Background/Purpose: HLA-B27/human β₂microglobulin transgenic rats develop spondyloarthritis (SpA) characterized by inflammation in the gut and peripheral joints by mechanisms that remain poorly defined, but do not require CD8+ T cells. HLA-B27 has a tendency to misfold and dimerize, and when upregulated, aberrant forms can accumulate in the endoplasmic reticulum (ER) and on the cell surface. ER accumulation can activate an unfolded protein response (UPR), which promotes pro-inflammatory cytokine production, and cell surface accumulation may trigger effector immune cells expressing certain killer immunoglobulin receptors (KIRs), both of which may contribute to disease. Here, we examined whether autophagy is involved in the degradation of HLA-B27 in transgenic rats and whether alteration of this pathway may affect accumulation of aberrant forms and the generation of ER stress.

Methods: Bone marrow-derived macrophages (MΦ) were obtained from HLA-B27-transgenic (TG), HLA-B7-TG and Lewis wild type (WT) rats and autophagy was induced with rapamycin treatment for 2 hours. Chloroquine or bafilomycin were used to block autophagic flux. The level of autophagy was measured by the amount of cytosolic microtubule-associated protein 1 light chain 3B-II (LC3B-II) expression detected by western blot (WB), flow cytometry (FACS) or immunofluorescence (IF). Expression of HLA-B27 was determined by WB analysis of cell lysates or after immunoprecipitation (IP) with the HC10 antibody. XBP1 mRNA splicing was used as an early marker to determine whether the UPR is activated after stimulation with IFNγ and TNFα in the presence or absence of bafilomycin or rapamycin.

Results: There was no difference in autophagy in HLA-B27-expressing MΦ compared to WT and HLA-B7-expressing cells based on WB and FACS analysis of LC3B-II expression upon stimulation of autophagy (rapamycin) or inhibition of autophagic flux (chloroquine). LC3B-II expression determined by IF showed a cytosolic vesicular pattern in all cells confirming LC3B-II localization in autophagic vesicles. However, inhibition of autophagic flux (bafilomycin) during upregulation of HLA expression with IFNγ resulted in an accumulation of HLA heavy chains, and in particular misfolded forms of HLA-B27. There was also significant exacerbation of ER stress (which itself is induced by blocking autophagic flux) as measured by XBP1 mRNA splicing. In contrast, activation of autophagy with rapamycin reduced the accumulation of aberrant forms of HLA-B27 induced by IFNγ, and resulted in diminished XBP1 splicing.

Conclusion: These results demonstrate that blocking autophagic flux causes ER stress, and that HLA-B27 expression exacerbates this response. More importantly, induction of autophagy can reduce the accumulation of misfolded forms of HLA-B27, and may alleviate ER stress. Current studies are assessing effects of autophagy on the cellular distribution of aberrant forms of HLA-B27. Alteration of the autophagic pathway may be a promising therapeutic tool worthy of further investigation in the treatment of SpA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/stimulation-of-autophagy-reduces-misfolded-hla-b27-in-a-rat-model-of-spondyloarthritis/