Objective. 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, ␤ 2 -microglobulin (␤ 2 m), 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.Methods. Rats with high transgene copy numbers of HLA-B27 heavy chain together with human ␤ 2 m (Hu␤ 2 m) 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/Hu␤ 2 mtransgenic rat lines with a healthy line, 283-2, carrying only the Hu␤ 2 m 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 Hu␤ 2 m was determined by flow cytometry. Disease manifestations were identified by clinical observation, histology, and measurement of cytokine mRNA.Results. The extra Hu␤ 2 m from the 283-2 line significantly reduced B27 misfolding and UPR triggering. Unexpectedly, however, F 1 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.Conclusion. 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.The spondylarthritides are a group of inflammatory rheumatic diseases characterized by axial and peripheral arthropathy and a variety of extraarticular lesions (1). In the prototype of these disorders, ankylosing spondylitis (AS), there is inflammation in the spinal and sacroiliac joints and in ligamentous attachments that, in severe cases, leads to bony fusion. The pathogenesis of spondylarthritis is poorly understood. Two factors strongly associated with these conditions are intestinal inflammation and the major histocompatibility complex (MHC) class I gene HLA-B27.With regard to the association with intestinal inflammation, the prevalence of inflammatory bowel disease (IBD) (both Crohn's disease and ulcerative colitis) is increased in individuals with AS. An even higher prevalence of IBD is found in individuals with other forms of spondylarthritis (2), and endoscopic studies with biopsy have shown a prevalence of microscopic inflammatory bowel lesions in 60-70% of patients

To test the hypothesis that HLA-B27 predisposes to disease by forming disulfide-linked homodimers, we examined rats transgenic for HLA-B27, mutant Cys67Ser 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 Cys67Ser 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. 125I-surface labeled B27 oligomers were immunoprecipitated with HC10, but not with a mAb to folded B27-β2-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.

The class I MHC allele HLA-B27 is highly associated with the human spondyloarthropathies, but the basis for this association remains poorly understood. Transgenic rats with high expression of HLA-B27 develop a multisystem inflammatory disease that includes arthritis and colitis. To investigate whether CD8αβ T cells are needed in this disease, we depleted these cells in B27 transgenic rats before the onset of disease by adult thymectomy plus short-term anti-CD8α mAb treatment. This treatment induced profound, sustained depletion of CD8αβ T cells, but failed to suppress either colitis or arthritis. To address the role of CD8α+β− cells, we studied four additional groups of B27 transgenic rats treated with: 1) continuous anti-CD8α mAb, 2) continuous isotype-matched control mAb, 3) the thymectomy/pulse anti-CD8α regimen, or 4) no treatment. Arthritis occurred in ∼40% of each group, but was most significantly reduced in severity in the anti-CD8α-treated group. In addition to CD8αβ T cells, two sizeable CD8α+β− non-T cell populations were also reduced by the anti-CD8α treatment: 1) NK cells, and 2) a CD4+CD8+CD11b/c+CD161a+CD172a+ monocyte population that became expanded in diseased B27 transgenic rats. These data indicate that HLA-B27-retricted CD8+ T cells are unlikely to serve as effector cells in the transgenic rat model of HLA-B27-associated disease, in opposition to a commonly invoked hypothesis concerning the role of B27 in the spondyloarthropathies. The data also suggest that one or more populations of CD8α+β− non-T cells may play a role in the arthritis that occurs in these rats.

Objective. HLA-B27 predisposes to spondylarthritis by an unknown mechanism. A logical candidate mechanism is through recognition of B27 by CD8؉ T cells. The purpose of this study was to examine the effects of a lack of CD8 on the spondylarthritis that develops in B27/human ␤ 2 -microglobulin (Hu␤ 2 m)-transgenic rats.Methods. A missense mutation in the CD8a gene that causes a loss of CD8␣ expression was identified in offspring of a male Sprague-Dawley rat that had been treated with the mutagen N-ethyl-N-nitrosourea. The mutation was crossed into B27/Hu␤ 2 m-transgenic lines on the Lewis background. CD8a -/-and CD8a ؉/-progeny were compared on a mixed SD-LEW background as well as after at least 10 backcrosses to LEW rats. CD8 function was assessed by generating cytolytic T lymphocytes (CTLs) against allogeneic DA strain antigens.Results. Homozygous mutant rats showed normal CD8a and CD8b messenger RNA levels but no detectable expression of either protein and an almost complete abrogation of the allogeneic CTL response. Two disease phenotypes previously observed in different B27/ Hu␤ 2 m-transgenic lines also occurred in the respective CD8a -/--transgenic rat lines. There was no significant difference in disease prevalence or severity between CD8a -/-rats and CD8a ؉/-rats. Conclusion. All of the previously described disease manifestations in HLA-B27/Hu␤ 2 m-transgenic rats arise in the absence of any functional CD8؉ T cells. It thus seems unlikely that classic T cell recognition of HLA-B27 is of primary importance in this animal model. The possibility of a secondary role of a CD8-dependent mechanism cannot be entirely excluded.

Objective Inhibition of inflammation and destruction but not osteoproliferation in spondyloarthritis (SpA) patients treated with anti-TNF raises the question of how these three processes are interrelated. This study aimed to analyze this relationship in a SpA rat model. Methods Histological spine and joint samples of HLA-B27/Huβ2m transgenic rats were analyzed for signs of spondylitis and destructive arthritis and semi-quantitatively scored as mild, moderate or severe inflammation. Results In spondylitis, mild inflamed sections displayed lymphocyte infiltration in connective tissue adjacent to the junction of the annulus fibrosus and vertebral bone but not at the enthesis. Moderate inflamed tissue samples contained osteoclasts eroding bone outside the cartilage endplate. In severe inflammation, the cartilage endplate and underlying bone marrow were also affected. End-stage disease was characterized by complete destruction of the intervertebral disc and vertebrae, with ongoing infiltration. Osteoproliferation was not observed in samples with no or mild inflammation, but was present at the edge of the vertebrae in moderate inflammation and persisted during severe inflammation and end-stage destruction. Osteoproliferation occurred at the border of inflammation, at a distance from bone destruction. A strong correlation between the extent of inflammation, destruction and osteoproliferation was observed. Arthritis displayed a similar pattern of synovial inflammation associated with bone destruction, and simultaneous but topographically distinct osteoproliferation starting from the periosteum. Conclusion Spondyloarthritis in HLA-B27/Huβ2m tg rats is characterized by destructive inflammatory pannus tissue rather than by enthesitis or osteitis. Destruction and osteoproliferation occur simultaneously but at distinct sites in joints with moderate to severe inflammation.

Objective Male rats transgenic for HLA-B27 and human-β2-microglobulin (Hu-β2m) spontaneously develop epididymo-orchitis preceding spondyloarthritis. In the specific B27/Hu-β2m transgenic cross (21-3x382-2)F1, only the males develop spondyloarthritis, and neither sex develops gut inflammation. We asked whether epididymo-orchitis and spondyloarthritis in the (21-3x382-2)F1 males are causally related, and we characterized the epididymo-orchitis. Methods B27/Hu-β2m (21-3x382-2)F1 transgenic males underwent bilateral, unilateral, or sham epididymo-orchiectomy between ages 36 and 125 d. Castrated rats were given testosterone replacement. Alternatively, the 21-3 and 283-2 transgene loci were crossed with a transgene inducing aspermatogenesis. Rats were observed for epididymo-orchitis, arthritis, and spondylitis. Results In unmanipulated transgenic rats, inflammation was first evident in the ductuli efferentes (DE, ducts linking rete testis to epididymis) as early as age 30 d. The inflammation was initially neutrophilic, and later became granulomatous. Serum anti-sperm and anti-testis cell antibodies appeared after age 70 d. Cells infiltrating the testes were predominantly CD4+ T cells and CD68+ or CD163+ macrophages. Quantitative PCR of DE, epididymis, and testis showed elevations of IFNγ, IL-10, andIL-17A. IL-12A, IL-22, IL-23A, and IL-23R were also examined in DE and found elevated. Remarkably, castration before 91 d of age completely prevented subsequent arthritis and spondylitis, as did transgene-induced azospermia. Conclusion In the (21-3x283-2)F1 HLA-B27/Hu-β2m transgenic rats, autoimmune epididymo-orchitis develops spontaneously at 30 d, the age when antigen-positive meiotic germ cells first exit the testis. Persistent testicular inflammation and/or antigenic stimulation are essential prerequisites to subsequent spondyloarthritis. Dysregulated innate immunity at immune privileged sites may be an essential mechanism triggering spondyloarthritis.

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 the 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 of Erap1 was permissive for the AS-like phenotype, 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.

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.