Functional interaction of STAT3 transcription factor with the coactivator NcoA/SRC1a.

Signal transducer and activator of transcription 3 (STAT3) transcription factors are cytoplasmic proteins that induce gene activation in response to cytokine receptor stimulation. Following tyrosine phosphorylation, STAT3 proteins dimerize, translocate to the nucleus, and activate specific target genes. This transcriptional activation by STAT3 proteins has been shown to require the recruitment of coactivators such as CREB-binding protein (CBP)/p300. In the present study, we show that steroid receptor coactivator 1, NcoA/SRC1a, originally identified as a nuclear receptor coactivator, also functions as a coactivator of STAT3 proteins. In coimmunoprecipitations, NcoA/SRC1a was found to associate with STAT3 following IL-6 stimulation of HepG2 hepatoma cells. Pull-down experiments indicated that the N-terminal part of NcoA/SRC1a associates with the activation domain of STAT3. Overexpression of NcoA/SRC1a or its SRC1e isoform enhanced transcriptional activation by STAT3 proteins in transient transfection experiments. This ability of NcoA/SRC1a to enhance STAT3 activity is dependent upon the presence of the CBP-interacting domain, activation domain 1. Using chromatin immunoprecipitation assays, we found that STAT3, NcoA/SRC1a, and CBP/p300 are simultaneously recruited to the p21(waf1) promoter following interleukin-6 stimulation. Taken together, these data suggest that CBP/p300 and NcoA/SRC1a may function in a common pathway to regulate STAT3 transcriptional activity

clc is co-expressed with clf or cntfr in developing mouse muscles

BACKGROUND: The ciliary neurotrophic factor (CNTF) receptor is composed of two signalling receptor chains, gp130 and the leukaemia inhibitory factor receptor, associated with a non-signalling CNTF binding receptor α component (CNTFR). This tripartite receptor has been shown to play important roles in the development of motor neurons, but the identity of the relevant ligand(s) is still not clearly established. Recently, we have identified two new ligands for the CNTF receptor complex. These are heterodimeric cytokines composed of cardiotrophin-like cytokine (CLC) associated either with the soluble receptor subunit cytokine-like factor-1 (CLF) or the soluble form of the binding receptor itself (sCNTFR). RESULTS: Here we show that, during development, clc is expressed in lung, kidney, vibrissae, tooth, epithelia and muscles during the period of development corresponding to when motoneuron loss is observed in mice lacking a functional CNTF receptor. In addition, we demonstrate that it is co-expressed at the single cell level with clf and cntfr, supporting the idea that CLC might be co-secreted with either CLF or sCNTFR. CONCLUSION: This expression pattern is in favor of CLC, associated either with CLF or sCNTFR, being an important player in the signal triggered by the CNTF receptor being required for motoneuron development

Astroglial expression of the P-glycoprotein is controlled by intracellular CNTF

BACKGROUND: The P-glycoprotein (P-gp), an ATP binding cassette transmembrane transporter, is expressed by astrocytes in the adult brain, and is positively modulated during astrogliosis. In a search for factors involved in this modulation, P-gp overexpression was studied in long-term in vitro astroglial cultures. RESULTS: Surprisingly, most factors that are known to induce astroglial activation in astroglial cultures failed to increase P-gp expression. The only effective proteins were IFNγ and those belonging to the IL-6 family of cytokines (IL-6, LIF, CT-1 and CNTF). As well as P-gp expression, the IL-6 type cytokines - but not IFNγ - stimulated the expression of endogenous CNTF in astrocytes. In order to see whether an increased intracellular level of CNTF was necessary for induction of P-gp overexpression by IL-6 type cytokines, by the same cytokines analysis was carried out on astrocytes obtained from CNTF knockout mice. In these conditions, IFNγ produced increased P-gp expression, but no overexpression of P-gp was observed with either IL-6, LIF or CT-1, pointing to a role of CNTF in the intracellular signalling pathway leading to P-gp overexpression. In agreement with this suggestion, application of exogenous CNTF -which is internalised with its receptor - produced an overexpression of P-gp in CNTF-deficient astrocytes. CONCLUSIONS: These results reveal two different pathways regulating P-gp expression and activity in reactive astrocytes, one of which depends upon the intracellular concentration of CNTF. This regulation of P-gp may be one of the long searched for physiological roles of CNTF

Tumour-derived leukaemia inhibitory factor is a major driver of cancer cachexia and morbidity in C26 tumour-bearing mice

BACKGROUND: Cancer cachexia is a metabolic wasting syndrome that is strongly associated with a poor prognosis. The initiating factors causing fat and muscle loss are largely unknown. Previously, we found that leukaemia inhibitory factor (LIF) secreted by C26 colon carcinoma cells was responsible for atrophy in treated myotubes. In the present study, we tested whether C26 tumour‐derived LIF is required for cancer cachexia in mice by knockout of Lif in C26 cells. METHODS: A C26 Lif null tumour cell line was made using CRISPR‐Cas9. Measurements of cachexia were compared in mice inoculated with C26 vs. C26^Lif−/− tumour cells, and atrophy was compared in myotubes treated with medium from C26 vs. C26^Lif−/− tumour cells. Levels of 25 cytokines/chemokines were compared in serum of mice bearing C26 vs. C26^Lif−/− tumours and in the medium from these tumour cell lines. RESULTS: At study endpoint, C26 mice showed outward signs of sickness while mice with C26^Lif−/− tumours appeared healthy. Mice with C26^Lif−/− tumours showed a 55–75% amelioration of body weight loss, muscle loss, fat loss, and splenomegaly compared with mice with C26 tumours (P < 0.05). The heart was not affected by LIF levels because the loss of cardiac mass was the same in C26 and C^26Lif−/− tumour‐bearing mice. LIF levels in mouse serum was entirely dependent on secretion from the tumour cells. Serum levels of interleukin‐6 and G‐CSF were increased by 79‐fold and 68‐fold, respectively, in C26 mice but only by five‐fold and two‐fold, respectively, in C26^Lif−/− mice, suggesting that interleukin‐6 and G‐CSF increases are dependent on tumour‐derived LIF. CONCLUSIONS: This study shows the first use of CRISPR‐Cas9 knockout of a candidate cachexia factor in tumour cells. The results provide direct evidence for LIF as a major cachexia initiating factor for the C26 tumour in vivo. Tumour‐derived LIF was also a regulator of multiple cytokines in C26 tumour cells and in C26 tumour‐bearing mice. The identification of tumour‐derived factors such as LIF that initiate the cachectic process is immediately applicable to the development of therapeutics to treat cachexia. This is a proof of principle for studies that when carried out in human cells, will make possible an understanding of the factors causing cachexia in a patient‐specific manner.This work was supported by NIAMS R01AR060217 to S. C. K. and R. W. J. and NIAMS R01 R01AR060209 to A. R. J., and by the Dudley Allen Sargent Research Fund. The authors certify that they comply with the ethical guidelines for publishing in the Journal of Cachexia, Sarcopenia and Muscle: update 2017.40 (R01AR060217 - NIAMS; R01 R01AR060209 - NIAMS; Dudley Allen Sargent Research Fund)Published versio

Expression of HLA-G by mast cells is associated with hepatitis C virus-induced liver fibrosis.

International audienceBACKGROUND AND AIMS: Infection by hepatitis C virus is a worldwide health problem. An inadequate Th2 cytokine response promotes the fibrosis-cirrhosis fate. Immune-modulating molecules favoring a Th2 profile, such as HLA-G molecules of the HLA class Ib family, may play a role in chronic hepatitis. HLA-G contributes to the escape of tumors, and their involvement in viral infections has been increasingly described. The aim of this work was to study the expression of HLA-G in the liver, its cellular source and its regulation in cases of chronic C hepatitis. METHODS: HLA-G cells in blocks of liver derived from patients infected with HCV were labeled by immunohistochemistry and enumerated. Double immunofluorescence allowed the identification of the cellular source. HLA-G secretion by a human mast cell line was quantified by ELISA after various stimulations. After treatment with IFN-α real-time PCR was performed to determine the kinetics of cytokine expression profiles, followed by heat map clustering analysis. RESULTS: The number of HLA-G + cells was significantly associated with the area of fibrosis. For the first time, we identify the HLA-G+ cells as being mast cells. HLA-G secretion was significantly induced in human mast cells stimulated by IL-10 or interferons of class I. The transcriptome of the secretome of this cell line stimulated by IFN-α revealed that i) the HLA-G gene is upregulated late, ii) T lymphocytes and NK cells are recruited. CONCLUSIONS: These findings suggest an autocrine loop in the genesis of HCV liver fibrosis, based on mast cells expressing HLA-G

Altered calcium influx of peripheral Th2 cells in pediatric Crohn’s disease: infliximab may normalize activation patterns

OBJECTIVE: Crohn's disease is a chronic inflammation of the gastrointestinal tract with an abnormal immune phenotype. We investigated how intracellular calcium kinetics of Th1 and Th2 lymphocytes alter upon specific inhibition of Kv1.3 and IKCa1 channels in pediatric Crohn's disease. STUDY DESIGN: Blood was taken from 12 healthy and 29 Crohn's disease children. Of those, 6 were switched to infliximab and re-sampled after the 4th infliximab treatment. Intracellular calcium levels were monitored using flow cytometry in the presence or absence of specific inhibitors of Kv1.3 and IKCa1 potassium channels. RESULTS: In Crohn's disease treated with standard therapy, calcium response during activation was higher than normal in Th2 cells. This was normalized in vitro by inhibition of Kv1.3 or IKCa1 potassium channels. After the switch to infliximab, potassium channel function and expression in Th2 lymphocytes were comparable to those in Th1 cells. CONCLUSION: These results may indicate that potassium channels are potential immune modulatory targets in Crohn's disease

Invariant NKT Cells Drive Hepatic Cytokinic Microenvironment Favoring Efficient Granuloma Formation and Early Control of Leishmania donovani Infection

The development of inflammatory granulomas around infected Kupffer cells is necessary for hepatic parasite clearance during visceral leishmaniasis. Invariant NKT (iNKT) cells are predominant T cells in the mouse liver and can synthesize large quantities of IL-4 and IFN-γ, two cytokines involved in granuloma formation. This study analyzed the role of iNKT cells in the hepatic immune response during Leishmania donovani infection, using a murine model of wild-type (WT) and iNKT cell-deficient (Jα18-/-) C57BL/6 mice sacrificed 15, 30 or 60 days post-infection. We recorded hepatic parasite loads, cytokine expression, and analyzed granulomatous response by immunohistochemistry and hepatic immune cell infiltration by flow cytometry. Whereas WT animals rapidly controlled the infection and developed an inflammatory response associated with a massive influx of iNKT cells observed by flow cytometry, Jα18-/- mice had significantly higher parasitic loads on all time points. This lack of control of parasite burden was associated with a delay in granuloma maturation (28.1% of large granulomas at day 60 versus 50.7% in WT). Cytokine transcriptome analysis showed that mRNA of 90/101 genes encoding chemokines, cytokines and their receptors, was underexpressed in Jα18-/- mice. Detection of IL-4 and TNF-α by ELISA in liver extracts was also significantly lower in Jα18-/- mice. Consistent with flow cytometry analysis, cytokinome profile in WT mice showed a bias of expression towards T cell-chemoattractant chemokines on D15, and displayed a switch towards expression of granulocytes and/or monocytes -chemoattractant chemokines on D60. In Jα18-/- mice, the significantly lower expression of CXCL5, MIP-2 and CCL2 mRNA was correlated with a defect in myeloperoxidase positive-cell attraction observed by immunohistochemistry and with a lower granulocyte and monocyte infiltration in the liver, as shown by flow cytometry. These data indicate that iNKT cells play a role in early and sustained pro-inflammatory cytokine response warranting efficient organization of hepatic granulomas and parasite clearance

The humoral pattern recognition receptor PTX3 is stored in neutrophil granules and localizes in extracellular traps

The long pentraxin (PTX) 3 is produced by macrophages and myeloid dendritic cells in response to Toll-like receptor agonists and represents a nonredundant component of humoral innate immunity against selected pathogens. We report that, unexpectedly, PTX3 is stored in specific granules and undergoes release in response to microbial recognition and inflammatory signals. Released PTX3 can partially localize in neutrophil extracellular traps formed by extruded DNA. Eosinophils and basophils do not contain preformed PTX3. PTX3-deficient neutrophils have defective microbial recognition and phagocytosis, and PTX3 is nonredundant for neutrophil-mediated resistance against Aspergillus fumigatus. Thus, neutrophils serve as a reservoir, ready for rapid release, of the long PTX3, a key component of humoral innate immunity with opsonic activity

Long-Lasting Immune Responses 4 Years after GAD-Alum Treatment in Children with Type 1 Diabetes

A phase II clinical trial with glutamic acid decarboxylase (GAD) 65 formulated with aluminium hydroxide (GAD-alum) has shown efficacy in preserving residual insulin secretion in children and adolescents with recent-onset type 1 diabetes (T1D). We have performed a 4-year follow-up study of 59 of the original 70 patients to investigate long-term cellular and humoral immune responses after GAD-alum-treatment. Peripheral blood mononuclear cells (PBMC) were stimulated in vitro with GAD65. Frequencies of naïve, central and effector memory CD4+ and CD8+ T cells were measured, together with cytokine secretion, proliferation, gene expression and serum GAD65 autoantibody (GADA) levels. We here show that GAD-alum-treated patients display increased memory T-cell frequencies and prompt T-cell activation upon in vitro stimulation with GAD65, but not with control antigens, compared with placebo subjects. GAD65-induced T-cell activation was accompanied by secretion of T helper (Th) 1, Th2 and T regulatory cytokines and by induction of T-cell inhibitory pathways. Moreover, post-treatment serum GADA titres remained persistently increased in the GAD-alum arm, but did not inhibit GAD65 enzymatic activity. In conclusion, memory T- and B-cell responses persist 4 years after GAD-alum-treatment. In parallel to a GAD65-induced T-cell activation, our results show induction of T-cell inhibitory pathways important for regulating the GAD65 immunity

T-cell and serological responses to Erp, an exported Mycobacterium tuberculosis protein, in tuberculosis patients and healthy individuals

<p>Abstract</p> <p>Background</p> <p>The identification of antigens able to differentiate tuberculosis (TB) disease from TB infection would be valuable. Cellular and humoral immune responses to Erp (Exported repetitive protein) – a recently identified <it>M. tuberculosis </it>protein – have not yet been investigated in humans and may contribute to this aim.</p> <p>Methods</p> <p>We analyzed the cellular and humoral immune responses to Erp, ESAT-6, Ag85B and PPD in TB patients, in BCG⁺individuals without infection, BCG⁺individuals with latent TB infection (LTBI) and BCG^-controls. We used lymphoproliferation, ELISpot IFN-γ, cytokine production assays and detection of specific human antibodies against recombinant <it>M. tuberculosis </it>proteins.</p> <p>Results</p> <p>We included 22 TB patients, 9 BCG⁺individuals without TB infection, 7 LTBI and 7 BCG^-controls. Erp-specific T cell counts were higher in LTBI than in the other groups. Erp-specific T cell counts were higher in LTBI subjects than TB patients (median positive frequency of 211 SFC/10⁶PBMC (range 118–2000) for LTBI subjects compared to 80 SFC/10⁶PBMC (range 50–191), p = 0.019); responses to PPD and ESAT-6 antigens did not differ between these groups. IFN-γ secretion after Erp stimulation differed between TB patients and LTBI subjects (p = 0.02). Moreover, LTBI subjects but not TB patients or healthy subjects produced IgG3 against Erp.</p> <p>Conclusion</p> <p>The frequencies of IFN-γ-producing specific T cells, the IFN-γ secretion and the production of IgG3 after Erp stimulation are higher in LTBI subjects than in TB patients, whereas PPD and ESAT-6 are not.</p