Background Spondyloarthritis (SpA) is strongly associated with HLA-B27, but the role of this MHC class I molecule in the disease pathogenesis remains largely unknown. Misfolding of HLA-B27 in the endoplasmic reticulum (ER) is described to induce an unfolded protein response (UPR), leading to increased production of pro-inflammatory cytokines upon TLR ligation. However, these data on HLA-B27-induced macrophage ER stress were generated in cell lines and animal models with strongly increased HLA-B27 expression and the relevance of this pathway in HLA-B27+ human SpA remains unknown.
Objectives The aim of the study was to investigate the effect of ER stress and HLA-B27 positivity on polarized human macrophages in HLA-B27+ SpA.
Methods To analyze the effects of ER stress on macrophage polarization, peripheral blood monocytes from healthy donors (HDs) were stimulated with thapsigargin (TG), polarized for 4 days by IFN-γ, IL-4, or IL-10, and subsequently phenotyped by flow cytometry. To assess the effect of ER stress on cytokine production, polarized peripheral blood derived macrophages (PBDMs) were submitted to ER stress by TG and subsequently stimulated with LPS. Finally, polarized PBDMs from HLA-B27- and HLA-B27+ SpA, rheumatoid arthritis (RA) and HDs were stimulated with LPS. The expression of ER stress markers (BiP, CHOP, ERdj4) and cytokines (IL-23, IL-12, IL-10) was measured by RT-PCR.
Results Stimulation of human monocytes with TG prior to in vitro polarization prevented the up-regulation of the macrophage (MΦ)IFN-γ marker CD64 (p<0.05), the MΦIL-4 marker CD200R (p<0.05), and the MΦIL-10 markers CD163 (p<0.001) and CD32 (p<0.05), indicating that ER stress impairs macrophage polarization. When MΦIFN-γ, MΦIL-4 and MΦIL-10 were incubated with TG, they showed up-regulation of BiP, CHOP and ERdj4. The relative expression of IL-23 in comparison with both IL-12 and IL-10 was significantly increased by TG in all three macrophage subsets (IL-23/IL-12 p<0.05 in MΦIFN-γ and p<0.01 in MΦIL-4; IL-23/IL-10 p<0.05 in MΦIL-4and MΦIL-10). LPS stimulation resulted in an important induction of IL-23, IL-12 and to a lesser extent IL-10 expression in MΦIFN-γ and MΦIL-4 (p<0.01), which could not further be up-regulated by TG. Analysis of in vitro polarized macrophages from SpA, RA and HD did not reveal differences in the expression of ER stress marker BiP, both prior and after activation with LPS. Stratification of SpA according to HLA-B27 status also did not reveal differences in BiP expression. However, LPS-stimulated MΦIFN-γ showed an increased relative expression of IL-23 and IL-12 versus IL-10 in HLA-B27+ SpA in comparison with HLA-B27- SpA (p<0.05), indicating a LPS-induced shift towards pro-inflammatory cytokine expression.
Conclusions Macrophages from HLA-B27+ SpA patients, and especially MΦIFN-γ, showed increased IL-23 and IL-12 versus IL-10 expression in comparison with macrophages from HLA-B27- SpA patients. However, these differences were not related to increased ER stress in HLA-B27+ SpA macrophages.
Disclosure of Interest None Declared