There is increasing evidence that release of endogenous nucleic acids may trigger autoimmune reactions crucially involved in the induction of systemic autoimmune diseases such as SLE or rheumatoid arthritis (RA). In recent years, endosomal Toll-like receptors (TLRs, i.e. TLR3, TLR7, TLR8 and TLR9) have been implicated in autoimmune processes due to their ability to recognize these nucleic acids.
To study the role of TLR7 and TLR9 in the pathogenesis of erosive arthritis by antagonizing these TLRs in rats with pristane-induced arthritis (PIA).
First, the inhibitory capacity of immunoregulatory sequences (IRS) known to antagonize TLR7 and/or TLR9 activation was investigated in cultured rat splenocytes by measuring production of pro-inflammatory cytokines. Subsequently, using the PIA model, these IRS were also tested for their efficiency to inhibit arthritis development in rats with PIA. The IRS’ were applied twice a week subcutaneously at the base of the tail, a non-inhibitory IRS and PBS served as control substances. Weight changes were measured during the experiment and arthritis was assessed using an established scoring system. Expression of TLRs was analyzed in paws, lymph nodes and spleen by Western blotting, RT-PCR and immunohistochemistry. Further, the impact of antagonizing TLR7/9 in osteoclastogenesis was analyzed by performing in vitro osteoclast assays.
IRS specific for TLR7, TLR9 or TLR7/9 inhibited in a dose-dependent manner production of pro-inflammatory cytokines in rat splenocytes pre-activated by TLR specific stimulators. However, neither the TLR7 specific inhibitor nor the inhibitor targeting both TLR7 and TLR9 showed an effect on incidence and severity of PIA. Remarkably however, antagonizing TLR9 solely led to delayed disease onset and reduced arthritis severity, which was accompanied by diminished TLR9 protein expression levels in paws and lymph nodes compared to placebo-treated control animals. Moreover, bone erosion was largely reduced in animals treated with the TLR9 antagonist. Furthermore, inhibition of TLR9 but not of TLR7 in an in vitro osteoclast formation assay diminished osteoclastogenesis significantly in a dose-dependent manner.
Our in-vitro and in-vivo results indicate a potential involvement of TLR9 not only in the initiation of inflammatory arthritis but also in the later phase of inflammatory bone loss pointing toward a hitherto unknown role for TLR9 in the regulation of osteoclast activity.
Disclosure: S. Herman, None; A. Fischer, None; M. Hoffmann, None; G. Steiner, None.