STAT1-dependent Manner Research Articles

Fibroblast growth factor pathway promotes glycolysis by activating LDHA and suppressing LDHB in a STAT1-dependent manner in prostate cancer

BackgroundThe initiation of fibroblast growth factor 1 (FGF1) expression coincident with the decrease of FGF2 expression is a well-documented event in prostate cancer (PCa) progression. Lactate dehydrogenase A (LDHA) and LDHB are essential metabolic products that promote tumor growth. However, the relationship between FGF1/FGF2 and LDHA/B-mediated glycolysis in PCa progression is not reported. Thus, we aimed to explore whether FGF1/2 could regulate LDHA and LDHB to promote glycolysis and explored the involved signaling pathway in PCa progression.MethodsIn vitro studies used RT‒qPCR, Western blot, CCK-8 assays, and flow cytometry to analyze gene and protein expression, cell viability, apoptosis, and cell cycle in PCa cell lines. Glycolysis was assessed by measuring glucose consumption, lactate production, and extracellular acidification rate (ECAR). For in vivo studies, a xenograft mouse model of PCa was established and treated with an FGF pathway inhibitor, and tumor growth was monitored.ResultsFGF1, FGF2, and LDHA were expressed at high levels in PCa cells, while LDHB expression was low. FGF1/2 positively modulated LDHA and negatively modulated LDHB in PCa cells. The depletion of FGF1, FGF2, or LDHA reduced cell proliferation, induced cell cycle arrest, and inhibited glycolysis. LDHB overexpression showed similar inhibitory effect on PCa cells. Mechanistically, we found that FGF1/2 positively regulated STAT1 and STAT1 transcriptionally activated LDHA expression while suppressed LDHB expression. Furthermore, the treatment of an FGF pathway inhibitor suppressed PCa tumor growth in mice.ConclusionThe FGF pathway facilitates glycolysis by activating LDHA and suppressing LDHB in a STAT1-dependent manner in PCa.

FAK suppresses antigen processing and presentation to promote immune evasion in pancreatic cancer

ObjectiveImmunotherapy for the treatment of pancreatic ductal adenocarcinoma (PDAC) has shown limited efficacy. Poor CD8 T-cell infiltration, low neoantigen load and a highly immunosuppressive tumour microenvironment contribute to this lack...

Clonal MDS/AML cells with enhanced TWIST1 expression reprogram the differentiation of bone marrow MSCs

Bone marrow-derived mesenchymal stem cells (BMMSCs) derived from myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients often show a shift in the balance between osteoblastogenesis and adipogenesis. It was suggested that BMMSCs can potentially undergo reprogramming or educational processes. However, the results of reprogrammed differentiation have been inconclusive. In this study, clinical samples, co-culture models and mouse models were employed to explore the association of MDS/AML clonal cells and BMMSCs differentiation. We found that clonal MDS/AML cells promoted adipogenic differentiation and inhibited osteogenic differentiation of BMMSCs, which in turn promoted MDS expansion. Mass spectrometry and cytokine array were used to identify the molecules to drive the BMMSCs differentiation in MDS/AML. Mechanistically, highly expressed transcription factor TWIST1 in clonal MDS/AML cells induces MDS/AML cells to secrete more IFN-γ, which can induce oxidative stress through STAT1-dependent manner, ultimately causing enhanced adipogenic differentiation and inhibited osteogenic differentiation in BMMSCs. Overall, our findings suggest that targeting the driving oncogenes in malignant clonal cells, such as TWIST1, may offer new therapeutic strategies by remodeling the surrounding bone marrow microenvironment in the treatment of MDS/AML and other hematopoietic malignancies.

Tertiary Lymphoid Tissues Are Microenvironments with Intensive Interactions between Immune Cells and Proinflammatory Parenchymal Cells in Aged Kidneys.

Ectopic lymphoid structures called tertiary lymphoid tissues (TLTs) develop in several kidney diseases and are associated with poor renal prognosis. However, the mechanisms underlying TLT expansion and their effect on renal regeneration remain unclear. The authors report that single-nucleus RNA sequencing and validation experiments demonstrate that TLTs potentially amplify inflammation in aged injured kidneys. Lymphocytes within TLTs promote proinflammatory phenotypes of the surrounding proximal tubules and fibroblasts within the TLTs via proinflammatory cytokine production. These proinflammatory parenchymal cells then interact with immune cells by chemokine or cytokine production. Such cell-cell interactions potentially increase inflammation, expand TLTs, and exacerbate kidney injury. These findings help illuminate renal TLT pathology and suggest potential therapeutic targets. Ectopic lymphoid structures called tertiary lymphoid tissues (TLTs) develop in several kidney diseases and are associated with poor renal prognosis. However, the mechanisms that expand TLTs and underlie exacerbation of kidney injury remain unclear. We performed single-nucleus RNA sequencing (snRNA-seq) on aged mouse kidneys with TLTs after ischemia-reperfusion injury. The results were validated using immunostaining, in situ hybridization of murine and human kidneys, and in vitro experiments. Using snRNA-seq, we identified proinflammatory and profibrotic Vcam1+ injured proximal tubules (PTs) with NF κ B and IFN-inducible transcription factor activation. VCAM1 + PTs were preferentially localized around TLTs and drove inflammation and fibrosis via the production of multiple chemokines or cytokines. Lymphocytes within TLTs expressed Tnf and Ifng at high levels, which synergistically upregulated VCAM1 and chemokine expression in cultured PT cells. In addition, snRNA-seq also identified proinflammatory and profibrotic fibroblasts, which resided within and outside TLTs, respectively. Proinflammatory fibroblasts exhibited STAT1 activation and various chemokine or cytokine production, including CXCL9/CXCL10 and B cell-activating factor, contributing to lymphocyte recruitment and survival. IFN γ upregulated the expression of these molecules in cultured fibroblasts in a STAT1-dependent manner, indicating potential bidirectional interactions between IFN γ -producing CXCR3 + T cells and proinflammatory fibroblasts within TLTs. The cellular and molecular components described in this study were confirmed in human kidneys with TLTs. These findings suggest that TLTs potentially amplify inflammation by providing a microenvironment that allows intense interactions between renal parenchymal and immune cells. These interactions may serve as novel therapeutic targets in kidney diseases involving TLT formation.

IFN-γ-STAT1-mediated NK2R expression is involved in the induction of antitumor effector CD8+ T cells in vivo.

The induction of antitumor effector T cells in the tumor microenvironment is a crucial event for cancer immunotherapy. Neurokinin receptor 2 (NK2R), a G protein-coupled receptor for neurokinin A (NKA), regulates diverse physiological functions. However, the precise role of NKA-NK2R signaling in antitumor immunity is unclear. Here, we found that an IFN-γ-STAT1 cascade augmented NK2R expression in CD8+ T cells, and NK2R-mediated NKA signaling was involved in inducing antitumor effector T cells in vivo. The administration of a synthetic analog of double-stranded RNA, polyinosinic-polycytidylic acid (poly I:C), into a liver cancer mouse model induced type I and type II IFNs and significantly suppressed the tumorigenesis of Hepa1-6 liver cancer cells in a STAT1-dependent manner. The reduction in tumor growth was diminished by the depletion of CD8+ T cells. IFN-γ stimulation significantly induced NK2R and tachykinin precursor 1 (encodes NKA) gene expression in CD8+ T cells. NKA stimulation combined with anti-CD3 monoclonal antibody (mAb) treatment significantly augmented IFN-γ and granzyme B production by CD8+ T cells compared with the anti-CD3 mAb alone in vitro. ERK1/2 phosphorylation and IκBα degradation in activated CD8+ T cells were suppressed under NK2R deficiency. Finally, we confirmed that tumor growth was significantly increased in NK2R-deficient mice compared with that in wild-type mice, and the antitumor effects of poly I:C were abolished by NK2R absence. These findings suggest that IFN-γ-STAT1-mediated NK2R expression is involved in the induction of antitumor effector T cells in the tumor microenvironment, which contributes to the suppression of cancer cell tumorigenesis in vivo. In this study, we revealed that IFN-γ-STAT1-mediated NK2R expression is involved in the induction of antitumor effector CD8+ T cells in the tumor microenvironment, which contributes to suppressing the tumorigenesis of liver cancer cells in vivo.

Abstract C019: FAK suppresses antigen processing and presentation to promote immune evasion in pancreatic cancer

Abstract Immunotherapy for the treatment of Pancreatic Ductal Adenocarcinoma (PDAC) has shown limited efficacy. Poor CD8 T-cell infiltration, low neoantigen load and a highly immunosuppressive tumor microenvironment are key factors that contribute to this lack of response. Here, we identify a novel role for Focal Adhesion Kinase (FAK) in regulating type-II interferon signalling in human and murine PDAC cells and tumors, leading to suppression of antigen processing and presentation pathways critical for T-cell tumor recognition. We find that FAK loss promotes expression of the immunoproteasome and MHC-I, resulting in a significant increase in the diversity of antigens presented by FAK-/- PDAC cells. We show that regulation of the immunoproteasome subunit Psmb8 (β5i) is a critical determinant of this response, optimising the physicochemical properties of the peptide repertoire for high-affinity binding to MHC-I. Expression of the immunoproteasome and MHC-I can be further amplified in a STAT1-dependent manner via co-depletion of FAK and STAT3, leading to extensive CD8 T-cell infiltration into PDAC tumors and further restraint of tumor growth. Regulation of antigen processing and presentation by FAK preferentially occurs in human and murine PDAC cells and tumors of the classical/progenitor molecular subtype, further suggesting that precision medicine approaches based on molecular subtyping should be integrated into current and future clinical testing of FAK targeted therapies for the treatment of pancreatic cancer. Citation Format: Marta Canel, Aleksandra Slawinska, David W. Lonergan, Ashwin A. Kallor, Rosie Upstill-Goddard, Catherine Davidson, Alex von Kriegsheim, Andrew V. Biankin, Adam Bryron, Javier Alfaro, Alan Serrels. FAK suppresses antigen processing and presentation to promote immune evasion in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C019.

C-Myc plays a key role in IFN-γ-induced persistence of Chlamydia trachomatis.

Chlamydia trachomatis (Ctr) can persist over extended times within their host cell and thereby establish chronic infections. One of the major inducers of chlamydial persistence is interferon-gamma (IFN-γ) released by immune cells as a mechanism of immune defence. IFN-γ activates the catabolic depletion of L-tryptophan (Trp) via indoleamine-2,3-dioxygenase (IDO), resulting in persistent Ctr. Here, we show that IFN-γ induces the downregulation of c-Myc, the key regulator of host cell metabolism, in a STAT1-dependent manner. Expression of c-Myc rescued Ctr from IFN-γ-induced persistence in cell lines and human fallopian tube organoids. Trp concentrations control c-Myc levels most likely via the PI3K-GSK3β axis. Unbiased metabolic analysis revealed that Ctr infection reprograms the host cell tricarboxylic acid (TCA) cycle to support pyrimidine biosynthesis. Addition of TCA cycle intermediates or pyrimidine/purine nucleosides to infected cells rescued Ctr from IFN-γ-induced persistence. Thus, our results challenge the longstanding hypothesis of Trp depletion through IDO as the major mechanism of IFN-γ-induced metabolic immune defence and significantly extends the understanding of the role of IFN-γ as a broad modulator of host cell metabolism.

Long Non-coding RNA TPT1-AS1 Suppresses APC Transcription in a STAT1-Dependent Manner to Increase the Stemness of Colorectal Cancer Stem Cells.

Cancer stem cells (CSCs) are the major culprits leading to a new level of complexity and the consequential therapy resistance and disease recurrence in colorectal cancer (CRC). This study focuses on the effect of long non-coding RNA (lncRNA) TPT1-AS1 and its associated molecules on the stemness maintenance of CRC stem cells. TPT1-AS1 was identified as a significantly upregulated gene in CRC using the GSE146587 dataset. Stem cells from CRC HCT116 and CACO2 cells were isolated. TPT1-AS1 was significantly highly expressed in the CSCs compared to non-stem cells. Downregulation of TPT1-AS1 reduced the stemness of the CRC stem cells. TPT1-AS1 recruited STAT1 to the promoter region of APC to suppress APC transcription. Further upregulation of STAT1 or downregulation of APC blocked the role of TPT1-AS1 silencing and restored the malignant behaviors of CSC stem cells. APC inactivated the Wnt/β-catenin pathway. Overexpression of STAT1 restored the levels of cyclin D1 and β-catenin in cells suppressed by TPT1-AS1 silencing. In summary, this work demonstrates that TPT1-AS1 recruits STAT1 to suppress APC transcription and increase the stemness of colorectal CSCs via Wnt/β-catenin activation.

Regulation of macrophage IFNγ-stimulated gene expression by the transcriptional coregulator CITED1.

Macrophages serve as a first line of defense against microbial pathogens. Exposure to interferon-γ (IFNγ) increases interferon-stimulated gene (ISG) expression in these cells, resulting in enhanced antimicrobial and proinflammatory activity. Although this response must be sufficiently vigorous to ensure the successful clearance of pathogens, it must also be carefully regulated to prevent tissue damage. This is controlled in part by CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 2 (CITED2), a transcriptional coregulator that limits ISG expression by inhibiting STAT1 and IRF1. Here, we show that the closely related Cited1 is an ISG, which is expressed in a STAT1-dependent manner, and that IFNγ stimulates the nuclear accumulation of CITED1 protein. In contrast to CITED2, ectopic CITED1 enhanced the expression of a subset of ISGs, including Ccl2, Ifit3b, Isg15 and Oas2. This effect was reversed in a Cited1-null cell line produced by CRISPR-based genomic editing. Collectively, these data show that CITED1 maintains proinflammatory gene expression during periods of prolonged IFNγ exposure and suggest that there is an antagonistic relationship between CITED proteins in the regulation of macrophage inflammatory function. This article has an associated First Person interview with the first author of the paper.

Immune-Mediated Reprogramming of Intestinal Stem Cells Drives STAT1-Dependent Myc Expression and Epithelial Regeneration in GI-Gvhd

Crypt apoptosis and regeneration are characteristic findings in GI-GVHD. Intestinal stem cells (ISCs) are critical for maintaining the intestinal epithelium, but their frequencies are reduced in experimental GVHD, and the mechanisms driving crypt regeneration in this context are poorly understood.To better understand the impact of GVHD on individual epithelial components, we performed single cell RNA sequencing (scRNAseq) of purified small intestine crypts from B6 mice during homeostasis and five days after B6 into B6 syngeneic (syn) or B10.Br into B6 allogeneic (allo) BMT. Sequenced cells were first partitioned into distinct clusters using PhenoGraph, and the cluster identities were annotated based on their gene expression profiles. Unsupervised clustering indicated multiple subpopulations within the various recognized crypt components (Fig 1A). While secretory lineages clustered similarly across experimental conditions, there was highly distinct clustering among ISC populations and striking dissimilarity between allo and syn ISCs. Quantification by computing the subpopulation's phenotypic distance, a composite score integrating both the number and amplitude of differentially expressed genes, indicated that ISCs demonstrated the greatest transcriptional change in response to GVHD among all crypt lineages (Fig 1B). Gene Set Enrichment Analysis (GSEA) highlighted activation of the interferon-γ (IFNγ) pathway in allo ISCs, and differential gene expression indicated that STAT1 was their most highly upregulated transcription factor. We then performed MHC-mismatched allo-BMT into STAT1-floxed x villin-Cre recipients. Consistent with a role in IFNγ-related toxicity, STAT1-deficient recipients initially demonstrated reduced GVHD pathology, as well as a reduction in proliferating Ki67 + cells (Fig 2A). However, the pathology reduction in STAT1-deficient recipients was transient, while reduction in crypt proliferation persisted. STAT1-deficient recipients ultimately demonstrated increased mortality after allo-BMT, indicating a complex response to epithelial STAT1 signaling in GVHD.While IFNγ can induce epithelial apoptosis and kill organoid cultures, organoid exposure to IFNγ augmented size even at IFNγ concentrations that did not impair viability in a STAT1-dependent manner, and co-culture with allo T cells augmented organoid size (Fig 2B, C). Moreover, cell cycle analysis showed augmentation of cell cycle in ISCs after IFNγ treatment in association with upregulation of cyclin D1 (Fig 2D), and human organoids also showed increased size in response to IFNγ treatment, further suggesting that this growth promotion was not simply a secondary response to tissue injury.In addition to the IFNγ signaling, GSEA of allo ISCs indicated activation of the Myc pathway. scRNAseq data showed specific upregulation of Myc in allo ISCs (Fig 3A). Myc expression within individual ISCs indicated that greater STAT1 expression and IFNγ signaling directly correlated with Myc expression in the same ISCs, providing a potential direct link between T-cell-derived IFNγ and ISC-dependent regeneration (Fig 3B). Additionally, intestinal organoid qPCR showed that Myc expression was upregulated after IFNγ treatment, and scRNAseq of IFNγ-treated organoids indicated this Myc upregulation was restricted to the ISC/progenitor compartment. Although Myc is downstream of Wnt signaling, which is critical for ISC maintenance, expression of the representative Wnt target gene Axin2 was downregulated after IFNγ treatment, and Irf1, a representative IFNγ/STAT1 target gene, was upregulated, suggesting that IFNγ/STAT1 signaling could replace Wnt/β-catenin as a driver of ISC Myc expression. We next examined Myc function and found that treatment with the Myc inhibitor 10058-F4 suppressed IFNγ-dependent organoid growth (Fig 3C). Finally, immunofluorescent staining showed Myc protein expression in intestinal crypts after allo-BMT in a STAT1-dependent manner (Fig 3D), and Myc inhibitor treatment in vivo suppressed crypt epithelial proliferation in mice with GVHD.In summary, we found that epithelial STAT1 contributes to crypt regeneration in GVHD by transmitting T-cell-derived JAK/STAT cytokine signaling to activate Myc expression in ISCs. Clinical use of JAK inhibitors in GVHD may inhibit this regenerative response, necessitating concurrent interventions aimed at restoring it. [Display omitted] DisclosuresHanash: Evive Biotech: Ended employment in the past 24 months.

Inhibition of Dot1L Alleviates Fulminant Hepatitis Through Myeloid-Derived Suppressor Cells

Background & AimsFulminant hepatitis (FH) is a clinical syndrome characterized by sudden and severe liver dysfunction. Dot1L, a histone methyltransferase, is implicated in various physiologic and pathologic processes, including transcription regulation and leukemia. However, the role of Dot1L in regulating inflammatory responses during FH remains elusive.MethodsPropionibacterium acnes (P. acnes)-primed, lipopolysaccharides (LPS)-induced FH was established in C57BL/6 mice and was treated with the Dot1L inhibitor EPZ-5676. Myeloid derived suppressor cells (MDSCs) were depleted by anti-Gr-1 antibody to evaluate their therapeutic roles in Dot1L treatment of FH. Moreover, peripheral blood of patients suffered with FH and healthy controls was collected to determine the expression profile of Dot1L-SOCS1-iNOS axis in their MDSCs.ResultsHere we identified that EPZ-5676, pharmacological inhibitor of Dot1L, attenuated the liver injury of mice subjected to FH. Dot1L inhibition led to decreased T helper 1 cell response and expansion of regulatory T cells (Tregs) during FH. Interestingly, Dot1L inhibition didn’t directly target T cells, but dramatically enhanced the immunosuppressive function of MDSCs. Mechanistically, Dot1L inhibition epigenetically suppressed SOCS1 expression, thus inducing inducible nitric oxide synthase (iNOS) expression in a STAT1-dependent manner. Moreover, in human samples, the levels of Dot1L and SOCS1 expression were upregulated in MDSCs, accompanied by decreased expression of iNOS in patients with FH, compared with healthy controls.ConclusionsAltogether, our findings established Dot1L as a critical regulator of MDSC immunosuppressive function for the first time, and highlighted the therapeutic potential of Dot1L inhibitor for FH treatment.

Counter Regulation of Spic by NF-κB and STAT Signaling Controls Inflammation and Iron Metabolism in Macrophages

SUMMARYActivated macrophages must carefully calibrate their inflammatory responses to balance efficient pathogen control with inflammation-mediated tissue damage, but the molecular underpinnings of this “balancing act” remain unclear. Using genetically engineered mouse models and primary macrophage cultures, we show that Toll-like receptor (TLR) signaling induces the expression of the transcription factor Spic selectively in patrolling monocytes and tissue macrophages by a nuclear factor κB (NF-κB)-dependent mechanism. Functionally, Spic downregulates pro-inflammatory cytokines and promotes iron efflux by regulating ferroportin expression in activated macrophages. Notably, interferon-gamma blocks Spic expression in a STAT1-dependent manner. High levels of interferon-gamma are indicative of ongoing infection, and in its absence, activated macrophages appear to engage a “default” Spic-dependent anti-inflammatory pathway. We also provide evidence for the engagement of this pathway in sterile inflammation. Taken together, our findings uncover a pathway wherein counter-regulation of Spic by NF-κB and STATs attune inflammatory responses and iron metabolism in macrophages.

Activation of the STAT1 Pathway Accelerates Periodontitis in Nos3-/- Mice

Early studies on the etiology and pathogenesis of hypertension have shown that it has a considerable association with inflammation and the immune response as well as periodontitis. Clinical studies have also shown that hypertension can promote the periodontal tissue destruction caused by periodontitis. However, the underlying mechanisms remain unclear. This study aimed to explore the possible mechanisms of how hypertension aggravates periodontitis. Treatment with or without the signal transducer and activator of transcription 1 (STAT1) inhibitor fludarabine was performed in an endothelial nitric oxide synthase gene knockout-related (Nos3-/-) mouse model with the hypertension phenotype of periodontitis induced by bacteria. Micro–computed tomography, immunohistochemistry, Western blot, quantitative reverse transcription polymerase chain reaction, immunofluorescence, and ELISA were performed. We demonstrated that Nos3-/--related hypertension increases bone resorption and periodontal destruction in periodontitis lesion areas, which can be inhibited by the STAT1 inhibitor. Experimental data also showed that Nos3-/- significantly increased macrophage infiltration and proinflammatory cytokine expression in the periodontitis lesion area, which is dependent on the angiotensin II–induced STAT1 pathway. Inhibition of STAT1 in vivo can decrease the expression of proinflammatory cytokines and macrophage infiltration. Furthermore, data in this study showed that Nos3-/--related hypertension further downregulated the STAT3 anti-inflammatory function and its downstream chemokine expression in a STAT1-dependent manner. By applying RAW 264.7 and L929 cell lines and monocytes isolated from Nos3-/- mice, we confirmed that activation of the STAT1 pathway inhibits STAT3 and its downstream pathway and promotes inflammatory cytokine expression in vitro. Collectively, our current study demonstrated that STAT1 plays an indispensable role in the Nos3-/--related hypertension with aggravation of periodontitis, suggesting that STAT1 may be a key target for the treatment of periodontitis with hypertension.

Combating viral contaminants in CHO cells by engineering innate immunity

Viral contamination in biopharmaceutical manufacturing can lead to shortages in the supply of critical therapeutics. To facilitate the protection of bioprocesses, we explored the basis for the susceptibility of CHO cells to RNA virus infection. Upon infection with certain ssRNA and dsRNA viruses, CHO cells fail to generate a significant interferon (IFN) response. Nonetheless, the downstream machinery for generating IFN responses and its antiviral activity is intact in these cells: treatment of cells with exogenously-added type I IFN or poly I:C prior to infection limited the cytopathic effect from Vesicular stomatitis virus (VSV), Encephalomyocarditis virus (EMCV), and Reovirus-3 virus (Reo-3) in a STAT1-dependent manner. To harness the intrinsic antiviral mechanism, we used RNA-Seq to identify two upstream repressors of STAT1: Gfi1 and Trim24. By knocking out these genes, the engineered CHO cells exhibited activation of cellular immune responses and increased resistance to the RNA viruses tested. Thus, omics-guided engineering of mammalian cell culture can be deployed to increase safety in biotherapeutic protein production among many other biomedical applications.

IL-27 promotes the expansion of self-renewing CD8+ T cells in persistent viral infection.

Chronic infection and cancer are associated with suppressed T cell responses in the presence of cognate antigen. Recent work identified memory-like CXCR5+ TCF1+ CD8+ T cells that sustain T cell responses during persistent infection and proliferate upon anti-PD1 treatment. Approaches to expand these cells are sought. We show that blockade of interferon type 1 (IFN-I) receptor leads to CXCR5+ CD8+ T cell expansion in an IL-27- and STAT1-dependent manner. IFNAR1 blockade promoted accelerated cell division and retention of TCF1 in virus-specific CD8+ T cells. We found that CD8+ T cell-intrinsic IL-27 signaling safeguards the ability of TCF1hi cells to maintain proliferation and avoid terminal differentiation or programmed cell death. Mechanistically, IL-27 endowed rapidly dividing cells with IRF1, a transcription factor that was required for sustained division in a cell-intrinsic manner. These findings reveal that IL-27 opposes IFN-I to uncouple effector differentiation from cell division and suggest that IL-27 signaling could be exploited to augment self-renewing T cells in chronic infections and cancer.

Receptor complex and signalling pathway of the two type II IFNs, IFN-γ and IFN-γrel in mandarin fish or the so-called Chinese perch Siniperca chuatsi

IFN-γ, as the sole member of mammalian type II IFN, is a multifunctional cytokine which exerts its effects through two distinct IFN-γ receptors, IFNGR1 and IFNGR2. However, in teleost fish, another IFN-γ homologous gene, namely IFN-γ related gene (IFN-γrel), has been identified. Although IFN-γ and IFN-γrel genes have been described in some fish species, many important aspects remain poorly understood in relation with their signalling and function. In the present study, IFN-γ and IFN-γrel, as well as their receptors, cytokine receptor family B (CRFB) 17, CRFB13, two of which are homologous to IFNGR1 in mammals, and CRFB6, homolomous to IFNGR2, have been characterized in mandarin fish, Siniperca chuatsi. It was revealed that the two type IFN members exhibit antiviral activity, and IFN-γ transduces downstream signalling through CRFB13 and CRFB6, while IFN-γrel interacts with CRFB17 to activate downstream signalling. Moreover, IFN-γ and IFN-γrel have been shown to exert antiviral biological activity in a STAT1-dependent manner. Intracellular domain analysis of CRFB17 and CRFB13 demonstrated that the Y386 tyrosine residue of CRFB13 is required for the activation of the IFN-γ-mediated biologic response, and the Y324 and Y370 residues in CRFB17 are required to activate IFN-γrel signalling.

Propagermanium, a CCR2 inhibitor, attenuates cerebral ischemia/reperfusion injury through inhibiting inflammatory response induced by microglia

CCR2 could recruit immune cells migrating into brain after ischemic stroke. It is unclear whether and why Propagermanium (PG, a CCR2 inhibitor) is able to protect against ischemic injury. Middle cerebral artery occlusion (MCAO) and reperfusion injury in C57BL/6 J male mice were performed in vivo to mimic ischemic stroke. Cultured BV2 microglia exposed to oxygen and glucose deprivation (OGD)/reoxygenation injury, LPS or IL-4 incubation were served in vitro. TTC staining, neurological score, brain water content, and MRI scan were performed to evaluate stroke outcome. Real time PCR, ELISA, and immunofluorescence were used to estimate inflammatory cytokines expression and releasing. Western blot was utilized to detect pSTAT1/STAT1 expression. Compared with MCAO mice, PG treatment significantly reduced infarction size and brain edema, improved neurological behavior at 72 h after MCAO. For inflammatory response, PG treatment inhibited inflammatory cytokines releasing, such as TNF-α, IFN-γ, IL-1β, IL-6, IL-12, IL-17, and IL-23. Further studies indicated that PG treatment downregulated mRNA expression of pro-inflammatory iNOS and CD86, and inhibited CD16 expressed in microglia. In vitro, PG incubation inhibited BV2 polarized to pro-inflammatory phenotype through STAT1 downregulation, while had no obvious effect on anti-inflammatory phenotype. Our observations suggest that CCR2 inhibitor PG downregulated pro-inflammatory microglia polarization for decreasing pro-inflammatory microglia phenotype marker, and thereafter inhibited inflammatory responses after MCAO in a STAT1-dependent manner.

Chronic virus infection compromises memory bystander T cell function in an IL-6/STAT1-dependent manner.

Chronic viral infections are widespread among humans, with ∼8-12 chronic viral infections per individual, and there is epidemiological proof that these impair heterologous immunity. We studied the impact of chronic LCMV infection on the phenotype and function of memory bystander CD8+ T cells. Active chronic LCMV infection had a profound effect on total numbers, phenotype, and function of memory bystander T cells in mice. The phenotypic changes included up-regulation of markers commonly associated with effector and exhausted cells and were induced by IL-6 in a STAT1-dependent manner in the context of chronic virus infection. Furthermore, bystander CD8 T cell functions were reduced with respect to their ability to produce inflammatory cytokines and to undergo secondary expansion upon cognate antigen challenge with major cell-extrinsic contributions responsible for the diminished memory potential of bystander CD8+ T cells. These findings open new perspectives for immunity and vaccination during chronic viral infections.

RIP1 Kinase Drives Macrophage-Mediated Adaptive Immune Tolerance in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDA) is characterized by immune tolerance and immunotherapeutic resistance. We discovered upregulation of receptor-interacting serine/threonine protein kinase 1 (RIP1) in tumor-associated macrophages (TAMs) in PDA. To study its role in oncogenic progression, we developed a selective small-molecule RIP1 inhibitor with high invivo exposure. Targeting RIP1 reprogrammed TAMs toward an MHCIIhiTNFα+IFNγ+ immunogenic phenotype in a STAT1-dependent manner. RIP1 inhibition in TAMs resulted in cytotoxic Tcell activation and T helper cell differentiation toward a mixed Th1/Th17 phenotype, leading to tumor immunity in mice and in organotypic models of human PDA. Targeting RIP1 synergized with PD1-and inducible co-stimulator-based immunotherapies. Tumor-promoting effects of RIP1 were independent of its co-association with RIP3. Collectively, our work describes RIP1 as a checkpoint kinase governing tumor immunity.

Metabolite-Sensing G Protein Coupled Receptor TGR5 Protects Host From Viral Infection Through Amplifying Type I Interferon Responses.

The metabolite-sensing G protein–coupled receptors (GPCRs) bind to various metabolites and transmit signals that are important for proper immune and metabolic functions. However, the roles of metabolite-sensing GPCRs in viral infection are not well characterized. Here, we identified metabolite-sensing GPCR TGR5 as an interferon (IFN)-stimulated gene (ISG) which had increased expression following viral infection or IFN-β stimulation in a STAT1-dependent manner. Most importantly, overexpression of TGR5 or treatment with the modified bile acid INT-777 broadly protected host cells from vesicular stomatitis virus (VSV), newcastle disease virus (NDV) and herpes simplex virus type 1 (HSV-1) infection. Furthermore, VSV and HSV-1 replication was increased significantly in Tgr5-deficient macrophages and the VSV distribution in liver, spleen and lungs was increased in Tgr5-deficient mice during VSV infection. Accordingly, Tgr5-deficient mice were much more susceptible to VSV infection than wild-type mice. Mechanistically, TGR5 facilitates type I interferon (IFN-I) production through the AKT/IRF3-signaling pathway, which is crucial in promoting antiviral innate immunity. Taken together, our data reveal a positive feedback loop regulating IRF3 signaling and suggest a potential therapeutic role for metabolite-sensing GPCRs in controlling viral diseases.