Changes in macrophage phenotype and induction of epithelial‐to‐mesenchymal transition genes following acute Achilles tenotomy and repair

Tendon injuries occur frequently in physically active individuals, but the clinical outcomes for these injuries can be poor. In many injured tissues the repair process is orchestrated by two types of cells, macrophages and fibroblasts. Macrophages, which have both pro‐inflammatory (M1) and anti‐inflammatory (M2) phenotypes, can directly participate in tissue remodeling and direct the response of other cells through the secretion of cytokines and growth factors. In many organ systems, epithelial cells can trans‐differentiate into fibroblasts, which can then regenerate damaged ECM. This process is triggered via activation of epithelial‐to‐mesenchymal transition (EMT) signaling programs. Most tendons are surrounded by sheets of epithelial cells, and these tissue layers could provide a source of fibroblasts to repair injured tendons. To gain greater insight into the biology of tendon repair, we performed a tenotomy and repair in Achilles tendons of adult rats and determined changes in macrophage phenotype, and ECM‐ and EMT‐related genes over a 4‐week time course. The results from this study suggest that changes in macrophage phenotype and activation of EMT‐related programs likely contribute to the degradation and subsequent repair of injured tendon tissue. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:944–951, 2014.     

Embryonic stem cell–derived M2‐like macrophages delay cutaneous wound healing

In adults, repair of deeply injured skin wounds results in the formation of scar tissue, whereas in embryos wounds heal almost scar‐free. Macrophages are important mediators of wound healing and secrete cytokines and tissue remodeling enzymes. In contrast to host defense mediated by inflammatory M1 macrophages, wound healing and tissue repair involve regulatory M2/M2‐like macrophages. Embryonic/fetal macrophages are M2‐like, and this may promote scar‐free wound healing. In the present study, we asked whether atopical application of ex vivo generated, embryonic stem cell–derived macrophages (ESDM) improve wound healing in mice. ESDM were tested side by side with bone marrow–derived macrophages (BMDM). Compared to BMDM, ESDM resembled a less inflammatory and more M2‐like macrophage subtype as indicated by their reduced responsiveness to lipopolysaccharide, reduced expression of Toll‐like receptors, and reduced bacterial phagocytosis. Despite this anti‐inflammatory phenotype in cell culture, ESDM prolonged the healing of deep skin wounds even more than BMDM. Healed wounds had more scar formation compared to wounds receiving BMDM or cell‐free treatment. Our data indicate that atopical application of ex vivo generated macrophages is not a suitable cell therapy of dermal wounds.     

Anti‐inflammatory effects of haptoglobin on LPS‐stimulated macrophages: Role of HMGB1 signaling and implications in chronic wound healing

Nonhealing wounds possess elevated numbers of pro‐inflammatory M1 macrophages, which fail to transition to anti‐inflammatory M2 phenotypes that promote healing. Hemoglobin (Hb) and haptoglobin (Hp) proteins, when complexed (Hb‐Hp), can elicit M2‐like macrophages through the heme oxygenase‐1 (HO‐1) pathway. Despite the fact that nonhealing wounds are chronically inflamed, previous studies have focused on non‐inflammatory systems, and do not thoroughly compare the effects of complexed vs individual proteins. We aimed to investigate the effect of Hb/Hp treatments on macrophage phenotype in an inflammatory, lipopolysaccharide (LPS)‐stimulated environment, similar to chronic wounds. Human M1 macrophages were cultured in vitro and stimulated with LPS. Concurrently, Hp, Hb, or Hb‐Hp complexes were delivered. The next day, 27 proteins related to inflammation were measured in the supernatants. Hp treatment decreased a majority of inflammatory factors, Hb increased many, and Hb‐Hp had intermediate trends, indicating that Hp attenuated overall inflammation to the greatest extent. From this data, Ingenuity Pathway Analysis software identified high motility group box 1 (HMGB1) as a key canonical pathway—strongly down‐regulated from Hp, strongly up‐regulated from Hb, and slightly activated from Hb‐Hp. HMGB1 measurements in macrophage supernatants confirmed this trend. In vivo results in diabetic mice with biopsy punch wounds demonstrated accelerated wound closure with Hp treatment, and delayed wound closure with Hb treatment. This work specifically studied Hb/Hp effects on macrophages in a highly inflammatory environment relevant to chronic wound healing. Results show that Hp—and not Hb‐Hp, which is known to be superior in noninflammatory conditions—reduces inflammation in LPS‐stimulated macrophages, and HMGB1 signaling is also implicated. Overall, Hp treatment on M1 macrophages in vitro reduced the inflammatory secretion profile, and also exhibited benefits in in silico and in vivo wound‐healing models.     

Pro‐inflammatory M1 macrophages promote Osteogenesis by mesenchymal stem cells via the COX‐2‐prostaglandin E2 pathway

Bone fractures are among the most common orthopaedic problems that affect individuals of all ages. Immediately after injury, activated macrophages dynamically contribute to and regulate an acute inflammatory response that involves other cells at the injury site, including mesenchymal stem cells (MSCs). These macrophages and MSCs work in concert to modulate bone healing. In this study, we co‐cultured undifferentiated M0, pro‐inflammatory M1, and anti‐inflammatory M2 macrophages with primary murine MSCs in vitro to determine the cross‐talk between polarized macrophages and MSCs and their effects on osteogenesis. After 4 weeks of co‐culture, MSCs grown with macrophages, especially M1 macrophages, had enhanced bone mineralization compared to MSCs grown alone. The level of bone formation after 4 weeks of culture was closely associated with prostaglandin E2 (PGE2) secretion early in osteogenesis. Treatment with celecoxib, a cyclooxygenase‐2 (COX‐2) selective inhibitor, significantly reduced bone mineralization in all co‐cultures but most dramatically in the M1‐MSC co‐culture. We also found that the presence of macrophages reduced the secretion of osteoprotegerin (OPG), the decoy RANKL receptor, suggesting that macrophages may indirectly modulate osteoclast activity in addition to enhancing bone formation. Taken together, these findings suggest that an initial pro‐inflammatory phase modulated by M1 macrophages promotes osteogenesis in MSCs via the COX‐2‐PGE2 pathway. Understanding the complex interactions between macrophages and MSCs provide opportunities to optimize bone healing and other regenerative processes via modulation of the inflammatory response. This study provides one possible biological mechanism for the adverse effects of non‐steroidal anti‐inflammatory drugs on fracture healing and bone regeneration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2378–2385, 2017.

     

Tetrandrine suppresses articular inflammatory response by inhibiting pro‐inflammatory factors via NF‐κB inactivation

Targeting activated macrophages using anti‐inflammatory phytopharmaceuticals has been proposed as general therapeutic approaches for rheumatic diseases. Besides macrophages, chondrocytes are another promising target of anti‐inflammatory agents. Tetrandrine is a major bisbenzylisoquinoline alkaloid isolated from Stephania tetrandrae S. Moore which has been used for 2,000 years as an antirheumatic herbal drug in China. Although, the anti‐inflammatory effect of tetrandrine has been demonstrated, the mechanism has not been clearly clarified. In this study, we designed a comprehensive anti‐inflammatory evaluation system for tetrandrine, including complete Freund's adjuvant (CFA)‐induced arthritis rat, LPS‐induced macrophage RAW 264.7 cells, and chondrogenic ATDC5 cells. The results showed that tetrandrine alleviated CFA‐induced foot swelling, synovial inflammation, and pro‐inflammatory cytokines secretion. Tetrandrine could inhibit IL‐6, IL‐1β, and TNF‐α expression via blocking the nuclear translocation of nuclear factor (NF)‐κB p65 in LPS‐induced RAW 264.7 cells. Moreover, ATDC5 cells well responded to LPS induced pro‐inflammatory mediators secretion and tissue degradation, and tetrandrine could also inhibit the production of nitric oxide and prostaglandin E₂, as well as the expression of matrix metalloproteinase (MMP)‐3 and tissue inhibitor of metalloproteinase (TIMP)‐1 via inhibiting IκBα phosphorylation and degradation. In conclusion, the results showed that one of the anti‐inflammatory mechanisms of tetrandrine was inhibiting IκBα and NF‐κB p65 phosphorylation in LPS‐induced macrophage RAW 264.7 cells and chondrogenic ATDC5 cells. Moreover, we introduce a vigorous in vitro cell screening system, LPS‐induced murine macrophage RAW 264.7 cells coupling chondrogenic ADTC5 cells, for screening anti‐rheumatic drugs. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1557–1568, 2016.     

Alternatively activated macrophages derived from THP‐1 cells promote the fibrogenic activities of human dermal fibroblasts

Macrophages play a key role in the wound healing process and can be divided into classically activated macrophages (M1) and alternatively activated macrophages (M2). Fibroblasts maintain the physical integrity of connective tissue, participate in wound closure as well as produce and remodel extracellular matrix. Macrophages have a close relationship with fibroblasts by increasing the production of matrix metalloproteinase‐1 (MMP‐1) for faster wound closure and remodeling and myofibroblast differentiation from fibroblasts. In this study, resting state (M0), M1 and M2 macrophages differentiated from the human monocytic THP‐1 cell line were used to co‐culture with human dermal fibroblasts (HDF) for 48, 96 and 144 hours to investigate the effect of macrophages subsets on the fibrogenic activity of fibroblasts. The differentiation and polarization from THP‐1 cells to M0, M1 and M2 macrophages were characterized by flow cytometry and cell cycle analysis. Cell sorting was performed to purify M0 and M2 macrophages. Cell proliferation, collagen synthesis, myofibroblast formation, gene expression of anti‐fibrotic and pro‐fibrotic factors, MMP‐1 activity, and cytokine concentration were investigated. Results showed differentiation of M0 and polarization of M1 and M2 macrophages. M2 macrophages promoted the fibrogenic activities of co‐cultured HDF by facilitating cell proliferation, increasing the collagen content, alpha‐smooth muscle actin expressed cells, expression of the pro‐fibrotic genes and concentration of M2 macrophage related factors, as well as decreasing the expression of the anti‐fibrotic genes and MMP‐1 activity. These findings reinforce the pro‐fibrotic role of M2 macrophages, suggesting therapeutic strategies in fibrotic diseases should target M2 macrophages in the future.     

Self‐synthesized extracellular matrix contributes to mature adipose tissue regeneration in a tissue engineering chamber

The development of an engineered adipose tissue substitute capable of supporting reliable, predictable, and complete fat tissue regeneration would be of value in plastic and reconstructive surgery. For adipogenesis, a tissue engineering chamber provides an optimized microenvironment that is both efficacious and reproducible; however, for reasons that remain unclear, tissues regenerated in a tissue engineering chamber consist mostly of connective rather than adipose tissue. Here, we describe a chamber‐based system for improving the yield of mature adipose tissue and discuss the potential mechanism of adipogenesis in tissue‐chamber models. Adipose tissue flaps with independent vascular pedicles placed in chambers were implanted into rabbits. Adipose volume increased significantly during the observation period (week 1, 2, 3, 4, 16). Histomorphometry revealed mature adipose tissue with signs of adipose tissue remolding. The induced engineered constructs showed high‐level expression of adipogenic (peroxisome proliferator‐activated receptor γ), chemotactic (stromal cell‐derived factor 1a), and inflammatory (interleukin 1 and 6) genes. In our system, the extracellular matrix may have served as a scaffold for cell migration and proliferation, allowing mature adipose tissue to be obtained in a chamber microenvironment without the need for an exogenous scaffold. Our results provide new insights into key elements involved in the early development of adipose tissue regeneration.     

Graft‐Infiltrating Macrophages Adopt an M2 Phenotype and Are Inhibited by Purinergic Receptor P2X7 Antagonist in Chronic Rejection

Macrophages exhibit diverse phenotypes and functions; they are also a major cell type infiltrating chronically rejected allografts. The exact phenotypes and roles of macrophages in chronic graft loss remain poorly defined. In the present study, we used a mouse heart transplant model to examine macrophages in chronic allograft rejection. We found that treatment of C57BL/6 mice with CTLA4 immunoglobulin fusion protein (CTLA4‐Ig) prevented acute rejection of a Balb/c heart allograft but allowed chronic rejection to develop over time, characterized by prominent neointima formation in the graft. There was extensive macrophage infiltration in the chronically rejected allografts, and the graft‐infiltrating macrophages expressed markers associated with M2 cells but not M1 cells. In an in vitro system in which macrophages were polarized into either M1 or M2 cells, we screened phenotypic differences between M1 and M2 cells and identified purinergic receptor P2X7 (P2x7r), an adenosine triphosphate (ATP)–gated ion channel protein that was preferentially expressed by M2 cells. We further showed that blocking the P2x7r using oxidized ATP (oATP) inhibited M2 induction in a dose‐dependent fashion in vitro. Moreover, treatment of C57BL/6 recipients with the P2x7r antagonist oATP, in addition to CTLA4‐Ig treatment, inhibited graft‐infiltrating M2 cells, prevented transplant vasculopathy, and induced long‐term heart allografts survival. These findings highlight the importance of the P2x7r–M2 axis in chronic rejection and establish P2x7r as a potential therapeutic target in suppression of chronic rejection.     

The clinical dynamic changes of macrophage phenotype and function in different stages of human wound healing and hypertrophic scar formation

The pathogenesis of hypertrophic scar (HS) is still poorly understood. Macrophages, especially the polarisation of that to M1 or M2, play a pivotal role in control of the degree of scar formation. Profiling of macrophage phenotypes in human specimens during long‐term period of wound healing and HS formation may provide valuable clinical evidence for understanding the pathology of human scars. Human wound and HS specimens were collected, the macrophage phenotype was identified by immunofluorescence, and biomarkers and cytokines associated with M1 and M2 macrophages were detected by RT‐PCR. The correlation between the macrophage phenotype and HS characteristics was analysed by linear regression analyses. We found excessive and persistent infiltration by M1 macrophages around the blood vessels in the superficial layer of the dermis at early wound tissues, whereas M2 macrophages predominated in later wound tissues and the proliferative phase of HS and were scattered throughout the dermis. The density of M1 macrophages was positively correlated with mRNA expression levels of tumour necrosis factor‐alpha (TNF‐α) and IL‐6. The density of M2 macrophages was positively correlated with ARG1 and negatively correlated with the duration of HS. The sequential infiltration by M1 macrophage and M2 macrophages in human wound and HS tissues was confirmed.     

TNFα From Classically Activated Macrophages Accentuates Epithelial to Mesenchymal Transition in Obliterative Bronchiolitis

Bronchiolitis obliterans syndrome is characterized by fibrotic obliteration of small airways which severely impairs graft function and survival after lung transplantation. Bronchial epithelial cells from the transplanted lung can undergo epithelial to mesenchymal transition and this can be accentuated by activated macrophages. Macrophages demonstrate significant plasticity and change phenotype in response to their microenvironment. In this study we aimed to identify secretory products from macrophages that might be therapeutic targets for limiting the inflammatory accentuation of epithelial to mesenchymal transition in bronchiolitis obliterans syndrome. TNFα, IL‐1β and IL‐8 are elevated in bronchoalveolar lavage from lung transplant patients prior to diagnosis of bronchiolitis obliterans syndrome. Classically activated macrophages secrete more TNFα and IL‐1β than alternatively activated macrophages and dramatically accentuate TGF‐β1‐driven epithelial to mesenchymal transition in bronchial epithelial cells isolated from lung transplant patients. Blocking TNFα, but not IL‐1β, inhibits the accentuation of epithelial to mesenchymal transition. In a pilot unblinded therapeutic intervention in five patients with progressive bronchiolitis obliterans syndrome, anti‐TNFα treatment improved forced expiratory volume in 1 second and 6‐min walk distances in four patients. Our data identify TNFα as a potential new therapeutic target in bronchiolitis obliterans syndrome deserving of a randomized placebo controlled clinical trial.     

Short-Term Overfeeding May Induce Peripheral Insulin Resistance Without Altering Subcutaneous Adipose Tissue Macrophages in Humans

OBJECTIVE

Chronic low-grade inflammation is a feature of obesity and is postulated to be causal in the development of insulin resistance and type 2 diabetes. The aim of this study was to assess whether overfeeding induces peripheral insulin resistance in lean and overweight humans, and, if so, whether it is associated with increased systemic and adipose tissue inflammation.

RESEARCH DESIGN AND METHODS

Thirty-six healthy individuals undertook 28 days of overfeeding by +1,250 kcal/day (45% fat). Weight, body composition, insulin sensitivity (hyperinsulinemic-euglycemic clamp), serum and gene expression of inflammation markers, immune cell activation, fat cell size, macrophage and T-cell numbers in abdominal subcutaneous adipose tissue (flow cytometry and immunohistochemistry) were assessed at baseline and after 28 days.

RESULTS

Subjects gained 2.7 ± 1.6 kg (P < 0.001) and increased fat mass by 1.1 ± 1.6% (P < 0.001). Insulin sensitivity decreased by 11% from 54.6 ± 18.7 to 48.9 ± 15.7 μmol/(kg of FFM)/min (P = 0.01). There was a significant increase in circulating C-reactive protein (P = 0.002) and monocyte chemoattractant protein-1 (P = 0.01), but no change in interleukin-6 and intercellular adhesion molecule-1. There were no changes in fat cell size, the number of adipose tissue macrophages or T-cells, or inflammatory gene expression and no change in circulating immune cell number or expression of their surface activation markers after overfeeding.

CONCLUSIONS

Weight gain-induced insulin resistance was observed in the absence of a significant inflammatory state, suggesting that inflammation in subcutaneous adipose tissue occurs subsequent to peripheral insulin resistance in humans.Chronic low-level inflammation may be a pivotal link between obesity, insulin resistance, and type 2 diabetes (1). Studies performed in mouse models of obesity and in humans have shown increases in circulating proinflammatory mediators including C-reactive protein (CRP), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) (2–4). Moreover, obesity is associated with increased macrophage accumulation in adipose tissue with the majority of macrophages being located in crownlike structures, where aggregates of macrophages surround dead adipocytes (5). Importantly, cytokines derived from adipose tissue macrophages inhibit the insulin signaling cascade and can contribute to systemic insulin resistance in humans (6–8). However, whether adipose tissue macrophage accumulation causes insulin resistance in obese humans remains unclear.Obesity is associated not only with an increase in the total number of macrophages in adipose tissue (9) but also with a change in their activation state, from the alternative M2 to classical M1 phenotype. M1 macrophages are typically proinflammatory and release tumor necrosis factor, IL-6, and MCP-1, whereas M2 macrophages express an anti-inflammatory gene profile, characterized by higher expression of CD206 and IL-10 (10). Recent data has shown that M1 phenotype macrophages are >4-fold increased in subcutaneous adipose tissue from morbidly obese individuals as compared with lean individuals, with surgery-induced weight loss resulting in a twofold decrease in the M1/M2 ratio due to a concomitant decrease in M1 and increase in M2 macrophages (11). Importantly, both the number of M1 macrophages and the M1/M2 ratio have been shown to correlate with whole-body insulin sensitivity (12). There have been no studies examining the effects of moderate weight gain on M1/M2 phenotype in humans.T-cells, with their activation and hyperpolarization into a proinflammatory T-helper 1 phenotype, may play an important role in initiating and perpetuating adipose tissue inflammation (13–16). In mouse models of obesity, T-cell infiltration in adipose tissue is observed after 5 weeks of high-fat diet (HFD) in conjunction with the induction of insulin resistance (14). Despite evidence linking T-cell infiltration to adipose tissue in animal models of obesity, there have been no studies examining T-cells in adipose tissue during overfeeding in humans. Furthermore, there is no data to clarify whether circulating immune cell activation is an early change contributing to chronic inflammation and development of insulin resistance or whether insulin resistance precedes immune activation.The aims of this study were to investigate 1) whether short-term high-fat overfeeding and modest weight gain affects insulin sensitivity and systemic low-grade inflammation, immune cell counts and activation, and inflammatory gene expression and 2) the relationships between these variables. Our data suggest that modest weight gain and peripheral insulin resistance induced by overfeeding occurs prior to subcutaneous adipose tissue macrophage infiltration and induction of a significant inflammatory state in humans.     

Pro‐healing effects of bilirubin in open excision wound model in rats

Bilirubin, a by‐product of heme degradation, has an important role in cellular protection. Therefore, we speculated that bilirubin could be of potential therapeutic value in wound healing. To validate the hypothesis, we used a full‐thickness cutaneous wound model in rats. Bilirubin (30 mg/kg) was administered intraperitoneally every day for 9 days. The surface area of the wound was measured on days 0, 2, 4, 7 and 10 after the creation of the wound. The granulation tissue was collected on day 10 post‐wounding for analysing various parameters of wound healing. Bilirubin treatment accelerated wound contraction and increased hydroxyproline and glucosamine contents. mRNA expression of pro‐inflammatory factors such as intercellular cell adhesion molecule‐1 (ICAM‐1) and tumour necrosis factor‐α (TNF‐α) were down‐regulated and that of anti‐inflammatory cytokine interleukin‐10 (IL‐10) was up‐regulated. The findings suggest that bilirubin could be a new agent for enhancing cutaneous wound healing.     

Vein wrapping promotes M2 macrophage polarization in a rat chronic constriction injury model

Although the therapeutic potential of vein wrapping (VW) for recurrent compressive neuropathy has been widely reported, the mechanisms underlying this technique have not been characterized. M2 macrophages induced by interleukin‐4 (IL‐4) or interleukin‐10 (IL‐10) have an anti‐inflammatory function and play an important role in peripheral nerve repair. To evaluate whether VW promotes M2 polarization, we divided chronic constriction injury (CCI) rats into untreated and VW (CCI + VW)‐treated groups. Pain withdrawal thresholds in both groups were evaluated using von Frey filaments. Expression of the anti‐inflammatory cytokines IL‐4 and IL‐10 in vein and nerve were quantified using real time polymerase chain reaction (RT‐PCR), and expression of the anti‐inflammatory M2 macrophage markers CD206 and arginase‐1 (Arg1) after VW was assessed by RT‐PCR and immunohistochemistry. To evaluate the effect of exogenous IL‐4 or IL‐10 on M2 macrophage‐marker expression, CD11b‐positive macrophages isolated from sciatic nerve were stimulated with recombinant IL‐4 and IL‐10. VW significantly increased the pain withdrawal threshold. IL‐4 and IL‐10 mRNA expression was higher in veins than in the sciatic nerve. VW significantly increased CD206 and Arg1 mRNA expression compared to the CCI group. The number of CD206‐ and Arg1‐immunoreactive cells in nerve bundles was twofold higher in the CCI + VW than CCI group. Application of exogenous IL‐4 doubled CD206 and Arg1 mRNA expression in CD11b‐positive macrophages. These results show that vein‐derived IL‐4 potentiates the benefit of VW through the activation of M2 macrophages in the sciatic nerve. Our results may help to optimize current procedures for treating recurrent compressive neuropathy. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2210–2217, 2018.

     

Response of human macrophages to wound matrices in vitro

Chronic wounds remain a major burden to the global healthcare system. Myriad wound matrices are commercially available but their mechanisms of action are poorly understood. Recent studies have shown that macrophages are highly influenced by their microenvironment, but it is not known how different biomaterials affect this interaction. Here, it was hypothesized that human macrophages respond differently to changes in biomaterial properties in vitro with respect to phenotype, including pro‐inflammatory M1, anti‐inflammatory M2a, known for facilitating extracellular matrix deposition and proliferation, and M2c, which has recently been associated with tissue remodeling. Using multiple donors, it was found that collagen scaffolds cross‐linked with 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide and N‐hydroxysuccinimide (EDC/NHS) promoted the least inflammatory phenotype in primary human macrophages compared with scaffolds cross‐linked with formaldehyde or glutaraldehyde. Importantly, gene expression analysis trends were largely conserved between donors, especially TNFa (M1), CCL22 (M2a), and MRC1 (M2a). Then the response of primary and THP1 monocyte‐derived macrophages to four commercially available wound matrices were compared—Integra Dermal Regeneration Template (Integra), PriMatrix Dermal Repair Scaffold (PriMatrix), AlloMend Acellular Dermal Matrix (AlloMend), and Oasis Wound Matrix (Oasis). Gene expression trends were different between primary and THP1 monocyte‐derived macrophages for all six genes analyzed in this study. Finally, the behavior of primary macrophages cultured onto the wound matrices over time was analyzed. Integra and Oasis caused down‐regulation of M2a markers CCL22 and TIMP3. PriMatrix caused up‐regulation of TNFa (M1) and CD163 (M2c) and down‐regulation of CCL22 and TIMP3 (both M2a). AlloMend caused up‐regulation in CD163 (M2c). Lastly, Oasis promoted the largest increase in the combinatorial M1/M2 score, defined as the sum of M1 genes divided by the sum of M2 genes. This preliminary study suggested that biomaterials influenced the wound microenvironment to affect macrophage phenotype.     

High‐Fat Diet–Induced Obesity Promotes Expansion of Bone Marrow Adipose Tissue and Impairs Skeletal Stem Cell Functions in Mice

Obesity represents a risk factor for development of insulin resistance and type 2 diabetes. In addition, it has been associated with increased adipocyte formation in the bone marrow (BM) along with increased risk for bone fragility fractures. However, little is known on the cellular mechanisms that link obesity, BM adiposity, and bone fragility. Thus, in an obesity intervention study in C57BL/6J mice fed with a high‐fat diet (HFD) for 12 weeks, we investigated the molecular and cellular phenotype of bone marrow adipose tissue (BMAT), BM progenitor cells, and BM microenvironment in comparison to peripheral adipose tissue (AT). HFD decreased trabecular bone mass by 29%, cortical thickness by 5%, and increased BM adiposity by 184%. In contrast to peripheral AT, BMAT did not exhibit pro‐inflammatory phenotype. BM progenitor cells isolated from HFD mice exhibited decreased mRNA levels of inflammatory genes (Tnfα, IL1β, Lcn2) and did not manifest an insulin resistant phenotype evidenced by normal levels of pAKT after insulin stimulation as well as normal levels of insulin signaling genes. In addition, BM progenitor cells manifested enhanced adipocyte differentiation in HFD condition. Thus, our data demonstrate that BMAT expansion in response to HFD exerts a deleterious effect on the skeleton. Continuous recruitment of progenitor cells to adipogenesis leads to progenitor cell exhaustion, decreased recruitment to osteoblastic cells, and decreased bone formation. In addition, the absence of insulin resistance and inflammation in the BM suggest that BMAT buffers extra energy in the form of triglycerides and thus plays a role in whole‐body energy homeostasis. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.     

Regulatory effects of dermal papillary pluripotent stem cells on polarization of macrophages from M1 to M2 phenotype in vitro

The M1:M2 macrophage ratio is important for spinal cord injury (SCI) repair. Bone marrow mesenchymal stem cells (BMSCs) can alter macrophage activation, promoting M1 to M2 macrophage conversion and SCI repair; however, clinical BMSC applications have limitations. Previously, we found DPCs to be superior to BMSCs in promoting tissue repair after SCI, which we hypothesized to be mediated by M1 to M2 macrophage conversion. We investigated the regulatory effect of DPCs on M1/M2 macrophage polarization. Dermal papilla cells (DPCs) were isolated from rat vibrissae and characterized. Bone marrow-derived macrophages (BMDMs) were isolated and identified based on specific marker expression, and stimulated to differentiate into M1 macrophages with GM-CSF, IFN-γ, and LPS. These cells were co-cultured with DPCs to evaluate the effect on macrophage differentiation. DPCs expressed dermal papillae-specific markers, including ALP and Sox2, had MSC-expression patterns like those of BMSCs, and were capable of multi-differentiation. BMDMs expressed ANAE and CD68. Three days after induction, differentiated cells exhibited morphology typical of M1-like macrophages and expressed the macrophage marker CD68 and the M1 macrophage markers iNOS, but lacked expression of the M2 macrophage marker CD206. Co-culture with DPCs resulted in a shift to anti-inflammatory M2-like macrophage differentiation, characterized by morphological changes typical of M2 macrophages, downregulation of the characteristic cytokine TNF-α and the proportion of iNOS⁺ cells, and upregulation of the characteristic cytokine IL-10 and the cell-surface marker CD206. The number of CD206-expressing M2 macrophages also increased. These findings demonstrate that DPCs reprogram macrophages to an anti-inflammatory M2 phenotype, which could improve adverse inflammatory microenvironments and promote tissue repair. Thus, DPCs may be an interesting alternative cell source and merit further investigation in applications for SCI therapy.     

Reduction of burn progression with topical delivery of (antitumor necrosis factor‐α)‐hyaluronic acid conjugates

In this study, we explored whether topical application of antibodies targeting tumor necrosis factor‐α (TNF‐α) or interleukin‐6 (IL‐6) conjugated to hyaluronic acid (HA) could reduce the extension of necrosis by modulating inflammation locally in a partial‐thickness rat burn model. Partial‐thickness to deep partial‐thickness burn injuries present significant challenges in healing, as these burns often progress following the initial thermal insult, resulting in necrotic expansion and increased likelihood of secondary complications. Necrotic expansion is driven by a microenvironment with elevated levels of pro‐inflammatory mediators, and local neutralization of these using antibody conjugates could reduce burn progression. Trichrome‐stained tissue sections indicated the least necrotic tissue in (anti‐TNF‐α)‐HA‐treated sites, while (anti‐IL‐6)‐HA‐treated sites displayed similar outcomes to saline controls. This was confirmed by vimentin immunostaining, which demonstrated that HA treatment alone reduced burn progression by nearly 30%, but (anti‐TNF‐α)‐HA reduced it by approximately 70%. At all time points, (anti‐TNF‐α)‐HA‐treated sites showed reduced tissue levels of IL‐1β compared to controls, suggesting inhibition of a downstream mediator of inflammation. Decreased macrophage infiltration in (anti‐TNF‐α)‐HA‐treated sites compared to controls was elucidated by immunohistochemical staining of macrophages, suggesting a reduction in overall inflammation in all time points. These results suggest that local targeting of TNF‐α may be an effective strategy for preventing progression of partial‐thickness burns.     

A Role for galectin‐3 in renal tissue damage triggered by ischemia and reperfusion injury

Ischemic‐reperfusion injury (IRI) triggers an inflammatory response involving neutrophils/macrophages, lymphocytes and endothelial cells. Galectin‐3 is a multi‐functional lectin with a broad range of action such as promotion of neutrophil adhesion, induction of oxidative stress, mastocyte migration and degranulation, and production of pro‐inflammatory cytokines. The aim of this study was evaluate the role of galectin‐3 in the inflammation triggered by IRI. Galectin‐3 knockout (KO) and wild type (wt) mice were subjected to 45 min of renal pedicle occlusion. Blood and kidney samples were collected at 6, 24, 48 and 120 h. Blood urea was analyzed enzymatically, while MCP‐1, IL‐6 and IL‐1β were studied by real‐time PCR. Reactive oxygen species (ROS) was investigated by flow cytometry. Morphometric analyses were performed at 6, 24, 48 and 120 h after reperfusion. Urea peaked at 24 h, being significantly lower in knockout animals (wt = 264.4 ± 85.21 mg/dl vs. gal‐3 KO = 123.74 ± 29.64 mg/dl, P = 0.001). Galectin‐3 knockout animals presented less acute tubular necrosis and a more prominent tubular regeneration when compared with controls concurrently with lower expression of MCP‐1, IL‐6, IL‐1β, less macrophage infiltration and lower ROS production at early time points. Galectin‐3 seems to play a role in renal IRI involving the secretion of macrophage‐related chemokine, pro‐inflammatory cytokines and ROS production.     

Diabetes‐impaired wound healing and altered macrophage activation: A possible pathophysiologic correlation

Macrophages play a critical role in wound healing and can be activated to two distinctive phenotypes in vitro: classical macrophage activation (caM) and alternative macrophage activation (aaM). This study investigated whether the impaired cutaneous repair observed in streptozotocin‐induced diabetic rats was associated with altered macrophage activation. Our results show that macrophage activation phenotypes could be observed in wound healing through double immunostaining. The caM macrophages appeared in the initial stage of wound healing, followed by aaM macrophages, which predominated in normal wounds. However, through examining markers associated with activation by immunoblotting and real‐time polymerase chain reaction (PCR), diabetic wounds demonstrated insufficient caM in the early stage but excessive aaM in the later proliferative phase. Moreover, the macrophage activation markers were correlated with the instructive T helper cell type 1 (Th1)/Th2 cytokines in both groups. It was indicated that changed macrophage activation might contribute to impaired healing in diabetes wounds, and that strategies for reverting this abnormal activation could be useful for enhancing the wound healing process.