S100A9 deletion in microglia/macrophages ameliorates brain injury through the STAT6/PPARγ pathway in ischemic stroke.

BACKGROUND: Microglia and infiltrated macrophages (M/M) are integral components of the innate immune system that play a critical role in facilitating brain repair after ischemic stroke (IS) by clearing cell debris. Novel therapeutic strategies for IS therapy involve modulating M/M phenotype shifting. This study aims to elucidate the pivotal role of S100A9 in M/M and its downstream STAT6/PPARγ signaling pathway in neuroinflammation and phagocytosis after IS. METHODS: In the clinical study, we initially detected the expression pattern of S100A9 in monocytes from patients with acute IS and investigated its association with the long-term prognosis. In the in vivo study, we generated the S100A9 conditional knockout (CKO) mice and compared the stroke outcomes with the control group. We further tested the S100A9-specific inhibitor paqunimod (PQD), for its pharmaceutical effects on stroke outcomes. Transcriptomics and in vitro studies were adopted to explore the mechanism of S100A9 in modulating the M/M phenotype, which involves the regulation of the STAT6/PPARγ signaling pathway. RESULTS: S100A9 was predominantly expressed in classical monocytes and was correlated with unfavorable outcomes in patients of IS. S100A9 CKO mitigated infarction volume and white matter injury, enhanced cerebral blood flow and functional recovery, and prompted anti-inflammation phenotype and efferocytosis after tMCAO. The STAT6/PPARγ pathway, an essential signaling cascade involved in immune response and inflammation, might be the downstream target mediated by S100A9 deletion, as evidenced by the STAT6 phosphorylation inhibitor AS1517499 abolishing the beneficial effect of S100A9 inhibition in tMCAO mice and cell lines. Moreover, S100A9 inhibition by PQD treatment protected against neuronal death in vitro and brain injuries in vivo. CONCLUSION: This study provides evidence for the first time that S100A9 in classical monocytes could potentially be a biomarker for predicting IS prognosis and reveals a novel therapeutic strategy for IS. By demonstrating that S100A9-mediated M/M polarization and phagocytosis can be reversed by S100A9 inhibition in a STAT6/PPARγ pathway-dependent manner, this study opens up new avenues for drug development in the field.

Myeloid Deletion of Cdc42 Protects Liver From Hepatic Ischemia-Reperfusion Injury via Inhibiting Macrophage-Mediated Inflammation in Mice.

BACKGROUND & AIMS: Hepatic ischemia-reperfusion injury (HIRI) often occurs in liver surgery, such as partial hepatectomy and liver transplantation, in which myeloid macrophage-mediated inflammation plays a critical role. Cell division cycle 42 (Cdc42) regulates cell migration, cytoskeleton rearrangement, and cell polarity. In this study, we explore the role of myeloid Cdc42 in HIRI. METHODS: Mouse HIRI models were established with 1-hour ischemia followed by 12-hour reperfusion in myeloid Cdc42 knockout (Cdc42mye) and Cdc42flox mice. Myeloid-derived macrophages were traced with RosamTmG fluorescent reporter under LyzCre-mediated excision. The experiments for serum or hepatic enzymic activities, histologic and immunologic analysis, gene expressions, flow cytometry analysis, and cytokine antibody array were performed. RESULTS: Myeloid deletion of Cdc42 significantly alleviated hepatic damages with the reduction of hepatic necrosis and inflammation, and reserved hepatic functions following HIRI in mice. Myeloid Cdc42 deficiency suppressed the infiltration of myeloid macrophages, reduced the secretion of proinflammatory cytokines, restrained M1 polarization, and promoted M2 polarization of myeloid macrophages in livers. In addition, inactivation of Cdc42 promoted M2 polarization via suppressing the phosphorylation of STAT1 and promoting phosphorylation of STAT3 and STAT6 in myeloid macrophages. Furthermore, pretreatment with Cdc42 inhibitor, ML141, also protected mice from hepatic ischemia-reperfusion injury. CONCLUSIONS: Inhibition or deletion of myeloid Cdc42 protects liver from HIRI via restraining the infiltration of myeloid macrophages, suppressing proinflammatory response, and promoting M2 polarization in macrophages.

STAT6 Deficiency Attenuates Myeloid Fibroblast Activation and Macrophage Polarization in Experimental Folic Acid Nephropathy.

Renal fibrosis is a pathologic feature of chronic kidney disease, which can lead to end-stage kidney disease. Myeloid fibroblasts play a central role in the pathogenesis of renal fibrosis. However, the molecular mechanisms pertaining to myeloid fibroblast activation remain to be elucidated. In the present study, we examine the role of signal transducer and activator of transcription 6 (STAT6) in myeloid fibroblast activation, macrophage polarization, and renal fibrosis development in a mouse model of folic acid nephropathy. STAT6 is activated in the kidney with folic acid nephropathy. Compared with folic-acid-treated wild-type mice, STAT6 knockout mice had markedly reduced myeloid fibroblasts and myofibroblasts in the kidney with folic acid nephropathy. Furthermore, STAT6 knockout mice exhibited significantly less CD206 and PDGFR-ß dual-positive fibroblast accumulation and M2 macrophage polarization in the kidney with folic acid nephropathy. Consistent with these findings, STAT6 knockout mice produced less extracellular matrix protein, exhibited less severe interstitial fibrosis, and preserved kidney function in folic acid nephropathy. Taken together, these results have shown that STAT6 plays a critical role in myeloid fibroblasts activation, M2 macrophage polarization, extracellular matrix protein production, and renal fibrosis development in folic acid nephropathy. Therefore, targeting STAT6 may provide a novel therapeutic strategy for fibrotic kidney disease.

Interleukin 4 promotes anti-inflammatory macrophages that clear cartilage debris and inhibits osteoclast development to protect against osteoarthritis.

OBJECTIVE: Osteoarthritis (OA), the leading cause of joint failure, is characterized by breakdown of articular cartilage and remodeling of subchondral bone in synovial joints. Despite the high prevalence and debilitating effects of OA, no disease-modifying drugs exist. Increasing evidence, including genetic variants of the interleukin 4 (IL-4) and IL-4 receptor genes, implicates a role for IL-4 in OA, however, the mechanism underlying IL-4 function in OA remains unknown. Here, we investigated the role of IL-4 in OA pathogenesis. METHODS: Il4-, myeloid-specific-Il4ra-, and Stat6-deficient and control mice were subjected to destabilization of the medial meniscus to induce OA. Macrophages, osteoclasts, and synovial explants were stimulated with IL-4 in vitro, and their function and expression profiles characterized. RESULTS: Mice lacking IL-4, IL-4Ra in myeloid cells, or STAT6 developed exacerbated cartilage damage and osteophyte formation relative to WT controls. In vitro analyses revealed that IL-4 downregulates osteoarthritis-associated genes, enhances macrophage phagocytosis of cartilage debris, and inhibits osteoclast differentiation and activation via the type I receptor. CONCLUSION: Our findings demonstrate that IL-4 protects against osteoarthritis in a myeloid and STAT6-dependent manner. Further, IL-4 can promote an immunomodulatory microenvironment in which joint-resident macrophages polarize towards an M2 phenotype and efficiently clear pro-inflammatory debris, and osteoclasts maintain a homeostatic level of activity in subchondral bone. These findings support a role for IL-4 modulation of myeloid cell types in maintenance of joint health and identify a pathway that could provide therapeutic benefit for osteoarthritis.

Loss of STAT6 leads to anchorage-independent growth and trastuzumab resistance in HER2+ breast cancer cells.

Approximately 20% of breast cancers are HER2-positive. Trastuzumab has improved patient outcomes significantly for these cancers. However, acquired resistance remains a major hurdle in the clinical management of these patients. Therefore, identifying molecular changes that cause trastuzumab resistance is worthwhile. STAT6 is a transcription factor that regulates a variety of genes involved in cell cycle regulation, growth inhibition, and apoptosis. STAT6 expression is lost in approximately 3% of breast cancers, but little work has been done in the context of trastuzumab resistance in breast cancer. In isogenic cell line pairs, we observed that trastuzumab-resistant cells expressed significantly lower levels of STAT6 compared to trastuzumab-sensitive cells. Therefore, in order to study the consequences of STAT6 loss in HER2+ breast cancer, we knocked out both alleles of the STAT6 gene using somatic cell gene targeting. Interestingly, loss of STAT6 resulted in anchorage-independent growth and changes in several genes involved in epithelial to mesenchymal transition. This study suggests that STAT6 may play a role in the pathophysiology of HER2+ human breast cancer.

Th1-Th2 Cross-Regulation Controls Early Leishmania Infection in the Skin by Modulating the Size of the Permissive Monocytic Host Cell Reservoir.

The impact of T helper (Th) 1 versus Th2 immunity on intracellular infections is attributed to classical versus alternative activation of macrophages leading to resistance or susceptibility. However, observations in multiple infectious settings demonstrate deficiencies in mediators of Th1-Th2 immunity, which have paradoxical or no impact. We report that prior to influencing activation, Th1/Th2 immunity first controls the size of the permissive host cell reservoir. During early Leishmania infection of the skin, IFN-γ- or STAT6-mediated changes in phagocyte activation were counteracted by changes in IFN-γ-mediated recruitment of permissive CCR2+ monocytes. Monocytes were required for early parasite expansion and acquired an alternatively activated phenotype despite the Th1 dermal environment required for their recruitment. Surprisingly, STAT6 did not enhance intracellular parasite proliferation, but rather modulated the size and permissiveness of the monocytic host cell reservoir via regulation of IFN-γ and IL-10. These observations expand our understanding of the Th1-Th2 paradigm during infection.

STAT6 Pathway Is Critical for the Induction and Function of Regulatory T Cells Induced by Mucosal B Cells.

B cells could convert naïve T cells into regulatory T cells (so-called Treg-of-B cells) which have the ability to treat animal models of inflammatory diseases, including allergic asthma, collagen-induced arthritis and colitis; however, the mechanisms of Treg-of-B cell generation remain unclear. In this study, we investigated the role of STAT6 in the generation of Treg-of-B (P) cells, which Treg cells were generated by Peyer's patch B cells (P stands for Peyer's patch). CD4+CD25- T cells from wild type, STAT6 knockout and IL-4 knockout mice were cocultured with wild type Peyer's patch B cells for Treg-of-B (P) cell generation. A murine asthmatic model was used to analyze the in vivo regulatory function of Treg-of-B (P) cells. The data demonstrated that STAT6 played a critical role in the generation of Treg-of-B (P) cells, which confirmed with STAT6-deficient T cells and the STAT6 inhibitor AS1517499. When STAT6 was lacking, Treg-of-B (P) cells exerted impaired suppressive ability with decreased LAG3 expression. Furthermore, Peyer's patch B cells played an essential role in regulatory T cell generation. In the absence of Peyer's patch B cells, T cells expressed decreased phosphorylated STAT6, which was followed by decreased LAG3 expression and impaired suppressive ability, suggesting that Peyer's patch B cells provided the critical signal to activate STAT6 phosphorylation in T cells. Moreover, STAT6 deficient Treg-of-B (P) cells could not alleviate inflammation in an animal model of asthma in vivo. IL-4 was downstream of phosphorylated STAT6 and maintained Treg-of-B (P) cell survival with increased expression of Bcl-2 and BclXL. We reported a novel finding that the STAT6-LAG3 signaling axis is important for the induction and function of Treg-of-B (P) cells.

Non-hematopoietic STAT6 induces epithelial tight junction dysfunction and promotes intestinal inflammation and tumorigenesis.

Enhanced gut permeability due to dysregulated epithelial tight junction is often associated with inflammatory bowel diseases (IBD), which have a greater risk for developing colorectal cancer. STAT6 activation was detected in inflamed colonic epithelium of active IBD patients, suggesting a role of epithelial STAT6 in colitis development. Here, we demonstrated that non-hematopoietic STAT6, but not hematopoietic STAT6, triggered DSS-induced colitis and subsequent tumorigenesis. This could be due to the enhancing-effect of STAT6 on gut permeability and microbiota translocation via interruption of epithelial tight junction integrity. Mechanistically, long-myosin light-chain kinase (MLCK1) was identified as a target of STAT6, leading to epithelial tight junction dysfunction and microbiota-driven colitis. Furthermore, neutralization of IL-13, which was primarily derived from type 2 innate lymphoid cells (ILC2) in a microbiota-dependent way, inhibited epithelial STAT6 activation and improved gut permeability and DSS-induced colitis. Importantly, pharmacological inhibition of STAT6 reduces murine intestinal tumor formation, and tumoral p-STAT6 levels positively correlated to the clinical stage and poor prognosis of human colorectal cancer. Thus, our study reveals a direct role of STAT6 in the disruption of epithelial tight junction integrity and colitis development, and suggests STAT6 as a potential therapeutic and prophylactic target for IBD and colitis-associated cancer.

Cutaneous permeability barrier function in signal transducer and activator of transcription 6-deficient mice is superior to that in wild-type mice.

BACKGROUND: Th2 cytokines exhibit a variety of inhibitory effects on permeability barrier function via signal transducer and activator of transcription 6 (STAT6). However, the role of STAT6 signaling on the construction and/or homeostasis of permeability barrier function in the physiological state has not been fully assessed. OBJECTIVE: We compared permeability barrier function between Stat6-deficient and wild-type C57BL/6 mice at steady state. METHODS AND RESULTS: Measurement of transepidermal water loss and quantitative penetration assay revealed that permeability barrier function was superior in Stat6-deficient mice. Accordingly, expressions of loricrin, acidic sphingomyelinase (aSMase) and ß-glucocerebrosidase (ß-GlcCer'ase) in epidermis and ceramide levels in stratum corneum were elevated in STAT6-deficient mice. On the other hands, up-regulations of loricrin, aSMase and ß-GlcCer'ase were not observed in 3-dimensionally cultured human keratinocytes transfected with siRNA for STAT6. Meanwhile, number of mast cells in the dermis was decreased in Stat6-deficient mice. CONCLUSIONS: These results suggest that STAT6 signaling negatively affects permeability barrier function in vivo, even in the physiological state. However, the superior permeability barrier function in Stat6-deficient mice may be a secondary effect exerted via cells other than keratinocytes, such as mast cells, since mast cells are known to influence permeability barrier function in vivo. Blockade of STAT6 signaling might be a strategy to augment the permeability barrier function.

Recirculating Th2 cells induce severe thymic dysfunction via IL-4/STAT6 signaling pathway.

Thymic involution happened early in life, but a certain ratio of activated CD4+ T cells will persistently recirculate into the thymus from the periphery and it have been suggested to be able to inhibit the development of embryonic thymocytes. Our present study was aimed to elucidate the specific mechanism how activated CD4+ T cells could influence upon developing thymocytes by using fetal thymic organ culture (FTOC) and kidney capsule transplantation. Our results demonstrated that Th2 cells were found to play a fundamental role in the inhibition of embryonic thymocyte development since a very low concentration of Th2 cells could obviously reduce the total number of thymocytes. And this effect was not tenable in other Th cell type. Notably, IL-4, the major cytokine secreted by Th2 cells, was suggested the key factor playing the inhibition role. In addition to reduced cell population, the proportion of double positive (DP) T cells was also heavily decreased. Furthermore, we demonstrated that it was the downstream effector signal transducer and activator of transcription 6 (STAT6) of IL-4 partially manipulate this inhibition. Together, these findings reveal a novel influence of Th2 cells re-entering the thymus on thymic involution.

Identification of targets of IL-13 and STAT6 signaling in polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a life-threatening, highly prevalent monogenic disease caused by mutations in polycystin-1 (PC1) in 85% of patients. We have previously identified a COOH-terminal cleavage fragment of PC1, PC1-p30, which interacts with the transcription factor STAT6 to promote transcription. STAT6 is aberrantly active in PKD mouse models and human ADPKD, and genetic removal or pharmacological inhibition of STAT6 attenuates disease progression. High levels of IL-13, a STAT6-activating cytokine, are found in the cyst fluid of PKD mouse models and increased IL-13 receptors in ADPKD patient tissue, suggesting that a positive feedback loop exists between IL-13 and STAT6 is activated in cystic epithelial cells and contributes to disease progression. In this study, we aimed to identify genes aberrantly regulated by STAT6 to better understand how increased IL-13/STAT6 signaling may contribute to PKD progression. We demonstrate that the expression of periostin, galectin-3, and IL-24 is upregulated in various forms of PKD and that their aberrant regulation is mediated by IL-13 and STAT6 activity. Periostin and galectin-3 have previously been implicated in PKD progression. We support these findings by showing that periostin expression is increased after IL-13 treatment in kidney epithelial cells, that galectin-3 expression is increased after injecting IL-13 in vivo and that IL-24 expression is upregulated by both IL-13 treatment and PC1-p30 overexpression in mouse and human kidney cells. Overall, these findings provide insight into the possible mechanisms by which increased IL-13/STAT6 signaling contributes to PKD progression and suggest potential therapeutic targets.

Aberrant Th2 inflammation drives dysfunction of alveolar macrophages and susceptibility to bacterial pneumonia.

The ubiquitin ligase, Itch, is required to prevent autoinflammatory disease in mice and humans. Itch-deficient mice develop lethal pulmonary inflammation characterized by the production of Th2 cytokines (for example, interleukin-4 (IL-4)); however, the contribution of Itch to immune defense against respiratory pathogens has not been determined. We found that Itch-deficient mice were highly susceptible to intranasal infection with the respiratory pathogen Klebsiella pneumoniae. Infected Itch-deficient mice exhibited increased immune cell infiltration, cytokine levels and bacterial burden in the respiratory tract compared with control mice. However, numbers of resident alveolar macrophages were reduced in the lungs from Itch-deficient mice both before and after infection. High levels of Th2 cytokines in the respiratory tract correlated with deceased alveolar macrophages, and genetic ablation of IL-4 restored alveolar macrophages and host defense to K. pneumoniae in Itch-deficient mice, suggesting that loss of alveolar macrophages occurred as a consequence of Th2 inflammation. Adoptive transfer of Itch-/- CD4+ T cells into Rag-/- mice was sufficient to drive reduction in numbers of Itch-replete alveolar macrophages. Finally, we found that Stat6 signaling downstream of the IL-4 receptor directly reduced fitness of alveolar macrophages when these cells were exposed to the Itch-/- inflamed respiratory tract. These data suggest that Th2 inflammation directly impairs alveolar macrophage fitness in Itch-/- mice, and elucidate a previously unappreciated link between Th2 cells, alveolar macrophages and susceptibility to bacterial infection.

GAB2 regulates type 2 T helper cell differentiation in humans.

Th2 cell differentiation involves complex changes in expression of multiple genes, many of which have poorly characterized roles. In a gene expression microarray analysis of human primary CD4+ effector T subsets, we identified that an adaptor protein, GAB2, was preferentially expressed in human Th2 cells. The role of GAB2 in human Th2 cells is unknown. Through analysis of primary and in vitro differentiated human T effector subsets, we confirmed that human Th2 cells preferentially expressed GAB2. Further analysis of public gene expression microarray data of STAT6-knockdowned Th2 cells indicated that GAB2 expression was regulated by IL-4 and STAT6. Both siRNA knockdown and ectopic expression of GAB2 in activated T cells showed that GAB2 positively regulated IL-4 and IL-13 expression in human Th2 cells. We hence identified the adaptor protein, GAB2, as an important novel regulator of the human Th2 immune response.

Lack of STAT6 Attenuates Inflammation and Drives Protection against Early Steps of Colitis-Associated Colon Cancer.

Colitis-associated colon cancer (CAC) is one of the most common malignant neoplasms and a leading cause of death. The immunologic factors associated with CAC development are not completely understood. Signal transducer and activator of transcription 6 (STAT6) is part of an important signaling pathway for modulating intestinal immune function and homeostasis. However, the role of STAT6 in colon cancer progression is unclear. Following CAC induction in wild-type (WT) and STAT6-deficient mice (STAT6-/-), we found that 70% of STAT6-/- mice were tumor-free after 8 weeks, whereas 100% of WT mice developed tumors. STAT6-/- mice displayed fewer and smaller colorectal tumors than WT mice; this reduced tumorigenicity was associated with decreased proliferation and increased apoptosis in the colonic mucosa in the early steps of tumor progression. STAT6-/- mice also exhibited reduced inflammation, diminished concentrations COX2 and nuclear ß-catenin protein in the colon, and decreased mRNA expression of IL17A and TNFα, but increased IL10 expression when compared with WT mice. Impaired mucosal expression of CCL9, CCL25, and CXCR2 was also observed. In addition, the number of circulating CD11b+Ly6ChiCCR2+ monocytes and CD11b+Ly6ClowLy6G+ granulocytes was both decreased in a STAT6-dependent manner. Finally, WT mice receiving a STAT6 inhibitor in vivo confirmed a significant reduction in tumor load as well as less intense signs of CAC. Our results demonstrate that STAT6 is critical in the early steps of CAC development for modulating inflammatory responses and controlling cell recruitment and proliferation. Thus, STAT6 may represent a promising target for CAC treatment. Cancer Immunol Res; 5(5); 385-96. ©2017 AACR.

STAT6 deficiency ameliorates Graves' disease severity by suppressing thyroid epithelial cell hyperplasia.

Signal transducer and activator of transcription 6 (STAT6) is involved in epithelial cell growth. However, little is known regarding the STAT6 phosphorylation status in Graves' disease (GD) and its role in thyroid epithelial cells (TECs). In this study, we found that STAT6 phosphorylation (p-STAT6) was significantly increased in TECs from both GD patients and experimental autoimmune Graves' disease mice and that STAT6 deficiency ameliorated GD symptoms. Autocrine IL-4 signalling in TECs activated the phosphorylation of STAT6 via IL-4 R engagement, and the downstream targets of STAT6 were Bcl-xL and cyclin D1. Thus, the IL-4-STAT6-Bcl-xL/cyclin D1 pathway is crucial for TEC hyperplasia, which aggravates GD. More importantly, in vitro and in vivo experiments demonstrated that STAT6 phosphorylation inhibited by AS1517499 decreased TEC hyperplasia, thereby reducing serum T3 and T4 and ameliorating GD. Thus, our study reveals that in addition to the traditional pathogenesis of GD, in which autoantibody TRAb stimulates thyroid-stimulating hormone receptors and consequently produces T3, T4, TRAb could also trigger TECs producing IL-4, and IL-4 then acts in an autocrine manner to activate p-STAT6 signalling and stimulate unrestricted cell growth, thus aggravating GD. These findings suggest that STAT6 inhibitors could be potent therapeutics for treating GD.

Prostaglandin I2 Suppresses Proinflammatory Chemokine Expression, CD4 T Cell Activation, and STAT6-Independent Allergic Lung Inflammation.

Allergic airway diseases are immune disorders associated with heightened type 2 immune responses and IL-5 and IL-13 production at the site of inflammation. We have previously reported that cyclooxygenase (COX) inhibition by indomethacin augmented allergic airway inflammation in a STAT6-independent manner. However, the key COX product(s) responsible for restraining indomethacin-mediated STAT6-independent allergic inflammation is unknown. In this study, using the mouse model of OVA-induced allergic airway inflammation, we identified that PGI2 receptor (IP) signaling was critical for indomethacin-induced, STAT6-independent proallergic effects. We demonstrated that IP deficiency increased inflammatory cell infiltration, eosinophilia, and IL-5 and IL-13 expression in the lung in a STAT6-independent manner. The augmented STAT6-independent allergic inflammation correlated with enhanced primary immune responses to allergic sensitization and elevated production of multiple inflammatory chemokines (CCL11, CCL17, CCL22, and CXCL12) in the lung after allergen challenge. We also showed that the PGI2 analogue cicaprost inhibited CD4 T cell proliferation and IL-5 and IL-13 expression in vitro, and IP deficiency diminished the stimulatory effect of indomethacin on STAT6-independent IL-5 and IL-13 responses in vivo. The inhibitory effects of PGI2 and the IP signaling pathway on CD4 T cell activation, inflammatory chemokine production, and allergic sensitization and airway inflammation suggest that PGI2 and its analogue iloprost, both Food and Drug Administration-approved drugs, may be useful in treating allergic diseases and asthma. In addition, inhibiting PGI2 signaling by drugs that either block PGI2 production or restrain IP signaling may augment STAT6-independent pathways of allergic inflammation.

IL-33 signaling is essential to attenuate viral-induced encephalitis development by downregulating iNOS expression in the central nervous system.

BACKGROUND: Viral encephalitis is a common cause of lethal infections in humans, and several different viruses are documented to be responsible. Rocio virus is a flavivirus that causes a severe lethal encephalitis syndrome in humans and also mice, providing an interesting model to study the CNS compartmentalized immune response. Interleukin 33 (IL-33), a member of the IL-1 family, is an immunomodulatory cytokine that is highly expressed in the CNS. However, the role of IL-33 on viral encephalitis remains unclear. Therefore, we aimed to explore how the IL-33/ST2 axis regulates the local immune response during Rocio virus infection. METHODS: Wild-type (WT), ST2 (ST2(-/-)), and nitric oxide synthase-deficient mice (iNOS(-/-)) and Stat6 (Stat6(-/-))-deficient mice were infected with different concentrations of the Rocio virus by intraperitoneal route, the cytokine mRNA level in CNS was analyzed by qPCR, and cellular immunophenotyping was performed on infected mice by the flow cytometry of isolated CNS mononuclear cells. RESULTS: We have shown that the mRNA expression of IL-33 and ST2 receptors is increased in the CNS of Rocio virus-infected WT mice and that ST2(-/-) mice showed increased susceptibility to infection. ST2 deficiency was correlated with increased tissue pathology, cellular infiltration, and tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ) mRNA levels and higher viral load in the CNS, compared with wild-type mice. The increased Th1 cytokine levels released in the CNS acted on infiltrating macrophages, as evidenced by flow cytometry characterization of cellular infiltrates, inducing the expression of iNOS, contributing to brain injury. Moreover, iNOS(-/-) mice were more resistant to Rocio virus encephalitis, presenting a lower clinical score and reduced mortality rate, despite the increased tissue pathology. CONCLUSIONS: We provide evidences of a specific role for IL-33 receptor signaling in nitric oxide induction through local IFN-γ modulation, suggesting that nitric oxide overproduction might have an important role in the progression of experimental viral encephalitis.

Local proliferation initiates macrophage accumulation in adipose tissue during obesity.

Obesity-associated chronic inflammation is characterized by an accumulation of adipose tissue macrophages (ATMs). It is generally believed that those macrophages are derived from peripheral blood monocytes. However, recent studies suggest that local proliferation of macrophages is responsible for ATM accumulation. In the present study, we revealed that both migration and proliferation contribute to ATM accumulation during obesity development. We show that there is a significant increase in ATMs at the early stage of obesity, which is largely due to an enhanced in situ macrophage proliferation. This result was obtained by employing fat-shielded irradiation and bone marrow reconstitution. Additionally, the production of CCL2, a pivotal chemoattractant of monocytes, was not found to be increased at this stage, corroborating with a critical role of proliferation. Nonetheless, as obesity proceeds, the role of monocyte migration into adipose tissue becomes more significant and those new immigrants further proliferate locally. These proliferating ATMs mainly reside in crown-like structures formed by macrophages surrounding dead adipocytes. We further showed that IL-4/STAT6 is a driving force for ATM proliferation. Therefore, we demonstrated that local proliferation of resident macrophages contributes to ATM accumulation during obesity development and has a key role in obesity-associated inflammation.

Cu,Zn-Superoxide Dismutase-Mediated Redox Regulation of Jumonji Domain Containing 3 Modulates Macrophage Polarization and Pulmonary Fibrosis.

M2 macrophages are implicated in the development of pulmonary fibrosis as they generate profibrotic signals. The polarization process, at least in part, is regulated by epigenetic modulation. Because Cu,Zn-superoxide dismutase-induced H2O2 can polarize macrophages to a profibrotic M2 phenotype, we hypothesized that modulation of the redox state of the cell is involved in the epigenetic modulation of the macrophage phenotype. In this study, we show that signal transducer and activator of transcription 6 (STAT6) regulates Jumonji domain containing (Jmjd) 3, a histone H3 lysine 27 demethylase, and mutation of a redox-sensitive cysteine in STAT6 attenuates jmjd3 expression. Moreover, Jmjd3 deficiency abrogates profibrotic M2 gene expression. Treatment with leflunomide, which reduces mitochondrial reactive oxygen species production and tyrosine phosphorylation, inhibits jmjd3 expression and M2 polarization, as well as development of a fibrotic phenotype. Taken together, these observations provide evidence that the redox regulation of Jmjd3 is a unique regulatory mechanism for Cu,Zn-superoxide dismutase-mediated profibrotic M2 polarization. Furthermore, leflunomide, which reduces reactive oxygen species production and tyrosine phosphorylation, may prove to be therapeutic in the treatment of asbestos-induced pulmonary fibrosis.