Ankylosing spondylitis macrophage production of higher levels of interleukin-23 in response to lipopolysaccharide without induction of a significant unfolded protein response.

Ankylosing spondylitis (AS) is an insidious spinal inflammatory disease that affects young adults and incurs a high rate of disability (1, 2). Inflammation can also involve the eyes, skin, gut, peripheral joints and tendinous insertions (entheses). AS is a complex genetic disease, with the presence of the MHC class I allele HLA-B27 conferring up to 40% of genetic risk; this allele is found in 90–95% of AS patients but only 8–9% of the Caucasian population (3–6). HLA-B27/human β2 microglobulin-expressing rats (B27-Tg) develop a spondyloarthritis-like disease involving gut inflammation and joint swelling that mimics human disease and highlights the importance of HLA-B27 as a causative factor (7). However, almost 40 years after the strong linkage to HLA-B27 was discovered, it is still not clear how this allele predisposes to disease (8). Although the physiologic role of MHC class I is to present intracellular peptides to CD8+ T cells, depletion of CD8+ T cells in the B27-Tg rat does not impact disease (9). One hypothesis for HLA-B27-related pathogenesis stems from the observation that HLA-B27 displays an unusual propensity to fold slowly and misfold during its biosynthesis (10, 11). HLA-B27 forms aberrant dimers that can be recognized by leukocytes on the cell surface and that accumulate in the endoplasmic reticulum (ER) (12–14). Accumulation of misfolded protein in the ER triggers a conserved intracellular stress response, the Unfolded Protein Response (UPR), that decreases protein production, enhances size and folding capacity of the ER, and degrades excess ER protein. The UPR profoundly alters cellular metabolism predominantly through the induction of gene expression (15). Evidence for HLA-B27 induction of the UPR, including up-regulation of binding immunoglobulin protein (BiP/GRP78), CCAAT/-enhancer-binding protein homologous protein (CHOP), and UPR-dependent splicing of the X-box binding protein-1 (XBP1) transcription factor mRNA, has been detected in cells transfected with HLA-B27 as well as in bone marrow macrophages from diseased B27-Tg animals (16). In the animal model, the magnitude of UPR induction correlated strongly with HLA-B27 expression; in pre-morbid animals, visualization of the UPR required acute up-regulation of MHC class I by inflammatory cytokines such as TNF-α and IFN (16, 17). The clinical relevance of these observations is supported by BiP over-expression in knee synovial fluid mononuclear cells from HLA-B27 positive spondyloarthropathy patients (18). However, the relationship between HLA-B27 triggered UPR and disease remains unclear: in B27-Tg rats with intermediate HLA-B27 expression, additional human β2 microglobulin induces arthritis and spondylitis in the absence of intestinal inflammation, while simultaneously reducing B27 misfolding and BiP expression evident in conconavalin A treated spleen cells.(19). Our previous studies suggested a link between HLA-B27, the UPR, and innate immune responses: macrophages stimulated with pharmacologic UPR inducers(e.g. tunicamycin)respond to Toll-like receptor (TLR) agonists such as lipopolysaccharide (LPS/endotoxin) with increased production of inflammatory cytokines, in particular IFN-β and IL-23 (20, 21). Bone marrow macrophages from diseased animals expressing UPR target genes also expressed type I IFN regulated genes (16). B27+ macrophages from transgenic rats stimulated in vitro with IFN-γ (to induce the UPR) produce increased IFN-β and IL-23 in response to LPS. IL-23 drives IL-17 production from pathogenic Th17 T cells and is implicated in multiple autoimmune and inflammatory diseases (22). Involvement of the IL-23/Th-17 axis in AS pathogenesis is further supported by studies in the B27-Tg rat colon, colonic biopsies from AS patients, and AS peripheral blood mononuclear cells (PBMC) (21, 23, 24). Macrophages pre-dominate in biopsies from early spondyloarthritis inflammatory lesions (25). Also, macrophages appear to be more susceptible to UPR induction than splenocytes in the B27-Tg rat model (17). For this study, we examined peripheral blood derived macrophages from AS patients to determine if they undergo an UPR and whether UPR induction correlates with increased cytokine/chemokine responses to LPS. Despite a 2-fold up-regulation of HLA-B by IFN-γ treatment, we did not observe significant UPR induction as measured by BiP, CHOP, and ERdj4 (an XBP1 gene target) expression. Except for IL-12 secretion, IFN-γ treatment minimized differences between patients and controls. In response to LPS alone, even in the absence of obvious UPR induction, AS patient macrophages produced strikingly greater levels of IL-23 (p=0.0007) than controls. These results have implications for AS pathogenesis and further support the development and application of anti-IL23 therapeutics to AS.