STAT1 Phosphorylation In Response Research Articles

DIAPH1-Deficiency is Associated with Major T, NK and ILC Defects in Humans

Loss of function mutations in Diaphanous related formin 1 (DIAPH1) are associated with seizures, cortical blindness, and microcephaly syndrome (SCBMS) and are recently linked to combined immunodeficiency. However, the extent of defects in T and innate lymphoid cells (ILCs) remain unexplored. Herein, we characterized the primary T, natural killer (NK) and helper ILCs of six patients carrying two novel loss of function mutation in DIAPH1 and Jurkat cells after DIAPH1 knockdown. Mutations were identified by whole exome sequencing. T-cell immunophenotyping, proliferation, migration, cytokine signaling, survival, and NK cell cytotoxicity were studied via flow cytometry-based assays, confocal microscopy, and real-time qPCR. CD4+ T cell proteome was analyzed by mass spectrometry. p.R351* and p.R322*variants led to a significant reduction in the DIAPH1 mRNA and protein levels. DIAPH1-deficient T cells showed proliferation, activation, as well as TCR-mediated signaling defects. DIAPH1-deficient PBMCs also displayed impaired transwell migration, defective STAT5 phosphorylation in response to IL-2, IL-7 and IL-15. In vitro generation/expansion of Treg cells from naïve T cells was significantly reduced. shRNA-mediated silencing of DIAPH1 in Jurkat cells reduced DIAPH1 protein level and inhibited T cell proliferation and IL-2/STAT5 axis. Additionally, NK cells from patients had diminished cytotoxic activity, function and IL-2/STAT5 axis. Lastly, DIAPH1-deficient patients’ peripheral blood contained dramatically reduced numbers of all helper ILC subsets. DIAPH1 deficiency results in major functional defects in T, NK cells and helper ILCs underlining the critical role of formin DIAPH1 in the biology of those cell subsets.Graphical The summary of findings are presented as a graphical abstract. DIAPH1 deficiency results in multiple defects in CD4+ T, Treg, NK cells and ILCs.

AMPK Amplifies IL2-STAT5 Signaling to Maintain Stability of Regulatory T Cells in Aged Mice.

AMP-activated protein kinase (AMPK), an important regulator of the aging process, is expressed in various immune cells. However, its role in regulatory T cell (Treg) stability during aging is poorly understood. Here, we addressed the role of AMPK in Treg function and stability during aging by generating Treg-specific AMPKα1 knockout mice. In this study, we found that AMPKα1-deficient Tregs failed to control inflammation as effectively as normal Tregs did during aging. AMPK knockout from Tregs reduces STAT5 phosphorylation in response to interleukin (IL)-2 stimulation, thereby destabilizing Tregs by decreasing CD25 expression. Thus, our study addressed the role of AMPK in Tregs in sensing IL-2 signaling to amplify STAT5 phosphorylation, which, in turn, supports Treg stability by maintaining CD25 expression and controlling inflamm-aging.

A novel anti-inflammatory role links the CARS2 locus to protection from coronary artery disease

Background and aimsGenome-wide association studies (GWAS) identified a coronary artery disease (CAD) risk locus on 13.q34 tagged by rs61969072 (T/G). This variant lies in an intergenic region, proximal to ING1, CARKD and CARS2 but its causal relationship to CAD is unknown. Methods and ResultsWe first demonstrated that rs61969072 and tightly linked single nucleotide polymorphisms (SNPs) associate with CARS2 but not ING1 or CARKD expression in carotid endarterectomy samples, with reduced CARS2 abundance in carriers of the CAD risk allele (G). THP-1 monocytes were differentiated and polarized to proinflammatory (M1) and anti-inflammatory (M2) macrophages. CARS2 gene expression decreased in M1 and increased in M2 macrophages, consistent with a role for CARS2 in inflammation. Gene expression profiling revealed an increase in pro-inflammatory markers in response to CARS2 siRNA knockdown in THP-1 derived macrophages, accompanied by an increased abundance of inflammatory cytokines in the cell supernatant. Functional enrichment analysis of impacted transcripts identified the anti-inflammatory IL10 signalling pathway. Western blot analysis of CARS2 silenced macrophages revealed reduced STAT3 phosphorylation in response to IL-10 and increased expression of LPS-induced genes that are repressed by IL-10, indicating a role for CARS2 in anti-inflammatory signalling. Finally, to simulate vessel wall conditions, macrophages, and smooth muscle cells (SMC) were maintained in co-culture. Significantly, CARS2 silencing in macrophages altered the SMC phenotype, decreasing expression of contractile genes and increasing expression of inflammatory genes. ConclusionsThese data highlight a novel anti-inflammatory novel role for CARS2 in human macrophages and SMCs that may underlie the protective effect of a common GWAS-identified variant.

Inhibition of Bromodomain and Extra Terminal (BET) Domain Activity Modulates the IL-23R/IL-17 Axis and Suppresses Acute Graft-Versus-Host Disease.

Acute graft-versus-host disease (GVHD) is the leading cause of non-relapse mortality following allogeneic hematopoietic cell transplantation. The majority of patients non-responsive to front line treatment with steroids have an estimated overall 2-year survival rate of only 10%. Bromodomain and extra-terminal domain (BET) proteins influence inflammatory gene transcription, and therefore represent a potential target to mitigate inflammation central to acute GVHD pathogenesis. Using potent and selective BET inhibitors Plexxikon-51107 and -2853 (PLX51107 and PLX2853), we show that BET inhibition significantly improves survival and reduces disease progression in murine models of acute GVHD without sacrificing the beneficial graft-versus-leukemia response. BET inhibition reduces T cell alloreactive proliferation, decreases inflammatory cytokine production, and impairs dendritic cell maturation both in vitro and in vivo. RNA sequencing studies in human T cells revealed that BET inhibition impacts inflammatory IL-17 and IL-12 gene expression signatures, and Chromatin Immunoprecipitation (ChIP)-sequencing revealed that BRD4 binds directly to the IL-23R gene locus. BET inhibition results in decreased IL-23R expression and function as demonstrated by decreased phosphorylation of STAT3 in response to IL-23 stimulation in human T cells in vitro as well as in mouse donor T cells in vivo. Furthermore, PLX2853 significantly reduced IL-23R+ and pathogenic CD4+ IFNγ+ IL-17+ double positive T cell infiltration in gastrointestinal tissues in an acute GVHD murine model. Our findings identify a role for BET proteins in regulating the IL-23R/STAT3/IL-17 pathway. Based on our preclinical data presented here, PLX51107 will enter clinical trial for refractory acute GVHD in a Phase 1 safety, biological efficacy trial.

A Novel Role for AMP-Activated Protein Kinase (AMPK) in Controlling T Cell Sensitivity to IL-6

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative treatment for high-risk leukemia and many non-malignant hematologic disorders. However, the use of alloHSCT as a therapy remains limited by acute graft-versus-host disease (GVHD), where activated donor T cells attack and destroy host tissues in the skin, gastrointestinal tract, and liver. We have previously shown that alloreactive T cells increase phosphorylation of the intracellular energy sensor AMP-activated protein kinase (AMPK) as early as day 3 post-transplant (levels 8-fold higher in alloreactive T cells compared to naive or syngeneic controls (p=0.0003)) and donor T cells lacking AMPK (AMPK KO) cause significantly less GVHD in both major- and minor-histocompatibility mismatch models of GVHD. At the same time, AMPK KO T cells equivalently clear leukemia cells and extend recipient survival in leukemia models with high tumor burden. Transplantation of cells lacking AMPK lowers the number of donor T cells recovered on day 7 post-transplant (3.15 +/- 0.49x106 versus1.87 +/- 0.53x106, p=0.0006, wildtype (wt) versus AMPKKO respectively) but interestingly, increases both the percentage and total number of regulatory T cells (Treg) (0.85 +/- 0.32x104 vs. 1.69 +/- 0.34x104, wt vs. KO, p=0.004).To understand these findings mechanistically, we sought to study the role of AMPK in donor Treg expansion and stabilization post-alloHSCT. Canonically, interleukin-2 (IL-2) signaling, through phosphorylation of Stat5, promotes Treg generation and stability, while inflammatory signals (e.g. IL-6 signaling through Stat3) are known to decrease Treg stability post-transplant. Studies testing IL-2 sensitivity in KO cells, or the degree of Stat5 phosphorylation post-transplant, were unrevealing. However, AMPK KO Tregs significantly decrease their sensitivity to IL-6, with a 50% reduction in phosphorylated-Stat3 levels following intermediate levels of cytokine stimulation (Figure 1A). To demonstrate the functional consequences of this difference in cytokine sensitivity, we exposed in vitro generated Treg to increasing concentrations of IL-6. As expected, the percentage of wt FoxP3+ cells decreased sharply with increasing IL-6 concentrations (decreasing from 50% to 14%), while AMPK KO Tregs were minimally affected over this dose range (47% versus 42%; Figure 1B, p<0.02).To determine if increased Treg stability was the predominant reason for improvements in GVHD seen with AMPK KO cells, CD25+ cells were removed from the donor inoculum of AMPK KO or wt mice by magnetic selection (to eliminate >90% of Treg), and then either CD25-replete or CD25-depleted sets of cells were transplanted into B6xDBA2 F1 recipients in a major MHC mismatch model of GVHD. Despite a decrease in AMPK KO Treg numbers on day 7 to levels commiserate with wt donors (Figure 2A), elimination of AMPK KO Treg had only a modest effect on GVHD severity (Figure 2B), implicating a substantial role for AMPK in the non-Treg compartment.To explore the role of AMPK in non-Tregs further, we investigated the sensitivity of unmanipulated wt and AMPK KO T cells to IL-6 stimulation. Similar to FoxP3+ cells, both naïve CD4 and CD8 T cells lacking AMPK experienced lower levels of Stat3 phosphorylation in response to graded concentrations of IL-6 (Figure 3A). Furthermore, if IL-6 was added to a 4-day culture with anti-CD3 stimulation, the number of wt T cells recovered after 96 hours increased >2.2-fold. In contrast, AMPK KO cells experienced minimal changes in T cell number over a wide range of IL-6 concentrations (Figure 3B).In conclusion, we define in these studies a novel role for AMPK in controlling donor T cell sensitivity to IL-6, both at the level of Stat3 phosphorylation, and in terms of functional consequences. Not only does this striking result elegantly explain our previous observation of increased Treg percentages, and decreased donor T cell numbers, following transplantation of AMPK KO cells, it continues to make the case that inhibition of AMPK represents an attractive clinical target for the amelioration of GVHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Restoration of type I interferon signaling in intrahepatically primed CD8+ T cells promotes functional differentiation.

Hepatitis B virus–specific (HBV-specific) CD8+ T cells fail to acquire effector functions after priming in the liver, but the molecular basis for the dysfunction is poorly understood. By comparing the gene expression profile of intrahepatically primed, dysfunctional HBV-specific CD8+ T cells with that of systemically primed, functional effector counterparts, we found that the expression of interferon-stimulated genes (ISGs) is selectively suppressed in the dysfunctional CD8+ T cells. The ISG suppression was associated with impaired phosphorylation of STAT1 in response to IFN-α treatment. Importantly, a strong induction of type I interferons (IFN-Is) in the liver facilitated the functional differentiation of intrahepatically primed HBV-specific CD8+ T cells in association with the restoration of ISGs’ expression in the T cells. These results suggest that intrahepatic priming suppresses IFN-I signaling in CD8+ T cells, which may contribute to the dysfunction. The data also suggest a therapeutic value of the robust induction of intrahepatic IFN-Is for the treatment of chronic HBV infection.

ISG15 protects human Tregs from interferon alpha-induced contraction in a cell-intrinsic fashion.

ObjectivesType I interferons (IFNs) inhibit regulatory T‐cell (Treg) expansion and activation, making them beneficial in antiviral responses, but detrimental in autoimmune diseases. Herein, we investigate the role of ISG15 in human Tregs in the context of refractoriness to type I IFN stimulation.MethodsISG15 expression and Treg dynamics were analysed in vitro and ex vivo from patients with chronic hepatitis C, with lupus and ISG15 deficiency.ResultsISG15 is expressed at high levels in human Tregs, renders them refractory to the IFN‐STAT1 signal, and protects them from IFN‐driven contraction. In vitro, Tregs from healthy controls upregulate ISG15 upon activation to higher levels than conventional CD4 T cells, and ISG15‐silenced Tregs are more susceptible to IFNα‐induced contraction. In human ISG15 deficiency, patient Tregs display an elevated IFN signature relative to Tregs from healthy control. In vivo, in patients with chronic hepatitis C, 2 days after starting pegIFN/ribavirin therapy, a stronger ISG15 inducibility correlates with a milder Treg depletion. Ex vivo, in systemic lupus erythematosus patients, higher levels of ISG15 are associated to reduced STAT1 phosphorylation in response to IFNα, and also to increased frequencies of Tregs, characterising active disease.ConclusionOur results reveal a Treg‐intrinsic role of ISG15 in dictating their refractoriness to the IFN signal, thus preserving the Treg population under inflammatory conditions.

Application of Flow Cytometry in the Diagnostics Pipeline of Primary Immunodeficiencies Underlying Disseminated Talaromyces marneffei Infection in HIV-Negative Children.

Talaromyces (Penicillium) marneffei is an AIDS-defining infection in Southeast Asia and is associated with high mortality. It is rare in non-immunosuppressed individuals, especially children. Little is known about host immune response and genetic susceptibility to this endemic fungus. Genetic defects in the interferon-gamma (IFN-γ)/STAT1 signaling pathway, CD40/CD40 ligand- and IL12/IL12-receptor-mediated crosstalk between phagocytes and T-cells, and STAT3-mediated Th17 differentiation have been reported in HIV-negative children with talaromycosis and other endemic mycoses such as histoplasmosis, coccidioidomycosis, and paracoccidioidomycosis. There is a need to design a diagnostic algorithm to evaluate such patients. In this article, we review a cohort of pediatric patients with disseminated talaromycosis referred to the Asian Primary Immunodeficiency Network for genetic diagnosis of PID. Using these illustrative cases, we propose a diagnostics pipeline that begins with immunoglobulin pattern (IgG, IgA, IgM, and IgE) and enumeration of lymphocyte subpopulations (T-, B-, and NK-cells). The former could provide clues for hyper-IgM syndrome and hyper-IgE syndrome. Flow cytometric evaluation of CD40L expression should be performed for patients suspected to have X-linked hyper-IgM syndrome. Defects in interferon-mediated JAK-STAT signaling are evaluated by STAT1 phosphorylation studies by flow cytometry. STAT1 hyperphosphorylation in response to IFN-α or IFN-γ and delayed dephosphorylation is diagnostic for gain-of-function STAT1 disorder, while absent STAT1 phosphorylation in response to IFN-γ but normal response to IFN-α is suggestive of IFN-γ receptor deficiency. This simple and rapid diagnostic algorithm will be useful in guiding genetic studies for patients with disseminated talaromycosis requiring immunological investigations.

Schizophrenia Patient Shows a Rare Interleukin 15 Receptor alpha Variant Disrupting Signal Transduction.

Schizophrenia is a complex and debilitating mental disorder with strong heritability. Its pathogenesis involves immune dysregulation. Interleukin 15 and interleukin 15 receptor alpha(IL-15Rα) are classical immune molecules. They also help maintain normal brain function, leading to our hypothesis that IL-15Rα gene(IL- 15RA) variants contribute to the pathogenesis of schizophrenia. We determine whether the genetic variants of IL-15RA are associated with the development and progression of schizophrenia and whether IL-15RA single nucleotide polymorphism(SNP) plays a key role in downstream signaling transduction. We sequenced IL-15RA exon from 132 Chinese schizophrenic patients and identified a rare variant(rs528238821) in a patient diagnosed with catatonic schizophrenia and ankylosing spondylitis(AS). We overexpressed this missense variant in cells driven by pBI-CMV vector. The cells showed attenuated STAT3 phosphorylation in response to interleukin15. IL-15RA mutation is rare in schizophrenic patients but interfered with IL- 15Rα intracellular signal transduction. Given the similarity of symptoms of catatonic schizophrenia and the known phenotype of IL-15Rα knockout mice, gene variation might offer diagnostic value for sub-types of schizophrenia.

PF‑114, a novel selective inhibitor of BCR‑ABL tyrosine kinase, is a potent inducer of apoptosis in chronic myelogenous leukemia cells.

A t(9;22) chromosomal translocation which forms the chimeric tyrosine kinase breakpoint cluster region (BCR)‑Abelson murine leukemia viral oncogene homolog1(ABL) is a key mechanism underlying the pathogenesis of chronic myelogenous leukemia(CML). Pharmacological inhibition of BCR‑ABL with imatinib (Gleevec) has been reported as an effective targeted therapy; however, mutations (including the kinase domain of ABL) suppress the efficacy of inhibitors. PF‑114, a derivative of the third generation BCR‑ABL inhibitor ponatinib, demonstrated a high inhibitory activity against wild-type and mutant BCR‑ABL forms, such as the clinically important T315I. Furthermore, PF‑114 exhibited preferential kinase selectivity, safety, notable pharmacokinetic properties and therapeutic efficacy in a murine model. Investigation into the mechanisms of CML cell death revealed an exceptional potency of PF‑114 (at low nanomolar concentrations) for the CML‑derived K562 cell line, whereas leukemia cell lines that lack the chimeric tyrosine kinase were markedly more refractory. The molecular ordering of events mechanistically associated with K562 cell death included the dephosphorylation of CrkL adaptor protein followed by inhibition of ERK1/2 and Akt, G1 arrest, a decrease of phosphorylated Bcl‑2‑associated death promoter, Bcl‑2‑like protein11, BH3 interacting‑domain death agonist, Bcl‑extra large and Bcl‑2 family apoptosis regulator, and reduced mitochondrial transmembrane potential. Increased AnnexinV reactivity, activation of caspases and poly(ADP‑ribose)polymerase cleavage were proposed to lead to internucleosomal DNA fragmentation. Thus, PF‑114 may be a potent inducer of apoptosis in CML cells. Nevertheless, activation of STAT3 phosphorylation in response to PF‑114 may permit cell rescue; thus, a combination of BCR‑ABL and STAT3 inhibitors should be considered for improved therapeutic outcome. Collectively, the targeted killing of BCR‑ABL‑positive cells, along with other beneficial properties, such as invivo characteristics, suggests PF‑114 as a potential candidate for analysis in clinical trials with CML patients.

SAT-151 Hyperinsulinemia-Driven Progressive Metabolic Dysfunction in Male Mice with Liver-Specific CEACAM1 Deletion

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) regulates insulin sensitivity by promoting hepatic insulin clearance. Mice with global null mutation (Cc1-/-) or with liver-specific inactivation (L-SACC1) of Ceacam1 gene display hyperinsulinemia due to impaired insulin clearance, insulin resistance, steatohepatitis, and obesity. To further investigate the primary role of hepatic CEACAM1 in insulin and lipid homeostasis independent of its potential effect in extra-hepatic tissues or without the confounding effect of the dominant-negative transgene, we examined whether hepatic-specific Ceacam1 deletion causes metabolic abnormalities starting with impairment of insulin clearance. To this end, we generated liver-specific Ceacam1 null mice (AlbCre+Cc1fl/fl) on C57BL/6 genetic background. Steady-state C-peptide to insulin molar ratio showed impairment of insulin clearance and hyperinsulinemia at 2 months, followed by hepatic insulin resistance (as measured by hyperinsulinemic-euglycemic clamp analysis) and steatohepatitis starting at 7 months of age. Relative to Cre and Flox control groups, AlbCre+Cc1fl/fl mutants developed visceral obesity and hyperphagia at 6-7 months of age, in parallel to hyperleptinemia and blunted hypothalamic STAT3 phosphorylation in response to an intraperitoneal injection of leptin. Together with hyperinsulinemia-driven rise in hypothalamic fatty acid synthase level and activity, this could contribute to hyperphagia and reduced physical activity. Pair-feeding experiment showed that hyperphagia caused systemic insulin resistance at 7-8 months of age, including blunted insulin signaling in white adipose tissue and lipolysis. Interestingly, hypothalamic Ceacam1 mRNA levels progressively decreased in controls and AlbCre+Cc1fl/fl mutants but to a higher extent in the nulls reaching more than 60% loss at ≥7 months of age, as a result of insulin resistance. Because Ceacam1 is expressed in POMC1 neurons, its significant reduction in this neuronal population could contribute to energy imbalance in AlbCre+Cc1fl/fl mutants. Collectively, these data validate the AlbCre+Cc1fl/fl mutant as an in vivo model to demonstrate the key role that impairment of hepatic insulin clearance plays in the pathogenesis of energy imbalance followed by systemic insulin resistance and lipolysis. This underscores the importance of the liver-hypothalamic axis in metabolic derangement.

3D type I collagen environment leads up to a reassessment of the classification of human macrophage polarizations

Macrophages have multiple roles in development, tissue homeostasis and repair and present a high degree of phenotypic plasticity embodied in the concept of polarization. One goal of macrophage biology field is to characterize these polarizations at the molecular level. To achieve this task, it is necessary to integrate how physical environment signals are interpreted by macrophages under immune stimulation. In this work, we study how a 3D scaffold obtained from polymerized fibrillar rat type I collagen modulates the polarizations of human macrophages and reveal that some traditionally used markers should be reassessed. We demonstrate that integrin β2 is a regulator of STAT1 phosphorylation in response to IFNγ/LPS as well as responsible for the inhibition of ALOX15 expression in response to IL-4/IL-13 in 3D. Meanwhile, we also find that the CCL19/CCL20 ratio is reverted in 3D under IFNγ/LPS stimulation. 3D also induces the priming of the NLRP3 inflammasome resulting in an increased IL-1β and IL-6 secretion. These results give the molecular basis for assessing collagen induced immunomodulation of human macrophages in various physiological and pathological contexts such as cancer.

Activation of naïve CD4+ T cells re-tunes STAT1 signaling to deliver unique cytokine responses in memory CD4+ T cells.

The cytokine IL-6 controls the survival, proliferation and effector characteristics of lymphocytes through activation of the transcription factors STAT1 and STAT3. While STAT3 activity is an ever-present feature of IL-6 signaling in CD4+ T cells, prior T-cell receptor activation limits the IL-6 control of STAT1 in effector and memory populations. Here we show that STAT1 phosphorylation in response to IL-6 was regulated by protein tyrosine phosphatases (PTPN2, PTPN22) expressed in response to the activation of naïve CD4+ T cells. Transcriptomic and chromatin immunoprecipitation-sequencing of IL-6 responses in naïve and effector memory CD4+ T cells showed how the suppression of STAT1 activation shaped the functional identity and effector characteristics of memory CD4+ T cells. Thus, protein tyrosine phosphatases induced by activation of naïve T cells determined the way activated or memory CD4+ T cells sensed and interpreted cytokine signals.

Hyperinsulinemia drives hepatic insulin resistance in male mice with liver-specific Ceacam1 deletion independently of lipolysis

BackgroundCEACAM1 regulates insulin sensitivity by promoting insulin clearance. Accordingly, global C57BL/6J.Cc1−/− null mice display hyperinsulinemia due to impaired insulin clearance at 2 months of age, followed by insulin resistance, steatohepatitis, visceral obesity and leptin resistance at 6 months. The study aimed at investigating the primary role of hepatic CEACAM1 in insulin and lipid homeostasis independently of its metabolic effect in extra-hepatic tissues. MethodsLiver-specific C57BL/6J.AlbCre+Cc1fl/fl mice were generated and their metabolic phenotype was characterized by comparison to that of their littermate controls at 2–9 months of age, using hyperinsulinemic-euglycemic clamp analysis and indirect calorimetry. The effect of hyperphagia on insulin resistance was assessed by pair-feeding experiments. ResultsLiver-specific AlbCre+Cc1fl/fl mutants exhibited impaired insulin clearance and hyperinsulinemia at 2 months, followed by hepatic insulin resistance (assessed by hyperinsulinemic-euglycemic clamp analysis) and steatohepatitis at ~ 7 months of age, at which point visceral obesity and hyperphagia developed, in parallel to hyperleptinemia and blunted hypothalamic STAT3 phosphorylation in response to an intraperitoneal injection of leptin. Hyperinsulinemia caused hypothalamic insulin resistance, followed by increased fatty acid synthase activity, which together with defective hypothalamic leptin signaling contributed to hyperphagia and reduced physical activity. Pair-feeding experiment showed that hyperphagia caused systemic insulin resistance, including blunted insulin signaling in white adipose tissue and lipolysis, at 8–9 months of age. ConclusionAlbCre+Cc1fl/fl mutants provide an in vivo demonstration of the key role of impaired hepatic insulin clearance and hyperinsulinemia in the pathogenesis of secondary hepatic insulin resistance independently of lipolysis. They also reveal an important role for the liver-hypothalamic axis in the regulation of energy balance and subsequently, systemic insulin sensitivity.

Effects of CKMT1 on radiosensitivity of nasopharyngeal carcinoma cells

Purpose: Radioresistance is an important factor for unsatisfactory prognosis in Nasopharyngeal carcinoma (NPC) patients. Ubiquitous mitochondrial creatine kinase (CKMT1) is always associated with malignancy in a variety of cancers. However, its significance in NPC progression and radiosensitivity remains unclear. The present study focused on investigating the effects of CKMT1 on NPC cell radiosensitivity.Material and methods: CKMT1 was overexpressed in NPC cell line CNE-1 or knocked out in CNE-2. Biological changes were detected after cells exposing to different doses of X-ray to determine the role of CKMT1 on NPC cell radiosensitivity.Results: CKMT1 promotes proliferation and migration in NPC cell lines CNE-1 and CNE-2. Overexpression of CKMT1 in CNE-1 cells enhanced colony formation rates, reduced G2/M phase cell cycle arrest, lowered apoptosis rate and c-PARP level, and elevated STAT3 phosphorylation level after radiation treatment. While knocking out CKMT1 using the CRISPR/Cas9 system in CNE-2 cells lowered colony formation rates, increased G2/M phase cell cycle arrest, apoptosis rates, and c-PARP levels, and decreased STAT3 phosphorylation in response to radiation treatment.Conclusions: NPC cells with higher CKMT1 exhibited lower radiosensitivity through promoting phosphorylation of STAT3. Our findings suggest that CKMT1 may be an alternative radiotherapeutic target in NPC therapy.

Nonstructural protein of severe fever with thrombocytopenia syndrome phlebovirus targets STAT2 and not STAT1 to inhibit type I interferon-stimulated JAK-STAT signaling

The nonstructural protein NSs of severe fever with thrombocytopenia syndrome phlebovirus blocks type I interferon (IFN)-stimulated JAK-STAT signaling. However, there is continuing controversy as to whether NSs targets STAT1 or STAT2 or both for this blockade. The present study was designed to gain a further understanding of the blockade mechanism. Immunoprecipitation experiments revealed a stronger interaction of NSs with STAT2 than with any other component constituting the JAK-STAT pathway. Expression of NSs resulted in the formation of cytoplasmic inclusion bodies (IBs), and affected cytoplasmic distribution of STAT2. STAT2 was relocated to NSs-induced IBs. Consequently, NSs inhibited IFN-α-stimulated tyrosine phosphorylation and nuclear translocation of STAT2. These inhibitory effects as well as the signaling blockade activity were not observed in NSs mutant proteins lacking the STAT2-binding ability. In contrast, NSs affected neither subcellular distribution nor phosphorylation of STAT1 in response to IFN-α and IFN-γ, demonstrating that NSs has little physical and functional interactions with STAT1. Taken together, these results suggest that NSs sequesters STAT2 into NSs-induced IBs, thereby blocking type I IFN JAK-STAT signaling.

Impaired IL‐10 Receptor‐mediated Suppression in Monocyte From Patients With Crohn Disease

Interleukin-10 (IL-10) is an immunoregulatory cytokine that has a central role in suppressing proinflammatory responses. Patients with deleterious mutations in interleukin (IL)-10 or IL-10 receptor (IL-10R) genes develop severe colitis and perianal disease in the first months of life. Whether IL-10R expression and signaling in pediatric- or adult-onset Crohn disease (CD) are altered is unknown. The objective of this study was to characterize IL-10R expression and IL-10R-mediated suppression in patients with CD. Monocytes were sorted from peripheral blood mononuclear cells of patients with CD and control subjects. IL-10R expression was determined by flow cytometry. Monocytes were stimulated with lipopolysaccharide (LPS) for 3 hours in the presence of different concentrations of IL-10 to determine IL-10-mediated suppression of tumor necrosis factor α production. Signaling through the IL-10R was evaluated by quantifying STAT3 phosphorylation in response to IL-10 stimulation. Forty-two subjects were enrolled in this study: 19 with CD and 23 controls. Stimulation of monocytes with LPS markedly increased IL-10R expression in both groups but to a much lower extent in patients with CD. In addition, IL-10-mediated suppression of TNFα production upon LPS stimulation and IL-10-induced STAT3 phosphorylation were attenuated in patients with CD versus controls. Finally, LPS-stimulated monocytes from patients with CD secreted significantly lower quantities of IL-10, compared with control monocytes. IL-10R expression and signaling are decreased in monocytes from patients with CD. Additional studies are required to assess whether similar patterns occur in other innate immune cells, especially in the gut, and whether disease activity, medical therapy, and genetic factors modulate these findings.

Tissue specific CD91+ APCs determine effectiveness of immunosurveillance mediated by HSPs

Abstract Gp96, an immunogenic Heat Shock Protein, is capable of eliciting potent immune responses against its chaperoned antigens and is being investigated as a potential cancer immunotherapy. Gp96 induces immune responses by activating antigen presenting cells (APCs) through CD91 which elicits downstream T and NK cell activation. T cell activation following gp96 is well understood, including the necessary APCs. Conversely, NK cell activation is less understood, even though NK cells are required for full T cell activation. We have determined that macrophages (MΦ) are capable of inducing IFNγ production by NK cells but, inversely, dendritic cells (DC) are not, despite similar expression of CD91. Thus, we investigated MΦ and DC production of soluble mediators of NK cell activation. IP10 was identified as a chemokine which is more greatly induced in MΦ than DC in response to gp96. Blocking the IP10 receptor, CXCR3, prevents IFNγ production by NK cells when cultured with gp96 activated MΦ. IP10 is STAT1 induced, thus we examined STAT1 phosphorylation (STAT1p) in MΦ and DC in response to gp96. STAT1 is phosphorylated in MΦ, but gp96 fails to induce STAT1p of tyrosine in DC. In STAT1 signaling, p38 is proximally upstream of STAT1p and interestingly, both MΦ and DC phosphorylate p38 to similar levels, suggesting that the difference between MΦ and DC is downstream of p38. There are several phosphatases downstream of CD91 which could be inhibiting STAT1p. To further support this, we inhibited tyrosine phosphatases using pervanadate, resulting in DC STAT1p in response to gp96. Collectively, these data indicate that gp96 activation of MΦ may result in a more robust immune response by activating NK cells which is a critical consideration for clinical applications.

Phosphorylation of STAT proteins by recombinant human IL-6 in immortalized human chondrocyte cell lines, T/C28a2 and C28/I2.

Two immortalized human juvenile chondrocyte cell lines, T/C28a2 and C28/I2, were employed to determine the extent to which recombinant human (rh) IL-6, a known cytokine activator of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway in many cell types, caused STAT proteins to be phosphorylated. The results showed that STAT3 was constitutively phosphorylated in the absence of rhIL-6 in T/C28a2 chondrocytes. However, C28/I2 chondrocytes treated with rhIL-6 caused STAT1, STAT3, and STAT5 to be phosphorylated without altering total unphosphorylated STAT proteins. STAT3 phosphorylation in response to rhIL-6 in T/C28a and C28/I2 chondrocytes was efficiently blocked by the JAK3-selective inhibitor WHI-P131 (Janex-1) and by soluble IL-6 receptor-α (sIL-6R). However, the combination of rhIL-6 and ruxolitinib, a JAK1/JAK2-selective inhibitor, was a less effective inhibitor of STAT protein activation. These findings showed that rhIL-6 activated STAT proteins in the C28/I2 chondrocyte cell line. STAT protein phosphorylation could be blocked by a JAK3-selective inhibitor or by the combination of rhIL-6 and sIL-6R.

Involvement of IKKα in STAT1 phosphorylation in antiviral signaling

Abstract RIG-I-like receptors (RLRs) sense non-self RNA, inducing type I IFNs. Type I IFN promotes the expression of IFN-stimulated genes (ISGs), which requires the activation of STAT1. We previously reported that dsRNA induced STAT1 phosphorylation via a type I IFN-independent pathway in addition to the well-known type I IFN-dependent pathway. IKKα is involved in antiviral signaling induced by dsRNA in the RLR-mediated antiviral signaling; however, its role remains uncertain. In this study, we investigated the role of IKKα in RLR signaling, which is essential for antiviral innate immune responses in non-professional antigen-presenting cells, such as epithelial cells. Silencing of IKKα markedly decreased the level of IFN-β and STAT1 phosphorylation in response to dsRNA. However, the inhibition of IKKα did not alter the RLR signaling-mediated dimerization of IRF3 or activation of NFκB. These results suggest a non-canonical role of IKKα in RLR signaling. In addition, STAT1 phosphorylation was inhibited by IKKα knockdown in IFNAR-deficient cells. These results suggest that the dual regulation of STAT1 by IKKα in the RLR-mediated antiviral signaling.