A single subcutaneous injection of pristane in DA rats leads to chronic relapsing arthritis similar to RA in humans. We now show that pristane injection also leads to an upregulated expression of TLRs, in particular TLR3. TLR3 was found to play a critical role in the development of arthritis as an injection of TLR3 ligand enhanced arthritis and downregulation of TLR3 through treatment with RNAi alleviated arthritis. And TLR3 expression was induced with pristane stimulation particularly in macrophages.
PIA is a T cell dependent disease, which can be transferred with αβT cells [42
], and there is no evidence so far arguing for a pathogenic role of B cells. In the human disease it is clear at present that many patients benefit from depletion of B cells through injection of antibodies to CD20. In contrast, in PIA antibodies to citrullinated proteins are not evoked and there is no evidence for pathogenic antibodies mediating the disease, unlike in the collagen-induced arthritis model. However, the immune response in PIA also shows some striking similarities with RA since rheumatoid factors and antibodies to RA33 are produced in both diseases [43
]. Importantly, a strong activation of an innate response is apparent in both RA and PIA, although in RA it has been possible to analyze the response only in already established disease. In RA synovial tissues a strong overexpression of TLR3 and 4 has been reported, whereas isolated macrophages show high expression of TLR2 and 4 [13
]. But there has been no study on TLRs in the PIA rat model so far.
A subcutaneous injection of mineral oil or phytol, structurally similar to pristane, leads to an accumulation of oil at the injection site but also a rapid spread of small volumes to draining lymph nodes and subsequently spreads systemically, reaching the spleen [44
]. Previous studies have shown that T cells isolated from spleens, less than eight days after pristane injection, transfer severe arthritis, showing that the critical arthritogenic change induced by pristane is present in the spleen [42
]. Therefore we selected the spleen to study the innate immune reaction after pristane injection.
Screening TLR mRNA expression at different time points after pristane injection, we found that expression of TLR1, 2, 3, 4 and 8 in the spleen is upregulated in the development stage of arthritis (d26), which is consistent with previous studies from arthritis models and RA patients [20
]. A compelling TLR is TLR3 as it was highly expressed uniquely in the early phase, and increased progressively with the development of the disease. It has been reported that TLR3 is overexpressed in synovial tissue from both early and longstanding RA [12
]. A possible pathogenic role of the upregulated TLR3 could be that it recognizes RNA released from necrotic synovial fluid cells, and then activates RA synovial fibroblast [14
]. TLR3 combined with TLR4, TLR7/8 is also involved in the regulation of DC activation and cytokine production in RA patients [12
]. It indicates that TLR3 in the spleen may play a vital role in PIA. To examine whether the observed TLR overexpression has functional consequences, downstream cytokines of TLR pathway, such as TNF-α, IL-6, IFN-γ and IFN-β mRNA expression in the spleen were determined. IFN-β expression was found to increase at both d6 and d26, whereas TNF-α and IL-6 expression only increased at d6, and the IFN-γ expression level did not change. These proinflammatory cytokines are known to be regulated by many TLRs via several intracellular pathways but are mainly associated with NF-κB. TLR3 and possibly TLR4 activate interferon regulatory factor 3 which regulates IFN-β expression [47
]. By flow cytometric staining we confirmed high TLR3 protein expression in splenocytes. The expression of TLR3 on splenocyte surfaces increased at d6 and d26, reflecting its mRNA expression pattern.
Based on the experimental data from the gene expression profile and TLR2, 3, 4 protein expression during the disease course, we suggest that TLR3 plays an important role in the pathogenesis of PIA. Such a role could be confirmed through in vivo
experiments with TLR3 ligands and RNAi. In a previous study, intra-articular treatment with polyI:C to rats caused a rapid and strong synovitis [48
], which suggested the important role of TLR3 signaling in arthritis pathogenesis. And in our research, the systemic treatment with polyI:C synergism with pristane showed that TLR3 ligand could exacerbate arthritis. This dominant role of the TLR3 ligand was also proved by pathology of the PIA rat ankles. The possible mechanism of polyI:C's effect on PIA could be that more TLR3 is activated by polyI:C, which triggers more severe inflammatory reaction in joints. Meanwhile, the results also emphasize the important roles of the ligand in arthritis pathogenesis. But at present, it is still not defined which endogenous ligand of TLR3 participates in PIA. Thus, priority should be given to exploring the probable ligand and investigating its roles in arthritis.
A crucial role of TLR3 in the PIA pathogenesis could also be confirmed by treatment with RNAi in vivo. The shRNA plasmids did not show any immunostimulatory effect in vivo because the shRNA-NC group shows no differences from the saline group in all indexes. The results indicated that the RNAi of TLR3 participated in the initiation of PIA significantly delayed the onset day and decreased arthritis severity. NO concentration of shRNA-TLR3 group was also significantly lower than that of the shRNA-NC and saline groups. The result validates the importance of TLR3 in PIA, however, the ligands and the functional consequences of TLR activation in PIA are not yet fully understood.
TLR3 expresses mainly on DC for humans but on macrophages for mice [49
]. It has been found that macrophages are involved in the pathology of K/BxN serum-induced arthritis [50
]. Macrophage-derived reactive oxygen species (ROS) participate in T cell selection, maturation and differentiation, and also mediate protection against arthritis by suppressing T cell activation [51
]. In addition, macrophages stimulated by excessive interleukin-15 may activate the characteristic autoreactive CD4+
T cells in RA [27
]. The increased macrophage activation may be mediated by TLRs in RA or other autoimmune inflammatory diseases [16
]. All findings indicate macrophages are a main player in the development of arthritis. We found that TLR3 positive macrophages were increased in the spleen of PIA rats, and cell surface TLR3 showed high expression in the initiation stage of arthritis. It suggests that cell surface TLR3 on macrophage, but not the intracellular expression, exerts a crucial influence on the pathogenesis of PIA.
Using a rat macrophage cell line, NR8383, we could confirm that TLR3, but not TLR4, was upregulated after stimulation with pristane, which excludes LPS contamination in the experiment. Furthermore, the mRNA expression of IFN-β, the main downstream cytokine of TLR3, also increased with the same tendency as that of TLR3. The increase of IFN-β and TNF-α was inhibited by anti-TLR3 antibody, suggesting that this induction was TLR3 dependent. The above mentioned data indicate TLR3 and its downstream molecules in macrophage were activated by pristane, and then took part in the pathogenesis of PIA. More TLR3 expression on macrophages could affect the cell function, such as the capacity of antigen presentation, ROS production, phagocytosis, secretion of cytokines and chemokines and so on. Macrophages with upregulated TLR3 may affect the balance of Th1/Th2 and the activations of autoreactive T cells and B cells [25
However, it is still unclear how pristane induces TLR3 expression. The interaction between TLR and alkanes like pristane is not completely known. It is reported that oxidized alkane polymers induced activation of the TLR1/2 pathway as its ligands. And pristane could augment the effect of TLR7 ligands but does not directly activate TLR7. In a PIA rat model, further study is needed as to whether pristane or its metabolism products are the ligand of TLR3, or if it induces TLR3 by another pathway. Further studies are also needed to understand underlying mechanisms as to how TLR3 activates joint-specific autoimmune T cells via macrophages.