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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 Ly6C Hi [Lin − (CD3 − CD19 − NK1.1 − Ter-119 − ) Ly6G − CD11b + ] and Ly6C Lo [Lin − (CD3 − CD19 − NK1.1 − Ter-119 − ) Ly6G − CD11b + ] monocyte/macrophage subsets in normal and diabetic wounds. Using flow-sorted tdTomato -labeled Ly6C Hi monocyte/macrophages, we show Ly6C Hi cells transition to a Ly6C Lo phenotype in normal wounds, whereas in diabetic wounds, there is a late, second influx of Ly6C Hi cells that fail transition to Ly6C Lo . The second wave of Ly6C Hi 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 Ly6C Hi 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 Ly6C Hi [Lin − Ly6G − CD11b + ] and Ly6C Lo [Lin − Ly6G − CD11b + ] cells from normal and diabetic wounds. Gene transcriptome profiling of diabetic wound Ly6C Hi 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 + Ly6C Hi monocyte/macrophages is a viable therapeutic strategy for inflammation in diabetic wounds.

Wound healing in diabetes is impaired due to failed resolution of inflammation. Macrophages play a significant role in the establishment of a regulated inflammatory response during wounding. Macrophage function is dictated by metabolism, which alters gene expression. Recent studies suggest that a fatty acid binding protein, FABP4, may control macrophage function in diabetes by altering metabolism. Thus, we examined whether FABP4 controls macrophage function and hence inflammation in diabetic wound healing. To investigate this, C57BL/6 mice were fed either a normal (12% saturated fat) diet or a high-fat (60% saturated fat) diet (HFD) for 12 weeks to induce physiologic “pre-diabetes.” Wounds were created and CD3-CD19-NK1.1-CD11b+ cells (macrophages) were isolated each day following injury and FABP4 expression was quantified by qPCR and Western blot. We found that HFD wound macrophages demonstrated a significant increase in FABP4 gene expression and protein production on day 3 post-injury compared with controls. To determine if FABP4 alters inflammatory gene expression in wound macrophages, we isolated wound macrophages with an FABP4 inhibitor, treated them, and analyzed for IL-1β and TNFα expression. IL1β and TNFα gene expression were significantly reduced (P Chromatin immunoprecipitation (ChIP) analysis of the FABP4 promoter in wound macrophages revealed a significant increase in H3K4 trimethylation, an activating mark, on the FABP4 promoter in diabetic wound macrophages suggesting that epigenetic regulation may play an important role in the differential expression of FABP4 in diabetic wounds. In conclusion, FABP4 appears to be upregulated in diabetic wound macrophages and contributes to increased macrophage inflammation. Modulation of FABP4 or its expression may help resolve inflammation in diabetic wounds and promote healing.

Macrophage (Mϕ) plasticity, allowing for transition of Mϕs from an inflammatory to a reparative phenotype, is critical for normal wound healing. In pathologic conditions, such as type 2 diabetes (T2D), wounds fail to heal due to impaired resolution of inflammation. The mechanism(s) responsible for the persistent inflammatory phenotype in T2D wounds are unclear. Prior studies have shown that the Toll-like receptor (TLR) 4 pathway regulates Mϕ-mediated inflammation in tissues. Growing evidence indicates that TLR4 is a versatile receptor binding a spectrum of ligands including non-microbial ligands such as saturated fatty acids (SFAs). Given the excess SFAs in T2D, the purpose of this study was to examine the role of the SFA palmitate on TLR4 signaling and Mϕ phenotype in diabetic wound healing. We have previously shown that Mϕs isolated from wounds in a murine model of glucose intolerance (diet-induced obesity; DIO) maintained on a 60% high fat diet (HFD) for 12-18 weeks, display increased levels of inflammatory cytokines (IL-1β, IL-12, and TNFα) at both a gene expression and protein level. In the current study, we found that blood monocytes and wound Mϕs from DIO mice display increased TLR4 receptors compared to control blood and wounds. To determine if altered metabolites in the diabetic environment impact Mϕ phenotype, bone marrow derived macrophages (BMDMs) were incubated in serum isolated from DIO or control mice. BMDMs incubated with DIO serum displayed a hyperinflammatory response following LPS stimulation. Further, stimulation with the metabolite palmitate produced significantly increased IL-1β expression in DIO BMDMs compared to controls suggesting that DIO BMDMs are programmed toward an inflammatory response. To determine the mechanism, we examined several epigenetic enzymes known to affect Mϕ polarization and found that palmitate stimulated expression of JMJD3, a histone demethylase, which increases inflammatory gene expression. In conclusion, these studies suggest that the diabetic milieu, specifically increased levels of the SFA palmitate, induces expression of the epigenetic enzyme, JMJD3, in Mϕs and this regulates inflammatory gene expression and cell function.

Background: Wound healing in chronic diseases, such as type 2 diabetes (T2D), is impaired due to dysregulated inflammation. Innate immune cells, particularly macrophages, play a significant role in regulated inflammation following tissue injury. After injury, CCR2+ monocytes are recruited to the peripheral wound. This recruitment is mediated in part by the CCR2 ligand, CCL2. Thus, we hypothesized that the CCL2/CCR2 interaction is vital for normal wound healing and appropriate inflammation, and that this signaling cascade is impaired in T2D. Methods: CCR2 -/- mice and littermate controls underwent 4mm hindlimb wounds, and wound closure was compared daily. Wound macrophages (CD3-CD19-NK1.1-CD11b+ cells) were analyzed on day 3 by flow cytometry for intracellular cytokine production. Adoptive transfer was performed using blood CD11b+ cells from WT C57BL/6 or CCR2 -/- mice isolated by magnetic sorting and transferred into CCR2 -/- mice via tail vein injection. Mice were then wounded and wound closure was compared between the two groups. C57BL/6 mice were maintained on normal or high fat diet for 12-14 weeks, wounds were created, and CD11b+ cells were isolated from wounds on day 2. ELISA for CCL2 was performed. Results: CCR2 -/- mice showed significantly impaired wound healing on days 2-7 compared with littermate controls. Macrophages isolated on day 3 from wounds of CCR2 -/- mice expressed significantly less inflammatory cytokines (IL-1β, TNF-α) by qPCR. Flow cytometry analysis revealed less Ly6C hi macrophages in the wounds, as well as macrophages that made significantly less IL-1β, NOS2, and TNF-α. When adoptive transfer was performed, wound healing was restored to normal in the mice that received WT compared to those that received CCR2 -/- CD11b+ cells (P< 0.01). Since CCR2 is important for normal wound inflammation, and we have previously shown that inflammation is impaired in the diet-induced obese (DIO) mice, we examined CCL2 in DIO wounds. CCL2 was significantly decreased in DIO wound macrophages on day 2. Conclusion: Appropriate CCR2/CCL2 interaction plays a crucial role in macrophage recruitment and regulated inflammation in normal wound healing. Impairment in CCR2/CCL2 signaling may be responsible, in part, for delayed early inflammation in T2D.