The spondylarthritides (SpA) comprise a group of diseases, including ankylosing spondylitis (AS), psoriatic arthritis (PsA), and reactive arthritis, that cause chronic joint inflammation and extraarticular inflammatory manifestations, including anterior uveitis, psoriasis, and the inflammatory bowel diseases (IBD) Crohn's disease and ulcerative colitis. SpA are thought to be triggered by an abnormal immune response to infection, and the >90% heritability of AS in twins suggests that this trigger is ubiquitous (1). There is evidence of involvement of innate and adaptive immunity in humans and rodent models with SpA. Recent genetic studies revealed common genes, including IL23R, IL12B, STAT3, and CARD9, to be associated with AS, psoriasis, and IBD, but not rheumatoid arthritis (RA) (2, 3). This suite of susceptibility genes is part of an inflammatory cascade downstream of the dectin 1/Syk pathway. Dectin 1, a receptor for β-glucan in antigen-presenting cells, promotes the expression of interleukin-1β (IL-1β), IL-12p35, IL-12p40, and IL-23p19 (4, 5). IL-23 is required for the expansion of IL-17+ cells, and signaling through the IL-23 receptor activates JAK-2 and STAT-3 (6, 7). It has been proposed that IL-17 immunity toward microorganisms may underpin the pathogenesis of SpA (8).
In this regard, some clinical studies have shown higher frequencies of peripheral blood IL-17+ T cells in patients with AS and those with psoriasis relative to healthy controls (9–11). However, Th17 cell frequencies are very low in peripheral blood, no differences in serum or intestinal IL-17 levels have been found between patients and controls (12, 13), and it is not clear whether SpA arises as a result of heightened Th17 responsiveness to an infectious trigger through gain-of-function polymorphisms and associated Th17-mediated autoimmunity (14), or as a result of defective Th17-mediated control of organisms colonizing epithelial and mucosal surfaces (15). Moreover, IL-17 at the inflammatory effector site in human SpA may derive predominantly from innate cells, such as mast cells and neutrophils (16, 17). Although IL-17 derived from the adaptive immune response may be produced in lymphoid organs, this is difficult to study in humans.
Sakaguchi and colleagues identified the SKG mouse strain, which develops spontaneous IL-17–dependent autoimmune inflammatory arthritis under conventional microbial conditions, initiated by pulmonary fungal infection and prevented by the antifungal agent amphotericin B (18). The SKG ZAP-70W163C mutation of the BALB/c mouse strain alters the sensitivity of developing thymocytes to both negative and positive selection in the thymus, enriching the peripheral repertoire with IL-17–skewed autoreactive T cells (19, 20). SKG mice with spontaneous disease developed multiple autoantibodies, including rheumatoid factor (RF) and anti–type II collagen, and disease was transferable by CD4+ T cells in an IL-17–dependent manner (18, 20).
Beta-glucan is a major component of bacterial and fungal cell walls, including Candida, Aspergillus, Saccharomyces, and Pneumocystis species, some of which were identified in the natural respiratory infection that occurs in SKG mice. Under specific pathogen–free (SPF) conditions, SKG mice remained healthy (21). However, under these conditions, intraperitoneal (IP) injection of β-glucan–containing products, including curdlan (1,3-β-glucan aggregates), laminarin (soluble 1,3-β- and some 1,6-β-glucans), and zymosan (containing β-glucans, mannan, chitin, and protein) was shown to induce inflammatory arthritis in these mice (21). Beta-glucan signaling through the dectin 1 receptor was identified as a key trigger of arthritis in this model. Another microbial cell wall component, mannan, also triggered peripheral arthritis in SKG mice, through a C5a-mediated mechanism (22). However, in each of these models, including mice housed under conventional conditions, the mice were not examined for extraarticular features of SpA, which are less clinically evident. Since curdlan triggers dectin 1, which is upstream of the gene cascade associated with human SpA, we investigated whether curdlan-treated SKG mice develop evidence of SpA, and the relationship of innate and adaptive autoimmunity to this process.
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The characteristic clinical, histologic, and radiographic features we describe herein indicate that after systemic exposure to curdlan, SKG mice develop a disease with multiple features that resemble those of human SpA. Whereas spontaneous autoimmune arthritis in SKG mice has been interpreted as RA-like (18), the clinical features we describe suggest that curdlan- and mannan-triggered disease more closely models SpA-like, rather than RA-like, disease. Pneumonitis, RF, and rheumatoid nodules were described in SKG mice with spontaneous arthritis and attributed to RA (18), but were not observed in the mice in our study after systemic curdlan exposure. It is unclear whether the lung microflora modified disease expression of spontaneous arthritis. It should be noted, however, that pneumonitis in SKG mice occurred in the context of fungal infection of the lung (21), and would not be expected after curdlan injection. Furthermore, the skin granuloma which was originally described as a rheumatoid nodule (18) did not have the typical central acellular necrobiosis normally attributed to this diagnosis.
The β-glucan molecular pattern is widespread in the environment in fungal cell walls, as well as in some plants, seaweeds, and bacteria. Of interest, in patients with Crohn's disease, antibody specificities toward laminarin, chitin, mannose, or mannan (anti–Saccharomyces cerevisiae antibodies [ASCAs]) components of yeast cell walls (including the commensal Candida albicans), the OmpC membrane determinant of Escherichia coli, or indeed a multibacterial membrane preparation prepared from common mucosa-associated microbiota were found to be increased (30, 31). ASCAs are also detected in 20–30% of patients with AS (32, 33). Those studies demonstrated immunogenicity of commensal yeasts and bacteria, potentially associated with systemic exposure in human IBD, underlining the strong clinical parallel with the terminal ileitis that is triggered by fungal or bacterial cell wall β-glucan in SKG mice.
The findings of the present study support the hypothesis that an interaction between innate control of microbial immunity and autoimmunity underlies the tissue-specificity of the initiation of arthritis and spondylitis in SKG mice. Proteoglycan and type II collagen–specific autoantibodies were induced after curdlan treatment in SKG mice, and CD4+ T cells transferred arthritis and spondylitis to recipient mice. Type II collagen–specific autoantibodies, which are associated with loss of T cell tolerance, were previously observed in SKG mice with spontaneous arthritis (18). The results of experiments in AIRE-deficient mice further support the conclusion that central tolerance mechanisms normally regulate type II collagen–specific autoimmunity (24). Thus, in SKG mice with deficient thymic negative selection and autoreactive peripheral CD4+ T cells (34), β-glucan signals rapidly induced peripheral and axial arthritis, and autoimmunity toward cartilage components. Consistent with this specificity, inflammatory disease in the SKG mice began at entheseal, fibrocartilaginous sites in the ankle and spine, and later progressed to dactylitis and erosive synovitis of the wrists, ankles, sacroiliac joints, and intervertebral discs. It has been proposed that entheses are particularly prone to microtrauma, giving rise to local stress, new vessel infiltration, and deposition of microbial components (35). Our findings suggest that this process may also contribute to the liberation of fibrocartilage autoantigens. Rodent models and humans with AS demonstrate CD4+ and CD8+ T cell autoreactivity toward aggrecan, and proteoglycan-immunized mice develop peripheral arthritis and spondylitis (36, 37).
Up to 75% of SpA patients may have subclinical terminal ileal inflammation based on microscopic analysis after colonoscopy (38). Given the likely role of infection in the pathogenesis of SpA, there has been speculation that gut infection or inflammation may act as a trigger for subsequent arthritis. The lack of histologic inflammation within the first weeks after curdlan injection in SKG mice does not exclude a role for the gut in arthritis development. Gut commensal microflora or specific classes of pathogens may be instrumental in the initiation of inflammatory peripheral arthritis (39). Moreover, HLA–B27–transgenic rats do not develop SpA in a germ-free environment (40). However, it has been proposed that self-reactive T cells primed to joint antigens may traffic to the gut and recognize or cross-react with local antigens, since T cells in Peyer's patches and lamina propria express adhesion molecules which allow adherence to high endothelial venules in both gut mucosa and synovial tissue, including α4β1 and lymphocyte function–associated antigen 1 (41). In SKG mice, histologic inflammation of the peripheral and axial joints preceded that of the gut by many weeks. Moreover, CD4+ T cells from curdlan-primed mice did not transfer ileitis, suggesting that joint-specific autoreactive T cells do not cross-react with self-antigens of the small intestine. Rather, Crohn's disease of the small intestine is likely to be driven by specific immune mechanisms which evolve subsequent to those driving arthritis.
The development of colitis in the mice that received CD4+ T cells in the present study is consistent with previous observations that colitis occurred after the transfer of SKG CD4+ T cells to nude mice. SKG Treg cells regulate poorly within the lymphopenic environment of the recipient, and the prevalence of colitis was greater after transfer of Treg-depleted SKG T cells (34). The capacity of CD4+ T cells from untreated SKG mice to transfer disease to immunodeficient recipients is consistent with the interpretation that regulatory populations control disease in untreated SKG mice under SPF conditions. Indeed, when SKG mice were crossed to ZAP-70−/− mice, thus increasing autoreactivity, investigators found that SKG/SKG ZAP-70+/− mice developed spontaneous arthritis under SPF conditions (34).
While infectious or pathogen-associated molecular pattern triggers amplify IL-6–dependent, IL-17–mediated inflammation through dectin 1 in BALB/c and SKG mice (20, 42), persistence of arthritis requires the ZAP-70 mutation, which decreases T cell receptor signaling and increases the autoreactivity of T cells in the peripheral repertoire. It is still unclear whether HLA–B27 contributes to human SpA through presentation of specific class I–restricted peptides, promotion of cellular stress with IL-17 induction through the unfolded protein response (43), or other noncanonical mechanisms (15). At least in SKG mice, presentation of specific HLA–B27–restricted peptides is not required for the development of SpA-like disease. From 5% to 25% of AS patients and up to 60% of undifferentiated SpA cases are HLA–B27 negative (44). Taken together, our data suggest that the autoimmune-prone SKG mouse strain is a model of rapid self (including cartilage) antigen priming of autoreactive CD4+ T cells triggered by the proinflammatory innate effects of β-glucan or mannan in an IL-23–dependent manner. Of interest, these T cells appear to have specificity for joint and spine, whereas ileitis appears to result from a slower and less penetrant CD4+ T cell–independent process triggered by β-glucan but not mannan signaling. Although IL-23 may be involved, other inflammatory factors, such as IL-17 production by innate immune cells, must also drive IBD in this model.
In support of our hypothesis regarding the development of arthritic disease in SKG mice, CD4+ T cells from humans heterozygous for the protective R381Q IL23R polymorphism were shown to have reduced IL-23–mediated STAT3 signaling relative to those homozygous for wild-type IL23R (45, 46). Human SpA is highly heritable, and genetic studies in the last 5 years have uncovered multiple associated genes, with individual small effect sizes in AS, psoriasis, PsA, and IBD. This means that predisposition to SpA in the population is associated with many possible genes with combined effects. A number of models have been described that recapitulate features of multisystem SpA, including HLA–B27–transgenic rats, TNFΔARE-transgenic mice overexpressing tumor necrosis factor, and SKG mice treated with β-glucan, as described herein (47, 48). Th17 cells were shown to be expanded, and CD4+ T cells to transfer disease in both SKG mice and HLA–B27–transgenic rats, whereas arthritis was found to be T cell independent in TNFΔARE mice (20, 49, 50). These observations demonstrate that multiple mechanisms can contribute to models with features of multisystem SpA. Similarly, as discussed above, it appears very likely that different mechanisms underlie arthritis and IBD in SKG mice treated with curdlan.
The polygenic nature of human SpA strongly suggests that multiple pathways will be involved in different patients depending on genetic background and environmental exposure, and that disease expression will likely involve more than one abnormal signal in order to overcome normal innate and adaptive immune regulation. However, the findings of the present study support the concept that genes enhancing IL-23 signaling, such as IL23R, IL12B, STAT3, and CARD9, predispose to a heightened and prolonged response to β-glucan through the dectin 1 receptor. Further elucidation of the mechanisms leading to disease in SKG mice will yield interesting hypotheses for translation to human disease.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
- ADDITIONAL DISCLOSURES
- 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. R. Thomas 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. Ruutu, Benham, Kikly, R. Thomas.
Acquisition of data. Ruutu, G. Thomas, Steck, Zinkernagel, Alexander, Velasco, Tran, Benham, Rehaume, Wilson, Davies.
Analysis and interpretation of data. Ruutu, G. Thomas, Steck, Degli-Esposti, Zinkernagel, Alexander, Strutton, Tran, Benham, Rehaume, Wilson, Pettit, Brown, McGuckin, R. Thomas.