IN HUMAN MYOBLASTS
2 The IL-17 and TNF α α cooperation enhances the hepatic and muscle inflammatory response
2.1 IL-17 and TNFα cooperate to enhance the expression and/or secretion of pro-inflammatory mediators
IL-17 alone is often poorly active. However, IL-17 can synergize with other cytokines such as TNFα, IL-1β or IFNγ, leading to increased production of pro-inflammatory mediators such as IL-6 and IL-8 by various cell types including synoviocytes or endothelial cells (Beringer et al., 2016; Chabaud et al., 1998; Hot et al., 2012). Nevertheless, the effects of the IL-17 and TNFα cooperation have not been investigated on liver and muscle cells whereas these two cytokines are involved in several liver and muscle disorders or systemic inflammatory diseases with hepatic and muscle complications (Beringer and Miossec, 2018; De Bleecker et al., 1999; Kuru et al., 2000; Schwabe and Brenner, 2006; Tournadre and Miossec, 2012). In this study, IL-17 and TNFα cooperate to promote systemic inflammation by increasing IL-6 secretion, which in turn, enhances the CRP expression in hepatocytes. IL-17 amplifies also the TNFα effect on the induction of IL-8, CCL20 and MCP-1 chemokine expression and/or secretion mainly through an IL-6 independent pathway in hepatocyte and LX-2 HSC cultures.
By promoting the neutrophil-attracting chemokine IL-8, the IL-17 and TNFα combination may have a crucial role in the initiation of liver inflammation. These in vitro results are consistent with the in vivo murine models of liver injury showing that IL-17 deletion or neutralization reduces neutrophil accumulation as well as the levels of inflammatory cytokines in liver (Furuya et al., 2015; He et al., 2013; Tan et al., 2013). It would be therefore interesting to investigate whether blocking both IL-17 and TNFα induce a further decrease of the immune cell recruitment and the release of pro-inflammatory mediators. In myoblast cultures, IL-17 and TNFα enhance also in synergy the IL-6 and CCL20 release. Since CCL20 attracts Th17 cells and IL-6 is involved in Th17 cell differentiation, the IL-17 and TNFα synergistic cooperation may contribute to the local Th17 cell induction in the liver and the muscles by increasing the IL-6 and CCL20 production.
2.2 Mechanisms of the IL-17 and TNFαα synergistic effects
Although the IL-17 and TNFα synergistic effects have been well described in several cell types, the mechanisms of this interaction on the IL-6 and IL-8 induction are not well established. Here, in HepaRG cells, first exposure to IL-17, but not to TNFα, is crucial for the initiation of the IL-17 and TNFα synergistic cooperation. Various mechanisms may act at several levels: at a receptor level, at a promoter level and at a post-transcriptional level. In synoviocytes, 17 up-regulated the TNFR2 and the blockade of its receptor reduced the IL-17 and TNFα synergistic cooperation on the CCL20 production (Zrioual et al., 2009). In HepaRG cells, IL-17 and TNFα together but not IL-17 alone induces the TNFR2 mRNA expression. The regulation of the TNFR2 appears therefore different between synoviocytes and HepaRG cells. Moreover, the TNFR2 mRNA lower in HepG2 and Huh7.5 cells than in HepaRG cells could explain the lack of response of the HepG2 and Huh7.5 cells to the IL-17 and TNFα synergistic effect on the IL-6 and IL-8 secretion. This data suggest that the TNFR2 could have a key role in the HepaRG cell inflammatory response induced by IL-17 and TNFα although the TNFR2 is preferentially activated by the transmembrane form of the TNFα (as seen in part 1.3.2). The use of a specific blocking anti-TNFR2 antibody could be useful to confirm its involvement in the IL-17 and TNFα cooperation.
Part of the IL-17 and TNFα cooperation may also occur at a gene transcription level.
Transcription factor-binding sites for NF-κB and C/EBP (CCAAT/enhancer-binding protein) in the IL-6 promoter were both involved in the IL-17 and TNFα synergistic effect on IL-6 in an osteoblastic cell line (Ruddy et al., 2004). The up-regulation of C/EBPδ by the IL-17 and TNFα stimulation and the increase of C/EBPδ recruitment to the promoter by TNFα may participate to the IL-17 and TNFα cooperative effect on the IL-6 induction (Gaffen et al., 2014; Ruddy et al., 2004; Zimmermann et al., 2015). In addition, by enhancing IκBζ mRNA expression in synergy, IL-17 and TNFα may induce a further increase of NF-κB activation since IκBζ acts as a NF-κB co-activator (Karlsen et al., 2010; Sparna et al., 2010;
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Post-transcriptional regulation is important to control cellular transcript abundance and, in turn, the levels of secreted proteins. IL-17 can increase the stability of mRNAs induced by the TNFα stimulation such as IL-6 and IL-8 mRNA (Hartupee et al., 2007; Henness et al., 2004, 2006). In this study, IL-17 enhances the IL-6 mRNA stability in HepaRG cells. As the 3’-untranslated region of the IL-6 mRNA contains adenylate and uridylate (AU)-rich elements, IL-17 may promote the binding of stabilizing AU-binding proteins over that of destabilizing AU-binding proteins, prolonging IL-6 mRNA half-life (Chowdhury et al., 2013). This IL-17 effect on the mRNA stabilization could therefore contribute to the IL-17 and TNFα synergistic effect on the IL-6 production.
Multiple mechanisms at different levels appear therefore involved in the IL-17 and TNFα synergistic effect. The contribution of these different mechanisms differs from one cell type to another as seen with the TNFR2. Moreover, in the same cell type, the mechanism involved varies between the gene or mRNA targets of IL-17 and TNFα. Indeed, NF-κB pathways participate in the secretion of IL-6 induced by the IL-17 and TNFα cooperation but not in the IL-8 secretion in HepaRG cells. In this way, the IL-17 and TNFα synergistic effect on the IκBζ up-regulation, which intensifies NF-κB activation, cannot be involved in the IL-17/TNFα-induced IL-8 production in HepaRG cells.
2.3 Potential contribution of the IL-17 and TNFαα synergistic cooperation in systemic inflammatory diseases
This study shows that the effects of TNFα at low concentration can be potentiate by IL-17 in the liver and the muscles. These results strengthen the potential important systemic role of 17 and TNFα in autoimmune and inflammatory diseases. However, the concentrations of IL-17 and TNFα used in our in vitro systems may no reflect the in vivo local concentrations in organs or tissues. Moreover, the range of cytokines is limited in our monocultures, which reduces the number and the diversity of the interactions between cytokines. Indeed, because cytokines act in networks, IL-17 and TNFα can interact with other cytokines to mediate additive, antagonistic or synergistic effects. For example, IL-17 can synergize with other cytokines such as IL-1β, IFNγ, IL-22 or GM-CSF, in addition to TNFα, to promote
inflammation (Beringer et al., 2016). For this reason, a cell-based assay has been developed at the laboratory to detect circulating bioactive IL-17 instead of circulating IL-17 levels to select the patients more susceptible to respond to an anti-IL-17 therapy (Ndongo-Thiam and Miossec, 2015).