Contribution of Type H Blood Vessels to Pathologic Osteogenesis and Inflammation in an Experimental Spondyloarthritis Model.

OBJECTIVE: Spondyloarthritis (SpA) is characterized by pathologic osteogenesis, inflammation, and extensive angiogenesis in axial and peripheral tissues. Current therapies effectively target inflammation, but these therapies lack efficacy in preventing pathologic osteogenesis. Transgenic mice overexpressing transmembrane tumor necrosis factor (tmTNF-Tg mice) exhibit SpA-like features. We hypothesized that type H blood vessels, which are implicated in osteogenesis, are increased and contribute to pathology in this experimental SpA model. METHODS: We analyzed ankles, femora, and vertebrae of tmTNF-Tg mice and nontransgenic littermates and tmTNF-Tg mice on either a TNF receptor type I (TNFRI)-deficient or TNF receptor type II (TNFRII)-deficient background for osteogenesis, angiogenesis, and inflammation using advanced imaging technologies at various stages of disease. RESULTS: Compared to nontransgenic littermates, tmTNF-Tg mice exhibited an increase in vertebral type H vessels and osteoprogenitor cells in subchondral bone. These features of increased angiogenesis and osteogenesis were already present before onset of clinical disease symptoms. Type H vessels and osteoprogenitor cells were in close proximity to inflammatory lesions and ectopic lymphoid structures. The tmTNF-Tg mice also showed perivertebral ectopic type H vessels and osteogenesis, an increased number of vertebral transcortical vessels, and enhanced entheseal angiogenesis. In tmTNF-Tg mice crossed on a TNFRI- or TNFRII-deficient background, no clear reduction in type H vessels was shown, suggesting that type H vessel formation is not exclusively mediated via TNFRI or TNFRII. CONCLUSION: The contribution of type H vessels to pathologic osteogenesis in experimental SpA advances our knowledge of the pathophysiology of this disease and may also provide a novel opportunity for targeted intervention.

Dual Blockade of TNF and IL-17A Inhibits Inflammation and Structural Damage in a Rat Model of Spondyloarthritis.

The tumor necrosis factor (TNF) and IL-23/IL-17 axes are the main therapeutic targets in spondyloarthritis. Despite the clinical efficacy of blocking either pathway, monotherapy does not induce remission in all patients and its effect on new bone formation remains unclear. We aimed to study the effect of TNF and IL-17A dual inhibition on clinical disease and structural damage using the HLA-B27/human ß2-microglobulin transgenic rat model of SpA. Immunized rats were randomized according to arthritis severity, 1 week after arthritis incidence reached 50%, to be treated twice weekly for a period of 5 weeks with either a dual blockade therapy of an anti-TNF antibody and an anti-IL-17A antibody, a single therapy of either antibody, or PBS as vehicle control. Treatment-blinded observers assessed inflammation and structural damage clinically, histologically and by micro-CT imaging. Both single therapies as well as TNF and IL-17A dual blockade therapy reduced clinical spondylitis and peripheral arthritis effectively and similarly. Clinical improvement was confirmed for all treatments by a reduction of histological inflammation and pannus formation (p < 0.05) at the caudal spine. All treatments showed an improvement of structural changes at the axial and peripheral joints on micro-CT imaging, with a significant decrease for roughness (p < 0.05), which reflects both erosion and new bone formation, at the level of the caudal spine. The effect of dual blockade therapy on new bone formation was more prominent at the axial than the peripheral level. Collectively, our study showed that dual blockade therapy significantly reduces inflammation and structural changes, including new bone formation. However, we could not confirm a more pronounced effect of dual inhibition compared to single inhibition.

Overexpression of Transmembrane TNF Drives Development of Ectopic Lymphoid Structures in the Bone Marrow and B Cell Lineage Alterations in Experimental Spondyloarthritis.

TNF is important in immune-mediated inflammatory diseases, including spondyloarthritis (SpA). Transgenic (tg) mice overexpressing transmembrane TNF (tmTNF) develop features resembling human SpA. Furthermore, both tmTNF tg mice and SpA patients develop ectopic lymphoid aggregates, but it is unclear whether these contribute to pathology. Therefore, we characterized the lymphoid aggregates in detail and studied potential alterations in the B and T cell lineage in tmTNF tg mice. Lymphoid aggregates developed in bone marrow (BM) of vertebrae and near the ankle joints prior to the first SpA features and displayed characteristics of ectopic lymphoid structures (ELS) including presence of B cells, T cells, germinal centers, and high endothelial venules. Detailed flow cytometric analyses demonstrated more germinal center B cells with increased CD80 and CD86 expression, along with significantly more T follicular helper, T follicular regulatory, and T regulatory cells in tmTNF tg BM compared with non-tg controls. Furthermore, tmTNF tg mice exhibited increased IgA serum levels and significantly more IgA+ plasma cells in the BM, whereas IgA+ plasma cells in the gut were not significantly increased. In tmTNF tg × TNF-RI-/- mice, ELS were absent, consistent with reduced disease symptoms, whereas in tmTNF tg × TNF-RII-/- mice, ELS and clinical symptoms were still present. Collectively, these data show that tmTNF overexpression in mice results in osteitis and ELS formation in BM, which may account for the increased serum IgA levels that are also observed in human SpA. These effects are mainly dependent on TNF-RI signaling and may underlie important aspects of SpA pathology.

Paradoxical Augmentation of Experimental Spondyloarthritis by RORC Inhibition in HLA-B27 Transgenic Rats.

Objective: IL-17A plays a major role in the pathogenesis of spondyloarthritis (SpA). Here we assessed the impact of inhibition of RAR related orphan receptor-γ (RORC), the key transcription factor controlling IL-17 production, on experimental SpA in HLA-B27 transgenic (tg) rats. Methods: Experimental SpA was induced by immunization of HLA-B27 tg rats with heat-inactivated Mycobacterium tuberculosis. Splenocytes obtained at day 7, 14 and 21 after immunization were restimulated ex vivo to assess the induction of pro-inflammatory cytokines. Rats were then prophylactically treated with a RORC inhibitor versus vehicle control. The biologic effect of RORC inhibition was assessed by pro-inflammatory cytokine expression in draining lymph nodes. Arthritis and spondylitis were monitored clinically, and the degree of peripheral and axial inflammation, destruction and new bone formation was confirmed by histology. Results: Ex vivo mRNA and protein analyses revealed the rapid and selective induction of IL-17A and IL-22 production by a variety of lymphocyte subsets upon disease induction in HLA-B27 tg rats. Prophylactic RORC inhibition in vivo suppressed the expression of IL-17A, IL17F, and IL-22 without affecting the expression of other T helper cell subset related genes. This biological effect did not translate into clinical efficacy as RORC inhibition significantly accelerated the onset of arthritis and spondylitis, and aggravated the clinical severity of arthritis. This worsening of experimental SpA was confirmed by histopathological demonstration of increased inflammation, destruction, and new bone formation. Conclusion: Despite a significant suppression of the IL-17 axis, RORC inhibitor treatment accelerates and aggravates experimental SpA in the HLA-B27 tg rat model.

Transmembrane TNF drives osteoproliferative joint inflammation reminiscent of human spondyloarthritis.

TNF plays a key role in immune-mediated inflammatory diseases including rheumatoid arthritis (RA) and spondyloarthritis (SpA). It remains incompletely understood how TNF can lead to different disease phenotypes such as destructive peripheral polysynovitis in RA versus axial and peripheral osteoproliferative inflammation in SpA. We observed a marked increase of transmembrane (tm) versus soluble (s) TNF in SpA versus RA together with a decrease in the enzymatic activity of ADAM17. In contrast with the destructive polysynovitis observed in classical TNF overexpression models, mice overexpressing tmTNF developed axial and peripheral joint disease with synovitis, enthesitis, and osteitis. Histological and radiological assessment evidenced marked endochondral new bone formation leading to joint ankylosis over time. SpA-like inflammation, but not osteoproliferation, was dependent on TNF-receptor I and mediated by stromal tmTNF overexpression. Collectively, these data indicate that TNF can drive distinct inflammatory pathologies. We propose that tmTNF is responsible for the key pathological features of SpA.

APRIL Induces a Novel Subset of IgA+ Regulatory B Cells That Suppress Inflammation via Expression of IL-10 and PD-L1.

Regulatory B cells (Bregs) are immunosuppressive cells that modulate immune responses through multiple mechanisms. The signals required for the differentiation and activation of these cells remain still poorly understood. We have already shown that overexpression of A PRoliferation-Inducing Ligand (APRIL) reduces the incidence and severity of collagen-induced arthritis (CIA) in mice. Furthermore, we have described that APRIL, but not BAFF, promoted IL-10 production and regulatory functions in human B cells. Therefore, we hypothesized that APRIL, but not BAFF, may be involved in the induction and/or activation of IL-10 producing Bregs that suppress inflammatory responses in vitro and in vivo. Here, we describe that APRIL promotes the differentiation of naïve human B cells to IL-10-producing IgA+ B cells. These APRIL-induced IgA+ B cells display a Breg phenotype and inhibit T cell and macrophage responses through IL-10 and PD-L1. Moreover, APRIL-induced IL-10 producing Bregs suppress inflammation in vivo in experimental autoimmune encephalitis (EAE) and contact hypersensitivity (CHS) models. Finally, we showed a strong correlation between APRIL and IL-10 in the inflamed synovial tissue of inflammatory arthritis patients. Collectively, these observations indicate the potential relevance of this novel APRIL-induced IgA+ Breg population for immune homeostasis and immunopathology.

mTOR Blockade by Rapamycin in Spondyloarthritis: Impact on Inflammation and New Bone Formation in vitro and in vivo.

Introduction: Spondyloarthritis (SpA) is characterized by inflammation, articular bone erosions and pathologic new bone formation. Targeting TNFα or IL-17A with current available therapies reduces inflammation in SpA, however, treatment of the bone pathology in SpA remains an unmet clinical need. Activation of the mammalian target Of rapamycin (mTOR) promotes IL-17A expression and osteogenesis. Therefore, the inhibition of mTOR (with rapamycin) could be a promising therapeutic avenue in SpA. Objectives: To investigate the effect of blocking mTOR on inflammation, bone erosions and new bone formation in SpA. Methods: Peripheral blood mononuclear cells (PBMCs) from patients with SpA were stimulated with anti-CD3/CD28 in the presence or absence of rapamycin and the resulting cytokine expression was assessed. Fibroblast-like synoviocytes (FLS) from SpA patients were assessed for osteogenic differentiation potential in conditions with TNFα, IL-17A, or TNFα plus IL-17A, in the presence or absence of rapamycin. HLA-B27/Huß2m transgenic rats were immunized with low dose heat-inactivated Mycobacterium tuberculosis (M. tub), treated with 1.5 mg/kg rapamycin prophylactically or therapeutically and monitored for arthritis and spondylitis. Histology and mRNA analysis were performed after 5 weeks of treatment to assess inflammation and bone pathology. Results:In vitro TNFα and IL-17A protein production by SpA PBMCs was inhibited in the presence of rapamycin. Rapamycin also inhibited osteogenic differentiation of human SpA FLS. Ex vivo analysis of SpA synovial biopsies indicated activation of the mTOR pathway in the synovial tissue of SpA patients. In vivo, prophylactic treatment of HLA-B27/Huß2m transgenic rats with rapamycin significantly inhibited the development and severity of inflammation in peripheral joints and spine (arthritis and spondylitis), with histological evidence of reduced bone erosions and new bone formation around peripheral joints. In addition, therapeutic treatment with rapamycin significantly decreased severity of arthritis and spondylitis, with peripheral joint histology showing reduced inflammation, bone erosions and new bone formation. IL-17A mRNA expression was decreased in the metacarpophalangeal joints after rapamycin treatment. Conclusion: mTOR blockade inhibits IL-17A and TNFα production by PBMCs, and osteogenic differentiation of FLS from patients with SpA in vitro. In the HLA-B27 transgenic rat model of SpA, rapamycin inhibits arthritis and spondylitis development and severity, reduces articular bone erosions, decreases pathologic new bone formation and suppresses IL-17A expression. These results may support efforts to evaluate the efficacy of targeting the mTOR pathway in SpA patients.

Interleukin-17A Inhibition Diminishes Inflammation and New Bone Formation in Experimental Spondyloarthritis.

OBJECTIVE: It remains unclear if and how inflammation and new bone formation in spondyloarthritis (SpA) are coupled. We undertook this study to assess the hypothesis that interleukin-17A (IL-17A) is a pivotal driver of both processes. METHODS: The effect of tumor necrosis factor (TNF) and IL-17A on osteogenesis was tested in an osteoblastic differentiation assay using SpA fibroblast-like synoviocytes (FLS) differentiated with dexamethasone, ß-glycophosphatase, and ascorbic acid. IL-17A blockade was performed in HLA-B27/human ß2 -microglobulin (hß2 m)-transgenic rats, which served as a model for SpA in both prophylactic and therapeutic settings. Inflammation and new bone formation were evaluated by micro-computed tomography imaging, histologic analysis, and gene expression profiling. RESULTS: TNF and IL-17A significantly increased in vitro osteoblastic differentiation. In vivo, prophylactic blockade of IL-17A significantly delayed spondylitis and arthritis development and decreased arthritis severity. Anti-IL-17A treatment was also associated with prevention of bone loss and periosteal new bone formation. Therapeutic targeting of IL-17A after the initial inflammatory insult also significantly reduced axial and peripheral joint inflammation. This treatment was again associated with a marked reduction in spinal and peripheral structural damage, including new bone formation. RNA sequencing of target tissue confirmed that IL-17A is a key driver of the molecular signature of disease in this model and that therapeutic anti-IL-17A treatment reversed the inflammatory signature and the selected gene expression related to bone damage. CONCLUSION: Both prophylactic and therapeutic inhibition of IL-17A diminished inflammation and new bone formation in HLA-B27/hß2 m-transgenic rats. Taken together with the ability of IL-17A to promote osteoblastic differentiation of human SpA FLS, these data suggest a direct link between IL-17A-driven inflammation and pathologic new bone formation in SpA.

The Initiation, but Not the Persistence, of Experimental Spondyloarthritis Is Dependent on Interleukin-23 Signaling.

IL-17A is a central driver of spondyloarthritis (SpA), its production was originally proposed to be IL-23 dependent. Emerging preclinical and clinical evidence suggests, however, that IL-17A and IL-23 have a partially overlapping but distinct biology. We aimed to assess the extent to which IL-17A-driven pathology is IL-23 dependent in experimental SpA. Experimental SpA was induced in HLA-B27/Huß2m transgenic rats, followed by prophylactic or therapeutic treatment with an anti-IL23R antibody or vehicle control. Spondylitis and arthritis were scored clinically and hind limb swelling was measured. Draining lymph node cytokine expression levels were analyzed directly ex vivo, and IL-17A protein was measured upon restimulation with PMA/ionomycin. Prophylactic treatment with anti-IL23R completely protected against the development of both spondylitis and arthritis, while vehicle-treated controls did develop spondylitis and arthritis. In a therapeutic study, anti-IL23R treatment failed to reduce the incidence or decrease the severity of experimental SpA. Mechanistically, expression of downstream effector cytokines, including IL-17A and IL-22, was significantly suppressed in anti-IL23R versus vehicle-treated rats in the prophylactic experiments. Accordingly, the production of IL-17A upon restimulation was reduced. In contrast, there was no difference in IL-17A and IL-22 expression after therapeutic anti-IL23R treatment. Targeting the IL-23 axis during the initiation phase of experimental SpA-but not in established disease-inhibits IL-17A expression and suppresses disease, suggesting the existence of IL-23-independent IL-17A production. Whether IL-17A can be produced independent of IL-23 in human SpA remains to be established.

Bone formation in ankylosing spondylitis during anti-tumour necrosis factor therapy imaged by 18F-fluoride positron emission tomography.

Objectives: Excessive bone formation is an important hallmark of AS. Recently it has been demonstrated that axial bony lesions in AS patients can be visualized using 18F-fluoride PET-CT. The aim of this study was to assess whether 18F-fluoride uptake in clinically active AS patients is related to focal bone formation in spine biopsies and is sensitive to change during anti-TNF treatment. Methods: Twelve anti-TNF-naïve AS patients [female 7/12; age 39 years (SD 11); BASDAI 5.5 ± 1.1] were included. 18 F-fluoride PET-CT scans were performed at baseline and in two patients, biopsies were obtained from PET-positive and PET-negative spine lesions. The remaining 10 patients underwent a second 18F-fluoride PET-CT scan after 12 weeks of anti-TNF treatment. PET scans were scored visually by two blinded expert readers. In addition, 18F-fluoride uptake was quantified using the standardized uptake value corrected for individual integrated whole blood activity concentration (SUVAUC). Clinical response to anti-TNF was defined according to a ⩾ 20% improvement in Assessment of SpondyloArthritis international Society criteria at 24 weeks. Results: At baseline, all patients showed at least one axial PET-positive lesion. Histological analysis of PET-positive lesions in the spine confirmed local osteoid formation. PET-positive lesions were found in the costovertebral joints (43%), facet joints (23%), bridging syndesmophytes (20%) and non-bridging vertebral lesions (14%) and in SI joints (75%). After 12 weeks of anti-TNF treatment, 18F-fluoride uptake in clinical responders decreased significantly in the costovertebral (mean SUVAUC -1.0; P < 0.001) and SI joints (mean SUVAUC -1.2; P = 0.03) in contrast to non-responders. Conclusions: 18F-fluoride PET-CT identified bone formation, confirmed by histology, in the spine and SI joints of AS patients and demonstrated alterations in bone formation during anti-TNF treatment.

Innate Immune Activation Can Trigger Experimental Spondyloarthritis in HLA-B27/Huß2m Transgenic Rats.

Spondyloarthritis (SpA) does not display the typical features of auto-immune disease. Despite the strong association with MHC class I, CD8+ T cells are not required for disease induction in the HLA-B27/Huß2m transgenic rats. We used Lewis HLA-B27/Huß2m transgenic rats [21-3 × 283-2]F1, HLA-B7/Huß2m transgenic rats [120-4 × 283-2]F1, and wild-type rats to test our hypothesis that SpA may be primarily driven by the innate immune response. In vitro, splenocytes were stimulated with heat-inactivated Mycobacterium tuberculosis and cytokine expression and production was measured. In vivo, male and female rats were immunized with 30, 60, or 90 µg of heat-inactivated M. tuberculosis and clinically monitored for spondylitis and arthritis development. After validation of the model, we tested whether prophylactic and therapeutic TNF targeting affected spondylitis and arthritis. In vitro stimulation with heat-inactivated M. tuberculosis strongly induced gene expression of pro-inflammatory cytokines such as TNF, IL-6, IL-1α, and IL-1ß, in the HLA-B27 transgenic rats compared with controls. In vivo immunization induced an increased spondylitis and arthritis incidence and an accelerated and synchronized onset of spondylitis and arthritis in HLA-B27 transgenic males and females. Moreover, immunization overcame the protective effect of orchiectomy. Prophylactic TNF targeting resulted in delayed spondylitis and arthritis development and reduced arthritis severity, whereas therapeutic TNF blockade did not affect spondylitis and arthritis severity. Collectively, these data indicate that innate immune activation plays a role in the initiation of HLA-B27-associated disease and allowed to establish a useful in vivo model to study the cellular and molecular mechanisms of disease initiation and progression.

Impact of the B Cell Growth Factor APRIL on the Qualitative and Immunological Characteristics of Atherosclerotic Plaques.

Studies on the role of B lymphocytes in atherosclerosis development, have yielded contradictory results. Whereas B lymphocyte-deficiency aggravates atherosclerosis in mice; depletion of mature B lymphocytes reduces atherosclerosis. These observations led to the notion that distinct B lymphocyte subsets have different roles. B1a lymphocytes exert an atheroprotective effect, which has been attributed to secretion of IgM, which can be deposited in atherosclerotic lesions thereby reducing necrotic core formation. Tumor necrosis factor (TNF)-family member 'A Proliferation-Inducing Ligand' (APRIL, also known as TNFSF13) was previously shown to increase serum IgM levels in a murine model. In this study, we investigated the effect of APRIL overexpression on advanced lesion formation and composition, IgM production and B cell phenotype. We crossed APRIL transgenic (APRIL-Tg) mice with ApoE knockout (ApoE-/-) mice. After a 12-week Western Type Diet, ApoE-/-APRIL-Tg mice and ApoE-/- littermates showed similar increases in body weight and lipid levels. Histologic evaluation showed no differences in lesion size, stage or necrotic area. However, smooth muscle cell (α-actin stain) content was increased in ApoE-/-APRIL-Tg mice, implying more stable lesions. In addition, increases in both plaque IgM deposition and plasma IgM levels were found in ApoE-/-APRIL-Tg mice compared with ApoE-/- mice. Flow cytometry revealed a concomitant increase in peritoneal B1a lymphocytes in ApoE-/-APRIL-Tg mice. This study shows that ApoE-/-APRIL-Tg mice have increased oxLDL-specific serum IgM levels, potentially mediated via an increase in B1a lymphocytes. Although no differences in lesion size were found, transgenic ApoE-/-APRIL-Tg mice do show potential plaque stabilizing features in advanced atherosclerotic lesions.

Insulin-Like Growth Factor I Does Not Drive New Bone Formation in Experimental Arthritis.

INTRODUCTION: Insulin like growth factor (IGF)-I can act on a variety of cells involved in cartilage and bone repair, yet IGF-I has not been studied extensively in the context of inflammatory arthritis. The objective of this study was to investigate whether IGF-I overexpression in the osteoblast lineage could lead to increased reparative or pathological bone formation in rheumatoid arthritis and/or spondyloarthritis respectively. METHODS: Mice overexpressing IGF-I in the osteoblast lineage (Ob-IGF-I+/-) line 324-7 were studied during collagen induced arthritis and in the DBA/1 aging model for ankylosing enthesitis. Mice were scored clinically and peripheral joints were analysed histologically for the presence of hypertrophic chondrocytes and osteocalcin positive osteoblasts. RESULTS: 90-100% of the mice developed CIA with no differences between the Ob-IGF-I+/- and non-transgenic littermates. Histological analysis revealed similar levels of hypertrophic chondrocytes and osteocalcin positive osteoblasts in the ankle joints. In the DBA/1 aging model for ankylosing enthesitis 60% of the mice in both groups had a clinical score 1<. Severity was similar between both groups. Histological analysis revealed the presence of hypertrophic chondrocytes and osteocalcin positive osteoblasts in the toes in equal levels. CONCLUSION: Overexpression of IGF-I in the osteoblast lineage does not contribute to an increase in repair of erosions or syndesmophyte formation in mouse models for destructive and remodeling arthritis.

Data showing non-conventional HLA-B27 expression in axial joints and gut tissue from B27 transgenic rats, and in frozen and paraffin-fixed synovial SpA tissue.

Data is presented showing expression of non-conventional (NC) heavy chain forms of B27 in synovial tissues from SpA patients. Data is presented showing the expression patterns of NC-B27 in joint, gastrointestinal and lymphoid tissues from B27 transgenic (TG(1)) rats with M. tuberculosis-induced SpA. Expression of NC-B27 was determined by immunohistochemistry and flow cytometry using HC10 and HD6 antibodies. These data are the extension of the data presented and discussed in "Non-conventional forms of HLA-B27 are expressed in Spondyloarthritis joints and gut tissue" (O. Rysnik, K. McHugh, L. M. van Duivenvoorde, M. N. van Tok, G. Guggino, J. D. Taurog, S. Kollnberger, F. Ciccia, D. L. Baeten, P. Bowness, 2016) [1].

Non-conventional forms of HLA-B27 are expressed in spondyloarthritis joints and gut tissue.

OBJECTIVES: Human leukocyte antigen (HLA)-B27 (B27) is the strongest genetic factor associated with development of Ankylosing Spondylitis and other spondyloarthropathies (SpA), yet the role it plays in disease pathogenesis remains unclear. We investigated the expression of potentially pathogenic non-conventional heavy chain forms (NC) of B27 in synovial and intestinal tissues obtained from SpA patients. We also determined the presence of NC-B27 in joints, lymphoid and gastrointestinal tissue from B27 transgenic (TG(1)) rats with M.tuberculosis-induced SpA. METHODS: Expression of NC-B27 in human SpA joints and gut and in (21-3 × 283-2)F1 HLA-B27/Huß2m rat tissue was determined by immunohistochemistry, flow cytometry and confocal microscopy analysis using HC10 and HD6 antibodies. RESULTS: Both HC10- and HD6-reactive HLA molecules were present in synovial tissue from SpA patients. Both NC-B27 and KIR3DL2, a ligand for NC-B27, were expressed in inflamed terminal ileal tissues in patients with early SpA. Infiltrating cells in inflamed joint tissues isolated from B27 TG(1) rats expressed high levels of NC-B27. NC-B27 were also expressed in joint-resident cells from ankle and tail joints of B27 TG(1) rats prior to clinical arthritis. The expression of NC-B27 on B27 TG(1) rat CD11b/c(+), CD8α(+), cells from spleens and LNs increased with animal age and disease progression. CONCLUSIONS: Non-conventional HLA class 1 molecules are expressed on resident and infiltrating cells in both synovial and GI tissues in human SpA. NC-B27 expression in joints and lymphoid tissues from B27 TG(1) rats prior to the onset of arthritis is consistent with the hypothesis that they play a pathogenic role in SpA.

Ectopic lymphoid neogenesis is strongly associated with activation of the IL-23 pathway in rheumatoid synovitis.

INTRODUCTION: The functional relevance of synovial ectopic lymphoid neogenesis (ELN) in rheumatoid arthritis (RA) remains unknown. As ELN correlates with the degree of tissue inflammation, we investigated whether ELN was associated with specific cytokine profiles. METHODS: Synovial ELN was determined by immunohistology and long CD21 isoform (CD21L) expression. Cytokine expression was determined by multiplex enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction (PCR) as well as immunohistology in synovial fluid (SF) (n = 44) and tissue (ST) (n = 108), respectively. Production of ELN-associated chemokines by fibroblast-like synoviocytes (FLS) was studied in vitro. RESULTS: Screening analysis of SF by multiplex ELISA showed higher protein levels of interleukin (IL)-23 (p = 0.018) and IL-17F (p = 0.028) in ELN+ versus ELN- samples. Other cytokines, including IL-17A, IL-6, and tumor necrosis factor (TNF)-α, were not different. The association between IL-23 and ELN was not biased by disease activity or other clinical features and was confirmed by higher IL-23 mRNA expression in ELN+ versus ELN- ST samples (p = 0.030), a correlation between IL-23 and CD21L expression in the same samples (r = 0.70 p < 0.0001), and a similar correlation in two independent ST sample sets (r = 0.778 p < 0.0001 and r = 0.817 p = 0.011). IL-23 p19 staining was neither restricted nor enhanced in close proximity of ectopic lymphoid follicles, and neither IL-23 nor IL-17A stimulation induced expression of the ELN-associated CC chemokine ligand, CCL21 and CXC chemokine ligand CXCL13, by FLS. Downstream of IL-23, CD21L expression was significantly associated with IL-17F, IL-21, and IL-22, but not IL-17A in two independent ST sample sets. CONCLUSIONS: Synovial ELN in RA is strongly associated with activation of the IL-23 pathway but not with IL-17A.

The cartilage protein melanoma inhibitory activity contributes to inflammatory arthritis.

OBJECTIVE: Melanoma inhibitory activity (MIA) is a small chondrocyte-specific protein with unknown function. MIA knockout mice (MIA(-/-)) have a normal phenotype with minor microarchitectural alterations of cartilage. Our previous study demonstrated that immunodominant epitopes of MIA are actively presented in an HLA-DR4-restricted manner in the inflamed RA joint. The objective of this study was to investigate the potential role of MIA as an autoantigen. METHODS: Collagen-induced arthritis (CIA) and anti-collagen antibody-induced arthritis (CAIA) were induced in MIA(-/-) mice. Anti-type II collagen (anti-CII) antibodies were measured by ELISA. T cell proliferation and cytokine production were assessed by flow cytometry. RESULTS: MIA(-/-) mice had a markedly reduced incidence and severity of CIA and CAIA compared with wild-type (WT) mice. Attenuation of disease was not related to defective binding of anti-CII antibodies to cartilage in the absence of MIA. However, MIA(-/-) mice had significantly reduced anti-CII IgG1 and IgG2a antibody levels accompanied by an increase in FoxP3-expressing CD25(+)CD4(+) regulatory T cells. This was paralleled by a significant reduction in CII-specific IFN-γ production by T cells in MIA(-/-) but not WT animals, suggesting a qualitative impact of MIA on the collagen-induced Th1 response. Furthermore, Ag-specific proliferation of T cells after restimulation with MIA in WT but not MIA(-/-) mice indicated the existence of MIA-specific T cells in the context of CIA. CONCLUSION: These data support a role for MIA as an autoantigen during arthritis development. Whether MIA can influence the balance of pathogenic vs regulatory responses in human RA remains to be investigated.