IMMUNEREGULATORY MECHANISMS IN MURINE EXPERIMENTAL AUTOIMMUNE NEURITIS

Objectives The aim of the present work was to determine the functional role of the TLR signaling cascade in P0106-125-induced murine experimental autoimmune neuritis (EAN). Therefore, we have addressed the specific impact of TLR2 and TLR4 as well as their associated adaptor proteins MyD88 and TRIF on EAN. Methods In TLR20/0, TLR40/0, MyD880/0, and TRIF0/0 mice as well as in co-isogenic C57BL/6J mice, which served as controls, P0106-125-induced murine EAN was established. Disease prevalence and activity were determined according to a well-established score. Animals were sacrificed at indicated time points to obtain the organ samples and blood samples for further analyses: histopathology, flow cytometry, immunoassays, qRT-PCR and Western Blot. Results Imm. TLR20/0, imm. TLR40/0, imm. MyD880/0, and imm. TRIF0/0 mice, respectively, revealed significantly attenuated disease activity and mild neuritis development as compared to imm. C57BL/6 control mice. Decreased systemic levels of the pro-inflammatory cytokines IL-6, IL-12p40, and IL-17A determined in the spleens of mutant strains were the result of an impaired NF-κB activation. Prolonged recoveryobserved in TLR2-deficient mice was associated with an impaired shift of macrophages from the M1 towards the M2 phenotype, impaired Tregs development as well as an attenuated differentiation of Th2 helper cells. Conclusion The present data emphasize the functional importance of TLR2, TLR4, MyD88, and TRIF molecules in murine model of autoimmune neuritis, marking them as important modulators in the immune network. Beside, these results shed new lights on potentially novel, promising therapeutic strategies for patients with the acute axonal form of Guillain-Barré syndrome.Ciljevi Cilj rada bio je odrediti funkciju TLR signalnih puteva u modelu P0106-125-induciranog EAN-a na mišu. Stoga smo odredili individualne uloge TLR2 i TLR4 molekula, kao i njihovih pridruženih adaptorskih proteina MyD88 i TRIF, u razvoju bolesti. Metode Eksperimentalni autoimuni neuritis uspostavljen je aplikacijom P0106-125 peptida u TLR20/0, TLR40/0, MyD880/0 i TRIF0/0 miševa ko-izogeničnih sa C57BL/6 miševima. P0106- 125–imunizirani C57BL/6 miševi bili su kontrolna skupina. Prevalencija i aktivnost bolesti određena je prema odgovarajućoj ljestvici. U određenim točkama mjerenja životinje su žrtvovane radi uzimanja uzoraka za daljnju analizu: histopatologija, protočna citometrija, imunološke metode, qRT-PCR i Western-Blot. Rezultati U imuniziranih TLR20/0, imuniziranih TLR40/0, imuniziranih MyD880/0 i imuniziranihTRIF0/0 miševa dokazan je značajno smanjeni razvoj bolesti te blagi neuritis u usporedbi sa C57BL/6 kontrolnom skupinom. Niske razine pro-upalnih citokina IL-6, IL12p40 i IL-17A određene u mutantnim skupinama rezultat su nedostatne NF-κB aktivacije te izostanka translokacije RelA podjedinice u jezgru. Produženi oporavak zabilježen u TLR2 deficijentnih miševa povezan je s oslabljenom polarizacijom makrofaga iz M1 u M2 fenotip te oslabljenim stvaranjem Treg stanica i oslabljenom Th2 diferencijacijom. Zaključak Priloženi rezultati naglašavaju važnost funkcije TLR2, TLR4, MyD88 i TRIF molekula u modelu autoimunog neuritisana u mišu, što ih čini ključnim regulatorima u mreži autoimunog odgovora. Naposlijetku, prikupljeni podaci mogli bi biti razmatrani u razvoju budućih terapijskih protokola za liječenje GBS-a