Background/Purpose: Spondyloarthritis (SpA) is an immune-mediated inflammatory disease strongly associated with HLA-B27 (B27). In a transgenic rat model where B27 causes SpA-like disease, CD8 T cells that recognize B27 peptides are not required. B27 has a tendency to misfold, and when upregulated in rat cells can cause endoplasmic reticulum (ER) stress resulting in an unfolded protein response (UPR). The UPR can promote cytokine production and activate autophagy, and may be important in SpA pathogenesis. Since B27 transgenic rats carry multiple transgene copies, an important question is whether B27 upregulation in human cells is sufficient to cause ER stress. We sought to determine whether B27 upregulation in peripheral blood mononuclear cells (PBMC) activates the UPR.

Methods: PBMC from 25 healthy controls (HCs) and 32 adult or pediatric SpA patients (27 B27 positive) meeting age-appropriate classification criteria for SpA were cultured for 20 hours without or with IFNγ, TNFα, or both (IT). Total RNA was isolated and expression of HLA-B and UPR-target genes (BiP, XBP1) was quantitated by real-time PCR. Relative gene expression was determined by normalizing to 3 housekeeping genes, and fold change calculated by comparing treated and untreated samples with statistical assessment by Mann-Whitney U test. Correlations were assessed by Spearman’s. XBP1 mRNA splicing was determined on agarose gels.

Results: HLA-B expression increased 1.9-fold with IFNγ in SpA PBMC and 1.6-fold in HCs (p<0.05 SpA vs. HCs). When treated with TNFα or IT, HLA-B expression in SpA patients increased 1.3-fold and 2.1-fold, respectively, compared to 1.1-fold and 1.9-fold in healthy controls (NS SpA vs. HCs). BiP expression in TNFα-treated PBMC increased 1.3-fold in SpA patients vs. 1.1-fold in HCs (p=0.05) while no significant difference between SpA patients and HCs was seen with IFNγ (1.3-fold and 1.2 fold) or IT (1.3-fold and 1.3-fold). We also asked whether individual fold changes in BiP correlated with HLA-B upregulation. With IFNγ and/or TNFα treatment, fold change in BiP and XBP1 positively correlated with fold change in HLA-B in SpA patients (p<0.005), while in HCs significant correlation was only observed in TNFa-treated PBMC (p<0.05, p<0.005, respectively). More importantly, the slopes of the correlation plots between HLA-B and BiP or XBP1 were 1.3-2.3 times greater for SpA patients than HCs. In addition, XBP1 splicing was significantly increased by cytokine treatments in patient samples but not in HCs (p<0.05). Subanalyses of B27-positive and negative SpA patients is inconclusive due to the low number of B27-negative patients.

Conclusion: Our results reveal greater upregulation of the UPR-target gene BiP as well as increased XBP1 mRNA splicing in response to cytokines in SpA patients compared to HCs. These preliminary results support the idea that low-level HLA-B27 upregulation may be sufficient to generate ER stress in certain cell types. Further exploration of cell types affected by this response, and additional conditions that may enhance B27 heavy chain accumulation, is necessary to better understand the potential role of ER stress in SpA pathogenesis.

 

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/evidence-for-enhanced-induction-of-unfolded-protein-response-target-genes-in-peripheral-blood-mononuclear-cells-from-spondyloarthritis-patients/