HLA-B27 Transgenic Mice Research Articles

Bacterial amyloid curli activates the host unfolded protein response via IRE1α in the presence of HLA-B27

ABSTRACT Salmonella enterica serovar Typhimurium (STm) causes gastroenteritis and can progress to reactive arthritis (ReA). STm forms biofilms in the gut that secrete the amyloid curli, which we previously demonstrated can trigger autoimmunity in mice. HLA-B27 is a genetic risk factor for ReA; activation of the unfolded protein response (UPR) due to HLA-B27 misfolding is thought to play a critical role in ReA pathogenesis. To determine whether curli exacerbates HLA-B27-induced UPR, bone marrow-derived macrophages (BMDMs) isolated from HLA-B27 transgenic (tg) mice were used. BMDMs treated with purified curli exhibited elevated UPR compared to C57BL/6, and curli-induced IL-6 was reduced by pre-treating macrophages with inhibitors of the IRE1α branch of the UPR. In BMDMs, intracellular curli colocalized with GRP78, a regulator of the UPR. In vivo, acute infection with wild-type STm increased UPR markers in the ceca of HLA-B27tg mice compared to C57BL/6. STm biofilms that contain curli were visible in the lumen of cecal tissue sections. Furthermore, curli was associated with macrophages in the lamina propria, colocalizing with GRP78. Together, these results suggest that UPR plays a role in the curli-induced inflammatory response, especially in the presence of HLA-B27, a possible mechanistic link between STm infection and genetic susceptibility to ReA.

The sacroiliac joint: An original and highly sensitive tool to highlight altered bone phenotype in murine models of skeletal disorders

Bone disorders may affect the skeleton in different ways, some bones being very impaired and others less severely. In translational studies using murine models of human skeletal diseases, the bone phenotype is mainly evaluated at the distal femur or proximal tibia. The sacroiliac joint (SIJ), which connects the spine to the pelvis, is involved in the balanced transfer of mechanical energy from the lumbar spine to the lower extremities. Because of its role in biomechanical stress, the SIJ is a region of particular interest in various bone diseases. Here we aimed to characterize the SIJ in several murine models to develop a highly reliable tool for studying skeletal disorders.We performed a 12-month in vivo micro-computed tomography (micro-CT) follow-up to characterize the SIJ in wild-type (WT) C57BL/J6 mice and compared the bone microarchitecture of the SIJ and the distal femur at 3 months by micro-CT and histology. To test the sensitivity of our methodology, the SIJ and distal femur were evaluated at 3 and 6 months, in 2 murine models of skeletal disorder, X-linked hypophosphatemia (Hyp mice) and HLA-B27 transgenic mice and compared to WT mice. A multimodal analysis was performed, using a combination of microCT and histological analysis.With the Hyp model, the SIJ displayed more bone microarchitecture alterations than the distal femur. Hyp mice showed a significant reduction in trabecular bone at both the distal femur and sacral slope as compared with WT mice, with a significant positive correlation between trabecular bone parameters of the distal femur and sacral side of the SIJ. Furthermore, trabecular bone parameters (Bone Volume/Total Volume (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular number (Tb.N), trabecular pattern factor (Tb.Pf)) were significantly increased compared to femoral parameters at the SIJ. The sacral articular cortical bone, which is indicative of osteoarticular lesions, was altered in Hyp mice. Interestingly, in accordance to previous studies, HLA-B27 transgenic mice did not show any osteoarticular lesions as compared with WT mice. Cortical bone parameters (thickness, porosity), as well as scoring performed with double blinding, did not show difference between the 2 genotypes.The characterization and evaluation of the SIJ surface appears very sensitive to emphasize alterations of bone and joint. The SIJ may represent a valuable tool to investigate both bone and local osteoarticular alterations in murine models of skeletal disorders and might be a relevant site for assessing the response to treatment of chronic bone diseases.

Rodent Models of Spondyloarthritis Have Decreased White and Bone Marrow Adipose Tissue Depots.

Bone marrow adipose tissue (BMAT) has recently been recognized as a distinct fat depot with endocrine functions. However, if and how it is regulated by chronic inflammation remains unknown. Here, we investigate the amount of white fat and BMAT in HLA-B27 transgenic rats and curdlan-challenged SKG mice, two well-established models of chronic inflammatory spondyloarthritis (SpA). Subcutaneous and gonadal white adipose tissue and BMAT was reduced by 65-70% and by up to 90% in both experimental models. Consistently, B27 rats had a 2-3-fold decrease in the serum concentrations of the adipocyte-derived cytokines adiponectin and leptin as well as a 2-fold lower concentration of triglycerides. The bone marrow of B27 rats was further characterized by higher numbers of neutrophils, lower numbers of erythroblast precursors, and higher numbers of IL-17 producing CD4+ T cells. IL-17 concentration was also increased in the serum of B27 rats. Using a cell culture model, we show that high levels of IL-17 in the serum of B27 rats negatively impacted adipogenesis (-76%), an effect that was reversed in the presence of neutralizing anti-IL-17 antibody. In summary, these findings show BMAT is severely reduced in two experimental models of chronic inflammatory SpA and suggest that IL-17 is involved in this process.

No evidence for a direct role of HLA-B27 in pathological bone formation in axial SpA

ObjectiveThe strong genetic association between HLA-B27 and ankylosing spondylitis has been known for over 40 years. HLA-B27 positivity is possibly associated with severity of ankylosis. We studied the in vitro...

Reactive Arthritis: From Clinical Features to Pathogenesis

Reactive arthritis (ReA) is a sterile synovitis which occurs after a gastrointestinal or urogenital infection. ReA belongs to Spondyloarthritis (SpA), a group of diseases that share several clinical and radiological features including familiar clustering, absence of rheumatoid factor and association with HLA-B27. Clinically, ReA is characterized by an asymmetric arthritis predominantly affecting the lower limbs, often associated with urethritis, conjunctivitis and other extraarticular symptoms. The ReA prevalence depends on the incidence of causative pathogens. The ReA diagnosis is based on clinical features and serological tests to evidence previous infection. Different treatment including antibiotics, disease modifying antirheumatic drugs (DMARs) and biologic agents has been recommended. Even though knowing that infections trigger the joint inflammation, the ReA pathogenesis remains to be poorly understood. Several animal models and in vitro studies have been used to elucidate the mechanisms involved in ReA development. In this sense, HLA-B27 transgenic rat or mice have been used to explain the role of this molecule in SpA aetiopathogenesis. Moreover, the infectious model of Yersinia-induced ReA in rodents has shed some lights on the relationship between host genetic susceptibility to infection and abnormal immune response in ReA development. Understanding the immune mediators triggering ReA will contribute to find a specific treatment for this arthritis. In this review, we focus on clinical features, epidemiology, treatment, and the different attempts to understand the pathogenesis of ReA.

HLA B27 et spondylarthropathies

The association between HLA-B27 and ankylosing spondylitis has been reported more than 35 years ago but its exact pathogenic role remains controversial. Various hypotheses have been proposed according to the results of various experimental approaches. The animal models involving HLA-B27 transgenic mice and rat have been crucial to demonstrate a direct involvement of B27 in disease pathogenesis. HLA-B27 misfolding is one of the most discussed pathogenic hypotheses, with a possible induction of inflammatory cytokines, including TNFα production and Th17 lymphocytes differentiation. Nevertheless, such hypothesis is not univocal. A better understanding of the pathogenic role of HLA-B27 would likely favour the development of targeted therapies in the future.

How does HLA-B27 affect susceptibility to spondyloarthropathy?

Human leukocyte antigen (HLA)-B27, in common with other HLA class I molecules, plays an important role in immunity to viral and bacterial infections by presenting foreign peptides to T lymphocytes. However, there are negative consequences for individuals who inherit HLA-B27, since this gene is strongly associated with susceptibility to a group of inflammatory joint diseases known as the spondyloarthropathies. A pathogenic role is suggested for the B27 molecule itself, rather than a closely linked gene, since HLA-B27 transgenic rats and mice exhibit features observed in spondyloarthropathy. This review will describe how recent research on the biology and function of the B27 molecule provides information on how B27 differs from other HLA class I molecules, and will discuss how these characteristics may lead to susceptibility to arthritis.

Animal models of HLA-B27-associated diseases: new outcomes

The HLA-B27 molecule is strongly associated with the spondyloarthropathies, a group of chronic inflammatory diseases, affecting the skeleton, the bowel and the skin. This association has been largely studied, but mechanisms of pathology remain unclear. The HLA-B27 transgenic rats develop a spontaneous disease that strikingly resembles human spondyloarthropathies, dependent of bacterial flora and implicating the immune system. The presence of CD4+ T cells is required, and antigen presenting cells (APC) expressing high levels of HLA-B27 likely play an important role. Indeed, APC are defective in naive T lymphocytes stimulation. This default appears to implicate the APC/T cells contact, and may result in a loss of tolerance toward microbial flora. Two models of skeletal inflammation linked to HLA-B27 have been developed in mice. The ANKENT mice develop a spontaneous ossifying enthesitis affecting ankle and tarsal joints, with increased frequency in the presence of an HLA-B27 transgene. The HLA-B27 transgenic mice lacking endogenous β 2 microglobulin develop arthritis of hind-paws. In this model, homodimers of B27 heavy chains could be implicated in the pathogenesis by presenting exogenous peptides to CD4 + T cells.

Strong memory CD8 + T cell responses against immunodominant and three new subdominant HLA-B27-restricted influenza A CTL epitopes following secondary infection of HLA-B27 transgenic mice

We previously showed that the known HLA-B27-restricted influenza A epitope identified from human studies, NP.383–391, was recognized by CTLs following influenza A infection of transgenic (Tg) HLA-B27/H2 class I-deficient (H2 DKO) mice. Here, we examined the kinetics of the primary NP.383–391-specific response in Tg HLA-B27/H2 DKO mice at the site of respiratory infection, along with the profile of additional influenza A epitopes recognized. While the temporal kinetics of the Tg HLA-B27/NP.383–391-specific CD8 + T cell response paralleled the H2-D b/NP.366–374-specific response of non-Tg H2 b mice, the magnitude was less. Using epitope prediction programs, we identified three novel B27-restricted influenza A epitopes, PB2.702–710, PB1.571–579, and PB2.368–376, recognized during both the primary and secondary response to infection. Although the secondary NP.383–391-specific response was dominant, PB1.571–579 and PB2.368–376 stimulated stronger proliferative expansion in memory T cells. Our results indicate a broader B27/influenza A CTL repertoire than previously known. Together with results for other HLA class I alleles, this information will become important in improving vaccine strategies for influenza A and other human pathogens.

Identification of Novel Human Aggrecan T Cell Epitopes in HLA-B27 Transgenic Mice Associated with Spondyloarthropathy

The pathology of ankylosing spondylitis, reactive arthritis, and other spondyloarthropathies (SpA) is closely associated with the human leukocyte class I Ag HLA-B27. A characteristic finding in SpA is inflammation of cartilage structures of the joint, in particular at the site of ligament/tendon and bone junction (enthesitis). In this study, we investigated the role of CD8+ T cells in response to the cartilage proteoglycan aggrecan as a potential candidate autoantigen in BALB/c-B27 transgenic mice. We identified four new HLA-B27-restricted nonamer peptides, one of them (no. 67) with a particularly strong T cell immunogenicity. Peptide no. 67 immunization was capable of stimulating HLA-B27-restricted, CD8+ T cells in BALB/c-B27 transgenic animals, but not in wild-type BALB/c mice. The peptide was specifically recognized on P815-B27 transfectants by HLA-B27-restricted CTLs, which were also detectable by HLA tetramer staining ex vivo as well as in situ. Most importantly, analysis of the joints from peptide no. 67-immunized mice induced typical histological signs of SpA. Our data indicate that HLA-B27-restricted epitopes derived from human aggrecan are involved in the induction of inflammation (tenosynovitis), underlining the importance of HLA-B27 in the pathogenesis of SpA.

HLA-B27 heavy chain homodimers are expressed in HLA-B27 transgenic rodent models of spondyloarthritis and are ligands for paired Ig-like receptors.

HLA-B27 transgenic rats and strains of HLA-B27-transgenic beta(2)-microglobulin (beta(2)m)-deficient mice develop a multisystem inflammatory disease affecting the joints, skin, and bowel with strong similarity to human spondyloarthritis. We show that HLA-B27 transgenic mice and rats express HC10-reactive, beta(2)m-free HLA-B27 homodimers (B27(2)) and multimers, both intracellularly and at the cell surface of leukocytes, including rat dendritic cells. Fluorescent-labeled tetrameric complexes of HLA-B27 homodimers (B27(2) tetramers) bind to populations of lymphocytes, monocytes, and dendritic cells. The murine (and probably rat) paired Ig-like receptors (PIRs) are ligands for B27(2). Thus, B27(2) tetramers stain RBL cells transfected with murine activating PIR-A4 and inhibitory PIR-B receptors. Murine PIR-A and -B can be immunoprecipitated from the RAW264.7 macrophage cell line, and murine PIR-A can be immunoprecipitated from the J774.A1 line using B27(2). B27(2) tetramer staining corresponds to the distribution of PIR expression on lymphoid and myeloid cells and on murine macrophage cell lines. B27(2) can induce TNF-alpha release from the J774.A1 macrophage cell line. The binding of B27(2) to PIR is inhibited by HC10, an mAb that ameliorates arthritis in HLA-B27(+) beta(2)m(-/-) mice. The expression and PIR recognition of B27(2) could explain the pathogenesis of rodent spondyloarthritis.

Animal models of HLA-B27-associated diseases.

The HLA-B27 molecule is strongly associated with the spondyloarthropathies (SpA), a group of inflammatory conditions affecting the skeleton, the skin and several mucosae. The mechanism of this association remains unknown, largely because the HLA-B27 molecule displays normal function. A disease that closely mimicks SpA arises spontaneously in HLA-B27 transgenic rats. This disease is dependent on the presence of a normal bacterial flora and implicates the immune system. The presence of both CD4+ T cells and antigen presenting cells (APCs) expressing high levels of HLA-B27, seems of critical importance in its pathogenesis, whereas CD8+ T cells are dispensable. The T cell stimulatory function of APCs is disturbed by the HLA-B27 molecule. This disease could result from a failure of tolerance, related in part to high level of B27 expression in professional APCs and to the immune response to gut bacteria. In contrast, HLA-B27 transgenic mice have usually remained healthy. However, two types of inflammatory conditions affecting the skeleton, which arise in mice of susceptible background after exposure to a conventional bacterial flora, are increased by an HLA-B27 transgene. The first is ANKENT, a spontaneous ankylosing enthesitis that affects ankle and/or tarsal joints of ageing mice; the second is a spontaneous arthritis of hindpaws developing in mice lacking endogenous mbeta2m. As in rats, the absence of CD8+ T cells in the latter model, argues against the "arthritogenic peptide" hypothesis. In these mbeta2m0 mice, B27 free heavy chain could be implicated in the pathogenesis of arthritis by presenting extracellular peptides to CD4+ T cells.

Fully functional HLA B27-restricted CD4+ as well as CD8+ T cell responses in TCR transgenic mice.

The strong association of HLA B27 with spondyloarthropathies contrasts strikingly with most autoimmune diseases, which are HLA class II associated and thought to be mediated by CD4+ T lymphocytes. By introducing a human-derived HLA B27-restricted TCR into HLA B27 transgenic mice, we have obtained a functional TCR transgenic model, GRb, dependent on HLA B27 for response. Surprisingly, HLA B27 supported CD4+ as well as CD8+ T cell responses in vivo and in vitro. Further, HLA B27-restricted CD4+ T cells were capable of differentiation into a range of Th1 and Th2 T cell subsets with normal patterns of cytokine expression. The transgenic T cells were also able to enhance clearance of recombinant vaccinia virus containing influenza nucleoprotein in vivo. This is the first description of a human HLA class I-restricted TCR transgenic line. The existence of CD4+ MHC class I-restricted T cells has significant implications for immune regulation in autoimmunity and, in particular, in HLA B27-associated arthritis. We believe that this model provides a novel system for the study of unusual T cell behavior in vivo.

Identification of HLA-B27-restricted peptides from the Chlamydia trachomatis proteome with possible relevance to HLA-B27-associated diseases.

The association of HLA-B27 with ankylosing spondylitis and reactive arthritis is the strongest one known between an MHC class I Ag and a disease. We have searched the proteome of the bacterium Chlamydia trachomatis for HLA-B27 binding peptides that are stimulatory for CD8(+) cells both in a model of HLA-B27 transgenic mice and in patients. This was done by combining two biomathematical computer programs, the first of which predicts HLA-B27 peptide binding epitopes, and the second the probability of HLA-B27 peptide generation by the proteasome system. After preselection, immunodominant peptides were identified by Ag-specific flow cytometry. Using this approach we have identified for the first time nine peptides derived from different C. trachomatis proteins that are stimulatory for CD8(+) T cells. Eight of these nine murine-derived peptides were recognized by cytotoxic T cells. The same strategy was used to identify B27-restricted chlamydial peptides in three patients with reactive arthritis. Eleven peptides were found to be stimulatory for patient-derived CD8(+) T cells, of which eight overlapped those found in mice. Additionally, we applied the tetramer technology, showing that a B27/chlamydial peptide containing one of the chlamydial peptides stained CD8(+) T cells in patients with Chlamydia-induced arthritis. This comprehensive approach offers the possibility of clarifying the pathogenesis of B27-associated diseases.

Identification of HLA‐B27–restricted cytotoxic T lymphocyte epitope from carcinoembryonic antigen

Characterization of epitopes recognized by cytotoxic T lymphocytes (CTLs) in the sequence of tumor antigens is an important step in the development of tumor therapies. Because carcinoembryonic antigen (CEA) is a protein expressed in a high number of epithelial tumors, it is an interesting target to study. We screened for the presence of HLA-B27-restricted CTL epitopes from CEA by studying the binding to HLA-B27 of 31 synthetic peptides predicted to bind to this molecule. This afforded 16 peptides with moderate or high binding affinity. Immunization of HLA-B27 transgenic mice with the best binder peptides yielded 4 immunogenic peptides: CEA(9-17), CEA(9-18), CEA(138-146) and CEA(360-369). However, splenocytes from mice immunized with a vaccinia virus-expressing CEA recognized only CEA(9-18). These CTLs were of the CD8(+) phenotype, which upon stimulation with peptide specifically produced IFN-gamma. Moreover, they did not cross-react against peptides of region 9-18 from proteins of the CEA family. Our results show that CEA(9-18) may induce specific CTL responses against CEA.

Spontaneous inflammatory disease in HLA-B27 transgenic mice does not require transporter of antigenic peptides.

HLA-B27 is strongly linked with a group of human diseases called spondyloarthropathies. Even though HLA-B27 as an MHC class I molecule would be expected to present endogenously processed peptides such as cytosolic or viral proteins, many of the B27-linked diseases begin after an infection with an enterobacteria, an exogenous antigen. In our previous studies, we have described development of spontaneous inflammatory disease in HLA-B27 transgenic mice expressing beta(2)m free heavy chains on the cell surface. In order to address the role of endogenous versus exogenous antigens and a role for Tap genes in the development of spontaneous diseases, mice lacking Tap-1 (knockout) were mated to HLA-B27/human beta(2)m transgenic mice. B27(+)/human beta(2)m(+) double-transgenic mice (without mouse beta(2)m) lacking the Tap-1 gene developed spontaneous inflammatory disease similar to wild-type Tap-1 gene-expressing counterparts. Our data demonstrate that peptide transporters (Tap) were not involved in the development of spontaneous inflammatory disease in B27(+)/human beta(2)m transgenic animals.

HLA-B27 transgenic mice are susceptible to collagen-induced arthritis: type II collagen as a potential target in human disease

HLA-B27 is highly linked with a group of human diseases called spondyloarthropathies (SpA). Many of these disorders begin after an infection with an enterobacteria. The symptoms seen in patients with spondyloarthropathies are inflammatory pain in the spine and asymmetrical arthritis of lower limbs. Additional symptoms related to SpA include inflammation in the eyes, bowel, and skin. The autoantigen(s) in SpA are not known. Proteins such as collagen and proteoglycans have been thought to be potent autoantigens in arthritidis including B27-associated human diseases. Type II collagen is a common denominator among eyes and joints, affected tissues in B27-linked diseases. Moreover, a few reports indicated CII specific T cells and antibodies in patients with spondyloarthropathies. We and others have previously described development of spontaneous arthritis and nail disease in HLA-B27 transgenic animals. To determine whether CII may be a target antigen in the B27-linked diseases, B27 + mβ 2m o/o (HLA-B27 transgenic mice lacking mouse β 2m with and without human β 2m) mice were immunized with type II collagen inside the barrier facility. Male HLA-B27 transgenic mice developed collagen-induced arthritis compared to transgene negative littermates or female counterparts. There was no difference in the incidence of arthritis in HLA-B27 transgenic mice with and without human β 2m. Our data suggest that β 2m free heavy chain of HLA-B27 may present soluble antigens such as type II collagen to trigger specific T cells contributing in the development of arthritis. Our data also suggest that CII may be a potential target antigen in the cartilage during the disease process.

Peptide binding α1α2 domain of HLA-B27 contributes to the disease pathogenesis in transgenic mice

Human spondyloarthropathies are strongly associated with a major histocompatibility complex (MHC) class I allele, HLA-B27. HLA-B27 transgenic mice and rats demonstrate many features of these diseases further confirming the role of HLA-B27 in disease. Yet the exact role of this molecule in disease pathogenesis is not clearly understood. We have previously reported spontaneous arthritis and nail disease in HLA-B27 transgenic mice lacking β 2-microglobulin (B27+β 2m o). These observations alongwith binding studies of B27 derived peptides to HLA-B27 molecule itself led to two hypotheses: (i) HLA-B27 derived peptide as a source of autoantigen; and (ii) HLA-B27 functions as an antigen presenting molecule. In this report, we confirm spontaneous disease in transgenic mice expressing a hybrid B27 molecule with α1α2 domain of B27 and α3 domain of mouse H-2K d. These mice developed spontaneous arthritis and nail disease when transferred from specific pathogen free barrier facility to the conventional area. Other control mice with MHC class I transgene (e.g., HLA-B7, HLA-Cw3, and H2-D d) did not develop such disease. In a MHC reassembly assay, binding of similar peptides to both wild type and hybrid B27 molecules was observed. In addition, the hybrid B27 molecule lacks at least one of the 3 proposed peptides from the third hypervariable (HV3) region of HLA-B27. These data strongly suggest that HLA-B27 molecule is an antigen presenting molecule rather than a peptide donor in the disease pathogenesis.

4 Animal models and in vitro models for the study of aetiopathogenesis of spondyloarthropathies

Among several animal models, HLA-B27 transgenic rodents proved useful for investigating the interplay between genetic factors and the bacterial environment in the aetiopathogenesis of the spondyloarthropathies (SpA). HLA-B27 transgenic rats spontaneously develop a multisystemic inflammatory disease resembling human SpA. This disease is dependent on the presence of a normal bacterial flora and implicates the immune system. The presence of both T cells and antigen-presenting cells expressing high levels of HLA-B27 seems of critical importance in its pathogenesis. HLA-B27 transgenic mice also develop arthritis, under the influence of the bacterial flora. In both types of model, CD8+ T cells seem not to be necessary, arguing against the 'arthritogenic peptide' hypothesis. In vitro models have been used to study the immune response against bacterial agents and the role of HLA-B27 in human SpA. It appears that an impaired immune response against bacteria could be involved in the triggering of human SpA. HLA-B27 could be implicated at the level of interaction between host cells and bacteria in the driving of a specific immune response against bacterial antigens or as a target of an autoimmune response.

PATHOGENESIS OF HUMAN LEUKOCYTE ANTIGEN B27–POSITIVE ARTHRITIS: Information from Clinical Materials

In the spondyloarthropathies human leukocyte antigen (HLA) B27 confers a strong genetic predisposition to the development and to the chronicity of disease after extra-articular infection with certain gram-negative bacteria. The close relationships between infection, HLA-B27, other genetic factors, and the host immune system, however, still are unexplained. HLA-B27-positive arthritis continues to be an area of intensive investigation in basic and clinical research. New animal models with HLA-B27 transgenic mice and rats, as well as recent developments in understanding the processes involved in signal transduction, cytokine production, and human T-lymphocyte activation, contribute to the development of new pathogenic models of the spondyloarthropathies. This article summarizes the current concepts of the cause and pathogenesis of the spondyloarthropathies resulting from studies of clinical materials. The host-microbial interplay in human disease, namely in bacteria-induced reactive arthritis, may eludicate principle disease mechanisms in acute disease and in the development of chronic autoimmune arthritis or ankylosing spondylitis.