Background: Regulatory T cells (Tregs) of patients with de novo autoimmune hepatitis (DAIH) secrete IL-17A & IFN-g; display T_H1 & T_H17 pehnotypes & are functionally impaired; monocytes drive differentiation of these Tregs towards T_H1-like Tregs in an IL-12 dependent manner. Aim: Identify molecular mechanisms through which Tregs of patients with DAIH differentiate toward pro-inflammatory phenotypes, via both cell-autonomous and non-cell-autonomous pathways. Methods: Blood was obtained from liver transplanted (LT) recipients with DAIH (n=6), LT recipients without DAIH who have normal graft function (LTC) (n=16), & healthy non-transplanted children (HC) (n=13). 1) LPS stimulated CD14⁺ monocytes were: (i) stained for intracellular IL-1β; (ii) RNA harvested for qRT-PCR for pro-IL-1β; (iii) protein harvested for western blot of inflammasome-associated proteins. 2) DNA harvested from sera for measurement of damage associated molecular patterns (DAMPs) by qRT-PCR. 3) ELISA performed on sera to measure TLR-specific DAMPs. 4) Tregs were FACS isolated and stimulated with PMA/ionomycin for 4-hrs &: (i) total RNA harvested for RNASeq & qPCR for ATF6, RORC, and STAT3; (ii) total protein harvested for western blot of same targets. Comparison of quantitative variables between 2 groups performed using Wilcoxon rank-sum test. P<0.05 statistically significant. Results: In patients with DAIH compared to HC and LTC: CD14⁺ monocytes display: 1) increased inflammasome activation upon LPS-stimulation (p<0.05), 2) increased pro-IL-1β & IL-1β (p<0.02) (p<0.03) respectively; Increased DAMPs in sera (qPCR p<0.005;ELISA p<0.05); and activation of ATF6 in Tregs as well as two of its targets, RORC & STAT3 (qPCR p<0.01; western blot; cleaved ATF6a p<0.05, RORC p<0.01, and phosphorylated STAT3 p<0.001, Figure 1).Conclusions: Endoplasmic reticulum stress synergizes with the innate immune signaling pathway, TLR, leading to cytokine production (Figure 2). This may explain the pro-inflammatory cytokine secretion observed in Tregs of patients with DAIH.

CITATION INFORMATION: Arterbery A, Martinez M, Lobritto S, Avitzur Y, Ekong U. Inflammasome, Unfolded Protein Response, and T Cell Immunity in DAIH. Am J Transplant. 2017;17 (suppl 3).

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