Background Endoplasmic reticulum aminopeptidase 1 (ERAP1) has recently been identified to be strongly associated with HLA-B27 positive AS. We have shown that ERAP1 variants cause changes in free heavy chain (FHC) expression on peripheral blood mononuclear cells from HLA-B27 positive patients as well as on B27-expressing C1R cells by in vitro assays. Unfolding of HLA-B27 and the formation of FHC can cause the release of inflammatory cytokines by triggering the unfolded protein response (UPR).
Objectives To generate an in vitro system with HLA-B27 expressing C1R (B-lymphoblastoid) cells that express different ERAP1 varaints. Using this system we will tested if ERAP1 variants asociated with AS can result in increase in UPR.
Methods Endogenous ERAP1 was silenced in C1R-HLA-B27 cells with ERAP1-shRNA (C1RERAP1sh). C1R cells with stable ERAP1-shRNA expression were identified by GFP expression and were selected with puromycin. Scrambled sequence shRNA was used as control. Western blot (WB) for ERAP1 suppression was done using ERAP1 antibody.
We then transfected either the common variant ERAP1 (ERAP1WT) or one of the two AS-associated ERAP1 variants, K528R or Q730E into the C1RERAP1sh cells. Lentivirus expression vector alone was used as control and exogenous ERAP1 expression was tracked with HA-tag. Stable cells expressing ERAP1WT or ERAP1-variants were selected by hygromycin. UPR was measured using PCR for spliced variants of XBP-1 and by qRT-PCR and western blot for BiP, CHOP and ATF-6.
Results Almost all C1R cells that were selected by antibiotics were GFP positive indicating stable ERAP1-shRNA expresion. Using WB we noted more than 90% suppression of ERAP1 and more than 75% suppression by qRT-PCR in C1RERAP1sh, compared to the cells with scrambled-sequence shRNA. Anti-HA WB showed uniform strong expression of ERAP1WT and variant forms of ERAP1 in the respective cell lines.
Spliced XBP1, a marker of UPR, was upregulated in the C1RERAP1sh cells. Re-introduction of ERAP1 (C1R-ERAP1WT cells) reduces the UPR response while C1R-ERAP1K528R and C1R-ERAP1Q730E cells expressing the ERAP1 variants had higher UPR activation compared to C1R-ERAP1WT cells. Other UPR markers including BiP, CHOP and ATF6 expression followed the same pattern with AS-associated variants leading to higher UPR.
Conclusions ERAP1 suppression leads to increased UPR. AS-associated ERAP1-variants, which are known to have reduced function, leads to more UPR compared to the common variant of ERAP1.
Disclosure of Interest None declared