Spondyloarthritis (SpA) comprises a spectrum of inflammatory disorders that predominantly affect spinal and sacroiliac joints, the prototypical form of which is ankylosing spondylitis (AS). Other characteristic manifestations that may variably combine with each other include peripheral joint arthritis and enthesitis, as well as several extraarticular features, including psoriasis, uveitis, and inflammatory bowel disease (). The precise molecular and cellular basis underlying the strong association of SpA with the class I major histocompatibility complex (MHC) antigen HLA–B27 remains uncertain today, 40 years after the initial description of the association, despite the ongoing efforts of many groups to explain it ([2, 3]).
Studies of HLA–B27/human β2-microglobulin (hβ2m)–transgenic rats (B27-transgenic rats) have advanced our understanding of this relationship. Several lines of B27-transgenic rats have proven to be suitable models of SpA, since B27-transgenic rats develop a spontaneous multisystem inflammatory disease that closely resembles human SpA (with arthritis, colitis, and psoriasiform skin lesions developing in rats with an intact gut microbiome) ([4, 5]).
In this model, cell transfer experiments have shown that bone marrow–derived cells (thought to be antigen-presenting cells [APCs]) that express high levels of HLA–B27 are necessary and sufficient to induce SpA in healthy rats () and that disease expression requires the intervention of CD4+ T cells (). Interestingly, professional APCs, such as dendritic cells (DCs) from disease-prone B27-transgenic rats, exhibit several strikingly abnormal functions, including impaired capacity to stimulate T cell responses, altered cytoskeletal dynamics, reduced expression of class II MHC molecules, and enhanced apoptotic death ([4, 8-10]). A strict correlation has been established between altered DC function, high expression of the HLA–B27/hβ2m transgene, and disease susceptibility across several transgenic lines (). Finally, aberrant function has been observed in DCs from premorbid B27-transgenic rats and from rats expressing the disease-associated HLA–B27/hβ2m–transgenic locus on a genetic background that is protective against disease development, thereby demonstrating that this defect was not a consequence of disease and could be a principal factor in the spontaneous development of SpA, e.g., by altering the interaction of DCs with CD4+ T cells ().
Consistent with the foregoing hypothesis, CD4+ T cells expressing a proinflammatory Th17 profile accumulate in B27-transgenic rats in parallel with disease development (), and DCs from B27-transgenic rats induce biased expansion of Th17 cells that could play a pathogenic role in SpA (). Moreover, we recently observed that Treg cell function was altered in B27-transgenic rats, as shown by a decreased ratio of interleukin-10 (IL-10) to IL-17 production, and that this imbalance favoring proinflammatory over antiinflammatory cytokine production was driven by the interaction of CD4+ T cells with DCs ().
Taken together, these data suggest a plausible link between aberrant DC function and the development of SpA in B27-transgenic rats. However, the molecular events that result from HLA–B27 expression in DCs and lead to functional impairment remain incompletely understood. In an attempt to more thoroughly investigate this issue, we analyzed the transcriptome, comparing ex vivo–purified CD103+CD4+ splenic DCs from B27-transgenic rats with those from control rats (transgenic for HLA–B7/hβ2m [B7-transgenic] or nontransgenic). Next, we compared our results with the results of a study of monocyte-derived macrophages from AS patients (). Strikingly, we observed that several genes induced by interferon (IFN) were similarly down-regulated in both disease-prone B27-transgenic rats and SpA patients, thereby highlighting a coordinate dysregulation of the IFN pathway in APCs that might be critical for disease development.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
- Supporting Information
Several hypotheses have been proposed to explain the striking association between SpA and HLA–B27. Investigations performed in B27-transgenic rat lines allowed us to address some of these associations. For example, the classic arthritogenic peptide hypothesis () was rendered unlikely, given that CD8+ T cells are not required for disease expression in these rats ([7, 24, 25]). Results of cell transfer experiments led us to speculate that APCs expressing high levels of HLA–B27 were required for disease induction, and that interaction with CD4+ T cells was also required ([6, 7]). It has also been proposed that proinflammatory Th17 cells are key effectors, since these cells accumulate in lymphoid and target organs in parallel with disease development ([12, 13, 26]). Such an interpretation is consistent with the results of recent experiments demonstrating that DCs from B27-transgenic rats favor a biased expansion of Th17 cells ([10, 13]). Moreover, B27-transgenic rat DCs seem to affect Treg cell differentiation and activation in such a way that they produce less IL-10 and more IL-17 (). These results provide a plausible explanation as to why HLA–B27 may favor a Th17-driven disease—an interpretation consistent with other recent data on SpA ().
However, the molecular mechanism by which B27-transgenic rat DCs may influence the differentiation of CD4+ T cells leading to this proinflammatory bias remains to be understood. It was previously reported that DCs from disease-prone rat lines exhibited altered functions (). Consistent across disease-prone rat lines, the decreased capacity to support T cell stimulation, even in antigen-independent assays, was an abnormal feature (). This phenomenon was linked to impaired formation of an immunologic synapse between B27-transgenic rat DCs and CD4+ T cells, which could itself be attributed to several molecular abnormalities, including a defective engagement of costimulatory molecules expressed on DCs with their partners on T cells, altered cytoskeletal dynamics, and reduced expression of class II MHC molecules (). We have also found evidence that the expression of the HLA–B27 molecule on the DC surface was likely responsible for these molecular abnormalities (). Similar defective capacity for T cell stimulation has now been shown among monocyte-derived DCs from AS patients ().
In the present study, we further investigated DC dysregulation linked to HLA–B27 expression. Our study focused on ex vivo–sorted CD103+CD4+ splenic DCs, since this population has been implicated in T cell activation (). Our findings of transcriptome analysis revealed coordinate aberrant gene expression patterns in DCs from B27-transgenic rats of the 33-3 line, with striking evidence of a “reverse” IFN signature. This result was confirmed in several different settings: among rats of various ages (starting at age 2 months, i.e., at disease onset), among highly purified DCs, and among rats of another HLA–B27 transgenic line, i.e., among (21-3 × 283-2)F1 rats, in which the phenotype closely mimics AS without gut inflammation. Moreover, we found that the signature in B27-transgenic rat DCs corresponded to the signature found in monocyte-derived macrophages from AS patients. This strengthened our observation and provided further evidence that the B27-transgenic rat is a faithful model of SpA.
In B27-transgenic rats of the 33-3 line, we further observed an overexpression of genes involved in ER stress processes (i.e., Calr, Pdia3, Pdia4, and Pdia6). This corroborated results of our previous proteomic study of CD103+ splenic DCs from the same line of rats, wherein we found enhanced expression of calreticulin and protein disulfide isomerase A3 (). This is consistent with cells having survived an unfolded protein response, such as the response initially described in bone marrow–derived macrophages from 33-3 rats (). HLA–B27 misfolding and the ensuing unfolded protein response have been proposed as triggering events explaining HLA–B27 pathogenicity in B27-transgenic rats by facilitating the transcription of IFNβ and/or IL-23p19 (). However, such a mechanism would not directly account for the aberrant DC function studied herein, since we found no evidence of up-regulation of those cytokines. Moreover, the evidence of ER stress was much weaker in DCs from arthritic (21-3 × 283-2)F1 rats, consistent with previous experiments, results of which showed an attenuated unfolded protein response in this line, presumably as a consequence of enhanced hβ2m expression ().
A group of innate immunity genes was also up-regulated. However, when FACS was used instead of magnetic selection to improve DC purification, this signature disappeared, indicating that this signature was secondary to contamination of DC samples by other cells. Similar signatures, attributed to tissue infiltration by neutrophils, macrophages, and mast cells, have been shown in human SpA studies (), suggesting that it reflected the ongoing disease process and could likely be attributed to Th17-mediated inflammation.
Thus, the down-regulated IFN pathway (including the gene for IFNγ itself) was the DC signature that was most relevant to SpA pathogenesis. This, combined with decreased IL-10 production by B27-transgenic rat DCs, might be a contributing factor to the drive behind the differentiation of CD4+ T cells into pathogenic Th17 cells, and might also affect Treg cells by decreasing the ratio of IL-10 to IL-17 production, thereby contributing to disease induction ([31, 32]). Accordingly, enhanced IL-17–mediated inflammation has been shown to result from IFNγ signaling deficiency in DCs through reciprocal modulation of osteopontin and IL-27 (). Thus, IFNγ induces IL-27, and conversely, it inhibits osteopontin production, with both effects contributing to the suppression of IL-17 production and the induction of IL-10 from T cells. Consistent with this interpretation, we observed a decreased expression of mRNA for Il27 in B27-transgenic rat DCs. Moreover, increased osteopontin was reported in AS (), and the IL27A gene has recently been shown to be associated with AS susceptibility ().
As another putative consequence of a reverse IFN signature in DCs, B27-transgenic rats might have an altered capacity to raise an appropriate immune response to infectious agents (). Indeed, heightened vulnerability to pathogenic Listeria monocytogenes was previously shown (). Such a mechanism could also account for the dysregulated microbiota that has been documented in the 33-3 line. Although it remains to be determined in this rat line whether it is a cause or consequence of gut inflammation (), recent data indicate that (21-3 × 283-2)F1 rats, which show no gut inflammation, also harbor a microbiome that is distinctly different from that in nontransgenic rats (ref., and Taurog JD, et al: unpublished observations).
We examined Socs1 and Socs3 expression levels, since both are essential inhibitors of Toll-like receptors (TLRs) and cytokine receptor cascades, including IFN receptor–mediated signal transduction. STAT-1 is hyperactivated, and Ifng and IFN-regulated genes are up-regulated in SOCS-1–deficient DCs. Conversely, SOCS-3–transduced DCs express low levels of IFNγ (). We found no difference in Socs1 expression between DCs from B27-transgenic rats and DCs from control rats, but we did observe overexpression of Socs3 in B27-transgenic rat DCs, thereby providing a possible explanation for the reverse IFN signature. Consistently, our transcriptome analysis showed moderate but significant decreased expression of several other genes associated with TLR and cytokine signaling (Irak2, Mp4k4, and Traf3) in DCs from B27-transgenic rats (data not shown).
Whether there is a causal link between HLA–B27 expression in DCs and Socs3 induction remains to be established. SOCS are induced in response to TLR and cytokine signaling by a negative feedback loop that engages signaling by the Tyro 3/Axl/Mer family of receptor tyrosine kinases (). In addition to their canonical function of antigen presentation, class I MHC molecules can be signaling receptors, mediating reverse signaling via association with other receptors or directly through aggregation, and can exert nonclassic functions ([41, 42]). Interestingly, class I MHC molecules expressed in APCs are involved, via reverse signaling, in negative regulation of TLR-triggered inflammatory responses mediated by the signaling molecule tumor necrosis factor receptor–associated factor 6 (). Thus, the singular biochemical properties of HLA–B27, including a tendency to form oligomers of heavy chains at the cell surface, as previously shown in B27-transgenic rat DCs (), could theoretically affect MHC-dependent feedback and influence downstream events, resulting in heightened SOCS-3 induction.
In summary, our findings indicate that expression of HLA–B27 in APCs may lead to a defect in IFN signaling, secondary to Socs3 induction. Given the critical role of the IFN pathway in the control of both inflammatory and regulatory responses by APCs, we postulate that this deficiency may have fundamental consequences and implications for SpA pathogenesis and treatment.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
- Supporting Information
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Breban had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Fert, Glatigny, Colbert, Taurog, Chiocchia, Araujo, Breban.
Acquisition of data. Fert, Glatigny, Letourneur, Jacques, Smith, Colbert, Araujo.
Analysis and interpretation of data. Fert, Cagnard, Letourneur, Jacques, Colbert, Chiocchia, Araujo, Breban.