Blockade of Store-Operated Calcium Entry Reduces IL-17/TNF Cytokine-Induced Inflammatory Response in Human Myoblasts

Muscle inflammation as in idiopathic inflammatory myopathies (IIM) leads to muscle weakness, mononuclear cell infiltration, and myofiber dysfunction affecting calcium channels. The effects of interleukin-17A (IL-17) and tumor necrosis factor-α (TNFα) on inflammation and calcium changes were investigated in human myoblasts. Human myoblasts were exposed to IL-17 and/or TNFα with/without store-operated Ca2+ entry (SOCE) inhibitors (2-ABP or BTP2). For co-cultures, peripheral blood mononuclear cells (PBMC) from healthy donors activated or not with phytohemagglutinin (PHA) were added to myoblasts at a 5:1 ratio. IL-17 and TNFα induced in synergy CCL20 and IL-6 production by myoblasts (>14-fold). PBMC-myoblast co-cultures enhanced CCL20 and IL-6 production in the presence or not of PHA compared to PBMC or myoblast monocultures. Anti-IL-17 and/or anti-TNFα decreased the production of IL-6 in co-cultures (p < 0.05). Transwell system that prevents direct cell-cell contact reduced CCL20 (p < 0.01) but not IL-6 secretion. IL-17 and/or TNFα increased the level of the ER stress marker Grp78, mitochondrial ROS and promoted SOCE activation by 2-fold (p < 0.01) in isolated myoblasts. SOCE inhibitors reduced the IL-6 production induced by IL-17/TNFα. Therefore, muscle inflammation induced by IL-17 and/or TNFα may increase muscle cell dysfunction, which, in turn, increased inflammation. Such close interplay between immune and non-immune mechanisms may drive and increase muscle inflammation and weakness

Interleukin-25 Produced by Synoviocytes Has Anti-inflammatory Effects by Acting As a Receptor Antagonist for Interleukin-17A Function

The production and function of cytokines are highly regulated. One mechanism is the balance between pro- and anti-inflammatory cytokines. As interleukin (IL)-17A and IL-25 share the IL-17RA receptor chain, we hypothesize that IL-25 acts as an IL-17A receptor antagonist and limits its pro-inflammatory effects. The production and expression kinetics of IL-25 and its receptor chains IL-17RA and RB were analyzed in rheumatoid synoviocytes alone or in coculture with peripheral blood mononuclear cells (PBMCs). The effects of autocrine or exogenous IL-25 on synoviocytes were investigated in the presence or not of an anti-IL-25 antibody. To study the regulatory effects of IL-25, synoviocytes and/or PBMCs were exposed to IL-25 before being treated with IL-17A and tumor necrosis factor alpha (TNF-α) alone or combined. IL-25, IL-6, and bioactive IL-17A were quantified in rheumatoid arthritis (RA) patient plasma. Synoviocytes expressed and secreted IL-25, and expressed the two chains of its receptor IL-17RA and IL-17RB. IL-17RB expression was increased by TNF-α. IL-25 production occurred at a delayed time point (5 days) after stimulation with IL-17A and TNF-α. Synoviocytes pretreated with IL-25 were less responsive to IL-17A and TNF-α. PBMCs exposed to IL-25 showed a decreased production of pro-inflammatory mediators, including IL-17A with a 57% decrease; p = 0.002. IL-25 levels were elevated in the plasma of RA patients compared to healthy subjects (p = 0.03). However, these levels are not high enough to inhibit the function of circulating IL-17A. In conclusion, it was shown for the first time that synoviocytes produce IL-25, specifically at late time points and that IL-25 acts as a regulator of IL-17A-driven inflammation, as indicated by in vitro results and in vivo, in a long-term RA patient follow-up. These results may be important when considering IL-17A inhibition

Drug-resistant and immune-escape HBV mutants in HIV-infected hosts.

International audienceHIV-HBV-coinfected patients require optimal control of viral replication in order to prevent the development of severe comorbidities, such as liver cirrhosis and hepatocellular carcinoma. The genetic diversity of HBV is a poorly investigated factor of such viral replication in HIV-infected hosts. HBV genome diversity can be differentiated into two major aspects: genotypic and phenotypic. Genotypic diversity is more related to the natural history of HBV infection and genotypes are mostly determined by geographical origin of the hosts. Phenotypic diversity arises from attempts to escape from host immune surveillance (that is, precore, core and basal core promoter mutants), selection resulting from the use of treatments with weak genetic barrier (that is, pol mutants), exposure to hepatitis B immunoglobulin (that is, 'immune-escape' S gene mutants) or treatment-induced mutations from overlapping genes (that is, pol mutants inducing 'vaccine-escape' S gene mutants). pol mutations typically lead to uncontrolled viral replication, whereas S gene mutations can significantly alter hepatitis B surface antigen synthesis and reduce binding to antibodies, which renders individuals who are vaccinated or cured of HBV infection susceptible to infection. For patients coinfected with HIV, hepatitis B treatment options that aim to reduce the risk of HBV mutations from emerging must be seriously considered, not only from clinical but also public health perspectives

Blockade of store-operated calcium entry reduces IL-17/TNF cytokine-induced Inflammatory response in human myoblasts

International audienceMuscle inflammation as in idiopathic inflammatory myopathies (IIM) leads to muscle weakness, mononuclear cell infiltration, and myofiber dysfunction affecting calcium channels. The effects of interleukin-17A (IL-17) and tumor necrosis factor-alpha (TNF alpha) on inflammation and calcium changes were investigated in human myoblasts. Human myoblasts were exposed to IL-17 and/or TNF alpha with/without store-operated Ca2+ entry (SOCE) inhibitors (2-ABP or BTP2). For co-cultures, peripheral blood mononuclear cells (PBMC) from healthy donors activated or not with phytohemagglutinin (PHA) were added to myoblasts at a 5:1 ratio. IL-17 and TNF alpha induced in synergy CCL20 and IL-6 production by myoblasts (> 14-fold). PBMC-myoblast co-cultures enhanced CCL20 and IL-6 production in the presence or not of PHA compared to PBMC or myoblast monocultures. Anti-IL-17 and/or anti-TNF alpha decreased the production of IL-6 in co-cultures (p < 0.05). Transwell system that prevents direct cell-cell contact reduced CCL20 (p < 0.01) but not IL-6 secretion. IL-17 and/or TNF alpha increased the level of the ER stress marker Grp78, mitochondrial ROS and promoted SOCE activation by 2-fold (p < 0.01) in isolated myoblasts. SOCE inhibitors reduced the IL-6 production induced by IL-17/TNF alpha. Therefore, muscle inflammation induced by IL-17 and/or TNF alpha may increase muscle cell dysfunction, which, in turn, increased inflammation. Such close interplay between immune and non-immune mechanisms may drive and increase muscle inflammation and weakness

06.01 Intra-articular injection of cadmium protects arthritic joints from inflammation and destruction

Background There has been no recent progress in the intra-articular treatment of joint inflammation. Rheumatoid arthritis (RA) synovium hyperplasia is sustained by the secretion of pro-inflammatory cytokines (IL-17/TNF-α), synergistically contributing to chronicity. Since inflammation up-regulates trans-membrane Zinc (Zn) importers, the effects of its binding-competitor Cadmium (Cd) were tested on synoviocytes, synovium explants and in a rat arthritis model in order to reduce hyperplasia and inflammation. Materials and methods After exposure to IL-17/TNF-α and Cd, Cd-kinetics and Cd-cell content were measured by ICP-MS, while Zn/Cd-transporter gene expression (Zrt-Irt-like protein-8, ZIP-8, importer and metallothioneins-1, MT-1, metal homeostasis regulators) by q-RT-PCR. Synoviocyte viability and apoptosis were measured by neutral red and annexin-V staining. IL-6 levels in synoviocyte and biopsy supernatants were measured by ELISA. Adjuvant induced arthritis rat model was used for in vivo Cd-injection into hind ankle joints. Clinical scores were evaluated. Immune cell recruitment was quantified after H and E staining. Micro-tomography and safarin-O staining were used to measure bone/cartilage loss. The potential Cd-spread was measured in different body reservoirs. Results After synoviocyte exposure to IL-17/TNF-α combination, ZIP-8 and MT-1s gene expressions increased up to 5.3\textpm3.1 fold and 5.0\textpm0.9 fold respectively, compared to the untreated condition (p0.05). Combined Cd-cytokine exposure further enhanced MT-1s expression up to 93.3\textpm32.1 fold. Through the transporter enhanced expression, Cd content in inflammatory synoviocytes increased two-fold. RA synoviocytes were sensitised toward apoptosis by exposure to the Cd/cytokine combination with an 80% reduction of cell viability in comparison to control (p0.01), after 5 days of culture. Moreover, Cd-cytokines association reduced IL-6 production in vitro (up to 83%, after 5 days, p0.05) and ex-vivo (up to 94%, after 8 days, p0.01). Intra-articular Cd injection improved arthritis, reducing clinical scores (arthritic score reduced from 4 to 2, p0.01), inflammatory cell recruitment (up to 50%, p0.01) and bone/cartilage destruction. The use of 1 ppm of Cd provided the best risk/benefit ratio, without toxic effects on other cell types and organs. Conclusion The anti-proliferative and anti-inflammatory properties of low-dose Cd may represent a new therapeutic approach for the local treatment of synovitis and hyperplasia in arthritis and other joint diseases

Protective effect of low dose intra-articular cadmium on inflammation and joint destruction in arthritis

International audienceSynovium hyperplasia characterizes joint diseases, such as rheumatoid arthritis (RA). The cytotoxic effect of low-dose Cadmium (Cd) was tested in vitro and ex vivo on synoviocytes, the mesenchymal key effector cells of inflammation and proliferation in arthritis. The anti-inflammatory and anti-proliferative effects of Cd were tested in vivo by intra-articular injection in the adjuvant induced arthritis rat joints, where the clinical scores and the consequences of arthritis were evaluated. Cell death through apoptosis was highly induced by Cd in inflammatory synoviocytes (80% reduction of cell viability, p \textless 0.01). TNF plus IL-17 cytokine combination induced a two-fold increase of Cd cell content by enhancing the ZIP-8 importer and the MT-1 homeostasis regulator expression. Addition of Cd reduced IL-6 production in TNF plus IL-17-activated synoviocytes (up to 83%, p \textless 0.05) and in ex-vivo synovium biopsies (up to 94%, p \textless 0.01). Cd-injection in rat joints improved arthritis, reducing clinical scores (arthritic score reduced from 4 to 2, p \textless 0.01), inflammatory cell recruitment (up to 50%, p \textless 0.01) and protecting from bone/cartilage destruction. This proof of concept study is supported by the limited Cd spread in body reservoirs, with low-dose Cd providing a safe risk/benefit ratio, without toxic effects on other cell types and organs