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.

The phosphatase DUSP2 controls the activity of the transcription activator STAT3 and regulates TH17 differentiation.

Deregulation of the TH17 subset of helper T cells is closely linked with immunological disorders and inflammatory diseases. However, the mechanism by which TH17 cells are regulated remains elusive. Here we found that the phosphatase DUSP2 (PAC1) negatively regulated the development of TH17 cells. DUSP2 was directly associated with the signal transducer and transcription activator STAT3 and attenuated its activity through dephosphorylation of STAT3 at Tyr705 and Ser727. DUSP2-deficient mice exhibited severe susceptibility to experimental colitis, with enhanced differentiation of TH17 cells and secretion of proinflammatory cytokines. In clinical patients with ulcerative colitis, DUSP2 was downregulated by DNA methylation and was not induced during T cell activation. Our data demonstrate that DUSP2 is a true STAT3 phosphatase that modulates the development of TH17 cells in the autoimmune response and inflammation.

IL-37 requires the receptors IL-18Rα and IL-1R8 (SIGIRR) to carry out its multifaceted anti-inflammatory program upon innate signal transduction.

Interleukin 37 (IL-37) and IL-1R8 (SIGIRR or TIR8) are anti-inflammatory orphan members of the IL-1 ligand family and IL-1 receptor family, respectively. Here we demonstrate formation and function of the endogenous ligand-receptor complex IL-37-IL-1R8-IL-18Rα. The tripartite complex assembled rapidly on the surface of peripheral blood mononuclear cells upon stimulation with lipopolysaccharide. Silencing of IL-1R8 or IL-18Rα impaired the anti-inflammatory activity of IL-37. Whereas mice with transgenic expression of IL-37 (IL-37tg mice) with intact IL-1R8 were protected from endotoxemia, IL-1R8-deficient IL-37tg mice were not. Proteomic and transcriptomic investigations revealed that IL-37 used IL-1R8 to harness the anti-inflammatory properties of the signaling molecules Mer, PTEN, STAT3 and p62(dok) and to inhibit the kinases Fyn and TAK1 and the transcription factor NF-κB, as well as mitogen-activated protein kinases. Furthermore, IL-37-IL-1R8 exerted a pseudo-starvational effect on the metabolic checkpoint kinase mTOR. IL-37 thus bound to IL-18Rα and exploited IL-1R8 to activate a multifaceted intracellular anti-inflammatory program.

The cytokine TGF-ß co-opts signaling via STAT3-STAT4 to promote the differentiation of human TFH cells.

Understanding the developmental mechanisms of follicular helper T cells (TFH cells) in humans is relevant to the clinic. However, the factors that drive the differentiation of human CD4+ helper T cells into TFH cells remain largely undefined. Here we found that transforming growth factor-ß (TGF-ß) provided critical additional signals for the transcription factors STAT3 and STAT4 to promote initial TFH differentiation in humans. This mechanism did not appear to be shared by mouse helper T cells. Developing human TFH cells that expressed the transcriptional repressor Bcl-6 also expressed RORγt, a transcription factor typically expressed by the TH17 subset of helper T cells. Our study documents a mechanism by which TFH cells and TH17 cells emerge together in inflammatory environments in humans, as is often observed in many human autoimmune diseases.

TLR10 (CD290) Is a Regulator of Immune Responses in Human Plasmacytoid Dendritic Cells.

TLRs are the most thoroughly studied group of pattern-recognition receptors that play a central role in innate immunity. Among them, TLR10 (CD290) remains the only TLR family member without a known ligand and clearly defined functions. One major impediment to studying TLR10 is its absence in mice. A recent study on TLR10 knock-in mice demonstrated its intrinsic inhibitory role in B cells, indicating that TLR10 is a potential drug target in autoimmune diseases. In this study, we interrogated the expression and function of TLR10 in human plasmacytoid dendritic cells (pDCs). We have seen that primary human pDCs, B cells, and monocytes constitutively express TLR10. Upon preincubation with an anti-TLR10 Ab, production of cytokines in pDCs was downregulated in response to stimulation with DNA and RNA viruses. Upon further investigation into the possible mechanism, we documented phosphorylation of STAT3 upon Ab-mediated engagement of TLR10. This leads to the induction of inhibitory molecule suppressor of cytokine signaling 3 (SOCS3) expression. We have also documented the inhibition of nuclear translocation of transcription factor IFN regulatory factor 7 (IRF7) in pDCs following TLR10 engagement. Our data provide the (to our knowledge) first evidence that TLR10 is constitutively expressed on the surface of human pDCs and works as a regulator of their innate response. Our findings indicate the potential of harnessing the function of pDCs by Ab-mediated targeting of TLR10 that may open a new therapeutic avenue for autoimmune disorders.

Defective sphingosine 1-phosphate receptor 1 (S1P1) phosphorylation exacerbates TH17-mediated autoimmune neuroinflammation.

Sphingosine 1-phosphate (S1P) signaling regulates lymphocyte egress from lymphoid organs into systemic circulation. The sphingosine phosphate receptor 1 (S1P1) agonist FTY-720 (Gilenya) arrests immune trafficking and prevents multiple sclerosis (MS) relapses. However, alternative mechanisms of S1P-S1P1 signaling have been reported. Phosphoproteomic analysis of MS brain lesions revealed S1P1 phosphorylation on S351, a residue crucial for receptor internalization. Mutant mice harboring an S1pr1 gene encoding phosphorylation-deficient receptors (S1P1(S5A)) developed severe experimental autoimmune encephalomyelitis (EAE) due to autoimmunity mediated by interleukin 17 (IL-17)-producing helper T cells (TH17 cells) in the peripheral immune and nervous system. S1P1 directly activated the Jak-STAT3 signal-transduction pathway via IL-6. Impaired S1P1 phosphorylation enhances TH17 polarization and exacerbates autoimmune neuroinflammation. These mechanisms may be pathogenic in MS.

Interleukin-15-Dependent T-Cell-like Innate Intraepithelial Lymphocytes Develop in the Intestine and Transform into Lymphomas in Celiac Disease.

The nature of gut intraepithelial lymphocytes (IELs) lacking antigen receptors remains controversial. Herein we showed that, in humans and in mice, innate intestinal IELs expressing intracellular CD3 (iCD3(+)) differentiate along an Id2 transcription factor (TF)-independent pathway in response to TF NOTCH1, interleukin-15 (IL-15), and Granzyme B signals. In NOTCH1-activated human hematopoietic precursors, IL-15 induced Granzyme B, which cleaved NOTCH1 into a peptide lacking transcriptional activity. As a result, NOTCH1 target genes indispensable for T cell differentiation were silenced and precursors were reprogrammed into innate cells with T cell marks including intracellular CD3 and T cell rearrangements. In the intraepithelial lymphoma complicating celiac disease, iCD3(+) innate IELs acquired gain-of-function mutations in Janus kinase 1 or Signal transducer and activator of transcription 3, which enhanced their response to IL-15. Overall we characterized gut T cell-like innate IELs, deciphered their pathway of differentiation and showed their malignant transformation in celiac disease.

CD5 Binds to Interleukin-6 and Induces a Feed-Forward Loop with the Transcription Factor STAT3 in B Cells to Promote Cancer.

The participation of a specific subset of B cells and how they are regulated in cancer is unclear. Here, we demonstrate that the proportion of CD5(+) relative to interleukin-6 receptor α (IL-6Rα)-expressing B cells was greatly increased in tumors. CD5(+) B cells responded to IL-6 in the absence of IL-6Rα. IL-6 directly bound to CD5, leading to activation of the transcription factor STAT3 via gp130 and its downstream kinase JAK2. STAT3 upregulated CD5 expression, thereby forming a feed-forward loop in the B cells. In mouse tumor models, CD5(+) but not CD5(-) B cells promoted tumor growth. CD5(+) B cells also showed activation of STAT3 in multiple types of human tumor tissues. Thus, our findings demonstrate a critical role of CD5(+) B cells in promoting cancer.

Antagonist anti-LIF antibody derived from naive human scFv phage library inhibited tumor growth in mice.

BACKGROUND: Leukemia inhibitory factor (LIF) is a multifunctional member of the IL-6 cytokine family that activates downstream signaling pathways by binding to the heterodimer consisting of LIFR and gp130 on the cell surface. Previous research has shown that LIF is highly expressed in various tumor tissues (e.g. pancreatic cancer, breast cancer, prostate cancer, and colorectal cancer) and promotes cancer cell proliferation, migration, invasion, and differentiation. Moreover, the overexpression of LIF correlates with poor clinicopathological characteristics. Therefore, we hypothesized that LIF could be a promising target for the treatment of cancer. In this work, we developed the antagonist antibody 1G11 against LIF and investigated its anti-tumor mechanism and its therapeutic efficacy in mouse models. RESULTS: A series of single-chain variable fragments (scFvs) targeting LIF were screened from a naive human scFv phage library. These scFvs were reconstructed in complete IgG form and produced by the mammalian transient expression system. Among the antibodies, 1G11 exhibited the excellent binding activity to human, cynomolgus monkey and mouse LIF. Functional analysis demonstrated 1G11 could block LIF binding to LIFR and inhibit the intracellular STAT3 phosphorylation signal. Interestingly, 1G11 did not block LIF binding to gp130, another LIF receptor that is involved in forming the receptor complex together with LIFR. In vivo, intraperitoneal administration of 1G11 inhibited tumor growth in CT26 and MC38 models of colorectal cancer. IHC analysis demonstrated that p-STAT3 and Ki67 were decreased in tumor tissue, while c-caspase 3 was increased. Furthermore, 1G11 treatment improves CD3+, CD4 + and CD8 + T cell infiltration in tumor tissue. CONCLUSIONS: We developed antagonist antibodies targeting LIF/LIFR signaling pathway from a naive human scFv phage library. Antagonist anti-LIF antibody exerts antitumor effects by specifically reducing p-STAT3. Further studies revealed that anti-LIF antibody 1G11 increased immune cell infiltration in tumor tissues.

Altered regulation of DPF3, a member of the SWI/SNF complexes, underlies the 14q24 renal cancer susceptibility locus.

Our study investigated the underlying mechanism for the 14q24 renal cell carcinoma (RCC) susceptibility risk locus identified by a genome-wide association study (GWAS). The sentinel single-nucleotide polymorphism (SNP), rs4903064, at 14q24 confers an allele-specific effect on expression of the double PHD fingers 3 (DPF3) of the BAF SWI/SNF complex as assessed by massively parallel reporter assay, confirmatory luciferase assays, and eQTL analyses. Overexpression of DPF3 in renal cell lines increases growth rates and alters chromatin accessibility and gene expression, leading to inhibition of apoptosis and activation of oncogenic pathways. siRNA interference of multiple DPF3-deregulated genes reduces growth. Our results indicate that germline variation in DPF3, a component of the BAF complex, part of the SWI/SNF complexes, can lead to reduced apoptosis and activation of the STAT3 pathway, both critical in RCC carcinogenesis. In addition, we show that altered DPF3 expression in the 14q24 RCC locus could influence the effectiveness of immunotherapy treatment for RCC by regulating tumor cytokine secretion and immune cell activation.

DOCK8 functions as an adaptor that links TLR-MyD88 signaling to B cell activation.

The adaptors DOCK8 and MyD88 have been linked to serological memory. Here we report that DOCK8-deficient patients had impaired antibody responses and considerably fewer CD27(+) memory B cells. B cell proliferation and immunoglobulin production driven by Toll-like receptor 9 (TLR9) were considerably lower in DOCK8-deficient B cells, but those driven by the costimulatory molecule CD40 were not. In contrast, TLR9-driven expression of AICDA (which encodes the cytidine deaminase AID), the immunoglobulin receptor CD23 and the costimulatory molecule CD86 and activation of the transcription factor NF-κB, the kinase p38 and the GTPase Rac1 were intact. DOCK8 associated constitutively with MyD88 and the tyrosine kinase Pyk2 in normal B cells. After ligation of TLR9, DOCK8 became tyrosine-phosphorylated by Pyk2, bound the Src-family kinase Lyn and linked TLR9 to a Src-kinase Syk-transcription factor STAT3 cascade essential for TLR9-driven B cell proliferation and differentiation. Thus, DOCK8 functions as an adaptor in a TLR9-MyD88 signaling pathway in B cells.

IL-6/STAT3 axis is hijacked by GCRV to facilitate viral replication via suppressing type â IFN signaling.

Grass carp reovirus (GCRV) infections and hemorrhagic disease (GCHD) outbreaks are typically seasonally periodic and temperature-dependent, yet the molecular mechanism remains unclear. Herein, we depicted that temperature-dependent IL-6/STAT3 axis was exploited by GCRV to facilitate viral replication via suppressing type Ⅰ IFN signaling. Combined multi-omics analysis and qPCR identified IL-6, STAT3, and IRF3 as potential effector molecules mediating GCRV infection. Deploying GCRV challenge at 18 °C and 28 °C as models of resistant and permissive infections and switched to the corresponding temperatures as temperature stress models, we illustrated that IL-6 and STAT3 expression, genome level of GCRV, and phosphorylation of STAT3 were temperature dependent and regulated by temperature stress. Further research revealed that activating IL-6/STAT3 axis enhanced GCRV replication and suppressed the expression of IFNs, whereas blocking the axis impaired viral replication. Mechanistically, grass carp STAT3 inhibited IRF3 nuclear translocation via interacting with it, thus down-regulating IFNs expression, restraining transcriptional activation of the IFN promoter, and facilitating GCRV replication. Overall, our work sheds light on an immune evasion mechanism whereby GCRV facilitates viral replication by hijacking IL-6/STAT3 axis to down-regulate IFNs expression, thus providing a valuable reference for targeted prevention and therapy of GCRV.

The ups and downs of STAT3 function: too much, too little and human immune dysregulation.

The STAT3 story has almost 30 years of evolving history. First identified in 1994 as a pro-inflammatory transcription factor, Signal Transducer and Activator of Transcription 3 (STAT3) has continued to be revealed as a quintessential pleiotropic signalling module spanning fields including infectious diseases, autoimmunity, vaccine responses, metabolism, and malignancy. In 2007, germline heterozygous dominant-negative loss-of-function variants in STAT3 were discovered as the most common cause for a triad of eczematoid dermatitis with recurrent skin and pulmonary infections, first described in 1966. This finding established that STAT3 plays a critical non-redundant role in immunity against some pathogens, as well as in the connective tissue, dental and musculoskeletal systems. Several years later, in 2014, heterozygous activating gain of function germline STAT3 variants were found to be causal for cases of early-onset multiorgan autoimmunity, thereby underpinning the notion that STAT3 function needed to be regulated to maintain immune homeostasis. As we and others continue to interrogate biochemical and cellular perturbations due to inborn errors in STAT3, we will review our current understanding of STAT3 function, mechanisms of disease pathogenesis, and future directions in this dynamic field.

Opposing regulation of the locus encoding IL-17 through direct, reciprocal actions of STAT3 and STAT5.

Interleukin 2 (IL-2), a cytokine linked to human autoimmune disease, limits IL-17 production. Here we found that deletion of the gene encoding the transcription factor STAT3 in T cells abrogated IL-17 production and attenuated autoimmunity associated with IL-2 deficiency. Whereas STAT3 induced IL-17 and the transcription factor RORγt and inhibited the transcription factor Foxp3, IL-2 inhibited IL-17 independently of Foxp3 and RORγt. STAT3 and STAT5 bound to multiple common sites across the locus encoding IL-17. The induction of STAT5 binding by IL-2 was associated with less binding of STAT3 at these sites and the inhibition of associated active epigenetic marks. 'Titration' of the relative activation of STAT3 and STAT5 modulated the specification of cells to the IL-17-producing helper T cell (T(H)17 cell) subset. Thus, the balance rather than the absolute magnitude of these signals determined the propensity of cells to make a key inflammatory cytokine.

IL-22 gets to the stem of colorectal cancer.

Cytokines can provide survival and proliferation signals to cancer cells, thus promoting tumor progression. In this issue of Immunity, Kryczek et al. (2014) reveal that interleukin-22 can also promote "stemness" in human colorectal cancer via transcription factor STAT3-mediated epigenetic regulation of stem cell genes.

IL-22(+)CD4(+) T cells promote colorectal cancer stemness via STAT3 transcription factor activation and induction of the methyltransferase DOT1L.

Little is known about how the immune system impacts human colorectal cancer invasiveness and stemness. Here we detected interleukin-22 (IL-22) in patient colorectal cancer tissues that was produced predominantly by CD4(+) T cells. In a mouse model, migration of these cells into the colon cancer microenvironment required the chemokine receptor CCR6 and its ligand CCL20. IL-22 acted on cancer cells to promote activation of the transcription factor STAT3 and expression of the histone 3 lysine 79 (H3K79) methytransferase DOT1L. The DOT1L complex induced the core stem cell genes NANOG, SOX2, and Pou5F1, resulting in increased cancer stemness and tumorigenic potential. Furthermore, high DOT1L expression and H3K79me2 in colorectal cancer tissues was a predictor of poor patient survival. Thus, IL-22(+) cells promote colon cancer stemness via regulation of stemness genes that negatively affects patient outcome. Efforts to target this network might be a strategy in treating colorectal cancer patients.

Serum- and Glucocorticoid-Inducible Kinase 1 Promotes Alternative Macrophage Polarization and Restrains Inflammation through FoxO1 and STAT3 Signaling.

Expression and activity of serum- and glucocorticoid-inducible kinase 1 (SGK1) are associated with many metabolic and inflammatory diseases. In this study, we report that SGK1 promotes alternative macrophage polarization and restrains inflammation in the infectious milieu of the gingiva. Inhibition of SGK1 expression or activity enhances characteristics of classically activated (M1) macrophages by directly activating the transcription of genes encoding iNOS, IL-12P40, TNF-α, and IL-6 and repressing IL-10 at message and protein levels. Moreover, SGK1 inhibition robustly reduces the expression of alternatively activated (M2) macrophage molecular markers, including arginase-1, Ym-1, Fizz1, and Mgl-1. These results were confirmed by multiple gain- and loss-of-function approaches, including small interfering RNA, a plasmid encoding SGK1, and LysM-Cre-mediated sgk1 gene knockout. Further mechanistic analysis showed that SGK1 deficiency decreases STAT3 but increases FoxO1 expression in macrophages under M2 or M1 macrophage-priming conditions, respectively. Combined with decreased FoxO1 phosphorylation and the subsequent suppressed cytoplasmic translocation observed, SGK1 deficiency robustly enhances FoxO1 activity and drives macrophage to preferential M1 phenotypes. Furthermore, FoxO1 inhibition abrogates M1 phenotypes, and STAT3 overexpression results in a significant increase of M2 phenotypes, indicating that both FoxO1 and STAT3 are involved in SGK1-mediated macrophage polarization. Additionally, SGK1 differentially regulates the expression of M1 and M2 molecular markers, including CD68 and F4/F80 and CD163 and CD206, respectively, and protects against Porphyromonas gingivalis-induced alveolar bone loss in a mouse model. Taken together, these results have demonstrated that SGK1 is critical for macrophage polarization and periodontal bone loss, and for the first time, to our knowledge, we elucidated a bifurcated signaling circuit by which SGK1 promotes alternative, while suppressing inflammatory, macrophage polarization.

ADAP Y571 Phosphorylation Is Required to Prime STAT3 for Activation in TLR4-Stimulated Macrophages.

Adhesion and degranulation-promoting adapter protein (ADAP), originally identified as an essential adaptor molecule in TCR signaling and T cell adhesion, has emerged as a critical regulator in innate immune cells such as macrophages; however, its role in macrophage polarization and inflammatory responses remains unknown. In this study, we show that ADAP plays an essential role in TLR4-mediated mouse macrophage polarization via modulation of STAT3 activity. Macrophages from ADAP-deficient mice exhibit enhanced M1 polarization, expression of proinflammatory cytokines and capacity in inducing Th1 responses, but decreased levels of anti-inflammatory cytokines in response to TLR4 activation by LPS. Furthermore, overexpression of ADAP enhances, whereas loss of ADAP reduces, the LPS-mediated phosphorylation and activity of STAT3, suggesting ADAP acts as a coactivator of STAT3 activity and function. Furthermore, the coactivator function of ADAP mostly depends on the tyrosine phosphorylation at Y571 in the motif YDSL induced by LPS. Mutation of Y571 to F severely impairs the stimulating effect of ADAP on STAT3 activity and the ability of ADAP to inhibit M1-like polarization in TLR4-activated mouse macrophages. Moreover, ADAP interacts with STAT3, and loss of ADAP renders mouse macrophages less sensitive to IL-6 stimulation for STAT3 phosphorylation. Collectively, our findings revealed an additional layer of regulation of TLR4-mediated mouse macrophage plasticity whereby ADAP phosphorylation on Y571 is required to prime STAT3 for activation in TLR4-stimulated mouse macrophages.

Role of IL-6-IL-27 Complex in Host Antiviral Immune Response.

The IL family of cytokines participates in immune response and regulation. We previously found that soluble IL-6 receptor plays an important role in the host antiviral response. In this study, we detected the IL-6-IL-27 complex in serum and throat swab samples from patients infected with influenza A virus. A plasmid expressing the IL-6-IL-27 complex was constructed to explore its biological function. The results indicated that the IL-6-IL-27 complex has a stronger antiviral effect than the individual subunits of IL-6, IL-27A, and EBV-induced gene 3. Furthermore, the activity of the IL-6-IL-27 complex is mainly mediated by the IL-27A subunit and the IL-27 receptor α. The IL-6-IL-27 complex can positively regulate virus-triggered expression of IFN and IFN-stimulated genes by interacting with adaptor protein mitochondrial antiviral signaling protein, potentiating the ubiquitination of TNF receptor-associated factors 3 and 6 and NF-κB nuclear translocation. The secreted IL-6-IL-27 complex can induce the phosphorylation of STAT1 and STAT3 and shows antiviral activity. Our results demonstrate a previously unrecognized mechanism by which IL-6, IL-27A, and EBV-induced gene 3 form a large complex both intracellularly and extracellularly, and this complex acts in the host antiviral response.

Thrombospondin-1 induced programmed death-ligand 1-mediated immunosuppression by activating the STAT3 pathway in osteosarcoma.

Thrombospondin-1 (TSP1) is generally assumed to suppress the growth of osteosarcoma through inhibiting angiogenesis; however, it is unclear whether TSP1 could affect the antitumor immunity against osteosarcoma. We aimed to explore the immune-related tumor-promoting effects of TSP1 and decipher its underlying mechanism. First, we identified that TSP1 regulated programmed death-ligand 1 (PD-L1) expression, which was related to the CD8+ T cells anergy in osteosarcoma cells. The exact role of PD-L1 in the immunosuppressive effect of TSP1 was then further confirmed by the addition of the PD-L1 neutralizing Ab. With the addition of PD-L1 neutralizing Abs during cocultivation, the inhibition of CD8+ T cells was abolished to a certain extent. Further mechanistic investigations showed that TSP1-induced PD-L1 upregulation was achieved by activation of the signal transducer and activator of transcription 3 (STAT3) pathway. In vivo experiments also indicated that TSP1 overexpression could promote the growth of primary lesions, whereas TSP1 knockdown effectively inhibits the growth of the primary lesion as well as lung metastasis by restoring the antitumor immunity. Thrombospondin-1 knockdown combined with PD-L1 neutralizing Ab achieved a more pronounced antitumor effect. Taken together, our study showed that TSP1 upregulates PD-L1 by activating the STAT3 pathway and, therefore, impairs the antitumor immunity against osteosarcoma.