The Human Leukocyte Antigen (HLA)-B27 Peptidome in Vivo, in Spondyloarthritis-susceptible HLA-B27 Transgenic Rats and the Effect of Erap1 Deletion.

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.

Relationship between inflammation, bone destruction, and osteoproliferation in the HLA-B27/human ß2 -microglobulin-transgenic rat model of spondylarthritis.

OBJECTIVE: Inhibition of inflammation and destruction, but not of osteoproliferation, in patients with spondylarthritis (SpA) treated with anti-tumor necrosis factor raises the question of how these three processes are interrelated. This study was undertaken to analyze this relationship in a rat model of SpA. METHODS: Histologic spine and joint samples from HLA-B27/human ß(2) -microglobulin (hß(2) m)-transgenic rats were analyzed for signs of spondylitis and destructive arthritis and semiquantitatively scored as showing mild, moderate, or severe inflammation. RESULTS: In rats exhibiting spondylitis, mildly inflamed sections displayed lymphocyte infiltration in connective tissue adjacent to the junction of the anulus fibrosus and vertebral bone but not at the enthesis. Moderately inflamed tissue samples contained osteoclasts eroding bone outside the cartilage end plate. In sections from rats with severe inflammation, the cartilage end plate 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 from rats with no or mild inflammation, but was present at the edge of the vertebrae in sections with 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. Sections from rats with arthritis displayed a similar pattern of synovial inflammation associated with bone destruction, and simultaneous but topographically distinct osteoproliferation starting from the periosteum. CONCLUSION: SpA in B27/hß(2) m-transgenic 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.

Autoimmune epididymoorchitis is essential to the pathogenesis of male-specific spondylarthritis in HLA-B27-transgenic rats.

OBJECTIVE: Male rats transgenic for HLA-B27 and human ß(2) -microglobulin (hß(2) m) spontaneously develop epididymoorchitis (EO) preceding the development of spondylarthritis (SpA). In the specific B27/hß(2) m-transgenic rat cross-strain (21-3 × 382-2)F(1) , only the males develop SpA, and neither sex develops gut inflammation. This study was undertaken to determine whether EO and SpA in male (21-3 × 382-2)F(1) rats are causally related. In addition, the primary characteristics of EO in this rat arthritis model were assessed. METHODS: Male B27/hß(2) m-transgenic (21-3 × 382-2)F(1) rats underwent bilateral, unilateral, or sham epididymoorchiectomy between ages 36 and 125 days. The 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 the development of EO, arthritis, and spondylitis. RESULTS: In unmanipulated transgenic rats, inflammation was first evident in the ductuli efferentes (DE; ducts linking the rete testis to epididymis) as early as age 30 days. The inflammation was initially neutrophilic, and later became granulomatous. Antisperm and anti-testis cell antibodies appeared in the rat serum after age 70 days. Cells infiltrating the testes were predominantly CD4+ T cells and CD68+ or CD163+ macrophages. Quantitative polymerase chain reaction of the DE, epididymis, and testis showed elevations in the levels of interferon-γ, interleukin-10 (IL-10), and IL-17A. In addition, levels of IL-12A, IL-22, IL-23A, and IL-23 receptor were found to be elevated in the DE. Remarkably, castration of the rats before age 91 days completely prevented the subsequent onset of arthritis and spondylitis, as did transgene-induced azospermia. CONCLUSION: Autoimmune EO develops spontaneously in HLA-B27/hß(2) m-transgenic (21-3 × 283-2)F(1) rats at age 30 days, the age when antigen-positive meiotic germ cells first exit the testis. Persistent testicular inflammation and/or antigenic stimulation are essential prerequisites for the subsequent development of SpA. Thus, dysregulated innate immunity at immune-privileged sites may be an essential mechanism triggering the onset of SpA.

Innate Immune Activation Can Trigger Experimental Spondyloarthritis in HLA-B27/Huß2m Transgenic 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/Huß2m transgenic rats. We used Lewis HLA-B27/Huß2m transgenic rats [21-3 × 283-2]F1, HLA-B7/Huß2m 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-1ß, 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.

HLA-B27 and human ß2-microglobulin affect the gut microbiota of transgenic rats.

The HLA-B27 gene is a major risk factor for clinical diseases including ankylosing spondylitis, acute anterior uveitis, reactive arthritis, and psoriatic arthritis, but its mechanism of risk enhancement is not completely understood. The gut microbiome has recently been shown to influence several HLA-linked diseases. However, the role of HLA-B27 in shaping the gut microbiome has not been previously investigated. In this study, we characterize the differences in the gut microbiota mediated by the presence of the HLA-B27 gene. We identified differences in the cecal microbiota of Lewis rats transgenic for HLA-B27 and human ß2-microglobulin (hß2m), compared with wild-type Lewis rats, using biome representational in situ karyotyping (BRISK) and 16S rRNA gene sequencing. 16S sequencing revealed significant differences between transgenic animals and wild type animals by principal coordinates analysis. Further analysis of the data set revealed an increase in Prevotella spp. and a decrease in Rikenellaceae relative abundance in the transgenic animals compared to the wild type animals. By BRISK analysis, species-specific differences included an increase in Bacteroides vulgatus abundance in HLA-B27/hß2m and hß2m compared to wild type rats. The finding that HLA-B27 is associated with altered cecal microbiota has not been shown before and can potentially provide a better understanding of the clinical diseases associated with this gene.

Spondylarthritis in HLA-B27/human beta2-microglobulin-transgenic rats is not prevented by lack of CD8.

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 beta(2)-microglobulin (Hubeta(2)m)-transgenic rats. METHODS: A missense mutation in the CD8a gene that causes a loss of CD8alpha 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/Hubeta(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/Hubeta(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/Hubeta(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.

Correlation between dendritic cell functional defect and spondylarthritis phenotypes in HLA-B27/HUMAN beta2-microglobulin-transgenic rat lines.

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

Additional human beta2-microglobulin curbs HLA-B27 misfolding and promotes arthritis and spondylitis without colitis in male HLA-B27-transgenic rats.

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, 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. METHODS: 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. RESULTS: 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. 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.

HLA-B27 in transgenic rats forms disulfide-linked heavy chain oligomers and multimers that bind to the chaperone BiP.

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.

CD8 alpha beta T cells are not essential to the pathogenesis of arthritis or colitis in HLA-B27 transgenic rats.

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 CD8alphabeta 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-CD8alpha mAb treatment. This treatment induced profound, sustained depletion of CD8alphabeta T cells, but failed to suppress either colitis or arthritis. To address the role of CD8alpha(+)beta(-) cells, we studied four additional groups of B27 transgenic rats treated with: 1) continuous anti-CD8alpha mAb, 2) continuous isotype-matched control mAb, 3) the thymectomy/pulse anti-CD8alpha regimen, or 4) no treatment. Arthritis occurred in approximately 40% of each group, but was most significantly reduced in severity in the anti-CD8alpha-treated group. In addition to CD8alphabeta T cells, two sizeable CD8alpha(+)beta(-) non-T cell populations were also reduced by the anti-CD8alpha 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 CD8alpha(+)beta(-) non-T cells may play a role in the arthritis that occurs in these rats.