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The necklace braid group $\mathcal{NB}_n$ is the motion group of the $n+1$ component necklace link $\mathcal{L}_n$ in Euclidean $\mathbb{R}^3$. Here $\mathcal{L}_n$ consists of $n$ pairwise unlinked Euclidean circles each linked to an auxiliary circle. Partially motivated by physical considerations, we study representations of the necklace braid group $\mathcal{NB}_n$, especially those obtained as extensions of representations of the braid group $\mathcal{B}_n$ and the loop braid group $\mathcal{LB}_n$. We show that any irreducible $\mathcal{B}_n$ representation extends to $\mathcal{NB}_n$ in a standard way. We also find some non-standard extensions of several well-known $\mathcal{B}_n$-representations such as the Burau and LKB representations. Moreover, we prove that any local representation of $\mathcal{B}_n$ (i.e. coming from a braided vector space) can be extended to $\mathcal{NB}_n$, in contrast to the situation with $\mathcal{LB}_n$. We also discuss some directions for future study from categorical and physical perspectives., Comment: 30 pages

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.

BACKGROUND: While Chronic Venous Insufficiency (CVI) and Varicose Veins (VVs) are a universally recognized problem, they are frequently under-appreciated as major contributors to long-term morbidity in the elderly despite the increasing prevalence with age. Prior studies have demonstrated that CVI and VV treatments in patients ≥ 65 yield an overall benefit, however, there has been little data if octogenarians are undergoing these procedures and with what success. As such, our objectives were to (1) investigate the procedures selected, (2) examine clinical outcomes following VV procedures in elderly patients ≥ 80 years old and (3) explore complication rates (both systemic and leg specific) following VV procedures in patients ≥ 80 years old. METHODS: We performed a retrospective review using the Vascular Quality Initiative Varicose Vein Registry (VQI VVR) of all VV procedures performed for ≥ C2 disease from January 2015 – February 2017. We divided all procedures into three age groups: patients < 65, patients ≥ 65 – 79, and patients ≥ 80 years of age. Statistical testing included Chi squared test for categorical variables and student t test for continuous variables. Two comparisons were performed. First comparing patients < 65 to ≥ 65 – 79 and second, comparing patients ≥ 65 – 79 to ≥ 80. RESULTS: There were a total of 12,262 procedures performed with 8,608 procedures in the < 65 group, 3,226 in the 65-79 group, and 428 procedures in patients ≥ 80 years old. A total of 22,050 veins were treated during the 12,262 procedures. Almost half of procedures (46.51%, N=5,703) had only one vein treated during a single procedure. Between age groups, the percentage of one vein treated increased as patient age increased, ranging from 45.39% (N=3,875) for patients < 65, 48.55% (N=1,555) for patients between 65-79, to 64.08% (N=273) for patients ≥ 80. Patients in the ≥ 80 group had an overall lower average BMI, were more likely to be on anticoagulation and undergo truncal procedures alone compared to the other groups. The ≥ 80 group had a significant improvement in both venous clinical severity scores (VCSS) (4.37 ± 4.16, p < .001) and patient reported outcomes (PROs) (8.79 ± 7.27, p < .001) from pre to post procedure. Overall complications were low in all age groups. The octogenarians had no higher risk of systemic complications. CONCLUSIONS: Vascular specialists are performing VV procedures in octogenarians and are more likely to perform truncal only therapy. Additionally, octogenarians have statistically significant improvement of VCSS and PROs with a low risk of complications despite more advanced venous disease at presentation.

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.

The healing of cutaneous wounds is dependent on the progression through distinct, yet overlapping phases of wound healing, including hemostasis, inflammation, proliferation, and resolution/remodeling. The failure of these phases to occur in a timely, progressive fashion promotes pathologic wound healing. The macrophage (MΦ) has been demonstrated to play a critical role in the inflammatory phase of tissue repair, where its dynamic plasticity allows this cell to mediate both tissue-destructive and -reparative functions. The ability to understand and control both the initiation and the resolution of inflammation is critical for treating pathologic wound healing. There are now a host of studies demonstrating that metabolic and epigenetic regulation of gene transcription can influence MΦ plasticity in wounds. In this review, we highlight the molecular and epigenetic factors that influence MΦ polarization in both physiologic and pathologic wound healing, with particular attention to 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.

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.

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]-1β, 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.

We unify and generalize several approaches to constructing braid group representations from finite groups, using iterated twisted tensor products. We provide some general characterizations and classification of these representations, focusing on the size of their images, which are typically finite groups. The well-studied Gaussian representations associated with metaplectic modular categories can be understood in this framework, and we give some new examples to illustrate their ubiquity. Our results suggest a relationship between the braiding on the G-gaugings of a pointed modular category $${\mathcal {C}}(A,Q)$$ and that of $${\mathcal {C}}(A,Q)$$ itself.

Macrophage (Mφ) plasticity, allowing the 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 Mφ inflammation. Although the mechanisms responsible for the persistent inflammatory phenotype in T2D wounds are unknown, recent investigations have revealed that cellular metabolites can alter Mφ function providing a link between metabolism and innate immunity. Further, there is increasing evidence that epigenetic mechanisms control Mφ function. The purpose of this study was to investigate if altered Mφ metabolism in T2D induces epigenetic modifications resulting in derangements in Mφ inflammation and impaired wound healing. Using the murine diet induce obesity (DIO) wound model, we demonstrate that diabetic bone marrow derived macrophages (BMDM) and wound Mφs display a hyperinflammatory phenotype following injury with increased expression of toll-like receptor (TLR) 4 signaling and proinflammatory cytokines (IL-1β and TNFα). Given that histone methylation has previously been shown by our group to influence Mφ phenotype, we examined H3K4 trimethylation on the TLR4 promoter and found a significant increase in DIO BMDMs and wound Mφs. To evaluate the impact of Mφ metabolites on inflammation we performed a LC-MS metabolite expression analysis demonstrating significant upregulation of the methyl donor, S-adenosyl methionine (SAM), which participates in histone methylation, and its rate limiting enzyme, MAT2a, in diabetic BMDMs. In summary, these results suggest that increased levels of the metabolite SAM in diabetic Mφs contribute to an inflammatory Mφ phenotype via increased H3K4 methylation at the TLR4 promoter. Disclosure F.M. Davis: None. A.D. denDekker: None. A.S. Kimball: None. A. Boniakowski: None. A. Joshi: None. M. ElAzzouny: Employee; Self; Agilent Technologies. C. Burant: Stock/Shareholder; Self; Metabolic Solutions Development Company. K. Gallagher: None. Funding National Institutes of Health

IMPACT: Our data reveal a histone modifying enzyme involved in regulating inflammation that may be a novel target for treating non-healing diabetic wounds. OBJECTIVES/GOALS: We investigate molecular mechanisms that regulate the inflammatory phenotype of macrophages in normal and diabetic wound healing. Our goal is to identify novel pathways that may be used to better treat diabetic patients with non-healing wounds. METHODS/STUDY POPULATION: We utilize normal and transgenic murine models on standard chow or high-diet to identify chromatin modifying enzymes involved in regulating macrophage function during wound healing. We validate our murine studies with human blood monocytes or wound macrophages from diabetic patients undergoing limb amputation surgery. RESULTS/ANTICIPATED RESULTS: We have identified the histone methyltransferase SETDB2 as a regulator inflammation in normal and diabetic wound macrophages. We found that SETDB2 was dependent on IFNβ singaling and that both IFNβ and Setdb2 expression were impaired in diabetic wound macrophages. Further, we show that SETDB2 regulates inflammatory response and immune cell trafficking pathways. We also show that SETDB2 genomic localization is dependent on *NFκΒ deposition of the promoter. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our results indicate that SETDB2 is a regulator of macrophage plasticity and that SETDB2 expression is impaired in diabetic wound macrophages leading to hyper-inflammatory response and delayed wound healing. These data provide a novel potential therapeutic pathway for treating non-healing diabetic wounds.

Control of inflammation is critical for the treatment of non-healing 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 post-injury. Under ‘pre-diabetic’ conditions, SIRT3 was decreased in wound macrophages and resulted in dysregulated inflammation. Further, we found that FABP4 regulates SIRT3 in human blood monocytes and inhibiting 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, as well as diabetic human monocytes, we demonstrate 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.

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.

Diabetic foot ulcerations (DFU) affect 25% of patients with diabetes mellitus during their lifetime and constitute a major health problem as they are often recalcitrant to healing due to a constellation of both intrinsic and extrinsic factors. The purpose of this review is to (1) detail the current mechanistic understanding of DFU formation and (2) highlight future therapeutic targets. From a molecular perspective, DFUs exhibit a chronic inflammatory predisposition. In addition, increased local hypoxic conditions and impaired cellular responses to hypoxia are pathogenic factors that contribute to delayed wound healing. Finally, recent evidence suggests a role for epigenetic alterations, including microRNAs, in delayed DFU healing due to the complex interplay between genes and the environment. In this regard, notable progress has been made in the molecular and genetic understanding of DFU formation. However, further studies are needed to translate preclinical investigations into clinical therapies.

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 post-injury. Flow cytometry analysis of wounds from CCR2(−/−) and wild type mice revealed a significant decrease in inflammatory, Ly6C(Hi) 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 wild type 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.

OBJECTIVE: Wound monocyte-derived macrophage plasticity controls the initiation and resolution of inflammation that are critical for proper healing, however, in diabetes, 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 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 pro-inflammatory and pro-fibrotic genes compared to 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 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.

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.

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) β, and under diabetic conditions, this IFNβ-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.

Venous thrombosis (VT), a leading cause of morbidity and mortality worldwide, has recently been linked to neutrophil activation and release of neutrophil extracellular traps (NETs) via a process called NETosis. The use of various in vivo thrombosis models and genetically modified mice has more precisely defined the exact role of NETosis in the pathogenesis of VT. Translational large animal VT models and human studies have confirmed the presence of NETs in pathologic VT. Activation of neutrophils, with subsequent NETosis, has also been linked to acute infection. This innate immune response, while effective for bacterial clearance from the host by formation of an intravascular bactericidal “net,” also triggers thrombosis. Intravascular thrombosis related to such innate immune mechanisms has been coined immunothrombosis. Dysregulated immunothrombosis has been proposed as a mechanism of pathologic micro- and macrovascular thrombosis in sepsis and autoimmune disease. In this focused review, we will address the dual role of NETs in the pathogenesis of VT and immunothrombosis.

Background: Venous thrombosis (VT) and the vein wall responses may be related to interleukin 6 (IL-6) signaling in humans and mice. Inflammatory and fibrotic effects of IL-6 largely signal through the trans pathway: IL-6/soluble IL-6Rα complex associates with ubiquitously expressed signal transducing protein gp130. We hypothesized that IL-6 signaling would differ depending upon thrombosis mechanism (complete v. partial stasis), and disruption of IL-6/IL-6Rα/gp130 axis would alter vein wall response to VT. Methods: Wild type C57BL/6 or IL-6-/- mice underwent induction of VT via inferior vena cava (IVC) stenosis (partial stasis) or ligation (complete stasis). Select WT mice received sgp130Fc chimera to inhibit trans pathway signaling. Vein wall, thrombus, and plasma were harvested at various time points and analyzed by PCR, western blot, ELISA or immunohistochemistry. Results: Compared to sham animals, those undergoing VT (complete or partial stasis) exhibited elevated vein wall IL-6 on post-VT day 4 (n=6-9, p Conclusions: Experimental VT stimulates IL-6 and downstream signaling processes and is dependent on the mechanism of thrombosis. Genetic deletion of IL-6 and selective disruption of trans-signaling pathway by sgp130 decreases vein wall inflammation and influences vein wall remodeling. Targeting of IL-6 trans-signaling pathway may represent a therapeutic option to treat vein wall inflammation post thrombosis.

Background: Wound healing is impaired in diabetes due to dysregulated inflammation. Previous studies by our group and others have demonstrated that inflammatory mediators are significantly increased in diabetic wounds, however, the mechanisms behind this hyper-inflammatory response remain largely unknown. As there is known to be high levels of endotoxin in type 2 diabetic (T2D) wounds, we aimed to assess the role of high fat diet (HFD) on Toll-Like Receptor 4 (TLR4) expression. We hypothesized that HFD/T2D would alter histone methylation, leading to increased receptor expression and enhanced inflammation. Methods: C57/BL6 mice were subjected to either normal or HFD for 12-16 weeks to induce the diet-induced obese (DIO) model of physiologic T2D. 4mm hindlimb wounds were created and healing was monitored daily using NIH ImageJ software. Bone marrow-derived macrophages (BMDM) were cultured and TLR4 expression measured by qPCR. Myeloid TLR4 protein expression was assessed in peripheral blood and wound cell isolates by analytical flow cytometry. Macrophages (NK1.1-/Ly6G-/CD11b+) were isolated using magnetic sorting and inflammatory cytokine expression determined with qPCR and bioplex. Chromatin Immunoprecipitation (ChIP) analysis at the NF-kB binding site on the TLR4 promoter was performed using antibodies to trimethylated Histone 3 Lysine 4 (H3K4me3). (N=~5 mice per group for all studies). Results: DIO mice demonstrated significantly delayed wound healing as compared to littermate controls and BMDM isolated from these mice revealed increased TLR4 gene expression. Correspondingly, peripheral blood and wound monocyte/macrophages showed increased TLR4 receptor levels by flow cytometry. ChIP analysis at the TLR4 promoter revealed significantly increased H3K4me3, a gene activating mark, in the HFD BMDM. Finally, macrophage wound isolates from DIO mice had elevated levels of inflammatory gene expression and cytokines. Conclusion: Enhanced H3K4me3 at the TLR4 promoter in diabetic macrophages may increase receptor expression and contribute to the hyper-inflammatory response seen in T2D. Further mechanistic studies are needed in order to examine the role of TLR4 in wound healing.

Wound healing in diabetic patients is impaired because of dysregulated innate immune response that results in a chronic inflammatory state. It has been shown by our group and others that metabolites can contribute to a state of chronic inflammation in diabetic tissues. In a murine model of Type-2 diabetes (diet-induced obese mice; DIO), we assessed role of uric acid in driving macrophage-mediated inflammation and delayed wound healing. Using liquid chromatography-mass spectrometry, we show that macrophages from DIO mice have reduced levels of xanthine, a precursor in the uric acid pathway. Xanthine is converted to uric acid via Xanthine oxidase/dehydrogenase (XOR) encoded by the Xdh gene. DIO macrophages express higher levels of Xdh and increased levels of uric acid, suggesting increased XOR activity. Further, exogenous addition of uric acid led to increased production of IL-1β by macrophages. Importantly, pharmacologic inhibition of XOR with Allopurinol resulted in reduced levels of IL-1β expression by macrophages. In order to examine this in vivo, DIO mice were treated with Allopurinol leading to significantly improved wound healing. In summary, our data suggest that the xanthine metabolic pathway can be targeted therapeutically to accelerate wound healing in diabetic patients.

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. There is increasing evidence that epigenetic-based mechanisms, including DNA hypomethylation, control Mϕ function. The cyclooxygenase (COX)-2/Prostaglandin E2 (PGE2) axis as well as upstream pathways including cytosolic phospholipase (c(PL)A2), involved in the release arachidonic acid, have been associated with chronic inflammation, however they have not been examined in diabetic wounds. The purpose of this study was to investigate if epigenetic modifications alter gene expression of the COX-2/PGE2 and c(PL)A2 pathway leading to derangements in macrophage inflammation in diabetes. Using the murine diet induce obesity (DIO) model, we evaluated diabetic wound healing compared with control mice. We demonstrate that TGFβ is elevated in DIO wound Mϕs in comparison to control wound Mϕs. Further, the increase in TGFβ stimulates miR-29B production in wound Mϕs resulting in hypomethylation and overexpression of the COX-2 gene. This results in diabetic wound Mϕs displaying increased levels of COX-2 which shunts amino acid metabolism towards the COX-2 pathway increasing PGE2 while limiting leukotriene synthesis via 5-lipoxygenase (5-LO) pathways. Lastly, c(PL)A2 appears to be upregulated in diabetic wound Mϕs and human diabetic monocytes. In summary, these results demonstrate that the COX-2/PGE2 pathway is increased in diabetic wound Mϕs by elevated TGFβ/miR-29b and results in elevated PGE2 that drives Mϕ-mediated inflammation preventing wound repair.

Macrophage (Mϕ) plasticity is essential for the repair and remodeling of wounds. However, under pathologic conditions such as type 2 diabetes (T2D), Mϕs exist in a chronic inflammatory state that drives continued tissue destruction and results in impaired wound healing. We have previously shown that Mϕs isolated from wounds in a murine model that mimics physiologic development of T2D (diet-induced obese mice; DIO) display increased levels of inflammatory cytokines at both a gene expression and protein level. Although the underlying mechanisms remain unclear, we and others have shown that Mϕ phenotype can be controlled by epigenetic modifications that alter gene expression. Our recent work provides evidence that the diabetic milieu alters gene expression and sets an “epigenetic signature” in myeloid cells that result in an exaggerated inflammatory phenotype and delayed wound healing in a murine model. Further, our recent work has found that the histone acetyl transferase, Males absent on the first (MOF), responsible for acetylation of lysine 16 on histone H4 (H4K16), was critical for expression of genes involved in fatty acid metabolism. In the present study, we find that wound Mϕs from DIO mice have increased expression of MOF at day 5, when Mϕs shift from a pro-inflammatory to an anti-inflammatory state within wound tissue. This change in MOF was accompanied by increased expression of components of MOF regulatory complexes, MSL1/2 and KANSL1. Our findings reveal a previously unreported role for MOF in promoting a pro-inflammatory state in innate immune cells. Moreover, our data suggest that the diabetic milieu promotes increased expression of MOF which, in turn, may contribute to maintaining the persistent inflammatory state in wound Mϕs.