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Type-2 innate immune responses that occur in airways and are accompanied by goblet-cell hyperplasia and mucus production are largely driven by interleukin-33 (IL-33) and natural helper (NH) cells, a member of group 2 innate lymphoid cells (ILC2s) and the major target of IL-33. Here we report that the corticosteroid resistance observed as a result of airway inflammation triggered by sensitization and exposure to allergen is induced via the IL-33/NH-cell axis. Thymic stromal lymphopoietin (TSLP) synthesized during airway inflammation plays a pivotal role in the induction of NH-cell corticosteroid resistance in vitro and in vivo , by controlling phosphorylation of STAT5 and expression of Bcl-xL in NH cells. Blockade of TSLP with a neutralizing antibody or blocking the TSLP/STAT5 signalling pathway with low molecular-weight STAT5 inhibitors such as pimozide restores corticosteroid sensitivity. Thus, the TSLP-STAT5 pathway could be a new therapeutic target in severe, corticosteroid-resistant asthma. Allergic airway inflammation in asthma can be treated with corticosteroids, but some patients remain unresponsive to this therapy. Here, Kabata et al . show that thymic stromal lymphopoietin contributes to the corticosteroid resistance during airway inflammation through its action on natural helper cells.

Inflammatory bowel disease (IBD) comprises chronic relapsing disorders of the gastrointestinal tract characterized pathologically by intestinal inflammation and epithelial injury. Here, we uncover a function of extracellular matrix protein 1 (ECM1) in promoting the pathogenesis of human and mouse IBD. ECM1 was highly expressed in macrophages, particularly tissue-infiltrated macrophages under inflammatory conditions, and ECM1 expression was significantly induced during IBD progression. The macrophagespecific knockout of ECM1 resulted in increased arginase 1 (ARG1) expression and impaired polarization into the M1 macrophage phenotype after lipopolysaccharide (LPS) treatment. A mechanistic study showed that ECM1 can regulate M1 macrophage polarization through the granulocyte-macrophage colony-stimulating factor/ STAT5 signaling pathway. Pathological changes in mice with dextran sodium sulfate-induced IBD were alleviated by the specific knockout of the ECM1 gene in macrophages. Taken together, our findings show that ECM1 has an important function in promoting M1 macrophage polarization, which is critical for controlling inflammation and tissue repair in the intestine.

T helper （Tn）-cell subsets, such as TH1 and TH17, mediate inflammation in both peripheral tissues and central nervous system. Here we show that STAT5 is required for T helper-cell pathogenicity in autoimmune neuroinflammation but not in experimental colitis. Although STAT5 promotes regulatory T cell generation and immune suppression, loss of STAT5 in CD4~ T cells resulted in diminished development of experimental autoimmune encephalomyelitis （EAE）, a mouse model of multiple sclerosis. Our results showed that loss of encephalitogenic activity of STAT5-deficient autoreaetive CD4＋ T cells was independent of IFN-7 or interleukin 17 （IL-17） production, but was due to the impaired expression of granulocyte-macrophage colony-stimulating factor （GM-CSF）, a crucial mediator of T-cell pathogenicity. We further showed that IL-7-activated STAT5 promotes the generation of GM-CSF-producing CD4＋ T cells, which were preferentially able to induce more severe EAE than TH17 or TH1 cells. Consistent with GM-CSF-produc- ing cells being a distinct subset of TH cells, the differentiation program of these cells was distinct from that of TH17 or TH1 cells. We further found that IL-3 was secreted in a similar pattern as GM-CSF in this subset of T, cells. In conclusion, the IL-7-STAT5 axis promotes the generation of GM-CSF/IL-3-producing T, cells. These cells display a distinct transcriptional profile and may represent a novel subset of T helper cells which we designate as TH-GM.

Osteosarcoma (OS) is the most common primary bone tumor that primarily affects children and adolescents. Studies suggested that dysregulation JAK/STAT signaling promotes the development of OS. Cells treated with pimozide, a STAT5 inhibitor suppressed proliferation and colony formation and induced sub G0/G1 cell cycle arrest and apoptosis. There was a reduction in cyclin D1 and CDK2 expression and Rb phosphorylation, and activation of Caspase-3 and PARP cleavage. In addition, pimozide suppressed the formation of 3-dimensional osteospheres and growth of the cells in the Tumor in a Dish lung organoid system. Furthermore, there was a reduction in expression of cancer stem cell marker proteins DCLK1, CD44, CD133, Oct-4, and ABCG2. More importantly, it was the short form of DCLK1 that was upregulated in osteospheres, which was suppressed in response to pimozide. We further confirmed by flow cytometry a reduction in DCLK1+ cells. Moreover, pimozide inhibits the phosphorylation of STAT5, STAT3, and ERK in OS cells. Molecular docking studies suggest that pimozide interacts with STAT5A and STAT5B with binding energies of −8.4 and −6.4 Kcal/mol, respectively. Binding was confirmed by cellular thermal shift assay. To further understand the role of STAT5, we knocked down the two isoforms using specific siRNAs. While knockdown of the proteins did not affect the cells, knockdown of STAT5B reduced pimozide-induced necrosis and further enhanced late apoptosis. To determine the effect of pimozide on tumor growth in vivo, we administered pimozide intraperitoneally at a dose of 10 mg/kg BW every day for 21 days in mice carrying KHOS/NP tumor xenografts. Pimozide treatment significantly suppressed xenograft growth. Western blot and immunohistochemistry analyses also demonstrated significant inhibition of stem cell marker proteins. Together, these data suggest that pimozide treatment suppresses OS growth by targeting both proliferating cells and stem cells at least in part by inhibiting the STAT5 signaling pathway.

Cytokines critically control immune responses, but how regulatory programs are altered to allow T cells to differentially respond to distinct cytokine stimuli remains poorly understood. Here, we have globally analyzed enhancer elements bound by IL-2–activated STAT5 and IL-21–activated STAT3 in T cells and identified Il2ra as the top-ranked gene regulated by an IL-2–activated STAT5-bound superenhancer and one of the top genes regulated by STAT3-bound superenhancers. Moreover, we found that STAT5 binding was rapidly superenriched at genes highly induced by IL-2 and that IL-2–activated STAT5 binding induces new and augmented chromatin interactions within superenhancer-containing genes. Based on chromatin interaction analysis by paired-end tag (ChIA-PET) sequencing data, we used CRISPR-Cas9 gene editing to target three of the STAT5 binding sites within the Il2ra superenhancer in mice. Each mutation decreased STAT5 binding and altered IL-2–induced Il2ra gene expression, revealing that individual elements within the superenhancer were not functionally redundant and that all were required for normal gene expression. Thus, we demonstrate cooperative utilization of superenhancer elements to optimize gene expression and show that STAT5 mediates IL-2–induced chromatin looping at superenhancers to preferentially regulate highly inducible genes, thereby providing new insights into the mechanisms underlying cytokine-dependent superenhancer function.

Many autoimmune diseases are characterized by the production of autoantibodies. The current view is that CD4 + T follicular helper (Tfh) cells are the main subset regulating autoreactive B cells. Here we report a CXCR5 + PD1 + Tfh subset of CD8 + T cells whose development and function are negatively modulated by Stat5. These CD8 + Tfh cells regulate the germinal center B cell response and control autoantibody production, as deficiency of Stat5 in CD8 T cells leads to an increase of CD8 + Tfh cells, resulting in the breakdown of B cell tolerance and concomitant autoantibody production. CD8 + Tfh cells share similar gene signatures with CD4 + Tfh, and require CD40L/CD40 and TCR/MHCI interactions to deliver help to B cells. Our study thus highlights the diversity of follicular T cell subsets that contribute to the breakdown of B-cell tolerance. B cell response and antibody production are generally facilitated by CD4 + follicular helper (Tfh) cells. Here the authors identify a subset of CXCR5 + PD1 + CD8 + Tfh cells that is normally suppressed by STAT5 signaling, so that STAT5 deficiency in mice increases the number of these CD8 + Tfh cells and induces concomitant production of autoantibodies.

B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This ‘alternate pathway’ of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties. B-1a B cells are innate-like cells with biased reactivity to bacteria and self-antigens. Here the authors show that reduced interleukin-7 in developing fetal liver-derived pro-B cells induces premature immunoglobulin κ rearrangement, alleviating the requirement for a pre-BCR selection stage and allowing the generation of autoreactive B1-a B cells.

Background In breast cancer, complex interactions between tumor cells and cells within the surrounding stroma, such as macrophages, are critical for tumor growth, progression, and therapeutic response. Recent studies have highlighted the complex nature and heterogeneous populations of macrophages associated with both tumor-promoting and tumor-inhibiting phenotypes. Defining the pathways that drive macrophage function is important for understanding their complex phenotypes within the tumor microenvironment. Signal transducer and activator of transcription (STAT) transcription factors, such as STAT5, are key regulators of immune cell function. The studies described here investigate the functional contributions of STAT5 to tumor-associated macrophage function in breast cancer. Methods Initial studies were performed using a panel of human breast cancer and mouse mammary tumor cell lines to determine the ability of tumor cell-derived factors to induce STAT5 activation in macrophages. Further studies used these models to identify soluble factors that activate STAT5 in macrophages. To delineate STAT5-specific contributions to macrophage function, a conditional model of myeloid STAT5 deletion was used for in vitro , RNA-sequencing, and in vivo studies. The effects of STAT5 deletion in macrophages on tumor cell migration and metastasis were evaluated using in vitro co-culture migration assays and an in vivo tumor cell-macrophage co-injection model. Results We demonstrate here that STAT5 is robustly activated in macrophages by tumor cell-derived factors and that GM-CSF is a key cytokine stimulating this pathway. The analysis of RNA-seq studies reveals that STAT5 promotes expression of immune stimulatory genes in macrophages and that loss of STAT5 in macrophages results in increased expression of tissue remodeling factors. Finally, we demonstrate that loss of STAT5 in macrophages promotes tumor cell migration in vitro and mammary tumor metastasis in vivo. Conclusions Breast cancer cells produce soluble factors, such as GM-CSF, that activate the STAT5 pathway in macrophages and drive expression of inflammatory factors. STAT5 deletion in myeloid cells enhances metastasis, suggesting that STAT5 activation in tumor-associated macrophages protects against tumor progression. Understanding mechanisms that drive macrophage function in the tumor microenvironment will ultimately lead to new approaches that suppress tumor-promoting functions while enhancing their anti-tumor functions.

Signal tranducer and activator of transcription 5 (STAT5) plays a critical role in mediating cellular responses following cytokine stimulation. STAT proteins critically signal via the formation of dimers, but additionally, STAT tetramers serve key biological roles, and we previously reported their importance in T and natural killer (NK) cell biology. However, the role of STAT5 tetramerization in autoimmune-mediated neuroinflammation has not been investigated. Using the STAT5 tetramer-deficient Stat5a-Stat5b N-domain double knockin (DKI) mouse strain, we report here that STAT5 tetramers promote the pathogenesis of experimental autoimmune encephalomyelitis (EAE). The mild EAE phenotype observed in DKI mice correlates with the impaired extravasation of pathogenic T-helper 17 (Th17) cells and interactions between Th17 cells and monocyte-derived cells (MDCs) in the meninges. We further demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF)–mediated STAT5 tetramerization regulates the production of CCL17 by MDCs. Importantly, CCL17 can partially restore the pathogenicity of DKI Th17 cells, and this is dependent on the activity of the integrin VLA-4. Thus, our study reveals a GM-CSF-STAT5 tetramer-CCL17 pathway in MDCs that promotes autoimmune neuroinflammation.

The Signal Transducer and Activator of Transcription (STAT)-5 proteins are required in immune regulation and homeostasis and play a crucial role in the development and function of several hematopoietic cells. STAT5b activation is involved in the expression of genes that participate in cell development, proliferation, and survival. STAT5a and STAT5b are paralogs and only human mutations in STAT5B have been identified leading to immune dysregulation and hematopoietic malignant transformation. The inactivating STAT5B mutations cause impaired post-natal growth, recurrent infections and immune dysregulation, whereas gain of function somatic mutations cause dysregulated allergic inflammation. These mutations are rare, and they are associated with a wide spectrum of clinical manifestations which provide a disease model elucidating the biological mechanism of STAT5 by studying the consequences of perturbations in STAT5 activity. Further, the use of Jak inhibitors as therapy for a variety of autoimmune and malignant disorders has increased substantially heading relevant lessons for the consequences of Jak/STAT immunomodulation from the human model. This review summarizes the biology of the STAT5 proteins, human disease associate with molecular defects in STAT5b, and the connection between aberrant activation of STAT5b and the development of certain cancers.