The class I protein HLA-B27 confers susceptibility to inflammatory arthritis in humans and when overexpressed in rodents for reasons that remain unclear. We demonstrated previously that HLA-B27 heavy chains (HC) undergo endoplasmic reticulum (ER)-associated degradation. We report here that HLA-B27 HC also forms two types of aberrant disulfide-linked complexes (dimers) during the folding and assembly process that can be distinguished by conformation-sensitive antibodies W6/32 and HC10. HC10-reactive dimers form immediately after HC synthesis in the ER and constitute at least 25% of the HC pool, whereas W6/32-reactive dimers appear several hours later and represent less than 10% of the folded HC. HC10-reactive dimers accumulate in the absence of tapasin or ␤ 2 -microglobulin, whereas W6/ 32-reactive dimers are not detected. Efficient formation of W6/32-reactive dimers appears to depend on the transporter associated with antigen processing, tapasin, and ␤ 2 -microglobulin. The unpaired Cys 67 and residues at the base of the B pocket that dramatically impair HLA-B27 HC folding are critical for the formation of HC10-reactive ER dimers. Although certain other alleles also form dimers late in the assembly pathway, ER dimerization of HLA-B27 may be unique. These results demonstrate that residues comprising the HLA-B27 B pocket result in aberrant HC folding and disulfide bond formation, and thus confer unusual properties on this molecule that are unrelated to peptide selection per se, yet may be important in disease pathogenesis.

Objective. To determine whether HLA-B27 misfolding and the unfolded protein response (UPR) result in cytokine dysregulation and whether this is associated with Th1 and/or Th17 activation in HLA-B27/human

The mechanism by which the MHC class I allele, HLA-B27, contributes to spondyloarthritis pathogenesis is unknown. In contrast to other alleles that have been examined, HLA-B27 has a tendency to form high m.w. disulfide-linked H chain complexes in the endoplasmic reticulum (ER), bind the ER chaperone BiP/Grp78, and undergo ER-associated degradation. These aberrant characteristics have provided biochemical evidence that HLA-B27 is prone to misfold. Recently, similar biochemical characteristics of HLA-B27 were reported in cells from HLA-B27/human β2-microglobulin transgenic (HLA-B27 transgenic) rats, an animal model of spondyloarthritis, and correlated with disease susceptibility. In this study, we demonstrate that the unfolded protein response (UPR) is activated in macrophages derived from the bone marrow of HLA-B27 transgenic rats with inflammatory disease. Microarray analysis of these cells also reveals an IFN response signature. In contrast, macrophages derived from premorbid rats do not exhibit a strong UPR or evidence of IFN exposure. Activation of macrophages from premorbid HLA-B27 transgenic rats with IFN-γ increases HLA-B27 expression and leads to UPR induction, while no UPR is seen in cells from nondisease-prone HLA-B7 transgenic or wild-type (nontransgenic) animals. This is the first demonstration, to our knowledge, that HLA-B27 misfolding is associated with ER stress that results in activation of the UPR. These observations link HLA-B27 expression with biological effects that are independent of immunological recognition, but nevertheless may play an important role in the pathogenesis of inflammatory diseases associated with this MHC class I allele.

Type I IFN are strongly induced upon engagement of certain pattern recognition receptors by microbial products, and play key roles in regulating innate and adaptive immunity. It has become apparent that the endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR), in addition to restoring ER homeostasis, also influences the expression of certain inflammatory cytokines. However, the extent to which UPR signaling regulates type I IFN remains unclear. Here we show that cells undergoing a UPR respond to TLR4 and TLR3 ligands, and intracellular dsRNA, with log-fold greater IFN-b induction. This synergy is not dependent on autocrine type I IFN signaling, but unexpectedly requires the UPR transcription factor X-box binding protein 1 (XBP-1). Synergistic IFN-b induction also occurs in HLA-B27/human b 2 m-transgenic rat macrophages exhibiting a UPR as a consequence of HLA-B27 up-regulation, where it correlates with activation of XBP-1 splicing. Together these findings indicate that the cellular response to endogenous 'danger' that disrupts ER homeostasis is coupled to IFN-b induction by XBP-1, which has implications for the immune response and the pathogenesis of diseases involving the UPR.Key words: HLA-B27 Á Interferons Á Protein misfolding Á Unfolded protein response IntroductionInitially identified as antiviral cytokines, type I IFN (IFN-a/b) are now recognized to have diverse immunoregulatory effects activating macrophages and NK cells, promoting T cell survival and dendritic cell (DC) maturation, and increasing the production of Th1-polarizing cytokines to mediate innate and adaptive immune responses [1]. Type I IFN also exert biological effects at low and even constitutive levels of expression [2]. For example, weak IFN-b-mediated signaling augments IFN-a/b induction in response to LPS through positive feedback involving autocrine/ paracrine up-regulation of IFN regulatory factor (IRF)7. In addition, low levels of IFN-a/b prime cells to respond to IFN-c and IL-6 by providing a phosphorylated type I IFN receptor 'niche' for commonly used signaling molecules in proximity to other cytokine receptor complexes [3,4]. Mice deficient in IFN-b are more susceptible to viral infection, have lower numbers of macrophages and mature B cells, and exhibit reduced bone mass [5], as IFN-b is a positive regulator of bone formation through inhibition of osteoclast differentiation [6]. Thus, even constitutive levels of this cytokine are important in osteo-immune homeostasis.IFN-b is produced by most cell types upon viral infection, and in large amounts by macrophages and DC, following engagement of pattern recognition receptors (PRR) by conserved molecular structures referred to as pathogen-associated molecular patterns [7, 8]. PRR that mediate IFN-b induction include cell surface TLR4 and endosomal TLR3 for LPS and dsRNA, respectively [9]. In both instances increased IFN-b gene transcription occurs via TLR/IL-1R domain-containing adapter inducing IFN-b (TRIF)-dependent IRF3 and NF-jB activation. PRR in the retinoic ...