Type I Interferon Activates PD-1 Expression through Activation of the STAT1-IRF2 Pathway in Myeloid Cells.

PD-1 (Programmed cell death protein 1) regulates the metabolic reprogramming of myeloid-derived suppressor cells and myeloid cell differentiation, as well as the type I interferon (IFN-I) signaling pathway in myeloid cells in the tumor microenvironment. PD-1, therefore, is a key inhibitory receptor in myeloid cells. However, the regulation of PD-1 expression in myeloid cells is unknown. We report that the expression level of PDCD1, the gene that encodes the PD-1 protein, is positively correlated with the levels of IFNB1 and IFNAR1 in myeloid cells in human colorectal cancer. Treatment of mouse myeloid cell lines with recombinant IFNß protein elevated PD-1 expression in myeloid cells in vitro. Knocking out IFNAR1, the gene that encodes the IFN-I-specific receptor, diminished the inductive effect of IFNß on PD-1 expression in myeloid cells in vitro. Treatment of tumor-bearing mice with a lipid nanoparticle-encapsulated IFNß-encoding plasmid (IFNBCOL01) increased IFNß expression, resulting in elevated PD-1 expression in tumor-infiltrating myeloid cells. At the molecular level, we determined that IFNß activates STAT1 (signal transducer and activator of transcription 1) and IRFs (interferon regulatory factors) in myeloid cells. Analysis of the cd279 promoter identified IRF2-binding consensus sequence elements. ChIP (chromatin immunoprecipitation) analysis determined that the pSTAT1 directly binds to the irf2 promoter and that IRF2 directly binds to the cd279 promoter in myeloid cells in vitro and in vivo. In colon cancer patients, the expression levels of STAT1, IRF2 and PDCD1 are positively correlated in tumor-infiltrating myeloid cells. Our findings determine that IFNß activates PD-1 expression at least in part by an autocrine mechanism via the stimulation of the pSTAT1-IRF2 axis in myeloid cells.

Neuroimmune pathways involvement in neurodegeneration of R6/2 mouse model of Huntington's disease.

Mechanisms of tissue damage in Huntington's disease (HD) involve excitotoxicity, mitochondrial damage, and neuroinflammation, including microglia activation. CD47 is a membrane protein that interacts with the inhibitory immunoreceptor SIRPα. Engagement of SIRPα by CD47 provides a downregulatory signal that inhibits host cell phagocytosis, promoting a "don't-eat-me" signal. These proteins are involved in the immune response and are downmodulated in inflammatory diseases. The involvement of inflammation and of the inflammasome in HD has already been described. In this study, we focused on other factors that can be involved in the unregulated inflammatory response that accelerates and exacerbate the neurodegenerative process in HD. Our results show that CD47 on striatal neurons decreased in HD mice, while it increased in wild type mice with age. SIRPα, on the other hand, was present in neurons in the wild type and increases in the R6/2 mice at all stages. Recruitment of SIRPα and binding to CD47 promotes the activation through phosphorylating events of non-receptor protein tyrosine phosphatase SHP-1 and SHP-2 in neurons and microglia. SHP phosphatases are able to curb the activity of NLRP3 inflammasome thereby reducing the detrimental effect of neuroinflammation. Such activity is mediated by the inhibition (dephosphorylation) of the proteins signal transducer and activator of transcription (STAT). We found that activated SHP-1 was present in microglia and neurons of WT mice at 5 and 13 weeks, increasing with time; while in R6/2 it was not localized in neurons but only in microglia, where it decreases with time. Consequently, STAT1 was overexpressed in neurons of R6/2 mice, as an effect of lack of modulation by SHP-1. Thus, our results shed light on the pathophysiology of neuronal damage, on one hand, paving the way toward a modulation of signal transducer proteins by specific inhibitors to achieve neuroprotection in HD, on the other.

The clinicopathological impact of tumor-associated macrophages in patients with cutaneous malignant melanoma.

BACKGROUND: Tumor-associated macrophages (TAMs) are an immune component of the cutaneous malignant melanoma (CMM) microenvironment and affect tumor growth. TAMs can polarize into different phenotypes, that is, proinflammatory M1 and anti-inflammatory M2 macrophages. However, the role of the macrophage phenotype in CMM remains unclear. METHODS: We examined 88 patients with CMM. Tissue microarrays were constructed, and the density of M1 and M2 macrophages was analyzed by immunohistochemistry. Immune cells coexpressing CD68 and phosphorylated signal transducer and activator of transcription 1 (pSTAT1) were considered M1 macrophages, whereas those coexpressing CD68 and c-macrophage activating factor (c-Maf) were defined as M2 macrophages. These TAMs were counted, and the relationships between the density of M1 and M2 macrophages and clinicopathological factors including prognosis were investigated. RESULTS: The CD68/c-Maf score ranged from 0 to 34 (median: 5.5). The patients were divided based on the median score into the CD68/c-Maf high (≥5.5) and low (<5.5) expression groups. Univariate and multivariate analyses revealed that CD68/c-Maf expression was an independent predictive factor for progression-free survival and an independent prognostic factor for overall survival. CD68/pSTAT1 expression was found in only two patients. CONCLUSION: We suggest that CD68/pSTAT1 coexpression is rarely observed in patients with CMM, and high CD68/c-Maf expression is a predictor of worse prognosis in these patients.

Downregulation of SOCS1 increases interferon-induced ISGylation during differentiation of induced-pluripotent stem cells to hepatocytes.

Background & Aims: Increased expression of IFN-stimulated gene 15 (ISG15) and subsequently increased ISGylation are key factors in the host response to viral infection. In this study, we sought to characterize the expression of ISG15, ISGylation, and associated enzymes at each stage of differentiation from induced pluripotent stem cells (iPSCs) to hepatocytes. Methods: To study the regulation of ISGylation, we utilized patient samples and in vitro cell culture models including iPSCs, hepatocytes-like cells, immortalized cell lines, and primary human hepatocytes. Protein/mRNA expression were measured following treatment with poly(I:C), IFNα and HCV infection. Results: When compared to HLCs, we observed several novel aspects of the ISGylation pathway in iPSCs. These include a lower baseline expression of the ISGylation-activating enzyme, UBE1L, a lack of IFN-induced expression of the ISGylation-conjugation enzyme UBE2L6, an attenuated activation of the transcription factor STAT1 and constitutive expression of SOCS1. ISGylation was observed in iPSCs following downregulation of SOCS1, which facilitated STAT1 activation and subsequently increased expression of UBE2L6. Intriguingly, HCV permissive transformed hepatoma cell lines demonstrated higher intrinsic expression of SOCS1 and weaker ISGylation following IFN treatment. SOCS1 downregulation in HCV-infected Huh 7.5.1 cells led to increased ISGylation. Conclusions: Herein, we show that high basal levels of SOCS1 inhibit STAT1 activation and subsequently IFN-induced UBE2L6 and ISGylation in iPSCs. Furthermore, as iPSCs differentiate into hepatocytes, epigenetic mechanisms regulate ISGylation by modifying UBE1L and SOCS1 expression levels. Overall, this study demonstrates that the development of cell-intrinsic innate immunity during the differentiation of iPSCs to hepatocytes provides insight into cell type-specific regulation of host defense responses and related oncogenic processes. Impact and implications: To elucidate the mechanism underlying regulation of ISGylation, a key process in the innate immune response, we studied changes in ISGylation-associated genes at the different stages of differentiation from iPSCs to hepatocytes. We found that high basal levels of SOCS1 inhibit STAT1 activation and subsequently IFN-induced UBE2L6 and ISGylation in iPSCs. Importantly, epigenetic regulation of SOCS1 and subsequently ISGylation may be important factors in the development of cell type-specific host defense responses in hepatocytes that should be considered when studying chronic infections and oncogenic processes in the liver.

Altered increase in STAT1 expression and phosphorylation in severe COVID-19.

The interferon pathway, a key antiviral defense mechanism, is being considered as a therapeutic target in COVID-19. Both, substitution of interferon and JAK/STAT inhibition to limit cytokine storms have been proposed. However, little is known about possible abnormalities in STAT signaling in immune cells during SARS-CoV-2 infection. We investigated downstream targets of interferon signaling, including STAT1, STAT2, pSTAT1 and 2, and IRF1, 7 and 9 by flow cytometry in 30 patients with COVID-19, 17 with mild, and 13 with severe infection. We report upregulation of STAT1 and IRF9 in mild and severe COVID-19 cases, which correlated with the IFN-signature assessed by Siglec-1 (CD169) expression on peripheral monocytes. Interestingly, Siglec-1 and STAT1 in CD14+ monocytes and plasmablasts showed lower expression among severe cases compared to mild cases. Contrary to the baseline STAT1 expression, the phosphorylation of STAT1 was enhanced in severe COVID-19 cases, indicating a dysbalanced JAK/STAT signaling that fails to induce transcription of interferon stimulated response elements (ISRE). This abnormality persisted after IFN-α and IFN-γ stimulation of PBMCs from patients with severe COVID-19. Data suggest impaired STAT1 transcriptional upregulation among severely infected patients may represent a potential predictive biomarker and would allow stratification of patients for certain interferon-pathway targeted treatments.

Maternal exposure to Di-n-butyl phthalate (DBP) aggravate gestational diabetes mellitus via FoxM1 suppression by pSTAT1 signalling.

The study focused on the toxicological effect of Di-n-butyl phthalate (DBP) on the expression of Phosphorylated signal transducer and activator of transcription 1 (pSTAT1) -regulated Forkhead box protein M1 (FoxM1), which might provide a new understanding of gestational diabetes mellitus (GDM) development and a potential target for treatment. Streptozotocin (STZ) (40 mg/kg) was introduced in maternal rats by intraperitoneal injection on gestation day 0 (GD 0) in the STZ and STZ + DBP groups. DBP was introduced in maternal rats by oral feeding in the STZ + DBP group over the following 3 days (750 mg/kg/day). The changes in fasting blood glucose level in rats were detected on GD 1 and GD 5. The insulin levels in maternal rats and PIBCs were measured on GD 18. The Oral Glucose Tolerance Test (OGTT) test was performed on GD 18 to check the stability of the GDM model. The primary islet ß cells (PIBCs) were established for in vitro experiments. We examined the FoxM1 and pSTAT1 expression in pancreas by immunohistochemistry. Real-time PCR and Western blot were used to detect the pSTAR1 and FoxM1 protein and mRNA gene expression levels in PIBCs. Cell Counting Kit-8 (CCK-8) and flow cytometric analysis was used to test the viability and apoptosis of cells. The results showed that the STZ + DBP group had higher glucose and lower insulin secretion levels than the other groups by both fasting test and OGTT. FoxM1 was significantly suppressed while pSTAT1 was highly expressed after DBP exposure. FoxM1 could be regulated by pSTAT1. DBP can influence the progression of GDM through its toxicological effect, which significantly increases the expression of pSTAT1 and suppresses FoxM1, causing a decline in ß cell viability.

Phosphorylation of STAT1 serine 727 enhances platinum resistance in uterine serous carcinoma.

Uterine serous carcinoma (USC) is a highly aggressive histological subtype of endometrial cancers harboring highly metastatic and chemoresistant features. Our previous study showed that STAT1 is highly expressed in USC and acts as a key molecule that is positively correlated with tumor progression, but it remains unclear whether STAT1 is relevant to the malicious chemorefractory nature of USC. In the present study, we investigated the regulatory role of STAT1 toward platinum-cytotoxicity in USC. STAT1 suppression sensitized USC cells to increase cisplatin-mediated apoptosis (p < 0.001). Furthermore, phosphorylation of STAT1 was prominently observed on serine-727 (pSTAT1-Ser727), but not on tyrosine-701, in the nucleus of USC cells treated with cisplatin. Mechanistically, the inhibition of pSTAT1-Ser727 by dominant-negative plasmid elevated cisplatin-mediated apoptosis by increasing intracellular accumulation of cisplatin through upregulation of CTR1 expression. TBB has an inhibitory effect on casein kinase 2 (CK2), which phosphorylate STAT1 at serine residues. Sequential treatment with TBB and cisplatin on USC cells greatly reduced nuclear pSTAT1-Ser727, enhanced intracellular accumulation of cisplatin, and subsequently increased apoptosis. Tumor load was significantly reduced by combination therapy of TBB and cisplatin in in vivo xenograft models (p < 0.001). Our results collectively suggest that pSTAT1-Ser727 may play a key role in platinum resistance as well as tumor progression in USC. Thus, targeting the STAT1 pathway via CK2 inhibitor can be a novel method for attenuating the chemorefractory nature of USC.

The assessment of correlation and prognosis among 18F-FDG uptake parameters, Glut1, pStat1 and pStat3 in surgically resected non-small cell lung cancer patients.

INTRODUCTION: To assess the correlation among 18F-FDG uptake, Glut1, pStat1 and pStat3, and to investigate the relationship between the prognosis and 18F-FDG uptake and these molecular markers in surgically resected non-small cell lung cancer (NSCLC) patients. RESULTS: Knockdown of Glut1 led to a significant increase in pStat1 expression. Glut1 expression positively correlated with the SUVmax, SUVmean, and TLG significantly (P<0.001). pStat3 expression negatively correlated with all PET parameters significantly (P<0.001). pStat1 had positive weak correlations with the SUVmax and SUVmean. All PET parameters and Glut1 were significantly associated with DFS (P<0.05). TLG, MTV, Glut1 and pStat1 were significantly associated with OS (P<0.05). CONCLUSION: pStat3 and Glut1 may be associated with 18F-FDG uptake mechanism. TLG, MTV, and Glut1 may be independent prognostic factors. METHODS: The SUVmax, SUVmean, MTV and TLG of primary lesions were calculated in 140 patients. The expressions of Glut1 and Stat pathway proteins in NSCLC cell lines were examined by immune blots. Excised tumor tissue was analyzed by immunohistochemistry. OS and DFS were evaluated by the Kaplan-Meier method. The difference in survival between subgroups was analyzed by log-rank test. The prognostic significance of clinicopathological, molecular and PET parameters was assessed by Cox proportional hazard regression analysis.

Removing a Cystein Group On Interferon Alpha 2b at Position 2 and 99 does Not Diminish Antitumor Activity of the Protein, Even Better.

Interferon alpha 2b is the only standard therapeutic protein for hepatitis virus infections. Further study demonstrated that this protein also posseses antitumor activity in several cancerous organs. One main pathway of this antitumor activity is mediated through antiproliferation as well as proapoptotic effects. Previously, we have successfully developed recombinant human interferon alpha 2b (rhIFNα2b) by using a synthetic gene. In addition, two mutein forms of rhIFNα2b were generated to improve the characteristics of this protein. Two point mutations showed better pharmacokinetic profiles than one point mutation as well as the native form. In the present study, this mutein form was studied for ist antitumor effect in vitro using HepG2 cells. As a comparison, the native form as well as a commercial rIFNα2b were used. Several parameters were investigated including the MTT assay, cell viability test, cell cycle using flow cytometric analysis, and the genes and protein expressions involved in cell growth. The latest was observed to study the mechanism of rhIFNα2b. There was no significant difference in the MTT assay and cell viability after cells were treated with both forms of rhIFNα2b. However, the mutein rhIFNα2b tended to show better proapoptotic activity reflected by flow cytometric data, protein expression of pSTAT1, and DNA expression of caspase 3.

Keratoacanthoma accompanied by multiple lung squamous cell carcinomas developing in a renal transplant recipient.

Keratoacanthoma (KA) is a benign keratinocytic neoplasm that spontaneously regresses after 3-6 months and shares features with well-differentiated squamous cell carcinoma (SCC). An increased incidence of both KA and non-melanoma skin tumor, including SCC, is seen among immunosuppressed, organ-transplant recipients. In this report we describe a case of KA accompanied by multiple lung SCCs developing in a renal transplant recipient.

Interferon alpha induced intrahepatic pSTAT1 inversely correlate with serum HCV RNA levels in chronic HCV infection.

BACKGROUND: The Jak-STAT signaling of hepatitis C virus (HCV) infected hepatocyte is critical for the antiviral action of endogenously produced interferon (IFN) as well as exogenously administered interferon alpha (IFN-α). The activation of cellular Jak-STAT signaling by IFN-α results in the phosphorylation and nuclear translocation of pSTAT1 and pSTAT2 proteins to induce antiviral gene transcription. Clinical studies show that chronic HCV patients with high viral load show poor response to interferon alpha and ribavirin combination therapy. AIM: We seek to determine whether the IFN-α induced activation of pSTAT1 and pSTAT2 in hepatocytes isolated from liver biopsy of patients chronically infected with hepatitis C virus could be related to the viral load. METHOD: Hepatocytes were isolated from liver biopsies of 18 chronic HCV patients using the collagen digestion method. Induction of pSTAT1 protein in the isolated hepatocyte was measured after IFN-α treatment. The fold change in the levels of pStat1 in the cell lysates due to IFN-treatment was measured by Western blot analysis followed by densitometry analysis. RESULTS: Results of our study indicate that IFN-α induced pSTAT1 levels vary in chronically infected hepatocytes from chronic HCV patients. Semi-quantitative analysis of the pSTAT1 bands revealed a median induction of 7.4-fold in non-infected primary hepatocytes and 2.3-fold in chronic hepatitis C patients (p < 0.001). Total STAT1 levels were not significantly different between treated and untreated primary hepatocytes. We also found a significantly inverse correlation between the intrahepatic pSTAT1 inductions with the serum HCV RNA levels. CONCLUSION: We have developed an antibody based Western blot detection method to measure intrahepatic pStat1 and pStat2 levels to assess the cellular response to exogenous IFN-alpha. Our results indicate that pStat1 activation is a good indicator to assess the level of HCV replication in chronic HCV patients.

Simple diagnosis of STAT1 gain-of-function alleles in patients with chronic mucocutaneous candidiasis.

CMCD is a rare congenital disorder characterized by persistent or recurrent skin, nail, and mucosal membrane infections caused by Candida albicans. Heterozygous GOF STAT1 mutations have been shown to confer AD CMCD as a result of impaired dephosphorylation of STAT1. We aimed to identify and characterize STAT1 mutations in CMCD patients and to develop a simple diagnostic assay of CMCD. Genetic analysis of STAT1 was performed in patients and their relatives. The mutations identified were characterized by immunoblot and reporter assay using transient gene expression experiments. Patients' leukocytes are investigated by flow cytometry and immunoblot. Six GOF mutations were identified, three of which are reported for the first time, that affect the CCD and DBD of STAT1 in two sporadic and four multiplex cases in 10 CMCD patients from Japan. Two of the 10 patients presented with clinical symptoms atypical to CMCD, including other fungal and viral infections, and three patients developed bronchiectasis. Immunoblot analyses of patients' leukocytes showed abnormally high levels of pSTAT1 following IFN-γ stimulation. Based on this finding, we performed a flow cytometry-based functional analysis of STAT1 GOF alleles using IFN-γ stimulation and the tyrosine kinase inhibitor, staurosporine. The higher levels of pSTAT1 observed in primary CD14(+) cells from patients compared with control cells persisted and were amplified by the presence of staurosporine. We developed a flow cytometry-based STAT1 functional screening method that would greatly facilitate the diagnosis of CMCD patients with GOF STAT1 mutations.

EGFR-mediated tumor immunoescape: The imbalance between phosphorylated STAT1 and phosphorylated STAT3.

The epidermal growth factor receptor (EGFR) supports the escape of malignant cells from immunosurveillance by inhibiting the activation of signal transducer and activator of transcription 1 (STAT1) while promoting that of STAT3. We have recently demonstrated that protein tyrosine phosphatase, non-receptor type 11 (PTNP11, best known as SHP2), a phosphatase that operates downstream of EGFR, is responsible for the dephosphorylation of active STAT1 and for the inhibition of the antigen-processing machinery (APM), hence favoring tumor immunoescape. Thus, EGFR signaling may skew the tumor microenvironment to suppress cellular immune responses.