Based on the B27-Tg rat spondyloarthritis model we hypothesized that increased HLA-B27 expression and misfolding during inflammation induces a UPR that renders innate immune cells (macrophages and dendritic cells) more pro-inflammatory and hyper-responsive to bacterial TLR agonists. UPR-driven cells secrete excessive IL-23 (Th17 activation) and type I IFN (20
). However, the transgenic rat highly over-expresses HLA-B27 and thus HLA-B27 misfolding effects would most likely be exaggerated. It is important to assess the UPR-TLR inflammatory model in human disease, where there are at most 2 copies of the HLA-B27 gene.
In this study we did not detect significantly increased UPR-regulated gene expression in AS patient macrophages relative to controls at baseline, nor did we observe significant up-regulation of classic UPR target genes following in vitro
IFN-γ and LPS stimulation. Recently, LPS was shown to activate IRE-1 dependent XBP1 splicing via the induction of an NADPH oxidase while suppressing activation of the PERK and ATF6 arms of the UPR (32
). In this report, 10 ng/mL LPS also tended to decrease mean UPR gene expression in AS patients. However, we did not detect increased UPR gene expression following IFN-γ stimulation even in the absence of the additional LPS treatment. The significance of the increase in ERdj4 mRNA following IFN-γ+LPS treatment in AS patients is unclear, since mean expression remained below that observed in control subject macrophages.
The simplest explanation for the difference in our findings and the B27-Tg animal studies lies in the magnitude of HLA-B up-regulation. In the report describing the correlation of UPR magnitude and HLA-B27 expression in transgenic rat macrophages, IFN-γ treatment up-regulated HLA-B27 four to ten fold; although even a two fold up-regulation of HLA-B27 by TNF-α treatment yielded a two-fold induction of BiP expression (17
). The mean two to three-fold HLA-B27 induction we observed may not have been sufficient to detect UPR induction in patient samples, particularly given patient to patient variability. Our small sample sizes may have been underpowered to detect subtle upregulation of gene expression. In addition, the representative UPR target genes we chose (BiP, CHOP, ERdj4) may not be the most sensitive indicators of an HLA-B27 UPR, although they are widely utilized to detect the UPR (15
). Alternatively, the UPR observed in the B27-Tg rats may depend upon dramatic HLA-B27 over-expression related to multiple transgene copy numbers, and thus be less relevant to human disease. However, the increased BiP expression found in the synovial cells from spondyloarthritis patients would argue against this possibility (18
). Another possibility is that factors in addition to IFN-γ activation and HLA-B27 up-regulation are required to induce a UPR in AS macrophages. Our in vitro
system may have been too simplistic compared to the in vivo
inflammatory milieu. Future examination of inflamed patient tissues may be more revealing. Related to all these factors, our results do not rule out a role for the UPR in pathogenesis.
The study by Tran et al. showing worse arthropathy in B27-Tg rats where the UPR has been modulated by β2 microglobulin throws into question the relationship between B27-related UPR and joint disease(19
). Multiple non-UPR hypotheses have sought to explain the striking contribution of HLA-B27 to genetic risk (8
). For instance, expanded numbers of natural killer and CD4 T cells that recognize cell surface HLA-B27 dimers have been identified in the circulation of AS patients (14
). Other efforts have focused on specific antigen presentation by HLA-B27 (33
). With some variability across studies, HLA-B27 has also been shown to alter survival and persistence of intracellular organisms through unclear mechanisms (8
). Ultimately, our data suggest that non-UPR, cytokine modulatory mechanisms may contribute towards a pro-inflammatory diathesis and that non-UPR hypotheses bear greater exploration.
The tendency of IFN-γ to minimize differences in inflammatory mediator production is consistent with our previous data showing a global decrease in IFN-γ regulated gene expression in AS patient macrophages that could be recovered with exogenous IFN-γ treatment (28
). This study and others have highlighted a relative deficit in the production of Th1 cytokines such as IFN-γ by AS patient cells (28
). However, the IL-12 (p70) data () suggests that under certain infectious or inflammatory conditions where IFN-γ is produced, AS patients could potentially mount an even more robust Th1 response than controls. Our findings of increased CXCL9, TNF-α and IL-10 in AS patient macrophages corroborate previous studies of patient samples (37
). Increased CXCL9, CXCL10/IP-10 and IL-12/23p40 are present in synovial fluid from patients with spondyloarthropathy (37
). Reports describing serum and PBMC from AS patients have demonstrated variable increases in TNF-α, IL-1β, IL-6, IL-8, VEGF, and IL-10(38
). What is unique to this study is removal of monocytes from the context of patient inflammation and medication. The in vitro
derivation and stimulation conditions are controlled; thus the increased cytokine/chemokine production most likely reflects an intrinsic, genetically determined property of AS macrophages.
The most striking finding from this study is that even in the absence of an obvious UPR, AS macrophages produced significantly more IL-23 in response to LPS alone. The one known HLA-B27 negative patient produced IL-23 close to the median AS patient level (335 pg/mL in response to 10 ng/mL LPS and 405 with 100 ng/mL LPS). The increased IL-23 was much more robust than the differences observed for TNF-α. ER stress induced CHOP has recently been shown to directly regulate IL-23 expression(41
). Although UPR and IL-23 production have also been closely associated in the B27-Tg rat model, our results suggest the potential for excess IL-23 production by patient macrophages in the absence of an overt UPR; in this study mean CHOP expression actually decreased following IFN-γ treatment and early during the LPS stimulation period (21
). In the recent report by Martinon et al., LPS stimulation decreased pharmacologically induced UPR target gene expression (BiP, CHOP, ERdj4) out to 9h(32
). In the present study we did not collect RNA after 24h of LPS, and thus cannot absolutely rule out late occurrence of an LPS-induced UPR.
Although this study is underpowered to assess the impact of HLA-B27 positivity in patients, our results support the hypothesis that AS macrophages produce an overabundance of IL-23 in response to TLR4 agonists in the infectious environment, thus predisposing to the development of inflammatory lesions. The IL-23/Th17 axis has become increasing recognized as a key component of anti-bacterial immunity, thus explaining genetic pressures to maintain risk alleles for a debilitating arthritic condition over the centuries (42
). Given the responses we observed to LPS, it will be important to determine whether macrophages from AS patients produce excess IL-23 in response to colonic and infectious organisms. The finding of robust IL-23 expression in subclinical gut inflammation from AS patients suggests this may be the case, however more defined studies will be helpful in elucidating pathogenesis(24
Our IL-23 results extend and support other recent studies examining the IL-23-Th17 axis in AS. Polymorphisms in the IL-23R were identified in genome wide association studies as susceptibility alleles for AS (43
). Increased IL-23 and IL-17 levels have been observed in the serum and cultured PBMC supernatants from AS patients with active disease (23
). AS patients also have a higher proportion of IL-17 producing CD4+ T cells in circulation (23
Anti-IL12p40 agents (e.g. ustekinumab), that block both IL-12-and IL-23-mediated effects, are highly efficacious in psoriasis, more than etanercept (a TNF-blocker) (45
). In this study, the overproduction of IL-23 by AS patient macrophages was much more impressive than that observed for TNF-α, even though TNF-blockers are currently the therapy of choice for AS (46
). The evidence from this study and others, implicating IL-23 expression in AS pathogenesis provides strong rationale for development of IL-23 blocking agents as treatment for AS.