Enhanced M2 Polarization Research Articles

Abstract LB-059: Hyperglycemia enhances cancer immune evasion by inducing alternative macrophage polarization through increased O-GlcNAcylation

Abstract Epidemiological studies have shown diabetes mellitus (DM) to significantly increase the risk for cancer and cancer progression. Hyperglycemia is the most important characteristic of DM and directly correlates with a poor prognosis in cancer patients. While enhanced availability of glucose can directly fuel tumor cell proliferation, the effect on the tumor immune microenvironment is less well known. Here, we demonstrate in vitro and in different murine tumor models that hyperglycemia induces an increased flux through the hexosamine biosynthetic pathway (HBP) in tumor-associated macrophages (TAMs) resulting in an upregulation of O-GlcNAcylation. Increased O-GlcNAcylation was associated with alternative M2 polarization of TAMs and reduced anti-tumor immunity. The effect of hyperglycemia on tumor growth was dependent on an intact adaptive immunity. Furthermore, inhibition of M2 polarization by the addition of anti-CSF1R antibodies abrogated the effect of hyperglycemia on tumor growth. Similarly, treatment of mice with inhibitors of the HBP or O-GlcNAcylation reduced M2 polarization of TAMs and reversed immune evasion of growing tumors. Finally, we find enhanced M2 polarization in chronically hyperglycemic patients with colorectal cancer compared to non-diabetic normoglycemic patients. Our findings provide a new and targetable mechanism of cancer immune evasion, advocating for strict control of hyperglycemia in cancer patients. Citation Format: Natalia Rodrigues Mantuano, Michal Stanczak, Isadora Oliveira, Alessandra Filardy, Ronan Silva, Alfred Zippelius, Adriane Regina Todeschini, Heinz Laubli. Hyperglycemia enhances cancer immune evasion by inducing alternative macrophage polarization through increased O-GlcNAcylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-059.

Trehalose enhances bone fracture healing in a rat sleep deprivation model.

The purpose of this study was to investigate whether sleep deprivation (SD) could delay bone fracture healing and evaluate the therapeutic effect of trehalose. Eighteen 300-350 g female Sprague-Dawley rats were created a mid-femoral transverse osteotomy in the right thigh and divided into three groups (i.e., group 1: fracture; group 2: fracture + SD; and group 3: fracture + SD + trehalose). Seven days after surgery, the rats in group 2 and group 3 were started to get sleep-deprived for 18 h per day for 3 weeks. The rats in group 3 were injected with trehalose intraperitoneally at 1 g/kg/d for 3 weeks. Radiological and histological analyses were used to assess fracture healing quality. Circulating cytokines were detected by the end of the study. The expression of M1 and M2 macrophage markers were measured by quantitative real-time polymerase chain reaction (qPCR). X-rays showed group 2 experienced much poorer fracture healing. Micro CT demonstrated that the bone quality of the fracture callus site in group 2 was much worse than that in groups 1 and 3. Both haematoxylin eosin (H&E) and Masson staining revealed that the bone fracture of the group 2 healed worse. Elisa results demonstrated that the interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) of the rats in group 2 were significantly higher. In vitro study showed that 100 mM trehalose enhanced the expression of M2 macrophage markers (Arg-1 and IL-10), and decreased M1 macrophage polarization through the decreasing expression of IL-6. The present study showed (SD) could delay bone fracture healing in a rat model. And, trehalose could promote the healing of delayed bone fracture union by down-regulating pro-inflammatory mediators and enhancing M2 polarization.

MTORC1-mediated macrophage polarization into M1 Contributes to Ehrlichia-induced sepsis

Abstract Macrophages heterogeneity that undergo dynamic changes in phenotype and function as well as reprograming of immune response to multiple intracellular bacterial infections is an important key to understand the host responses to infection. Ehrlichia is an obligate Gram-negative intracellular bacterium that cause potentially fatal human monocytotropic ehrlichiosis (HME). We utilized murine models of mild and fatal ehrlichiosis caused by Ehrlichia muris and Ixodes Ovatus Ehrlichia (IOE), respectively, to evaluate the macrophages polarization to inflammatory (M1) and regenerative (M2). Our data show that in vivo and in vitro infections of macrophages with IOE significantly polarize macrophages towards M1, while EM infection enhances M2 polarization. M1 polarization of macrophages in IOE-infected mice was associated with recruitment and/or expansion of granzyme B+ neutrophils, NK cells, IL-17+ NKT cells, and IL-17+ CD4 Th17 cells compared to EM infection. We previously identified mTORC1 (mammalian target of rapamycin complex-1), a negative regulator of autophagy and the key mediator for immunopathology during IOE infection. We found that M1 polarization in the livers of IOE-infected mice is dependent on mTORC1 activation. Blockade of mTORC1 activation with rapamycin decreased frequency of Th17, and enhanced autophagy, which in turn decreased inflammation as well as the pathogenic immune response during lethal IOE infection. Together, these data reveal a key role of mTORC1 in M1 polarization of macrophages, which likely promotes Ehrlichia-induced sepsis. Understanding the dynamic changes of macrophage polarization and how it affects immune responses to infection is crucial for development novel therapeutic strategies.

5, 7, 2', 4', 5'-Pentamethoxyflavanone regulates M1/M2 macrophage phenotype and protects the septic mice.

Flavonoids have been reported to exert protective effect against many inflammatory diseases, while the underlying cellular mechanisms are still not completely known. In the present study, we explored the anti-inflammation activity of 5, 7, 2', 4', 5'-pentamethoxyflavanone (abbreviated as Pen.), a kind of polymethoxylated flavonoid, both in vitro and in vivo experiments. Pen. was showed no obvious toxicity in macrophages even at high dosage treatment. Our results indicated that Pen. significantly inhibited both mRNA and protein level of proinflammatory cytokines, IL-1β, IL-6, TNF-α and iNOS, which was characteristic expressed on M1 polarized macrophages. These effects of Pen. were further confirmed by diminished expression of CD11c, the M1 macrophage surface marker. Further researches showed that the mechanism was due to that Pen. downregulated the activity of p65, key transcription factor for M1 polarization. On the other hand, Pen. also enhanced M2 polarization with upregulation of anti-inflammatory factors and increase of M2 macrophage surface markers, which lead to the balance of M1 and M2 macrophages. Moreover, in vivo research verified that Pen. treatment alleviated LPS-induced sepsis in mice by increasing survival rate, decreasing inflammatory cytokines and improving lung tissue damage. In summary, our results suggested that Pen. modulated macrophage phenotype via suppressing p65 signal pathway to exert the anti-inflammation activity.

Sex Differences in Human Monocyte/Macrophages in Response to IL‐4

BackgroundAllergic asthma is a common chronic inflammatory disease of the lungs that affects 10% of the population in developed countries. Asthma shows a sex difference with more boys than girls diagnosed before puberty. In adulthood, women are twice as likely to have asthma than men. Women can also have increased disease severity and be less responsive to corticosteroid treatment. Sex hormones therefore may be important in asthma. Asthmatics have more and a greater percentage of M2 macrophages in their lungs than healthy individuals. Mouse studies have also demonstrated that M2 macrophages contribute to worse allergic lung inflammation. Furthermore, estrogen (E2) stimulates the production of M2 macrophage‐related cytokines that promote lung remodeling. How sex steroids affect monocyte‐macrophages to enhance M2 polarization is still unknown.Aim of the studyTo determine the molecular basis of sex differences and E2/androgen modulation of IL‐4‐induced M2 macrophage differentiation.ResultsOur preliminary data showed that both human monocytes and monocyte derived‐macrophages (MDM) from asthmatic donors upregulated M2 genes (CCL22, CCL17, TGM2, and MMP12) than cells from healthy donors, on IL‐4 stimulation. Moreover, expression of CCL17, TGM2, and CD200R was higher in MDM from asthmatic donors than in monocytes. IL‐4‐induced CCL17 and MMP12 shown a trend to greater expression in asthmatic female than male MDM. To explain the differences in IL‐4 responsiveness, we measured IL‐4 receptors (IL‐4R) on monocytes from men and women. Expression of γC, part of the type‐I IL‐4R complex, was greater on the surface of monocytes from healthy female compared to male donors. Conversely, IL‐13Rα2, an inhibitory receptor for IL‐13, was higher on male monocytes. To determine how responsive macrophages may be to E2 stimulation, we quantified estrogen receptor (ER) expression in MDM from healthy and allergic donors. MDM from allergic donors expressed more mRNA for ERα and less ERβ (linked to inhibition of E2 responses) and AR compared with MDM from a healthy donor. These data match the increased expression of ERα we measured in alveolar macrophages from female mice compared to male mice during allergic lung inflammation. Expression of CD206 greater on IL‐4‐stimulated cells in the presence of E2, mirroring our findings of increased M2 gene expression in mouse macrophages treated with E2 in vitro and in ovariectomized female mice implanted with E2 in vivo.ConclusionsOur preliminary results suggest that MDM from asthmatic donors are more sensitive to IL‐4 with enhanced M2 polarization. Sex differences in IL‐4R expression suggest increased IL‐4/‐13 responsiveness in female compared to male monocytes. ERα expression is higher in allergic MDMs and likely promotes enhanced M2 phenotype in the presence of E2. E2 promoted M2 polarization in human and mouse macrophages. Understanding the differences in monocyte‐macrophage responses between women and men and the effect of sex steroids on asthma can lead to developing better therapies for asthma according to the sex of the patient.Support or Funding InformationNIH grant R01 HL124477This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neuroprotective Effects of BHDPC, a Novel Neuroprotectant, on Experimental Stroke by Modulating Microglia Polarization

This study mainly investigated the therapeutic effects of BHDPC on ischemic stroke and its underlying mechanisms. In vivo, the transient middle cerebral artery occlusion (MCAO) was used to induce ischemic model. In vitro, oxygen and glucose deprivation/reperfusion (OGD/R)-induced ischemic stroke in BV-2 microglia and primary neurons, and bEnd.3 mouse cerebral microvascular endothelial cells (ECs) were also used. First, we found that BHDPC exerts considerable neuroprotection against MCAO-induced ischemic injury to mice via alleviating neurological deficits and brain infarcts, inhibiting neuronal cell loss and apoptosis, and attenuating blood-brain barrier disruption and tight junction protein changes. Next, we observed that BHDPC significantly reduced microglial M1 activation but enhanced M2 polarization in MCAO-induced ischemic brain. Further experiments in vitro indicated that BHDPC suppressed microglial activation but promoted M2 microglial polarization in OGD/R-induced BV-2 microglia. In addition, conditioned medium (CM) experiments showed that CM from BHDPC-treated BV-2 microglia provided protections against OGD/R-induced ischemic damage in primary neurons and bEnd.3 ECs. Moreover, we found that BHDPC actions on microglial inflammation were associated with the inactivation of NF-κB signaling. Interestingly, we also found that BHDPC enhanced phosphorylation of protein kinase A (PKA) and cAMP-response element-binding protein (CREB). The pharmacological inhibition or gene knockdown of PKA/CREB signaling diminished BHDPC-promoted microglial M2 polarization. In summary, BHDPC conferred neuroprotection against ischemic injury in experimental stroke models. Modulating microglial activation and polarization contributes to BHDPC-mediated neuroprotective actions, which in part were mediated by nuclear factor kappa B and PKA/CREB signaling pathway.

Podophyllotoxin and Rutin Modulate M1 (iNOS+) Macrophages and Mitigate Lethal Radiation (LR) Induced Inflammatory Responses in Mice.

Accidental exposure to lethal doses of Gamma radiation leads to the systemic inflammatory syndrome which causes mortality. In view of this, management of hemopoietic syndrome by modulating pro-inflammatory response in clinically manageable time period seems to be the most appropriate strategy for encountering radiation induced damage and recovery. As both tissue and peripheral macrophages are critical for the management of radiation induced injuries, we have unraveled the immunomodulatory potential of radioprotective formulation (G-003M) on peripheral macrophages populations in this study. G-003M inhibited lethal radiation induced NO and Th1 effector cytokines in the exposed macrophages indicating its M1 dim polarizing capacity. In similar lines, conditioning of mice with G-003M before lethal irradiation (LR) inhibited LR induced titre of Th1 effector cytokines in both serums as well as in lung, small intestine, and spleen tissue confirming its immunomodulatory potential. G-003M potentially down modulated inflammatory response in LPS induced inflammatory model and enhanced M2 polarization of iNOS+ M1 effector macrophages providing a molecular hint on G-003M mechanism of action on macrophages. These observations revealed that G-003M potentially modulate pro-inflammatory programming of macrophages and mitigate radiation-induced inflammatory stress which is believed to contribute significantly to radioprotective attribute of G-003M. In this study, we demonstrate that Rutin and Podophyllotoxin drive M1dim/M2 polarization of LR primed macrophages apart from protecting DNA from radiation. These drugs have the capacity to programme innate immune cells like macrophages which may be involved in homeostasis during recovery.

The NADPH Oxidase Nox4 controls polarization of macrophages in a NFκB-dependent manner.

The family of NADPH oxidases represents a source of reactive oxygen species (ROS). Within this family, Nox4 is special, as it constitutively produces H2O2. The loss of Nox4 promotes inflammation. A major cellular component of inflammation is the macrophage population, which divides in several subpopulations with pro-inflammatory M1 and wound-healing M2 macrophages being extremes of the functional spectrum of these cells. As controversial findings on Nox4 expression in macrophages exist, we hypothesize that Nox4 expression is only present in a subpopulation of macrophages, where it may determine the phenotype. In a murine inflammation-driven fibro sarcoma model Nox4-deficiency results an increase in the expression of pro-inflammatory genes and cytokines and higher amounts of pro-inflammatory Ly6C+ macrophages in the tumors. Ex vivo, bone marrow-derived macrophages deficient in Nox4 displayed a reduced M2 polarization whereas M1 markers were elevated. ROS levels where augmented in Nox4-/- cells polarized to M1 accompanied by a higher expression of Nox2. Mechanistically, Nox4-deficiency reduces STAT6 activation and promotes NFκB activity, with the latter being responsible for the higher level of Nox2 in Nox4-deficient M1-polarized macrophages. Collectively the data obtained in this study propose an anti-inflammatory role of Nox4 in macrophages. This is achieved by an enhanced M2 polarization and a suppression of NFκB activity.

Androgen and Androgen Receptor as Enhancers of M2 Macrophage Polarization in Allergic Lung Inflammation.

Allergic asthma is a disease initiated by a breach of the lung mucosal barrier and an inappropriate Th2 inflammatory immune response that results in M2 polarization of alveolar macrophages (AM). The number of M2 macrophages in the airway correlates with asthma severity in humans. Sex differences in asthma suggest that sex hormones modify lung inflammation and macrophage polarization. Asthmatic women have more M2 macrophages than asthmatic men and androgens have been used as an experimental asthma treatment. In this study, we demonstrate that although androgen (dihydrotestosterone) reconstitution of castrated mice reduced lung inflammation in a mouse model of allergic lung inflammation, it enhanced M2 polarization of AM. This indicates a cell-specific role for androgens. Dihydrotestosterone also enhanced IL-4-stimulated M2 macrophage polarization in vitro. Using mice lacking androgen receptor (AR) in monocytes/macrophages (ARfloxLysMCre), we found that male but not female mice exhibited less eosinophil recruitment and lung inflammation due to impaired M2 polarization. There was a reduction in eosinophil-recruiting chemokines and IL-5 in AR-deficient AM. These data reveal an unexpected and novel role for androgen/AR in promoting M2 macrophage polarization. Our findings are also important for understanding pathology in diseases promoted by M2 macrophages and androgens, such as asthma, eosinophilic esophagitis, and prostate cancer, and for designing new approaches to treatment.

Glibenclamide Reduces Primary Human Monocyte Functions Against Tuberculosis Infection by Enhancing M2 Polarization.

Tuberculosis (TB) is a global public health problem, which is caused by Mycobacterium tuberculosis (Mtb). Type 2 diabetes mellitus (T2DM) is one of the leading predisposing factors for development of TB after HIV/AIDS. Glibenclamide is a widely used anti-diabetic drug in low and middle-income countries where the incidence of TB is very high. In a human macrophage cell line, glibenclamide, a K+ATP-channel blocker, promoted alternative activation of macrophages by enhancing expression of the M2 marker CD206 during M2 polarization. M2 macrophages are considered poorly microbicidal and associated with TB susceptibility. Here, we investigated the effect of glibenclamide on M1 and M2 phenotypes of primary human monocytes and further determined whether specific drug treatment for T2DM individuals influences the antibacterial function of monocytes in response to mycobacterial infection. We found that glibenclamide significantly reduced M1 (HLA-DR+ and CD86+) surface markers and TNF-α production on primary human monocytes against mycobacterial infection. In contrast, M2 (CD163+ and CD206+) surface markers and IL-10 production were enhanced by pretreatment with glibenclamide. Additionally, reduction of bactericidal activity also occurred when primary human monocytes from T2DM individuals who were being treated with glibenclamide were infected with Mtb in vitro, consistent with the cytokine responses. We conclude that glibenclamide reduces M1 and promotes M2 polarization leading to impaired bactericidal ability of primary human monocytes of T2DM individuals in response to Mtb and may lead to increased susceptibility of T2DM individuals to TB and other bacterial infectious diseases.

Improvement of Adipose Macrophage Polarization in High Fat Diet-Induced Obese GHSR Knockout Mice.

Purpose Adipose tissue inflammation is the key linking obesity to insulin resistance. Over 50% of the interstitial cells in adipose tissue are macrophages, which produce inflammatory cytokines and therefore play an important role in the progression of insulin resistance. Within this classification view, macrophage biology is driven by two polarization phenotypes, M1 (proinflammatory) and M2 (anti-inflammatory). The unique functional receptor of ghrelin, growth hormone secretagogue receptor (GHSR), is a classic seven-transmembrane G protein-coupled receptor that is linked to multiple intracellular signaling pathways. Knockout of GHSR improves the obesity and glucose metabolic disorders, suggesting a crucial role of ghrelin activity in insulin resistance. Here, we discussed whether macrophage polarization phenotypes in adipose tissue were changed in GHSR knockout (GHSR-/-) mice. Methods GHSR-/- mice were fed with normal chow diet (NCD) or high fat diet (HFD). Markers of different macrophage polarization phenotypes were detected by real-time RT-PCR. Results The size of adipocytes decreased and interstitial cells, especially infiltrated macrophages, reduced in epididymal adipose tissue of GHSR-/- mice fed with HFD. Compared with wild type mice, the mRNA levels of inflammatory adipokines such as resistin, IL-6, and PAI-1 were significantly lower in epididymal adipose tissue of GHSR-/- mice, whereas anti-inflammatory adipokine, adiponectin, was significantly higher. M1 markers, MCP-1, TNF-α, and iNOS, were significantly lower in epididymal adipose tissue of GHSR-/- mice, whereas M2 markers, Arg-1, Mgl-1, were Mrc1, were significantly higher. Conclusion The GHSR-/- mice fed with HFD showed suppressed adipose inflammation, reduced macrophage infiltration, and enhanced M2 polarization of macrophages in adipose tissue, which improved insulin sensitivity.

Modulating Macrophage Polarization through CCR2 Inhibition and Multivalent Engagement.

Excessive or prolonged recruitment of inflammatory monocytes to damaged tissue can significantly worsen patient outcomes. Monocytes migrate to sites of tissue inflammation in response to high local concentrations of CCL2, a chemokine that binds to and signals through the CCR2 receptor. While the role of CCR2 in cellular migration is well studied, it is unclear how CCR2 inhibition affects macrophage polarization and if multivalency can increase downstream signaling effects. Using affinity selection with a phage library, we identified a novel single-chain variable fragment (scFv) (58C) that binds specifically and with high affinity to the N-terminal domain of CCR2 ( KD = 59.8 nM). The newly identified 58C-scFv bound to native CCR2 expressed on macrophages and MDA-MB-231 cells, inhibited migration, and induced a pro-inflammatory M1-phenotype in macrophages. The M1/M2 macrophage phenotype ratio for monomeric 58C-scFv was significantly increased over the negative control by 1.0 × 104-fold (iNOS/Arg-1), 5.1 × 104-fold (iNOS/Mgl2), 3.4 × 105-fold (IL-6/Arg-1), and 1.7 × 106-fold (IL-6/Mgl2). The multivalent display of 58C-scFv on liposomes further reduced migration of both cell types by 25-40% and enhanced M1 polarization by 200% over monomeric 58C-scFv. These studies demonstrate that CCR2 inhibition polarizes macrophages toward an inflammatory M1 phenotype, and that multivalent engagement of CCR2 increases the effects of 58C-scFv on polarization and migration. These data provide important insights into the role of multivalency in modulating binding, downstream signaling, and cellular fate.

Unexpected role for androgen and androgen receptor as enhancers of M2 macrophage polarization

Abstract Allergic asthma is an inappropriate Th2 immune response in the lungs to innocuous inhaled antigens. This results in M2 polarization of alveolar macrophages (AM), which number correlates with asthma severity in humans. Sex differences in asthma suggest that sex hormones modify lung inflammation and macrophage polarization. Since estrogen enhances M2 polarization and androgens have been used as experimental asthma therapy, we hypothesized that androgens [testosterone and dihydrotestosterone (DHT)] and androgen receptor (AR) suppress M2 macrophage polarization. Here, we demonstrate that although androgen reconstitution in castrated mice reduced inflammation of the bronchoalveolar space in a mouse model of allergic lung inflammation, it enhanced the production of the eosinophil-recruiter and M2 marker YM1 in AM, indicating a cell-specific role for androgens. DHT also enhanced IL-4-induced M2 macrophage polarization in vitro. Using mice lacking the AR in monocytes/macrophages (ARfloxLysMCre), we found that ARfloxLysMCre males had less eosinophil recruitment and lung inflammation in our model of allergic lung inflammation. No differences were found in females. This decreased inflammation was due to impaired M2 polarization and diminished production of eosinophil-recruiting chemokines by AM rather to a regulatory profile of AM lacking AR. Furthermore, macrophages lacking AR did not show an increase in IL-4-induced M2 polarization in vitro with DHT treatment. These data reveal an unexpected role for androgen/AR in promoting M2 macrophage polarization, and suggest possible mechanisms to counteract diseases that are promoted by M2 macrophages and affect mainly men, such as eosinophilic esophagitis and prostate cancer.

MicroRNA let-7c-5p improves neurological outcomes in a murine model of traumatic brain injury by suppressing neuroinflammation and regulating microglial activation

MicroRNAs (miRNAs) are a class of non-coding small RNAs that regulate the expression of target genes. They derive from pre-miRNAs that are enzymatically processed by dicer to ∼22 nucleotide mature miRNAs. Members of the pre-miRNA lethal-7 (let-7) are known to regulate cell proliferation and apoptosis. Here, we showed that the level of let-7c-5p, a key member of the let-7 family, was rapidly reduced in the traumatically injured foci in brains of adult C57BL/6J mice and gradually recovered to the pre-injury level 14 days after traumatic brain injury (TBI) induction. This finding led us to test whether upregulating let-7c-5p in murine cerebral tissue by intracerebroventricular injection (ICV) of let-7c-5p mimic could improve the outcomes of mice subjected to controlled cortical impact (CCI). We found that let-7c-5p overexpression attenuated TBI-induced neurological dysfunction and brain edema. The improvements were attributed to let-7c-5p-mediated inhibiting neuroinflammation and attenuation of microglia/macrophage activation, both inhibiting M1 polarization and enhancing M2 polarization. In vitro experiments, we observed that let-7c-5p was decreased in primary microglia activated by LPS treatment or oxygen/glucose deprivation (OGD). Transfection of let-7c-5p mimic suppressed the release of inflammatory mediators in cultured activated primary microglia. In addition, the expressions of caspase-3, a let-7c-5p putative target gene, and the PKC-δ which mediates effect of caspase-3 were inhibited by let-7c-5p in a murine model of TBI. Taken together, these results define the biological activities of cerebral let-7c-5p and delineate its therapeutic potential for improving the neurological outcome of TBI.

Glyburide and retinoic acid synergize to promote wound healing by anti-inflammation and RIP140 degradation

Chronic inflammation underlies the development of metabolic diseases and individuals with metabolic disease often also suffer from delayed wound healing due to prolonged inflammation. Resolving inflammation provides a therapeutic strategy in treating metabolic diseases. We previously showed that during an anti-inflammatory response when macrophages were alternatively (M2) polarized, retinoic acid (RA) dramatically activated arginase 1 gene (Arg1), a gene crucial for wound healing. Here we report that a widely used sulfonylurea drug for type 2 diabetes mellitus (T2DM), glyburide, enhances the anti-inflammatory response and synergizes with RA to promote wound healing. Our data also delineate the mechanism underlying glyburide’s anti-inflammatory effect, which is to stimulate the degradation of a pro-inflammatory regulator, Receptor Interacting Protein 140 (RIP140), by activating Ca2+/calmodulin-dependent protein kinase II (CamKII) that triggers specific ubiquitination of RIP140 for degradation. By stimulating RIP140 degradation, glyburide enhances M2 polarization and anti-inflammation. Using a high-fat diet induced obesity mouse model to monitor wound healing effects, we provide a proof-of-concept for a therapeutic strategy that combining glyburide and RA can significantly improve wound healing. Mechanistically, this study uncovers a new mechanism of action of glyburide and a new pathway modulating RIP140 protein degradation that is mediated by CamKII signaling.

Plaque Calcification During Atherosclerosis Progression and Regression.

Plaque calcification develops by the inflammation-dependent mechanisms involved in progression and regression of atherosclerosis. Macrophages can undergo two distinct polarization states, that is, pro-inflammatory M1 phenotype in progression and anti-inflammatory M2 phenotype in regression. In plaque progression, predominant M1 macrophages promote the initial calcium deposition within the necrotic core of the lesions, called as microcalcification, through not only vesicle-mediated mineralization as the result of apoptosis of macrophages and vascular smooth muscle cells (VSMCs), but also VSMC differentiation into early phase osteoblasts. On the other hand, in plaque regression M2 macrophages are engaged in the healing response to plaque inflammation. In association with the resolution of chronic inflammation, M2 macrophages may facilitate macroscopic calcium deposition, called as macrocalcification, through induction of osteoblastic differentiation and maturation of VSMCs. Oncostatin M, which has been shown to promote osteoblast differentiation in bone, may play a pivotal role in the development of plaque calcification. Clinically, two types of plaque calcification have distinct implications. Macrocalcification leads to plaque stability, while microcalcification is more likely to be associated with plaque rupture. Statin therapy, which reduces cardiovascular mortality, has been shown to exert its dual actions on plaque morphology, that is, regression of atheroma and increment of macroscopic calcium deposits. Statins may facilitate the healing process against plaque inflammation by enhancing M2 polarization of macrophages. Vascular calcification has pleiotropic properties as pro-inflammatory “microcalcification” and anti-inflammatory “macrocalcification”. The molecular mechanisms of this process in relation with plaque progression as well as plaque regression should be intensively elucidated.

Estrogen Signaling Contributes to Sex Differences in Macrophage Polarization during Asthma.

Allergic asthma is a chronic Th2 inflammation in the lungs that constricts the airways and presents as coughing and wheezing. Asthma mostly affects boys in childhood and women in adulthood, suggesting that shifts in sex hormones alter the course of the disease. Alveolar macrophages have emerged as major mediators of allergic lung inflammation in animal models as well as humans. Whether sex differences exist in macrophage polarization and the molecular mechanism(s) that drive differential responses are not well understood. We found that IL-4-stimulated bone marrow-derived and alveolar macrophages from female mice exhibited greater expression of M2 genes in vitro and after allergen challenge in vivo. Alveolar macrophages from female mice exhibited greater expression of the IL-4Rα and estrogen receptor (ER) α compared with macrophages from male mice following allergen challenge. An ERα-specific agonist enhanced IL-4-induced M2 gene expression in macrophages from both sexes, but more so in macrophages from female mice. Furthermore, IL-4-stimulated macrophages from female mice exhibited more transcriptionally active histone modifications at M2 gene promoters than did macrophages from male mice. We found that supplementation of estrogen into ovariectomized female mice enhanced M2 polarization in vivo upon challenge with allergen and that macrophage-specific deletion of ERα impaired this M2 polarization. The effects of estrogen are long-lasting; bone marrow-derived macrophages from ovariectomized mice implanted with estrogen exhibited enhanced IL-4-induced M2 gene expression compared with macrophages from placebo-implanted littermates. Taken together, our findings suggest that estrogen enhances IL-4-induced M2 gene expression and thereby contributes to sex differences observed in asthma.

RORα Induces KLF4-Mediated M2 Polarization in the Liver Macrophages that Protect against Nonalcoholic Steatohepatitis

The regulation of M1/M2 polarization in liver macrophages is closely associated with the progression of nonalcoholic steatohepatitis (NASH); however, the mechanism involved in this process remains unclear. Here, we describe the orphan nuclear receptor retinoic-acid-related orphan receptor α (RORα) as a key regulator of M1/M2 polarization in hepatic residential Kupffer cells (KCs) and infiltrated monocyte-derived macrophages. RORα enhanced M2 polarization in KCs by inducing the kruppel-like factor 4. M2 polarization was defective in KCs and bone-marrow-derived macrophages of the myeloid-specific RORα null mice, and these mice were susceptible to HFD-induced NASH. We found that IL-10 played an important role in connecting the function of M2 KCs to lipid accumulation and apoptosis in hepatocytes. Importantly, M2 polarization was controlled by a RORα activator, JC1-40, which improved symptoms of NASH. Our results suggest that the M2-promoting effects of RORα in liver macrophages may provide better therapeutic strategies against NASH.

Dual role of SIRPα in macrophage activation: inhibiting M1 while promoting M2 polarization via selectively activating SHP-1 and SHP-2 signal

Abstract Macrophages polarization into proinflammatory M1 or alternative M2 states, which plays a central role in inflammation and host defense, is tightly controlled by multiple regulatory molecules. Here we examined the role of signal regulatory protein α (SIRPα), which negatively regulates leukocyte responses, in regulating macrophage polarization and function. Compared to WT macrophages, macrophage obtained from SIRPα deficient (SIRPα−/−) mice demonstrated an enhanced response to M1 induction but were refractory to M2 induction, as indicated by the increased expression of M1-related markers CD86, MHC-II, iNOS, IL-1β, IL-12 and TNF-α under M1-skewed LPS/IFNγ treatment but the defective expression of anti-inflammatory mediators CD206, Arg1, IL-10 and TGFβ during IL-4 induced M2 activation. Furthermore, ligating SIRPα by CD47 extracellular domain (mCD47-AP) suppressed M1 polarization but enhanced M2 polarization in WT macrophages, whereas had no effect on SIRPα−/− macrophage polarization. Interestingly, under M1 and M2 activation, macrophage SIRPα was found to be tyrosine-phosphorylated and selectively associated with SHP-1 and SHP-2, respectively. Mechanistic studies indicated that SIRPα deficiency promoted M1 but attenuated M2 polarization of macrophages by enhancing the PI3k/Akt2 signaling pathway. Overall, our findings demonstrated for the first time that SIRPα plays an important role in modulating macrophage polarization, inhibiting M1 while promoting M2 phenotypic polarization through selectively activating SHP-1 and SHP-2 signaling.

Estrogen contributes to sex differences in M2-polarization during asthma

Abstract Asthma is a chronic inflammation of the airways that affects over 300 million people worldwide. Asthma exhibits sex differences, affecting mostly boys in childhood and women in adulthood. Alveolar macrophages have emerged as major mediators of allergic lung inflammation. Macrophages express receptors that recognize and respond to estrogen. We hypothesized that estrogen enhances M2 polarization of alveolar macrophages and thereby contributes to asthma. We found increased expression of the canonical mouse M2 marker YM1 by alveolar macrophages from allergic female mice compared to male mice. Consistently, bone marrow derived macrophages (BMMs) from female mice exhibited higher levels of YM1 and ARG1 than did BMMs from male mice following stimulation with IL-4. This sex difference is reflected on the epigenetic level as M2 promoters in BMMs from female mice exhibited enhanced acetylated H3 compared to BMMs from male mice following stimulation with IL-4. Pretreatment of BMMs from female mice with estrogen receptor ligands enhanced IL-4-induced M2-gene expression. Using ovariectomized (OVx) and LysMCREERaflox/floxmice, we show that an absence of either estrogen or the estrogen receptor-a (ERa) impairs M2 polarization following challenge with OVA. Thus, macrophages exhibit sex differences in their ability to polarize to an M2-phenotype in vitro and in vivo. Estrogen and ERa engagement augment IL-4-induced M2-gene expression. Together these data suggest that sex and hormonal factors contribute to sex differences in macrophage responses during asthma. Understanding the role of estrogen signaling in M2-polarization is paramount for identifying novel therapeutic targets to better treat women with asthma.