Extracellular vesicles from neural stem cells transfer IFN-γ via Ifngr1 to activate Stat1 signaling in target cells.

The idea that stem cell therapies work only via cell replacement is challenged by the observation of consistent intercellular molecule exchange between the graft and the host. Here we defined a mechanism of cellular signaling by which neural stem/precursor cells (NPCs) communicate with the microenvironment via extracellular vesicles (EVs), and we elucidated its molecular signature and function. We observed cytokine-regulated pathways that sort proteins and mRNAs into EVs. We described induction of interferon gamma (IFN-γ) pathway in NPCs exposed to proinflammatory cytokines that is mirrored in EVs. We showed that IFN-γ bound to EVs through Ifngr1 activates Stat1 in target cells. Finally, we demonstrated that endogenous Stat1 and Ifngr1 in target cells are indispensable to sustain the activation of Stat1 signaling by EV-associated IFN-γ/Ifngr1 complexes. Our study identifies a mechanism of cellular signaling regulated by EV-associated IFN-γ/Ifngr1 complexes, which grafted stem cells may use to communicate with the host immune system.

In cardiac fibroblasts, interferon-beta attenuates differentiation, collagen synthesis, and TGF-ß1-induced collagen gel contraction.

Cardiac fibroblasts (CF) play a key role in the homeostasis of the extracellular matrix in cardiac tissue and are newly recognized as inflammatory supporter cells. Besides, CF-to-Cardiac myofibroblast differentiation is commanded by TGF-b, through SMAD signaling pathways, and these last cells are strongly implicated in cardiac fibrosis. In the heart IFN-ß is produced by CF; however, the role of IFN-ß, STAT proteins, and STAT-homo or heterodimers in the regulation of CF function with or without a fibrotic environment is unknown. CF were isolated from hearts of adult rats, and by western blot analysis we studied STAT1, STAT2, and STAT3 phosphorylation and through specific siRNA against these proteins we analyzed their role in CF functions such as differentiation (α-SMA expression); and pro-collagen type-I synthesis and secretion expression levels; collagen gels contraction and CF migration. In cultured adult rats CF, IFN-ß increases phosphorylation of STAT1, STAT2, and STAT3. Both STAT1 and STAT2 were involved in decreasing α-SMA and CF migration induced by TGF-ß1. Also, IFN-ß through STAT1 regulated pro-collagen type-I protein expression levels, and collagen gels contraction induced by TGF-ß1. STAT3 was not involved in any effects of IFN-ß studied. In conclusion, IFN-ß through STAT1 and STAT2 shows antifibrotic effects on CF TGF-ß1-treated, whereas STAT3 did not participate in such effect.

IL-22Ra1 is induced during influenza infection by direct and indirect TLR3 induction of STAT1.

BACKGROUND: Influenza attacks the epithelium of the lung, causing cell death and disruption of the epithelial barrier leading to fluid buildup in the lung and impairment of gas exchange. Limited treatment options for severe influenza pneumonia prioritize the need for the discovery of effective therapies. IL-22 is a cytokine that promotes tissue integrity and has strong promise as a treatment option. While research has been focused on the cytokine itself, there is limited understanding of the regulation of the IL-22 receptor (IL-22Ra1) at the epithelial surface during infection. METHODS: IL-22Ra1 levels were measured by qRT-PCR, western blot and immunofluorescence following H1N1 influenza infection (A/PR/8/34 H1N1) or synthetic TLR3 mimetic, Poly (I:C). Regulation of the receptor was determined using STAT inhibitors (STAT1, STAT3 and PanSTAT inhibitors), TLR3 inhibition, and neutralization of interferon alpha receptor 2 (IFNAR2). Significance was determined by a p-value of greater than 0.05. Significance between two groups was measured using unpaired t-test and significance between more than two groups was measured using one-way ANOVA with Tukey Multiple Comparison Test. RESULTS: Here we show both in vivo and in vitro that IL-22Ra1 was induced as early as 24 h after influenza (H1N1 PR8) infection. This induction was triggered by toll-like receptor 3 (TLR3) as a TLR3 mimetic [Poly (I:C)] also induced IL-22Ra1 and inhibition of endosomal formation required for TLR3 function inhibited this process. This upregulation was dependent upon IFNß signaling through STAT1. Importantly, induction of IL-22Ra1 significantly increased IL-22 signaling as evidenced by pSTAT3 levels following IL-22 treatment. CONCLUSION: Collectively, these data suggest epithelial cells may optimize the beneficial effects of IL-22 through the induction of the IL-22 receptor during viral infection in the lung.

The influence of ustekinumab on expression of STAT1, STAT3, STAT4, SOCS2, and IL17 in patients with psoriasis and in a control.

The aim of this study was to assess changes in the expression of STAT1, STAT3, STAT4, SOCS2, and IL17 in psoriatic patients under ustekinumab treatment and in healthy volunteers. The study group consisted of 14 patients suffering from psoriasis vulgaris qualified for ustekinumab therapy (4 women, 10 men) The control group consisted of 14 healthy volunteers (7 women, 7 men), their whole blood was used as a material in this study. A quantitative reverse transcription polymerase chain assay was used to amplify analyzed genes. To indicate the differences in expression of selected genes in the test and control groups, the Kruskal-Wallis and the post hoc Dunn's test was carried out. After 40 weeks of observation of the effectiveness of ustekinumab in patients with psoriasis, the expression of STAT1, STAT3, STAT4, IL17, and SOCS2 was silenced. Statistic differences in expression were observed for STAT3 (40 vs. 0 weeks, p < .05; 0 week vs. C, p < .05) and SOCS2 (0 week vs. C, p < .05). Patients with psoriasis vulgaris have higher levels of STAT1, STAT3, STAT4, SOCS2, and IL17 expression compared to healthy individuals. On the other hand, the treatment of ustekinumab lasting 40 weeks caused a decrease in the transcriptional activity of the analyzed genes.

Molecular mechanism and structural basis of gain-of-function of STAT1 caused by pathogenic R274Q mutation.

Gain-of-function (GOF) mutations in the STAT1 gene are critical for the onset of chronic mucocutaneous candidiasis (CMC) disease. However, the molecular basis for the gain of STAT1 function remains largely unclear. Here, we investigated the structural features of STAT1 GOF residues to better understand the impact of these pathogenic mutations. We constructed STAT1 alanine mutants of the α3 helix residues of the coiled-coil domain, which are frequently found in CMC pathogenic mutations, and measured their transcriptional activities. Most of the identified GOF residues were located inside the coiled-coil domain stem structure or at the protein surface of the anti-parallel dimer interface. Unlike those, Arg-274 was adjacent to the DNA-binding domain. In addition, Arg-274 was found to functionally interact with Gln-441 in the DNA-binding domain. Because Gln-441 is located at the anti-parallel dimer contact site, Gln-441 reorientation by Arg-274 mutation probably impedes formation of the dimer. Further, the statistical analysis of RNA-seq data with STAT1-deficient epithelial cells and primary T cells from a CMC patient revealed that the R274Q mutation affected gene expression levels of 66 and 76 non-overlapping RefSeq genes, respectively. Because their transcription levels were only slightly modulated by wild-type STAT1, we concluded that the R274Q mutation increased transcriptional activity but did not change dramatically the repertoire of STAT1 targets. Hence, we provide a novel mechanism of STAT1 GOF triggered by a CMC pathogenic mutation.

Increased STAT1 Expression in High Grade Serous Ovarian Cancer Is Associated With a Better Outcome.

OBJECTIVE: Recently it has been demonstrated that constitutively activated signal transducer and activator of transcription 1 (STAT1) gene expression may act as a biomarker of ovarian cancer chemotherapy response. In this study, our objective was to validate the use of STAT1 immunohistochemistry as a prognostic biomarker for disease outcome using a cohort derived from Latin America. METHODS: We evaluated a cohort of Brazilian high-grade serous ovarian cancer, comprising 65 patients with outcome data covering more than 5 years to determine the prognostic and predictive value of STAT1 expression levels. High-grade serous ovarian cancer tumors were used to construct a tissue microarray. Exploratory analyses were conducted on clinical, histopathological, and STAT1 expression data that included descriptive statistics and Pearson correlative analyses. Survival curves for disease-free survival and overall survival were obtained by the Kaplan-Meier method, and the significance of homogeneity between the classes was assessed by log-rank statistics (Mantel-Cox). RESULTS: High expression of STAT1 in tumors was significantly associated with improved disease-free survival (P = 0.0256) and overall survival (P = 0.0193). Proportional hazards regression analysis showed STAT1 expression had an independent effect on both disease-free survival (P = 0.0358) and overall survival (P = 0.0469). CONCLUSIONS: These findings from a Brazilian cohort of patients with ovarian cancer reinforce the association of high STAT1 expression with better response to chemotherapy, providing additional validation of this protein as both a prognostic and predictive biomarker. Collectively, these results together with other recently published studies increase the feasibility of using the STAT1 pathway for the development of novel immunomodulator drugs that could enhance response to treatment.

The Inflammatory Transcription Factors NFκB, STAT1 and STAT3 Drive Age-Associated Transcriptional Changes in the Human Kidney.

Human kidney function declines with age, accompanied by stereotyped changes in gene expression and histopathology, but the mechanisms underlying these changes are largely unknown. To identify potential regulators of kidney aging, we compared age-associated transcriptional changes in the human kidney with genome-wide maps of transcription factor occupancy from ChIP-seq datasets in human cells. The strongest candidates were the inflammation-associated transcription factors NFκB, STAT1 and STAT3, the activities of which increase with age in epithelial compartments of the renal cortex. Stimulation of renal tubular epithelial cells with the inflammatory cytokines IL-6 (a STAT3 activator), IFNγ (a STAT1 activator), or TNFα (an NFκB activator) recapitulated age-associated gene expression changes. We show that common DNA variants in RELA and NFKB1, the two genes encoding subunits of the NFκB transcription factor, associate with kidney function and chronic kidney disease in gene association studies, providing the first evidence that genetic variation in NFκB contributes to renal aging phenotypes. Our results suggest that NFκB, STAT1 and STAT3 underlie transcriptional changes and chronic inflammation in the aging human kidney.

Interferon alpha receptors and STAT1 as therapy predictors of a sustained virological response in hepatitis C/B co-infection.

OBJECTIVE: : To determine the expression of interferon alpha receptors 1 and 2 along with signal transducer and activator of transcription-1 in peripheral blood mononuclear cells of both hepatitis C mono-infected and hepatitis C and B co-infected patients, and to assess whether these targeted genes predict sustained virological response to interferon therapy. METHODS: This cross-sectional study was carried out at the Army Medical College, Rawalpindi, Pakistan, from January 2012 to December 2015, and comprised hepatitis C mono-infected and hepatitis C and B co-infected patients. The patients were divided into groups 1 and 2. Group-1a and group-2a consisted of mono-infected and co-infected sustained responders, while group-1b and group-2b had mono-infected and co-infected non sustained responders. Peripheral blood mononuclear cells from healthy controls were also quantified for these subunits. Target gene expressions were studied by retro-transcription of respective messenger ribonuclieic acid extracted from the cells followed by polymerase chain reaction amplification. RESULTS: Of the 191 subjects, there were 20(10.5%) in group-1a, 35(18.3%) in group-2a, 65(34%) in group-1b and 51(26.7%) in group-2b. The remaining 20(10.5%) were controls. Overall, 106 (55.5%) were males and 85 (44.5%) were females. Interferon alpha receptor 1 expression in groups 1a and 2a was significantly higher compared to groups 1b (p=0.018) and 2b (p 0.031). Signal transducer and activator of transcription-1 protein expression showed no significant difference (p=0.062 and p=0.519). No difference in expression was measured between the two sets of groups with regard to interferon alpha receptor 2 expression (p=0.278 and p=0.590). CONCLUSIONS: Our results show that levels of IFNAR-1 mRNA expression may be a good predictor for IFN-related anti-viral activity in both HCV mono infected and HCV/HBV co-infected patients.

Targeting Jurkat T Lymphocyte Leukemia Cells by an Engineered Interferon-Alpha Hybrid Molecule.

BACKGROUND/AIMS: Adult T-cell leukemia/lymphoma (ATL) is a very aggressive T cell malignancy that carries a poor prognosis, primarily due to its resistance to chemotherapy and to life-threatening infectious complications. Interferon-alpha (IFNα) has been used in combination with the anti-retroviral drug zidovudine to treat patients with ATL. However, the efficacy of long-term therapy is significantly limited due to the systemic toxicity of IFNα. METHODS: We utilized phage display library screening to identify short peptides that specifically bind to Jurkat T lymphocyte leukemia cells. By fusing the Jurkat-binding peptide to the C-terminus of IFNα, we constructed an engineered chimeric IFNα molecule (IFNP) for the treatment of ATL. RESULTS: We found that IFNP exhibited significantly higher activity than wild type IFNα in inhibiting the growth of leukemia cells and inducing cell blockage at the G0/G1 phase. The synthetic IFNP molecule exerted its antitumor activity by upregulating the downstream genes involved in the STAT1 pathway and in apoptosis. Using a cell receptor binding assay, we showed that this Jurkat-binding peptide facilitated the binding affinity of IFNα to the cell surface type I IFN receptor. CONCLUSION: The isolated Jurkat-binding peptide significantly potentiates the therapeutic activity of IFNα in T lymphocyte leukemia cells. The engineered IFNP molecule may prove to a novel antitumor approach in the treatment of patients with ATL.

Age-Associated Failure To Adjust Type I IFN Receptor Signaling Thresholds after T Cell Activation.

With increasing age, naive CD4 T cells acquire intrinsic defects that compromise their ability to respond and differentiate. Type I IFNs, pervasive constituents of the environment in which adaptive immune responses occur, are known to regulate T cell differentiation and survival. Activated naive CD4 T cells from older individuals have reduced responses to type I IFN, a defect that develops during activation and that is not observed in quiescent naive CD4 T cells. Naive CD4 T cells from young adults upregulate the expression of STAT1 and STAT5 after activation, lowering their threshold to respond to type I IFN stimulation. The heightened STAT signaling is critical to maintain the expression of CD69 that regulates lymphocyte egress and the ability to produce IL-2 and to survive. Although activation of T cells from older adults also induces transcription of STAT1 and STAT5, failure to exclude SHP-1 from the signaling complex blunts their type I IFN response. In summary, our data show that type I IFN signaling thresholds in naive CD4 T cells after activation are dynamically regulated to respond to environmental cues for clonal expansion and memory cell differentiation. Naive CD4 T cells from older adults have a defect in this threshold calibration. Restoring their ability to respond to type I IFN emerges as a promising target to restore T cell responses and to improve the induction of T cell memory.

9p21 DNA variants associated with coronary artery disease impair interferon-γ signalling response.

Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in the 9p21 gene desert associated with coronary artery disease (CAD) and type 2 diabetes. Despite evidence for a role of the associated interval in neighbouring gene regulation, the biological underpinnings of these genetic associations with CAD or type 2 diabetes have not yet been explained. Here we identify 33 enhancers in 9p21; the interval is the second densest gene desert for predicted enhancers and six times denser than the whole genome (P < 6.55 × 10(-33)). The CAD risk alleles of SNPs rs10811656 and rs10757278 are located in one of these enhancers and disrupt a binding site for STAT1. Lymphoblastoid cell lines homozygous for the CAD risk haplotype show no binding of STAT1, and in lymphoblastoid cell lines homozygous for the CAD non-risk haplotype, binding of STAT1 inhibits CDKN2BAS (also known as CDKN2B-AS1) expression, which is reversed by short interfering RNA knockdown of STAT1. Using a new, open-ended approach to detect long-distance interactions, we find that in human vascular endothelial cells the enhancer interval containing the CAD locus physically interacts with the CDKN2A/B locus, the MTAP gene and an interval downstream of IFNA21. In human vascular endothelial cells, interferon-γ activation strongly affects the structure of the chromatin and the transcriptional regulation in the 9p21 locus, including STAT1-binding, long-range enhancer interactions and altered expression of neighbouring genes. Our findings establish a link between CAD genetic susceptibility and the response to inflammatory signalling in a vascular cell type and thus demonstrate the utility of genome-wide association study findings in directing studies to novel genomic loci and biological processes important for disease aetiology.

Identification and characterization of differentially expressed genes from human microglial cell samples infected with Japanese encephalitis virus.

BACKGROUND & OBJECTIVES: Limited studies have been reported on Japanese encephalitis (JE) with reference to microarray data analysis. The present study involved an in silico approach for identification and characterization of differentially expressed genes in human microglial cell (CHME3) samples, infected with P20778 strain of Japanese encephalitis virus (JEV). METHODS: Gene expression data (GSE57330) belonging to mRNA expression profile of CHME3 cells infected with JEV, was downloaded from the gene expression omnibus (GEO) database, processed and normalized by robust multichip averaging (RMA) method using affy packages of R. The Bayes method was used to correct multiple testing. The log fold change (logFC > 1) and p< 0.05 were used as cut-off to identify differentially expressed genes (DEGs). The newly identified hub genes were set at the centre for construction of protein-protein interaction network using search tool for the retrieval of interacting genes/proteins (STRING) database considering human genome as reference. Gene ontology and pathway enrichment analysis of the hub gene and its associated genes were performed using STRING and DAVID tool. RESULTS: Microarray data analysis revealed that STAT1 gene was down-regulated during JEV infection. STAT1 gene was found to interact with tyrosine protein kinase family members, and showed strong interaction with JAK1 and JAK2 genes. INTERPRETATION & CONCLUSION: The identified transcription factors and the binding sites in the promoter region of STAT1 gene might act as potential drug targets in near future.

Alleviation of spinal cord injury by Ginkgolide B via the inhibition of STAT1 expression.

Ginkgolide B has been known to inhibit cell apoptosis by modulating multiple cytokines and plays an important role in neuroprotection. Signal transducer and activator of transcription 1 (STAT1) has been studied in a spinal cord injury (SCI) model. However, the role of Ginkgolide B in SCI treatment remains unclear. This study investigated the potential mechanism of Ginkgolide B using an SCI rat model. SD rats were used to generate an SCI model followed by Ginkgolide B injection (4 mg/kg) for 14 days. Spinal cord tissue samples were examined using hematoxylin and eosin (H&E) staining. The expression of STAT1 was determined by western blot. Using a dyskinesia scale, intervention with Ginkgolide B significantly decreased the severity of SCI. H&E staining revealed less nuclear condensation and cell necrosis in SCI rats after treatment with Ginkgolide B. STAT1 expression was significantly increased in SCI model rats, but was lower after Ginkgolide B treatment. Therefore, Ginkgolide B can effectively inhibit STAT1 expression and alleviate SCI.

A distinct pre-existing inflammatory tumour microenvironment is associated with chemotherapy resistance in high-grade serous epithelial ovarian cancer.

BACKGROUND: Chemotherapy resistance is a major determinant of poor overall survival rates in high-grade serous ovarian cancer (HGSC). We have previously shown that gene expression alterations affecting the NF-κB pathway characterise chemotherapy resistance in HGSC, suggesting that the regulation of an immune response may be associated with this phenotype. METHODS: Given that intrinsic drug resistance pre-exists and is governed by both tumour and host factors, the current study was performed to examine the cross-talk between tumour inflammatory microenvironment and cancer cells, and their roles in mediating differential chemotherapy response in HGSC patients. Expression profiling of a panel of 184 inflammation-related genes was performed in 15 chemoresistant and 19 chemosensitive HGSC tumours using the NanoString nCounter platform. RESULTS: A total of 11 significantly differentially expressed genes were found to distinguish the two groups. As STAT1 was the most significantly differentially expressed gene (P=0.003), we validated the expression of STAT1 protein by immunohistochemistry using an independent cohort of 183 (52 resistant and 131 sensitive) HGSC cases on a primary tumour tissue microarray. Relative expression levels were subjected to Kaplan-Meier survival analysis and Cox proportional hazard regression models. CONCLUSIONS: This study confirms that higher STAT1 expression is significantly associated with increased progression-free survival and that this protein together with other mediators of tumour-host microenvironment can be applied as a novel response predictive biomarker in HGSC. Furthermore, an overall underactive immune microenvironment suggests that the pre-existing state of the tumour immune microenvironment could determine response to chemotherapy in HGSC.

Evidence for postinitiation regulation of mRNA biogenesis in tuberculosis.

Mycobacterium tuberculosis infection alters macrophage gene expression and macrophage response to IFN-γ, a critical host defense cytokine. However, regulation of these changes is poorly understood. We report discordance of changes in nascent transcript and total nuclear RNA abundance for the transcription factors STAT1 and IRF1, together with lack of effect on their RNA half-lives, in human THP-1 cells infected with M. tuberculosis and stimulated with IFN-γ. The results indicate that negative postinitiation regulation of mRNA biogenesis limits the expression of these factors, which mediate host defense against M. tuberculosis through the cellular response to IFN-γ. Consistent with the results for STAT1 and IRF1, transcriptome analysis reveals downregulation of postinitiation mRNA biogenesis processes and pathways by infection, with and without IFN-γ stimulation. Clinical relevance for regulation of postinitiation mRNA biogenesis is demonstrated by studies of donor samples showing that postinitiation mRNA biogenesis pathways are repressed in latent tuberculosis infection compared with cured disease and in active tuberculosis compared with ongoing treatment or with latent tuberculosis. For active disease and latent infection donors from two populations (London, U.K., and The Gambia), each analyzed using a different platform, pathway-related gene expression differences were highly correlated, demonstrating substantial specificity in the effect. Collectively, the molecular and bioinformatic analyses point toward downregulation of postinitiation mRNA biogenesis pathways as a means by which M. tuberculosis infection limits expression of immunologically essential transcription factors. Thus, negative regulation of postinitiation mRNA biogenesis can constrain the macrophage response to infection and overall host defense against tuberculosis.

Jak2/Stat1 pathway mediated tetrahydrobiopterin up-regulation contributes to nitric oxide overproduction in high-glucose cultured rat mesangial cells.

Nitric oxide (NO) is crucial for the progression of early diabetic nephropathy (DN). It is important to clarify the mechanism for the production of NO in mesangial cells (MCs). In this study, the amounts/activities of related factors such as reactive oxygen species (ROS), NO, 3 isoforms of nitric oxide synthase (NOS), tetrahydrobiopterin (BH4), GTP cyclohydrolase I (GTPCH I), Jak2, and Stat1 were determined using high-glucose cultured rat MCs. The results showed that the production of BH4 under oxidative stress was strongly stimulated by its rate-limiting enzyme GTP cyclohydrolase, which increased the expression and activity of inducible NOS to facilitate NO synthesis. Furthermore, the relative quantities of activated-Jak2 and activated-Stat1 were increased. Therefore, Jak2/Stat1 pathway mediated BH4 up-regulation can contribute to excessive NO in high-glucose cultured MCs. Our results will be helpful for screening new targets to improve the therapy for early DN.

[Search of (-)-Cytisine Derivatives as Potential Inhibitors of NF-κB and STAT1].

Design and synthesis ofnew derivatives of (-)-cytisine with a wide spectrum of pharmacological activity, represents the potential therapeutic interest for development of drug candidates for neurodegenerative disorders, inflammatory diseases, and treatment of nicotine addiction. We used HEK293 cell line transiently transfected with N F-κB and STATI luciferase reporter constructs to screen the (-)-cytisine derivatives for their potency to modulate basal and induced NF-κB and STAT1 activity. Currently, NF-κB, STAT1 and components of their signaling pathways are considered as attractive targets for pharmacological intervention, primarily in chronic inflammation, cancer, autoimmune, neurodegenerative and infectious diseases. The library of compounds included the derivatives of (-)-cytisine with amino-, amide-, thio- and carboxamide groups at 3, 5 and 12 position of the starting molecule, as well as some bimolecular derivatives. Our experimental data revealed compounds with moderate activating as well as inhibitory effects for basal NF-κB and STATI activity (IC50 or EC50 values are mainly in the micromolar range). The structure-activity relationship analysis demonstrated that the character of activity (activation or inhibition of NFκ-B and STAT1) is determined by the topology of the substituents at the (-)-cytisine molecule, whereas the nature of the substituents mainly contributes to severity of the effect (introduction of aromatic and adamantyl substituents, as well as thionyl or keto groups are of the principal importance). When evaluating the effect of (-)-cytisine derivatives on activity of NF-κB and STATI, induced by specific agents (TNFα and IFNγ, respectively) we observed that some compounds inhibited basal and stimulated activity of NF-κB and STAT1, another compounds showed the dual effect (an increase of basal- and a decrease of stimulated NF-κB activity) and several compounds increase both basal and induced activity of NF-κB and STAT1. Thus, obtained results suggest that one of the possible mechanisms of biological action of (-)-cytisine derivatives is their ability to influence the components of NF-κB and STAT1-dependent signaling pathways.

Regulation and activity of secretory leukoprotease inhibitor (SLPI) is altered in smokers.

A hallmark of cigarette smoking is a shift in the protease/antiprotease balance, in favor of protease activity. However, it has recently been shown that smokers have increased expression of a key antiprotease, secretory leukoprotease inhibitor (SLPI), yet the mechanisms involved in SLPI transcriptional regulation and functional activity of SLPI remain unclear. We examined SLPI mRNA and protein secretion in differentiated nasal epithelial cells (NECs) and nasal lavage fluid (NLF) from nonsmokers and smokers and demonstrated that SLPI expression is increased in NECs and NLF from smokers. Transcriptional regulation of SLPI expression was confirmed using SLPI promoter reporter assays followed by chromatin immunoprecipitation. The role of STAT1 in regulating SLPI expression was further elucidated using WT and stat1(-/-) mice. Our data demonstrate that STAT1 regulates SLPI transcription in epithelial cells and slpi protein in the lungs of mice. Additionally, we reveal that NECs from smokers have increased STAT1 mRNA/protein expression. Finally, we demonstrate that SLPI contained in the nasal mucosa of smokers is proteolytically cleaved but retains functional activity against neutrophil elastase. These results demonstrate that smoking enhances expression of SLPI in NECs in vitro and in vivo, and that this response is regulated by STAT1. In addition, despite posttranslational cleavage of SLPI, antiprotease activity against neutrophil elastase is enhanced in smokers. Together, our findings show that SLPI regulation and activity is altered in the nasal mucosa of smokers, which could have broad implications in the context of respiratory inflammation and infection.

Interferon and interferon-inducible gene activation in patients with type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease that is thought to be triggered by environmental factors in genetically susceptible individuals. Enteroviruses have been mentioned as the most probable induction component of the disease. Nevertheless, the literature is controversial regarding the association of T1D with viral infection and first-line antiviral defence components, for example type I interferons (IFNs). Our aim was to test the hypothesis that an abnormality in IFN-stimulated gene patterns may cause a failure in immunological tolerance and, thereby, initiate T1D as an autoimmune disorder. We studied material from 64 T1D and 36 control subjects, divided into two age groups: <10 years and ≥10 years old. Using a relative gene expression method, we observed a lower expression of interferon-induced helicase 1 (IFIH1) and other type I IFN-induced genes in the blood cells of T1D subjects, especially subjects under 10 years old, in spite of their higher IFN levels as measured by the pSTAT1-inducing capacity of their sera. Likewise, freshly purified CpG-stimulated cells from T1D patients showed significantly lower upregulation of IFN-induced genes, that is IFIH1 and CXCL10, compared to cells from the control group. The identified dysregulation in the IFN-α-induced antiviral response in T1D patients, especially in early childhood, could be one of the factors affecting T1D development.

High STAT1 mRNA levels but not its tyrosine phosphorylation are associated with macrophage infiltration and bad prognosis in breast cancer.

BACKGROUND: STAT1 has been attributed a function as tumor suppressor. However, in breast cancer data from microarray analysis indicated a predictive value of high mRNA expression levels of STAT1 and STAT1 target genes belonging to the interferon-related signature for a poor response to therapy. To clarify this issue we have determined STAT1 expression levels and activation by different methods, and investigated their association with tumor infiltration by immune cells. Additionally, we evaluated the interrelationship of these parameters and their significance for predicting disease outcome. METHODS: Expression of STAT1, its target genes SOCS1, IRF1, CXCL9, CXCL10, CXCL11, IFIT1, IFITM1, MX1 and genes characteristic for immune cell infiltration (CD68, CD163, PD-L1, PD-L2, PD-1, CD45, IFN-γ, FOXP3) was determined by RT-PCR in two independent cohorts comprising 132 breast cancer patients. For a subset of patients, protein levels of total as well as serine and tyrosine-phosphorylated STAT1 were ascertained by immunohistochemistry or immunoblotting and protein levels of CXCL10 by ELISA. RESULTS: mRNA expression levels of STAT1 and STAT1 target genes, as well as protein levels of total and serine-phosphorylated STAT1 correlated with each other in neoplastic tissue. However, there was no association between tumor levels of STAT1 mRNA and tyrosine-phosphorylated STAT1 and between CXCL10 serum levels and CXCL10 expression in the tumor. Tumors with increased STAT1 mRNA amounts exhibited elevated expression of genes characteristic for tumor-associated macrophages and immunosuppressive T lymphocytes. Survival analysis revealed an association of high STAT1 mRNA levels and bad prognosis in both cohorts. A similar prognostically relevant correlation with unfavorable outcome was evident for CXCL10, MX1, CD68, CD163, IFN-γ, and PD-L2 expression in at least one collective. By contrast, activation of STAT1 as assessed by the level of STAT1-Y701 phosphorylation was linked to positive outcome. In multivariate Cox regression, the predictive power of STAT1 mRNA expression was lost when including expression of CXCL10, MX1 and CD68 as confounders. CONCLUSIONS: Our study confirms distinct prognostic relevance of STAT1 expression levels and STAT1 tyrosine phosphorylation in breast cancer patients and identifies an association of high STAT1 levels with elevated expression of STAT1 target genes and markers for infiltrating immune cells.