Leonie M. van Duivenvoorde

Autoimmune epididymoorchitis is essential to the pathogenesis of male-specific spondylarthritis in HLA-B27-transgenic rats.

OBJECTIVE Male rats transgenic for HLA-B27 and human β(2) -microglobulin (hβ(2) m) spontaneously develop epididymoorchitis (EO) preceding the development of spondylarthritis (SpA). In the specific B27/hβ(2) m-transgenic rat cross-strain (21-3 × 382-2)F(1) , only the males develop SpA, and neither sex develops gut inflammation. This study was undertaken to determine whether EO and SpA in male (21-3 × 382-2)F(1) rats are causally related. In addition, the primary characteristics of EO in this rat arthritis model were assessed. METHODS Male B27/hβ(2) m-transgenic (21-3 × 382-2)F(1) rats underwent bilateral, unilateral, or sham epididymoorchiectomy between ages 36 and 125 days. The castrated rats were given testosterone replacement. Alternatively, the 21-3 and 283-2 transgene loci were crossed with a transgene inducing aspermatogenesis. Rats were observed for the development of EO, arthritis, and spondylitis. RESULTS In unmanipulated transgenic rats, inflammation was first evident in the ductuli efferentes (DE; ducts linking the rete testis to epididymis) as early as age 30 days. The inflammation was initially neutrophilic, and later became granulomatous. Antisperm and anti-testis cell antibodies appeared in the rat serum after age 70 days. Cells infiltrating the testes were predominantly CD4+ T cells and CD68+ or CD163+ macrophages. Quantitative polymerase chain reaction of the DE, epididymis, and testis showed elevations in the levels of interferon-γ, interleukin-10 (IL-10), and IL-17A. In addition, levels of IL-12A, IL-22, IL-23A, and IL-23 receptor were found to be elevated in the DE. Remarkably, castration of the rats before age 91 days completely prevented the subsequent onset of arthritis and spondylitis, as did transgene-induced azospermia. CONCLUSION Autoimmune EO develops spontaneously in HLA-B27/hβ(2) m-transgenic (21-3 × 283-2)F(1) rats at age 30 days, the age when antigen-positive meiotic germ cells first exit the testis. Persistent testicular inflammation and/or antigenic stimulation are essential prerequisites for the subsequent development of SpA. Thus, dysregulated innate immunity at immune-privileged sites may be an essential mechanism triggering the onset of SpA.

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

IntroductionThe 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.MethodsSynovial 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.ResultsScreening 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.ConclusionsSynovial ELN in RA is strongly associated with activation of the IL-23 pathway but not with IL-17A.

Review: animal models as a tool to dissect pivotal pathways driving spondyloarthritis

Animal models have strongly contributed to an understanding of the disease mechanisms relevant to human arthritis, including rheumatoid arthritis (RA), osteoarthritis, and spondyloarthritis (SpA). Nevertheless, animal models rarely replicate human rheumatic diseases that develop spontaneously, and they fail to encompass the complexity of their human counterparts. Indeed, many animal models, either those that are genetically modified or those that are experimentally induced, will mirror the activation of only a single pathway of interest or only certain disease manifestations. They should thus be considered “pathway models” or “mechanism of disease models” rather than “disease equivalent models.” Moreover, not only activation of the pathways but also the tissue and immunologic context in which they are operating determine the pathologic outcome and phenotype that may also contribute to major differences between rodent models and human diseases (1). Therefore, it is crucial to define which model is relevant for which aspect of the corresponding human disease in order to increase the relevance and predictive value of preclinical work in animals. This is particularly pertinent to a field in which the diversity of SpA phenotypes sharing peripheral, axial, and extraarticular manifestations (e.g., psoriasis, uveitis, and inflammatory bowel disease [IBD]) with different degrees of severity precludes the validity of using a single animal model for the study of human SpA. Additional factors that deserve consideration are 1) the uncertainty regarding whether the pathogenesis of SpA involves activation of a single unifying pathway or several distinct pathways driving heterogeneous disease manifestations; 2) the complex and incompletely understood picture of structural damage in SpA, involving a combination of bone and cartilage destruction and pathologic new bone formation; and 3) the fact that many new models mimicking aspects of human SpA have recently been developed. Therefore, we sought to provide a review of the most commonly used animal models of SpA. By systematically describing the clinical features, histopathology (Tables 1 and 2), and pathophysiology of animal models, we provide practical guidelines for selecting the most appropriate model with which to study a specific research question in the field of SpA.

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 (TG1) 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/Huβ2m 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 TG1 rats expressed high levels of NC-B27. NC-B27 were also expressed in joint-resident cells from ankle and tail joints of B27 TG1 rats prior to clinical arthritis. The expression of NC-B27 on B27 TG1 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 TG1 rats prior to the onset of arthritis is consistent with the hypothesis that they play a pathogenic role in SpA.

A2.15 Relative Overexpression of Transmembrane Versus Soluble TNF in Human and Experimental Spondyloarthritis

Background Macrophages and their pro-inflammatory cytokines, including TNF, are pivotal mediators of chronic synovitis in rheumatoid arthritis (RA) as well as spondyloarthritis (SpA). Despite similar levels of synovial macrophage infiltration and similar clinical responses to TNF blockade in both diseases, SpA is characterised by a more pronounced infiltration with alternatively activated CD163+ macrophages and ongoing osteoproliferation. This study aimed to investigate whether these differences were related to a differential expression and/or function of TNF between both diseases. Methods Expression of transmembrane TNF (tmTNF) and soluble TNF (sTNF) was measured in IFN-γ, IL-4 or IL-10 polarised macrophages obtained from healthy donors. Expression of TNF and its receptors was measured in synovial fluid (SF) and synovial tissue biopsies (ST) of actively inflamed knee joints of SpA and RA patients. Mice transgenically overexpressing tmTNF (TgA86) were evaluated for spondylitis and arthritis. Results In vitro polarisation with IL-10 specifically induced the expression of CD163 on macrophages, mimicking the phenotype in SpA synovitis. IL-10 and IL-4 polarised macrophages secreted less TNF than classically, IFN-γ-polarised macrophages (p < 0.05). In contrast, IL-4 polarised macrophages expressed more tmTNF compared to the IFN-γ polarised macrophages (p < 0.05), with a similar trend for IL-10 polarised macrophages. In line with these in vitro data, the sTNF SF levels were significantly lower in SpA compared to RA (p = 0.01) despite similar TNF mRNA levels in ST. This was not related to higher expression of TNF receptors as both TNFR1 and TNFR2 were similarly expressed in ST, both at protein and mRNA levels. On the contrary, the SF levels of sTNFR1 and sTNFR2, which are both cleaved from the cell membrane by the same enzyme as tmTNF, were even decreased in SpA versus RA. Investigating the potential pathophysiological role of relative overexpression of tmTNF versus sTNF in vivo, clinical analysis revealed that tmTNF transgenic mice developed arthritis, resulting in deformation and loss of grip strength, and spondylitis as evidenced by crinkled tails with a 100% incidence. Axial and peripheral joint inflammation was confirmed by histology. In contrast to mouse strains overexpressing sTNF, tmTNF tg mice did not develop systemic disease and weight loss and showed clear signs of osteoproliferation on histology. Conclusions tmTNF is relatively overexpressed by CD163+ alternatively polarised macrophages in SpA synovitis and leads to an axial and peripheral SpA phenotype in transgenic mice.

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

! The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Rheumatology. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com RHEUMATOLOGY Rheumatology 2018;57:770 doi:10.1093/rheumatology/key034 Advance Access publication 3 February 2018

Comment on: ‘Spondyloarthritis: may the force be with you?’, the Editorial by McGonagle et al

We read with great interest the editorial by McGonagle et al 1 on the recent study by Jacques et al. 2 Jacques et al describe a series of elegant experiments in TNFΔARE mice demonstrating that mechanical unloading by tail suspension alleviates peripheral arthritis and explore the molecular mechanisms underlying this phenomenon. McGonagle et al begin their editorial by stating that Jacques et al demonstrated that experimental spondyloarthritis (SpA) might be a biomechanically triggered process. This conclusion is surprising as Jacques et al did not perform any experiments supporting this suggestion. On the contrary, their experiments used the opposite approach and indicated that mechanical unloading alleviates tumour necrosis factor (TNF)-induced arthritis. This is emphasised …

Innate Immune Activation Can Trigger Experimental Spondyloarthritis in HLA-B27/Huβ2m 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/Huβ2m transgenic rats. We used Lewis HLA-B27/Huβ2m transgenic rats [21-3 × 283-2]F1, HLA-B7/Huβ2m 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-1β, 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.

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.

Altered BANK1 expression is not associated with humoral autoimmunity in chronic joint inflammation

OBJECTIVE The presence of disease-specific autoantibodies in RA but not spondyloarthritis (SpA) suggests that B-cell tolerance is preserved in the latter condition despite chronic joint inflammation. Which factors control B-cell tolerance vs autoimmunity in chronic arthritis remains incompletely understood. As single nucleotide polymorphisms in the B-cell scaffold protein with ankyrin repeats (BANK1) gene have recently been associated with various autoantibody-positive autoimmune diseases including RA, we explored whether altered expression of BANK1 was associated with humoral autoimmunity in arthritis. METHODS Peripheral B-cell subsets and inflamed synovial tissue were obtained from active SpA and RA. Quantitative PCR was used to assess the expression of full-length BANK1 and delta2 BANK1, a splice variant lacking exon 2 that counteracts BANK1 function. B-cell subsets, autoantibody titres and clinical disease were monitored upon CIA induction in Bank1 knockout (KO) mice. RESULTS Whereas full-length BANK1 was not differentially expressed, the BANK1 delta2 splicing variant was decreased in naïve peripheral B cells as well as in synovial tissue of SpA compared with RA. However, no differences were observed in seropositive vs seronegative RA. Performing functional analysis in mice, we found no differences in B-cell subsets and anti-collagen antibodies upon CIA induction between Bank1 KO mice and littermate controls. Accordingly, the incidence and severity of clinical disease were not altered in Bank1 KO mice. CONCLUSION This study did not reveal a major role for BANK1 in humoral autoimmunity in chronic arthritis. The decreased levels of BANK1 delta2 in SpA, however, warrant more detailed analysis of the functional consequences of an altered BANK1/BANK1 delta2 balance.