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Title: Role of TLR receptors in reversion of immune tolerance induced by epicutaneous aplication of antigen

Abstract:

It is commonly known that antigen deposition on the mucosa, e.g. mucosa of gastrointestinal tract, results in local mucosal immunity but induction of a state of peripheral tolerance. For many years, the skin was considered to be an organ where immune responses such as contact hypersensitivity (CS) were easily induced. However, skin as a site for the induction of tolerance has received very little attention. Because skin and mucosa have similar roles in our bodies, it is possible that under definied conditions, epicutaneous application of an antigen may induce a strong local CS response as well as induce peripheral tolerance. Experiments performed at the Department of Human Developmental Biology at Jagiellonian University School of Medicine showed that indeed, epicutaneous application of a protein antigen prior to sensitization with a hapten resulted in significant inhibition of CS reaction to the hapten. The inhibition of the skin response was shown to be mediated by TCRαβ+ CD4+ CD8+ T suppressor cells (Ts). Both in vivo and in vitro studies revealed that skin induced Ts cells are antigen non-specific. The mechanism of skin induced suppression relies on the action of the anti-inflammatory cytokine TGF-β. Employing animal models of multiple sclerosis and rheumatoid arthritis it was shown that skin induced suppression resulted in significant inhibition of inflammatory process and subsequent amelioration of disease. Moreover, skin induced suppression was found to effectively delay allogeneic graft rejection. Therefore, skin induced immune suppression may be a new therapeutic strategy to treat inflammatory disease. However, the lack of antigen specificity of skin induced tolerance gives rise to the risk that generalized immunosuppression might also inhibit helpful anti-microbial responses. In contrast to the experimental models described above, where purified protein antigen applied alone to induce tolerance, adminitration of antigen along with pathogen associated molecular patterns (PAMPs) recognized by pattern recognition receptors (PRRs) e.g. TLR may play a crucial role not only in the induction of an immune response but also in overcoming tolerance. In this dissertation, the role of TLR ligands in reversing skin induced immune suppression is described. Experiments performed using a model of CS in CBA/J mice showed that antigen non-specific suppression induced via epicutaneous immunization with protein antigen could be reversed when animals were simultaneously immunized with protein antigen and PAMPs. As was shown skin induced contrasuppression could be achieved after epicutaneous immunization with protein antigen TNP-Ig together with bacterial lysates (Staphylococcus aureus, Staphylococcus epidermidis oraz Propionibacterium acnes) or complete Freund’s adjuvant containing Mycobacterium tuberculosis. Similar effects were observed after epicutaneous immunization with purified TLR ligands: TLR2 (peptidoglycan, lipoteichoic acid and zymosan A), TLR3 (dsRNA – polyI:C), TLR4 (LPS) and TLR9 (ODN CpG). Reversal of skin-induced suppression by TLR ligands is dose dependant and the optimal working dose of TLR4 ligand (LPS ) is 100 ng/mouse. Based on the experiments described above, the most commonly used TLR4 ligand, LPS from E. coli, was used in subsequent experiments. Reversal of suppression was shown to not be strain dependent and can be induced independently on MHC haplotype, as was demonstrated by using mouse strains other than CBA/J (H-2k) e.g. BALB/c (H-2d) and C57Bl/6 (H-2b) Reversal of skin induced immunosupression is dependent on functional TLR4 receptor and the adaptor protein MyD88, as LPS did not overcome tolerance in C3H/HeJ (TLR4-mutant mice) and MyD88-/- (mice lacking an adaptor protein MyD88) mice. Adoptive cell transfer experiments showed that reversal of suppression by LPS is via the induction of contrasuppressor cells in axillary and inginual lymph nodes. ; Negative selection experiments with monoclonal antibodies and rabbit complement revealed that these cells are TCRαβ+ CD4+ lymphocytes (contasuppressor T cell, Tcs). Experiments in an active immunization and adoptive transfer of CS employing three non-crossreacting antigens TNP-Ig, OX-Ig and OVA showed that contrasuppression induced via epicutaneous immunization with an antigen and LPS is antigen specific. In the final section of this dissertation, the mechanism of contrasuppression induced via epicutaneous application of TNP-Ig with LPS is discussed. Four different approaches were used: measurement of cytokine production in vitro, induction of Tcs cells in IL-6-/-, IL-12-/-, IFN-γ-/- and control mice (adoptive transfer of CS), neutralization of IFN-γ at the time of induction of T effector, Tcs and Ts cells (adoptive transfer of CS) and replacement of Tcs by rIL-12 (adoptive transfer of CS). These experiments showed that contrasuppression is IFN-γ and IL-12 dependent, whereas IFN-γ is required for induction of Tcs cells that activate antigen presenting cells to release IL-12, which protects T effector cells from the action of Ts cells. This dissertation describes a novel method to reverse skin induced suppression via epicutaneous immunization with an antigen and PAMP. Because of its effectiveness, ease of induction and non invasiveness, this route of immunization may be effective for new vaccines and anti-cancer therapy.

Level of degree:

2 - studia doktoranckie

Degree discipline:

immunologia

Degree grantor:

Wydział Lekarski

Promoter:

Marian Szczepanik

Date issued:

2007

Format:

application/pdf

Identifier:

oai:dl.cm-uj.krakow.pl:1018

Call number:

ZB-107976

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tylko w bibliotece

Location of original object:

Biblioteka Medyczna Uniwersytetu Jagiellońskiego - Collegium Medicum

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Last modified:

Jun 27, 2019

In our library since:

Nov 21, 2012

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All available object's versions:

http://dl.cm-uj.krakow.pl:8080/publication/1018

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ZB-107976 Jun 27, 2019
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