Ingrid Fert

Proinflammatory Th17 cells are expanded and induced by dendritic cells in spondylarthritis-prone HLA–B27–transgenic rats

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.

Dendritic cells from spondylarthritis‐prone HLA–B27–transgenic rats display altered cytoskeletal dynamics, class II major histocompatibility complex expression, and viability

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.

Correlation between dendritic cell functional defect and spondylarthritis phenotypes in HLA–B27/HUMAN β2‐microglobulin–transgenic rat lines

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.

Reverse Interferon Signature Is Characteristic of Antigen‐Presenting Cells in Human and Rat Spondyloarthritis

In HLA–B27–transgenic rats, the development of a disorder that mimics spondyloarthritis (SpA) is highly correlated with dendritic cell (DC) dysfunction. The present study was undertaken to analyze the underlying mechanisms of this via transcriptome analysis.

The HLA-B27 transgenic rat, a model of spondyloarthritis, has decreased bone mineral density and increased RANKL to osteoprotegerin mRNA ratio.

Objective. Bone metabolism in spondyloarthritis (SpA) is not well elucidated. We investigated alterations in bone in the HLA-B27 transgenic rat, a model of SpA. Methods. Femur, tibia, and lumbar vertebral bodies of disease-prone HLA-B27 transgenic, healthy HLA-B7 transgenic, and nontransgenic control rats were used for bone histomorphometric and dual energy x-ray absorptiometry (DEXA) analysis. Serum levels of type I collagen C-telopeptides (CTX), N-terminal propeptide of type I procollagen (P1NP), and osteocalcin, as well as receptor activator of nuclear factor-κ B ligand (RANKL) and osteoprotegerin (OPG), were measured. RNA was isolated from the bone tissue of the femura to analyze gene expression of RANKL, OPG, and osteocalcin. Results. Histomorphometric analysis indicated a significant decrease in bone volume as well as trabecular number and thickness in the HLA-B27 rats. Trabecular separation was increased. Numbers of osteoblasts, osteoclasts, and osteoid volume were not altered significantly. The decrease in bone mineral density was confirmed using DEXA. Levels of RANKL mRNA were significantly increased in the bone tissue of HLA-B27 transgenic rats, resulting in an increased RANKL to OPG ratio. Osteocalcin mRNA expression was also significantly elevated in bone of HLA-B27 rats. Serum levels of CTX, RANKL, OPG, P1NP, and osteocalcin did not differ significantly. Conclusion. Our data indicate that, similarly to SpA in humans, HLA-B27 transgenic rats show a reduced bone mass, and suggest an involvement of the RANKL/OPG system in the mechanism of bone loss in this disease. This model may be adequate to study osteoporosis in SpA.

HLA–B27 Subtype Oligomerization and Intracellular Accumulation Patterns Correlate With Predisposition to Spondyloarthritis

Mechanisms underlying the striking association of spondyloarthritis (SpA) with the class I major histocompatibility complex molecule HLA–B27 remain poorly understood. SpA‐like disease develops spontaneously in B*2705‐transgenic rats, in conjunction with high HLA–B27 expression levels. This study was undertaken to examine the effects of increased expression of HLA–B27 alleles that are differentially associated with SpA on oligomerization and intracellular redistribution.

Loss of bone strength in HLA-B27 transgenic rats is characterized by a high bone turnover and is mainly osteoclast-driven

OBJECTIVE Although osteopenia is frequent in spondyloarthritis (SpA), the underlying cellular mechanisms and association with other symptoms are poorly understood. This study aimed to characterize bone loss during disease progression, determine cellular alterations, and assess the contribution of inflammatory bowel disease (IBD) to bone loss in HLA-B27 transgenic rats. METHODS Bones of 2-, 6-, and 12-month-old non-transgenic, disease-free HLA-B7 and disease-associated HLA-B27 transgenic rats were examined using peripheral quantitative computed tomography, μCT, and nanoindentation. Cellular characteristics were determined by histomorphometry and ex vivo cultures. The impact of IBD was determined using [21-3 x 283-2]F1 rats, which develop arthritis and spondylitis, but not IBD. RESULTS HLA-B27 transgenic rats continuously lost bone mass with increasing age and had impaired bone material properties, leading to a 3-fold decrease in bone strength at 12 months of age. Bone turnover was increased in HLA-B27 transgenic rats, as evidenced by a 3-fold increase in bone formation and a 6-fold increase in bone resorption parameters. Enhanced osteoclastic markers were associated with a larger number of precursors in the bone marrow and a stronger osteoclastogenic response to RANKL or TNFα. Further, IBD-free [21-3 x 283-2]F1 rats also displayed decreased total and trabecular bone density. CONCLUSIONS HLA-B27 transgenic rats lose an increasing amount of bone density and strength with progressing age, which is primarily mediated via increased bone remodeling in favor of bone resorption. Moreover, IBD and bone loss seem to be independent features of SpA in HLA-B27 transgenic rats.