Background/Purpose: HLA-B27 predisposes to ankylosing spondylitis (AS), an immune-mediated inflammatory disease associated with osteitis, bone loss, and dysregulated bone formation, most notably in the axial skeleton. Although the role of HLA-B27 is incompletely understood, it is thought to exert upstream pro-inflammatory effects. Recently however, it was shown that HLA-B27 promotes osteoclast development from monocytes in HLA-B27 transgenic rats. Given that MHC class I molecules can also be expressed in osteoblasts (OBs), we examined whether HLA-B27 alters the response of OBs to pro-inflammatory cytokines IFNγ and TNFα during differentiation and mineralization in vitro.

Methods: Wild type (WT), HLA-B7 (B7), and HLA-B27 (B27) transgenic rat calvarial OBs were harvested and differentiated in osteogenic medium for up to 3 weeks. Cultures were treated with rat IFNγ (100 ng/mL), TNFα (30 ng/mL) or both cytokines for up to 5 days of the 3-week culture period upon pre-calcified nodule formation, and collected for evaluation of gene (real-time PCR) and protein (immunoblotting) expression, and mineralization (alizarin red staining).

Results: IFNγ had no effect on mineralization while TNFα inhibited mineralization similarly for all 3 genotypes in a time- and dose-dependent manner. TNFα also inhibited mineralization of IFNγ-treated WT and B7 OBs, similar to TNFα alone. In contrast, B27 OBs were refractory to the inhibitory effects of TNFα when cells were co-treated with IFNγ, exhibiting 3-fold higher mineralization than WT and B7 controls. HLA-B was synergistically upregulated at the mRNA and protein level by co-treatment with IFNγ and TNFα in both B7 and B27 OBs. Despite comparable upregulation of HLA-B27 and HLA-B7, only B27-expressing OBs exhibited activation of the unfolded protein response (UPR) as evidenced by induction of GRP78/BiP and CHOP, along with increased XBP1 mRNA splicing. B27-expressing OBs were responsive to TNFα as shown by RUNX2 degradation. However, RUNX2 degradation was incomplete in IFNγ/TNFα-treated B27-expressing OBs and RUNX2 binding to STAT1 was reduced compared to both WT and B7 controls.

Conclusion: This is the first demonstration to our knowledge that HLA-B27 expression can alter OB function as demonstrated in this transgenic animal model. Upregulation of HLA-B27 caused ER stress and activation of the UPR. In addition to its traditional role to maintain ER homeostasis, the UPR is also involved in development and differentiation and is activated during osteogenesis. Additionally, the ER chaperone BiP has been shown to affect mineralization processes, and IFNγ-activated STAT1 can inhibit mineralization through cytosolic sequestration of RUNX2. While the precise mechanism remains unclear, our studies strongly suggest that HLA-B27-induced UPR activation can affect OB function. The possibility that HLA-B27 expression and upregulation could interfere with TNFα-induced inhibition of bone formation might explain why patients with AS exhibit axial progression and develop ankylosis despite active inflammation.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/hla-b27-expression-prevents-tnf%ce%b1-induced-inhibition-of-bone-formation-in-vitro-in-ifn%ce%b3tnf%ce%b1-treated-osteoblasts/