Increased autophagy in fibroblast-like synoviocytes leads to immune enhancement potential in rheumatoid arthritis

Abstract

The incidence of rheumatoid arthritis (RA) has been reported to be correlated with a disorder of immunregulation. Rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) play an important role in regulating the local immune microenvironment. However, the potential mechanism of RA-FLS in regulating the immnue response is not clearly understood. In this study, we demonstrated that the expression of HIF-1α was significantly up-regulated in rheumatoid arthritis tissue which indicated that the hypoxia condition in the microenvironment. We also observed that RA-FLSs demonstrated the potential to up-regulate immune activation. Meanwhile, the level of autophagy increased in RA-FLSs compared with control group. Besides that, the expression of IL-6 was up-regulated not only in RA-FLSs but also in the fibroblasts that treated with hypoxia condition. Accordingly, we found that autophagy inhibitiors could effectively inhibit the immune activation function of RA-FLSs medicated by IL-6. Taken together, the results we demonstrated above indicated that the hypoxia microenvironment could effectively induce the incidence of autophagy and then lead to the immune activation function of RA-FLSs medicated by IL-6.

Keywords: IL-6; autophagy; fibroblast-like synoviocytes; rheumatoid arthritis.

Figure 1. The expression of HIF-1α was significantly up-regulated in rheumatoid arthritis tissue

A. IHC was employed to detect the expression of HIF-1α in rheumatoid arthritis tissues. B. Lactic acid detection assay was used to examine the hypoxic state in rheumatoid arthritis tissues. Data represent three independent experiments with SD. Statistically significant differences are shown (*P < 0.05; **P<0.01).

Figure 2. RA-FLSs demonstrated the potential to up-regulate immune activation

A-B. CCK-8 assay was used to detect the cell viability of lymphocytes. C-D. PI/Annexin V-FITC assay was used to examine the apoptosis of the lymphocytes by flow cytometry. Data represent three independent experiments with SD. Statistically significant differences are shown (*P < 0.05; **P<0.01).

Figure 3. The level of autophagy increased significantly in RA-FLSs

A. N-FLSs were transfected with GFP-tagged LC3; after 24 hours transfection, the N-FLSs cultured in hypoxia condition (1% O₂) for 8 hours and then were observe by a fluorescence microscope. RA-FLSs were transfected with GFP-tagged LC3; after 24 hours transfection, the RA-FLSs were observed by a fluorescence microscope. Arrows show the punctate GFP-LC3 in the cytoplasm (×1000). B. The average percentages of GFP-LC3II cells. C. Immunfluorescence was employed to observe the expression of p62 in RA-FLSs and N-FLSs. N-FLSs were obtained from control rat model (×1000). D. The average percentages of GFP-LC3II cells. E. Western blot were used to examine the expression of LC-3II and p62.

Figure 4. Autophagy inhibitors could effectively inhibit the immune activation function of RA-FLSs

A. CCK-8 assay was used to detect the cell viability of lymphocytes B. PI/Annexin V-FITC assay was used to examine the apoptosis of the lymphocytes by flow cytometry with or without autophagy inhibitor 3-MA and CQ. Data represent three independent experiments with SD. Statistically significant differences are shown (*P < 0.05; **P<0.01).

Figure 5. The expression of IL-6 was up-regulated not only in RA-FLSs but also in the fibroblasts that treated with hypoxia condition

A. ELISA was employed to detect the expression of IL-6 in the conditioned mediums obtained from RA-FLSs and N-FLSs; B and D. CCK-8 assay was used to detect the cell viability of lymphocytes with or without inhibiting the expression of IL-6 by shRNA in RA-FLSs; C and F. PI/Annexin V-FITC assay was used to examine the apoptosis of the lymphocytes by flow cytometry with or without inhibiting the expression of IL-6 by shRNA in RA-FLSs. Data represent three independent experiments with SD. Statistically significant differences are shown (*P < 0.05; **P<0.01, NS means no significant).

Figure 6. Autophagy mediated the up-regulation of IL-6 in RA-FLSs

A and C. Realtime PCR was used to detect the expression of IL-6 in N-FLSs and RA-FLSs; B and D. ELISA was employed to detect the expression of IL-6 in the conditioned mediums obtained from RA-FLSs and N-FLSs Data represent three independent experiments with SD. Statistically significant differences are shown (*P < 0.05; **P<0.01, NS means no significant).