The Role of Autophagy in the Degradation of Misfolded HLA-B27 Heavy Chains.

OBJECTIVE: To determine whether autophagy is involved in the degradation of misfolded HLA-B27 in experimental spondyloarthritis.

METHODS: Bone marrow-derived macrophages from HLA-B27/human β2 -microglobulin (hβ2 m)-transgenic rats were incubated in the presence or absence of interferon-γ and proteasome or autophagy inhibitors. Immunoprecipitation, immunoblotting, and immunofluorescence analysis were used to measure HLA-B27 heavy chains and autophagy. Autophagy was induced using rapamycin. Macrophages from HLA-B7/hβ2 m-transgenic and wild-type rats were used as controls.

RESULTS: HLA-B27-expressing macrophages showed phosphatidylethanolamine-conjugated microtubule-associated protein 1 light chain 3B levels similar to those in both control groups, before and after manipulation of autophagy. Blocking autophagic flux with bafilomycin resulted in the accumulation of misfolded HLA-B27 dimers and oligomers as well as monomers, which was comparable with the results of blocking endoplasmic reticulum-associated degradation (ERAD) with the proteasome inhibitor bortezomib. HLA-B7 monomers also accumulated after blocking each degradation pathway. The ubiquitin-to-heavy chain ratio was 2-3-fold lower for HLA-B27 than for HLA-B7. Activation of autophagy with rapamycin rapidly eliminated ~50% of misfolded HLA-B27, while folded HLA-B27 or HLA-B7 monomeric heavy chains were minimally affected.

CONCLUSION: This study is the first to demonstrate that both autophagy and ERAD play roles in the elimination of excess HLA class I heavy chains expressed in transgenic rats. We observed no evidence that HLA-B27 expression modulated the autophagy pathway. Our results suggest that impaired ubiquitination of HLA-B27 may play a role in the accumulation of misfolded disulfide-linked dimers, the elimination of which can be enhanced by activation of autophagy. Manipulation of the autophagy pathway should be further investigated as a potential therapeutic target in spondyloarthritis.