Spondylarthritis (SpA) is an inflammatory rheumatic disorder characterized by axial and peripheral enthesitis and/or arthritis and frequent extraarticular manifestations, such as uveitis, psoriasis, and inflammatory bowel diseases (Crohn's disease or ulcerative colitis). All skeletal and extraarticular features of SpA are thought to be determined by shared genetic factors, dominated by the class I major histocompatibility complex (MHC) allele HLA–B27 (1). Although the strong association between HLA–B27 and SpA was first described more than 35 years ago, the mechanism of this association has remained unexplained until now (2).
Evidence that HLA–B27 plays a direct role in determining SpA pathogenesis came from the description of an HLA–B27–transgenic rat model. A multisystem inflammatory disorder mimicking the most striking aspects of SpA (rat SpA), including ankylosing spondylitis, peripheral arthritis, psoriatic lesions, and ulcerative colitis, arises spontaneously in several lines of rats transgenic for HLA–B*2705 and human β2-microglobulin (hβ2m), but not in the control HLA–B*0702/hβ2m–transgenic line (3, 4). The expression of rat SpA is determined by several factors, including high levels of both HLA–B27 and hβ2m transgenes, a specific rat genetic background, and the presence of normal microbial flora (5, 6). Importantly, the role of the immune system in this model was highlighted in several ways. First, rat SpA was induced in nontransgenic recipients by transferring immature hematopoietic cells, but not mature lymph node (LN) cells, from disease-prone rats, indicating that a hematopoietic-derived cell is critical for disease induction (7). Second, nude HLA–B27–transgenic rats, which lack thymically derived T cells, were protected against disease but promptly developed rat SpA upon reconstitution with CD4+ T cells or thymus engraftment, whereas reconstitution with CD8+ T cells was relatively inefficient (6, 8). Moreover, experiments involving CD8+ T cell depletion confirmed that CD8+ T cells were not required for the development of rat SpA (9, 10).
Based on the results described above, we postulated that rat SpA could arise as a consequence of an interaction between antigen-presenting cells (APCs) expressing high levels of HLA–B27 and CD4+ T cells, and carried out experiments to examine the consequences of such interaction. Interestingly, these experiments revealed several aberrant functions in APCs from HLA–B27/hβ2m–transgenic rat lines. Most notably, mature splenic and LN dendritic cells (DCs) exhibited a decreased capacity to stimulate an allogeneic or a syngeneic T cell response, which paralleled disease susceptibility in a broad variety of lines, and was not a consequence of the inflammatory disease (11–13). This altered function could be linked to a defective engagement of costimulatory molecules, such as CD86, and also to a decreased capacity to form an antigen-independent immunologic synapse with CD4+ T cells (12, 14). Other aberrant functions in HLA–B27–transgenic rat DCs included altered cytoskeletal dynamics, decreased class II MHC expression, and enhanced apoptotic susceptibility, all potentially contributing to their decreased stimulatory function (15).
DCs are professional APCs that are capable of activating naive CD4+ T cells to differentiate and to proliferate. However, besides promoting T cell responses to antigen, evidence indicates that DCs play a role in establishing tolerance toward self antigens and in the maintenance of peripheral tolerance (16). Accordingly, spontaneous inflammation in this model could result from a breakdown of peripheral tolerance.
Upon stimulation by DCs, naive CD4+ T cells are known to differentiate into distinct lineages of effector cells. While interleukin-12 (IL-12) drives the development of Th1 cells, producing predominantly interferon-γ (IFNγ) and IL-2 and eliciting cell-mediated immunity against intracellular pathogens (17), Th2 cells differentiate in response to IL-4; produce IL-4, IL-5, and IL-13; and are involved in the humoral response against parasites and allergy (17, 18). More recently, Th17 cells, a subset of T cells producing IL-17A, IL-17F, and IL-22, have been identified (19–21) and shown to be critical for the induction of several autoimmune disease models, such as collagen-induced arthritis and experimental autoimmune encephalomyelitis (22, 23). Th17 cells differentiate in mice in response to transforming growth factor β (TGFβ) and IL-6 or IL-21 (24) and differentiate in humans in response to a mixture of cytokines, including IL-1β, IL-6, tumor necrosis factor α (TNFα), IL-23, and TGFβ (25, 26). Finally, a CD4+ Treg cell population expressing the FoxP3 transcription factor has also been described (27). This Treg cell population appears to inhibit the proliferation of all of the former effector Th cells (28).
In the present study, we examined the phenotype of CD4+ T cells in a disease-prone HLA–B27–transgenic rat line and the putative influence of DCs from these rats on the differentiation of CD4+ T cells. First, we observed that CD4+ T cells with a proinflammatory Th17 phenotype accumulate in B27-transgenic rats, in parallel with the development of rat SpA. Furthermore, we showed that Th17 cells were preferentially induced and expanded by DCs from B27-transgenic rats, by a contact-dependent mechanism that may involve a previously described defective engagement of costimulatory molecules (12, 14).
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A critical role for T cells in the B27-transgenic rat model of SpA was previously established (7). Furthermore, several lines of evidence strongly support a direct role for CD4+ T cells, whereas CD8+ T cells appear to be dispensable (8–10). However, the phenotype of CD4+ T cells that might drive the disease process has not been previously thoroughly ascertained. In the present study, we observed an expansion of CD4+ T cells in the LNs draining the sites of inflammation in B27-transgenic rats. Interestingly, the frequency of CD4+ T cells has also been shown to be increased in the peripheral blood of SpA patients (32).
Among CD4+ T cells, the expansion was most striking in the CD4+CD25+ subset, which may correspond to activated or Treg cells. We showed a preferential expansion of FoxP3− activated cells compared to FoxP3+ Treg cells. We had previously shown that DCs from B27-transgenic rats have a decreased capacity to activate naive CD4+ T cells, by a mechanism implying defective costimulatory function (12, 14). We had proposed that such abnormal function could predominantly impair the capacity of B27-transgenic rat APCs to activate Treg cells. Our present results corroborate this hypothesis by showing an unbalanced control of CD4+ T cell activation.
We next characterized the cytokines produced by CD4+ T cells in the LNs draining the sites of inflammation. Notably, we observed a preferential expansion of TNFα- and IL-17A–producing cells, which paralleled disease development, likely corresponding to Th17 cells (20, 26, 33). Such an interpretation is strongly supported by the increased transcription of Rorc, IL-21, and IL-22 (which are involved in the Th17 lineage) in the LN CD4+ T cells from B27-transgenic rats (34, 35), and by the detection of IL-17A–positive mononuclear cells in arthritic joints from B27-transgenic rats (although we cannot exclude the possibility that at least some of these IL-17–producing cells were not T cells). Accordingly, up-regulation of IL-17A in CD4+ T cells from the colonic lamina propria was recently shown in the same B27-transgenic rats (36). We also detected increased levels of IL-17A in the serum of the B27-transgenic rats, similar to findings reported in human SpA (37). All of these findings support a direct role of Th17 cells in this model, although this remains to be formally demonstrated by experiments such as those involving cell transfer. (Of note, it has been impossible until now to purify live Th17 cells in the rat.)
Former studies of T cell cytokines produced in the B27-transgenic rat model of SpA demonstrated an early increase in IFNγ and IL-2 levels in the inflammatory colonic mucosa, suggesting a Th1-mediated disorder (5). Interestingly, in the present study the proportion of Th1 cells producing IFNγ alone and the expression levels of IFNγ and Tbx21 transcripts in LN CD4+ T cells from B27-transgenic rats were comparable to that in controls, suggesting that Th1 cells may not be the most critical T cells mediating the disease process. Similar observations have been reported in the context of human SpA (38). Nevertheless, we also detected a minor population of CD4+ T cells that produced IFNγ, TNFα, and IL-17A together, which was specific to the B27-transgenic rats and appeared late during the disease process. A similar phenotype of polyfunctional T cells producing both Th1 and Th17 cytokines has previously been described as selectively enriched in the peripheral blood of SpA patients (38, 39).
The role of Th17 cells has recently emerged as pivotal in several models of autoimmune and inflammatory diseases (40). Our results suggest that Th17 cells could contribute to both intestinal and joint inflammation in the B27-transgenic rat model of SpA. Such an interpretation is consistent with recent findings in human SpA. Genetics studies have identified several polymorphisms in the IL23R gene, which codes for a receptor involved in Th17 cell differentiation, as being associated with susceptibility to ankylosing spondylitis (41). Furthermore, the expansion of cells producing both TNFα and IL-17A is consistent with the striking efficacy of anti-TNFα agents, both in the B27-transgenic rat model and in human SpA (42, 43). In contrast, anti-rat IL-17 treatment failed to prevent disease in B27-transgenic rats (Glatigny S, et al: unpublished observations). Nevertheless, this result does not rule out a role for Th17 cells in rat SpA pathogenesis, since we observed an increased number of IL-17–producing T cells in the LNs of the anti–IL-17–treated rats. It is known that IL-17 negatively regulates its own production by way of negative feedback (44). Therefore, blocking IL-17 in the B27-transgenic rat led to an increase in the proportion of Th17 cells, which could still exert a pathogenic effect via the production of other mediators.
We next showed that interaction between DCs from B27-transgenic rats and CD4+ T cells from control rats contributes to the expansion of Th17 cells, despite their defective capacity to support a T cell– proliferative response (11–13). Furthermore, those IL-17A–producing cells were detected among the T cells that divided the most, suggesting that the B27-transgenic rat DCs stimulated potentially autoreactive Th17 cells, similar to what has been described with DCs isolated from human psoriatic lesions (45).
Regarding the mechanism responsible for the biased Th17 cell induction by B27-transgenic rat DCs, Transwell experiments indicated that it was not explained by a difference in soluble factor production, such as an excess of IL-23 or TGFβ, or a lack of IFNγ (results not shown). Alternatively, blocking the interaction between CD86 and CD28 during DC–T cell coculture resulted in an increased induction of Th17 cells in the nontransgenic DC condition. This effect was not correlated with the level of T cell proliferation, which was inhibited by the anti-CD86 mAb, but was conversely enhanced by the anti-CD28 superagonist mAb JJ316. Taken together, these results are consistent with a critical role played by the defective costimulatory capacity of B27-transgenic rat DCs in the biased induction of Th17 cells and could link aberrant characteristics of B27-transgenic rat DCs to their putative pathogenic role in this model.
In conclusion, these results provide a mechanism that could explain how defective function in HLA–B27–transgenic rat DCs would contribute to disease development by skewing CD4+ T cell differentiation toward a proinflammatory over a regulatory phenotype. Whether similar mechanisms also apply to SpA in humans remains to be demonstrated. However, it is worth noting that a defective stimulatory capacity of autologous and heterologous CD4+ T cells has been observed in human DCs from HLA–B27–positive ankylosing spondylitis patients (46).
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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. Glatigny, Fert, Lories, Chiocchia, Breban.
Acquisition of data. Glatigny, Fert, Blaton, Lories, Araujo.
Analysis and interpretation of data. Glatigny, Fert, Blaton, Lories, Araujo, Chiocchia, Breban.