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Title: Evaluation ofnegative regulation of immune response induced via epicutaneous application of antigen in animal model of rheumatoid arthritis (CIA)


Rheumatoid arthritis (RA) an example of an autoimmune disease. While not uncommon, with a prevalence of 1% worldwide, the pathogenesis of RA is not clear yet. At present it is thought that the pathogenesis of RA is the result of an inflammatory response mediated by CD4+ Th1 cells that recognize unidentified antigens that are present in joints. Following antigen-induced activation, self-specific Th1 cells release cytokines that initiate inflammatory processes in the joint. This leads to polimorphonuclear and macrophage accumulation and subsequent destruction of cartilage and erosion of bone with extra-articular manifestations. Recently, there is a growing evidence for a role for Th17 lymphocytes in autoimmunity including RA and that this population of helper cells may be more important to the pathogenesis of RA than Th1 cells. Research on autoimmune disease pathogenesis, and development and of new drugs is possible thanks to animal models that mimic human diseases. Collagen-induced arthritis (CIA) in genetically prone strains of mice, rats, rabbits or rhesus monkeys has been used as an experimental model of RA, as they share many histological and immunological characteristics. CIA can be induced by subcutaneous (s.c.) injection of collagen II in complete Freund’s adjuvant (CFA). There are many drugs that are used in therapy of autoimmune diseases including RA. It is noteworthy that, apart from drugs as gold salts, sulphasalazine, corticosteroids, there are medicines that interfere with immune processes. These include cyclosporine A, antibodies that neutralize TNF-α or IL-1R antagonists. However, most of these inhibit immune response non-specifically and cause many side effects. For many years there have been numerous efforts to create a vaccine that can protect from autoimmune diseases including RA. One possible method relies on the induction of oral tolerance. In an animal model it was shown that feeding with collagen induced strong tolerance in the periphery, resulting in significantly reduced symptoms of CIA. Similar observations were also made in experimental autoimmune encephalomyelitis (EAE), another autoimmune disease mediated by Th1/Th17 lymphocytes. However, clinical trials in humans showed no benefit in RA or multiple sclerosis patients fed with collagen or myelin basic protein, respectively. Although skin is considered to be an organ where immune responses are easily induced, little attention has been given to skin induced tolerance. Wang, et. al. showed that epicutaneous (EC) application of the protein antigen OVA resulted in allergic dermatitis accompanied by the appearance of IL-4 secreting cells. In addition, Herrick, et al. showed that EC immunization with protein antigen induced a Th2-mediated animal model of asthma. These data may suggest that, similar to mucosal exposure, EC immunization could induce peripheral tolerance when special conditions are fulfilled. Previous work carried out in the Department of Human Developmental Biology, Jagiellonian University College of Medicine both in CS and EAE models showed that EC immunization with protein antigen strongly suppressed the induction of the T cell mediated immune response. Further work employing allogeneic skin grafts showed that EC immunization with a protein antigen delays graft rejection. In the current study, It was investigated if EC immunization with a soluble protein antigen via a gauze skin patch could ameliorate the inflammatory response during CIA. It was shown that EC immunization with collagen II (COLL II) prior to induction of CIA reduced disease severity in susceptible DBA/1 mice. The optimal dose of COLL II that inhibits CIA was found to be between 30 and 100 μg/animal. The decrease in disease severity correlated with milder histological changes, reduced MPO activity in joint tissue, decreased production of pathogenic anti-type II collagen IgG2a antibody and reduced level of anti-cyclic citrulinated peptide (anti CCP) IgG antibodies. ; Further experiments showed that suppressive activity can be adoptively transferred by cells isolated from the inguinal and auxiliary lymph node cells and spleens of EC immunized mice. Suppression could be transferred with as 4x105 peripheral lymph node cells. Further, the effects of skin-induced suppression was found to decline with time. However, neither mesenteric lymph node cells or thymocytes could transfer EC induced tolerance. Depletion experiments using anti-TCRβ or anti-TCRδ monoclonal antibodies (mAb) and complement revealed that EC induced suppressor cells belong to the population of TCRαβ+ lymphocytes. To identify the phenotype of EC induced regulatory cells more precisely, total lymph node cells from mice EC tolerized with COLL II were depleted of either CD4 or CD8 T cells by treatment with mAb and complement prior to transfer into syngeneic DBA/1 recipients. Protection was abrogated when either CD4 or CD8 T cells were depleted. Combining CD4-depleted lymphocytes with CD8-depleted lymphocytes, thereby reconstituting the populations of single-positive cells, did not restore protection from disease, suggest that EC-induced T regulatory cells co-express both CD4 and CD8 coreceptors. In summary, these data suggest that EC immunization with COLL II induces a population of regulatory T cells with a TCRαβ+ CD4+ CD8+ phenotype. Surprisingly, suppression was not specific to antigen, as EC immunization with any tested protein antigen (COLL II, OVA or MBP) prior to induction of CIA reduced disease severity. To determine the mechanism of EC induced suppression in CIA, the expression of pro- and anti-inflammatory cytokines by lymph node cells isolated from treated mice was investigated. These experiments showed that EC immunization with COLL II does not affect production of IL-4, IL-6, IL-10, IL-17E, TGF-β or IFN-γ when compared to DPBS-treated animals. However, production of IL-17A and TNF-α was significantly increased. In vivo neutralization of IL-17 abolished EC induced protection from the disease, which correlated with increased MPO activity and upregulated production of anti-COLL II IgG2a antibodies. In the final part of the study, it was shown that transfer of EC induced T regulatory cells either before or after induction of CIA suppressed disease. Moreover, EC treatment of mice with COLL II after first signs of CIA caused partial recovery from disease, suggesting that this method might be useful to treat ongoing disease. In Summary, induction of immune tolerance via EC immunization could potentially be used to prevent and possibly treat RA and other autoimmune disorders. The noninvasive application of protein antigen to the skin and the ability to induce antigen nonspecific T regulatory cells makes EC immunization an attractive potential therapy.

Level of degree:

2 - studia doktoranckie

Degree discipline:

choroby narządów ruchu

Degree grantor:

Wydział Nauk o Zdrowiu


Marian Szczepanik

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Biblioteka Medyczna Uniwersytetu Jagiellońskiego - Collegium Medicum

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Oct 15, 2019

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Nov 21, 2012

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ZB-113858 Oct 15, 2019


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