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.

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.

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 type 2 diabetes (T2D). Further, in pre-diabetic 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.

Development of the immune system can be influenced by diverse extrinsic and intrinsic factors that influence the risk of disease. Severe early life respiratory syncytial virus (RSV) infection is associated with persistent immune alterations. Previously, our group had shown that adult mice orally supplemented with Lactobacillus johnsonii exhibited decreased airway immunopathology following RSV infection. Here, we demonstrate that offspring of mice supplemented with L. johnsonii exhibit reduced airway mucus and Th2 cell–mediated response to RSV infection. Maternal supplementation resulted in a consistent gut microbiome in mothers and their offspring. Importantly, supplemented maternal plasma and breastmilk, and offspring plasma, exhibited decreased inflammatory metabolites. Cross-fostering studies showed that prenatal Lactobacillus exposure led to decreased Th2 cytokines and lung inflammation following RSV infection, while postnatal Lactobacillus exposure diminished goblet cell hypertrophy and mucus production in the lung in response to airway infection. These studies demonstrate that Lactobacillus modulation of the maternal microbiome and associated metabolic reprogramming enhance airway protection against RSV in neonates.