The spondylarthritides (SpA), including ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis, and arthritis associated with inflammatory bowel disease, cause chronic inflammation of the large peripheral and axial joints, eyes, skin, ileum, and colon. Genetic studies reveal common candidate genes for AS, PsA, and Crohn's disease, including IL23R, IL12B, STAT3, and CARD9, all of which are associated with interleukin-23 (IL-23) signaling downstream of the dectin 1 β-glucan receptor. In autoimmune-prone SKG mice with mutated ZAP-70, which attenuates T cell receptor signaling and increases the autoreactivity of T cells in the peripheral repertoire, IL-17–dependent inflammatory arthritis developed after dectin 1–mediated fungal infection. This study was undertaken to determine whether SKG mice injected with 1,3-β-glucan (curdlan) develop evidence of SpA, and the relationship of innate and adaptive autoimmunity to this process.
SKG mice and control BALB/c mice were injected once with curdlan or mannan. Arthritis was scored weekly, and organs were assessed for pathologic features. Anti–IL-23 monoclonal antibodies were injected into curdlan-treated SKG mice. CD4+ T cells were transferred from curdlan-treated mice to SCID mice, and sera were analyzed for autoantibodies.
After systemic injection of curdlan, SKG mice developed enthesitis, wrist, ankle, and sacroiliac joint arthritis, dactylitis, plantar fasciitis, vertebral inflammation, ileitis resembling Crohn's disease, and unilateral uveitis. Mannan triggered spondylitis and arthritis. Arthritis and spondylitis were T cell– and IL-23–dependent and were transferable to SCID recipients with CD4+ T cells. SpA was associated with collagen- and proteoglycan-specific autoantibodies.
Our findings indicate that the SKG ZAP-70W163C mutation predisposes BALB/c mice to SpA, resulting from innate and adaptive autoimmunity, after systemic β-glucan or mannan exposure.
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.
MATERIALS AND METHODS
Mice, induction of disease, and reagents.
BALB/c and BALB-SCID mice were supplied by Animal Research Centre. SKG mice were obtained from S. Sakaguchi (University of Kyoto, Kyoto, Japan) and bred in house. SKG mice were rederived into 2 separate SPF facilities and maintained under SPF conditions. All experiments were approved by the University of Queensland animal ethics committee. Disease was induced between 6 and 10 weeks of age using either 3 mg curdlan (derived from Alcaligenes faecalis variety myxogenes; Wako) administered IP or subcutaneously (SC) into either the base of the tail or the neck scruff, 3 mg curdlan administered by oral gavage, or 20 mg mannan administered IP. Mice were then monitored for 7–12 weeks. The doses used have been described previously (21, 22). In some experiments, 58 μg of mouse anti-mouse anti–IL-23 monoclonal antibody (mAb; developed and supplied by Eli Lilly) or isotype control mAb was given IP 1 day before curdlan injection and then weekly until mice were killed.
At experimental end points, organs from control and treated mice were fixed in formalin and embedded in paraffin. Mouse joint sections were first decalcified in EDTA. Enucleated eyes were fixed in 4% paraformaldehyde plus 1% glutaraldehyde and stored in 70% ethanol, then rehydrated before embedding in paraffin. Four-micrometer sections were cut and stained with hematoxylin and eosin (H&E), Martius Scarlet Blue, and active tartrate-resistant acid phosphatase (TRAP), or with mAb directed against Mac-2, osteocalcin, type I collagen, and anti-rat IgG2a revealed with horseradish peroxidase. Photographs were taken with a Nikon 5RS-08-0 microscope and NIS-Elements software. Mouse eyes were photographed using an Olympus BX60 microscope with a DP70 camera. Sections were analyzed, and inflammation was scored by at least 2 independent researchers (KA and ARP) who were blinded with regard to treatment group.
Scoring of clinical and histologic signs.
Clinical features in the mice were monitored weekly and scored by the same observer, who was blinded with regard to treatment, as follows: 0 = no swelling or redness, 0.1 = swelling or redness of digits, 0.5 = mild swelling and/or redness of wrists or ankle joints, and 1 = severe swelling of the larger joints. The scores of the affected joints were summed; the maximum possible score was 6. Histologic features of the mouse joint were scored on a scale of 1–4, where 1 = few infiltrating immune cells, 2 = 1–2 small patches of inflammation, 3 = inflammation throughout the ankle joint, and 4 = inflammation in soft tissue/entheses/fasciitis. Histologic features of the tail were scored on a scale of 1–4, where 1 = few infiltrating immune cells, 2 = mild inflammation of the discs or along the vertebrae (0–30% of discs), 3 = inflammation on the discs and/or along the vertebrae (30–70% of discs), and 4 = inflammation in >70% of the discs and along the vertebrae. The mouse intestine was scored 1 for the presence or 2 for the absence of ileitis or colitis.
T cell transfer.
CD3+CD4+ T cells were purified from the mouse spleen and lymph nodes by flow cytometric cell sorting using a MoFlo flow cytometer (Beckman Coulter), and 106 sorted cells were transferred intravenously to each SCID recipient. The postsort purity of CD3+CD4+ T cells was 98%. After 12 weeks, mouse organs were stained with H&E and scored.
Sera from untreated BALB/c or SKG mice, and from mice that had been treated with curdlan 4 or 8 weeks previously, were analyzed for IgG reactivity to type II collagen, proteoglycan, or mouse IgG-RF by enzyme-linked immunosorbent assay as previously described (18, 23, 24).
Radiographs were obtained with a Kodak in vivo animal imager. Formalin-fixed mouse bones were stored in a 70% ethanol solution and then scanned in a micro–computed tomography (micro-CT) scanner (Scanco Medical), with a resolution of 12 μm for the wrists and 18 μm for the spine. Bone mineral density (BMD), bone mineral content (BMC), total mass, and fat mass were determined by dual x-ray absorptiometry (DXA) using a Piximus densitometer (GE Lunar). Quality control and calibration were carried out within 24 hours prior to each scanning period using the phantom and procedures supplied by GE. The precision of the machine was measured, and the coefficients of variation for fat, BMD, and BMC were 2.3%, 0.2%, and 0.5%, respectively, for in vivo measurements.
Student's unpaired 2-tailed t-tests, with an alpha value of 0.05, were used to assess whether the means of 2 normally distributed groups differed significantly. Mann-Whitney tests (unpaired) were used for means that were not normally distributed or for sample sizes of <10. One-way analysis of variance (ANOVA) was used to compare normally distributed means, and two-way ANOVA was used to analyze treatment effects on clinical scores over time. Bonferroni post-test for multiple comparisons was then used to compare multiple means. Results are shown as the mean ± SEM.
Systemic curdlan exposure triggers SpA-like disease in SKG mice.
SKG mice were rederived into 2 separate SPF facilities at the University of Queensland. Under SPF conditions, SKG mice remained healthy, and none of the mice developed spontaneous arthritis for up to 1 year of observation. We treated SKG mice maintained under SPF conditions with 3 mg IP curdlan and scored joint inflammation weekly. Curdlan-treated BALB/c mice were used as controls. After curdlan administration, all SKG mice developed progressive inflammatory arthritis of the ankles and wrists (Figures 1A, N, and O), and swelling of the soft tissue of the feet (Figure 1B), all of which were more severe in females. Dactylitis occurred in 40–50% of the curdlan-treated mice (Figures 1B and C). We also observed reduced mobility, deformity or bumps along the tail, hunching of the upper body, conjunctivitis, and weight loss in curdlan-treated mice, but no diarrhea (Figure 1A). BALB/c mice developed arthritis less frequently than did SKG mice, and arthritis in BALB/c was much milder and nonprogressive (Figures 1N and O). The small intestine (jejunum and ileum), but not the colon, was thickened, with stricturing, in the curdlan-treated SKG mice (Figures 1D and E). The clinical pattern was identical in SKG mice treated with IP curdlan and those treated with SC curdlan, whether delivered SC to the base of the tail or to the neck scruff. However, disease did not develop after oral gavage of curdlan (Figure 1P). Results were identical after curdlan injection into mice bred at each SPF location.
Radiography and micro-CT of the paws and spine of SKG mice, performed 12 weeks after IP curdlan treatment, demonstrated deformity, loss of intervertebral disc height, severe erosion, and dystrophic new bone formation typical of SpA, comprising multiple osteophytes (Figures 1F–M). See Supplementary Videos 1 and 2, available on the Arthritis & Rheumatism web site at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131). The interphalangeal joints of the paws of the curdlan-treated mice demonstrated periarticular erosions and deformity (Figures 1I and M). These results show that severe inflammatory and erosive arthritis, spondylitis, and ileitis develop in SKG mice after systemic delivery of curdlan.
Histologic analysis of the mice over the first 5 weeks showed that inflammation appeared first in the ankle and small joints of the feet and at the bony insertion of the Achilles tendon, in the plantar fascia, and around the intervertebral discs, beginning 1 week after curdlan injection (Figures 2A—C). After 10 weeks, enthesitis of the Achilles tendon, plantar fasciitis (Figure 2I), arthritis of the sacroiliac joint (Figure 2K), arthritis of the adjacent lumbar and upper tail vertebral facet joint (Figure 2D), and inflammation of the intervertebral discs in the tail (including “skip lesions,” where adjacent discs were unaffected or inflamed) (Figures 2E–G) were evident in all curdlan-treated SKG mice. At this time point, severe synovitis and progressive destruction were detected in the ankles, wrists, feet, and digits of the mice (Figure 2J). Figure 2J shows synovitis of the small joints of the feet. Although typical psoriatic skin lesions did not develop, there was thickening of the epidermis and dermal inflammation (Figure 2L), sometimes with small dermal granulomas. Ear clipping in the curdlan-treated mice caused severe local inflammation, suggestive of a Koebner phenomenon (25).
Consistent with the histologic changes and with the radiologic evidence of erosion and new bone formation observed in the curdlan-treated SKG mice, immunohistochemical staining of the inflammatory infiltrate of the ankle joint, enthesis at the Achilles tendon insertion, and soft tissue of the foot in these mice revealed abundant Mac-2+ macrophages (Figure 3A) relative to the ankles of the healthy control (untreated) SKG mice (Figure 3D). Type I collagen staining of the bone matrix demonstrated that the normally smooth outline of the periosteum (Figure 3E) was replaced by extensive erosion and new bone formation in the curdlan-treated mice (Figure 3B). As expected, sites of bone erosion and new bone formation had greater numbers of associated TRAP+ multinucleated osteoclasts relative to the control (Figures 3C and F). These results indicate that, similar to other animal models of inflammatory arthritis, a macrophage-driven synovial inflammatory response leads to bone erosion and remodeling with new bone formation at affected sites.
Total body (excluding head and tail tip) BMD, BMC, and bone area were quantified using DXA in healthy BALB/c and SKG mice and in arthritic SKG mice that had been treated with curdlan 12 weeks earlier. BMD was reduced in arthritic SKG mice relative to healthy SKG mice, but bone area was increased (Figures 3G and H), similar to patients with AS, who demonstrate both osteoporosis and increased bone mass due to syndesmophyte formation in the spine (26). Consistent with chronic inflammation and osteopenia, fat mass was reduced in arthritic SKG mice (Figure 3K), as is observed in patients with AS (27).
Extraarticular manifestations triggered by β-glucan in SKG mice.
Of the curdlan-treated SKG mice, 50–60% developed small intestine inflammation (predominantly in the ileum) 10–12 weeks after curdlan injection. Transmural inflammation, crypt elongation, stricturing, abscesses, and granuloma formation were observed as discontinuous lesions in the ileum, as occur in Crohn's disease (Figures 4A–D). In severe cases, the jejunum was also affected, with occasional vasculitic lesions (Figure 4E), but colitis was not detected at any time point. The exact temporal relationship between IBD and arthritis in SpA patients remains elusive. After systemic injection of curdlan into SKG mice, peripheral and axial histologic inflammation developed simultaneously within 7 days in females and 35 days in males (Figure 2A), while histologic inflammation was not detected in the gut between 7 and 35 days after curdlan treatment (data not shown), and indeed only occurred some weeks later.
Approximately 25–40% of SpA patients have acute unilateral anterior uveitis. By 12 weeks after curdlan injection, 25% of the SKG mice developed unilateral uveitis, characterized by inflammatory infiltrate and fibrotic deposits in the anterior chamber and around the ciliary body in the affected eyes (Figures 4F–I). In addition to the overt iridocyclitis observed in the treated SKG mice (Figure 4F), adjacent vitritis was detected histologically (Figure 4H), but the retina remained unaffected. No inflammatory disease was present in any other major organ in the mice, including the lung, liver, spleen, thymus, salivary gland, colon, pancreas, kidney, central nervous system, or heart (data not shown). Thus, the curdlan-treated mice did not have the systemic multiorgan disease seen in some human autoimmune syndromes.
Role of innate and adaptive immunity in the development of inflammatory disease in SKG mice.
The shared human SpA susceptibility genes IL23R, IL12B, and CARD9 contribute to an inflammatory cascade downstream of dectin 1, the β-glucan receptor (4). Given the dectin 1–dependent multiorgan inflammatory disease resembling SpA triggered by β-glucan in SKG mice, we hypothesized that IL-23 plays a major role in disease development. SKG mice were treated with curdlan alone or were treated with either anti–IL-23 mAb or isotype control mAb beginning 1 day before IP curdlan treatment. Inhibition of IL-23 suppressed the development of peripheral arthritis (Figure 5A). Histologic scoring after 8 weeks of treatment showed that arthritis and spondylitis were absent in mice treated with anti–IL-23 mAb, while ileitis was observed in equivalent proportions of mice in each group. These data demonstrate the dependence of arthritis and spondylitis on IL-23 triggered by β-glucan in SKG mice.
CD4+ T cell–mediated autoimmunity has been shown to underpin spontaneous arthritis development in SKG mice housed under clean conventional conditions. To determine the role of autoreactive CD4+ T cells in the inflammatory disease triggered by curdlan, 106 CD4+ T cells purified by flow cytometric cell sorting from the spleen and lymph nodes of naive SKG mice, curdlan-treated SKG mice, or curdlan-treated BALB/c control mice were transferred to lymphopenic SCID recipients. Curdlan was delivered IP to treated donor mice 7 days before sorting. To determine the requirement for T cells in disease expression, SCID mice were injected with IP curdlan or saline. All recipients of CD4+ T cells from naive or curdlan-treated SKG mice developed arthritis and spondylitis (Figures 5B and C).
We found no evidence of fungal infection in SCID recipients, or treated or untreated SKG or BALB/c mice, as determined by serum β-glucan levels or periodic acid–Schiff staining of lung sections (data not shown). SKG CD4+ T cells were necessary and sufficient for the development of SpA, as evidenced by the fact that arthritis did not develop after transfer of BALB/c CD4+ T cells or after curdlan treatment of SCID mice in the absence of transferred T cells (Figures 5B and C). None of the mice developed diarrhea. None of the mice had ileitis, but all of the recipients of CD4+ T cells from curdlan-treated or untreated SKG mice, but not from BALB/c mice, developed colitis (scored as either present or absent) (Figure 5C). Histologically, transmural inflammation, crypt elongation, and loss of colonic epithelial integrity and goblet cells were evident (data not shown).
Entheseal, intervertebral disc, and sacroiliac joint fibrocartilaginous tissue contains potential autoantigens, including type II collagen and proteoglycan (aggrecan) (28). In support of the notion of CD4+ T cell–mediated autoimmunity, curdlan-treated SKG mice developed antiproteoglycan and anti–type II collagen autoantibodies within 4 weeks of curdlan treatment (Figure 5C), but they did not develop RF (data not shown). Moreover, antiproteoglycan and anti–type II collagen autoantibody levels increased in SCID mouse recipients after CD4+ T cell transfer. These data demonstrate the development of autoreactive CD4+ T cell–mediated and IL-23–dependent arthritis and spondylitis, but not ileitis, in SKG mice after systemic exposure to curdlan.
Multiple cell wall components trigger inflammatory arthritis and spondylitis in SKG mice.
After administration of either mannan or β-glucan to SKG mice, disease severity depends on C5a receptor signaling. C5a was found to amplify cytokine production by macrophages, and thus amplify IL-17+ T cells (29). After β-glucan injection, C5a is likely to amplify signals downstream of dectin 1, since anti–dectin 1 mAb prevents disease expression (21). To determine the features of SpA that develop through this mechanism, we treated SKG mice with 20 mg mannan and assessed histologic and clinical features for 12 weeks (Figure 6). Mice developed peripheral arthritis and spondylitis that was histologically similar to disease induced by curdlan. Of interest, clinical scores were lower in mannan-treated mice than in curdlan-treated mice, and disease severity was equivalent in the male and female mice treated with mannan. However, we detected neither ileitis nor iritis in response to mannan. Thus, multiple microbial cell wall components independently trigger inflammatory disease resembling SpA in SKG mice.
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.
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.
Authors Kikly and Davies are employees of Eli Lilly.
We thank Shimon Sakaguchi (Osaka University, Osaka, Japan) for supplying mice and for helpful discussions and Tibor Glant (Rush University, Chicago, IL) for supplying proteoglycan extract.