Transcription Factor Activity Regulating Macrophage Heterogeneity during Skin Wound Healing.

Monocytes and macrophages (Mos/Mϕs) play diverse roles in wound healing by adopting a spectrum of functional phenotypes; however, the regulation of such heterogeneity remains poorly defined. We enhanced our previously published Bayesian inference TF activity model, incorporating both single-cell RNA sequencing and single-cell ATAC sequencing data to infer transcription factor (TF) activity in Mos/Mϕs during skin wound healing. We found that wound Mos/Mϕs clustered into early-stage Mos/Mϕs, late-stage Mϕs, and APCs, and that each cluster showed differential chromatin accessibility and differential predicted TF activity that did not always correlate with mRNA or protein expression. Network analysis revealed two highly connected large communities involving a total of 19 TFs, highlighting TF cooperation in regulating wound Mos/Mϕs. This analysis also revealed a small community populated by NR4A1 and NFKB1, supporting a proinflammatory link between these TFs. Importantly, we validated a proinflammatory role for NR4A1 activity during wound healing, showing that Nr4a1 knockout mice exhibit decreased inflammatory gene expression in early-stage wound Mos/Mϕs, along with delayed wound re-epithelialization and impaired granulation tissue formation. In summary, our study provides insight into TF activity that regulates Mo/Mϕ heterogeneity during wound healing and provides a rational basis for targeting Mo/Mϕ TF networks to alter phenotypes and improve healing.

Monocyte/Macrophage Heterogeneity during Skin Wound Healing in Mice.

Monocytes (Mos)/macrophages (Mϕs) orchestrate biological processes critical for efficient skin wound healing. However, current understanding of skin wound Mo/Mϕ heterogeneity is limited by traditional experimental approaches such as flow cytometry and immunohistochemistry. Therefore, we sought to more fully explore Mo/Mϕ heterogeneity and associated state transitions during the course of excisional skin wound healing in mice using single-cell RNA sequencing. The live CD45+CD11b+Ly6G- cells were isolated from skin wounds of C57BL/6 mice on days 3, 6, and 10 postinjury and captured using the 10x Genomics Chromium platform. A total of 2813 high-quality cells were embedded into a uniform manifold approximation and projection space, and eight clusters of distinctive cell populations were identified. Cluster dissimilarity and differentially expressed gene analysis categorized those clusters into three groups: early-stage/proinflammatory, late-stage/prohealing, and Ag-presenting phenotypes. Signature gene and Gene Ontology analysis of each cluster provided clues about the different functions of the Mo/Mϕ subsets, including inflammation, chemotaxis, biosynthesis, angiogenesis, proliferation, and cell death. Quantitative PCR assays validated characteristics of early- versus late-stage Mos/Mϕs inferred from our single-cell RNA sequencing dataset. Additionally, cell trajectory analysis by pseudotime and RNA velocity and adoptive transfer experiments indicated state transitions between early- and late-state Mos/Mϕs as healing progressed. Finally, we show that the chemokine Ccl7, which was a signature gene for early-stage Mos/Mϕs, preferentially induced the accumulation of proinflammatory Ly6C+F4/80lo/- Mos/Mϕs in mouse skin wounds. In summary, our data demonstrate the complexity of Mo/Mϕ phenotypes, their dynamic behavior, and diverse functions during normal skin wound healing.

Enhanced Proliferation of Ly6C+ Monocytes/Macrophages Contributes to Chronic Inflammation in Skin Wounds of Diabetic Mice.

Diabetic wounds are characterized by persistent accumulation of proinflammatory monocytes (Mo)/macrophages (MΦ) and impaired healing. However, the mechanisms underlying the persistent accumulation of Mo/MΦ remain poorly understood. In this study, we report that Ly6C+F4/80lo/- Mo/MΦ proliferate at higher rates in wounds of diabetic mice compared with nondiabetic mice, leading to greater accumulation of these cells. Unbiased single cell RNA sequencing analysis of combined nondiabetic and diabetic wound Mo/MΦ revealed a cluster, populated primarily by cells from diabetic wounds, for which genes associated with the cell cycle were enriched. In a screen of potential regulators, CCL2 levels were increased in wounds of diabetic mice, and subsequent experiments showed that local CCL2 treatment increased Ly6C+F4/80lo/- Mo/MΦ proliferation. Importantly, adoptive transfer of mixtures of CCR2-/- and CCR2+/+ Ly6Chi Mo indicated that CCL2/CCR2 signaling is required for their proliferation in the wound environment. Together, these data demonstrate a novel role for the CCL2/CCR2 signaling pathway in promoting skin Mo/MΦ proliferation, contributing to persistent accumulation of Mo/MΦ and impaired healing in diabetic mice.

Reduced apoptosis of monocytes and macrophages is associated with their persistence in wounds of diabetic mice.

Monocytes and macrophages (Mo/MΦ) rapidly accumulate in skin wounds after injury, then disappear as healing progresses. However, the mechanisms underlying their ultimate fate in wounds remain to be elucidated. Here, we show that apoptosis of Mo/MΦ parallels their reduction as wound healing progresses in non-diabetic mice. scRNAseq analysis confirmed enriched apoptosis GO pathways on day 6 post-injury in wound Mo/MΦ from non-diabetic mice. In contrast, there was significantly less Mo/MΦ apoptosis in wounds from diabetic mice, particularly in the pro-inflammatory Ly6C+ population, which may contribute to persistent Mo/MΦ accumulation and chronic inflammation. scRNAseq analysis implicated TNF, MAPK, Jak-STAT, and FoxO signaling pathways in promoting wound Mo/MΦ apoptosis in non-diabetic mice while cell proliferation related pathways appeared to be activated in diabetic mice. These novel findings indicate that reduced apoptosis is a contributor to persistent Mo/MΦ accumulation in diabetic wounds. These findings also highlight pathways that may regulate Mo/MΦ apoptosis during wound healing, which could be targeted to help resolve inflammation and improve healing.

Skin Wounding-Induced Monocyte Expansion in Mice Is Not Abrogated by IL-1 Receptor 1 Deficiency.

The aim of this study was to determine whether skin wounding induces monocyte (Mo) expansion in bone marrow and whether IL-1R1 signaling regulates this process. Our data show that skin wounding increases myeloid lineage-committed multipotent progenitors (MPP3 subset) and Mo in bone marrow, but this expansion is not impaired in Il1r1-/- mice. We also demonstrate that M-CSF-induced differentiation of myeloid progenitors into Mo is not impaired by the loss of IL-1R1 ex vivo, indicating that IL-R1 deficiency does not abrogate myeloid progenitor differentiation potential. In addition, we observed modestly delayed wound closure in Il1r1-/- mice associated with higher frequency of Ly6Clo Mo in the circulation at baseline and in wounds early after injury. Thus, in contrast to other models of inflammation that involve IL-1R1-dependent monopoiesis, our results demonstrate that skin wounding induces Mo progenitor and Mo expansion independently of IL-1R1 signaling.

Diet-Induced Obesity Increases Monocyte/Macrophage Proliferation during Skin Wound Healing in Mice.

Obesity is associated with low-grade chronic inflammation and impaired glucose metabolism, both of which are detrimental to wound healing. C-C motif chemokine receptor 2 (CCR2) plays an important role in cell recruitment during healing, and our recent studies revealed the significance of CCR2-CCL2 signaling in promoting the proliferation of pro-inflammatory monocytes/macrophages in wounds. Therefore, we sought to determine whether diet-induced obesity increases monocyte/macrophage proliferation and their accumulation in skin wounds. We first confirmed that wound closure was delayed in obese CCR2RFP/+ mice fed with a high-fat diet (HFD) compared to mice fed with a normal diet (ND). Using in vivo imaging and flow cytometry analysis, we found that HFD mice had significantly increased accumulation of CCR2+ monocytes/macrophages, particularly pro-inflammatory CCR2+Ly6C+ cells in wounds compared to their ND counterparts. Importantly, HFD mice exhibited an increased proliferation of wound CCR2+Ly6C+ compared to ND mice. Together, our data suggest that obesity leads to an increased proliferation and accumulation of pro-inflammatory CCR2+Ly6C+ monocytes/macrophages in skin wounds, which may contribute to delayed healing.

Proliferation of monocytes and macrophages in homeostasis, infection, injury, and disease.

Monocytes (Mo) and macrophages (Mφ) play important roles in the function of tissues, organs, and systems of all animals during homeostasis, infection, injury, and disease. For decades, conventional wisdom has dictated that Mo and Mφ are end-stage cells that do not proliferate and that Mφ accumulation in tissues is the result of infiltration of Mo from the blood and subsequent differentiation to Mφ. However, reports from the early 1900s to the present describe evidence of Mo and Mφ proliferation in different tissues and contexts. The purpose of this review is to summarize both historical and current evidence for the contribution of Mφ proliferation to their accumulation in different tissues during homeostasis, infection, injury, and disease. Mφ proliferate in different organs and tissues, including skin, peritoneum, lung, heart, aorta, kidney, liver, pancreas, brain, spinal cord, eye, adipose tissue, and uterus, and in different species including mouse, rat, rabbit, and human. Mφ can proliferate at different stages of differentiation with infiltrating Mo-like cells proliferating in certain inflammatory contexts (e.g. skin wounding, kidney injury, bladder and liver infection) and mature resident Mφ proliferating in other inflammatory contexts (e.g. nematode infection, acetaminophen liver injury) and during homeostasis. The pathways involved in stimulating Mφ proliferation also may be context dependent, with different cytokines and transcription factors implicated in different studies. Although Mφ are known to proliferate in health, injury, and disease, much remains to be learned about the regulation of Mφ proliferation in different contexts and its impact on the homeostasis, injury, and repair of different organs and tissues.

Non-epitaxial growth of highly oriented transition metal dichalcogenides with density-controlled twin boundaries.

Twin boundaries (TBs) in transition metal dichalcogenides (TMDs) constitute distinctive one-dimensional electronic systems, exhibiting intriguing physical and chemical properties that have garnered significant attention in the fields of quantum physics and electrocatalysis. However, the controlled manipulation of TBs in terms of density and specific atomic configurations remains a formidable challenge. In this study, we present a non-epitaxial growth approach that enables the controlled and large-scale fabrication of homogeneous catalytically active TBs in monolayer TMDs on arbitrary substrates. Notably, the density achieved using this strategy is six times higher than that observed in convention chemical vapor deposition (CVD)-grown samples. Through rigorous experimental analysis and multigrain Wulff construction simulations, we elucidate the role of regulating the metal source diffusion process, which serves as the key factor for inducing the self-oriented growth of TMD grains and the formation of unified TBs. Furthermore, we demonstrate that this novel growth mode can be readily incorporated into the conventional CVD growth method by making a simple modification of the growth temperature profile, thereby offering a universal approach for engineering of grain boundaries in two-dimensional materials.

Proliferation of Ly6C+ monocytes/macrophages contributes to their accumulation in mouse skin wounds.

Monocytes and macrophages (Mo/MΦ) play critical roles in all phases of skin wound healing. The majority of these cells are thought to be recruited from blood Mo; however, the role local proliferation of Mo/MΦ in the wound has not been defined. Therefore, we tested the hypothesis that local proliferation of Mo and/or MΦ contributes to their accumulation during wound healing. Male C57Bl/6 mice (N = 4-9/group) were subjected to excisional skin wounding. Proliferating Mo/MΦ (F4/80+Ki67+) were observed in wound cryosections, peaking on day 5 post-wounding. Cell cycle analysis on cells isolated from skin tissue revealed that wounding increased both the number and percentage of inflammatory Ly6C+F4/80lo/- Mo/MΦ in the S/G2/M phases, peaking on day 6 post-wounding. In contrast, more mature Ly6C-F4/80+ cells were found predominantly in the G0 phase with less than 1% cells in S/G2/M phase following injury. In peripheral blood, Mo were very rarely found in the S/G2/M phase, suggesting that the wound environment triggered the Ly6C+F4/80lo/- Mo proliferative response. Furthermore, injury induced several potential regulators of proliferation in wounds, including IL-1ß and IL-6, and wound Mo/MΦ expressed surface receptors for these cytokines. However, wound Mo/MΦ proliferation was not altered in IL-1R1 knockout (KO) or IL-6 KO mice. In summary, our findings indicate that proliferation contributes to Mo/MΦ accumulation in wounds and, contrary to findings in other pathophysiologic conditions, Ly6C+/F4/80lo/- Mo/MΦ proliferate during skin wound healing whereas mature Ly6C-F4/80+ MΦ do not.

A 124-plex Microhaplotype Panel Based on Next-generation Sequencing Developed for Forensic Applications.

Microhaplotypes are an emerging type of forensic genetic marker that are expected to support multiple forensic applications. Here, we developed a 124-plex panel for microhaplotype genotyping based on next-generation sequencing (NGS). The panel yielded intralocus and interlocus balanced sequencing data with a high percentage of effective reads. A full genotype was determined with as little as 0.1 ng of input DNA. Parallel mixture experiments and in-depth comparative analyses were performed with capillary-electrophoresis-based short tandem repeat (STR) and NGS-based microhaplotype genotyping, and demonstrated that microhaplotypes are far superior to STRs for mixture deconvolution. DNA from Han Chinese individuals (n = 256) was sequenced with the 124-plex panel. In total, 514 alleles were observed, and the forensic genetic parameters were calculated. A comparison of the forensic parameters for the 20 microhaplotypes with the top Ae values in the 124-plex panel and 20 commonly used forensic STRs showed that these microhaplotypes were as effective as STRs in identifying individuals. A linkage disequilibrium analysis showed that 106 of the 124 microhaplotypes were independently hereditary, and the combined match probability for these 106 microhaplotypes was 5.23 × 10-66. We conclude that this 124-plex microhaplotype panel is a powerful tool for forensic applications.

Multiphasic Regulation of Systemic and Peripheral Organ Metabolic Responses to Cardiac Hypertrophy.

BACKGROUND: Reduced fat oxidation in hypertrophied hearts coincides with a shift of carnitine palmitoyl transferase I from muscle to increased liver isoforms. Acutely increased carnitine palmitoyl transferase I in normal rodent hearts has been shown to recapitulate the reduced fat oxidation and elevated atrial natriuretic peptide message of cardiac hypertrophy. METHODS AND RESULTS: Because of the potential for reduced fat oxidation to affect cardiac atrial natriuretic peptide, and thus, induce adipose lipolysis, we studied peripheral and systemic metabolism in male C57BL/6 mice model of transverse aortic constriction in which left ventricular hypertrophy occurred by 2 weeks without functional decline until 16 weeks (ejection fraction, -45.6%; fractional shortening, -22.6%). We report the first evidence for initially improved glucose tolerance and insulin sensitivity in response to 2 weeks transverse aortic constriction versus sham, linked to enhanced insulin signaling in liver and visceral adipose tissue (epididymal white adipose tissue [WAT]), reduced WAT inflammation, elevated adiponectin, mulitilocular subcutaneous adipose tissue (inguinal WAT) with upregulated oxidative/thermogenic gene expression, and downregulated lipolysis and lipogenesis genes in epididymal WAT. By 6 weeks transverse aortic constriction, the metabolic profile reversed with impaired insulin sensitivity and glucose tolerance, reduced insulin signaling in liver, epididymal WAT and heart, and downregulation of oxidative enzymes in brown adipose tissue and oxidative and lipogenic genes in inguinal WAT. CONCLUSIONS: Changes in insulin signaling, circulating natriuretic peptides and adipokines, and varied expression of adipose genes associated with altered insulin response/glucose handling and thermogenesis occurred prior to any functional decline in transverse aortic constriction hearts. The findings demonstrate multiphasic responses in extracardiac metabolism to pathogenic cardiac stress, with early iWAT browning providing potential metabolic benefits.

Activin in acute pancreatitis: Potential risk-stratifying marker and novel therapeutic target.

Acute Pancreatitis is a substantial health care challenge with increasing incidence. Patients who develop severe disease have considerable mortality. Currently, no reliable predictive marker to identify patients at risk for severe disease exists. Treatment is limited to rehydration and supporting care suggesting an urgent need to develop novel approaches to improve standard care. Activin is a critical modulator of inflammatory responses, but has not been assessed in pancreatitis. Here, we demonstrate that serum activin is elevated and strongly correlates with disease severity in two established murine models of acute pancreatitis induced by either cerulein or IL-12 + IL-18. Furthermore, in mice, inhibition of activin conveys survival benefits in pancreatitis. In addition, serum activin levels were measured from a retrospective clinical cohort of pancreatitis patients and high activin levels in patients at admission are predictive of worse outcomes, indicated by longer overall hospital and intensive care unit stays. Taken together, activin is a novel candidate as a clinical marker to identify those acute pancreatitis patients with severe disease who would benefit from aggressive treatment and activin may be a therapeutic target in severe acute pancreatitis.

Lipodystrophy and severe metabolic dysfunction in mice with adipose tissue-specific insulin receptor ablation.

OBJECTIVE: Insulin signaling plays pivotal roles in the development and metabolism of many tissues and cell types. A previous study demonstrated that ablation of insulin receptor (IR) with aP2-Cre markedly reduced adipose tissues mass and protected mice from obesity. However, multiple studies have demonstrated widespread non-adipocyte recombination of floxed alleles in aP2-Cre mice. These findings underscore the need to re-evaluate the role of IR in adipocyte and systemic metabolism with a more adipose tissue-specific Cre mouse line. METHODS: We generated and phenotyped a new adipose tissue-specific IR mouse model using the adipose tissue-specific Adipoq-Cre line. RESULTS: Here we show that the Adipoq-Cre-mediated IR KO in mice leads to lipodystrophy and metabolic dysfunction, which is in stark contrast to the previous study. In contrast to white adipocytes, absence of insulin signaling does not affect development of marrow and brown adipocytes, but instead is required for lipid accumulation particularly for the marrow adipocytes. Lipodystrophic IR KO mice have profound insulin resistance, hyperglycemia, organomegaly, and impaired adipokine secretion. CONCLUSIONS: Our results demonstrate differential roles for insulin signaling for white, brown, and marrow adipocyte development and metabolic regulation.

Increased adiposity in annexin A1-deficient mice.

Production of Annexin A1 (ANXA1), a protein that mediates the anti-inflammatory action of glucocorticoids, is altered in obesity, but its role in modulation of adiposity has not yet been investigated. The objective of this study was to investigate modulation of ANXA1 in adipose tissue in murine models of obesity and to study the involvement of ANXA1 in diet-induced obesity in mice. Significant induction of ANXA1 mRNA was observed in adipose tissue of both C57BL6 and Balb/c mice with high fat diet (HFD)-induced obesity versus mice on chow diet. Upregulation of ANXA1 mRNA was independent of leptin or IL-6, as demonstrated by use of leptin-deficient ob/ob mice and IL-6 KO mice. Compared to WT mice, female Balb/c ANXA1 KO mice on HFD had increased adiposity, as indicated by significantly elevated body weight, fat mass, leptin levels, and adipocyte size. Whereas Balb/c WT mice upregulated expression of enzymes involved in the lipolytic pathway in response to HFD, this response was absent in ANXA1 KO mice. A significant increase in fasting glucose and insulin levels as well as development of insulin resistance was observed in ANXA1 KO mice on HFD compared to WT mice. Elevated plasma corticosterone levels and blunted downregulation of 11-beta hydroxysteroid dehydrogenase type 1 in adipose tissue was observed in ANXA1 KO mice compared to diet-matched WT mice. However, no differences between WT and KO mice on either chow or HFD were observed in expression of markers of adipose tissue inflammation. These data indicate that ANXA1 is an important modulator of adiposity in mice, with female ANXA1 KO mice on Balb/c background being more susceptible to weight gain and diet-induced insulin resistance compared to WT mice, without significant changes in inflammation.

New pathways to control inflammatory responses in adipose tissue.

Obesity is characterized by the presence of chronic inflammation in adipose tissue, particularly in the visceral compartment, that has been causally linked to development of obesity-associated comorbidities. This link can be either direct or indirect, through induction of insulin resistance. This review summarizes recent evidence on potential pharmacological targets of adipose tissue inflammation, with emphasis on mediators that are being studied for intervention in chronic inflammatory diseases and are therefore viable therapeutical candidates. Specifically, we discuss evidence on the role of the inflammasome and its downstream products as a potential target for anti-inflammatory strategies as well as T regulatory (Treg) cells and mediators involved in the resolution phase of inflammation such as resolvins, protectins, annexin A1 (ANXA1) and galectins as potential targets for novel agonist therapies.

Increased adiposity, dysregulated glucose metabolism and systemic inflammation in Galectin-3 KO mice.

Obesity and type 2 diabetes are associated with increased production of Galectin-3 (Gal-3), a protein that modulates inflammation and clearance of glucose adducts. We used Lean and Diet-induced Obese (DIO) WT and Gal-3 KO mice to investigate the role of Gal-3 in modulation of adiposity, glucose metabolism and inflammation. Deficiency of Gal-3 lead to age-dependent development of excess adiposity and systemic inflammation, as indicated by elevated production of acute-phase proteins, number of circulating pro-inflammatory Ly6C(high) monocytes and development of neutrophilia, microcytic anemia and thrombocytosis in 20-week-old Lean and DIO male Gal-3 KO mice. This was associated with impaired fasting glucose, heightened response to a glucose tolerance test and reduced adipose tissue expression of adiponectin, Gal-12, ATGL and PPARγ, in the presence of maintained insulin sensitivity and hepatic expression of gluconeogenic enzymes in 20-week-old Gal-3 KO mice compared to their diet-matched WT controls. Expression of PGC-1α and FGF-21 in the liver of Lean Gal-3 KO mice was comparable to that observed in DIO animals. Impaired fasting glucose and altered responsiveness to a glucose load preceded development of excess adiposity and systemic inflammation, as demonstrated in 12-week-old Gal-3 KO mice. Finally, a role for the microflora in mediating the fasting hyperglycemia, but not the excessive response to a glucose load, of 12-week-old Gal-3 KO mice was demonstrated by administration of antibiotics. In conclusion, Gal-3 is an important modulator of glucose metabolism, adiposity and inflammation.