HLA-B27 misfolding and spondyloarthropathies.

HLA-B27 plays a central role in the pathogenesis of many spondyloarthropathies and in particular ankylosing spondylitis. The observation that the HLA-B27 heavy chain has a tendency to misfold has raised the possibility that associated diseases may belong in a rapidly expanding category of protein misfolding disorders. The synthesis of the HLA-B27 heavy chain, assembly with beta2m and the loading of peptide cargo, occurs in the endoplasmic reticulum (ER) before transport to the cell surface. The evidence indicates that misfolding occurs in the ER prior to b2m association and peptide optimization and is manifested in the formation of aberrant inter- and intra-chain disulfide bonds and accumulation of heavy chain bound to the chaperone BiP. Enhanced accumulation ofmisfolded heavy chains during the induction of class I expression by cytokines, can cause ER stress resulting in activation of the unfolded protein response (UPR). Effects of UPR activation on cytokine production are beginning to emerge and may provide important missinglinks between HLA-B27 misfolding and spondyloarthritis. In this chapter we will review what has been learned about HLA-B27 misfolding in human cells and in the transgenic rat model of spondyloarthritis-like disease, considering it in the context of other protein misfolding disorders. These studies provide a framework to support much needed translational work assessing HLA-B27 misfolding and UPR activation in patient-derived material, its consequences for disease pathogenesis and ultimately how and where to focus intervention strategies.

HLA-B27 misfolding and the unfolded protein response augment interleukin-23 production and are associated with Th17 activation in transgenic rats.

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 beta(2)-microglobulin (Hubeta(2)m)-transgenic rats, an animal model of spondylarthritis. METHODS: Cytokine expression in lipopolysaccharide (LPS)-stimulated macrophages was analyzed in the presence and absence of a UPR induced by chemical agents or by HLA-B27 up-regulation. Cytokine expression in colon tissue and in cells purified from the lamina propria was determined by real-time reverse transcription-polymerase chain reaction analysis, and differences in Th1 and Th17 CD4+ T cell populations were quantified after intracellular cytokine staining. RESULTS: Interleukin-23 (IL-23) was found to be synergistically up-regulated by LPS in macrophages undergoing a UPR induced by pharmacologic agents or by HLA-B27 misfolding. IL-23 was also increased in the colon tissue from B27/Hubeta(2)m-transgenic rats concurrently with the development of intestinal inflammation, and IL-17, a downstream target of IL-23, exhibited robust up-regulation in a similar temporal pattern. IL-23 and IL-17 transcripts were localized to CD11+ antigen-presenting cells and CD4+ T cells, respectively, from the colonic lamina propria. Colitis was associated with a 6-fold expansion of CD4+ IL-17-expressing T cells. CONCLUSION: The IL-23/IL-17 axis is strongly activated in the colon of B27/Hubeta(2)m-transgenic rats with spondylarthritis-like disease. HLA-B27 misfolding and UPR activation in macrophages can result in enhanced induction of the pro-Th17 cytokine IL-23. These results suggest a possible link between HLA-B27 misfolding and immune dysregulation in this animal model, with implications for human disease.

Subtype-specific peripheral blood gene expression profiles in recent-onset juvenile idiopathic arthritis.

OBJECTIVE: To identify differences in peripheral blood gene expression between patients with different subclasses of juvenile idiopathic arthritis (JIA) and healthy controls in a multicenter study of patients with recent-onset JIA prior to treatment with disease-modifying antirheumatic drugs (DMARDs) or biologic agents. METHODS: Peripheral blood mononuclear cells (PBMCs) from 59 healthy children and 136 patients with JIA (28 with enthesitis-related arthritis [ERA], 42 with persistent oligoarthritis, 45 with rheumatoid factor [RF]-negative polyarthritis, and 21 with systemic disease) were isolated from whole blood. Poly(A) RNA was labeled using a commercial RNA amplification and labeling system (NuGEN Ovation), and gene expression profiles were obtained using commercial expression microarrays (Affymetrix HG-U133 Plus 2.0). RESULTS: A total of 9,501 differentially expressed probe sets were identified among the JIA subtypes and controls (by analysis of variance; false discovery rate 5%). Specifically, 193, 1,036, 873, and 7,595 probe sets were different in PBMCs from the controls compared with those from the ERA, persistent oligoarthritis, RF-negative polyarthritis, and systemic JIA patients, respectively. In patients with persistent oligoarthritis, RF-negative polyarthritis, and systemic JIA subtypes, up-regulation of genes associated with interleukin-10 (IL-10) signaling was prominent. A hemoglobin cluster was identified that was underexpressed in ERA patients but overexpressed in systemic JIA patients. The influence of JAK/STAT, ERK/MAPK, IL-2, and B cell receptor signaling pathways was evident in patients with persistent oligoarthritis. In systemic JIA, up-regulation of innate immune pathways, including IL-6, Toll-like receptor/IL-1 receptor, and peroxisome proliferator-activated receptor signaling, were noted, along with down-regulation of gene networks related to natural killer cells and T cells. Complement and coagulation pathways were up-regulated in systemic JIA, with a subset of these genes being differentially expressed in other subtypes as well. CONCLUSION: Expression analysis identified differentially expressed genes in PBMCs obtained early in the disease from patients with different subtypes of JIA and in healthy controls, providing evidence of immunobiologic differences between these forms of childhood arthritis.

HLA-B27 misfolding and spondyloarthropathies.

HLA-B27 plays a central role in the pathogenesis of many spondyloarthropathies and in particular ankylosing spondylitis. The observation that the HLA-B27 heavy chain has a tendency to misfold has raised the possibility that associated diseases may belong in a rapidly expanding category of protein misfolding disorders. The synthesis of the HLA-B27 heavy chain, assembly with beta(2)m and the loading of peptide cargo, occurs in the endoplasmic reticulum (ER) before transport to the cell surface. The evidence indicates that misfolding occurs in the ER prior to beta(2)m association and peptide optimization and is manifested in the formation of aberrant inter- and intra-chain disulfide bonds and accumulation of heavy chain bound to the chaperone BiP. Enhanced accumulation of misfolded heavy chains during the induction of class I expression by cytokines, can cause ER stress resulting in activation of the unfolded protein response (UPR). Effects of UPR activation on cytokine production are beginning to emerge and may provide important missing links between HLA-B27 misfolding and spondyloarthritis. In this chapter we will review what has been learned about HLA-B27 misfolding in human cells and in the transgenic rat model of spondyloarthritis-like disease, considering it in the context of other protein misfolding disorders. These studies provide a framework to support much needed translational work assessing HLA-B27 misfolding and UPR activation in patient-derived material, its consequences for disease pathogenesis and ultimately how and where to focus intervention strategies.

Endoplasmic reticulum stress and the unfolded protein response are linked to synergistic IFN-beta induction via X-box binding protein 1.

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-beta 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-beta induction also occurs in HLA-B27/human beta(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-beta induction by XBP-1, which has implications for the immune response and the pathogenesis of diseases involving the UPR.

Follistatin-like protein-1 is a novel proinflammatory molecule.

While analyzing gene expression in collagen-induced arthritis, we discovered that a poorly characterized gene, follistatin-like protein 1 (FSTL-1), is highly overexpressed in mouse paws during early arthritis, especially at the interface of synovial pannus and eroding bone. In this study, we show that FSTL-1 is a novel proinflammatory molecule with a previously unrecognized role in inflammation. Transfection of FSTL-1 into macrophages and fibroblasts leads to up-regulation of proinflammatory cytokines, including IL-1beta, TNF-alpha, and IL-6. Overexpression of FSTL-1 in mouse paws by gene transfer results in severe paw swelling and arthritis.

HLA-B27 misfolding in transgenic rats is associated with activation of the unfolded protein response.

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 beta2-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-gamma 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.

Proteasome inhibition enhances AAV-mediated transgene expression in human synoviocytes in vitro and in vivo.

To explore the potential applicability of recombinant adeno-associated virus (rAAV) vectors in the treatment of rheumatoid arthritis (RA), primary human fibroblast-like synoviocytes (FLS) derived from patients with RA were infected with rAAV encoding mouse IL-10 under the control of the CMV promoter. Addition of the proteasome inhibitor carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (zLLL) to the cultures dramatically enhanced expression of the IL-10 transgene, in a dose-dependent manner. The increased expression was transient, peaking at 3 days and returning to near baseline by 7 days. The enhancement was observed even when zLLL was added 13 days after infection with rAAV. The effect of zLLL was not specific to either the mIL-10 transgene or the CMV promoter, as similar findings were observed using an rAAV construct encoding alpha1-anti-trypsin under the control of the chick beta-actin promoter or GFP, driven by the CMV promoter. Transgene expression could be repeatedly induced by reexposure to zLLL. Transgene mRNA levels increased in parallel with protein levels. Transgene expression could also be repeatedly induced in vivo by administering zLLL to SCID mice previously injected with rAAV-infected FLS. These data demonstrate that proteasome inhibition can dramatically enhance transgene expression in human RA FLS following infection with rAAV and suggest a possible approach to regulating synovial transgene expression in vivo.

DNA microarray analysis reveals novel gene expression profiles in collagen-induced arthritis.

Global gene expression was analyzed in early and late collagen-induced arthritis (CIA). Of 8734 cDNAs analyzed, 330 were induced and 55 downregulated greater than twofold in early or late disease. Hierarchical clustering of these 385 cDNAs demonstrated five distinct expression patterns differentiating early from late disease and correlating with histopathologic changes in the paw. Of the 385 cDNAs, 185 are known, characterized genes, the majority of which are not described as playing a role in arthritis. However, several of these genes are involved in pathological processes relating to arthritis, including apoptosis, inflammation, and cellular proliferation. One interesting gene, follistatin-like gene, is highly expressed along the margin of contact between inflammatory synovial pannus and eroding bone, suggesting a role in joint destruction. These results demonstrate that global gene expression profiles distinguish early and late CIA and reveal several genes novel to arthritis the further characterization of which will advance our understanding of arthritis.