Publisher Correction: The transcriptional coactivator TAZ regulates reciprocal differentiation of TH17 cells and Treg cells.

In the version of this article initially published, the institution name for affiliation 3 (Maryland Anderson Cancer Center) was incorrect. The correct institution is MD Anderson Cancer Center. The error has been corrected in the HTML and PDF versions of the article.

Corrigendum: The transcriptional coactivator TAZ regulates reciprocal differentiation of TH17 cells and Treg cells.

This corrects the article DOI: 10.1038/ni.3748.

The transcriptional coactivator TAZ regulates reciprocal differentiation of TH17 cells and Treg cells.

An imbalance in the lineages of immunosuppressive regulatory T cells (Treg cells) and the inflammatory TH17 subset of helper T cells leads to the development of autoimmune and/or inflammatory disease. Here we found that TAZ, a coactivator of TEAD transcription factors of Hippo signaling, was expressed under TH17 cell-inducing conditions and was required for TH17 differentiation and TH17 cell-mediated inflammatory diseases. TAZ was a critical co-activator of the TH17-defining transcription factor RORγt. In addition, TAZ attenuated Treg cell development by decreasing acetylation of the Treg cell master regulator Foxp3 mediated by the histone acetyltransferase Tip60, which targeted Foxp3 for proteasomal degradation. In contrast, under Treg cell-skewing conditions, TEAD1 expression and sequestration of TAZ from the transcription factors RORγt and Foxp3 promoted Treg cell differentiation. Furthermore, deficiency in TAZ or overexpression of TEAD1 induced Treg cell differentiation, whereas expression of a transgene encoding TAZ or activation of TAZ directed TH17 cell differentiation. Our results demonstrate a pivotal role for TAZ in regulating the differentiation of Treg cells and TH17 cells.

Adult Connective Tissue-Resident Mast Cells Originate from Late Erythro-Myeloid Progenitors.

Tissue-resident mast cells are associated with many inflammatory and physiological processes. Although mast cells arise from the yolk sac, the exact ontogeny of adult mast cells remains unclear. Here we have investigated the hematopoietic origin of mast cells using fate-mapping systems. We have shown that early erythro-myeloid progenitors (EMPs), late EMPs, and definitive hematopoietic stem cells (HSCs) each gave rise to mast cells in succession via an intermediate integrin ß7+ progenitor. From late embryogenesis to adult, early EMP-derived mast cells were largely replaced by late EMP-derived cells in most connective tissues except adipose and pleural cavity. Thus, mast cells with distinct origin displayed tissue-location preferences: early EMP-derived cells were limited to adipose and pleural cavity and late EMP-derived cells dominated most connective tissues, while HSC-derived cells were a main group in mucosa. Therefore, embryonic origin shapes the heterogeneity of adult mast cells, with diverse functions in immunity and development.

Cholesterol Homeostatic Regulator SCAP-SREBP2 Integrates NLRP3 Inflammasome Activation and Cholesterol Biosynthetic Signaling in Macrophages.

Cholesterol metabolism has been linked to immune functions, but the mechanisms by which cholesterol biosynthetic signaling orchestrates inflammasome activation remain unclear. Here, we have shown that NLRP3 inflammasome activation is integrated with the maturation of cholesterol master transcription factor SREBP2. Importantly, SCAP-SREBP2 complex endoplasmic reticulum-to-Golgi translocation was required for optimal activation of the NLRP3 inflammasome both in vitro and in vivo. Enforced cholesterol biosynthetic signaling by sterol depletion or statins promoted NLPR3 inflammasome activation. However, this regulation did not predominantly depend on changes in cholesterol homeostasis controlled by the transcriptional activity of SREBP2, but relied on the escort activity of SCAP. Mechanistically, NLRP3 associated with SCAP-SREBP2 to form a ternary complex which translocated to the Golgi apparatus adjacent to a mitochondrial cluster for optimal inflammasome assembly. Our study reveals that, in addition to controlling cholesterol biosynthesis, SCAP-SREBP2 also serves as a signaling hub integrating cholesterol metabolism with inflammation in macrophages.

Bile Acids Control Inflammation and Metabolic Disorder through Inhibition of NLRP3 Inflammasome.

Reciprocal interactions between the metabolic system and immune cells play pivotal roles in diverse inflammatory diseases, but the underlying mechanisms remain elusive. The activation of bile acid-mediated signaling has been linked to improvement in metabolic syndromes and enhanced control of inflammation. Here, we demonstrated that bile acids inhibited NLRP3 inflammasome activation via the TGR5-cAMP-PKA axis. TGR5 bile acid receptor-induced PKA kinase activation led to the ubiquitination of NLRP3, which was associated with the PKA-induced phosphorylation of NLRP3 on a single residue, Ser 291. Furthermore, this PKA-induced phosphorylation of NLRP3 served as a critical brake on NLRP3 inflammasome activation. In addition, in vivo results indicated that bile acids and TGR5 activation blocked NLRP3 inflammasome-dependent inflammation, including lipopolysaccharide-induced systemic inflammation, alum-induced peritoneal inflammation, and type-2 diabetes-related inflammation. Altogether, our study unveils the PKA-induced phosphorylation and ubiquitination of NLRP3 and suggests TGR5 as a potential target for the treatment of NLRP3 inflammasome-related diseases.

Tespa1 is involved in late thymocyte development through the regulation of TCR-mediated signaling.

Signaling via the T cell antigen receptor (TCR) during the CD4(+)CD8(+) double-positive developmental stage determines thymocyte selection and lineage commitment. Here we describe a previously uncharacterized T cell-expressed protein, Tespa1, with critical functions during the positive selection of thymocytes. Tespa1(-/-) mice had fewer mature thymic CD4(+) and CD8(+) T cells, which reflected impaired thymocyte development. Tespa1 associated with the TCR signaling components PLC-γ1 and Grb2, and Tespa1 deficiency resulted in attenuated TCR signaling, as reflected by defective activation of the Erk-AP-1 and Ca(2+)-NFAT pathways. Our findings demonstrate that Tespa1 is a component of the TCR signalosome and is essential for T cell selection and maturation through the regulation of TCR signaling during T cell development.

NPAT Supports CD8+ Immature Single-Positive Thymocyte Proliferation and Thymic Development.

Thymocytes need to proliferate into a significant cell mass to allow a subsequent selection process during the double-positive (DP) stage. However, it is not clear at what stage this massive cell proliferation occurs. Immature CD8 single-positive (ISP) cells are a well-defined thymocyte subpopulation. However, the function of this cell subset has not yet been characterized. In this study, we analyzed the transcription pattern of mouse ISP cells and observed higher expression levels of cell cycling genes. We also found out that ISP cells exhibited the highest cell proliferative capacity among thymocytes in different developmental stages. Nuclear protein ataxia-telangiectasia (NPAT/p220) is one of the highly expressed cell cycling genes in ISP cells, which is known to play a critical role in coordinating histone gene expression necessary for rapid cell proliferation. Selective deletion of NPAT at the ISP stage led to reduced thymus size and significant loss of DP cells, secondary to reduced histone gene expression and impaired ISP cell proliferation capacity. A block of thymocyte development at the ISP stage was also observed, which was due to increased IL-7R expression. Continuous IL-7R signal served as a compensating mechanism for cell proliferation upon NPAT deletion, but in turn inhibited the expression of transcription factors TCF-1 and LEF-1, which is essential for the transition of ISP to DP cells. In summary, our study revealed the proliferation capacity of the ISP subpopulation during thymocyte differentiation as well as a vital role of NPAT in this developmental stage.

huARdb: human Antigen Receptor database for interactive clonotype-transcriptome analysis at the single-cell level.

T-cell receptors (TCRs) and B-cell receptors (BCRs) are critical in recognizing antigens and activating the adaptive immune response. Stochastic V(D)J recombination generates massive TCR/BCR repertoire diversity. Single-cell immune profiling with transcriptome analysis allows the high-throughput study of individual TCR/BCR clonotypes and functions under both normal and pathological settings. However, a comprehensive database linking these data is not yet readily available. Here, we present the human Antigen Receptor database (huARdb), a large-scale human single-cell immune profiling database that contains 444 794 high confidence T or B cells (hcT/B cells) with full-length TCR/BCR sequence and transcriptomes from 215 datasets. All datasets were processed in a uniform workflow, including sequence alignment, cell subtype prediction, unsupervised cell clustering, and clonotype definition. We also developed a multi-functional and user-friendly web interface that provides interactive visualization modules for biologists to analyze the transcriptome and TCR/BCR features at the single-cell level. HuARdb is freely available at https://huarc.net/database with functions for data querying, browsing, downloading, and depositing. In conclusion, huARdb is a comprehensive and multi-perspective atlas for human antigen receptors.

Protein phosphatase 2A propels follicular T helper cell development in lupus.

Follicular helper T (Tfh) cells are important for generating humoral immune responses by helping B cells form germinal centers (GCs) and the production of high-affinity antibodies. However, aberrant Tfh cell expansion also contributes to the generation of self-reactive autoantibodies and promotes autoantibody-mediated autoimmune diseases such as systemic lupus erythematosus (SLE). Protein phosphatase 2A catalytic subunit alpha isoform (PP2A Cα) expression levels are elevated in peripheral T cells of SLE patients and positively correlate with autoantibody titers and disease activity. Here, we demonstrate a critical role of PP2A in Tfh differentiation by using T cell restricted PP2A Cα deficient mice. We observed impaired Tfh differentiation and GC response in two different classical Tfh induction models. Mechanistic studies revealed that downregulation of protein translation of the Tfh lineage transcription factor BCL6 in PP2A deficient T cells. Importantly, we found that PP2A deficiency by either gene knockout or chemical inhibition alleviated lupus severity in mice. Lastly, we confirmed a positive correlation between PP2A Cα and BCL6 protein levels in human CD4+ T cells from patients with SLE. In summary, our study revealed a critical role of PP2A in regulating Tfh cells and suggests it is a potential therapeutic target for lupus.

MDA5 expression is associated with TGF-ß-induced fibrosis: potential mechanism of interstitial lung disease in anti-MDA5 dermatomyositis.

OBJECTIVES: This study aimed to investigate the high-resolution CT (HRCT) characteristics of anti-melanoma differentiation-associated gene 5 (MDA5) antibody positive dermatomyositis-associated interstitial lung disease (anti-MDA5 DM-ILD), and to clarify the underlying mechanisms of the clinical phenomenon. METHODS: Clinical data and HRCT patterns were compared between anti-MDA5 DM-ILD (n = 32) and antisynthetase syndrome-associated ILD (ASS-ILD) (n = 29). RNA sequencing of whole-blood samples from the two groups, and in vitro experiments using human embryonic lung fibroblasts (HELFs) were conducted to explore the potential mechanisms of the clinical findings. RESULTS: The anti-MDA5 DM-ILD subset had a significantly higher incidence of rapidly progressive ILD (RPILD) than ASS-ILD (65.6% vs 37.9%; P = 0.031). The relative percentage of the lung fibrosis HRCT pattern was significantly lower in the anti-MDA5 DM-ILD group, especially the RPILD subgroup (P = 0.013 and 0.003, respectively). RNA sequencing detected the upregulated genes including interferon-induced helicase C domain 1 (encoding MDA5), and a trend towards downregulated expression of TGF-ß signalling components in anti-MDA5 DM-ILD. In vitro culture of HELFs revealed that upregulated expression of MDA5 in HELFs was correlated with the downregulated expression of alpha smooth muscle actin, connective tissue growth factor, collagen I and collagen III by suppressing the TGF-ß signalling pathway. CONCLUSIONS: Anti-MDA5 DM-ILD patients have significantly less lung fibrosis and elevated MDA5 expression. The upregulated expression of MDA5 has relations with the suppression of the pro-fibrotic function of fibroblasts via the TGF-ß signalling pathway, which may partially explain the mechanism of the clinical phenomenon.

Phosphatase PP2A is essential for TH17 differentiation.

Phosphatase PP2A expression levels are positively correlated to the clinical severity of systemic lupus erythematosus (SLE) and IL17A cytokine overproduction, indicating a potential role of PP2A in controlling TH17 differentiation and inflammation. By generating a mouse strain with ablation of the catalytic subunit α of PP2A in peripheral mature T cells (PP2A cKO), we demonstrate that the PP2A complex is essential for TH17 differentiation. These PP2A cKO mice had reduced TH17 cell numbers and less severe disease in an experimental autoimmune encephalomyelitis (EAE) model. PP2A deficiency also ablated C-terminal phosphorylation of SMAD2 but increased C-terminal phosphorylation of SMAD3. By regulating the activity of RORγt via binding, the changes in the phosphorylation status of these R-SMADs reduced Il17a gene transcription. Finally, PP2A inhibitors showed similar effects on TH17 cells as were observed in PP2A cKO mice, i.e., decreased TH17 differentiation and relative protection of mice from EAE. Taken together, these data demonstrate that phosphatase PP2A is essential for TH17 differentiation and that inhibition of PP2A could be a possible therapeutic approach to controlling TH17-driven autoimmune diseases.

Protein phosphatase 2A has an essential role in promoting thymocyte survival during selection.

The development of thymocytes to mature T cells in the thymus is tightly controlled by cellular selection, in which only a small fraction of thymocytes equipped with proper quality of TCRs progress to maturation. It is pivotal to protect the survival of the few T cells, which pass the selection. However, the signaling events, which safeguard the cell survival in thymus, are not totally understood. In this study, protein Ser/Thr phosphorylation in thymocytes undergoing positive selection is profiled by mass spectrometry. The results revealed large numbers of dephosphorylation changes upon T cell receptor (TCR) activation during positive selection. Subsequent substrate analysis pinpointed protein phosphatase 2A (PP2A) as the enzyme responsible for the dephosphorylation changes in developing thymocytes. PP2A catalytic subunit α (Ppp2ca) deletion in the T cell lineage in Ppp2caflox/flox-Lck-Cre mice (PP2A cKO) displayed dysregulated dephosphorylation of apoptosis-related proteins in double-positive (DP) cells and caused substantially decreased numbers of DP CD4+ CD8+ cells. Increased levels of apoptosis in PP2A cKO DP cells were found to underlie aberrant thymocyte development. Finally, the defective thymocyte development in PP2A cKO mice could be rescued by either Bcl2 transgene expression or by p53 knockout. In summary, our work reveals an essential role of PP2A in promoting thymocyte development through the regulation of cell survival.

Dimethyl Itaconate-Loaded Nanofibers Rewrite Macrophage Polarization, Reduce Inflammation, and Enhance Repair of Myocardic Infarction.

Cellular metabolism plays a major role in the regulation of inflammation. The inflammatory macrophages undergo a wide-range of metabolic rewriting due to the production of significant amount of itaconate metabolite from cis-aconitate in the tricarboxylic acid cycle. This itaconate molecule has been recently described as a promising immunoregulator. However, its function and mode of action on macrophages and tissue repair and regeneration are yet unclear. Herein, the itaconate-derivative dimethyl itaconate (DMI) suppresses the IL-23/IL-17 inflammatory axis-associated genes and promotes antioxidant nuclear factor erythroid 2-related factor 2 target genes. The poly-ε-caprolactone (PCL)/DMI nanofibers implanted in mice initially maintain inflammation by suppressing anti-inflammatory activity and particular inflammation, while at later stage promotes anti-inflammatory activity for an appropriate tissue repair. Furthermore, the PCL/DMI nanofiber patches show an excellent myocardial protection by reducing infarct area and improving ventricular function via time-dependent regulation of myocardium-associated genes. This study unveils potential DMI macrophage modulatory functions in tissue microenvironment and macrophages rewriting for proper tissue repair.

Tespa1 plays a role in the modulation of airway hyperreactivity through the IL-4/STAT6 pathway.

BACKGROUND: Thymocyte-expressed, positive selection-associated 1 (Tespa1) is a critical signaling molecule in thymocyte development. This study aimed to investigate the regulatory effect of Tespa1 on mast cells in the pathogenesis of asthma and its relationship with the interleukin (IL)-4/signal transducers and activators of transcription 6 (STAT6) signaling pathway. METHODS: Tespa1 mRNA expression analysis and IgE levels were carried out using the induced sputum of 33 adults with stable asthma and 36 healthy controls. Tespa1-knockout mice (Tespa1-/-, KO) and C57BL/6 background (wild-type, WT) mice were sensitized and treated with ovalbumin (OVA) to establish an asthma model. Pathological changes, number and activity of mast cells, and changes in activation of the IL-4/STAT6 pathway in lung tissue were detected. The changes of tryptase expression and STAT6 activation after mast cell gene knockout were analyzed in vitro. The changes of enzyme expression and STAT6 activation after mast cell gene knockout were analyzed in vitro. The association between the Tespa1 and p-STAT6 was analyzed by co-immunoprecipitation method. RESULTS: Compared with the healthy controls, Tespa1 expression was decreased, and IgE levels were elevated in the sputum of asthmatic patients. Animal experiments showed that Tespa1-/- mice exhibited more severe inflammation, higher quantity of goblet cells and mast cells in the bronchium, and greater expression of mast cell tryptase, which is induced by ovalbumin, than WT mice. And IL-4, IL-13, phospho-Janus kinase 1, and p-STAT6 expressions presented a higher increase in the Tespa1-/- mouse model than in the WT mouse model. Further in vitro studies confirmed that IL-4 could more significantly promote tryptase and p-STAT6 activities in Tespa1-/- mast cells than their WT counterparts. Correlation analysis results showed a negative correlation between Tespa1 and p-STAT6. Co-immunoprecipitation results demonstrated an association between Tespa1 and p-STAT6. CONCLUSIONS: Altogether, our results indicate that Tespa1 can negatively regulate mast cell activity, and this event is related to the mast cell IL-4/STAT6 signaling pathway and could be therapeutically exploited to treat asthma attacks.

Misshapen/NIK-related kinase (MINK1) is involved in platelet function, hemostasis, and thrombus formation.

The sterile-20 kinase misshapen/Nck-interacting kinase (NIK)-related kinase 1 (MINK1) is involved in many important cellular processes such as growth, cytoskeletal rearrangement, and motility. Here, with MINK1-deficient (MINK1(-/-)) mice, we showed that MINK1 plays an important role in hemostasis and thrombosis via the regulation of platelet functions. In the tail-bleeding assay, MINK1(-/-) mice exhibited a longer bleeding time than wild-type (WT) mice (575.2 ± 59.7 seconds vs 419.6 ± 66.9 seconds). In a model of ferric chloride-induced mesenteric arteriolar thrombosis, vessel occlusion times were twice as long in MINK1(-/-) mice as in WT mice. In an in vitro microfluidic whole-blood perfusion assay, thrombus formation on a collagen matrix under arterial shear conditions was significantly reduced in MINK1(-/-) platelets. Moreover, MINK1(-/-) platelets demonstrated impaired aggregation and secretion in response to low doses of thrombin and collagen. Furthermore, platelet spreading on fibrinogen was largely hampered in MINK1(-/-) platelets. The functional differences of MINK1(-/-) platelets could be attributed to impaired adenosine 5'-diphosphate secretion. Signaling events associated with MINK1 appeared to involve extracellular signal-regulated kinase, p38, and Akt. Hence, MINK1 may be an important signaling molecule that mediates mitogen-activated protein kinase signaling and participates in platelet activation and thrombus formation.

A novel size-based sorting mechanism of pinocytic luminal cargoes in microglia.

Microglia are the resident immune cells in the CNS and play diverse roles in the maintenance of CNS homeostasis. Recent studies have shown that microglia continually survey the CNS microenvironment and scavenge cell debris and aberrant proteins by phagocytosis and pinocytosis, and that reactive microglia are capable to present antigens to T cells and initiate immune responses. However, how microglia process the endocytosed contents and evoke an immune response remain unclear. Here we report that a size-dependent selective transport of small soluble contents from the pinosomal lumen into lysosomes is critical for the antigen processing in microglia. Using fluorescent probes and water-soluble magnetic nanobeads of defined sizes, we showed in cultured rodent microglia, and in a cell-free reconstructed system that pinocytosed proteins become degraded immediately following pinocytosis and the resulting peptides are selectively delivered to major histocompatibility complex class II (MHC-II) containing lysosomes, whereas undegraded proteins are retained in the pinosomal lumen. This early size-based sorting of pinosomal contents relied on the formation of transient tunnel between pinosomes and lysosomes in a Rab7- and dynamin II-dependent manner, which allowed the small contents to pass through but restricted large ones. Inhibition of the size-based sorting markedly reduced proliferation and cytokine release of cocultured CD4(+) T cells, indicating that the size-based sorting is required for efficient antigen presentation by microglial cells. Together, these findings reveal a novel early sorting mechanism for pinosomal luminal contents in microglial cells, which may explain how microglia efficiently process protein antigens and evoke an immune response.

Inhibition of follicular T-helper cells by CD8(+) regulatory T cells is essential for self tolerance.

The ability to produce vigorous immune responses that spare self tissues and organs depends on the elimination of autoreactive T and B cells. However, purging of immature and mature self-reactive T and B cells is incomplete and may also require the involvement of cells programmed to suppress immune responses. Regulatory T cells (T(reg)) belonging to the CD4(+) T-cell subset may have a role in preventing untoward inflammatory responses, but T-cell subsets programmed to inhibit the development of autoantibody formation and systemic-lupus-erythematosus-like disease have not yet been defined. Here we delineate a CD8(+) regulatory T-cell lineage that is essential for the maintenance of self tolerance and prevention of murine autoimmune disease. Genetic disruption of the inhibitory interaction between these CD8(+) T cells and their target Qa-1(+) follicular T-helper cells results in the development of a lethal systemic-lupus-erythematosus-like autoimmune disease. These findings define a sublineage of CD8 T cells programmed to suppress rather than activate immunity that represents an essential regulatory element of the immune response and a guarantor of self tolerance.

Glatiramer acetate ameliorates inflammatory bowel disease in mice through the induction of Qa-1-restricted CD8⁺ regulatory cells.

Inflammatory bowel diseases (IBDs) are complex multifactorial immunological disorders characterized by dysregulated immune reactivity in the intestine. Here, we investigated the contribution of Qa-1-restricted CD8(+) Treg cells in regulating experimental IBD in mice. We found that CD8(+) T cells induced by T-cell vaccination ameliorated the pathological manifestations of dextran sulfate sodium induced IBD when adoptively transferred into IBD mice. In addition, CD8(+) cell suppressive activity was induced by vaccination with glatiramer acetate (GA), an FDA-approved drug for multiple sclerosis (MS). We next showed that GA-induced CD8(+) Treg cells worked in a Qa-1-dependent manner and their suppressive activity depends on perforin-mediated cytotoxicity. Finally, we confirmed the role of CD4(+) T cells in dextran sulfate sodium induced colitis progression, and clarified that GA-induced CD8(+) T cells exerted their therapeutic effects on colitis by targeting pathogenic CD4(+) T cells. Our results reveal a new regulatory role of Qa-1-restricted CD8(+) Treg cells in IBD and suggest their induction by GA vaccination as a potential therapeutic approach to IBD.