Inhibition of adjuvant-induced arthritis by nasal administration of novel synthetic peptides from heat shock protein 65

Rheumatoid arthritis (RA) is a systemic autoimmune disease, characterized by the presence of inflammatory synovitis and destruction of joint cartilage and bone. This disease leads to severe pain and reduces life expectancy. The underlying immunologic mechanisms and etiologic factors responsible for the initiation and development of RA are not yet fully understood, but it is well established that inflammation and bone destruction plays crucial roles in RA. RA is specifically characterized by enhanced production of tumor necrosis factor (TNF)-alpha, interferon-gamma (IFN) - gamma, interleukin (IL)-6 and IL-17, and in the affected joints [1–3]. Therefore, therapeutic agents which possess anti-inflammatory and immunosuppressant activity will provide important benefits and promises in the treatment of RA. Non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin and celecoxib, represent an effective therapy for treating RA. Disappointingly, this kind of medicine has side-effects including gastric ulcer and the cardiovascular risk, which has limited its use [4–6]. Therefore, there is still an unmet need for improved therapies for RA.

Heat shock proteins (HSPs) are ubiquitous housekeeping proteins found in virtually all living organisms. HSPs have been a subject of expanding interest in human and experimentally induced autoimmune diseases, both as potential antigens and as intracellular chaperones involved in peptide binding to human leucocyte antigens. Previous studies have reported that nasal administration of mycobacterial HSP60 peptide induces highly effective protection against adjuvant-induced arthritis (AA), an experimental arthritis model with close histopathologic resemblance to RA [7, 8]. Notably, our recent study has demonstrated that the synthetic peptides from HSP65 inhibit pro-inflammatory cytokine secretion by peripheral blood mononuclear cells from RA patients [9].

However, there is no scientific evidence to prove the effectiveness of the synthetic peptides from HSP65 on RA in the vivo model. In the present study, we employed AA rat model and treated the rats with Heat shock protein- derived peptide (HP). To investigate the therapeutic effect of these peptides, we detected arthritis severity, footpad swelling, and anti-inflammatory effects of these peptides.

RA is characterized by synovial inflammation, destruction of cartilage and bone, severe joint pain, and ultimately life-long crippling. In the present study, the synthetic peptides derived from HSP65 were studied for their efficacy on RA treatment employing an adjuvant-induced arthritis rat model, which is extensively used for studies regarding the pathogenesis of autoimmune arthritis as well as for the validation of the efficacy of anti-rheumatic drugs because it has various similarities to human RA [12–14]. Administrations of three peptides from the day of disease onset significantly inhibited the arthritic clinical scoring and paw swelling. We also observed dampening effects on TNF-alpha concomitant with dramatic increases in IL-4 production in the serum after peptides treatment, which was relevant to inhibiting arthritic progression. These findings were positively consistent with the results of histological examinations, which reduced cellular infiltration and synovium hyperplasia in the peptide-treated groups.

The disease onset in rats commenced from day 10 and reached maximum on day 28 in the adjuvant induced (disease control) arthritic rats. The synthetic peptides from HSP65 efficiently inhibited the progression of AA with a significant reduction of the clinical score. The most important index for evaluation of arthritis is the footpad swelling, and these peptides showed the ability to inhibit the paw swelling of AA rats. Inflammatory cellular infiltration and prominent synovial hyperplasia were observed in H&E tissue sections of arthritic control animals. In contrast, the histopathological changes were restored to normal stage after giving treatment with three peptides.

Pro-inflammatory cytokines TNF-alpha, IL-1bata, and IFN-gamma produced by macrophages and other immune cells are of decisive importance in the pathogenesis and progression of RA [1–3]. Among the T cell, the T-helper (Th1) cells secrete TNF- alpha and IFN-gamma, whereas the counter-regulatory Th2 cells secrete IL-4 and IL-10 [7]. The role of Th1-Th2 balance plays an important role in regulation of autoimmunity [15, 16]. In view of the critical role of pro-inflammatory cytokines in RA, scientists intend to design drugs for suppression of this disease via anti-inflammation. There is substantial evidence demonstrating the importance of TNF-alpha in the inflammatory process of RA [9, 17], and this cytokine is better value to assess the degree of the arthritis and also good targets for treating the disease. Drugs that block pro-inflammatory cytokines TNF-alpha can improve outcomes for RA [18]. However, the anticytokine biologics are costly in the treatment of RA [19]. In the current study, the serum levels of TNF-alpha in arthritic rats were dramatically up-regulated as compared to control rats and these levels were markedly reduced after treatment with the synthetic peptides. Of note, HP-R2 and HP-R3 also increased levels of IL-4, resulting in an overall anti-inflammatory effect. IL-4 has been identified as a crucial anti-inflammatory cytokine in RA [20–23]. It is important to detect other cytokines such as IFN-gamma and IL-10. Our recent study has demonstrated that the secretion of IFN-gamma by peripheral blood mononuclear cells from RA patients was significantly suppressed by these synthetic peptides [9]. The peptides tended to increase IL-10, although the effect did not reach statistical significance. It was suggested that these peptides conferred the ability in treatment of RA via the inhibition of inflammation response.

It has been shown that disease-protective peripheral T cell tolerance can be induced through oral or nasal administration of an antigen [8]. Successful experiments in RA rat models have shown promising results using HSP peptides to modulate the arthritis disease [8, 24, 25]. Based on previous studies [25, 26] and our current results, the administration of HSP65 appears to generate suppressor or regulatory T cells that may down-regulate the autoimmune responses by the release of suppressor cytokines, and as a result, the inflammation is suppressed. Compared with orally induced tolerance, nasally induced tolerance has been shown to be equally or, in the case of a peptide antigen, even more effective in curbing experimental autoimmune diseases [8, 27]. Similar to oral tolerance, three distinct mechanisms (e.g., clonal deletion, clonal anergy, and active suppression) are possibly operational in nasal induced tolerance [24, 27].

Recent study has demonstrated that an altered peptide ligand derived from HSP60 induces the activation of T cells by modifying cell cycle phase’s distribution of CD4+ T cells from RA patients [26]. Thus, CD4+ T cells may be involved in the immunomodulatory effects elicited by the three HSP peptides in the present study. Further investigations are clearly required to detect the immunophenotype of the immune cells and elucidate which cells are HSP65 targets.

In conclusion, the results of the present study suggest that synthetic peptides containing a T cell-dominant epitope could efficiently inhibit the progression of AA with a significant reduction of the clinical and histopathogic score. This effect was associated with a suppression of TNF-alpha production and an increase of IL-4 production, which may indicate the presence of a Th2 type tolerogenic mechanism. The present study has led to the identification of a promising peptide candidate for RA treatment. Besides the possibility to use them as a therapeutic drug, they can be used as tools to define the antigen (epitope).

These results suggest that the synthetic peptides derived from HSP65 induce highly effective protection against AA, which is mediated in part by down-regulation of inflammatory cytokines, and support the view that the synthetic peptides is a potential therapy for RA that may help to diminish both joint inflammation and destruction.