown that macrophage plasticity during wound repair is partly regulated epigenetically and that diabetes alters this epigenetic regulation and contributes to a sustained inflammatory state. Epigenetic regulation is also known to regulate keratinocyte and fibroblast function during wound repair. In this review, we provide an introduction to the epigenetic mechanisms that regulate tissue repair and highlight recent findings that demonstrate, how epigenetic events are altered during the course of diabetic wound healing.

Murine macrophage chemokine receptor CCR2 plays a crucial role in macrophage recruitment and regulated inflammation in wound healing

Macrophages play a critical role in the establishment of a regulated inflammatory response following tissue injury. Following injury, CCR2+ monocytes are recruited from peripheral blood to wound tissue, and direct the initiation and resolution of inflammation that is essential for tissue repair. In pathologic states where chronic inflammation prevents healing, macrophages fail to transition to a reparative phenotype. Using a murine model of cutaneous wound healing, we found that CCR2‐deficient mice (CCR2−/−) demonstrate significantly impaired wound healing at all time points postinjury. Flow cytometry analysis of wounds from CCR2−/− and WT mice revealed a significant decrease in inflammatory, Ly6CHi recruited monocyte/macrophages in CCR2−/− wounds. We further show that wound macrophage inflammatory cytokine production is decreased in CCR2−/− wounds. Adoptive transfer of mT/mG monocyte/macrophages into CCR2+/+ and CCR2−/− mice demonstrated that labeled cells on days 2 and 4 traveled to wounds in both CCR2+/+ and CCR2−/− mice. Further, adoptive transfer of monocyte/macrophages from WT mice restored normal healing, likely through a restored inflammatory response in the CCR2‐deficient mice. Taken together, these data suggest that CCR2 plays a critical role in the recruitment and inflammatory response following injury, and that wound repair may be therapeutically manipulated through modulation of CCR2.

PC200 A Deacetylase Enzyme, Sirtuin 3, Plays a Major Role in Macrophage Inflammation and Wound Healing

Objectives: Diabetic patients suffer an increased risk of restenosis and late stent thrombosis after angioplasty, complications that are related to a defective re-endothelialization. Dipeptidyl peptidase-4 inhibitors have been suggested to exert direct effect on endothelial and smooth muscle cells (SMCs). Therefore, the objective was to study whether the dipeptidyl peptidase-4 inhibitor linagliptin could influence vascular repair and accelerate re-endothelialization after arterial injury in healthy and diabetic animals. Methods: Diabetic Goto-Kakizaki and healthy Wistar rats were subjected to arterial injury and treated with linagliptin or vehicle. Vessel wall healing was monitored noninvasively using ultrasound imaging and upon sacrifice with Evans blue staining and immunohistochemistry. The effect of linagliptin on SMCs was also studied in vitro. Results: We observed a delay in the healing response in the diabetic animals compared with the Wistar controls. Goto-Kakizaki rats had more pronounced intimal hyperplasia, which affected the lumen diameter. We found that linagliptin reduced the proliferation and dedifferentiation of SMCs in vitro and modulated the inflammatory response in the SMCs after arterial injury in vivo. However, these effects of linagliptin did not affect the neointima formation or the re-endothelialization under normal and diabetic conditions. Conclusions: Although linagliptin did not influence vessel wall healing, it appears to possess a desirable antiproliferative influence on SMCs in vitro and an anti-inflammatory effect in vivo. These pharmacologic properties might carry a potential significance for favorable outcome after vascular interventions in diabetic patients.