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Background: Spondyloarthritis (SpA) is a chronic inflammatory disease affecting primarily axial and peripheral joints and sometimes also extra-articular organs, such as the gut. Rats transgenic for HLA-B27 and human β2-microglobulin (B27-Tg rat) develop clinical manifestations resembling human disease. In this model, it has been shown that CD103 + conventional dendritic cells (cDCs) exhibited altered functions, likely promoting SpA development. CD4 - cDC subpopulation expressing XCR1, a chemokine receptor involved in their migration, have been described to be tolerogenic in steady state. Thus, in this study, we wished to examine the fate of XCR1 + cDCs in this animal model of SpA., Methods: cDC populations were isolated from the spleen, mesenteric lymph nodes (MLN), and colonic lamina propria from B27-TG and control nontransgenic (NTG) and/or HLA-B7 transgenic rats after collagenase digestion and density gradient and characterized with flow cytometry or real-time PCR. Migration of cDCs from intestinal mucosa to MLN was assessed, using TLR-7 stimulation with Resiquimod., Results: We observed a reduced frequency of cCD4 - DCs in B27-Tg rats, as compared to control rats. Furthermore, such decrease was not due to excessive death of CD4 - cDCs in B27-Tg rats. Interestingly, we observed a decrease frequency of the XCR1 + subpopulation among CD4 - cDCs in the spleen, MLN, and lamina propria from B27-Tg rats. Finally, after TLR-7 stimulation, the migration of XCR1 + cDCs to MLN was proportionally reduced in B27-Tg rats., Conclusion: Our results demonstrate for the first time a decreased proportion of the tolerogenic XCR1 + cDC subpopulation in SpA target organs in B27-Tg rat, which may affect the maintenance of self-tolerance and control of inflammation.

Objective: Several polymorphisms in ERAP1 are strongly associated with susceptibility to spondyloarthritis (SpA). The combination of rs17482078, rs10050860, and rs30187 results in the construction of 3 major haplotypes that are associated with SpA (the "protective" haplotype T/T/C, the "neutral" haplotype C/C/C, and the "susceptibility" haplotype C/C/T). The aim of the present study was to determine whether such haplotypes might affect endoplasmic reticulum aminopeptidase 1 (ERAP-1) messenger RNA (mRNA) expression, protein level, and/or enzymatic activity in antigen-presenting cells, a type of cell that is potentially relevant to disease pathogenesis., Methods: Monocyte-derived dendritic cells (DCs) were generated in 2 cohorts (a discovery cohort and a replication cohort) comprising a total of 23 SpA patients and 44 healthy controls. Lymphoblastoid B cell lines were established from individuals who were homozygous for the risk, the neutral, or the protective ERAP1 haplotype, respectively. In those samples, we investigated the relationship between ERAP1 haplotypes and mRNA expression level. We also used Western blot analysis to measure the relative protein expression of ERAP-1 and a fluorogenic assay to measure its enzymatic activity., Results: In monocyte-derived DCs, there was a strong association between ERAP1 haplotypes and the ERAP-1 mRNA expression level, with higher levels in subjects harboring the susceptibility haplotype (P = 0.001 and P = 5.6 × 10(-7) in the discovery and replication cohorts, respectively). In lymphoblastoid B cell lines, we observed a significant correlation between haplotype risk score and ERAP1 transcript or protein level (P = 0.003, ρ = 0.92 for both). Enzymatic activity followed a similar trend both in monocyte-derived DCs and in lymphoblastoid B cell lines., Conclusion: These data provide strong evidence that SpA-associated ERAP1 polymorphisms affect the level of gene expression in antigen-presenting cells. How increased production/activity of ERAP-1 may influence susceptibility to SpA remains to be determined., (© 2015, American College of Rheumatology.)

Introduction: This study aimed to compare the functional capacity and gene expression profile of monocyte-derived dendritic cells (MD-DCs) in HLA-B27+ axial spondyloarthritis (SpA) patients and healthy controls., Methods: MD-DCs were differentiated with interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) for seven days, starting from purified CD14+ monocytes and stimulated with lipopolysaccharide (LPS) for six and twenty four hours. Their capacity to stimulate allogeneic CD4+ T cells from unrelated healthy donor was tested. Transcriptomic study was performed with Affymetrix HuGene 1.0 ST microarrays. Gene expression levels were compared between patients and controls using a multivariate design under a linear model (LIMMA). Real-time quantitative PCR (qRT-PCR) was performed for validation of the most striking gene expression differences., Results: The stimulatory capacity of allogeneic CD4+ T cells by MD-DCs from SpA patients was decreased. Transcriptomic analysis revealed 81 genes differentially expressed in MD-DCs between SpA patients and controls (P <0.01 and fold-change <0.66 or >1.5). Four selected genes were validated by q, Rt-Pcr: ADAMTS15, CITED2, F13A1 and SELL. Expression levels of ADAMTS15 and CITED2, encoding a metallopeptidase and a transcription factor, respectively, were inversely correlated with each other (R = 0.75, P = 0.0003). Furthermore, in silico analysis identified several genes of the Wnt signaling pathway having expression co-regulated with CITED2., Conclusion: This study revealed altered function and gene expression pattern in MD-DCs from HLA-B27+ axial SpA. Co-expression study showed an inverse correlation between ADAMTS15 and CITED2. Moreover, the Wnt signaling pathway appeared as deregulated in SpA MD-DCs, a finding which may be connected to Th17-driven inflammatory responses.

Objective: In rats transgenic for human HLA-B27 and β(2) -microglobulin (B27-transgenic rats), colitis and peripheral inflammation develop spontaneously. Therefore, B27-transgenic rats provide a model of spondylarthritis. Because inflammation in these rats requires CD4+ T lymphocytes and involves intestinal pathology, we hypothesized that dendritic cells (DCs) that migrate from the intestine and control CD4+ T cell differentiation would be aberrant in B27-transgenic rats., Methods: Migrating intestinal lymph DCs were collected via thoracic duct cannulation from B27-transgenic and control (HLA-B7-transgenic or nontransgenic) rats. The phenotypes of these DCs and of mesenteric lymph node DCs were assessed by flow cytometry. The ability of DCs to differentiate from bone marrow precursors in vitro was also assessed., Results: Lymph DCs showed increased activation and, strikingly, lacked the specific DC population that is important for maintaining tolerance to self-antigens. This population of DCs was also depleted from the mesenteric lymph nodes of B27-transgenic rats. Furthermore, in vitro culture of DCs from bone marrow precursors revealed a defect in the ability of B27-transgenic rats to produce DCs of the migratory phenotype, although the DCs that were generated induced enhanced interleukin-17 (IL-17) production from naive CD4+ T cells., Conclusion: We describe 2 different mechanisms by which HLA-B27 may contribute to inflammatory disease: increased apoptotic death of B27-transgenic DCs that normally function to maintain immunologic tolerance and enhanced IL-17 production from CD4+ T cells stimulated by the surviving B27-transgenic DCs., (Copyright © 2012 by the American College of Rheumatology.)

Objective: HLA-B27/human β2-microglobulin-transgenic (B27-transgenic) rats, a model of spondylarthritis (SpA), develop spontaneous colitis and arthritis under conventional conditions. CD4+ T cells are pivotal in the development of inflammation in B27-transgenic rats. This study was undertaken to characterize the phenotype of CD4+ T cells in this model and to determine whether dendritic cells (DCs) induce proinflammatory T cells., Methods: The phenotype of CD4+ T cells from rat lymph nodes (LNs) draining the sites of inflammation was analyzed by flow cytometry. Immunostaining was used to detect interleukin-17 (IL-17)-producing cells in the rat joints. DCs from B27-transgenic or control rats (transgenic for HLA-B7 or nontransgenic) were cocultured with control CD4+ T cells and stimulated with anti-T cell receptor α/β., Results: IL-17A- and tumor necrosis factor α (TNFα)-producing CD4+ T cells were expanded in mesenteric and popliteal LNs from B27-transgenic rats. The accumulation of Th17 cells correlated with disease development, in contrast to Th1 or Treg cells. IL-17-positive mononuclear cells were detected in the arthritic joints of B27-transgenic rats but not in the joints of control rats. Finally, in vitro cocultures demonstrated that Th17 cells were preferentially induced and expanded by DCs from B27-transgenic rats, by a process that may involve defective engagement of costimulatory molecules., Conclusion: Our findings indicate that expanded CD4+ T cells in B27-transgenic rats exhibit a proinflammatory Th17 phenotype characterized by IL-17A and TNFα production. Furthermore, this population is preferentially induced by DCs from B27-transgenic rats. These data point toward an induction of Th17 cells as a possible pathogenic mechanism in this model of SpA. However, their pathogenic role still needs to be shown., (Copyright © 2012 by the American College of Rheumatology.)

Objective: Spondylarthritis (SpA) is characterized by spinal and peripheral joint inflammation, frequently combined with extraarticular manifestations. Despite the well-established association of SpA with the class I major histocompatibility complex (MHC) allele HLA-B27, there are still different, parallel hypotheses on the relationship between HLA-B27 and disease mechanisms. The present study was undertaken to investigate several characteristics of mature dendritic cells (DCs), which are believed to be essential for triggering disease in a model of SpA in HLA-B27-transgenic rats., Methods: We combined different whole-proteome approaches (2-dimensional polyacrylamide gel electrophoresis and iTRAQ) to define the most aberrant molecular processes occurring in spleen DCs. Videomicroscopy and flow cytometry were used to confirm both cytoskeletal and class II MHC expression deficiencies., Results: Our proteome studies provided evidence of up-regulation of proteins involved in class I MHC loading, and unfolded protein response, along with a striking down-regulation of several cytoskeleton-reorganizing proteins. The latter result was corroborated by findings of deficient motility, altered morphology, and decreased immunologic synapse formation. Furthermore, class II MHC surface expression was reduced in DCs from B27-transgenic rats, and this could be linked to differences in class II MHC-induced apoptotic sensitivity. Finally, we found reduced viability of the CD103+CD4- DC subpopulation, which likely exerts tolerogenic function., Conclusion: Taken together, our findings have different important implications regarding the physiology of B27-transgenic rat DCs, which have a putative role in spontaneous disease in these rats. In particular, the reduced motility and viability of putatively tolerogenic CD4+ DCs could play an important role in initiating the inflammatory process, resulting in SpA.

Objective: To examine the functional capacity of dendritic cells (DCs) from a panel of HLA-B27/human beta2-microglobulin (Hubeta2m)-transgenic rat lines and crosses with varying susceptibilities to spondylarthritis (SpA)-like disease., Methods: Mature splenic DCs were isolated from HLA-B27-transgenic, HLA-B7-transgenic, and/or Hubeta2m-transgenic rats and tested for support of allogeneic proliferation, compared with nontransgenic controls (all male rats on Lewis background). Graded numbers of DCs were cultured with allogeneic lymph node CD4+ T cells (dark agouti background). Proliferation was assayed by incorporation of tritiated deoxythymidine after 2-4 days of culture., Results: Allogeneic proliferation stimulated by DCs from the healthy HLA-B27/Hubeta2m-transgenic line 21-3 and from the healthy Hubeta2m-transgenic line 283-2 was weakly decreased (21-3) or close to normal (283-2) as compared with that observed with control nontransgenic Lewis rat DCs. In contrast, the ability of DCs from (21-3 x 283-2)F1 rats, which develop a dramatic SpA phenotype, to stimulate allogeneic proliferation was markedly defective. When DC-induced allogeneic proliferation was compared among different transgenic lines and crosses with distinct levels of susceptibility to SpA-like disease, stimulatory capacity was inversely correlated with disease susceptibility., Conclusion: In HLA-B27/Hubeta2m-transgenic rats, a defective functional capacity of DCs correlates with susceptibility to SpA. Since it was previously demonstrated that defective DC function is not a consequence of disease, it could well be a principal factor in the spontaneous development of SpA in these lines.

Objective: To investigate the molecular mechanism responsible for the reduced capacity of dendritic cells (DCs) from HLA-B27-transgenic rats to form conjugates with naive T cells., Methods: We monitored interactions between DCs derived from HLA-B27-transgenic, HLA-B7-transgenic control, and nontransgenic rats and naive CD4+ T cells. Chemoattraction was studied in Transwell assays, and the formation of an immunologic synapse was examined by videomicroscopy and electron microscopy. Involvement of specific molecules in the defective interaction was examined in antibody-blocking assays., Results: T cells migrated normally toward B27 DCs, but upon contact, the frequency of T cells undergoing a Ca2+ response was decreased, indicating impaired immunologic synapse formation. The immunologic synapse formed between B27 DCs and T cells appeared to be normal, as assessed by electron microscopy and by the Ca2+ response. Blocking lymphocyte function-associated antigen 1 on T cells or blocking activated leukocyte cell adhesion molecules on DCs inhibited an equivalent proportion of conjugates from forming between B27 or control DCs and T cells, whereas blocking CD86 on DCs and blocking CD28, CD2, or CD4 on T cells inhibited a greater number of conjugates from forming with control DCs, indicating specific involvement of costimulatory molecules in the reduced formation of conjugates with B27 DCs. Mature B27 molecules on the DC surface were responsible for this decreased formation of conjugates., Conclusion: In the HLA-B27-transgenic rat model of spondylarthropathy, mature B27 molecules expressed by DCs impair the formation of an antigen-independent immunologic synapse with naive CD4+ T cells by interfering with the engagement of costimulatory molecules. This phenomenon could potentially affect the production and/or maintenance of regulatory T cells and contribute to the expansion of pathogenic CD4+ T cells.