CMTM4 inhibits gastric tumorigenesis and metastasis.

Background: CKLF-like MARVEL transmembrane domain-containing 4 (CMTM4) is involved in immune regulation and tumor progression; however, its role in gastric cancer (GC) remains unclear. This study explored the role and mechanism of CMTM4 in GC. Methods: Immunohistochemistry was used to analyze CMTM4 expression in human gastric biopsied cells from patients with GC (N=23) or chronic superficial gastritis (N=23). To investigate the function of CMTM4 in GC cells, the gene CMTM4 was knocked down and overexpressed in human gastric adenocarcinoma cell line AGS. The gene CMTM4 was overexpressed in AGS cells and human gastric cell line SGC7901. Cell Counting Kit 8 (CCK-8) and cell clonogenic assays were used to analyze the proliferation of the GC cells. Flow cytometry was used to analyze the effects of CMTM4 on apoptosis and the cell cycle. Wound healing and transwell assays were used to analyze the migration and invasion of the gastric cells, respectively. The mechanism of CMTM4 in GC cells was explored using the tandem mass tags (TMTs) proteome and verified by western blot analysis. Results: CMTM4 expression was more downregulated in the human GC tissues than the gastritis tissues. CMTM4 overexpression significantly inhibited the proliferation, migration, and invasion of the GC cells, whereas CMTM4 knockdown enhanced gastric cell proliferation (P>0.05), migration (P>0.05), and invasion (P>0.05). Flow cytometry showed that CMTM4 promoted apoptosis and resulted in G1/S arrest in the GC cells. In addition, the proteome and western blot results showed that STAT1 was significantly upregulated, and the STAT1 signaling pathways were enriched in the GC cells overexpressing CMTM4. Conclusions: Our results suggest that CMTM4 plays a tumor-suppressive role in GC and may affect the growth, migration, and invasion of GC cells through the STAT1 signaling pathway. CMTM4 might have potential value as a prognosis marker and potential therapeutic target for GC therapy.

Recapitulating primary immunodeficiencies with expanded potential stem cells: Proof of concept with STAT1 gain of function.

BACKGROUND: Inborn errors of immunity (IEI) often lack specific disease models and personalized management. Signal transducer and activator of transcription (STAT)-1 gain of function (GoF) is such example of an IEI with diverse clinical phenotype with unclear pathomechanisms and unpredictable response to therapy. Limitations in obtaining fresh samples for functional testing and research further highlights the need for patient-specific ex vivo platforms. OBJECTIVE: Using STAT1-GoF as an example IEI, we investigated the potential of patient-derived expanded potential stem cells (EPSC) as an ex vivo platform for disease modeling and personalized treatment. METHODS: We generated EPSC derived from individual STAT1-GoF patients. STAT1 mutations were confirmed with Sanger sequencing. Functional testing including STAT1 phosphorylation/dephosphorylation and gene expression with or without Janus activating kinase inhibitors were performed. Functional tests were repeated on EPSC lines with GoF mutations repaired by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) editing. RESULTS: EPSC were successfully reprogrammed from STAT1-GoF patients and expressed the same pluripotent makers as controls, with distinct morphologic differences. Patient-derived EPSC recapitulated the functional abnormalities of index STAT1-GoF patients with STAT1 hyperphosphorylation and increased expression of STAT1 and its downstream genes (IRF1, APOL6, and OAS1) after IFN-γ stimulation. Addition of ruxolitinib and baricitinib inhibited STAT1 hyperactivation in STAT1-GoF EPSC in a dose-dependent manner, which was not observed with tofacitinib. Corrected STAT1 phosphorylation and downstream gene expression were observed among repaired STAT1-GoF EPSC cell lines. CONCLUSION: This proof-of-concept study demonstrates the potential of our patient-derived EPSC platform to model STAT1-GoF. We propose this platform when researching, recapitulating, and repairing other IEI in the future.

Corrigendum: Transcriptional inhibition of STAT1 functions in the nucleus alleviates Th1 and Th17 cell-mediated inflammatory diseases.

[This corrects the article DOI: 10.3389/fimmu.2022.1054472.].

A Case of STAT1 Mutations in Chronic Mucocutaneous Candidiasis Diagnosed in an Adult.

A 30-year-old man presented with oral candidiasis and a history of lung abscess. He experienced recurring oral and skin candidiasis in childhood but spent long periods without any infections. Therefore, immunodeficiency was suspected. T and B lymphocyte and natural killer cell counts as well as immunoglobulin levels were normal. Human immunodeficiency virus test results were negative. Therefore, we suspected chronic mucocutaneous candidiasis (CMC). The signal transducer and activator of transcription (STAT) mutation, the leading cause of CMC, was detected by exome sequencing. Most cases of STAT-1 mutations are diagnosed in childhood, but a few are diagnosed in adulthood because Candida infections may not be severe.

MST4 kinase regulates immune thrombocytopenia by phosphorylating STAT1-mediated M1 polarization of macrophages.

Primary immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disorder in which macrophages play a critical role. Mammalian sterile-20-like kinase 4 (MST4), a member of the germinal-center kinase STE20 family, has been demonstrated to be a regulator of inflammation. Whether MST4 participates in the macrophage-dependent inflammation of ITP remains elusive. The expression and function of MST4 in macrophages of ITP patients and THP-1 cells, and of a macrophage-specific Mst4-/- (Mst4ΔM/ΔM) ITP mouse model were determined. Macrophage phagocytic assays, RNA sequencing (RNA-seq) analysis, immunofluorescence analysis, coimmunoprecipitation (co-IP), mass spectrometry (MS), bioinformatics analysis, and phosphoproteomics analysis were performed to reveal the underlying mechanisms. The expression levels of the MST4 gene were elevated in the expanded M1-like macrophages of ITP patients, and this elevated expression of MST4 was restored to basal levels in patients with remission after high-dose dexamethasone treatment. The expression of the MST4 gene was significantly elevated in THP-1-derived M1 macrophages. Silencing of MST4 decreased the expression of M1 macrophage markers and cytokines, and impaired phagocytosis, which could be increased by overexpression of MST4. In a passive ITP mouse model, macrophage-specific depletion of Mst4 reduced the numbers of M1 macrophages in the spleen and peritoneal lavage fluid, attenuated the expression of M1 cytokines, and promoted the predominance of FcγRIIb in splenic macrophages, which resulted in amelioration of thrombocytopenia. Downregulation of MST4 directly inhibited STAT1 phosphorylation, which is essential for M1 polarization of macrophages. Our study elucidates a critical role for MST4 kinase in the pathology of ITP and identifies MST4 kinase as a potential therapeutic target for refractory ITP.

Cancer cell-derived extracellular vesicles drive pre-metastatic niche formation of lymph node via IFNGR1/JAK1/STAT1-activated-PD-L1 expression on FRCs in head and neck cancer.

OBJECTIVE: The aim of this study is to evaluate the role of FRCs regulated by cancer cell-derived extracellular vesicles (CEVs) played in pre-metastatic niche (PMN) formation of lymph node (LN). MATERIALS AND METHODS: The FRCs in sixty fresh cervical LNs from 20 patients were evaluated by flow cytometric analysis. Cells in LN with or without metastasis were analyzed by single-cell RNA sequencing (scRNA-seq). CEVs were isolated from the culture supernatant of primarily cultured cancer cells and cocultured with FRCs. Mass Spectrometry was used to identify LN metastasis related protein in CEVs. The activation of IFNGR1/JAK1/STAT1-activated-PD-L1 pathway in FRCs was detected by western blotting. FRCs were co-cultured with CD8+ T lymphocytes to confirm the cytotoxicity assay of FRCs. RESULTS: The proportion of fibroblastic reticular cells (FRCs) was significantly higher in micro-metastatic LN in head and neck squamous cell carcinoma patients (HNSCC, p < 0.05) and scRNA-seq analysis further showed a high focus of extracellular vesicles-related pathway on FRCs in LN with metastasis (p < 0.05). Interferon gamma receptor 1 (IFNGR1) in CEVs can be engulfed by FRCs and promote PD-L1 expression on FRCs via JAK1-STAT1 pathway, resulting in an increased CD8+ T cell exhaustion. CONCLUSION: IFNGR1, originated from cancer cell-derived extracellular vesicles, promote PD-L1 expression on FRCs and subsequent CD8+ T cell exhaustion via JAK1-STAT1 activation, which facilitate pre-metastatic niche formation and tumor metastasis in sentinel lymph node in HNSCC.

Chicken-derived MERTK protein inhibits Newcastle disease virus replication by increasing STAT1 phosphorylation in DF-1 cells.

The receptor tyrosine kinases TYRO3, AXL, and MERTK (TAM) are transmembrane proteins associated with the regulation of the innate immune response. In this study, the role of the chicken-derived MERTK protein (chMertk) in the regulation of the type I interferon (IFN) signaling pathway and its antiviral effect were investigated in vitro. Newcastle disease (ND) caused by the Newcastle disease virus (NDV) is able to widely spread in chickens and give rise to massive losses in the poultry industry around the world. We found that the overexpression of the exogenous chMertk upregulated the STAT1 phosphorylation and the expression of IFN-stimulated gene IFITM3 and significantly reduced the NDV titer (p < 0.05). A mutation assay showed that three tyrosine residues (Y739, Y743, and Y744) in chMertk promoted STAT1 phosphorylation and inhibited NDV replication. However, the chicken-derived E3 ubiquitin ligase CBL significantly negatively regulated chMertk expression, thus attenuating STAT1 phosphorylation. chMertk function was restored by the ubiquitin-proteasome inhibitor MG132, demonstrating that chMertk was controlled by Casitas B-lineage proto-oncogene (CBL) ubiquitination and degradation. Together, these results suggested that chMertk participated in regulating the immune responses to NDV infection, and that CBL significantly downregulated the expression of chMertk through its ubiquitination and degradation, to maintain cellular homeostasis. Overall, our study provided new insights into the role of chMertk in regulating the innate immune response and its anti-NDV activity.

Chinese Pedigree of Chronic Mucocutaneous Candidiasis Due to STAT1 Gain-of-Function Mutation: A Case Study and Literature Review.

OBJECTIVE: To further elucidate the clinical, immunological and genetic features of chronic mucocutaneous candidiasis (CMC) due to STAT1 GOF mutation in the Chinese population. METHODS: Clinical data for a proband were collected, and pedigree analyses were performed. Whole-exome sequencing and targeted Sanger sequencing were conducted to explore genetic factors of a Chinese pedigree involving inherited CMC. RESULTS: An autosomal dominant CMC pedigree was identified, and both the proband and his father had mucocutaneous Candida infections without involvement of other systems. A rare mutation (c.T1175C) in STAT1 was detected in this CMC pedigree. Multiple sequence alignment revealed that the amino acid position of this mutation (p.M392T) is evolutionarily conserved in vertebrate species. Serum IFN-α was elevated in patients harbouring the mutation. A total of 10 publications reporting 26 CMC patients with STAT1 GOF mutations were retrieved by literature review, and the most common mutation found in previously reported Chinese patients is T385M in the DNA-binding domain. CONCLUSIONS: STAT1 GOF mutation at c.T1175C (p.M392T) may lead to mucocutaneous Candida infections and an increase in serum IFN-α. T385M in the DNA-binding domain is the most common STAT1 GOF mutation found in the Chinese population.

Single-cell RNA sequencing combined with whole exome sequencing reveals the landscape of the immune pathogenic response to chronic mucocutaneous candidiasis with STAT1 GOF mutation.

Chronic mucocutaneous candidiasis (CMC) is characterized by recurrent or persistent infections with Candida of the skin, nails, and mucous membranes (e.g., mouth, esophagus, and vagina). Compared with that of other infectious diseases, the immune pathogenic mechanism of CMC is still poorly understood. We identified a signal transducer and activator of transcription 1 gain-of-function (c.Y289C) mutation in a CMC patient. Single-cell transcriptional profiling on peripheral blood mononuclear cells from this patient revealed decreases in immature B cells and monocytes. Further analysis revealed several differentially expressed genes related to immune regulation, including RGS1, TNFAIP3, S100A8/A9, and CTSS. In our review of the literature on signal transducer and activator of transcription 1 gain-of-function (c.Y289C) mutations, we identified seven cases in total. The median age of onset for CMC (n=4, data lacking for three cases) was 10.5 years (range: birth to 11 years), with an average onset age of 8 years. There were no reports linking tumors to the c.Y289C mutation, and the incidence of pre-existing clinical disease in patients with the c.Y289C mutation was similar to previous data.

Unraveling the Role of Guanylate-Binding Proteins (GBPs) in Breast Cancer: A Comprehensive Literature Review and New Data on Prognosis in Breast Cancer Subtypes.

At least one member of the Guanylate-Binding Protein (GBP) family of large interferon-induced GTPases has been classified as both a marker of good prognosis and as a potential drug target to treat breast cancers. However, the activity of individual GBPs appears to not just be tumor cell type-specific but dependent on the growth factor and/or cytokine environment in which the tumor cells reside. To clarify what we do and do not know about GBPs in breast cancer, the current literature on GBP-1, GBP-2, and GBP-5 in breast cancer has been assembled. In addition, we have analyzed the role of each of these GBPs in predicting recurrence-free survival (RFS), overall survival (OS), and distance metastasis-free survival (DMFS) as single gene products in different subtypes of breast cancers. When a large cohort of breast cancers of all types and stages were examined, GBP-1 correlated with poor RFS. However, it was the only GBP to do so. When smaller cohorts of breast cancer subtypes grouped into ER+, ER+/HER2-, and HER2+ tumors were analyzed, none of the GBPs influenced RFS, OS, or DMSF as single agents. The exception is GBP-5, which correlated with improved RFS in HER2+ breast cancers. All three GBPs individually predicted improved RFS, OS, and DMSF in ER- breast cancers, regardless of the PR or HER2 status, and TNBCs.

Chronic Mucocutaneous Candidiasis due to Signal Transducer and Activator of Transcription 1 (STAT 1) Mutation in an Indian Patient - A Case Report.

Chronic mucocutaneous candidiasis (CMC) is a primary immunodeficiency due to defect in various genes leading to an increase in susceptibility to skin and mucosal infection. Mutation in signal transducer and activator of transcription 1 (STAT 1) gene being the most common cause of CMC can lead to increased risk of infections, multisystem abnormalities, and malignancy. We describe a 27 year old Indian woman with clinical features of CMC including esophageal stenosis, gangrene of the finger, endocrinological and immunological abnormalities and STAT1 mutation (p.Leu407Val). She was treated with antifungals which led to symptomatic improvement.

Transcriptional inhibition of STAT1 functions in the nucleus alleviates Th1 and Th17 cell-mediated inflammatory diseases.

T helper 1 cells (Th1 cells) and T helper 17 cells (Th17 cells) play pivotal roles in the pathogenesis of various autoimmune diseases, including psoriasis and inflammatory bowel disease (IBD). Signal transducer and activator of transcription 1 (STAT1) regulates the Th1 and Th17 cell lineage commitment at an early stage and maintains their immunological functions in vitro and in vivo. The previous strategies to block STAT1 functions to treat autoimmune diseases inhibit Th1 cell activity but simultaneously cause hyper-activation of Th17 cells. Herein, to modulate the functions of pathogenic Th1 and Th17 cells without genetic modification in normal physiological conditions, we generated the nucleus-deliverable form of the transcription modulation domain of STAT1 (ndSTAT1-TMD), which can be transduced into the nucleus of the target cells in a dose- and time-dependent manner without affecting the cell viability and T cell activation signaling events. ndSTAT1-TMD significantly blocked the differentiation of naïve CD4+ T cells into Th1 or Th17 cells via competitive inhibition of endogenous STAT1-mediated transcription, which did not influence Th2 and Treg cell differentiation. When the gene expression profile of Th1 or Th17 cells after ndSTAT1-TMD treatment was analyzed by mRNA sequencing, the expression of the genes involved in the differentiation capacity and the immunological functions of Th1 or Th17 cells were substantially reduced. The therapeutic potential of ndSTAT1-TMD was tested in the animal model of psoriasis and colitis, whose pathogenesis is mainly contributed by Th1 or/and Th17 cells. The symptoms and progression of psoriasis and colitis were significantly alleviated by ndSTAT1-TMD treatment, comparable to anti-IL-17A antibody treatment. In conclusion, our study demonstrates that ndSTAT1-TMD can be a new therapeutic reagent for Th1/17 cell-mediated autoimmune diseases by modulating the functions of pathogenic Th1 and Th17 cells together.

Precision Medicine in the Treatment of Primary Immune Deficiency Patients With Disorders of Immune Dysregulation.

There are now more than 450 described monogenic germline mutations for inborn errors of immunity that result in the loss of expression, loss of function (LOF), or gain in function (GOF) of the encoded protein. Molecular characterization of these inborn errors of immunity has not only allowed us to characterize on a genetic basis these immune deficiency disorders but has provided a better understanding of the immunobiology of these inborn errors of immunity. More recently, these advances have allowed us to apply targeted therapy or precision medicine in their treatment. Of particular interest related to this review are those inborn errors of immunity that result in gain-of-function (GOF) genetic abnormalities. Many of these inborn errors of immunity fall into a new category referred to as diseases of immune dysregulation in which many of the patients not only exhibit an increased susceptibility to infection but also have a clinical phenotype associated with autoimmune processes and lymphoproliferative disease.

Luteolin inhibits respiratory syncytial virus replication by regulating the MiR-155/SOCS1/STAT1 signaling pathway.

BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infection in infants, children, immunocompromised adults, and elderly individuals. Currently, there are few therapeutic options available to prevent RSV infection. The present study aimed to investigate the effects of luteolin on RSV replication and the related mechanisms. MATERIAL AND METHODS: We pretreated cells and mice with luteolin before infection with RSV, the virus titer, expressions of RSV-F, interferon (IFN)-stimulated genes (ISGs), and production of IFN-α and IFN-ß were determined by plaque assay, RT-qPCR, and ELISA, respectively. The activation of Janus kinase (JAK)-signal transducer and activator of transcription 1 (STAT1) signaling pathway was detected by Western blotting and luciferase assay. Proteins which negatively regulate STAT1 were determined by Western blotting. Then cells were transfected with suppressor of cytokine signaling 1 (SOCS1) plasmid and virus replication and ISGs expression were determined. Luciferase reporter assay and Western blotting were performed to detect the relationship between SOCS1 and miR-155. RESULTS: Luteolin inhibited RSV replication, as shown by the decreased viral titer and RSV-F mRNA expression both in vitro and in vivo. The antiviral activity of luteolin was attributed to the enhanced phosphorylation of STAT1, resulting in the increased production of ISGs. Further study showed that SOCS1 was downregulated by luteolin and SOCS1 is a direct target of microRNA-155 (miR-155). Inhibition of miR-155 rescued luteolin-mediated SOCS1 downregulation, whereas upregulation of miR-155 enhanced the inhibitory effect of luteolin. CONCLUSION: Luteolin inhibits RSV replication by regulating the miR-155/SOCS1/STAT1 signaling pathway.

The pathogenic T387A missense mutation in the gene encoding signal transducer and activator of transcription 1 exhibits a differential gene expression profile.

Heterozygous gain-of-function (GOF) mutations in the interferon-driven transcription factor STAT1 (signal transducer and activator of transcription 1) cause chronic mucocutaneous candidiasis (CMC). In this study, we characterized the molecular basis of a CMC-associated missense mutation by introducing a threonine-to-alanine exchange in the STAT1 DNA-binding domain at position 387. This substitution had previously been described in a CMC patient with suppurative eyelid infection and cutaneous abscesses, which are both unusual symptoms in this immunodeficiency. The STAT1-T387A mutant generated was compared to the wild-type protein and, in addition, to the missense mutant in the neighbouring position 386. Our results showed that the T387A mutant displayed distinct properties different from the wild-type molecule, namely elevated levels of tyrosine phosphorylation in conjunction with increased DNA-binding activity, hyperactive transcriptional regulation, and prolonged nuclear accumulation. The elevated tyrosine phosphorylation of the T387A mutant did not result in an increased mRNA production above the level of the wild-type molecule for all transcripts tested, indicating that the transcriptional activity of this mutant is largely gene-dependent. Nonetheless, these data demonstrate that the pathogenic T387A mutation associated with an atypical CMC symptomatology is biochemically similar to other well-characterized GOF mutants, while the H386A mutant was indistinguishable from the wild-type molecule. Our findings are in line with the assumption that the phenotype of this dominant STAT1 GOF mutation probably results from a disturbed shift in the equilibrium between the parallel and antiparallel dimer conformation, which is required for physiological gene activation.

STAT1-avtiviated LINC00961 regulates myocardial infarction by the PI3K/AKT/GSK3ß signaling pathway.

Myocardial infarction (MI) remains a severe cardiac disease because of its high incidence and mortality worldwide. A growing body of recent investigations have confirmed that LINC00961 acts as a tumor suppressor in diverse malignancies. However, the biological significance of LINC00961 and its molecular mechanism in MI are still elusive. Hypoxia is the leading cause of MI and induces myocardial injury. In this study, we found the upregulated expression of LINC00961 in cardiomyocytes H9c2 after hypoxia treatment. Knockdown of LINC00961 ameliorated hypoxia-induced cell injury by facilitating cell viability while repressing cell apoptosis. The significant increase of signal transducer and activator of transcription 1 (STAT1) expression and phosphorylation levels was observed in hypoxia-induced cells and proved to exacerbate hypoxia injury. In addition, STAT1 transcriptionally activated LINC00961 by binding to LINC00961 promoter. Furthermore, our results validated that suppressing LINC00961 contributed to the remarkable diminution in the phosphorylation levels of phosphoinositide 3-kinases (PI3K), AKT, and glycogen synthase kinase-3ß (GSK3ß). Inhibition of PI3K/AKT signaling or GSK3ß pathway rescued the effects of LINC00961 knockdown on the hypoxia-induced injury of cardiomyocytes. Namely, we concluded that STAT1-avtiviated LINC00961 accelerated MI via the PI3K/AKT/GSK3ß pathway, which may provide clues for the treatment of patients with MI.

Dichotomal functions of phosphorylated and unphosphorylated STAT1 in hepatocellular carcinoma.

Interferons (IFNs) with antiviral and immune-stimulatory functions have been widely used in prevention and treatment of hepatocellular carcinoma (HCC). Signal transducer and activator of transcription 1 (STAT1) is a key element of the IFN signaling, and the function of STAT1 is critically determined by its phosphorylation state. This study aims to understand the functions of phosphorylated (p-) and unphosphorylated (u-) STAT1 in HCC. We found that u-STAT1 is significantly elevated in patient HCC tumor tissues and predominantly expressed in cytoplasm; while p-STAT1 is absent. Loss of u-STAT1 potently arrested cell cycle and inhibited cell growth in HCC cells. Induction of p-STAT1 by IFN-α treatment effectively triggers the expression of interferon-stimulated genes (ISGs), but has moderate effect on HCC cell growth. Interestingly, both u-STAT1 and p-STAT1 are induced by IFN-α, through with distinct time-dependent process. Furthermore, the ISG induction patterns mediated by p-STAT1 and u-STAT1 are also distinct. Importantly, artificial blocking of the induction of u-STAT1, but not p-STAT1, sensitizes HCC cells to treatment of IFNs. Therefore, p-STAT1 and u-STAT1 exert dichotomal functions and coordinately regulate the responsiveness to IFN treatment in HCC. KEY MESSAGES: STAT1 is upregulated and predominantly presented as u-STAT1 in HCC, while p-STAT1 is absent. U-STAT1 sustains but p-STAT1 inhibits HCC growth. The dynamic change of phosphorylation state of STAT1 control the responsiveness to IFN treatment.

Inhibition of STAT1 sensitizes radioresistant nasopharyngeal carcinoma cell line CNE-2R to radiotherapy.

Radioresistance remains a major obstacle for clinicians in the treatment of nasopharyngeal carcinoma (NPC). Others and we have reported that signal transducer and activator of transcription 1 (STAT1) may be as an important gene for resistance to radiation. However, the relationship between STAT1 and radioresistance is still elusive. In this study, by constitutive silencing STAT1 in human radioresistant nasopharyngeal carcinoma CNE-2R cell line, we showed that inhibition of STAT1 enhanced radiosensitivity of CNE-2R. Furthermore, knockdown of STAT1 led to growth suppression and apoptosis promotion in vitro and in vivo. Moreover, cells with low STAT1 expression increased G2/M phase and decreased S phase at 2Gy. These result revealed that knockdown of stat1 expression could sensitizes the CNE-2R to radiotherapy, But the exact mechanism needs to be further clarified.

The C-terminal tail of polycystin-1 regulates complement factor B expression by signal transducer and activator of transcription 1.

Inhibition of the overactivated alternative complement pathway in autosomal dominant polycystic kidney disease (ADPKD) retards disease progression in animal models; however, it remains unknown how complement factor B (CFB) is upregulated in ADPKD. Here, we showed that the overexpression of CFB in cystic kidneys is associated with increased JAK2/STAT1 activity and enhanced expression of the polycystin-1 C-terminal tail (PC1-CTT). Overexpression or blockage of STAT1 increased or decreased CFB expression and CFB promoter activity. Moreover, overexpression of PC1-CTT induced JAK2/STAT1 activation and CFB upregulation in renal tubular epithelial cells. Furthermore, PC1-CTT overexpression increased human CFB promoter activity, whereas dominant negative STAT1 plasmids or mutation of putative STAT1 responsive elements decreased PC1-CTT-induced CFB promoter activity. The effect of CFB on macrophage differentiation was tested on a mouse macrophage cell line. Bioactive CFB dose dependently promoted macrophage M2 phenotype conversion. In addition, conditioned media from renal epithelial cells promoted macrophage M2 phenotype conversion which was blocked by STAT1 inhibition in a dose-dependent manner. Conditioned media from PC1-CTT-transfected renal epithelial cells further promoted macrophage M2 phenotype conversion, which was suppressed by fludarabine or a CFB antibody. In addition, we show that NF-κB acts downstream of PC1-CTT and may partly mediate PC1-CTT-induced CFB expression. In conclusion, our study reveals possible mechanisms of CFB upregulation in ADPKD and a novel role of PC1-CTT in ADPKD-associated inflammation. Furthermore, our study suggests that targeting STAT1 may be a new strategy to prevent inflammation in the kidney of patients with ADPKD.

IFNα signaling through PKC-θ is essential for antitumor NK cell function.

We have previously shown that the development of a major histocompatibility complex class I (MHC-I)-deficient tumor was favored in protein kinase C-θ knockout (PKC-θ-/-) mice compared to that occurring in wild-type mice. This phenomenon was associated with scarce recruitment of natural killer (NK) cells to the tumor site, as well as impaired NK cell activation and reduced cytotoxicity ex vivo. Poly-inosinic:cytidylic acid (poly I:C) treatment activated PKC-θ in NK cells depending on the presence of a soluble factor produced by a different splenocyte subset. In the present work, we sought to analyze whether interleukin-15 (IL-15) and/or interferon-α (IFNα) mediate PKC-θ-dependent antitumor NK cell function. We found that IL-15 improves NK cell viability, granzyme B expression, degranulation capacity and interferon-γ (IFNγ) secretion independently of PKC-θ. In contrast, we found that IFNα improves the degranulation capability of NK cells against target cancer cells in a PKC-θ-dependent fashion both ex vivo and in vivo. Furthermore, IFNα induces PKC-θ auto-phosphorylation in NK cells, in a signal transduction pathway involving both phosphatidylinositol-3-kinase (PI3K) and phospholipase-C (PLC) activation. PKC-θ dependence was further implicated in IFNα-induced transcriptional upregulation of chemokine (C-X-C motif) ligand 10 (CXCL10), a signal transducer and activator of transcription-1 (STAT-1)-dependent target of IFNα. The absence of PKC-θ did not affect IFNα-induced STAT-1 Tyr701 phosphorylation but affected the increase in STAT-1 phosphorylation on Ser727, attenuating CXCL10 secretion. This connection between IFNα and PKC-θ in NK cells may be exploited in NK cell-based tumor immunotherapy.