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

Abstract

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.

Keywords: inflammatory myopathies; interleukin-17; myoblasts; store-operated calcium entry; tumor necrosis factor-α.

Figure 1

Synergistic effect of IL-17 and TNFα on the CCL20 and IL-6 production by myoblasts. Myoblasts were treated with IL-17 (50 ng/mL) and/or TNFα (1 ng/mL) for 48 h. IL-6 and CCL20 secretion by myoblasts was quantified by ELISA (A,B). Data are the mean of 5–8 independent experiments ± SEM; ^*p < 0.05, ^**p < 0.01 vs. control untreated condition and #p < 0.05, ^**p < 0.01 vs. other inflammatory conditions.

Figure 2

IL-17 and TNFα increase ER stress and mitochondrial ROS in myoblasts. Myoblasts were treated with IL-17 (50 ng/mL) and/or TNFα (1 ng/mL) for 24 h. Expression of BiP/Grp78 protein was measured by western-blot and the band density was normalized with tubulin expression (A,B). Mitochondrial oxidative stress measurements (ROS) of human myoblasts was measured with the fluorescence intensity of CellRox Dye, using 40x objective of a confocal microscope Nikon A1r, scale bar 70 μm (C,D). Data are the mean of 4 to 7 independent experiments ± SEM, ^***p < 0.001 and ^****p < 0.0001 vs. control untreated condition.

Figure 3

PBMC-myoblast interaction induces a strong production CCL20 and IL-6. PBMC and myoblasts were cultured alone or in co-culture at a ratio of 5 PBMCs for 1 myoblast for 48 h in the presence or not of PHA (5 μg/mL). CCL20 and IL-6 secretion by myoblasts was quantified by ELISA (A–F). The contribution of direct cell-cell contact was investigated with a cell culture permeable insert (C,D). PBMCs were pre-incubated for 24 h in presence or not of PHA and then exposed or not to an anti-IL-17 antibody and/or an anti-TNFα antibody for 3 h before being added to the myoblast cultures. Data are expressed as CCL20 and IL-6 supernatant level percentages compared to the non-activated pre-incubated PBMC—myoblast co-cultures (E,F). Data are the mean of 6–14 independent experiments ± SEM; ^*p < 0.05, ^**p < 0.01 vs. control co-culture condition.

Figure 4

IL-17 and TNFα increase store-operated calcium entry. Myoblasts were treated with IL-17 (50 ng/mL) and/or TNFα (1 ng/mL). mRNA levels of STIM1 and ORAI1 at 12 h was expressed as fold changes compared to control (A,B). ORAI1 and STIM1 protein was measured by western-blot and the band density was normalized with the tubulin expression. (C–F) Representative image of STIM1 puncta in human myoblast treated with IL-17 (50 ng/mL) and TNFα (1 ng/mL) for 24 h. Image Correlation Spectroscopy (ICS) analysis of STIM1 puncta (left inset) mean density of puncta (μm2). (right inset) mean surface of puncta (puncta/μm2). Data are the mean of 3 independent experiments with cells from 3 different donors ± SEM; ^*p < 0.05 vs. control untreated condition, scale bar 3 μm (G,H). SOCE was measured by using Fura2-AM dye in human myoblasts. Cells were imaged on an epifluorescence microscope using a 40x objective. Trace is a representative measurement of SOCE in IL17/TNFα treated myoblasts. (I) Slope analysis of the SOCE (J) Data are the mean of 4–5 independent experiments ± SEM; ^*p < 0.05 and ^****p < 0.00001, vs. control untreated condition. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 5

Inhibition of SOCE reduces IL-6 production induced by the IL-17/TNFα combination. Myoblasts were stimulated with IL-17 and/or TNFα in presence or not of the SOCE inhibitor 2-APB (10, 25, and 50 μM) or BTP2 (10 and 20 μM) for 48 h. CCL20 and IL-6 secretion by myoblasts was quantified by ELISA (A,B). Data are the mean of 7 independent experiments ± SEM; ^**p < 0.01 vs. control condition [medium alone (A) and medium+DMSO (B)] and #p < 0.05, ##p < 0.01 vs. other inflammatory conditions.

Figure 6

IL-17 and TNFα mediate muscle damage and weakness through immune and non-immune pathways in myoblasts. The immune cell infiltration in IIM constitutes a local source of cytokines and promotes the cell-cell interactions. IL-17 mainly produced by Th17 cells, and TNFα act in synergy on myoblasts to increase IL-6 and CCL20 secretion. Because IL-6 is involved in the Th17 cell differentiation and CCL20 in dendritic and Th17 cell recruitment, IL-6 and CCL20 mediate a positive feedback loop promoting local IL-17 production. IL-17 and TNFα induce also non-immune pathways with ROS production, ER stress and SOCE activation. The IL-17/TNFα effect of mitochondrial dysfunction, ER stress, and SOCE activation are probably closely linked. SOCE and calcium dysregulation contribute to the IL-6 release induced by IL-17/TNFα. CCL20, chemokine (C-C motif) ligand 20; DC, dendritic cells; ER, endoplasmic reticulum; IL, interleukin; ROS, reactive oxygen species; STIM, stromal interacting molecule; TNFα, tumor necrosis factor-α.