Adaptive immunity

Aminopeptidases play a key role in the processing of cytoplasmic proteins for presentation in the context of MHC class I molecules on the cell surface. The function of the MHC class I pathway is to present a sample of cytoplasmic proteins to the immune system so that peptides derived from tumours or intracellular infections can be detected by cognate CD8 T cells. Aminopeptidases take N-terminal extended peptides that are produced by the proteasome and trim them to the length required for optimal presentation in the context of MHC class I molecules.

Genetic loci containing four aminopeptidases have been associated with AS (ankylosing spondylitis). They are endoplasmic reticulum aminopeptidase 1 (ERAP1, encoded by the ERAP1 gene), endoplasmic reticulum aminopeptidase 2 (ERAP2, encoded by the ERAP2 gene), insulin regulated aminopeptidase (IRAP, encoded by the LNPEP gene) and puromycin-sensitive aminopeptidase (PSA, encoded by the NPEPPS gene) . Although genetic associations at a locus cannot always be pinpointed to specific genes, the evidence that ERAP1 and ERAP2 are causal genes in AS is compelling. There are strong associations with non-synonymous SNPs in the gene sequence and strong functional candidates identified for both of these aminopeptidases . The haplotype containing LNPEP – associated with AS – is on the same haplotype containing ERAP2 and may reflect linkage disequilibrium with the ERAP2 association. The functional impact of the arthritis-associated genetic variants in ERAP1 has been elucidated in detail. The 2 haplotypes of ERAP1 that are strongly associated with AS both carry variants that map to crucial regions of the protein and reduce the trimming activity of the enzyme . This decrease in trimming capacity corresponds to protection from AS .

Evidence from mice suggests the ERAP1 is important for fighting intracellular infections such as Chlamydia. Mice only have one endoplasmic reticulum aminopeptidase, called ERAAP (endoplasmic reticulum associated with antigen processing). Mice deficient in ERAAP are more susceptible to infection with Toxoplasma, an intracellular pathogen, than their ERAAP sufficient controls .

Besides its antigen processing function, three other functions of ERAP1 have been identified, which could impact on immune control of microorganisms. ERAP1 is secreted by macrophages stimulated by LPS (lipopolysaccharide) and IFN-γ . LPS is an intrinsic part of the cell wall of some microorganisms and acts as a PAMP (pathogen associated molecular pattern) to stimulate the innate immune system. IFN-γ is secreted in response to a range of infections by microorganisms. It was shown that secreted ERAP1 enhanced phagocytosis. ERAP1 has also been shown to cleave cell surface cytokine receptors, including IL-6, TNF and IL-1 . Soluble cytokine receptors may act as decoy receptors for these cytokines, modulating inflammatory arthritis as well as the response to invading microorganisms. However, it has been shown that in AS patients that the levels of these receptors do not vary according to ERAP1 genotype . Although ERAP1 could play multiple potential roles in the response to infection, it is likely that the ERAP1 association with AS relates to its antigen processing function. This is because a gene–gene interaction has been noted to occur with HLA-B27 . SNPs in ERAP1 are only associated with AS when the HLA-B27 classical allele is also present . Similar associations with ERAP1 have been noted in psoriasis and Behçet’s disease but with different MHC classical alleles in each case .

Two important functional variants of the ERAP2 aminopeptidase have been identified. In one of these, no ERAP2 protein is produced in cells homozygous for the G allele of rs2248374. In the same way that decreased ERAP1 function is protective for AS, loss of ERAP2 variant is also protective for AS . The other variant changes the enzyme’s activity and specificity for peptides, but is in almost complete LD, and therefore this version of the enzyme would almost never be made. Both ERAP1 and ERAP2 trim peptides, but have different substrate specificities. Their role in response to infection would expose them to selective pressure. Consistent with this, Andres and colleagues described signatures of balancing selection in both ERAP1 and ERAP2 across human populations . Differing genotypes of ERAP2 not only affect enzyme levels but also affect the amount of surface MHC molecules. Knockdown of ERAP1 in human cells increased cell surface MHC molecules, and knockdown in mouse cells decreased cell surface MHC expression . While the explanation for incongruity between species is not clear, a range of evidence indicates that alteration in aminopeptide function affects class I MHC presentation. This is turn would impact the response to exogenous microorganisms and host fitness to control intracellular organisms such as Chlamydia.

The haplotype harbouring the ERAP2 gene also contains the gene encoding IRAP. IRAP is an integral component of the cross-presentation machinery of dendritic cells . Cross presentation traffics exogenous antigen into the endogenous pathway, resulting in MHC class I-restricted antigen presentation. This process is important for the control of tumours, and infectious agents that do not infect antigen presenting cells such as viruses and intracellular obligate pathogens . Exactly which micro-organisms might be relevant to AS are not yet clear. However the biological plausibility of the link between IRAP and intracellular pathogens warrants further investigation of this pathway.

Innate immunity

Adaptive immunity

Aminopeptidases play a key role in the processing of cytoplasmic proteins for presentation in the context of MHC class I molecules on the cell surface. The function of the MHC class I pathway is to present a sample of cytoplasmic proteins to the immune system so that peptides derived from tumours or intracellular infections can be detected by cognate CD8 T cells. Aminopeptidases take N-terminal extended peptides that are produced by the proteasome and trim them to the length required for optimal presentation in the context of MHC class I molecules.

Genetic loci containing four aminopeptidases have been associated with AS (ankylosing spondylitis). They are endoplasmic reticulum aminopeptidase 1 (ERAP1, encoded by the ERAP1 gene), endoplasmic reticulum aminopeptidase 2 (ERAP2, encoded by the ERAP2 gene), insulin regulated aminopeptidase (IRAP, encoded by the LNPEP gene) and puromycin-sensitive aminopeptidase (PSA, encoded by the NPEPPS gene) . Although genetic associations at a locus cannot always be pinpointed to specific genes, the evidence that ERAP1 and ERAP2 are causal genes in AS is compelling. There are strong associations with non-synonymous SNPs in the gene sequence and strong functional candidates identified for both of these aminopeptidases . The haplotype containing LNPEP – associated with AS – is on the same haplotype containing ERAP2 and may reflect linkage disequilibrium with the ERAP2 association. The functional impact of the arthritis-associated genetic variants in ERAP1 has been elucidated in detail. The 2 haplotypes of ERAP1 that are strongly associated with AS both carry variants that map to crucial regions of the protein and reduce the trimming activity of the enzyme . This decrease in trimming capacity corresponds to protection from AS .

Evidence from mice suggests the ERAP1 is important for fighting intracellular infections such as Chlamydia. Mice only have one endoplasmic reticulum aminopeptidase, called ERAAP (endoplasmic reticulum associated with antigen processing). Mice deficient in ERAAP are more susceptible to infection with Toxoplasma, an intracellular pathogen, than their ERAAP sufficient controls .

Besides its antigen processing function, three other functions of ERAP1 have been identified, which could impact on immune control of microorganisms. ERAP1 is secreted by macrophages stimulated by LPS (lipopolysaccharide) and IFN-γ . LPS is an intrinsic part of the cell wall of some microorganisms and acts as a PAMP (pathogen associated molecular pattern) to stimulate the innate immune system. IFN-γ is secreted in response to a range of infections by microorganisms. It was shown that secreted ERAP1 enhanced phagocytosis. ERAP1 has also been shown to cleave cell surface cytokine receptors, including IL-6, TNF and IL-1 . Soluble cytokine receptors may act as decoy receptors for these cytokines, modulating inflammatory arthritis as well as the response to invading microorganisms. However, it has been shown that in AS patients that the levels of these receptors do not vary according to ERAP1 genotype . Although ERAP1 could play multiple potential roles in the response to infection, it is likely that the ERAP1 association with AS relates to its antigen processing function. This is because a gene–gene interaction has been noted to occur with HLA-B27 . SNPs in ERAP1 are only associated with AS when the HLA-B27 classical allele is also present . Similar associations with ERAP1 have been noted in psoriasis and Behçet’s disease but with different MHC classical alleles in each case .

Two important functional variants of the ERAP2 aminopeptidase have been identified. In one of these, no ERAP2 protein is produced in cells homozygous for the G allele of rs2248374. In the same way that decreased ERAP1 function is protective for AS, loss of ERAP2 variant is also protective for AS . The other variant changes the enzyme’s activity and specificity for peptides, but is in almost complete LD, and therefore this version of the enzyme would almost never be made. Both ERAP1 and ERAP2 trim peptides, but have different substrate specificities. Their role in response to infection would expose them to selective pressure. Consistent with this, Andres and colleagues described signatures of balancing selection in both ERAP1 and ERAP2 across human populations . Differing genotypes of ERAP2 not only affect enzyme levels but also affect the amount of surface MHC molecules. Knockdown of ERAP1 in human cells increased cell surface MHC molecules, and knockdown in mouse cells decreased cell surface MHC expression . While the explanation for incongruity between species is not clear, a range of evidence indicates that alteration in aminopeptide function affects class I MHC presentation. This is turn would impact the response to exogenous microorganisms and host fitness to control intracellular organisms such as Chlamydia.

The haplotype harbouring the ERAP2 gene also contains the gene encoding IRAP. IRAP is an integral component of the cross-presentation machinery of dendritic cells . Cross presentation traffics exogenous antigen into the endogenous pathway, resulting in MHC class I-restricted antigen presentation. This process is important for the control of tumours, and infectious agents that do not infect antigen presenting cells such as viruses and intracellular obligate pathogens . Exactly which micro-organisms might be relevant to AS are not yet clear. However the biological plausibility of the link between IRAP and intracellular pathogens warrants further investigation of this pathway.

Innate immunity

HLA-B27 and spondyloarthritis (SpA)

Several potential mechanisms are proposed to explain the strong genetic association between HLA-B27 and SpA. Each strongly implicates microorganism involvement. Firstly molecular mimicry is proposed between an infectious HLA-B27-restricted ligand and innate PAMPs or DAMPs . For example ReA, which is triggered by gastrointestinal or genitourinary infections, including Salmonella and Chlamydia , was proposed to be autoimmune because cross-reactivity between bacterial and self-antigens was hypothesized. However, the very low burden of antigen-specific auto-reactive T cells identified by HLA-B27 tetramers in Chlamydia-triggered reactive arthritis does not support molecular mimicry between Chlamydia trachomatis dominant epitopes and human self-proteins .

Secondly a cellular response to PAMPs has been proposed. This alternative paradigm postulates that a pathogenic intracellular microbe disseminates from a mucosal portal of entry to the joint, where PAMPs drive an inflammatory response. A third hypothesis is based on evidence that MHC class I molecules assemble in the ER (endoplasmic reticulum), whereby a series of essential interactions between newly-synthesized heavy chains and resident ER proteins occurs . Newly synthesized HLA-B27 is slow to fold and shows an aberrant ability to associate with β2-microglobulin; thus under inflammatory pressure HLA-B27 heavy chain misfolds and accumulates in the ER . Under such conditions the UPR (unfolded protein response) ensues, as ER resident chaperones are further induced to facilitate folding, known as ER stress. ER stress may lead to activation of NF-κB and downstream pathways.

A major step in the understanding of disease mechanisms in SpA was recently achieved when Sherlock and colleagues showed that IL-23 over-expression was sufficient to induce SpA by acting on entheseal resident T cells producing IL-22 and IL-17, which in turn activated osteoblast-mediated bone remodelling. These data corroborate Th17 activation observed in patients suffering from SpA.

In splenic macrophages from HLA-B27 transgenic rats and in ileal tissue from SpA-susceptible SKG mice, ER stress is associated with up-regulation of IL-23 expression . ER stress, in combination with TLR agonists, has been shown to enhance expression of IL-23p19 mRNA and to augment secretion of IL-23 . Infection of myeloid cells with C. trachomatis – a well recognized pathogen associated with SpA – results in the expression of the ER stress-induced transcription factor, C/EBP homologous protein (CHOP); subsequently CHOP binds to the IL-23p19 promoter . This suggests further links between ER stress, HLA-B27 misfolding, IL-23 and infection.

While ER stress decreased after IL-23 neutralization in curdlan-treated SKG mice, IL-23 production in SpA was not correlated with ER stress induced by HLA-B27 misfolding in patients . Although analysis of gut biopsies from HLA-B27-positive patients suffering from SpA and Crohn’s disease provided evidence for HLA-B27 misfolding in the gut, autophagy rather than the UPR was associated with the production of IL-23 . Therefore autophagy has emerged as an important pathway involved in immune dysregulation of inflamed gut tissue in HLA-B27 positive patients suffering from SpA . It is not yet clear why autophagy is increased in these patients. Possible explanations are that autophagy is a compensatory mechanism to avoid cell death triggered by ER stress, or it may be a mechanism by which antigens derived from intracellular pathogens are processed for cross-presentation.

HLA-B27 and bacterial infections

The interaction between HLA-B27 and bacterial infection has been extensively studied for decades, providing new insights into the role of HLA-B27 in SpA. Some authors have suggested that Propionibacterium acnes triggers the syndrome of SAPHO (synovitis, acne, pustulosis, hyperostosis and osteitis) . However, there is as yet insufficient evidence to cement this relationship. In contrast, ReA is the prototype of sterile inflammatory diseases induced by bacterial infection within the SpA spectrum. C. trachomatis is an obligate intracellular pathogen causing the most frequent type of ReA worldwide, as it is the most common bacterial sexually transmitted disease. Approximately 4–15% of individuals with genital C. trachomatis infection subsequently develop ReA .

The term ‘reactive’ in ReA describes how this disease was traditionally considered a non-septic, autoimmune reaction occurring in the joint in response to a bacterial infection, based on the fact that no pathogen could be cultured from the arthritic joint. Numerous investigators have accurately described ReA in the context of venereal and gut infections. However the molecular mechanisms are still unclear. Recent epidemiologic studies show that 30–50% patients suffering from ReA are HLA-B27 positive .

Of interest, HLA-B27-positive patients suffering ReA displayed a lower concentration of IFN-γ in synovial fluid than HLA-B27-negative patients . Since IFN-γ is a critical cytokine for the clearance of C. trachomatis, this suggests that HLA-B27-positive patients suffering ReA are less likely to clear the genital infection and control Chlamydia dissemination into the joint. In support of this concept, an increased incidence of ReA has been described in HLA-B27-negative patients infected with HIV, who display impaired T cell function and Th1 cytokine production. Together these data suggest a critical role for T cell mediated control of the bacterial load in human ReA.

An outbreak of salmonellosis was reported in Ontario, Canada, primarily due to consumption of contaminated bean sprouts , in which approximately 600 infected individuals developed acute Salmonella enteritidis gastroenteritis. A total of 62.5% of those infected reported extra-intestinal symptoms consistent with ReA. Of interest, HLA-B27 was three-fold more common amongst those infected than amongst healthy controls. However the incidence of HLA-B27 in patients with Salmonella infection was similar in those who did and did not develop symptoms of ReA , suggesting that HLA-B27 may render individuals more susceptible to Salmonella gastroenteritis, but not to the development of ReA once infected. Another study suggested that disease activity was increased in HLA-B27-positive patients suffering from ReA, and therefore that HLA-B27-positive patients are more likely to be diagnosed than HLA-B27-negative patients .

Analysis of the arthritogenic bacterial immunopeptidome in HLA-B27-positive human cells identified bacterial peptides that are endogenously processed and presented by HLA-B27 . The HLA-B27-restricted T cell epitope from C. trachomatis was derived from DNA primase . Since C. trachomatis could be found in both healthy joints and arthritic states , it is proposed that C. trachomatis infection induces chronic or recurrent arthritic flares through ongoing presentation of bacterial antigens to pathogenic antigen-specific T cells. Whether infection triggers a pathological polarization of the immune response in patients with underlying genetic susceptibility with consequent inflammatory pathology, is currently unclear.

Indeed, bacterial infections may trigger symptoms of SpA through the activation of innate immunity. TLRs recognize PAMPs and are activated by DAMPs implicated in SpA. The engagement of TLRs results in the activation of the NF-κB pathway, which promotes secretion of pro-inflammatory cytokines and activation of antigen-presenting cells. Activation of TLR2 led to synergistic secretion of both IL-23 and IL-12p70 in patients suffering from SpA . Moreover, HLA-B27 transgenic rats had altered functional TLR expression and an intrinsic defect in IL-10 production in response to TLR ligands . Microbial-β-glucan-treated TLR4-deficient SKG mice had reduced ileitis severity , and SKG mice infected intravaginally with Chlamydia muridarum , a mouse-adapted Chlamydia species, developed TLR2-dependent arthritis, with spondylitis and psoriasiform dermatitis, but without inflammatory bowel disease . In this model, C. muridarum DNA was found in myeloid cells outside the genital tract in both SKG and BALB/c mice, however SKG mice had decreased antigen-specific T cell IFN-γ and IL-17A, with a concomitant increase in TNF production , which supports the mechanisms for human ReA described above, through presentation of antigen to T cells with a lower capability for infection control and higher inflammatory potential. Interventions that reduced bacterial load and thus PAMP load, including antibiotics, also reduced arthritis in SKG mice. Consistent with this, in a phase II trial in patients with chronic C. trachomatis -induced ReA, antibiotic combinations of rifampicin and azithromycin, or rifampicin and doxycycline decreased disease activity and bacterial load .

Bacterial products such as Yersinia outer membrane protein induce TLR expression in joints, which results in a local inflammatory response . TLR2 expression was also increased in monocytes of psoriatic arthritis patients , suggesting that Gram-positive bacteria could induce higher inflammatory responses in this disease. The upregulation of TLR2 expression provides support for the role of the innate immune system in the pathogenesis of psoriatic arthritis . Moreover, genetic variants of TLR2 (rs5743708, R753Q) have been associated with acute ReA following infection with S. enteritidis , suggesting that effects of more than one microorganism may contribute to the inflammatory pathogenesis.

HLA-B27 and microbiota

SpA is a family of diseases that includes AS, ReA, psoriatic arthritis and inflammatory bowel disease. These diseases share some clinical features and considerable similarity in genetic susceptibility both within and outside the MHC. Much evidence implicates an altered gut microbiome in the pathogenesis of inflammatory bowel disease and SpA in rodent models and in patients . The cecal microbiota of Lewis rats transgenic for HLA-B27 and human β2-microglobulin displayed an increased proportion of Prevotella species (spp.) and a decrease in Rikenellaceae spp. and Bacteroides vulgatis , compared with wild-type Lewis rats . Furthermore, arthritis and colitis do not develop in these transgenic rats in the germ-free environment .

ZAP-70 ^W163C point-mutant SKG mice have reduced T cell receptor signalling. After intraperitoneal microbial β-glucan injection, they develop IL-23-dependent arthritis, spondylitis, enthesitis, uveitis, psoriasis-like skin disease and Crohn’s-like ileitis . IL-17A and IL-22, which are induced by IL-23, drive enthesitis but play opposing roles in the development of ileitis, in that IL-17A is pathogenic whilst IL-22 is protective . Spontaneous arthritis develops when SKG mice are housed under conventional conditions, however disease development requires a β-glucan trigger through the dectin-1 receptor, under specific pathogen-free conditions, suggesting that microbiota content directly influences the course of disease . SKG mice housed under germ-free conditions have reduced arthritis and spondylitis incidence and severity and do not develop ileitis . Cohousing of SKG and wild type BALB/c mice demonstrated that the SKG mutation affects the gut microbiota before and after disease onset, and that the microbiota influence ileitis severity . From these studies it is clear that the host genetic background influences microbiota composition, but how changes in the microbiota are related to SpA development and whether disease development is independently influenced by genetic background warrants further investigation.

Environmental influences, potentially impacting through microbiota composition, contribute to an absence of AS in the Fula people who inhabit the Gambia in West Africa, despite a moderate prevalence (6%) of HLA-B27 . This contrasts with AS epidemiology elsewhere in the world, in which the prevalence of AS is proportional to the HLA-B27 carriage rate in the community. This raises the possibility that local environment can influence the relationship between host genetics and microbial immunity, leading to protection from disease.

A lower abundance of “beneficial” bacteria was recently found in patients suffering from psoriatic arthritis compared to healthy controls . Interestingly, the profile of gut microbiota in psoriatic arthritis was similar to that reported in inflammatory bowel diseases . Anti-inflammatory cytokines, such as IL-22 or IL-10, in response to stimulation with microbiota or microbial metabolites may represent a mechanism by which microbiota prevent or control gut and joint inflammation . Beta-glucan-treated SKG mice have an IL-23-dependent reduction of goblet cells and mucin (Muc2) expression in the ileum, which is not observed in BALB/c mice or germ-free SKG mice . Therefore both genetic background and the microbiota contribute to a reduced epithelial barrier in SKG mice. Bacterial mucin degradation provides an important source of short-chain fatty acids required for regulatory T cell differentiation and for further mucin production . However, reduced mucin may change the homeostasis of the microbiota – so-called dysbiosis – and greater exposure of pathogenic organisms to the host. Pathogenic organisms within the microbiota may thus trigger the UPR or autophagy, leading to the production of pro-inflammatory cytokines, as well as recruitment of the adaptive immune system .

It is hoped that continued research in this area will provide a robust scientific rationale for the use of specific diet, antibiotics, probiotics and/or prebiotics, i.e. chemicals that induce the growth and/or activity of immunomodulatory microbiota in chronic inflammatory bowel disease and SpA.

HLA-B27 and viral infection

Several studies assessed the role of HLA-B27 in the context of HIV (human immunodeficiency virus) and HCV (hepatitis C virus) infections . A protective role of HLA-B27 has been proposed, providing new insights into genetic predisposition in viral infections. After acute HCV infection, whether asymptomatic or causing hepatitis characterized by jaundice, fatigue and raised liver enzymes, approximately one third of patients develop chronic HCV infection, predisposing to liver cirrhosis and hepatocellular carcinoma. Considering the critical role for CD8+ cytotoxic T cells in viral infections, the impact of MHC class I-restricted responses and clearance of the infection has been studied. Relative to other MHC class I alleles, HLA-B27 was associated with increased spontaneous HCV clearance, low viral loads, slow disease progression and stable CD4+ T cell counts in HIV infection .

Various mechanisms have been proposed to explain the protective role of HLA-B27 in chronic viral infections . Firstly, the immunodominant epitopes provoking the most intense immune responses towards HIV and HCV have been shown to trigger a very efficient HLA-B27-restricted CD8+ T cell toxicity. Secondly, HLA-B27 affects antigen presenting cell function, as dendritic cells from donors bearing this allele have altered function and reduced antigen presenting capacity . Thirdly, CD8+ T cells from HLA-B27 patients are more likely to escape regulatory T cell suppression, and may therefore display sustained cytotoxicity .