Endogenous stress proteins as targets for anti-inflammatory T cells

Abstract

Stress proteins such as heat shock proteins (Hsp) are important controllers of both cellular and immune homeostasis. Enhanced Hsp expression can be observed in virtually every inflammatory condition and has been proposed by us and others to lead to local activation of Hsp-specific anti-inflammatory T cells. Amongst the various Hsp, particularly Hsp70 is highly stress inducible. But stress inducibility of Hsp70 is decreased at increasing age. Therefore, we hypothesized in this thesis that when expression of the self-antigen is reduced, Hsp mediated immunoregulation fails and that boosting either Hsp-specific T cell responses or HSP expression itself, can restore or enhance immune homeostasis. First we explored in in vitro systems if the immune system can actually respond to enhanced Hsp70 levels. Using flow cytometry, Hsp70-specific CD4+ T cell hybridomas and reporter cell lines we found that Hsp70 is highly stress inducible in immune cells and that Hsp70-specific T cells can recognize enhanced Hsp70 expression. Furthermore, this strategy yielded the identity of several new food-derived enhancers of stress-induced Hsp70 expression, amongst them carvacrol, found in thyme and oregano. Then we proceeded to investigate the effect of boosting Hsp70-specific T cell responses with exogenous Hsp70 in the proteoglycan-induced arthritis (PGIA model). We demonstrate that intraperitoneal immunization with mycobacterial (Mt) Hsp70 suppresses PGIA. Moreover, we provide evidence for the mechanism of Hsp-specific regulatory T cells by our finding that IL-10 is essential for suppression. Since mucosal antigen administration will be an appealing way of tolerance induction, we studied the cellular basis of mucosal tolerance in the PGIA model and confirmed that also nasal administration of Mt Hsp70 ameliorates PGIA. Subsequently, the immunodominant T cell epitopes of Mt Hsp70 were identified. One of the highly conserved epitopes (peptide C1) was found to induce IL-10 and to suppress PGIA upon intranasal administration. We uncovered in addition, that this peptide is a good binder (in silico) to the human HLA and that human CD4+ T cell can recognize the peptide, illustrating the relevance for clinical application in humans. To boost Hsp70 expression directly, Hsp70 levels were manipulated with the food constituent carvacrol. Carvacrol potently enhanced stress-induced Hsp70 expression both in vitro and upon intragastric administration in vivo. Up-regulation of Hsp70 was sensed by Hsp70-specific T cell hybridomas. Also in vivo we show that carvacrol enhances Hsp70-specific T cell responses. Subsequently, intragastric administration of carvacrol suppressed PGIA and this protection could be transferred to naïve recipients with T cells, thus providing the first evidence that boosting endogenous Hsp can be translated into immune regulation. Furthermore, this data demonstrate that enhancing immune fitness through food components such as carvacrol is feasible and would allow relatively easy and safe intervention means. In summary the findings presented in this thesis show that boosting Hsp70 expression or the Hsp70-specific T cell response can modulate the immune system and enhance immune homeostasis, thereby illustrating that endogenous Hsp can function as targets for anti-inflammatory T cells, which will contribute to development of new or refinement of existing intervention strategies to treat autoimmune disease.