Abstract
Purpose: :
Mouse Experimental Autoimmune Uveoretinitis (EAU) is a CD4+ Th1 cell mediated disease used to model human sight-threatening posterior uveitis. We have previously shown that LPS-activated IL10-producing bone marrow-derived dendritic cells (DC) suppress EAU in C57BL/6 mice via T regulatory cell (Treg) induction. In this study we explored mechanisms of how LPS-induced signaling pathways in DC were activated to promote tolerance.
Methods: :
EAU was induced in C57BL/6 mice by subcutaneous injection of interphotoreceptor retinoid binding protein (IRBP) peptide (1-20) emulsified in complete Freund's adjuvant (CFA), with the addition of pertussis toxin administered intraperitoneally. EAU severity was measured 28 days post immunization by endoscopic fundoscopy and histology.Bone marrow-derived semi-mature tolerogenic DC were differentiated with GM-CSF and stimulated with a high dose of LPS (1µg/ml) for 24h. DC were stimulated with LPS for 1h and the phosphorylation state of proteins in the Toll-like receptor 4 (TLR 4) pathway was examined by Western bloting. In addition, we explored the localization of NF-kB and IRF-3 by immunocytochemistry. The expression of cytokines in tolerogenic DC was determined by ELISA, Cytometric Bead Array and PCR. The maturation status of DC was evaluated using flow cytometry.The effect of DC on EAU was studied by injecting DC into the nape skin and 24h later EAU was induced as described above.
Results: :
LPS-activated DC "vaccination" effectively attenuated expression of EAU as measured by clinical ocular fundus examination and by histology. Both MyD88-dependent and -independent routes were activated by LPS treatment of DC for 1h. Tolerogenic DC activated with LPS for 24h became selectively refractory to further maturation with LPS in that LPS desensitisation suppressed LPS-induced IRF-3 phosphorylation and nuclear translocation. Moreover JNK, p38 and MAPKAPK-2 phosphorylation levels were reduced. However, NF-kB phosphorylation level was not affected. Interestingly, the percentage of DC expressing TLR 4 was decreased 24h after initial stimulation with LPS.
Conclusions: :
Subcutaneous administration of LPS activated DC producing IL10 decreases EAU severity in C57BL/6 mice. The mechanism of EAU inhibition by DC depends on CD4+CD25+Foxp3+ Treg induction. In DC LPS signals by both MyD88 dependent and independent pathways. Interestingly, our results show that TBK-1/IRF-3 signaling is down-regulated in tolerogenic DC, suggesting that activation of this pathway may prevent DC tolerogenicity.
Keywords: uveitis-clinical/animal model • immune tolerance/privilege • signal transduction