ERAP1 genetic variations associated with HLA-B27 interaction and disease severity of syndesmophytes formation in Taiwanese ankylosing spondylitis

Ankylosing spondylitis (AS) is a chronic inflammatory arthritis that preferably affects the sacroiliac and spine joints in young males. Chronic inflammation in joints causes the alteration of joint architecture with new bone formations, and joint fusions may occur consequently [1, 2]. The unique structural changes of syndesmophyte formation and ankylosis of the vertebrae are the primary causes of early severe work disability of AS patients during disease progressions [1–4]. Multiple factors and complex biological interactions may be involved in AS development [5–8].

Twin and family studies revealed a substantial proportion of heritability in AS susceptibility. Genetic studies indicate that HLA-B27 in the MHC (major histocompatibility complex) locus confers the greatest risk to AS susceptibility. However, genome wide association studies (GWAS) also reveal that the non-MHC genes contribute to the AS disease process [4, 5, 8–10]. Several genes and genetic regions have now been discovered and known to be associated with AS susceptibility and severity [8–10]. Of note, there were conflicting association results between Caucasians and Asians [9–12].

Endoplasmic reticulum aminopeptidase 1 (ERAP1) (also known as aminopeptidase regulator of TNFR1 shedding 1 or ARTS1) is an important non-MHC gene associated with AS in genetic studies [9, 10]. Functionally, ERAP1 facilitates the antigen peptide loading onto the major histocompatibility complex class I (MHC I) through trimming the fragmented antigen peptides into the optimal length for peptide/MHC I (pMHC I) complex formation, which is necessary for effective immune responses [13–17]. In addition, ERAP1 involves the shedding of pro-inflammatory cytokine receptors for TNFα, IL-1, and IL-6 [18–20]. ERAP1 SNPs are associated with AS in several ethnic populations [9–12, 21–26], but it is unknown whether ERAP1 SNPs have a role in AS development in Taiwanese. The aim of the present study was to examine whether the ERAP1 SNPs are associated with AS susceptibility and have effects on AS disease severity of syndesmophyte development in Taiwanese.

Complex interactions between environmental factors and host immune responses are the origins for AS development [4, 5]. It is clear that genetic factors influence the immune responses and progression of AS. The current study demonstrated the associations of EARP1 SNPs with the AS susceptibility in Taiwanese. Aminopeptidases play a central role in the generation of MHC class I-binding peptides through processing and trimming peptide. As an IFNγ-induced aminopeptidase, ERAP1 breaks down protein antigen precursors and trims the peptides fragments into the suitable length for peptide/MHC I complex formation in ER [13, 14, 30]. In mice, ERAP1 deficiency led to the reduced MHC I expression in splenocytes and the decreased viral peptide presentation by MHC I on fibroblasts [31]. The generation of effective CD8 T cell responses was compromised in ERAP1 deficient mice as self and foreign antigen presentations were disrupted [31–36]. In humans, over-expression of ERAP1 was observed in AS patient dendritic cells (DCs) [37]. ERAP1 also enhances phagocytic activity of human macrophages through generating active peptides [38]. Functionally, ERAP1 may have a substantial role in the AS pathogenesis. Our current study demonstrated the associations of ERAP1 SNPs with the AS susceptibility in Taiwanese, indicating that ERAP1 has a role in AS development in Taiwanese

We observed that >90% of AS patients are HLA-B27 carriers. Our data confirmed that HLA-B27 plays the most critical role in AS disease progression. HLA-B27 may influence AS development through three possible mechanisms: I) HLA-B27 may preferentially bind distinctive arthritogenic peptides, II) inappropriately folded HLA-B27 heavy chain without a docking antigen peptide may lead to the unfolded protein responses (UPRs), and III) HLA-B27 may have a tendency to be expressed as empty MHC I heavy chain homodimers [4, 39]. However, the precise mechanisms underlying the critical role of HLA-B27 in AS development remain to be elucidated [40].

Of great importance, immune surveillance of the CD8 T cell to environmental insults, such as bacterial and viral infections, requires the interaction between T cell receptor (TCR) and peptide/MHC I complex. ERAP1 trims protein antigens to fit for MHC I including HLA-B27. The stability of antigen peptide/MHC I complexes is influenced by both ERAP1 activities and peptide binding groove sequences of MHC I [41]. Very recently, Evans et al. demonstrated the gene-gene interaction between ERAP1 SNP rs30187T allele and HLA-B27 positivity in the pathogenesis of AS [42]. Our study provided further evidence that ERAP1 SNPs are indeed associated with the HLA-B27 positivity in Taiwanese AS patients. Our data support the notion that ERAP1 and HLA-B27 have synergistic roles in AS pathogenesis in humans. Our findings also suggest that abnormal antigen processing by ERAP1 and antigen presentation by HLA-B27 may be critical pathways in AS development. On the other hand, AS patients negative for HLA-B27 may develop pathologic immune responses through the other unidentified biological pathways.

The disease course of AS is heterogeneous and the genetic factors are believed to influence disease susceptibility and severity. Early syndesmophyte formations in AS patients indicate radiologic progression and characteristic structure changes that lead to the decreased spinal mobility, functional impairments and work disability in long-term disease courses [4, 43]. Sacroiliac joint biopsy examinations frequently revealed significant new bone formations and bony ankylosis in AS patients [44]. Spinal inflammation and bone remodeling may be two important factors in the progress of ankylosis but the precise pathogenesis of AS remains unknown [44, 45]. The current study demonstrated that ERAP1 cSNPs rs27044 and rs30187 are modestly associated with AS syndesmophyte formation, suggesting that the ERAP1 cSNPs may affect AS disease severity. Syndesmophyte of AS is more likely to develop at sites of previous inflammation, indicating ankylosis development is strongly correlated with inflammation [44]. The ERAP1 protein also participates in the regulation of proinflammatory cytokine receptor functions. ERAP1 cleaves membrane-associated TNFR1, IL-6R, and interleukin 1 receptor II (IL-1RII) and causes the shedding of those cytokine receptors. Some cellular and molecular signal pathways that regulate the development of hematopoietic cell and bone homeostasis are shared by the immune system and bone development [45]. IL6 and TNFα cytokine networks may affect the Th17 cell development and the plasticity of T cell differentiation, which are critical in the pathogenesis of AS. The serum cytokine receptor levels in AS patients are correlated with the levels ESR and CRP, which are indicators of AS inflammatory activities. Nevertheless, the serum cytokine receptor levels in patients with AS are not influenced by ERAP1 SNPs [46]. Animal models have even suggested that inflammation and new bone formation are uncoupled processes [47, 48]. Clinically, various anti-TNF therapies suppress the inflammation process but do not retard the structural progression according to modified Stoke's Ankylosing Spondylitis Spinal Score (mSASSS) [49–51]. These findings indicate the syndesmophyte development may largely attribute to the intrinsic genetic effects of ERAP 1 on p/MHC I complex formation.

UPRs can cause the activation of NFκB, which could enhance downstream proinflammatory gene expression and promote inflammations. Genetic variations of HLA-B27 and ERAP1 have functional roles in the misfolding and UPR of the heavy chain [4, 40]. HLA-B27 misfolding is triggered in the oxidizing environment in endoplasmic reticulum (ER) by exposing cysteine residues within the heavy chain [40]. ERAP1 is required for efficient enzymatic activities that suggest the polymorphisms at sites remote from the catalytic sites might modify this association [52]. ERAP1 processes peptide substrates with the optimal sizes for MHC I (40, 41). Crystallography analyses suggest that the coding SNP rs30187 changes the amino acid residue that may affect the catalytic activity [53]. Notably, the alleles and genotypes of ERAP1 SNPs in Taiwanese and other Asians showed different distribution and opposite risk results in AS susceptibility compared to Caucasians [9–12, 21–26]. In addition, ERAP1 SNP rs30187C allele carriers (CC+CT) were found to be associated with higher baseline radiographic severity based on mSASSS on univariate analysis [54]. The current study demonstrated that both SNP rs30187T and rs27044G allele carrier were modestly associated with AS disease severity of syndesmophyte formation. Functional studies of ERAP1 SNPs have identified that the rs30187C allele carries less biological enzyme activity, which may decrease aberrant peptide processing and HLA-B27 presentation [42], the rs27044C allele carrier genotype demonstrated significantly higher free heavy chain (FHC) expression but lower intact HLAB27 complexes/FHCs ratio [55], and ERAP1 SNPs (rs30187 and rs27044) showed specific peptide substrate sequence interaction [52]. Moreover, large multifunctional peptidase 2 (LMP2) rs17587 SNP has been demonstrated to associate with AS radiographic severity [54]. These findings indicated multiple gene interactions are involved in the complexity of AS disease susceptibility and severity, and no definite functional roles of ERAP1 cSNPs rs30187 and rs27044 in antigen processing can fully answer the discrepancy results in different ethnic backgrounds. Nevertheless, other ERAP1 SNPs may also affect UPR and subsequent immune responses. Further functional studies are required to understand the precise roles of ERAP1 SNPs contribute to the AS pathogenesis.

In this cross sectional study, some AS patients may not receive enough follow-up in the syndesmophyte formation assessment, which is the main limitation of our study. Therefore, future longitudinal studies are required to estimate the effect of ERAP1 SNPs on the bamboo spine development in large AS patient cohorts.

This study provided further evidence that ERAP1 interaction with HLA-B27 is involved in the development of AS and disease severity, which emphasized the critical role of pMHC I pathway genes in the pathogenesis of AS. Identification of the functional causal alleles of ERAP1 provides a new avenue in understanding the molecular mechanisms of AS pathogenesis, which may lead to novel treatment approaches.

The authors would like to thank Shin Chu Blood Donor Center for sample collection. This study was supported by grants from Chang Gung Memorial Hospital (No. CMRPG381082) and National Science Council, Taiwan (NMRPG-97-2314-B182A-025-MY3).