Nimman Satumtira

Inflammatory disease in HLA-B27 transgenic rats

Summary: A spontaneous inflammatory disease in rats transgenic for HLAB27 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 B2 7 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‐y, 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.

CD8αβ 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 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.

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 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.

Spondylarthritis in HLA–B27/human β2-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 β2‐microglobulin–transgenic rat lines

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

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 β(2) -microglobulin (hβ(2) m) spontaneously develop epididymoorchitis (EO) preceding the development of spondylarthritis (SpA). In the specific B27/hβ(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/hβ(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/hβ(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.

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.

Innate Immune Activation Can Trigger Experimental Spondyloarthritis in HLA-B27/Huβ2m 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/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.

FRI0164 Innate Immune Stimulation Triggers Altered IL-1A/B Gene Expression and Experimental Spondyloarthritis in Hla-B27/Huβ2M Transgenic Rats

Background Spondyloarthritis (SpA) does not display the typical features of autoimmune diseases such as female predominance, presence of autoantibodies and clinical response to T- and/or B cell targeting biologicals. Despite the strong association with MHC class I, CD8 T cells are not required for disease induction in the HLA-B27/huβ2m transgenic rat model. Moreover, the capability of HLA-B27 to misfold and thereby induce endoplasmatic reticulum stress and the direct recognition of HLA-B27 homodimers by NK cells suggest pathogenic mechanisms which may be independent of classical acquired immune responses. Therefore, we and others propose that SpA may be primarily driven by the innate immune response. Objectives Using the HLA-B27/huβ2m transgenic rat model [1], we investigated this hypothesis by studying the effect of innate immune stimulation on ex vivo cytokine expression and in vivo development of arthritis and spondylitis. Methods Splenocytes and bone marrow cells isolated from HLA-B27/huβ2m transgenic rats, HLA-B7/huβ2m transgenic control rats and Lewis wild-type rats, were stimulated for 6 hours with 50 ng/ml LPS, 5 μg/ml zymosan or 5 μg/ml Mycobacterium tuberculosis. TNF, IL-1, IL-6, IL-10 and IL-23p19 expression was measured by qPCR. For in vivo analysis, six week old male and female HLA-B27/huβ2m transgenic rats and HLA-B7/huβ2m transgenic control rats were immunized with low doses (30, 60 or 90 ug) of M. tuberculosis in incomplete Freunds adjuvant. Rats were followed up for 60 days and scored clinically for arthritis and spondylitis. Results In vitro stimulation of splenocytes with zymosan and with M. tuberculosis, but not with LPS, strongly induced gene expression of pro-inflammatory cytokines such as TNF, IL-1alpha, IL-1beta and IL-6 in all 3 rat strains. IL-1alpha and IL-1beta, but not TNF or IL-6, were increased in the HLA-B27/huβ2m transgenic cells as compared to both HLA-B7/huβ2m transgenic and wild-type controls upon ex vivo stimulation. IL-10 and IL23p19 expression could not be detected in any of the groups after stimulation. In vivo, non-immunized HLA-B27/huβ2m transgenic males spontaneously develop arthritis and spondylitis after 4-6 months of age with an incidence of 70% and 40%, respectively, whereas female rats did not develop disease. Immunization of 6 weeks old male HLA-B27/huβ2m transgenic rats with 30 μg of M. tuberculosis was sufficient to induce development of arthritis and spondylitis within 2-3 weeks with an incidence of 80-100%. Moreover, HLA-B27/huβ2m transgenic females also developed spondylitis and arthritis with the same disease onset, severity and incidence when immunized with 60 μg of M. tuberculosis. Control rats were less sensitive to these low doses of M. tuberculosis. Conclusions The transgenic over-expression of HLA-B27/Hub2m increases the sensitivity to innate immune stimulation as evidenced by increased IL-1alpha and IL-1beta expression ex vivo and development of arthritis and spondylitis in vivo. These data indicate that innate immune activation can trigger experimental SpA. References Tran TM et. al. Arthritis Rheum 2006; 54(4):1317-27 Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4108