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Summary Hepatic stellate cells (HSCs) have a central role in liver inflammation and fibrosis by producing inflammatory and fibrotic mediators. Their activation is regulated through direct cell–cell interactions, but also through systemic and local effects of soluble factors such as cytokines. The effects of the proinflammatory cytokines interleukin (IL)-17 and tumor necrosis factor (TNF)-α and cell interactions with hepatocytes on HSC activation were assessed. Human HSC and HepaRG cells were exposed to IL-17 and/or TNF-α. IL-17 and TNF-α contribution from immune cells was determined in a co-culture model with phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMC), HSC and/or hepatocytes. IL-17 enhanced TNF-α effects on the induction of IL-6, IL-1β, and the chemokine IL-8, chemokine (C-C motif) ligand 20 (CCL20) and monocyte chemoattractant protein-1 (MCP-1) expression/secretion in isolated HSC cultures. HSC–hepatocyte interactions did not enhance IL-6, IL-8 and CCL20 production compared to hepatocyte alone. However, HSC–hepatocyte interactions increased C-reactive protein expression. IL-17 and/or TNF-α had no direct profibrotic effects on collagen 1 α1, tissue inhibitor of matrix metalloproteinase (TIMP) and matrix metalloproteinase (MMP) 2 gene expression, whereas mRNA levels of MMP3, an enzyme involved in matrix destruction, were up-regulated in HSCs. The use of specific inhibitors of IL-17 and TNF-α indicated their contribution to the strong increase of IL-6 and IL-8 production induced by PBMC, HSC and/or hepatocyte interactions. As chronic liver inflammation leads to liver fibrosis, IL-17 and/or TNF-α neutralization can be of interest to control liver inflammation and therefore its effects on fibrosis.

Les mécanismes des maladies inflammatoires font intervenir les cytokines principales de d’inflammation IL-1, TNF, IL-6 et IL-17, qui sont les cibles thérapeutiques des biothérapies. Elles contribuent à l’atteinte locale de maladies différentes sur le plan clinique. A côté de ces aspects locaux, les cytokines ont des effets systémiques par leur action sur le foie, le tissu adipeux, le muscle et le système cardio-vasculaire. Toutes ces maladies inflammatoires ont en commun une augmentation du risque cardio-vasculaire. Dans la population générale, les mêmes concepts sont applicables, avec un lien entre une élévation même modeste de la CRP et le risque cardio-vasculaire. Plus récemment, la tempête cytokinique des formes sévères du COVID-19 a montré que les interactions synergiques des cytokines d’abord décrites in vitro se retrouvent renforcées dans le tableau clinique avec atteinte multiple et sévère des organes clés. Dans ces contextes, le contrôle de l’inflammation par ciblage des cytokines est une nouvelle option thérapeutique à visée vasculaire, avec déjà des résultats importants pour l’IL-1.

Career situation of first and presenting author Assistant. Introduction Dendritic cells (DCs) are central regulators of the balance between immunity and tolerance, and alteration of the specialized DCs system is a common feature in chronic inflammatory diseases, such as rheumatoid arthritis (RA). In RA, synovial tissues are characterized by aberrant distribution of DCs populations, with altered phenotype. However, an in-depth characterization of the altered phenotype and functions of DCs in RA has been hampered by a lack of specific DCs markers and relevant in vitro culture model. Objectives To perform an in-depth phenotypic characterization of DCs found in RA joints with an extensive panel of monoclonal antibodies specific of DCs, and to establish a relevant in vitro culture model to generate DCs resembling to those found in joints. Methods Flow cytometry was used for analysis of cell surface molecules expressed on DCs found in RA synovial fluid (SF). Staining was performed with a panel of 100 mouse monoclonal antibodies specific of DCs, generated by immunization with different subsets of human DCs. Phenotype of DCs from synovial fluid was then compared to the phenotype of monocytes co-cultured in vitro with synoviocytes from RA patients, in presence or not of synovial fluid, or cytokines. Results In RA SF patients, two main DCs subsets were identified, with distinct surface protein phenotypes. One subset was characterized by markers of plasmacytoid DCs (CD303, CD123), whereas the other subset expressed markers of myeloid/conventional DCs (CD11c, DCIR). This last subset of DCs expressed some markers found on inflammatory DCs, the main inducers of Th17 cells in RA joints. Surprisingly, the two subsets of DCs expressed also surface proteins shared by synoviocytes. Culture of human monocytes with synoviocytes and/or combination of pro-inflammatory cytokines (TNF, M-CSF, IL-4) yielded DCs with a phenotype close to the phenotype of conventional DCs found in RA synovial fluid. Conclusions We uncovered previously unreported phenotypic heterogeneity of DCs in a chronic inflammatory context, and characterized two main subsets of DCs. The next steps will be to understand the consequences of this heterogeneity on the pathogenicity of the disease. This will be facilitated by our relevant in vitro culture model that allows the generation of DCs, closely resembling those found in joints of patients with RA. Disclosure of Interest None declared.

Career situation of first and presenting author Assistant. Introduction Cryoglobulins (CG) are immunoglobulins (Ig) that precipitate in vitro at cold temperature and dissolve at 37°C, they are classified in 3 types. Type I CG are monoclonal Ig of IgM or IgG isotype. Type II and III are mixed CG: type II CG associate a monoclonal with polyclonal Ig, type III CG associate polyclonal Ig. Rheumatoid factor activity (RF) of mixed CG leads to the formation of immune complexes that can precipitate and result in vasculitis. Objectives The purpose of this study was to update information on CG biological characteristics in a large population recruited from all clinical departments of a French University Hospital. Methods This retrospective study was conducted from January 2010 to December 2016. Patients included had at least one serum sample for CG detection and characterization: CG isotype and clonality, concentration and RF (IgM anti-IgG) in the cryoprecipitate; and serum complement exploration. Associated pathologies were determined by biological markers of gammopathy, viral infection and autoimmunity. Results A total of 13439 patients were included, 1675 (12,5%) of whom had positive CG. In case of negative CG detection, 2213 patients were retested and CG was detected on the new sample for 196/2213 patients (8.9%). Type I CG was found in 9.3% (155/1675), type II CG in 47% (788/1675), and type III CG in 43.7% (732/1675) of patients. In type I CG, IgM CG was more frequent than IgG CG, but in lower concentration (p=0.02). For mixed CG, 34.8% of HCV+ tested patients had CG, less than 5% of CG was associated with HBV+ or HIV+ serology. Mixed CG were found in 25.4% of patients with anti dsDNA, anti-SSA60 or anti-CCP autoantibodies (Ab). Mixed CG were positive for 87/333 (26.1%) patients with anti-dsDNA Ab, 74/447 (16.6%) patients with anti-SSA60 Ab, and for 19/155 (12.3%) patients with anti-CCP Ab. Both the cryoprecipitate and the serum were positive for RF in 21.6% of type II CG and 10.1% of type III CG. C3, C4 and/or CH50 decrease was found in 23.6% of serum with CG vs 3.2% of CG-negative serum (p Conclusions In this largest cohort even studied, with patients from all clinical specialties, CG distribution and characteristics were described with minimal selection bias. Despite strict pre-analytical conditions, negative CG detection must be repeated according to clinical context. Mixed CG are more frequently detected in patients positive for HCV or anti-dsDNA Ab. CG with RF activity form immune complexes that precipitate in vessels and activate complement system, responsible for cryoglobulinemic vasculitis. Disclosure of Interest None declared.

Career situation of first and presenting author Post-doctoral fellow. Introduction IL-17A has effects on a growing number of cell types, including synoviocytes, skin fibroblasts or endothelial cells. IL-17A is now a therapeutic target in several inflammatory diseases, including psoriasis, PsA and SPA. IL-17F is the closest member of IL-17A in the IL-17 family and it shares IL-17A biological activities. It is now of interest to target both IL-17A and F. Furthermore, IL-17A and IL-17F can synergize with other cytokines such as TNF or IL-1. Objectives The aim of this study is to compare in the same experiments, the effect of IL-17A and IL-17F, in combination or not with TNF and IL-1β, on pro-inflammatory cytokine production (IL-6 and IL-8) first by RA synoviocytes, then extended to other cell types. Methods Cells (RA synoviocytes, psoriatic skin fibroblasts, human umbilical vein endothelial cells, myoblasts and hepatocytes) were cultured in presence or not of: IL-17A alone, IL-17F alone, TNF alone, IL-1β alone or combinations, IL-17A/TNF, IL-17A/IL-1β, IL-17A/TNF/IL-1β, IL-17F/TNF, IL-17F/IL-1β IL-17F/TNF/IL-1β. After 48 hour of culture, supernatants were recovered and IL-6 and IL-8 levels were measured by ELISA. A data analysis of microarrays, done on synoviocytes, was realized. Results IL-17A and IL-17F alone increased significantly both IL-6 and IL-8 production by synoviocytes, with a higher efficiency for IL-17A. For IL-6, TNF and IL-1β alone had the largest effect on myoblasts, increasing 5 fold the secretion, while for IL-8 it was on skin fibroblasts, increasing at least 5 fold the production. The synergy between IL-17A and TNF was observed on all cells for IL-6 secretion, and for IL-8, only endothelial cells did not show a synergistic effect. For IL-17F and TNF, except endothelial cells, a synergy was also observed, however less powerful than IL-17A/TNF. The effect of the combination IL-17A/IL-1β or IL-17F/IL-1β was more dependent on the cell type, with an additive effect for synoviocytes, and a synergistic effect for hepatocytes. The effect of combination of the three cytokines could induce an additive effect as for synoviocytes or a synergistic effect as for skin fibroblasts. The analysis of microarrays showed 1346 regulated probes, with a measured modulation of more than 2 fold, and 279 probes more than 4 fold, with a stronger effect of IL-17A/TNF combination on cell signalization compared to IL-17F/TNF or each cytokine alone. Conclusions IL-17A and IL-17F act in a similar way on the different cell types, by inducing pro-inflammatory cytokine secretion. The difference was the intensity of the response, where IL-17A induced a higher effect than IL-17F. IL-17A and IL-17F activities were potentiated by the combination with TNF and IL-1β. The effect of these combinations on cytokine production was dependent on cell types. These differences may explain different response to treatment according to the primary cell type involved in disease. Disclosure of Interest None declared.