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Additional human β2-microglobulin curbs HLA–B27 misfolding and promotes arthritis and spondylitis without colitis in male HLA–B27–transgenic rats
Abstract
Ankylosing spondylitis and related spondylarthritides are associated with HLA-B27, and also with intestinal inflammation, by unknown mechanisms. The folded HLA-B27 molecule is a trimer of heavy chain, beta2-microglobulin (beta2m), and short peptide. However, B27 heavy chain has an unusual propensity to misfold and trigger the unfolded protein response (UPR). This study was initiated to test the hypothesis that B27 misfolding plays a role in the pathogenesis of spondylarthritis.
Rats with high transgene copy numbers of HLA-B27 heavy chain together with human beta2m (Hubeta2m) spontaneously develop colitis, peripheral arthritis, and occasional spondylitis, and those with lower transgene copy numbers remain healthy. We crossed disease-prone and healthy HLA-B27/Hubeta2m-transgenic rat lines with a healthy line, 283-2, carrying only the Hubeta2m transgene. HLA-B27 assembly was assessed by pulse-chase analysis of B27 molecules, and UPR triggering was assessed by measuring BiP/Grp78 messenger RNA (mRNA) in splenic concanavalin A blasts. Surface expression of B27 and Hubeta2m was determined by flow cytometry. Disease manifestations were identified by clinical observation, histology, and measurement of cytokine mRNA.
The extra Hubeta2m from the 283-2 line significantly reduced B27 misfolding and UPR triggering. Unexpectedly, however, F1 male offspring of the healthy 21-3 line crossed with the 283-2 line showed a high prevalence, severity, and duration of arthritis and spondylitis, in the absence of colitis. The arthropathy showed many features characteristic of human spondylarthritis.
These results suggest that B27 misfolding is associated with intestinal inflammation, but that neither B27 misfolding nor intestinal inflammation is critical to the development of B27-associated arthropathy.
... In lines bearing the highest Semin Immunopathol numbers of copies of HLA-B27 transgene, the phenotype affects both sexes and mimics severe form of AIBD, by combining peripheral arthritis-more prevalent in males-with UC and psoriatic skin changes. Moreover, increasing the number of hβ2m transgene copies resulted in heightened prevalence and severity of arthritis [11,12]. Other line with fewer copies of HLA-B27 but a high number of hβ2m transgene copies develop a phenotype more reminiscent of AS, restricted to males and to osteo-articular tissue, including spinal and sacroiliac joint inflammation with osteoproliferation, in addition to peripheral arthritis [12,44]. ...
... Moreover, increasing the number of hβ2m transgene copies resulted in heightened prevalence and severity of arthritis [11,12]. Other line with fewer copies of HLA-B27 but a high number of hβ2m transgene copies develop a phenotype more reminiscent of AS, restricted to males and to osteo-articular tissue, including spinal and sacroiliac joint inflammation with osteoproliferation, in addition to peripheral arthritis [12,44]. In both cases, males uniformly develop early epididymoorchitis that seems to be a critical event, at least for the second phenotype since orchiectomy prevents its development [45]. ...
... As expected, the result was a decrease of UPR markers, but contrary to the prediction that UPR was a triggering event in rat-SpA, disease was not attenuated. Even more, both severity and frequency of arthritis worsened-as already mentioned above [12,54]. Therefore, UPR may not be the critical molecular mechanism by which HLA-B27 triggers rat-SpA. ...
Understanding the complex mechanisms underlying a disorder such as spondyloarthritis (SpA) may benefit from studying animal models. Several suitable models have been developed, in particular to investigate the role of genetic factors predisposing to SpA, including HLA-B27, ERAP1, and genes related to the interleukin (IL)-23/IL-17 axis. One of the best examples of such research is the HLA-B27 transgenic rat model that fostered the emergence of original theories regarding HLA-B27 pathogenicity, including dysregulation of innate immunity, contribution of the adaptive immune system to chronic inflammation, and influence of the microbiota on disease development. Very recently, a new model of HLA-B27 transgenic Drosophila helped to expand further some of those theories in an unexpected direction involving the TGFβ/BMP family of mediators. On the other hand, several spontaneous, inducible, and/or genetically modified mouse models—including SKG mouse, TNFΔARE mouse and IL-23-inducible mouse model of SpA—have highlighted the importance of TNFα and IL-23/IL-17 axis in the development of SpA manifestations. Altogether, those animal models afford not only to study disease mechanism but also to investigate putative therapeutic targets.
... In particular the heavy chain β2 microglobulin is prone to misfolding [49,[51][52][53] leading to the activation of the UPR [54]. Interestingly, studies that aimed to link the extent of β2 microglobulin misfolding to UPR activation, inflammation, and SpA development in HLA B27-transgenic rats demonstrated that the introduction of additional copies of the human β2-microglobulin gene, which reduced HLA-B*27 misfolding and UPR activation, led to an increase in the incidence and severity of arthritis [55], rather than the expected decrease. Interestingly, these studies led to the creation of rat strains with arthritic disease that closely resembled human spondylarthritis but showed no evidence of gut inflammation, indicating that spondylarthritis and IBD, particularly Crohn's disease, may involve separate disease mechanisms [55]. ...
... Interestingly, studies that aimed to link the extent of β2 microglobulin misfolding to UPR activation, inflammation, and SpA development in HLA B27-transgenic rats demonstrated that the introduction of additional copies of the human β2-microglobulin gene, which reduced HLA-B*27 misfolding and UPR activation, led to an increase in the incidence and severity of arthritis [55], rather than the expected decrease. Interestingly, these studies led to the creation of rat strains with arthritic disease that closely resembled human spondylarthritis but showed no evidence of gut inflammation, indicating that spondylarthritis and IBD, particularly Crohn's disease, may involve separate disease mechanisms [55]. These studies challenged the view that HLA-B*27 contributes to spondylarthritis through misfolding, formation of heavy chain β2 microglobulin dimers, and activation of the UPR, but supported the idea that UPR triggering is associated with inflammatory disease mechanisms that can lead to gut inflammation in SpA patients [55]. ...
... Interestingly, these studies led to the creation of rat strains with arthritic disease that closely resembled human spondylarthritis but showed no evidence of gut inflammation, indicating that spondylarthritis and IBD, particularly Crohn's disease, may involve separate disease mechanisms [55]. These studies challenged the view that HLA-B*27 contributes to spondylarthritis through misfolding, formation of heavy chain β2 microglobulin dimers, and activation of the UPR, but supported the idea that UPR triggering is associated with inflammatory disease mechanisms that can lead to gut inflammation in SpA patients [55]. Consistent with this view, studies of tissues of AS patients did not suggest that UPR and ER stress was directly associated with inflammation and HLA-B*27 misfolding [56,57]. ...
Spondyloarthritis comprises a group of inflammatory diseases of the joints and spine, with various clinical manifestations. The group includes ankylosing spondylitis, reactive arthritis, psoriatic arthritis, arthritis associated with inflammatory bowel disease, and undifferentiated spondyloarthritis. The exact etiology and pathogenesis of spondyloarthritis are still unknown, but five hypotheses explaining the pathogenesis exist. These hypotheses suggest that spondyloarthritis is caused by arthritogenic peptides, an unfolded protein response, HLA-B*27 homodimer formation, malfunctioning endoplasmic reticulum aminopeptidases, and, last but not least, gut inflammation and dysbiosis. Here we discuss the five hypotheses and the evidence supporting each. In all of these hypotheses, HLA-B*27 plays a central role. It is likely that a combination of these hypotheses, with HLA-B*27 taking center stage, will eventually explain the development of spondyloarthritis in predisposed individuals.
... Generating an animal model that replicates pathological changes of human disease is crucial to developing therapeutics. One of the best animal models described is the rat HLA-B27 model [1]. Although this models has all three phenotypes of the disease, namely inflammatory aggregates, bone loss, and bone gain, only 30%-50% of males develop these phenotypes the time until disease onset and the severity of lesions vary among individuals [1]. ...
... One of the best animal models described is the rat HLA-B27 model [1]. Although this models has all three phenotypes of the disease, namely inflammatory aggregates, bone loss, and bone gain, only 30%-50% of males develop these phenotypes the time until disease onset and the severity of lesions vary among individuals [1]. IL-23 is also a culprit in this disease; others have shown that in vivo expression of IL-23 is sufficient to phenocopy the human disease, with development of enthesitis and entheseal new bone development [2]. ...
... One caveat with the already well-established HLA-B27/Huβ2m rats is that these animals have low incidence of disease and variable time until disease onset [1]. To understand whether these limitations are present in the proposed IL27RA -/-p53 R172H/+ mouse model, a large number of mice were examined to delineate the incidence, locations, frequency, and variability within the group. ...
Spondyloarthropathies, the second most frequently occurring form of chronic inflammatory arthritis, affects young adults in particular. However, a proper model with which to study the biology of this disease and to develop therapeutics is lacking. One of the most accepted animal models for this disease uses HLA-B27/Hu-β2m transgenic rats; however, only 30%-50% of male HLA-B27/Hu-β2m rats develop spontaneous, clinically apparent spondylitis and have a variable time until disease onset. Here, we report a high-incidence, low-variation spontaneous mouse model that delineates how the combination of inflammatory cytokine interleukin-27 (IL-27) signaling deficiency and mitogenic signaling (mutant p53R172H) in vivo, leads to bone loss in the vertebral bodies and ossification of the cartilage in the intervertebral discs. In this human disease–like mouse model, bone loss and pathogenic bone development are seen as early as 4 months of age in the absence of inflammatory aggregates in the enthesis or intervertebral disc.
... The second model with less HLA-B27 copy numbers but additional copy numbers of Huβ2m was first described in 2006. These rats, in the absence of colitis or other systemic inflammation, developed spontaneous spondylitis and arthritis at the age of 9 months, with an increased, but still low, incidence (40% of the male rats while females remain healthy) (19,20). Studies assessing the potential role of HLA-B27 in the activation of pathogenic T cells demonstrated that CD4 + T cells from the original HLA-B27 transgenic rats efficiently induced SpA symptoms (21), while CD8 + T cells were not required for disease in both the original and the second HLA-B27 transgenic rat model (22,23). ...
... In contrast, rederiving the original HLA-B27 transgenic rats in a germ-free environment prevented the inflammatory phenotype (24), which fits with the human observation that gastrointestinal infections with, amongst others, Salmonella, Shigella, and Campylobacter, can trigger SpA in HLA-B27-positive individuals (25). Furthermore, in the second HLA-B27 transgenic rat model epididymo-orchitis is preceding the development of spondylitis and arthritis in 100% of the rats (19), while orchiectomy completely prevents spontaneous spondylitis and arthritis development in these rats (20). These data suggest that danger signals such as GI pathogens and testicular tissue inflammation trigger disease in HLA-B27 transgenic rats, we hypothesized that the presence of HLA-B27 would increase the sensitivity toward innate immune activation and lower the threshold for spondylitis and arthritis development. ...
... Male HLA-B27 tg rats spontaneously develop spondylitis and arthritis with an incidence of 40 and 70%, respectively, and a variable disease onset between 4 and 9 months of age. Female rats on the other hand are not capable of spontaneous spondylitis and arthritis development (19). Based on the upregulation of pro-inflammatory cytokine production in vitro, we hypothesized that in vivo activation of the innate immune system would trigger spondylitis and arthritis development in the HLA-B27 tg 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.
... The HLA-B27 transgenic rat lines are bred on a Lewis background and express human HLA-B27 and h 2m (Hammer et al., 1990;Tran et al., 2006). The first line was described in 1990, where 'spontaneous' spondyloarthritis-like disease and IBD occurred in 100% of rats. ...
... However, the frequency of peripheral arthritis was low and axial arthritis minimal (Hammer et al., 1990). Subsequently, a second line was created in which the frequency of HLA-B27:h 2m was altered (Tran et al., 2006). In this model, the extra h 2m caused more frequent and severe arthritis and spondylitis, which better resembled AS; however, this coincided with reduced gut disease and unfolded protein response. ...
... Up until recently, the rat model was the best animal model of disease. Unfortunately, study in rats is somewhat hampered due to fewer reagents when compared to those available for studies in mice (Tran et al., 2006). As such for many years, researchers have been hunting for a good mouse model of disease. ...
Animal models are used for the study of a number of human autoimmune diseases, including multiple sclerosis, diabetes, rheumatoid arthritis, systemic lupus erythematosis and spondyloarthropathies. Induced, spontaneous and genetically manipulated animal models can be described in terms of their parallels to human disease and as valuable tools for the development of potential therapies. Studies in animal models have led to a number of important discoveries, which have increased our understanding of the pathogenesis of autoimmune disease, including the roles played by regulatory T cells and T H 17 cells. In addition, important therapeutic advances have emerged as a result of studies of immune intervention in animal models of autoimmunity. For example, tumour necrosis factor (TNF) blocking drugs, which are widely used for the treatment of rheumatic diseases, were developed following pre‐clinical testing in animal models.
Key Concepts
Animal models may be either spontaneously occurring or induced as a result of genetic manipulation, immunisation with a self‐antigen or triggered by pathogen‐associated molecular patterns (PAMPs) in genetically susceptible hosts.
No animal model completely mimics human disease.
Animal models can be used to delineate common mammalian immunological mechanisms, test novel therapeutic concepts and understand mechanisms of drug action, but therapeutic efficacy therein is not predictive of response in clinical trials.
The use of transgenic and knockout strains facilitates the identification of key genes that contribute to disease susceptibility and pathogenesis.
... We obtained 6-12-week-old male and female HLA-B27/Huβ2m transgenic rats weighing 250-300 g from the Rheumatology Team of the University of Texas Southwestern Medical Center (Dallas, TX, USA) [16] . ...
... This rat strain possesses 20 copies of HLA-B27 and 50 copies of the Hu-β2m microglobulin gene [16][17][18] and is the most representative available animal model of AS. All animal breeding and experiments were carried out in a speci c pathogen-free laboratory of the Animal Experiment Center of Guangxi Medical University. ...
Objective: We aimed to verify whether mechanical growth factor (MGF) may be an effective target for treating ankylosing spondylitis.
Methods: FOXP3 expression was measured in Treg cells from healthy male subjects administered MGF. A rat model of ankylosing spondylitis was established, and the level of ankylosing spondylitis-related factors (tumor necrosis factor [TNF]-α, interleukin [IL]-2, and IL-10) was measured.
Results: We found that the proliferation and total number of Treg cells, as well as FOXP3 expression, significantly increased in the MGF-treated groups compared with those in the control. The level of inflammation, bone destruction, and new bone formation significantly decreased in rats treated with MGF compared with those in the control group. TNF-α expression significantly decreased, whereas the IL-2 and IL-10 levels significantly increased in the MGF group compared with those in the control.
Conclusions: MGF may delay disease progression in ankylosing rats by inducing FOXP3 expression, promoting FOXP3+ Treg cell proliferation and differentiation, reducing TNF-α expression, and increasing IL-10 and IL-2 expression.
... We obtained 6-12-week-old male and female HLA-B27/Huβ2m transgenic rats weighing 250-300 g from the Rheumatology Team of the University of Texas Southwestern Medical Center (Dallas, TX, USA) [16]. ...
... This rat strain possesses 20 copies of the HLA-B27 gene and 50 copies of the Hu-β2m microglobulin gene [16][17][18], and is the most representative available animal model of AS. All animal breeding and experiments were carried out in the speci c pathogenfree laboratory of the Animal Experiment Center of Guangxi Medical University and were approved by the Institutional Ethics Committee. ...
Background
To verify that mechanical growth factor (MGF) may be an effective target for treating ankylosing spondylitis. MethodsFOXP3 expression was measured in Treg cells from healthy male subjects treated with MGF. A rat model of ankylosing spondylitis was established, and the levels of ankylosing spondylitis-related factors (tumor necrosis factor [TNF]-α, interleukin [IL]-2, and IL-10) were measured. ResultsWe found that the proliferation and total number of Treg cells, as well as FOXP3 expression levels, increased significantly in the MGF-treated groups versus in the control. The levels of inflammation, bone destruction, and new bone formation were significantly decreased in animals treated with MGF compared to in the control group. TNF-α expression decreased significantly, while IL-2 and IL-10 levels increased significantly in the MGF group compared to in the control. ConclusionsMGF may delay disease progression in ankylosing rats by inducing FOXP3 expression, promote FOXP3+ Treg cell proliferation and differentiation and reducing the expression of TNF-α and increasing the expression of IL-10 and IL-2.
... The HLA-B27/human β 2 -microglobulin (hβ 2 m)-transgenic rat line 21-3 and the hβ 2 mtransgenic line 283-2, originally produced at University of Texas Southwestern Medical Center (Dallas, TX), were crossed to obtain disease-prone [21-3 × 283-2] F1 rats bearing 20 copies of HLA-B*27:05 and 50 copies of hβ 2 m, and disease-free 21-3 rats (20 copies of HLA-B*27:05 and 15 copies of hβ 2 m) and 283-2 rats (35 copies of hβ 2 m), all on a Lewis background (14). Nontransgenic littermates were used as controls. ...
... Interestingly, association of HLA-B*27:05 heavy chain oligomers with BiP was previously shown in splenocytes from rats of the 33-3 line, but not in HLA-B*07:02-transgenic rats (29). Moreover, increasing expression of hβ 2 m by crossing HLA-B27-transgenic rat lines with the 283-2 line promoted arthritis development and contributed to decreased expression of BiP messenger RNA (14). Given that increased expression of hβ 2 m was associated with a reduction in folded B27 molecules and hβ 2 m at the cell surface, it can be hypothesized that the increased level of hβ 2 m production led to its accumulation with misfolded HLA-B27 heavy chain in ER vesicles and contributed to dampening the level of BiP, by dampening UPR. ...
Objective
It was previously shown that HLA–B27 subtypes predisposing to spondyloarthritis (SpA), i.e., B*27:02, B*27:05, and B*27:07, displayed an increased propensity to form intracellular oligomers and to accumulate at a high density in cytoplasmic vesicles, as compared to the non–SpA‐associated HLA–B*07:02 and HLA–B*27:06. This study was undertaken to characterize the nature and content of HLA–B–containing vesicles and to further examine their relevance to SpA predisposition.
Methods
Vesicles containing HLA–B proteins were detected in transfected HeLa cells and in cells from SpA patients or HLA–B27/human β2‐microglobulin (hβ2m)–transgenic rats, by microscopy. The nature and content of HLA–B–containing vesicles were characterized in colocalization experiments with appropriate markers.
Results
The SpA‐associated HLA–B*27:04 subtype accumulated at higher levels (P < 10⁻⁵) in cytoplasmic vesicles compared to HLA–B*27:06, from which it differs only by 2 substitutions, reinforcing the correlation between vesicle formation and SpA predisposition. Colocalization studies showed that those vesicles contained misfolded HLA–B heavy chain along with β2m and endoplasmic reticulum (ER) chaperones (calnexin, calreticulin, BiP, glucose‐regulated protein 94‐kd) and belonged to the ER but were distinct from the peptide‐loading complex (PLC). Similar vesicles were observed in immune cells from HLA–B27+ SpA patients, in greater abundance than in healthy controls (P < 0.01), and in dendritic cells from HLA–B27/hβ2m transgenic rats, correlating with SpA susceptibility.
Conclusion
Accumulation of misfolded HLA–B heavy chain along with β2m and ER chaperones into ER‐derived vesicles distinct from the PLC is a characteristic feature of HLA–B27 subtypes predisposing to SpA. This phenomenon could contribute to HLA–B27 pathogenicity, via a noncanonical mechanism.
... Studies have found that HLA-B27 transgenic rats can develop not only SpA but also peripheral arthritis, colitis, uveitis, and skin diseases. Environmental factors (such as gut microbiota) and other genetic factors determine the exact phenotype [12,13]. The genome-wide association study (GWAS) found that endoplasmic reticulum aminopeptidase 1 (ERAP1), ERAP2, and IL-23R are susceptibility factors for SpA in addition to HLA-B27 and that the IL-23/IL-17 pathway is very important in the SpA pathogenesis [14][15][16]. ...
The aim was to study the genetic correlation and causal relationship between spondyloarthritis (SpA) and blood metabolites based on the large-scale genome-wide association study (GWAS) summary data. The GWAS summary data (3966 SpA and 448,298 control cases) of SpA were from the UK Biobank, and the GWAS summary data (486 blood metabolites) of human blood metabolites were from a published study. First, the genetic correlation between SpA and blood metabolites was analyzed by linkage disequilibrium score (LDSC) regression. Next, we used Mendelian randomization (MR) analysis to perform access causal relationship between SpA and blood metabolites. Random effects inverse variance weighted (IVW) was the main analysis method, and the MR Egger, weighted median, simple mode, and weighted mode were supplementary methods. The MR analysis results were dominated by the random effects IVW. The Cochran’s Q statistic (MR-IVW) and Rucker’s Q statistic (MR Egger) were used to check heterogeneity. MR Egger and MR pleiotropy residual sum and outlier (MR-PRESSO) were used to check horizontal pleiotropy. The MR-PRESSO was also used to check outliers. The “leave-one-out” analysis was used to assess whether the MR analysis results were affected by a single SNP and thus test the robustness of the MR results. Finally, we identified seven blood metabolites that are genetically related to SpA: X-10395 (correlation coefficient = −0.546, p = 0.025), pantothenate (correlation coefficient = −0.565, p = 0.038), caprylate (correlation coefficient = −0.333, p = 0.037), pelargonate (correlation coefficient = −0.339, p = 0.047), X-11317 (correlation coefficient = −0.350, p = 0.038), X-12510 (correlation coefficient = −0.399, p = 0.034), and X-13859 (Correlation coefficient = −0.458, p = 0.015). Among them, X-10395 had a positive genetic causal relationship with SpA (p = 0.014, OR = 1.011). The blood metabolites that have genetic correlation and causal relationship with SpA found in this study provide a new idea for the study of the pathogenesis of SpA and the determination of diagnostic indicators.
... Однако эта теория была поставлена под сомнение исследованиями на трансгенных крысах, у которых процесс накопления неправильно свернутых тяжелых цепей был остановлен за счет повышения экспрессии человеческого β2-микроглобулина. При этом степень тяжести клинических проявлений суставных поражений не изменилась [60]. ...
The literature review presents the characteristics of the human leukocyte antigen (HLA)-B27 as a factor contributing to the development of psoriatic arthritis. HLA-B27 is a class I surface antigen encoded by the major histocompatibility complex (MHC) B locus located on chromosome 6. The main function is to present antigenic peptides to the CD8+ T-cells. HLA-B27 is the most important genetic biomarker for psoriatic arthritis, as it provides phenotypic differentiation in the patient population. The prevalence of HLA-B27 in various population groups are presented. The structural features of the HLA-B27 molecule are described. The characteristics of methods for detecting HLA-B27 status and determining its subtypes are given. The main mechanisms of the HLA-B27 polymorphism influence on the development of psoriatic arthritis are considered, and hypotheses are analyzed that explain the pathogenic effect of HLA-B27: the arthritogenic peptide hypothesis, the misfolding hypothesis, the HLA-B27 heavy chain homodimer formation hypothesis. The features of the clinical manifestations and course of HLA-B27-positive psoriatic arthritis are presented, allowing the use of HLA-B27 to predict the development of psoriatic joint damage.
... HLA-B27 can also misfold due to its B pocket structure, which may trigger the unfolded protein response leading to endoplasmic reticulum stress, inflammation, and apoptosis ( Figure IC) [1]. Some non-disease-associated subtypes of HLA-B27 fold more efficiently [1], but reducing misfolding in HLA-B27 transgenic rats was not protective against arthritis [108]. ...
The pathogenesis of human leukocyte antigen (HLA)-B27-associated diseases such as acute anterior uveitis (AAU) and ankylosing spondylitis (AS) remains poorly understood, though Gram-negative bacteria and subclinical bowel inflammation are strongly implicated. Accumulating evidence from animal models and clinical studies supports several hypotheses, including HLA-B27-dependent dysbiosis, altered intestinal permeability, and molecular mimicry. However, the existing literature is hampered by inadequate studies designed to establish causation or uncover the role of viruses and fungi. Moreover, the unique disease model afforded by AAU to study the gut microbiota has been neglected. This review critically evaluates the current literature and prevailing hypotheses on the link between the gut microbiota and HLA-B27-associated disease. We propose a new potential role for HLA-B27-driven altered antibody responses to gut microbiota in disease pathogenesis and outline recommendations for future well-controlled human studies, focusing on AAU.
... Intriguingly, a healthy HLA-B27 rat line expressing less transgene copies, when provided with extra human b2m exhibited a severe arthritis but no colitis. Such observations suggest HLA-B27 misfolding may have a predominant role in intestinal epithelial cellular inflammatory responses (86). Our studies could explain why, even in the presence of a high affinity HLA-B27 binding peptide, gastrointestinal symptoms were not alleviated in HLA-B27 transgenic rats and that these clinical features may be more associated with HLA-B27 misfolding. ...
Peptide-loaded Major Histocompatibility Complex (pMHC) class I molecules can be expressed in a single chain trimeric (SCT) format, composed of a specific peptide fused to the light chain beta-2 microglobulin (β2m) and MHC class I heavy chain (HC) by flexible linker peptides. pMHC SCTs have been used as effective molecular tools to investigate cellular immunity and represent a promising vaccine platform technology, due to their intracellular folding and assembly which is apparently independent of host cell folding pathways and chaperones. However, certain MHC class I HC molecules, such as the Human Leukocyte Antigen B27 (HLA-B27) allele, present a challenge due to their tendency to form HC aggregates. We constructed a series of single chain trimeric molecules to determine the behaviour of the HLA-B27 HC in a scenario that usually allows for efficient MHC class I molecule folding. When stably expressed, a pMHC SCT incorporating HLA-B27 HC formed chaperone-bound homodimers within the endoplasmic reticulum (ER). A series of HLA-B27 SCT substitution mutations revealed that the F pocket and antigen binding groove regions of the HLA-B27 HC defined the folding and dimerisation of the single chain complex, independently of the peptide sequence. Furthermore, pMHC SCTs can demonstrate variability in their association with the intracellular antigen processing machinery.
... In light of this data, we hypothesize that early, profound and sustained suppression of inflammation is essential to arrest or prevent structural damage. This hypothesis is consistent with previous observations in the non-immunized HLA-B27/Huβ2m tg rats where no new bone formation is observed in the absence of inflammation [51][52][53], and in the tmTNF tg mice where the prevention of inflammation via the knockout of TNFR1 also results in a reduction of new bone formation [37]. Follow-up studies in this rat model utilizing dual blockade therapy with various treatment regimens such as early versus late treatment, sustained versus intermittent, or sub-optimal versus optimal dosages of both inhibitors will help to elucidate a full understanding of the underlying pathological mechanisms, and help determine the ideal timing for therapeutic intervention to stop inflammation and prevent any subsequent structural damage. ...
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 β2-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.
... There are 2 types of AxSpA; one is radiographic and second is non-radiographic and subjects with AxSpA can have inflammation in non-articular sites. With regard to animal models, it has been shown in HLA-B27 rats with overexpression of human β2-microglobulin that these animals developed the clinical (arthritis and dactylitis) and histopathologic features (synovitis, spondylitis, enthesitis, fibrosis, and bony proliferation) of the arthritic disease resemble human SpA to a greater degree than has been observed in the high-copy HLA-B27/ Huβ2m (3). It has also been shown that older male DBA/1 mice develop spontaneous entheseal cell proliferation and infiltration, progressively leading to endochondral bone formation similar to human SpA (4). ...
Many mouse models of rheumatoid arthritis have been identified, but only a limited number are present for axial spondyloarthritis (AxSpA). Collagen Ab-induced arthritis (CAIA) is one of the most widely used mouse models of arthritis, and it is complement-dependent. We found that mice developing CAIA also developed spinal lesions similar to those found in AxSpA. To induce CAIA, mice were injected intraperitoneally at day 0 with anti-collagen Abs, followed by LPS injection at day 3. CAIA mice demonstrated a significant kyphosis through the spine, as well as hypertrophic cartilage and osseous damage of the intravertebral joints. Immunohistochemical staining of the kyphotic area revealed increased complement C3 deposition and macrophage infiltration, with localization to the intravertebral joint margins. Near Infrared (NIR) in vivo imaging showed that anti-collagen Abs conjugated with IRDye® 800CW not only localized to cartilage surface in the joints but also to the spine in arthritic mice. We report here a novel preclinical mouse model in which, associated with the induction of CAIA, mice also exhibited salient features of AxSpA; this new experimental model of AxSpA may allow investigators to shed light on the local causal mechanisms of AxSpA bone and soft tissue changes as well as treatment.
... For many years, the traditional pathophysiological framework for SpA has been the arthritogenic-peptide theory, which proposes that HLA-B27 presents self-peptides that resemble pathogen-derived peptides to CD8-restricted T lymphocytes [14,15]. However, this hypothesis has been seriously challenged by two independent reports showing that CD8 T cells were not needed for disease in HLA-B27 transgenic rats [16,17]. New hypotheses argue for an autoinflammatory rather than an autoimmune origin, because HLA-B27 has a role in triggering innate immune responses rather than its canonical role of antigen presentation [14,17]. ...
Objectives
Systemic-onset juvenile idiopathic arthritis (SJIA) and adult-onset Still’s disease (AOSD) are the same sporadic systemic auto-inflammatory disease. Spondyloarthritis (SpA) is a group of inflammatory non-autoimmune disorders. We report the observations of eight patients with SJIA/AOSD who also presented features of SpA during their disease evolution and estimate the prevalence of SpA in SJIA/AOSD.
Methods
This was a retrospective national survey of the departments of paediatric and adult rheumatology and internal medicine. To be included, SJIA patients had to fulfil the ILAR criteria, AOSD patients the Yamaguchi or Fautrel criteria, and all patients the ASAS classification criteria for axial or peripheral SpA, ESSG criteria for spondyloarthropathy or CASPAR criteria for psoriatic arthritis. The data were collected with a standardized form.
Results
Eight patients (five adults) were identified in one paediatric and two adult departments. In all but one patient, SpA manifestations occurred several years after SJIA/AOSD onset (mean delay 6.2 ± 3.8 years). Two patients had peripheral and three axial SpA, and four later exhibited psoriatic arthritis and one SAPHO syndrome. The prevalence of SpA in an adult cohort of 76 patients with AOSD was 6.58% (95% CI [2.17–14.69]), greater than the prevalence of SpA in the French general population (0.3%, 95%CI [0.17–0.46]). The prevalence of SpA in an SJIA cohort of 30 patients was 10% (95%CI [2.11–26.53]), more than that reported in the general population of industrialized countries, estimated at 0.016% to 0.15%.
Conclusion
Whilst the temporal disassociation between SpA and AOSD in most cases might suggest a coincidental finding, our work raises the possibility of an SpA AOSD spectrum overlap that needs further study.
... A noticeable difference was observed as AS macrophages secreted more IL-23 (median 265 pg/ml in AS patients versus 9 pg/ml in controls; P = 0.0007), IL-12p70, IL-10, CXCL9 and TNFα than control macrophages providing a logical support for developing IL-23 directed drugs. Contrary to previous studies of the HLA-B27-transgenic rat model of AS (67), no signifi cant UPR induction was detected in AS patients which led them to believe that the relationship between UPR and infl ammatory cytokine production remains unclear (68). ...
Ankylosing spondylitis (AS) is a chronic inflammatory arthritis which involves mainly the spine and the sacroiliac joints, but also the peripheral joints and non-articular structures. IL-12 and IL-23 are heterodimeric cytokines sharing a common p40 subunit. Studies showed than IL-12 is essential for Th1 differentiation while IL-23 promotes a particular subset of T cells characterized by IL-17 production, called Th17. Since the association of IL-23R with AS was reported, the scientific interest has focused on identifying new single nucleotide polymorphisms (SNPs), present in the coding genes for IL-12/IL-23 subunits and their receptors, that increase the susceptibility for AS. Another point of interest for researchers was to investigate if the identified gene variants modify the gene expression of the respective interleukins and/or their receptors, whether that refers to their mRNA expression, their serum levels or the expression of the receptors on the surface of T cells. Given the increasing amount of data that suggests the pivotal role of the IL-23/Th17 axis in AS pathophysiology, new promising therapies, designed to interfere with these pathways, are now in development.
... In this study, we aimed to assess RORC as potential therapeutic target in SpA by studying the effects of a small molecule RORC inhibitor in experimental SpA in HLA-B27 transgenic (tg) rats, a well validated model for human SpA (25)(26)(27). As we previously showed that both initiation and disease persistence in this model is partially but not completely inhibited by IL-17A blockade (28), we used this model to assess how RORC inhibition affects a panel of IL-17 related cytokines in vivo and if this biological effect translates into clinical efficacy. ...
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.
... Although these data have indicated that AS/SpA could be the result of incorrectly folded HLA-B27 accumulating in the ER, contributing to ER stress and consequent inflammatory responses, there is no direct evidence in human SpA for this mechanism. Furthermore, a study performed in B27-transgenic rats by introducing copies of human β2-microglobulin gene reduced B27 misfolding and alleviated colitis, but exacerbated arthritic features (Tran et al., 2006). Studies in AS human tissues did not show convincing evidence for ER stress and UPR. ...
La spondylarthrite (SpA) est une famille de maladies inflammatoires chroniques apparentées (CID), et se caractérise par une inflammation de la colonne vertébrale et des articulations sacro-iliaques, caractéristiques de la forme axiale de cette maladie (AxSpA). Des études d'association pangénomique (GWAS) ont identifié des locus génétiques associés à la susceptibilité à la maladie de la SpA. GWAS a également fourni la preuve d'un rôle clé pour les voies de signalisation immunitaire dans la pathogenèse de la SpA, car de nombreux loci identifiés sont associés à des gènes immunitaires. En particulier, les GWAS ont suggéré un rôle pour l'axe interleukine-23 / interleukine-17 (IL-23 / IL-17) dans la pathogenèse de plusieurs CID. Cependant, pour la plupart des loci associés, le mécanisme par lequel ils affectent la pathogenèse et les populations de cellules immunitaires dans lesquelles ils agissent ne sont toujours pas connus. L’objectif de ce projet est de comprendre les mécanismes moléculaires de la pathogenèse de la maladie en étudiant la fonction des cellules immunitaires et la réponse aux traitement biologique dans la SpA.Pour comprendre le rôle des loci de susceptibilité dans les CID, nous avons conçu un panel d'expression génique nCounter® (NanoString Technologies): le panel Autoimmune Discovery Consortium. Ce panel a été créé en utilisant les loci rapportés de GWAS dans 9 CID, avec l'ajout de plusieurs gènes immunitaires, tels que les gènes de cytokine et de chimiokine. Nous avons utilisé ce panel pour déterminer le modèle d'expression des «gènes GWAS» dans les populations de cellules immunitaires isolées de 50 patients SpA axiaux (AxSpA). Les profils d'expression génique spécifiques au type de cellule ont ensuite été corrélés avec le génotype du patient pour identifier les locus de caractères quantitatifs d'expression (eQTL). L'analyse de l'expression génique a montré qu'environ 80% des gènes associés aux CID étaient exprimés dans des sous-ensembles de cellules T (CD4+ et CD8+, à l'état de repos et activé), ce qui implique leur rôle dans la maladie. L'étude eQTL a révélé un cluster sur le chromosome 11, y compris des SNP qui affectent l'expression de la CTSW (cathepsin W), une peptidase lysosomale impliquée dans l'activité cytotoxique des cellules T CD8+. En outre, nous avons démontré que le facteur de transcription NFATC2 (Nuclear factor of activated T-cells, cytoplasmic 2) se lie de manière allélique spécifique à l'eQTL rs12225345, soutenant son rôle dans la régulation CTSW dans les lymphocytes T. Ces données illustrent comment la liaison allélique spécifique des facteurs de transcription pourrait contribuer à la régulation des gènes associés à la maladie et jouer un rôle dans la pathogenèse de la maladie.Dans la deuxième partie de ce projet, nous avons étudié le mécanisme moléculaire de la pathogenèse AxSpA et l'impact des traitement biologiques (inhibiteurs du TNF, TNFi) sur les réponses immunitaires des patients axSpA. Pour étudier le rôle de l'axe IL-23 / IL-17 dans la pathogenèse de AxSpA, nous avons caractérisé les cellules immunitaires qui produisent l'IL-17A chez les patients AxSpA. Nous avons comparé la capacité de production d'IL-17 de sous-ensembles cellulaires des bras innés (MAIT, γδT et polynucléaires neutrophiles) et adaptatifs (cellules T CD4+ et CD8+) du système immunitaire. Nous avons identifié les cellules MAIT comme les principaux producteurs d'IL-17 dans AxSpA. Nous avons également observé que les lymphocytes innés et adaptatifs expriment des gènes appartenant à la voie IL-23 / IL-17 et des gènes précédemment associés à la sensibilité SpA.
... Over time, the HLA-B27 transgenic (tg) rat model has progressed from the HLA-B27/ human b2 microglobulin (hb2m) (tg) Lewis (21-4H) rats, characterized by orchitis, colitis and hind limbs arthritis, with psoriasis in up to 50% of the rats, to the HLA-B27/hb2m-tg F344 (33-3) rats with similar clinical manifestations but with earlier disease onset. This was followed by the HLA-B27/hb2m-tg Lewis rats (21-3 x 283-2)F1 line with lower HLA-B27 copy numbers, in which all male rats develop orchitis, followed by the development of arthritis (4-6 months age) in 70% of all male rats, and spondylitis (7-9 months age) in 50% of them (41). These rats also show signs of peripheral and axial new bone formation. ...
Spondyloarthritis (SpA) is a spectrum of chronic inflammatory joint diseases that frequently presents with inflammation of the axial skeleton, peripheral joints, entheses, skin, and gut. Understanding SpA pathogenesis has been proven challenging due to the limited availability of human target tissues. In recent years, the interleukin (IL)-23/IL-17 pathway has been implicated in the pathogenesis of SpA, in addition to the Tumor Necrosis Factor Alpha (TNF-α) cytokine. The underlying molecular mechanisms by which the IL-23/IL-17 pathway triggers disease initiation, both in the joints as well as at extra-musculoskeletal sites, are not precisely known. Animal models that resemble pathological features of human SpA have provided possibilities for in-depth molecular analyses of target tissues during various phases of the disease, including the pre-clinical initiation phase of the disease before arthritis and spondylitis are clinically present. Herein, we summarize recent insights gained in SpA animal models on the role of the IL-23/IL-17 pathway in immune activation across affected sites in SpA, which include the joint, entheses, gut and skin. We discuss how local activation of the IL-23/IL-17 axis may contribute to the development of tissue inflammation and the onset of clinically manifest SpA. The overall aim is to provide the reader with an overview of how the IL-23/IL-17 axis could contribute to the onset of SpA pathogenesis. We discuss how insights from animal studies into the initiation phase of disease could instruct validation studies in at-risk individuals and thereby provide a perspective for potential future preventive treatment.
... Studies have shown that in the gut and synovial tissues, β2 microglobulin (β2m), a noncovalent part of the MHC-I complex, reduces HLA-B*27 proper folding, thereby activating the interleukin-23/interleukin-17 (IL-23/IL-17) pathway. Activation of the IL-23/IL-17 pathway leads to inflammation of spine and peripheral joints, implying that this process is associated with intestinal inflammation [12,13]. In addition to this genetic influence, many studies have shown the importance of the gut microenvironment, and particularly the gut microbiome in SpA, so it was reasonable to assume that there is a gut-joint axis in the pathogenesis of these diseases [14,15]. ...
Background:
We aimed to investigate possible association between the HLA-B*35 allele and peripheral arthritis, tenosynovitis and enthesitis.
Methods:
Ultrasound of peripheral joints and tendons was performed in 72 HLA-B*35 positive patients with preliminary diagnosis of undifferentiated axial form of spondyloarthitis and joint and tendon pain. Patients with other known types of axial and peripheral spondyloarthritis were excluded as well as patients with other known types of arthritis.
Results:
Pathological changes were found in the joints of 33 (46%) patients and on the tendons in 13 (18%) patients. The most common ultrasound findings were joint effusion and synovial proliferation with positive power Doppler signal grade 1. The most common ultrasound finding in patients with painful tendons was tenosynovitis. A higher disease activity and an increased incidence of elevated CRP (≥5 mg/L) were more often observed in the group with positive ultrasound findings.
Conclusion:
In this study, we showed that the HLA-B*35 allele could be a potential risk factor for developing peripheral arthritis, but not for tenosynovits and enthesitis in patients with the undifferentiated axial form of spondyloarthritis. This result may influence the follow up of these patients, especially since it gives us an opportunity to consider the use of different types of DMARDs in the treatment of these patients.
... These findings provide possible links between HLA-B * 27 misfolding and dysregulation of immune responses in SpA patients. However, inconsistent with the HLA-B * 27 misfolding-UPR theory, increasing β2m copy numbers (which promotes HLA-B * 27 folding in the ER and mitigates UPR) enhanced the severity of AS symptoms in HLA-B * 27 transgenic rats (77). These results argue against the crucial role of HLA-B * 27 misfolding in the ER for SpA development. ...
Heritability of Spondyloarthritis (SpA) is highlighted by several familial studies and a high association with the presence of human leukocyte antigen (HLA)-B*27. Though it has been over four decades since the association of HLA-B*27 with SpA was first determined, the pathophysiological roles played by specific HLA-B*27 allotypes are not fully understood. Popular hypotheses include the presentation of arthritogenic peptides, triggering of endoplasmic reticulum (ER) stress by misfolded HLA-B*27, and the interaction between free heavy chains or heavy chain homodimers of HLA-B*27 and immune receptors to drive IL-17 responses. Several non-HLA susceptibility loci have also been identified for SpA, including endoplasmic reticulum aminopeptidases (ERAP) and those related to the IL-23/IL-17 axes. In this review, we summarize clinical aspects of SpA including known characteristics of gut inflammation, enthesitis and new bone formation and the existing models for understanding the association of HLA-B*27 with disease pathogenesis. We also examine newer insights into the biology of HLA class I (HLA-I) proteins and their implications for expanding our understanding of HLA-B*27 contributions to SpA pathogenesis.
... When overexpressed along with human β 2 -microglobulin (hβ 2 m) in rats, HLA-B*27 causes a spondyloarthritis-like inflammatory phenotype that involves predominantly the gastrointestinal tract and peripheral joints but does not require CD8+ T cells [4,5]. This phenotype can be modified when additional hβ 2 m is overexpressed, resulting in more severe peripheral arthritis and axial inflammation [6]. Interestingly, additional hβ 2 m overexpression generates the axial inflammatory phenotype even in rats that have lower levels of HLA-B*27 expression (due to lower transgene copy number) and are healthy prior to the introduction of additional hβ 2 m. ...
Establishing a clear role for HLA-B*27 in the pathogenesis of spondyloarthritis continues to be challenging. Aberrant properties of the heavy chain as well as a potential role presenting arthritogenic peptides continue to be pursued as plausible mechanisms. Recent studies implicate HLA-B*27 in aberrant bone formation. An unanticipated cell surface interaction between HLA-B*27 and the bone morphogenetic protein pathway receptor subunit ALK2 may augment TGFβ superfamily signaling pathways, increasing responsiveness to Activin A and TGFβ. This has the potential to increase bone formation as well as Th17 T cell development, presenting an attractive model to explain several aspects of axial and peripheral spondyloarthritis. In a separate study, intracellular effects of misfolded HLA-B*27 implicate this mechanism in increased osteoblast mineralization and bone formation. HLA-B*27 expression in early osteoblasts activates unfolded protein response-mediated X-box binding protein-1 mRNA splicing and induction of the retinoic acid receptor-β gene, with downstream increases in expression of tissue non-specific alkaline phosphatase. Increased TNAP expression in osteoblasts was linked to increased mineralization in vitro and bone formation in vivo. In the ongoing search for evidence of arthritogenic peptides, high-throughput TCR (T cell receptor) sequencing has provided evidence for reduced clonal expansion and increased TCR diversity in ankylosing spondylitis. In addition to two common CD8+ TCR sequences identified in one study, similar CD8 and CD4 TCR motifs were found in another study. Further work will be needed to shed light on the nature of the peptide-HLA class I complex recognized by these T cells and its role in disease.
... Understanding that exosomes would get liberated in the form of Extra Cellular Vesicles(ECV), they might depict a significant mechanism of intra cellular talk [22,68]. Hence , the totally folded MHC-1 dimers in the exosomes might transfer signals towards the resident cells in enthuses for stimulating inflammation, that might result in changes in the joint architecture as well as new bone On dissociation of the HLA-B27 dimers, β2m might collect as well as get trapped or locked within the synovium, where they may bind to the collagen as well as develop amyloid deposits or crosstalk with synovial fibroblasts, hence stimulating the formation as well as liberation of proteins generating this breakdown, ultimately causing AS [70]. This is labelled as β2m deposition posit. ...
After getting a patient of spondyloarthropathy not fitting into any of the classical group of axialspondyloarthritis(axSpA) spondyloarthritis)like rheumatoid arthritis,ankylosing spondylitis but having severe pain and positive HLAB27, our interest got rekindled into the role of HLAB-27 in the pathophysiology of these spondyloarthropathies .With the latest classification axSpA gets classified in the form of an intermediate in between autoimmunity as well as auto inflammatory disease with this second terminology used in parallel with the innate immune impairment.
... In order to generate M.tub-induced HLA-B27/Huβ2m transgenic rats (HLA-B27 tg rats), the Tg(HLA-B * 2705, B2M)21-3Reh and Tg(B2M)283-2Reh Lewis rat lines (30) were bred and housed at the animal research institute of AMC. F1 (21-3 × 283-2) male and female rats were used for experiments. ...
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/Huβ2m 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/Huβ2m 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.
... 6 This could have been the case in Drosophila that lacks tapasin and transporters associated with antigen processing, required for optimal folding of MHC-I molecules. 21 However, given that MHC-I misfolding is reduced in the presence of hβ2m, 34 and that neither expression of HLA-B alone nor its coexpression with hβ2m induced ER stress in transgenic Drosophila, it is unlikely that MHC-I misfolding would account for the observed phenotypes. Moreover, the level of folded HLA-B molecules at the cell surface was greater in HLA-B27/hβ2m than HLA-B7/hβ2m transgenic wing imaginal discs. ...
Objectives
The human leucocyte antigen (HLA)-B27 confers an increased risk of spondyloarthritis (SpA) by unknown mechanism. The objective of this work was to uncover HLA-B27 non-canonical properties that could explain its pathogenicity, using a new Drosophila model.
Methods
We produced transgenic Drosophila expressing the SpA-associated HLA-B*27:04 or HLA-B*27:05 subtypes, or the non-associated HLA-B*07:02 allele, alone or in combination with human β2-microglobulin (hβ2m), under tissue-specific drivers. Consequences of transgenes expression in Drosophila were examined and affected pathways were investigated by the genetic interaction experiments. Predictions of the model were further tested in immune cells from patients with SpA.
Results
Loss of crossveins in the wings and a reduced eye phenotype were observed after expression of HLA-B*27:04 or HLA-B*27:05 in Drosophila but not in fruit flies expressing the non-associated HLA-B*07:02 allele. These HLA-B27-induced phenotypes required the presence of hβ2m that allowed expression of well-folded HLA-B conformers at the cell surface. Loss of crossveins resulted from a dominant negative effect of HLA-B27 on the type I bone morphogenetic protein (BMP) receptor saxophone (Sax) with which it interacted, resulting in elevated mothers against decapentaplegic (Mad, a Drosophila receptor-mediated Smad) phosphorylation. Likewise, in immune cells from patients with SpA, HLA-B27 specifically interacted with activin receptor-like kinase-2 (ALK2), the mammalian Sax ortholog, at the cell surface and elevated Smad phosphorylation was observed in response to activin A and transforming growth factor β (TGFβ).
Conclusions
Antagonistic interaction of HLA-B27 with ALK2, which exerts inhibitory functions on the TGFβ/BMP signalling pathway at the cross-road between inflammation and ossification, could adequately explain SpA development.
... While transgenic rats expressing human HLA-B27 show increased pro-inflammatory cytokine production by macrophages [18,56], reduction of ER stress by reducing HLA-B27 misfolding through additional trans-gene expression of beta2microglobulin in rats prevents intestinal inflammation in these animals. Interestingly, these rats still develop axial and peripheral arthritis [57], which highlights the specific importance of ER stress in controlling immune regulation in the intestine. Taken together, we identified that ER stress abrogates the immunosuppressive effect of IL-10, which is mediated by inhibition of STAT3 phosphorylation. ...
Objective and design
To determine whether ER stress affects the inhibitory pathways of the human immune system, particularly the immunosuppressive effect of IL-10 on macrophages.
Material or subjects
In vitro stimulation of human monocyte-derived macrophages.
Treatment
Cells were stimulated with TLR ligands and IL-10, while ER stress was induced using thapsigargin or tunicamycin.
Methods
mRNA expression was determined using qPCR, while cytokine protein production was measured using ELISA. Protein expression of receptors and transcription factors was determined using flow cytometry. Student’s t test was used for statistics.
Results
While under normal conditions IL-10 potently suppresses pro-inflammatory cytokine production by LPS-stimulated macrophages, we demonstrate that ER stress counteracts the immunosuppressive effects of IL-10, leading to increased pro-inflammatory cytokine production. We identified that ER stress directly interferes with IL-10R signaling by reducing STAT3 phosphorylation on Tyr705, which thereby inhibits the expression of SOCS3. Moreover, we show that ER stress also inhibits STAT3 activation induced by other receptors such as IL-6R.
Conclusions
Combined, these data uncover a new general mechanism by which ER stress promotes inflammation. Considering its potential involvement in the pathogenesis of diseases such as Crohn’s disease and spondyloarthritis, targeting of this mechanism may provide new opportunities to counteract inflammation.
... We hypothesized that IL-17A is a key mediator linking inflammation to new bone formation in SpA. To test this hypothesis, we performed in vitro human osteoblastic differentiation assays and in vivo IL-17A blockade in Mycobacterium tuberculosis-induced disease in B27/hβ 2 mtransgenic rats (9,25). ...
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, β‐glycophosphatase, and ascorbic acid. IL‐17A blockade was performed in HLA–B27/human β2‐microglobulin (hβ2m)–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/hβ2m–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.
... This strain does not develop colitis or arthritis (although males develop epididymo-orchitis) unless 35 additional copies of hb 2 m (283-2 transgene locus) are present (21-3x283-2). The 21-3x283-2 rats with extra hb 2 m overexpression develop severe arthritis for reasons that remain unclear but remain free from gastrointestinal disease [142]. This study did not completely resolve effects of HLA-B*27 from those of hb 2 m on gut microbiota, although it suggested that HLA-B*27/hb 2 m co-expression had different effects compared to HLA-B7/hb 2 m. ...
The mechanism by which HLA-B*27 predisposes to spondyloarthritis remains unresolved. Arthritogenic peptides have not been defined in humans and are not involved in experimental models of spondyloarthritis. Aberrant properties of HLA-B*27 can activate the IL-23/IL-17 axis in HLA-B*27 transgenic rats and humans. In HLA-B*27-independent rodent models, spondyloarthritis can be driven by IL-23 triggering entheseal-resident CD4⁻/CD8⁻ T cells or CD4⁺ Th17 T cells. These findings point toward noncanonical mechanisms linking HLA-B*27 to the disease and provide a potential explanation for HLA-B*27-negative spondyloarthritis. Gut microbial dysbiosis may be important in the development of spondyloarthritis. HLA-B*27-induced changes in gut microbiota are complex and suggest an ecological model of dysbiosis in rodents. The importance of the IL-23/IL-17 axis in ankylosing spondylitis has been demonstrated by studies showing efficacy of IL-17. Although deciphering the precise role(s) of HLA-B*27 in disease requires further investigation, considerable progress has been made in understanding this complex relationship.
... Considering this emerging evidence that the respective biologies of IL-23 and IL-17 are not always linear but instead partially overlapping, the aim of the present study was to assess to what extend IL-23 is required to drive IL-17-dependent pathology in SpA. To examine this, we used the HLA-B27/Huβ2m transgenic rat as a model for HLA-B27-associated peripheral and axial joint pathology (34). We previously demonstrated that this model not only displays the inflammatory features of SpA but also the prototypical remodeling structural phenotype (35). ...
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/Huβ2m 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.
... In an effort to more directly address the role of the UPR in these rats, one study interbred HLA-B27 transgenic rats with human beta-2 microglobulin overexpressing rats to stabilize and aid in HLA-B27 folding. This breeding did indeed reduce misfolding in Con-A stimulated splenocytes, although macrophages and tissue UPR were not assessed (235,236). Surprisingly, these animals developed more severe arthritis, without changes to their colitis. This study suggests the role of HLA-B27-linked UPR may be discordant in the joints and the gut during SpA and raises further questions regarding HLA-B27 misfolding, UPR, and disease pathogenesis. ...
Protein folding in the endoplasmic reticulum (ER) is an essential cell function. To safeguard this process in the face of environmental threats and internal stressors, cells mount an evolutionarily conserved response known as the unfolded protein response (UPR). Invading pathogens induce cellular stress that impacts protein folding, thus the UPR is well situated to sense danger and contribute to immune responses. Cytokines (inflammatory cytokines and interferons) critically mediate host defense against pathogens, but when aberrantly produced, may also drive pathologic inflammation. The UPR influences cytokine production on multiple levels, from stimulation of pattern recognition receptors, to modulation of inflammatory signaling pathways, and the regulation of cytokine transcription factors. This review will focus on the mechanisms underlying cytokine regulation by the UPR, and the repercussions of this relationship for infection and autoimmune/autoinflammatory diseases. Interrogation of viral and bacterial infections has revealed increasing numbers of examples where pathogens induce or modulate the UPR and implicated UPR-modulated cytokines in host response. The flip side of this coin, the UPR/ER stress responses have been increasingly recognized in a variety of autoimmune and inflammatory diseases. Examples include monogenic disorders of ER function, diseases linked to misfolding protein (HLA-B27 and spondyloarthritis), diseases directly implicating UPR and autophagy genes (inflammatory bowel disease), and autoimmune diseases targeting highly secretory cells (e.g., diabetes). Given the burgeoning interest in pharmacologically targeting the UPR, greater discernment is needed regarding how the UPR regulates cytokine production during specific infections and autoimmune processes, and the relative place of this interaction in pathogenesis.
... [41] Rats transgenic for the mutant HLA-B*27:05-C67S (line 133-1) were as previously described. [28,29,45] Rat spleens were resected after sacrificing with CO 2 and immediately frozen in liquid nitrogen for storage at -80°C. ...
The HLA-B*27 peptidome has drawn significant attention due to the genetic association between some of the HLA-B*27 alleles and the inflammatory rheumatic disease Ankylosing Spondylitis (AS), for which a comprehensive biological explanation is still lacking. This study aims to expand the known limits of the HLA-B*27 peptidome to facilitate selection and testing of new peptides, possibly involved in the disease. The HLA peptidomes of HeLa and C1R cell lines stably transfected with the AS-associated HLA-B*27:05 allele, the non-associated HLA-B*27:09 allele, or their Cysteine 67 to Serine mutants (C67S), were analyzed on a very large scale. In addition, the peptidomes of HLA-B*27:05 and HLA-B*27:05-C67S were analyzed from the spleens of rats transgenic for these alleles. The results indicate that C67S mutation increases the percentage of peptides with glutamine or lysine at their P2 position (P2-Lys), in both HLA-B*27:05 and HLA-B*27:09. Furthermore, a small fraction of HLA-B*27 peptides contains lysine at their second position (P2), in addition to the more commonly found peptides with arginine (P2-Arg), or the less common glutamine (P2-Gln) located at this anchor position. Overall these data indicate that peptides with P2-Lys should be considered as real ligands of HLA-B*27 molecules and taken into account while looking for putative peptides implicated in the AS.
... Human β2m stabilises the HLA-B27 heavy chain when expressed at the cell surface. Overexpression of additional human β2m in HLA-B27/human β2m transgenic rats promotes arthritis and spondylitis, 19 suggesting an important role for this human molecule in the pathogenesis of AS. Mouse β2m, however, could inhibit the possible inflammatory and osteogenic properties of HLA-B27 as it is seen that HLA-B27 transgenic mice do not show inflammatory disease 15 20 whereas HLA-B27 expression in a mouse β2m-deficient mouse model induces inflammatory arthritis of the hind paws and nail changes. ...
Objective
The 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 and in vivo impact of HLA-B27 in models of chondrogenesis and osteogenesis.
Methods
Different in vitro differentiation systems were used to mimic endochondral and direct bone formation. ATDC5 cells and primary human periosteum-derived cells (hPDCs) were transduced with lentiviral vectors expressing HLA-B27 or HLA-B7. These cells and limb bud cells (from HLA-B27 transgenic and wild-type (WT) mice) were cultured in micromasses. To study direct osteogenesis in hPDCs, cells were cultured as monolayers and stimulated with osteogenic media. Chondrogenesis (COL2, ACAN, COL10) and osteogenesis (OSC, ALP, RUNX2) marker expression was studied by quantitative RT-PCR. Colorimetric tests were performed to measure proteoglycans, mineralization and collagens. Collagen antibody-induced arthritis (CAIA) was induced in HLA-B27 transgenic and WT mice. Clinical scoring and µCTs were performed. Statistical analyses were performed by two-way ANOVA.
Results
There was no difference in chondrogenesis markers or in colorimetric tests between HLA-B27⁺ and HLA-B7⁺ micromasses. Expression of osteogenesis markers and Alizarin red staining was comparable in the HLA-B27⁺ and the HLA-B7⁺ hPDCs in monolayers. HLA-B27 transgenic mice showed more severe arthritis compared with WT mice in the CAIA model. µCT analysis showed no increased bone formation in HLA-B27 transgenic mice.
Conclusion
HLA-B27 seems to enhance joint inflammation in the CAIA model. We could not document a direct effect of HLA-B27 on chondrogenesis or osteogenesis.
... The misfolded HLA-B27 heavy chains tend to accumulate in the endoplasmic reticulum (ER), triggering ER stress, which leads to the activation of the unfolded protein response (UPR) and the NF-κB pathway which, in turn, leads to the release of pro-inflammatory cytokines, such as TNF-α, IL-1, IL-6, mainly by monocytes/macrophages, thus favouring the inflammatory process [16]. Animal disease models argue both in favour and against this hypothesis [17,18]. The cell surface HLA-B27 homodimers hypothesis is, instead, based on the observation that HLA-B27 homodimers produced at the cell surface bind to specific receptors expressed on NK cells, T-lymphocytes, and myelomonotic cells producing an immunomodulatory effect [19]. ...
The term spondyloarthritis (SpA) is used to describe a group of multifactorial chronic inflammatory diseases characterized by a predisposing genetic background and clinical manifestations typically involving the sacroiliac joint. The absence of pathognomonic clinical and/or laboratory findings generally results in a delay in diagnosis and, consequently, in treatment. In addition, 20–40% of SpA patients are non-responders to tumor necrosis factor (TNF) inhibitor therapies. Given these considerations, it is important to identify biomarkers that can facilitate the diagnosis and assessment of disease activity. As inflammation plays a key role in the pathogenesis of SpA, inflammatory mediators have been investigated as potential biomarkers for diagnosing the disease and predicting response to therapy. Some investigators have focused their attention on the role of matrix metalloproteinases (MMPs), which are known to be markers of synovial inflammation that is generated in the joint in reaction to inflammatory stimuli. Several studies have been carried out to verify if serum MMPs levels could be useful to diagnose SpA, to assess disease severity, and to predict response to TNF inhibitor therapy. The current review focuses on MMPs’ role in SpA pathogenesis, diagnosis and therapeutic implications.
... This is termed the 'β2m deposition' hypothesis. It was further hypothesized that β2m expression levels may be associated with AS pathogenesis, as spondylitis was successfully induced in a novel HLA-B27/β2m transgenic rat model expressing increased levels of β2m (60). This lead to the proposed 'β2m over-expression' hypothesis (60). ...
The study of ankylosing spondylitis (AS) has made significant progress over the last decade. Genome-wide association studies have identified and further substantiated the role of susceptibility genes outside the major histocompatibility complex locus. However, human leukocyte antigen (HLA)‑B27 has been suggested to be important in the pathogenesis of AS, contributing to ~20.1% of AS heritability. The current review will present the classical and non‑classical forms of HLA-B27, as well as their pathogenic roles, and further discuss the hypotheses regarding the potential pathogenesis of AS. In addition, the association between the pathogenic role of HLA‑B27 and inflammatory indexes, including the interleukin-23/‑17 axis will be investigated to provide novel insights into the pathogenesis of AS. The aim of the present review is to provide an update of the current research into the pathogenesis of AS, and provide a comprehensive description of the pathogenic role of HLA-B27 in AS.
... The second hypothesis concerns HLA-B27 heavy chain misfolding prior to binding of β2-M and peptide, resulting in unfolded protein responses (UPR), which subsequently may lead to an inflammatory cytokine release [200,203]. Interestingly, modulation of β2-M expression levels in the HLA-B27 transgenic rat model of SpA did profoundly affect the phenotype of the disease [204][205][206], confirming the potential importance of this molecule in SpA. Therefore, serum β2-M expression levels should be further explored as potential biomarker in SpA. ...
Introduction: Early diagnosis, monitoring of disease activity, prediction of treatment response, and structural outcome remain major challenges in spondyloarthritis (SpA). Biomarkers could play a role in addressing these challenges, but in SpA there is a lack of suitable biomarkers.
Areas covered: As SpA is clinically and pathophysiologically closely related to psoriasis and inflammatory bowel disease (IBD), we reviewed in literature, the value of serum biomarkers in these conditions with the aim to find potential candidates for assessing SpA.
Expert commentary: Candidates of interest were antimicrobial peptides, including serum human beta defensin-2 (hBD-2) and lipocalin-2 (LCN-2), and class-1 MHC molecule beta2-microglobulin. Since these biomarkers are relevant in psoriasis and/or IBD from a pathophysiological point of view, and may play a role in the pathogenesis of SpA, we recommend further exploration of their value as biomarker in the diagnosis and prognosis of SpA.
Murine models have played an indispensable role in the understanding of rheumatic and musculoskeletal disorders (RMD), elucidating the genetic, endocrine and biomechanical pathways involved in joint pathology and associated pain. To date, the available models in RMD can be classified as induced or spontaneous, both incorporating transgenic alternatives that improve specific insights. It is worth noting that the selection of the most appropriate model together with the evaluation of their specific characteristics and technical capabilities are crucial when designing the experiments. Furthermore, it is also imperative to consistently adhere to the ethical standards concerning animal experimentation. Recognizing the inherent limitation that any model can entirely encapsulates the complexity of the pathophysiology of these conditions, the aim of this review is to provide an updated overview on the methodology of current murine models in major arthropathies and their immune-mediated pathways, addressing to basic, translational and pharmacological research in joint damage and pain.
Psoriasis is an autoimmune, chronic, inflammatory skin condition mediated by T cells. It differs from other inflammatory conditions by causing significant alterations in epidermal cell proliferation and differentiation that are both complicated and prominent. The lack of an appropriate animal model has significantly hindered studies into the pathogenic mechanisms of psoriasis since animals other than humans typically do not exhibit the complex phenotypic features of human psoriasis. A variety of methods, including spontaneous mutations, drug-induced mutations, genetically engineered animals, xenotransplantation models, and immunological reconstitution approaches, have all been employed to study specific characteristics in the pathogenesis of psoriasis. Although some of these approaches have been used for more than 50 years and far more models have been introduced recently, they have surprisingly not yet undergone detailed validation. Despite their limitations, these models have shown a connection between keratinocyte hyperplasia, vascular hyperplasia, and a cell-mediated immune response in the skin. The xenotransplantation of diseased or unaffected human skin onto immune-compromised recipients has also significantly aided psoriasis research. This technique has been used in a variety of ways to investigate the function of T lymphocytes and other cells, including preclinical therapeutic studies. The design of pertinent in vivo and in vitro psoriasis models is currently of utmost concern and a crucial step toward its cure. This article outlines the general approach in the development of psoriasis-related animal models, aspects of some specific models, along with their strengths and limitations.
The mechanisms underlying the genetic association between HLA-B27 and ankylosing spondylitis, including the contribution of endoplasmic reticulum aminopeptidase 1 (ERAP1), continue to elude the field. New findings support the involvement of the unfolded protein response and highlight the therapeutic potential and limitations of targeting ERAP1 in this disease.
The chronic inflammatory disease ankylosing spondylitis (AS) is marked by back discomfort, spinal ankylosis, and extra-articular symptoms. In AS, inflammation is responsible for both pain and spinal ankylosis. However, the processes that sustain chronic inflammation remain unknown. Despite the years of research conducted to decipher the intricacy of AS, little progress has been made in identifying the signaling events that lead to the development of this disease. T cells, an immune cell type that initiates and regulates the body’s response to infection, have been established to substantially impact the development of AS. T lymphocytes are regarded as a crucial part of adaptive immunity for the control of the immune system. A highly coordinated interaction involving antigen-presenting cells (APCs) and T cells that regulate T cell activation constitutes an immunological synapse (IS). This first phase leads to the controlled trafficking of receptors and signaling mediators involved in folding endosomes to the cellular interface, which allows the transfer of information from T cells to APCs through IS formation. Discrimination of self and nonself antigen is somatically learned in adaptive immunity. In an autoimmune condition such as AS, there is a disturbance of self/nonself antigen discrimination; available findings imply that the IS plays a preeminent role in the adaptive immune response. In this paper, we provide insights into the genesis of AS by evaluating recent developments in the function of vesicular trafficking in IS formation and the targeted release of exosomes enriched microRNAs (miRNA) at the synaptic region in T cells.
Human leucocyte antigen B*27 (HLA-B*27) is strongly associated with inflammatory diseases of the spine and pelvis (for example, ankylosing spondylitis (AS)) and the eye (that is, acute anterior uveitis (AAU))¹. How HLA-B*27 facilitates disease remains unknown, but one possible mechanism could involve presentation of pathogenic peptides to CD8⁺ T cells. Here we isolated orphan T cell receptors (TCRs) expressing a disease-associated public β-chain variable region–complementary-determining region 3β (BV9–CDR3β) motif2–4 from blood and synovial fluid T cells from individuals with AS and from the eye in individuals with AAU. These TCRs showed consistent α-chain variable region (AV21) chain pairing and were clonally expanded in the joint and eye. We used HLA-B*27:05 yeast display peptide libraries to identify shared self-peptides and microbial peptides that activated the AS- and AAU-derived TCRs. Structural analysis revealed that TCR cross-reactivity for peptide–MHC was rooted in a shared binding motif present in both self-antigens and microbial antigens that engages the BV9–CDR3β TCRs. These findings support the hypothesis that microbial antigens and self-antigens could play a pathogenic role in HLA-B*27-associated disease.
Objective
We undertook this study to examine the functional basis for epistasis between endoplasmic reticulum aminopeptidase 1 (ERAP1) and HLA–B27 in experimental spondyloarthritis (SpA).
Methods
ERAP1–knockout rats were created using genome editing and bred with HLA–B27/human β2‐microglobulin–transgenic (HLA–B27‐Tg) rats and HLA–B7‐Tg rats. The effects of ERAP1 deficiency on HLA allotypes were determined using immunoprecipitation and immunoblotting, flow cytometry, allogeneic T cell proliferation assays, and gene expression analyses. Animals were examined for clinical features of disease, and tissue was assessed by histology.
Results
ERAP1 deficiency increased the ratio of folded to unfolded (β2m‐free) HLA–B27 heavy chains, while having the opposite effect on HLA–B7. Furthermore, in rats with ERAP1 deficiency, HLA–B27 misfolding was reduced, while free HLA–B27 heavy chain dimers on the cell surface and monomers were increased. The effects of ERAP1 deficiency persisted during up‐regulation of HLA–B27 and led to a reduction in endoplasmic reticulum stress. ERAP1 deficiency reduced the prevalence of arthritis in HLA–B27‐Tg rats by two‐thirds without reducing gastrointestinal inflammation. Dendritic cell abnormalities attributed to the presence of HLA–B27, including reduced allogeneic T cell stimulation and loss of CD103‐positive/major histocompatibility complex class II–positive cells, were not rescued by ERAP1 deficiency, while excess Il23a up‐regulation was mitigated.
Conclusion
ERAP1 deficiency reduced HLA–B27 misfolding and improved folding while having opposing effects on HLA–B7. The finding that HLA–B27‐Tg rats had partial protection against SpA in this study is consistent with genetic evidence that loss‐of‐function and/or reduced expression of ERAP1 reduces the risk of ankylosing spondylitis. Functional studies support the concept that the effects of ERAP1 on HLA–B27 and SpA may be a consequence of how peptides affect the biology of this allotype rather than their role as antigenic determinants.
Development of animal models of inflammatory articular disorders, although not completely reproducing the entire spectrum of clinical manifestations seen in human disorders, has significantly contributed to a better understanding of the complex interplay of genetic, immunologic, and environmental factors playing a role in pathogenesis. Several animal models, either spontaneous, genetically engineered, or induced, are available and have allowed delineation of the individual or combine contribution of T-, B-, macrophage, dendritic, neutrophil, and mast cells, cytokine pathways, and their interaction with the microbiota. In addition, information obtained has led to the development of innovative therapeutic approaches. An overview of the most commonly used animal models to study spondyloarthritis (SpA), especially reactive arthritis, follows.
Ankylosing spondylitis (AS), a common type of spondyloarthropathy, is a chronic inflammatory autoimmune disease that mainly affects spine joints, causing severe, chronic pain; additionally, in more advanced cases, it can cause spine fusion. Significant progress in its pathophysiology and treatment has been achieved in the last decade. Immune cells and innate cytokines have been suggested to be crucial in the pathogenesis of AS, especially human leukocyte antigen (HLA)‑B27 and the interleukin‑23/17 axis. However, the pathogenesis of AS remains unclear. The current study reviewed the etiology and pathogenesis of AS, including genome-wide association studies and cytokine pathways. This study also summarized the current pharmaceutical and surgical treatment with a discussion of future potential therapies.
Animal models of human diseases are used to study a broad array of autoimmune disorders including spondyloarthritis (SpA). Various animal models, either genetically engineered, spontaneous or induced, have been described to address the fundamental pathological features of human SpA. With the advent of novel techniques for manipulations in animal models, it has become possible to delineate critical pathways involved in SpA disease mechanisms and to develop novel therapeutics. In this chapter, we discuss animal models to study the fundamental characteristics of human SpA. None of the animal models developed exactly mimics human diseases, but some of the models closely resemble the group of human SpA diseases. The SKG mouse, in particular, harbours a point mutation in the ZAP-70 gene yielding reduced T-cell receptor (TCR) signalling, develops multi-organ inflammation under microbial influence, mimics human SpA disease pathogenesis and is a promising tool for designing new therapeutics against SpA. We describe the contribution of animal models to the earliest known disease mechanisms, including how attenuated T cell signalling contributes to disease pathogenesis as a result of genetic or environmental predisposition. We also discuss recent advances in the understanding of key cytokines that drive SpA pathophysiology and the crosstalk of gut microbiota with host genetic determinants of immune function and finally the potential future prospects to study these animal models to translate our knowledge of pathogenesis into clinical practice.
Objectives:
Spondyloarthritis (SpA) encompasses a group of disorders including ankylosing spondylitis, psoriatic arthritis, reactive arthritis, and enteropathic arthritis. SpA pathogenesis is still not well understood. Animal models are important for studying disease mechanisms and identifying new therapeutic agents. Recently, a β-glucan-induced SKG mouse was used as an animal model for SpA. The aim of this study was to evaluate the clinical and molecular characteristics of a zymosan-induced SKG mouse.
Methods:
Zymosan was injected intraperitoneally into SKG mice. Clinical arthritis scores were measured, and fluorine-18 fluorodeoxyglucose (18F-FDG) small-animal positron emission tomography/computed tomography (PET/CT) was performed to quantify joint inflammation. Histologic features of the joints, intestines, skin, and eyes were evaluated. Inflammatory cytokine and Wnt inhibitor expression was measured in mouse serum.
Results:
Zymosan exposure triggered SpA-like diseases in SKG mice, including peripheral arthritis, spondylitis, dactylitis, enteritis, and psoriatic skin lesions. 18F-FDG uptake was significantly higher in the zymosan-treated mice compared with controls. The expression of tumor necrosis factor α, interleukin (IL)-6, and Dickkopf-1 increased significantly, while IL-4 and sclerostin expression decreased significantly in zymosan-induced mice compared with control mice.
Conclusions:
Zymosan-induced SKG mice developed articular and extra-articular features as well as molecular changes that resembled those of human SpA. These findings suggest that the zymosan-induced SKG mouse is a good animal model to reflect the complex features of human SpA.
HLA-B27 is a class I major histocompatibility (MHC-I) allele that confers susceptibility to the rheumatic disease ankylosing spondylitis (AS) by an unknown mechanism. ERAP1 is an aminopeptidase that trims peptides in the endoplasmic reticulum for binding to MHC-I molecules. ERAP1 shows genetic epistasis with HLA-B27 in conferring susceptibility to AS. Male HLA-B27 transgenic rats develop arthritis and serve as an animal model of AS, whereas female B27 transgenic rats remain healthy. We used large-scale quantitative mass-spectrometry to identify over 15,000 unique HLA-B27 peptide ligands, isolated after immunoaffinity purification of the B27 molecules from the spleens of HLA-B27 transgenic rats. Heterozygous deletion of ERAP1, which reduced ERAP1 level to less than half, had no qualitative or quantitative effects on the B27 peptidome. Homozygous deletion of ERAP1 affected approximately one third of the B27 peptidome, but left most of the B27 peptidome unchanged, suggesting the possibility that some of the HLA-B27 immunopeptidome is not processed in the presence of ERAP1. Deletion on ERAP1 was permissive for the AS-like phenotype. Deletion of ERAP1 increased mean peptide length, and increased the frequency of C-terminal hydrophobic residues and of N-terminal Ala, Ser or Lys. The presence of ERAP1 increased the frequency of C-terminal Lys and Arg, of Glu and Asp at intermediate residues, and of N-terminal Gly. Several peptides of potential interest in AS pathogenesis, previously identified in human cell lines, were isolated. However, rats susceptible to arthritis had B27 peptidomes similar to those of non-susceptible rats, and no peptides were found to be uniquely associated with arthritis. Whether specific B27-bound peptides are required for AS pathogenesis remains to be determined. Data are available via ProteomeXchange with identifier PXD005502.
We have previously produced lines of rats transgenic for HLA-B27 and human beta 2-microglobulin (h beta 2m) that develop a progressive inflammatory disease sharing many clinical and histologic features with the B27-associated human spondyloarthropathies, including gut and male genital inflammation, arthritis, and psoriasiform skin lesions. Other transgenic lines that express lower levels of B27 and h beta 2m remain healthy. To investigate the cellular basis for the multisystem inflammatory disease in these rats, we transferred lymphoid cell populations from disease-prone transgenic lines to irradiated disease-resistant transgenic and nontransgenic recipients. In recipients of cells from two different disease-prone lines, successful transfer required engraftment of bone marrow cells. Transfer of disease with fetal liver cells suggested that neither mature effector cells nor active disease in the donors was necessary for induction of disease in the recipients. Remission of the spontaneous disease in irradiated transgenic rats was induced by engraftment of nontransgenic bone marrow. These results suggest that the expression of HLA-B27 in bone marrow-derived cells alone is sufficient for the development of B27-associated disease, and that disease transfer requires engraftment of a bone marrow precursor cell for which mature cells in spleen or in lymph node cannot substitute.
The association between spondyloarthropathy and Crohn's disease is well known. A risk for evolution to Crohn's disease has already been shown in the subgroup of patients with spondyloarthropathy associated with chronic gut inflammation.
To investigate whether the reported polymorphisms in the CARD15 gene, a susceptibility gene for Crohn's disease, are associated with the presence of preclinical intestinal inflammation observed in spondyloarthropathies.
104 patients with spondyloarthropathies were studied. All underwent ileocolonoscopy with biopsies between 1983 and 2004. The prevalence of three single nucleotide polymorphisms in the CARD15 gene (R702W, G908R, and 1007fs) was assessed using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR); the patients were compared with an ethnically matched Crohn's disease population and a control population.
The carrier frequency of R702W, G908R, or 1007fs variants in the spondyloarthropathy populations (20%) was similar to the control population (17%), but increased to 38% in the spondyloarthropathy subgroup with chronic gut inflammation. This frequency was significantly higher than in the other spondyloarthropathy subgroups (p = 0.001) or the control group (p = 0.006), but not different from the Crohn's disease group (49%) (NS). This indicates that CARD15 polymorphisms are associated with a higher risk for development of chronic gut inflammation.
CARD15 gene polymorphisms clearly identify a subgroup of patients with spondyloarthropathies associated with chronic intestinal inflammation.
Molecular mimicry is discussed as a possible mechanism that may contribute to the development of autoimmune diseases. It could also be involved in the differential association of the human major histocompatibility subtypes HLA-B(*)2705 and HLA-B(*)2709 with ankylosing spondylitis. These two subtypes differ only in residue 116 of the heavy chain (Asp in B(*)2705 and His in B(*)2709), but the reason for the differential disease association is not understood. Using x-ray crystallography, we show here that the viral peptide pLMP2 (RRRWRRLTV, derived from latent membrane protein 2 (residues 236-244) of Epstein-Barr virus) is presented by the B(*)2705 and B(*)2709 molecules in two drastically deviating conformations. Extensive structural similarity between pLMP2 and the self-peptide pVIPR (RRKWRRWHL, derived from vasoactive intestinal peptide type 1 receptor (residues 400-408)) is observed only when the peptides are presented by B(*)2705 because of a salt bridge between Arg(5) of both peptides and the subtype-specific heavy chain residue Asp(116). Combined with functional studies using pLMP2/pVIPR-cross-reactive cytotoxic T cell lines and clones, together with target cells presenting these peptides or a modified peptide analogue, our results reveal that a pathogen-derived peptide can exhibit major histocompatibility complex class I subtype-dependent, drastically distinct binding modes. Furthermore, the results demonstrate that molecular mimicry between pLMP2 and pVIPR in the HLA-B27 context is an allele-dependent property.
Reports of the use of HLA-B27/peptide tetrameric complexes to study peptide-specific CD8+ T cells in HLA-B27+-related diseases are rare. To establish HLA-B27 tetramers we first compared the function of HLA-B27 tetramers with HLA-A2 tetramers by using viral epitopes. HLA-B27 and HLA-A2 tetramers loaded with immunodominant peptides from Epstein-Barr virus were generated with comparable yields and both molecules detected antigen-specific CD8+ T cells. The application of HLA-B27 tetramers in HLA-B27-related diseases was performed with nine recently described Chlamydia-derived peptides in synovial fluid and peripheral blood, to examine the CD8+ T cell response against Chlamydia trachomatis antigens in nine patients with Chlamydia-triggered reactive arthritis (Ct-ReA). Four of six HLA-B27+ Ct-ReA patients had specific synovial T cell binding to at least one HLA-B27/Chlamydia peptide tetramer. The HLA-B27/Chlamydia peptide 195 tetramer bound to synovial T cells from three of six patients and HLA-B27/Chlamydia peptide 133 tetramer to synovial T cells from two patients. However, the frequency of these cells was low (0.02-0.09%). Moreover, we demonstrate two methods to generate HLA-B27-restricted T cell lines. First, HLA-B27 tetramers and magnetic beads were used to sort antigen-specific CD8+ T cells. Second, Chlamydia-infected dendritic cells were used to stimulate CD8+ T cells ex vivo. Highly pure CD8 T cell lines could be generated ex vivo by magnetic sorting by using HLA-B27 tetramers loaded with an EBV peptide. The frequency of Chlamydia-specific, HLA-B27 tetramer-binding CD8+ T cells could be increased by stimulating CD8+ T cells ex vivo with Chlamydia-infected dendritic cells. We conclude that HLA-B27 tetramers are a useful tool for the detection and expansion of HLA-B27-restricted CD8+ T cells. T cells specific for one or more of three Chlamydia-derived peptides were found at low frequency in synovial fluid from HLA-B27+ patients with Ct-ReA. These cells can be expanded ex vivo, suggesting that they are immunologically functional.
A number of inflammatory disease states occur with greatly increased frequency in individuals inheriting the human major histocompatibility complex class I allele HLA-B27. In a minority of cases, namely those with B27-associated reactive arthritis, there is good evidence that the disease state is triggered by infection with an enteric or genitourinary bacterial pathogen. For the majority of B27-associated disease, no definite pathogenetic role for bacteria has been established. However, in these latter cases intestinal inflammation can often be demonstrated, and it sometimes occupies a major part of the clinical picture. Rats transgenic for B27 are known to develop a disorder resembling B27-associated human disease, with prominent intestinal, joint, skin, and male genital inflammatory lesions. We report here that B27 transgenic rats raised in a germfree environment do not develop inflammatory intestinal or peripheral joint disease, whereas the skin and genital inflammatory lesions are unaffected by the germfree state. These findings support the concept that gut and joint inflammation are pathogenetically closely related, and they provide direct evidence that the commensal gut flora play an important role in the pathogenesis of B27-associated gut and joint inflammation.
We have previously produced lines of rats transgenic for HLA-B27 and human beta 2-microglobulin (h beta 2m) that develop a progressive inflammatory disease sharing many clinical and histologic features with the B27-associated human spondyloarthropathies, including gut and male genital inflammation, arthritis, and psoriasiform skin lesions. Other transgenic lines that express lower levels of B27 and h beta 2m remain healthy. To investigate the cellular basis for the multisystem inflammatory disease in these rats, we transferred lymphoid cell populations from disease-prone transgenic lines to irradiated disease-resistant transgenic and nontransgenic recipients. In recipients of cells from two different disease-prone lines, successful transfer required engraftment of bone marrow cells. Transfer of disease with fetal liver cells suggested that neither mature effector cells nor active disease in the donors was necessary for induction of disease in the recipients. Remission of the spontaneous disease in irradiated transgenic rats was induced by engraftment of nontransgenic bone marrow. These results suggest that the expression of HLA-B27 in bone marrow-derived cells alone is sufficient for the development of B27-associated disease, and that disease transfer requires engraftment of a bone marrow precursor cell for which mature cells in spleen or in lymph node cannot substitute.
To investigate the role of the class I MHC molecule HLA-B27 in the spondyloarthropathies, we produced rats transgenic for HLA-B27 and human beta 2-microglobulin. Of five lines bearing > 1 copy of each transgene and showing hemizygous expression of both transgenes, two (lines 21-4H and 33-3) developed spontaneous inflammatory disease that closely resembled B27-associated human disease. Two lines, 21-4L and 25-6, remained healthy even when homozygous for the transgene locus, whereas the 21-3 line, bearing the third highest transgene copy number, developed disease similar to that of the 21-4H and 33-3 lines only when homozygous for the transgene locus. The disease-prone lines showed higher expression of B27--thymic mRNA in utero, splenic mRNA by 5 days of age, and splenic cell surface protein by the time of disease onset--than the disease-resistant lines. Disease susceptibility thus appeared to correlate with gene copy number and the quantity of B27 in lymphoid cells. The increase in the amount of B27 protein did not appear to be simply a consequence of the inflammatory disease because 1) there was no similar change in endogenous RT1 class I expression; 2) no alteration of B27 expression occurred in 21-4H rats with adjuvant-induced arthritis; 3) in rats with inflammatory disease transgenic for HLA-A2 and the 21-4H transgene locus, A2 expression was the same as in healthy rats transgenic for A2 but not B27; and 4) the transgenes in disease-prone and disease-resistant lines were equally susceptible to induction by IFN-gamma. Immunocytochemistry of the distal colon, an early site of inflammation, showed that the B27 Ag is expressed at high levels in cells of the lamina propria, but not at all in colonic epithelial cells. Taken together, the data suggest that the B27 transgene is expressed in a copy number dependent, position-independent manner in lymphoid tissue and that disease results from the expression of B27 above a critical threshold level.
Genetic and environmental factors are important in the pathogenesis of clinical and experimental chronic intestinal inflammation. We investigated the influence of normal luminal bacteria and several groups of selected bacterial strains on spontaneous gastrointestinal and systemic inflammation in HLA-B27 transgenic rats. Rats maintained germfree for 3-9 mo were compared with littermates conventionalized with specific pathogen-free bacteria. Subsequently, germfree transgenic rats were colonized with groups of five to eight bacteria that were either facultative or strictly anaerobic. Transgenic germfree rats had no gastroduodenitis, colitis, or arthritis, but developed epididymitis and dermatitis to the same degree as conventionalized rats. Colonic proinflammatory cytokine expression was increased in transgenic conventionalized rats but was undetectable in germfree and nontransgenic rats. Colitis progressively increased over the first 4 wk of bacterial exposure, then plateaued. Only transgenic rats colonized with defined bacterial cocktails which contained Bacteroides spp. had colitis and gastritis. Normal luminal bacteria predictably and uniformly induce chronic colonic, gastric and systemic inflammation in B27 transgenic F344 rats, but all bacterial species do not have equal activities.
The MHC class I protein HLA-B27 is strongly associated with susceptibility to spondyloarthropathies and can cause arthritis when expressed in rats and mice, implying a direct role in disease pathogenesis. A prominent hypothesis to explain this role suggests that the unique peptide binding specificity of HLA-B27 confers an ability to present arthritogenic peptides. The B pocket, a region of the peptide binding groove that is an important determinant of allele-specific peptide binding, is thought to be critical for arthritogenicity. However, this hypothesis remains unproven. We show that in addition to its role in peptide selection, the B pocket causes a portion of the pool of assembling HLA-B27 heavy chains in the endoplasmic reticulum to misfold, resulting in their degradation in the cytosol. The misfolding phenotype is corrected by replacing the HLA-B27 B pocket with one from HLA-A2. Our results suggest an alternative to the arthritogenic peptide hypothesis. Misfolding and its consequences, rather than allele-specific peptide presentation, may underlie the strong link between the HLA-B27 B pocket and susceptibility to spondyloarthropathies.
In this study, we examined expression of the major histocompatibility complex (MHC) class I molecules and of the mRNAs for MHC class I-mediated antigen presentation machinery molecules in highly purified C57BL/6 mouse testicular germ cells. Although H2 class I molecules were not detected on the cell surface, small amounts of the molecules were detected inside the cells by flow cytometric and immunoprecipitation analyses. Interferon (IFN)gamma treatment did not affect expression of the molecules on the surface by flow cytometric analysis. In addition, expression of mRNAs for MHC class I-mediated antigen presentation machinery molecules in the germ cells was analyzed by reverse transcription-polymerase chain reaction analysis. The faint constitutive expression of mRNAs for H2-K, beta2-microglobulin (beta2m), tapasin, and calnexin was observed in the cells. IFNgamma treatment up-regulated expression of mRNAs for H2-K, beta2m, TAP1 (a peptide transporter), and LMP2 (a proteosomal subunit). These findings indicate that impaired expression of MHC class I molecules on the cell surface of mouse testicular germ cells is mainly caused by the post-transcriptional regulatory mechanism.
The Sa system is a recently described immune system that has a specificity and positive predictive value of nearly 100% for rheumatoid arthritis (RA) in Asia, Europe and the Americas. Its sensitivity of 30-40% suggests that it identifies a subset of RA patients. Anti-Sa antibodies are present from disease onset and are predictive of disease severity. The immune reactants are plentiful in the target tissue: antigen is present in the synovium, IgG antibody in the fluid. Immunologically, Sa is a hapten-carrier antigen in which vimentin is the carrier and citrulline is the hapten. The citrullination of vimentin is closely related to apoptosis, and citrullinated vimentin is extremely sensitive to digestion by the ubiquitous calpains. Nevertheless, Sa is found in only a few cell lines. Calpastatin, the natural specific inhibitor of calpains, is also a RA-associated, albeit non-specific, autoimmune system. Is it possible that calpain-related apoptotic pathways could be prominent in cells containing Sa? The task is to reconcile the specificity of Sa/citrullinated proteins in a multifactorial and polygenic disease such as RA.
The epithelial cells of the gastrointestinal tract are exposed to toxins and infectious agents that can adversely affect protein folding in the endoplasmic reticulum (ER) and cause ER stress. The IRE1 genes are implicated in sensing and responding to ER stress signals. We found that epithelial cells of the gastrointestinal tract express IRE1beta, a specific isoform of IRE1. BiP protein, a marker of ER stress, was elevated in the colonic mucosa of IRE1beta(-/-) mice, and, when exposed to dextran sodium sulfate (DSS) to induce inflammatory bowel disease, mutant mice developed colitis 3-5 days earlier than did wild-type or IRE1beta(+/-) mice. The inflammation marker ICAM-1 was also expressed earlier in the colonic mucosa of DSS-treated IRE1beta(-/-) mice, indicating that the mutation had its impact early in the inflammatory process, before the onset of mucosal ulceration. These findings are consistent with a model whereby perturbations in ER function, which are normally mitigated by the activity of IRE1beta, participate in the development of colitis.
Our goal in the present work was to determine whether male patients with untreated hypogonadism have an increased risk of developing rheumatic/autoimmune disease (RAD), and, if so, whether there is a relation to the type of hypogonadism. We carried out neuroendocrine, genetic, and rheumatologic investigations in 13 such patients and 10 healthy male 46,XY normogonadic control subjects. Age and body mass index were similar in the two groups. Nine of the 13 patients had hypergonadotropic hypogonadism (five of whom had Klinefelter's syndrome [karyotype 47,XXY]) and 4 of the 13 had hypogonadotropic hypogonadism (46,XY). Of these last four, two had Kallmann's syndrome and two had idiopathic cryptorchidism.
Eight (61%) of the 13 patients studied had RADs unrelated to the etiology of their hypogonadism. Of these, four had ankylosing spondylitis and histocompatibility B27 antigen, two had systemic lupus erythematosus (in one case associated with antiphospholipids), one had juvenile rheumatoid arthritis, and one had juvenile dermatomyositis. In comparison with the low frequencies of RADs in the general population (about 0.83%, including systemic lupus erythematosus, 0.03%; dermatomyositis, 0.04%; juvenile rheumatoid arthritis, 0.03%; ankylosing spondylitis, 0.01%; rheumatoid arthritis, 0.62%; and other RAD, 0.1%), there were surprisingly high frequencies of such disorders in this small group of patients with untreated hypogonadism (P < 0.001) and very low serum testosterone levels (P = 0.0005). The presence of RADs in these patients was independent of the etiology of their hypogonadism and was associated with marked gonadal failure with very low testosterone levels.
The class I protein HLA-B27 confers susceptibility to inflammatory arthritis in humans and when overexpressed in rodents for reasons that remain unclear. We demonstrated previously that HLA-B27 heavy chains (HC) undergo endoplasmic reticulum (ER)-associated degradation. We report here that HLA-B27 HC also forms two types of aberrant disulfide-linked complexes (dimers) during the folding and assembly process that can be distinguished by conformation-sensitive antibodies W6/32 and HC10. HC10-reactive dimers form immediately after HC synthesis in the ER and constitute at least 25% of the HC pool, whereas W6/32-reactive dimers appear several hours later and represent less than 10% of the folded HC. HC10-reactive dimers accumulate in the absence of tapasin or beta(2)-microglobulin, whereas W6/32-reactive dimers are not detected. Efficient formation of W6/32-reactive dimers appears to depend on the transporter associated with antigen processing, tapasin, and beta(2)-microglobulin. The unpaired Cys(67) and residues at the base of the B pocket that dramatically impair HLA-B27 HC folding are critical for the formation of HC10-reactive ER dimers. Although certain other alleles also form dimers late in the assembly pathway, ER dimerization of HLA-B27 may be unique. These results demonstrate that residues comprising the HLA-B27 B pocket result in aberrant HC folding and disulfide bond formation, and thus confer unusual properties on this molecule that are unrelated to peptide selection per se, yet may be important in disease pathogenesis.
The strong association of the HLA class 1 allele HLA B27 with ankylosing spondylitis (AS) has been recognized for over 25 yr, however the pathogenic mechanism linking HLA B27 with AS and other spondyloarthropathies remains a mystery. We now know that the principal natural function of HLA B27 is an immunologic one, namely to bind antigenic peptides and then present them to T lymphocytes. I have shown that HLA B27 functions as an excellent antigen-presenting molecule in both spondyloarthropathy patients and healthy individuals. A working molecular model of how T cells recognize HLA B27 has been generated and tested. Evidence that T cells have a role in spondyloarthritis has also been found. First, expanded populations of T lymphocytes were found in both the blood and synovial fluid of patients with reactive arthritis (ReA). Secondly, a strong cytotoxic T-cell response to an HLA B27-restricted peptide epitope from Chlamydia trachomatis was found in a patient with ReA. This peptide, derived from a bacterium known to trigger ReA, is thus a candidate 'arthritogenic' peptide. We have also found evidence that HLA B27 has an unusual cell biology compared with other HLA molecules. HLA B27 demonstrates an unusual ability to form heavy chain homodimers in vitro. Dimerization is dependent upon disulphide bonding through an unpaired cysteine at position 67. Remarkably these dimers lack beta2 microglobulin, previously thought to be an essential component of all mature MHC class 1 molecules. HLA B27 homodimer formation has also been demonstrated in certain cell lines in vivo, and preliminary data suggest that significant numbers of T cells from patients with spondyloarthropathy express a ligand for HLA B27 homodimers. These findings have extended our understanding of the beneficial immunologic function of HLA B27, and have also led us to propose the testable new hypothesis that HLA B27 heavy chain dimerization may be involved in the pathogenesis of spondyloarthritis.
The human HLA-B27 class I molecule exhibits a strong association with the inflammatory arthritic disorder ankylosing spondylitis
and other related arthropathies. Major histocompatibility complex class I heavy chains normally associate with β2-microglobulin and peptide in the endoplasmic reticulum before transit to the cell surface. However, an unusual characteristic
of HLA-B27 is its ability to form heavy chain homodimers through an unpaired cysteine at position 67 in the peptide groove.
Homodimers have previously been detected within the ER and at the cell surface, but their mechanism of formation and role
in disease remain undefined. Here we demonstrate, in the rat C58 thymoma cell line and in human HeLa cells transfected with
HLA-B27, that homodimer formation involves not only cysteine at position 67 but also the conserved structural cysteine at
position 164. We also show that homodimer formation can be induced in the non-disease-associated HLA class I allele HLA-A2
by slowing its assembly rate by incubation of cells at 26 °C, suggesting that homodimer formation in the endoplasmic reticulum
may occur as a result of the slower folding kinetics of HLA-B27. Finally, we report an association between unfolded HLA-B27
molecules and immunoglobulin-binding protein at the cell surface.
We have determined the sequence of a 4-Mb interval on rat chromosome 20p12 that encompasses the rat major histocompatibility complex (MHC). This is the first report of a finished sequence for a segment of the rat genome and constitutes one of the largest contiguous sequences thus far for rodent genomes in general. The rat MHC is, next to the human MHC, the second mammalian MHC sequenced to completion. Our analysis has resulted in the identification of at least 220 genes located within the sequenced interval. Although gene content and order are well conserved in the class II and class III gene intervals as well as the framework gene regions, profound rat-specific features were encountered within the class I gene regions, in comparison to human and mouse. Class I region-associated differences were found both at the structural level, the number, and organization of class I genes and gene families, and, in a more global context, in the way that evolution worked to shape the present-day rat MHC.
To define the genetic basis of susceptibility to ankylosing spondylitis (AS), especially non-major histocompatibility complex (MHC) genes.
The study group comprised 244 affected sibling pairs from 180 pedigrees of primarily European ancestry. Sibling pairs were concordant for AS by the modified New York criteria and had available sacroiliac radiographs. The subjects were genotyped for 400 markers in ABI PRISM linkage map MD-10 and for 17 additional markers on chromosomes 6p, 6q, and 11q (including HLA-B, DRB1, DQA1, DQB1, and DPB1 alleles). Two-point and multipoint nonparametric linkage (NPL) analyses were conducted using the NPL statistic and 1-parameter allele-sharing model logarithm of odds (LOD) scores, calculated using the Allele-Sharing Model (ASM) computer program.
Linkage of the MHC region was supported by both 2-point and multipoint analyses, with the strongest peak (45.90 cM) in the MHC at the HLA-DRB1 locus (NPL score 8.720, ASM LOD score 20.49; P = 6.8 x 10(-20) for 2-point analysis). A second region was found to have positive linkage at the q arm of chromosome 6 (D6S441) in 2-point analysis; this was supported by a 39.13-cM region (135.58-174.71 cM) in multipoint analysis, with the smallest P value (4.2 x 10(-3)) at 166.39 cM. A third region was found on chromosome 11q, with the strongest evidence for linkage for D11S4094 at 123 cM (NPL score 2.235, ASM LOD score 1.939) and, on transmission disequilibrium test analysis, D11S4090 at 105.74 cM (P = 6.2 x 10(-5)). Linkage in this area was supported by multipoint analysis, spanning 22.19 cM continuously from 101.68 cM to 123.87 cM, with the strongest peak at 112.33 cM (P = 0.014); this was confirmed by subsequent fine mapping studies.
Thus, this genome-wide scan implicates, in addition to the MHC, regions outside the MHC in AS susceptibility, especially on chromosomes 6q and 11q.
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.
Spondyloarthritis tends to cluster in families and, to a great extent, is associated with human leukocyte antigen (HLA) B27.
In fact, the population frequency of spondyloarthritis in most groups is proportional to that of HLA-B27. But the frequency
of HLA-B27 in the population-at-large far exceeds that of spondyloarthritis, suggesting other genetic factors also are operative.
Other major histocompatibility complex genes have been implicated, especially HLA-DR, though linkage to HLA-B27 confounds
the analysis of this in many studies. Genome-wide scans have implicated regions on chromosomes 2q, 6p, 6q, 10q, 11q, 16q,
17q, and 19q in ankylosing spondylitis, on 4, 6p, and 17q in psoriasis, and on 7q and 16q in inflammatory bowel disease. The
search for non-major histocompatibility complex candidate genes has been complicated by inadequate power, because of the small
effect they exert on overall disease susceptibility, although recent studies are revealing promising candidates that must
be confirmed by other groups.
To explain the strong association between HLA-B27 and ankylosing spondylitis, we suggest that the release of β2-microglobulin (β2m) from a subpopulation of cell surface-expressed HLA-B27 molecules leads to β2m-deposition within synovia and to the initiation of an inflammatory process, which culminates in destructive spondyloarthropathy.
Modern technological innovations have advanced our understanding of the genetic basis of spondyloarthritis. In ankylosing spondylitis (AS), where the major histocompatibility complex (MHC) accounts for nearly half of the predisposition, most comes from HLA-B27, for which 65 subtypes are now recognised, although other genes are also at work including HLA-B60 (B*40:01). Other genes have been identified, including those involved in peptide editing for loading onto class I MHC molecules (ERAP1) and cytokine genes such as interleukin 1A (IL-1A) and those involved in the Th17 network (IL-23R, an association seen primarily in Caucasians) and others. In acute anterior uveitis, these associations are also seen as well as a region on chromosome 9p and genes whose confirmation is under way. Psoriasis and psoriatic arthritis fall into this disease spectrum, with the largest region of susceptibility coming from the MHC (most likely HLA-C, ie, C*06:02 although additional influences are also being implicated), and most of the other genetic susceptibility coming from genes involved in cytokine production, specifically genes in the Th17 pathway (IL-12B, IL-23A and IL-23R, the latter, like in AS, not seen in Asians), genes in the nuclear factor κB pathway (TNFAIP3 and TNIP1) and genes in the Th2 pathway (IL-4 and IL-13). Given that more than half of patients with AS have evidence on colonoscopy of at least occult inflammatory bowel disease (IBD), it is not surprising that shared genetic influences are operative. In IBD, genes important in the innate immune response (NOD2), autophagy (ATG6L1) and regulation of the IL-23 pathway (IL-23R) play a role in disease susceptibility.
The levels of immunosuppressive activity and the presence of MHC antigens and leukocytes were studied in the immature and the sexually mature rat testis. The immunosuppressive activities were measured from high-molecular weight (greater than 5 kDa) fractions of testis extracts using the protectin bioassay. The presence of MHC antigens and leucocytes was studied using the indirect immunoperoxidase method. In the immature rats, clusters of class I MHC antigen positive cells and a few cells expressing class II MHC antigen were present in the testicular interstitium. In the sexually mature rats, all the cells were MHC I+, and MHC II+ cells were numerous in the testicular interstitium. The seminiferous epithelium was MHC-negative in both the immature and the sexually mature testis. W3/25+ leukocytes were present in the interstitium and the tubular wall in both the immature and the sexually mature rat testis, but not in the seminiferous epithelium at any age. At 20-30 days of age, the testicular extracts were neutral or slightly stimulated 3H-TdR incorporation into peripheral blood lymphocytes, but at 44-60 days of age they inhibited lymphocyte proliferation significantly. In gel filtration, a peak of immunosuppressive activity was observed at approximately 400 kDa (protectin A) in both 20- and 60-days-old rat testes. A smaller peak was present at approximately 200 kDa in both age groups. This study shows that the testicular immunoregulatory microenvironment is different in the immature and the sexually mature rats. This may be important in such age-dependent human diseases as mumps orchitis and the testicular relapses of acute lymphoblastic leukemia.
Humans who have inherited the human class I major histocompatibility allele HLA-B27 have a markedly increased risk of developing the multi-organ system diseases termed spondyloarthropathies. To investigate the role of B27 in these disorders, we introduced the B27 and human beta 2-microglobulin genes into rats, a species known to be quite susceptible to experimentally induced inflammatory disease. Rats from one transgenic line spontaneously developed inflammatory disease involving the gastrointestinal tract, peripheral and vertebral joints, male genital tract, skin, nails, and heart. This pattern of organ system involvement showed a striking resemblance to the B27-associated human disorders. These results establish that B27 plays a central role in the pathogenesis of the multi-organ system processes of the spondyloarthropathies. Elucidation of the role of B27 should be facilitated by this transgenic model.
The expression and function of HLA antigens in mice single transgenic for HLA-B27.2 (sTGM-B27.2) or double transgenic (dTGM) for HLA-B27.2 and human beta 2-microglobulin (h beta 2m) were compared. B27.2 could be well detected on the cell membrane of lymphocytes of sTGM. However, the expression in sTGM was much lower than in dTGM mice. Nevertheless, also in sTGM mice, the B27-transgene product possessed all functional properties of a class I HLA molecule. This was shown by the recognition and induction of antibodies and cytotoxic T cells, by the induction of "allo"-immunity, including skin graft rejection, and by the ability to present viral antigens. In dTGM, the expression of B27 on peripheral blood lymphocytes, spleen and lymphnode cells was comparable to H-2. However, on thymocytes, a relatively lower expression of HLA than H-2 was observed. This low expression of B27 on thymocytes is in concert with the observation that B27 is expressed only in the medulla of the thymus and not detectable in the cortex.
The human genomic clone pb2m13 contains a functional beta 2-microglobulin (B2m) gene, which upon transfection is readily expressed in murine fibroblasts. Here we report the nucleotide sequence of the human beta 2m gene and of a nearly full length cDNA clone. A comparison with the murine beta 2m gene reveals that exon/intron boundaries are absolutely conserved. In the protein-coding regions the similarity is 70%. As far as intron sequences of the murine beta 2m gene are available, no significant similarity between human and murine genes is observed. The transcriptional start site of the human beta 2m gene was determined by S1 mapping, and comparison with the nearly full length cDNA clone now defines the transcriptional unit of the beta 2m gene. In the 5' region of the gene strong clustering of the usually underrepresented CpG dinucleotide is found resembling a similar overrepresentation in the 5' regions of the major histocompatibility complex class I genes.
Two glucose-regulated proteins, GRP78 and GRP94, are major constituents of the endoplasmic reticulum (ER) of mammalian cells. These proteins are synthesized constitutively in detectable amounts under normal growth conditions; they can also be induced under a variety of conditions of stress including glucose starvation and treatment with drugs that inhibit cellular glycosylation, with calcium ionophores or with amino-acid analogues. Unlike the closely-related heat shock protein (HSP) family, the GRPs are not induced significantly by high temperature. Recently, GRP78 has been identified as the immunoglobulin heavy chain binding protein (BiP) (ref. 5 and Y.K. et al., in preparation) which binds transiently to a variety of nascent, wild-type secretory and transmembrane proteins and permanently to malfolded proteins that accumulate within the ER. We have tested the hypothesis that the presence of malfolded proteins may be the primary signal for induction of GRPs by expressing wild-type and mutant forms of influenza virus haemagglutinin (HA) in simian cells. Only malfolded HAs, whose transport from the ER is blocked, induced the synthesis of GRPs 78 and 94. Additional evidence is presented that malfolding per se, rather than abnormal glycosylation, is the proximal inducer of this family of stress proteins.
During the last 50 years there has been an obvious change in the relationship between Reiter's syndrome and spondarthritis, probably due to the introduction of antibiotics. Postgonorrhoeic prostatovesiculitis was formerly common: Romanus' spondylitics in the 1940s had a history of gonorrhoea in 35% of cases and 50% of my patients with chronic uro-arthritis in the 1950s had had gonorrhoea. Urogenital syndromes nowadays rarely develop into ankylosing spondylitis; on the other hand, sacroiliitis is still a rather common late sequela, especially in females, however often asymptomatic. The HLA-B27 tissue type is much less frequent in the urogenital syndromes than in ankylosing spondylitis. Accordingly one may postulate that patients with HLA-B27 negative sacroiliitis run a small risk that the disease will progress to ankylosing spondylitis.
146 men with rheumatoid factor-negative (sero-negative) arthritis, i.e., 97 patients with ankylosing spondylitis, 36 patients with Reiter's syndrome, and 13 patients with reactive arthritis, were examined for infections of the urogenital tract by following recently established criteria.
74 patients (50.7%) had infections of the male adnexes: 3 patients suffered from balanitis, 14 patients from urethritis, 49 patients from prostatitis, 1 patient from epididymitis, and 7 patients from urinary tract infection. Balanitis and urethritis were almost exclusively associated with Reiter's syndrome. In 37 of 97 patients with ankylosing spondylitis, a urogenital tract infection, mainly a prostatitis (31 patients), was detected. The microorganisms isolated most frequently from patients suffering from urethritis and prostatitis, were Chlamydia trachomatis and Ureaplasma urealyticum.
Ethylenediaminetetraacetic acid (EDTA) solution is used to decalcify bone specimens for histological examination. Sodium hydroxide (NaOH) has been used to dissolve EDTA and to bring EDTA solutions to neutral pH. This solution, however, requires several weeks to decalcify bone specimens. We investigated a new decalcification fluid using concentrated ammonium hydroxide (NH4OH) to dissolve EDTA and to adjust the pH to neutral. Decalcification was performed using a magnetic stirrer with and without vacuum, or with a sonic cleaner. Decalcification end point was confirmed using both the weight loss and X-ray methods. After decalcification, specimens were processed through paraffin and sections were stained with hematoxylin and eosin. Decalcification employing NH4OH required an average of six days. Light microscopy indicated good retention of cellular detail.
Ankylosing spondylitis (AS) and spondylarthropathies (SAP), proposed immune diseases, present sexual preponderance: men are mostly affected. It is known that androgens are decreased in systemic immune disorders. We have investigated two aspects: gonadal--with 13 parameters, and entheso-osteoarthritic--with 10 parameters, by an original methodological semiquantitative analysis. All the parameters were divided into five degrees; each degree was pointed from 0 to 5, and the total and final scores were obtained. In this way differences and correlations could be performed between all 23 parameters. We have studied 30 SAP patients in inflammatory attack, 4 SAP patients out of the inflammatory attack and 16 control subjects; all were men and in fertile age. Between the gonadal status of SAP patients vs the control group there is a significant difference concerning: the degree of testosterone (1.81 vs 0.22, p < 0.005) and testes trophicity (1.5 vs 0.35, p < 0.01); marked differences have been recorded for integrative scores: total (12.18 vs 6.21, p < 0.02), final (1.07 vs 0.57, p < 0.01) and general degree score (1.7 vs 1.18, p < 0.01). Testosteronemia has been different, too: 7.38 vs 23.25 nmol/l, p < 0.01. Between SAP patients in and out of the inflammatory attack there are no significant differences. A significant positive correlation between gonadal axis status degree and entheso-osteoarthritic status degree has been obtained by Spearman rank test: r = 0.41, p < 0.05. Our new methodological analysis allows to change qualitative criteria in mathematical used quantitative data, for performing correlations between so different fields: rheumatological and endocrinological. SAP patients (in inflammatory attack, and out of the inflammatory attack) have a certain degree of hypogonadism, which does not represent a specific disease but suggests a specific spondylarthropathic background.
To study prospectively the clinical evolution of different forms of spondyloarthropathy (SpA) in relation to the evolution of gut histology in consecutive ileocolonoscopic biopsy specimens.
Ileocolonoscopy was performed in 49 patients with SpA (34 men, 15 women). They also underwent clinical, laboratory, and radiological examinations. Two to 9 years later, a 2nd and sometimes a 3rd or 4th ileocolonoscopy was performed, and the other examinations were repeated.
At first ileocolonoscopy, 34 patients (69%) showed inflammatory gut lesions. At the 2nd ileocolonoscopy, 16 patients (32%) were in clinical remission; none were found to have inflammatory gut lesions. Of the 33 patients with persistent locomotor inflammation, 14 had persistent inflammatory gut lesions, predominantly the chronic type. Of these 14 patients, 6 had developed inflammatory bowel disease (IBD). None of the 15 patients with an initially normal gut histology had gut inflammation at 2nd examination. Of the 9 with initially acute lesions, 3 developed chronic lesions (1 Crohn's disease). Initial chronic lesions in 25 patients persisted in 9, of whom 5 had developed IBD. Seven of the 19 patients with non-SpA ankylosing spondylitis (non-AS-SpA) developed ankylosing spondylitis (AS); all had initially presented inflammatory gut lesions, which persisted at 2nd examination. In the 11 patients with more than 2 consecutive ileocolonoscopies, clinical remission was always associated with normal gut histology, and flares of the joint disease were related temporally to the reappearance of gut inflammation.
This study demonstrates the close relationship between gut and locomotor inflammation in SpA. Clinical remission was always associated with normal gut histology, whereas active locomotor inflammation was usually associated with the presence of gut inflammation. Absence of gut inflammation in the SpA is a good prognostic indicator, since gut inflammation or IBD never develops in these patients. Evolution of non-AS-SpA to full blown AS or of uncomplicated SpA to a form of IBD was always associated with gut inflammation at disease onset.
To study prospectively the clinical evolution of different forms of spondyloarthropathy (SpA) (excluding inflammatory bowel disease, IBD): reactive arthritis, undifferentiated SpA, and ankylosing spondylitis (AS).
Ileocolonoscopy was performed on 217 patients with SpA (149 men, 68 women). They also underwent clinical, laboratory, and radiological examinations. Two to 9 years later, 123 patients (84 men, 39 women) who had been regularly monitored were reviewed and given the same examinations. For the remaining 94 patients clinical data were obtained by telephone.
At the time of clinical review, 53 (43%) of the regularly monitored patients were in clinical remission. The remission rate was higher in patients with non-ankylosing spondylitis SpA (non-As-SpA) than in patients with AS (19%). Fourteen patients with non-AS-SpA had developed AS; 4 of them also had IBD. IBD was also found in 4 patients with AS and in 3 patients from the telephone group. The prevalence of HLA-B27 was significantly higher in all SpA subgroups, while HLA-BW62 was elevated in the undifferentiated SpA. At review, HLA-B27 was significantly more prevalent in patients with persistent locomotor inflammation compared to patients in clinical remission, while HLA-BW62 was predominant in the latter group.
Patients with SpA, especially those with non-AS-SpA, have a good longterm prognosis. However, patients with non-AS-SpA may develop AS. Six percent of the patients with SpA in whom manifestations of IBD are absent will develop this disease. This confirms the hypothesis that some of these patients with SpA initially have a form of subclinical Crohn's disease, of which locomotor inflammation is the only clinical expression. HLA-B27 positivity predisposes to a more severe course of locomotor inflammation, while HLA-BW62 has a protective effect but is associated with gut inflammation.
Human major histocompatibility complex class I allele HLA-B27 is associated with a group of diseases called spondyloarthropathies. In reactive arthritis (ReA), the disease is triggered by certain infections, e.g. gastroenteritis caused by Salmonella. The host/microbe interaction is abnormal in susceptible individuals leading to inefficient elimination of arthritis-triggering bacteria, fragments of them, or both, after the initial infection. Using transfected human monocytic U937 cell lines, we demonstrate that the expression of the HLA-B27 antigen does not influence the uptake of S. enteritidis into U937 cells in vitro. Interestingly, HLA-B27 remarkably impairs the elimination of S. enteritidis within the HLA-B27 transfected U937 cells. The impaired elimination of ReA-triggering microbes by HLA-B27+ monocytes may offer an explanation for the persistence of ReA-triggering microbes in susceptible HLA-B27+ individuals. This modulation of the host/microbe interaction by HLA-B27 may have an important role in the pathogenesis of ReA.
One hundred and thirty-four male and 32 female patients with ankylosing spondylitis and 33 women with pure ileitis terminalis Crohn were examined. The study protocol included a medical-rheumatological examination and thorough investigation for genitourinary infection. Urethroadnexitis was found in 37/134 male patients (2 patients suffered from balanitis, 17 patients from urethritis, 18 patients from prostatitis, and 2 patients from epididymitis), 15/32 female patients (11 of them had urethritis and in 4 cases urethritis associated with vaginitis) and 5/33 women with ileitis terminalis (every case with urethritis). The microorganism isolated most frequently from patients with genitourinary infection was Chlamydia trachomatis. The majority of patients with genitourinary infection were HLA-B27 positive. Nevertheless, the following conclusions can be reached: (1) evidence of Chlamydia trachomatis infection is frequent in male and female patients with ankylosing spondylitis, (2) patients with genitourinary infection tend to have HLA-B27, and (3) furthermore, presence of genitourinary infection was not significantly associated with chronic illness.
Unlabelled:
A spontaneous inflammatory disease in rats transgenic for HLA-B27 resembles the B27-associated human spondyloarthropathies. Colitis and arthritis, the two most important features, require T cells, gut bacteria, and high expression of B27 in bone marrow-derived cells. Control rats with HLA-B7 remain healthy. Most rats with HLA-Cw6 (associated with psoriasis vulgaris) remain healthy; a minority develop mild and transient disease. Rats with a mutant B27 with a Cys67-->Ser substitution resemble wild-type B27 transgenics, but with a lower prevalence of arthritis. A similar phenotype is seen in B27 rats co-expressing a viral peptide that binds B27. Disease-prone LEW but not F344 B27 rats develop high serum IgA levels concurrent with disease progression. Colitis is associated with high interferon-gamma, arthritis with high interleukin-6. Disease is similar in B27 LEW, F344, and PVG rats, but the DA background is protective.
Conclusions:
The spondyloarthropathy-like disease in rats is specific for HLA-B27 but does not require Cys67. Arthritis but not colitis is particularly sensitive to B27 peptide-binding specificity. Genetic background exerts a strong influence, but some phenotypic differences exist between permissive strains that do not influence disease susceptibility. The data favor a role for B27 peptide presentation in arthritis, but other mechanisms to explain the role of B27 have not been excluded.
This study quantifies the effect of afterload and preload changes and of temperature on interbeat interval variability of the intact isolated heart. Ventricular pressure pulse records were obtained from isolated working rat hearts. The variability of interbeat intervals (BIs) was quantified by C90, the central 90% range of the BIs during 10 min periods; predominant frequencies were searched for by power spectral analysis. At 37 degrees C the BI lengths oscillated pseudo-randomly with BI variability C90< or =4 ms. Alternating signs of consecutive BI differences were predominant, and no peaks. were seen in the power spectra. Changes in end-diastolic and aortic pressure had little effect. From 37 degrees C down to 27 degrees C the variability increased about sevenfold, run phase length became randomly distributed, and individual, time-variant peaks occurred in the power spectra. BI variability vanished during atrial pacing. We conclude that: (1) effective mutual synchronization with minimal fluctuation happens within the sino-atrial node of intact rat hearts at body temperature, and synchronization is not affected even by extreme changes in pre- and afterload, (2) the sino-atrial node is the sole source of BI variability in the intact isolated rat heart, (3) low temperature hampers this functional organization which can be reestablished by sinus node accelerating agents (isoprenaline, theophylline), (4) decreasing frequency by N6-Cyclopentyladenosine at normothermia also increases BI variability but less pronouncedly than hypothermia does.
To determine the overall prevalence of spondyloarthropathy (SpA) among patients with inflammatory bowel disease (IBD) [Crohn's disease (CD) and ulcerative colitis (UC)].
One hundred three consecutive patients with IBD from a gastroenterology unit were questioned and examined for SpA symptoms. Patients previously diagnosed with SpA were excluded. All patients were questioned and examined for SpA symptoms such as inflammatory back pain, joint swelling, enthesitis, and psoriasis or a specific family history. Radiographs were taken of all sacroiliac joints. HLA loci A, B, C, and DR were determined in all patients.
Thirty-nine percent of the patients with IBD had clinical articular manifestations: 30% had inflammatory back pain, 10% had synovitis, and 7% had a peripheral enthesopathy. The majority (90%) of patients with rheumatic complaints fulfilled the classification criteria for SpA and 10% fulfilled the criteria for ankylosing spondylitis. Asymptomatic sacroiliitis was found in an additional 18% of the patients. Moreover, sacroiliitis, symptomatic or asymptomatic, was related to the disease duration. HLA-B27 conferred an additional risk for inflammatory low back pain in patients with IBD.
Articular involvement in IBD can be classified as SpA. The appearance of SpA occurs irrespective of the extent of the bowel disease. Moreover, asymptomatic sacroiliac involvement is a common manifestation in IBD and it is related to disease duration, suggesting evidence for a related pathogenic mechanism.
Spondyloarthropathies (SpA) are a group of related disorders with common clinical and genetic characteristics. The prototype disease in this group is ankylosing spondylitis; other entities include reactive arthritis, psoriatic arthritis, and arthritis in patients with inflammatory bowel disease. Over recent years, there has been a special interest in the relation between spondylitis/synovitis and gut inflammation in patients with SpA. Two thirds of patients with undifferentiated SpA show histologic signs of gut inflammation, and a fraction of these patients go on to develop clinically overt Crohn's disease. In this review, the authors will focus on 1) the growing evidence that has been provided that gut inflammation in SpA is immunologically related to Crohn's disease, based on the molecular characterization of the inflammation (lymphocyte homing markers and ligands, T cell cytokines, macrophage markers, and serology); and 2) on the therapeutic implications resulting from this concept. The recent introduction and positioning of anti-tumor necrosis factor-alpha therapy in patients with ankylosing spondylitis and other types of SpA is, in large part, based on this concept.
Previous work suggested that expanded CD8+ T-cell clones in the synovial fluid (SF) of HLA-B27+ patients with reactive arthritis (ReA) preferentially use the T-cell receptor variable region (TCRBV) 1, similar CDR3 sequences, and joining region (BJ) 2S3. To determine the range of conservation and disease-specificity of CDR3-sequences, we analyzed the TCRBV1-J2S3 repertoire from 33 healthy HLA-B27+ individuals, patients with various types of spondyloarthropathies (SpA), and with rheumatoid arthritis (RA) by CDR3-spectratyping. After collection and database submission of all available TCRB-CDR3 from HLA-B27-restricted or SpA-derived T cells, we systematically screened the entire human sequence database for sequences similar to the B27/SpA-related CDR3. Spectratyping revealed expanded T cell clones using conserved TCRBV1J2S3 in the SF from 5/6 of the patients with acute ReA but not among the controls. In database searches, 50 HLA-B27 or SpA-related CDR3-sequences generated similar clusters of matched sequences, and matched reciprocally. Identical or closely related sequences were identified in 15 different individuals and a canonical ReA-associated TCRB was defined [BV1-CASSVG(V/I/L)(Y/F)STDTQYF-J2S3]. All but one patient-derived conserved sequences originated from acute stage ReA-patients, and were not present among approximately 3800 other human TCRB sequences in the database. Five of the conserved sequences originated from T cell clones that recognized uninfected cells in an HLA-B27-restricted fashion, implying a role of HLA-B27-restricted CD8+ T cells specific for a ubiquitous self- or cross-reactive microbial determinant in the early phase of ReA. Related sequences were independently identified in four different laboratories. The consensus TCRB motif could be a helpful diagnostic marker in HLA-B27-associated 'undifferentiated arthritis'.
During the assembly of MHC class I molecules with peptide, a series of transient interactions are made with endoplasmic reticulum-resident chaperones and MHC class I-specific accessory molecules. These interactions culminate in the trafficking of MHC class I molecules to the cell surface and presentation of peptide to CD8+ T lymphocytes. Recent studies have revealed just how important these early interactions are, and how they influence the quality control of assembly and the optimisation of peptide–ligand binding.
To explain the strong association between HLA-B27 and ankylosing spondylitis, we suggest that the release of beta(2)-microglobulin (beta(2)m) from a subpopulation of cell surface-expressed HLA-B27 molecules leads to beta(2)m-deposition within synovia and to the initiation of an inflammatory process, which culminates in destructive spondyloarthropathy.
Enteropathic arthritis is a form of arthritis associated with the chronic inflammatory bowel diseases, ulcerative colitis, and Crohn's disease. This form of arthritis is classified as one of the group of seronegative spondyloarthropathies, which also includes psoriatic arthritis, reactive arthritis, and idiopathic ankylosing spondylitis. Joint involvement also occurs with other gastrointestinal diseases such as Whipple's disease, celiac disease, and following intestinal bypass surgery for morbid obesity. In these conditions, abnormal bowel permeability and immunologic and genetic influences are probably involved in the pathogenesis of the joint disease, although the exact mechanisms remain uncertain.
As an MHC class I protein, the disease association of HLA-B27 with inflammatory arthritis has been widely assumed to imply a role for the T cell antigen receptor (TCR) in disease. However, in addition to their classical antigen-presenting role, HLA class I proteins are recognised by members of the killer immunoglobulin receptor (KIR) and leukocyte immunoglobulin-like receptor (LILR/ILT/LIR) families. Unusual properties of HLA-B27 include an ability of free heavy chains (FHC) to reach the cell surface in the absence of beta2m and to maintain their peptide-binding groove in vitro. This review describes immunomodulatory receptors that recognise HLA class I, and the recognition of HLA-B27 in both the classical beta2m-associated and beta2m-independent forms by members of the KIR and LILR families. Alternative recognition of different forms of HLA-B27 by leukocyte receptors could influence the function of cells from both innate and adaptive immune systems, and may indicate a role for various leukocyte populations in HLA-B27-associated inflammatory disease.
In the thirty years since the initial discovery of a striking association between HLA-B27 and susceptibility to ankylosing spondylitis, numerous hypotheses have been proposed to explain the role of this molecule in the pathogenesis of spondyloarthropathies. In the past few years the focus has shifted from one centered largely on the physiological peptide-presenting function of HLA-B27, to include ideas based on aberrant aspects of its immunobiology. This has been driven in part by results from animal models of HLA-B27-associated disease where CD8+ T cells do not appear to be playing a major role in pathogenesis. In addition, the HLA-B27 heavy chain is unusual in that it has a tendency to misfold in the endoplasmic reticulum and to form disulfide linked heavy chain dimers that can be expressed on the cell surface. Although the data suggest misfolding and cell surface dimerization are fundamentally different processes, it appears that certain structural features of the heavy chain are common to both. Potential links between these aberrant characteristics of HLA-B27 and inflammatory disease are discussed in this and other reviews in this issue. Herein we consider how protein misfolding affects cell function through the activation of an 'unfolded protein response' and/or an 'ER overload response', and the potential impact on the immune system. Despite significant advances in the treatment of spondyloarthropathies over the past few years, a better understanding of pathogenesis is likely to improve outcome by identifying ways to provide greater and more sustained clinical responses.
To test the hypothesis that HLA-B27 predisposes to disease by forming disulfide-linked homodimers, we examined rats transgenic for HLA-B27, mutant Cys(67)Ser HLA-B27, or HLA-B7. In splenic Con A blasts from high transgene copy B27 lines that develop inflammatory disease, the anti-H chain mAb HC10 precipitated four bands of molecular mass 78-105 kDa and additional higher molecular mass material, seen by nonreducing SDS-PAGE. Upon reduction, all except one 78-kDa band resolved to 44 kDa, the size of the H chain monomer. The 78-kDa band was found to be BiP/Grp78, and the other high molecular mass material was identified as B27 H chain. Analysis of a disease-resistant low copy B27 line showed qualitatively similar high molecular mass bands that were less abundant relative to H chain monomer. Disease-prone rats with a Cys(67)Ser B27 mutant showed B27 H chain bands at 95 and 115 kDa and a BiP band at 78 kDa, whereas only scant high molecular mass bands were found in cells from control HLA-B7 rats. (125)I-surface labeled B27 oligomers were immunoprecipitated with HC10, but not with a mAb to folded B27-beta(2)-microglobulin-peptide complexes. Immunoprecipitation of BiP with anti-BiP Abs coprecipitated B27 H chain multimers. Folding and maturation of B27 were slow compared with B7. These data indicate that disulfide-linked intracellular H chain complexes are more prone to form and bind BiP in disease-prone wild-type B27 and B27-C67S rats than in disease-resistant HLA-B7 rats. The data support the hypothesis that accumulation of misfolded B27 participates in the pathogenesis of B27-associated disease.
To reveal the cause of the impaired elimination of Salmonella enteritidis in HLA-B27-transfected human monocytic cells and to study whether the B pocket of HLA-B27 contributes to these modulatory effects.
Stable U937 cell transfectants expressing HLA-A2, B27, or different forms of B27 with amino acid substitutions in the B pocket were prepared. Mock-transfected cells were prepared using the antibiotic resistance vector (pSV2neo) alone. Cells were differentiated, infected with S enteritidis, and the number of live intracellular S enteritidis organisms was determined using the colony-forming unit method. To visualize intracellular S enteritidis, the bacteria were transformed with green fluorescent protein (GFP), and studied by confocal microscopy.
Cells expressing wild-type HLA-B27 were more permissive of intracellular replication of S enteritidis compared with mock-transfected or A2-transfected controls. Cells expressing B27 with an altered B pocket composition having either 6 amino acid substitutions (B27.A2B; substitutions H9F, T24A, E45M, I66K, C67V, and K70H) or a single substitution (B27.E45M) were no longer permissive of S enteritidis replication. In contrast, cells expressing B27 with the single substitution of F for H at position 9 (B27.H9F) retained their permissiveness. Studies using GFP-transformed S enteritidis confirmed that the increase in the amount of intracellular bacteria in B27-expressing cells was due to replication of the bacteria.
Our data indicate that HLA-B27 expression modulates the host-microbe interaction that results in an impaired capacity of monocytes to resist intracellular replication of S enteritidis. The phenotype is dependent on glutamic acid at position 45 in the B pocket and, thus, may be due to properties of the B27 heavy chain that are related to this residue. The ability of HLA-B27 to confer susceptibility to Salmonella-triggered reactive arthritis may occur, at least in part, through these modulatory effects.