Toll-like receptor 3 upregulation in macrophages participates in the initiation and maintenance of pristane-induced arthritis in rats

Abstract

Introduction: Toll-like receptors (TLRs) are involved in both innate and adaptive immune responses and are likely to play a complex role in the pathogenesis of human rheumatoid arthritis (RA) and experimental arthritis. The objective of this study was to identify the key TLR in pristane-induced arthritis (PIA), a rat model for RA, and to clarify its roles in the initiation and maintenance of arthritis.

Methods: Arthritis in DA rats was induced by pristane and the severity was evaluated by macroscopic and microscopic score systems. Spleen TLR and cytokine expression was detected at different time points by real-time polymerase chain reaction (PCR) and flow cytometry. Polyinosine-polycytidylic acid (polyI:C, a ligand of TLR3) or TLR3 specific short-hairpin RNA plasmid for RNA interference was administrated to PIA rats in vivo. Serum nitrogen oxide concentration was determined by Griess method, and tumor necrosis factor alpha (TNF-alpha) was determined by L929 biotest. In splenic macrophages, TLR3 expression was measured by flow cytometry. A rat macrophage cell line (NR8383) was stimulated by pristane, and anti-TLR3 antibody were used to block TLR3 pathway. TLR3 and cytokine expression in NR8383 were detected by real-time PCR.

Results: By screening the TLR expression profile in spleen of DA rats after pristane injection, we found that TLR3 was the most early and prominently upregulated TLR. Both TLR3 mRNA and protein expression of spleen were upregulated at 6 and 26 days after pristane injection. Furthermore, administration of polyI:C exacerbated, whereas RNA interference targeting TLR3 ameliorated, the arthritis. Particularly, TLR3 expression was induced in splenic macrophages of PIA rats, and also in the NR8383 cell line after pristane stimulation in a dose- and time- dependent manner. Upregulation of interferon beta (IFN-beta) and TNF-alpha by pristane stimulation was blocked by anti-TLR3 antibody in NR8383.

Conclusions: TLR3 plays a pivotal role in the initiation and development of PIA which may dependent on macrophage. These findings are useful to understand the pathogenesis of RA and may provide an intriguing therapeutic opportunity for RA.

Figure 1

TLR and cytokine expression profile of spleen from DA rats with pristane-induced arthritis. The mRNA expression (a) of TLR1-9 at d0, 6 and 26, (b) of TLR3 at d0, 2, 6, 12 and 26, (c) and of cytokines at d0, 6 and 26 in spleen of rats was measured by Real-time quantitative PCR (n = 8 to 10 for each time point). Relative mRNA expression was compared with β-actin. Protein expression of TLRs in spleen was detected by FACS at d0, 6 and 26 (n = 3 for each time point). The positive splenocyte proportion (d) of cell surface and intracellular TLR3, and cell surface TLR2 and TLR4 is showed in the course of arthritis. Data were collected after correction for isotype-matched IgG control. Values are shown as means ± SEM. Levels of significance between the pristane treated group (6, 26 days) and untreated group (0 day) were calculated by using Mann-Whitney U test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Figure 2

More severity, higher serology index and increased histopathological scores of PIA caused by polyI:C treatment. The arthritis clinical indexes (a) including clinical score, max clinical score, onset day, mid-paw perimeter, ankle perimeter; and serum NO and TNF-α concentration at d26 (b) were compared among the polyI:C, LPS and saline groups. (c), In representative histological images of ankle joints, the ankle joints in the control group and PIA groups treated with saline or ligands were stained by H&E. (d), For the histological analysis of synovitis, joint destruction, new bone and cartilage formation and the total histological index, the various scores were quantified among PIA groups treated with saline and ligands. Data are presented as means ± SEM. * represents the comparison between ligands treated groups and saline group in A and D, or between ligands treated groups and control group in B. But ^# between ligands treated groups as marked. Levels of significance were calculated using Mann-Whitney U test (n = 9 for each group, */^# P < 0.05, **/^## P < 0.01, ***/^### P < 0.001).

Figure 3

Attenuated severity and reduced and a serum NO concentration of PIA caused by RNAi of TLR3 in vivo. Four TLR3-shRNA plasmids and a negative control plasmid were transfected into NR8383 cells. mRNA ((a), 24 hours after transfection) and protein ((b), 48 hours after transfection) expression levels of TLR3 were detected by RT-PCR and FACS respectively, and shRNA3-TLR3 (shRNA-TLR3) shows the best RNAi efficacy. The plasmids of shRNA-TLR3 and shRNA-NC were used to treat PIA rats, and GFP mRNA expression (c) in plasmid treatment groups except in the saline group was successfully amplified by RT-PCR. (d), TLR3 expression of the spleen at d20 was detected by Real-time quantitative PCR. Relative mRNA expression was compared with the housekeeping gene β-actin. The arthritis clinical indexes (e) including clinical score, max clinical score and onset day; and serum NO concentration at d26 (f) were compared among three group PIA rats treated with saline, shRNA-NC and shRNA-TLR3, respectively. Data are presented as means ± SEM. * represents the comparison between the shRNA treated group and the saline group, and ^# between the shRNA-TLR3 and shRNA-NC groups. Levels of significance were calculated by using Mann-Whitney U test (n = 8 for each group, *^/# P < 0.05).

Figure 4

TLR3 expression was induced in macrophages with pristane stimulation. In splenic macrophages, TLR3 protein expression was detected by FACS at 0, 6 and 26 days after pristane injection. The proportion and mean fluorescence intensity (MFI) of cell surface and intracellular TLR3 were detected on His36⁺ (anti-rat macrophage) gated TLR3⁺ cells from splenocytes, and representative histograms (a) are showed in the course of arthritis. Data were collected after correction for isotype-matched IgG control. NR8383 cells were stimulated with a series of concentrations of pristane (0.1 μ, 1 μ, 10 μ, 100 μ, 1 m, 10 mM as theoretical pristane concentration), and TLR3 and TLR4 mRNA expression was detected at 48 hours after stimulation (b). The expression of TLR3 increased with a raised dosage of pristane (r² = 0.78) by Pearson correlation analysis. With 100 μM pristane stimulation, TLR3 (c) and IFN-β, TNF-α (d) mRNA expression was measured at 0, 8, 24, 48 and 72 hours. (e) NR8383 pretreated with anti-TLR3 or isotype control antibodies were stimulated by 100 μM pristane or 10 μg/ml polyI:C, and IFN-β, TNF-α mRNA expression was measured at 24 hours. mRNA expression level was detected by Real-time quantitative PCR, and data are presented as means ± SEM of four replicated determinations from three independent experiments. * represents the comparison with the control group (Blank in B and E, and 0 hour in C and D), and levels of significance were calculated by using Student's T test (* P < 0.05, ** P < 0.01, *** P < 0.001).