IL-17-mediated mitochondrial dysfunction impairs apoptosis in rheumatoid arthritis synovial fibroblasts through activation of autophagy

Abstract

Fibroblast-like synoviocytes (FLSs) are a major cell population of the pannus that invades cartilage and bone in rheumatoid arthritis (RA). FLS resistance to apoptosis is a major characteristic of RA. The aims of this study were to investigate the effects of interleukin-17 (IL-17) and IL-17-producing T helper (Th17) cells on resistance to apoptosis in FLSs from RA patients (RA FLSs) and their roles in mitochondrial dysfunction and autophagy. Mitochondrial function was assessed in RA FLSs and FLSs from osteoarthritis patients (OA FLSs). FLSs were treated with IL-17 and their morphological features, respiratory level and mitochondrial gene expression were measured. The effects of IL-17 and Th17 cells on the relationship between autophagy and apoptosis were evaluated by measuring the expression of apoptosis-related genes using sodium nitroprusside or 3-methyladenine. The mitochondria of FLSs isolated from RA and osteoarthritis patients displayed different morphological and physiological features. RA FLSs exhibited greater autophagosome formation and greater dysfunction of mitochondrial respiration compared with OA FLSs. IL-17 induced mitochondrial dysfunction and autophagosome formation in RA FLSs, suggesting that they were resistant to apoptosis. Autophagy-related antiapoptosis induced by IL-17 was restored by inhibition of autophagy, suggesting a relationship between mitochondrial dysfunction and cell survival in RA FLSs. Th17 cells and IL-17 increased autophagy of RA FLSs by causing mitochondrial dysfunction. Our findings suggest that, in RA, interactions between RA FLSs and Th17 cells may be involved in the tumorous growth of FLSs and the formation of pannus in joints.

Figure 1

Mitochondrial dysfunction increases in RA FLSs. (a) Transmission electron microscopy images of FLSs from OA and RA patients. Arrowheads indicate mitochondria. Scale: 2 μm (upper panels), 0.5 μm (lower panels). (b) OA and RA FLSs were immunostained with MitoTracker Red CMXROS (red), anti-α-tubulin (green), and DAPI (nuclei, blue). quantification of perinuclear mitochondrial distance in confocal micrographs of OA and RA FLSs. Data represent means±S.D. of five different culture experiments. The mitochondrial membrane potential was measured using JC-1 dye. (c) Confocal microscopy and (d) flow cytometry analysis. A representative density plot shows the OA and RA FLSs. (e) OCR in OA and RA FLSs (data are representative of three experiments). Quantification of mitochondrial respiration in OA and RA FLSs. The data are expressed as the mean±S.D. (or S.E.M.). *P<0.05, **P<0.01, ***P<0.001

Figure 2

Pathogenesis of RA is mediated by IL-17. FLSs were cultured with IL-17 at concentrations of 0, 2 or 10 ng/ml for 12 h (n=5). (a) FLSs were immunostained with MitoTracker Red CMXROS (red), anti-α-tubulin (green) and DAPI (nuclei, blue), and the perinuclear mitochondrial distance was quantified in confocal micrographs. A representative density plot shows the RA FLSs. Data represent the mean±S.D. ***P<0.001. Mitochondrial membrane potential was measured using JC-1 dye for confocal microscopy (b) or flow cytometry analysis (c)

Figure 3

IL-17 disrupts mitochondrial respiration in RA FLSs. (a) The expression of OxPhos complex subunits in FLSs was analysed by western blotting. A representative figure is shown. (b) OCR in FLSs in the presence or absence of IL-17 (10 ng/ml). The data are representative of three independent experiments. Quantification of mitochondrial respiration in FLSs. (c) The expression level of mitochondrial OxPhos genes were determined using RT-qPCR in FLS incubated in the presence or absence of IL-17 (10 ng/ml) for 12 h. The data are expressed as the mean±S.D. (or S.E.M.). *P<0.05, **P<0.01, ***P<0.001

Figure 4

Th17 disrupts mitochondrial respiration in RA FLSs. FLSs isolated from RA patients were co-cultured with or without Th17 cells for 12 h. (a) The mitochondrial membrane potential was measured using JC-1 dye and confocal microscopy. Green indicates decreased membrane potential. (b) Mitochondria were immunostained with MitoTracker Red CMXROS (red) and confocal microscopy images were captured. (c) The OCR was measured in FLSs co-cultured with or without supernatant collected from Th17 cells or from Th17 cells that had been co-cultured with FLS. Data are representative of three experiments. Quantification of mitochondrial respiration in RA FLSs. (d) Relative gene expression associated with mitochondrial OxPhos in RA FLSs. The data are expressed as the mean±S.D. (or S.E.M.). *P<0.05, **P<0.01, ***P<0.001

Figure 5

IL-17 induces autophagy by causing mitochondrial dysfunction in RA FLSs. (a) RA FLSs were cultured in the presence or absence of IL-17 (10 ng/ml) and transmission electron microscopy was performed. Arrowheads indicate autophagosomes. The right panels show magnified autophagosomes in RA FLSs cultured in the presence of IL-17. The number of autophagosomes in FLSs was quantified on the electron microscopy images. The data in the graph on the right are expressed as mean±S.D. of five different experiments. (b) OA FLSs and RA FLSs were immunostained for the autophagosome marker with anti-LC3 antibody and analysed by confocal microscopy. Scale: 20 μm. (c) Expression of LC3 in RA FLSs cultured with 3-MA (1 mM) in the presence or absence of IL-17 (10 ng/ml) was analysed by Western blotting. The LC3-II/LC3-I ratio was quantified. (d) The gene expression of ATG5 or LC3 associated with autophagy in FLSs cultured in the presence or absence of IL-17 (10 ng/ml) was quantified using RT-qPCR. The data are expressed as the mean±S.D. *P<0.05, **P<0.01, ***P<0.001

Figure 6

Induction of IL-17-mediated autophagy increases antiapoptosis in RA FLSs. (a) Apoptosis was induced by treatment of FLSs with 1 mM SNP. The degree of apoptosis was assessed by flow cytometry using propidium iodide and Annexin V staining. (c) Inhibition of autophagy with 3-MA decreased the expression of BCL-2 in RA FLSs in the presence of IL-17 (10 ng/ml). FLSs from RA were immunostained using anti-BCL2 antibody as the antiapoptosis marker and analysed by confocal microscopy. Scale: 20 μm. (b, d) The viability of synoviocytes were determined using a CCK-8 assay kit. (e) RT-qPCR was performed to quantify the relative expression of genes associated with apoptosis or antiapoptosis in RA FLSs cultured in the presence or absence of IL-17 (10 ng/ml) for 12 h. The data are expressed as the mean±S.D. *P<0.05, **P<0.01, ***P<0.001