Soluble HMGB1 Is a Novel Adipokine Stimulating IL-6 Secretion through RAGE Receptor in SW872 Preadipocyte Cell Line: Contribution to Chronic Inflammation in Fat Tissue

International audienceLow-grade inflammation (LGI) is a central phenomenon in the genesis of obesity and insulin-resistance characterized by IL-6 in human serum. Whereas this LGI was initially thought to be mainly attributed to macrophage activation, it is now known that pre-adipocytes and adipocytes secrete several adipokines including IL-6 and participate to LGI and associated pathologies. In macrophages, HMGB1 is a nuclear yet secreted protein and acts as a cytokine to drive the production of inflammatory molecules through RAGE and TLR2/4. In this paper we tested the secretion of HMGB1 and the auto-and paracrine contribution to fat inflammation using the human preadipocyte cell line SW872 as a model. We showed that 1) human SW872 secreted actively HMGB1, 2) IL-6 production was positively linked to high levels of secreted HMGB1, 3) recombinant HMGB1 boosted IL-6 expression and this effect was mediated by the receptor RAGE and did not involve TLR2 or TLR4. These results suggest that HMGB1 is a major adipokine contributing to LGI implementation and maintenance, and can be considered as a target to develop news therapeutics in LGI associated pathologies such as obesity and type II diabetes. Citation: Nativel B, Marimoutou M, Thon-Hon VG, Gunasekaran MK, Andries J, et al. (2013) Soluble HMGB1 Is a Novel Adipokine Stimulating IL-6 Secretion through RAGE Receptor in SW872 Preadipocyte Cell Line: Contribution to Chronic Inflammation in Fat Tissue. PLoS ONE 8(9): e76039

Regulation of type I-interferon responses in the human epidermal melanocyte cell line SKMEL infected by the Ross River alphavirus

International audienceMelanocytes are melanin-producing cells and with emerging innate immune functions including the expression of antiviral interferon-type I cytokines. We herein ascertained the susceptibility of the human melanocytes to Ross River alphavirus (RRV) infection and analyzed the subsequent immune responses. We demonstrated for the first time that (1) SKMEL-28 melanocyte cell line was susceptible to RRV infection and displaying major cytopathic activities and (2) RRV interfered with the interferon-type I response by altering nuclear translocation of pSTAT1 and pSTAT2 in infected SKMEL-28. These results suggest that the human melanoma cell line SKMEL-28 is a valuable model to analyze the mechanisms involved in severe skin manifestations and melanocyte’s immunity at the portal of entry of major infection by arboviruses

Deciphering the differential response of two human fibroblast cell lines following Chikungunya virus infection

Abstract Background Chikungunya virus (CHIKV) is an arthritogenic member of the Alphavirus genus (family Togaviridae) transmitted by Aedes mosquitoes. CHIKV is now known to target non hematopoietic cells such as epithelial, endothelial cells, fibroblasts and to less extent monocytes/macrophages. The type I interferon (IFN) response is an early innate immune mechanism that protects cells against viral infection. Cells express different pattern recognition receptors (including TLR7 and RIG-I) to sense viruses and to induce production of type I IFNs which in turn will bind to their receptor. This should result in the phosphorylation and translocation of STAT molecules into the nucleus to promote the transcription of IFN-stimulated antiviral genes (ISGs). We herein tested the capacity of CHIKV clinical isolate to infect two different human fibroblast cell lines HS 633T and HT-1080 and we analyzed the resulting type I IFN innate immune response. Methods Indirect immunofluorescence and quantitative RT-PCR were used to test for the susceptibility of both fibroblast cell lines to CHIKV. Results Interestingly, the two fibroblast cell lines HS 633T and HT-1080 were differently susceptible to CHIKV infection and the former producing at least 30-fold higher viral load at 48 h post-infection (PI). We found that the expression of antiviral genes (RIG-I, IFN-β, ISG54 and ISG56) was more robust in the more susceptible cell line HS 633T at 48 h PI. Moreover, CHIKV was shown to similarly interfere with the nuclear translocation of pSTAT1 in both cell lines. Conclusion Critically, CHIKV can control the IFN response by preventing the nuclear translocation of pSTAT1 in both fibroblast cell lines. Counter-intuitively, the relative resistance of HT-1080 cells to CHIKV infection could not be attributed to more robust innate IFN- and ISG-dependent antiviral responses. These cell lines may prove to be valuable models to screen for novel mechanisms mobilized differentially by fibroblasts to control CHIKV infection, replication and spreading from cell to cell.</p

List of primers used in this study for RT-PCR on SW872 RNA.

<p>List of primers used in this study for RT-PCR on SW872 RNA.</p

HMGB1 expression and localization in the human preadipocyte cell line SW872.

<p>A) Expression of HMGB1 by RT-PCR from total RNA obtained from SW872 cell after 12h of culture, with (+) and without (−) Reverse Transcriptase (RT). B) Detection of HMGB1 protein in different cell fraction by Western blot analysis. β-actin is a cytoplasmic protein (Cy) and lamin B a nuclear protein (Nu). C) Immunofluorescence of HMGB1 in SW872 cells (X 200). Cells were incubated with mouse anti-HMGB1 primary antibody (1:200), Alexa Fluor 594 linked secondary antibody (1∶1000) and DAPI. Scale bar indicated 50 µm.</p

Secreted HMGB1 controls IL-6 production in SW872 cells.

<p>IL-6 levels were determined by ELISA from SW872 cells A) treated with increasing concentrations of recombinant HMGB1 for 5 h (n = 4) or B) treated or not with rHMGB1 with a dose of 1 µg.mL⁻¹ for 5 h in presence of either an irrelevant control rabbit IgG (CTL) or a rabbit monoclonal anti-HMGB1 (abHMGB1) (n = 4). Detection of IL-6 secretion in SW872 cells treated for 5 h with C) ethyl-pyruvate (n = 3) or D) glycyrrhizin (n = 3). All values are expressed as means +/− SD. Degrees of significance are indicated in the figure captions as follow, * p<0.05, ** p<0.01, *** p<0.001.</p

HMGB1 mediates IL-6 release through RAGE.

<p>A) TLR2, TLR4, RAGE and HMGB1 mRNA expression was assessed by RT-PCR from total RNA obtained from SW872 cell after 12h in culture, with (+) and without (−) Reverse Transcriptase (RT). B) Cell surface expression of TLR2, TLR4 and RAGE were analyzed by FACS C) SW872 cells were treated with rHMGB1 1 µg.mL⁻¹ for 5 h with control mouse IgG (CTL), TLR2 or TLR4 blocking antibody (n = 4). D) SW872 cells were treated as in (C) except that control goat IgG (CTL) or goat polyclonal anti-RAGE were used (n = 4). All values are expressed as means +/− SD and degrees of significance are indicated in the figure captions as follow, * p<0.05, ** p<0.01, *** p<0.001 and ns  =  not significant.</p

HMGB1 shRNA dowregulates IL6 secretion.

<p>HMGB1 mRNA expression or protein expression were assessed respectively by RT-PCR (A) or Western blot (B) after 12h in culture of stably SW872 infected with scrumble shRNA (Sh CTL) or HMGB1 shRNA (sh HMGB1) constructs. C) Detection of IL-6 secretion in stably SW872 infected cells with scramble shRNA (Sh CTL) or HMGB1 shRNA (sh HMGB1) constructs after 5h of media renewal (n = 3). All values are expressed as means +/− SD. Degrees of significance are indicated in the figure captions as follow *** p<0.001.</p