Fabiana Paladini

Interaction between ERAP1 and HLA-B27 in ankylosing spondylitis implicates peptide handling in the mechanism for HLA-B27 in disease susceptibility

Ankylosing spondylitis is a common form of inflammatory arthritis predominantly affecting the spine and pelvis that occurs in approximately 5 out of 1,000 adults of European descent. Here we report the identification of three variants in the RUNX3, LTBR-TNFRSF1A and IL12B regions convincingly associated with ankylosing spondylitis (P < 5 × 10−8 in the combined discovery and replication datasets) and a further four loci at PTGER4, TBKBP1, ANTXR2 and CARD9 that show strong association across all our datasets (P < 5 × 10−6 overall, with support in each of the three datasets studied). We also show that polymorphisms of ERAP1, which encodes an endoplasmic reticulum aminopeptidase involved in peptide trimming before HLA class I presentation, only affect ankylosing spondylitis risk in HLA-B27–positive individuals. These findings provide strong evidence that HLA-B27 operates in ankylosing spondylitis through a mechanism involving aberrant processing of antigenic peptides.

The interplay between the geographic distribution of HLA-B27 alleles and their role in infectious and autoimmune diseases: A unifying hypothesis

Due to its strong association with Ankylosing Spondylitis and the other Spondyloarthropathies, the HLA-B27 family of alleles and, in particular, the ancestral HLA-B*2705, has been the object of numerous studies. More recently, some novel interesting features have emerged such as the ability of HLA-B27 to confer resistance to the progression of HIV infection and to promote a spontaneous CD8+ T cell-mediated viral clearance of HCV. The co-occurrence of these protective and pathogenic features suggests a common ground, i.e. to promote a more pronounced immune/inflammatory response leading to an effective clearance of some pathogens on one side and to autoimmunity on the other. This might be due to the antigen presenting properties and/or to the co-inheritance of gene variants that contribute to an altered homeostasis in case of microbial infections or tissue injury. The existence of conserved HLA haplotypes have since long been thought to result from a selective pressure by some pathogens that have edited the immune response genes. The peculiar distribution of the ancestor HLA-B*2705 along a latitude-dependent gradient and the opposite distribution of their variants have suggested a correlation with endemic malaria. In this respect, Sardinia, a small Mediterranean island plagued by malaria, represents an interesting laboratory since its population is enriched in conserved HLA haplotypes and several genetic studies have disclosed their correlation with infectious and autoimmune diseases.

Age‐dependent association of idiopathic achalasia with vasoactive intestinal peptide receptor 1 gene

Abstract  Idiopathic achalasia is a rare disorder of the oesophagus of unknown aetio‐pathogenesis characterized by a myenteric inflammation, aperistalsis and insufficient lower oesophageal sphincter relaxation. Vasoactive intestinal peptide (VIP), present in the myenteric plexus, is involved in smooth muscle relaxation and acts as an anti‐inflammatory cytokine. The human VIP receptor 1 gene (VIPR1) is highly polymorphic and may play a role in idiopathic achalasia. One hundred and four consecutive patients and 300 random controls from the same geographic area were typed for five SNPs mapping in the VIPR1 gene. Patients with idiopathic achalasia show a significant difference in allele, genotype and phenotype distribution of SNP rs437876 mapping in intron 4. This association, however, was almost entirely due to the group of patients with late disease onset (P = 0.0005). These results strongly suggest that idiopathic achalasia is a heterogeneous disease with a different aetiology in cases with early or late disease onset.

A functional polymorphism of the vasoactive intestinal peptide receptor 1 gene correlates with the presence of HLA-B * 2705 in Sardinia

The association of HLA-B27 with ankylosing spondylitis (AS) is the strongest among all inflammatory diseases. However, the exact role of these molecules in disease pathogenesis is still unknown. The existence of HLA-B27 variants rarely found in patients introduces a further level of complexity. It is now accepted that other genes of minor impact contribute to modify disease susceptibility and these genes might be diverse in different populations depending on the genetic background. We report here a study performed in Sardinia, an outlier population in which two major HLA-B27 subtypes are present, B *2705 strongly associated with AS and B *2709 which is not, and show the co-occurrence of the B *2705 allele with a single nucleotide polymorphism (SNP) mapping at 3′-UTR of the receptor 1 (VIPR1) for the vasoactive intestinal peptide (VIP), a neuropeptide with anti-inflammatory properties. This same SNP is associated with a different kinetics of down-modulation of the VIPR1 mRNA in monocytes after exposure to lipopolysaccharide (P=0.004). This particular setting, HLA-B *2705 and a functional polymorphism in VIPR1 gene, might be due to a founder effect or might be the result of a selective pressure. Irrespectively, the consequent downregulation of this receptor in the presence of a ‘danger’ signal might influence susceptibility to AS.

Citrullination-dependent Differential Presentation of a Self-peptide by HLA-B27 Subtypes

Inflammatory processes are accompanied by the posttranslational modification of certain arginine residues within proteins to yield citrulline, although it is largely unknown how this modification influences antigen presentation. We employed crystallographic and functional studies to investigate whether the exchange of arginine to citrulline affects the display of a peptide by two human major histocompatibility antigen class I subtypes, HLA-B*2705 and HLA-B*2709. Both differ only in residue 116 within the peptide binding groove despite their differential association with ankylosing spondylitis, an inflammatory rheumatic disorder. The crystal structures described here show that a modified self-peptide, pVIPR-U5 (RRKWURWHL; U = citrulline), is presented by the two HLA-B27 molecules in distinct conformations. These binding modes differ not only drastically from each other but also from the conformations exhibited by the non-citrullinated peptide in a given subtype. The differential reactivity of HLA-B27-restricted cytotoxic T cells with modified or unmodified pVIPR supports the structural findings and shows that the presentation of citrullinated peptides has the potential to influence immune responses.

HLA-E gene polymorphism associates with ankylosing spondylitis in Sardinia

IntroductionAnkylosing spondylitis (AS) is a severe, chronic inflammatory disease strongly associated with HLA-B27. The presence of additional HLA risk factors has been suggested by several studies. The aim of the current study is to assess the occurrence of an additional HLA susceptibility locus in the region between HLA-E and HLA-C in the Sardinian population.Methods200 random controls, 120 patients with AS and 175 HLA-B27 positive controls were genotyped for six single nucleotide polymorphisms (SNPs) spanning the HLA region between HLA-E and HLA-C loci previously shown to harbour an additional susceptibility locus for AS. Allele, genotype and haplotype frequencies were compared.ResultsThe data confirm our previous finding of a significant increase in patients with AS of allele A at SNP rs1264457 encoding for an Arg at the functional HLA-E polymorphism (Arg128/Gly128). This was due to a remarkable increase in the frequency of genotype A/A in patients vs HLA-B27-matched controls (51% vs 29%; P for trend: 5 × 10-5). Genotype distribution of three other SNPs mapping in genes (GNL1, PRR3 and ABCF-1) close to HLA-E and showing high LD with it, was also significantly skewed. Accordingly, haplotype distribution was also remarkably different. The frequency of the haplotype AAGA, is 42% in random controls, increases to 53% in the HLA-B27-positive controls, and reaches 68% in patients with AS (P values: 2 × 10-11 vs random and 3 × 10-4 vs HLA-B27 controls).ConclusionsThere is a strong association between the presence of a haplotype in genes mapping between HLA-E and HLA-C and AS due to an increase of homozygous markers in patients. The strongest association however, is with the HLA-E functional polymorphism rs1264457. Since HLA-E is the ligand for the NKG2A receptor, these data point to the natural killer (NK) activity as possible player in the pathogenesis of AS.

The expression of vasoactive intestinal peptide receptor 1 is negatively modulated by microRNA 525-5p

Background The human Vasoactive Intestinal Peptide (VIP) is a neurokine with effects on the immune system where it is involved in promoting tolerance. In this context, one of its receptors, VPAC1, has been found to be down-modulated in cells of the immune network in response to activating stimuli. In particular, the bacterial liposaccaride (LPS), a strong activator of the innate immune system, induces a rapid decrease of VPAC1 expression in monocytes and this event correlates with polymorphisms in the 3′-UTR of the gene. Methodology/Principal Findings MicroRNA 525-5p, having as putative target the 3′-UTR region of VPAC1, has been analysed for its expression in monocytes and for its role in down-modulating VPAC1 expression. We report here that miR-525-5p is promptly up-regulated in LPS-treated monocytes. This microRNA, when co-transfected in 293T cells together with a construct containing the 3′-UTR of the VPAC1 gene, significantly reduced the luciferase activity in a standard expression assay. The U937 cell line as well as primary monocytes enforced to express miR-525-5p, both down-modulate VPAC1 expression at similar extent. Conclusions/Significance Our results show that the response to an inflammatory stimulus elicits in monocytes a rapid increase of miR-525-5p that targets a signaling pathway involved in the control of the immune homeostasis.

CD8+ T-cell mediated self-reactivity in HLA-B27 context as a consequence of dual peptide conformation.

HLA-B2709 does not predispose for Ankylosing Spondylitis although it differs from B2705, the most common and AS-associated subtype in different ethnic groups, only for the substitution His116Asp. Therefore, a productive approach to elucidate the molecular mechanisms of the disease could be the comparison of these alleles. B2705 has been shown to display certain self-peptides enriched in basic residues i.e., pVIPR and pGR, in a dual conformation and this is accompanied by the presence of specific cytotoxic T cells in patients with AS. In this study, we convalidate our previous observation that B2709 healthy subjects do not possess primary reactivity towards pVIPR while showing a prompt CD8+ T cell response driven by pGR. Notably, in the B2709 context of presentation, pVIPR assumes only a single conformation in contrast with pGR which is dimorphic. These results suggest a possible general connection between the occurrence of double peptide conformation and the property of inducing specific autoimmune responses.

Regulation and trafficking of the HLA-E molecules during monocyte- macrophage differentiation

HLA‐E is a nonclassical HLA‐class I molecule whose best known role is to protect from the natural killer cells. More recently, an additional function more similar to that of classical HLA‐class I molecules, i.e., antigen presentation to T cells, is emerging. However, much remains to be explored about the intracellular trafficking of the HLA‐E molecules. With the use of 3 different cellular contexts, 2 monocytic cell lines, U937 and THP1, and peripheral blood monocytes, we show here a remarkable increase of HLA‐E during monocyte‐macrophage differentiation. This goes independently from the classical HLA‐class I, the main source of HLA‐E‐specific peptides, which is found strongly up‐regulated upon differentiation of peripheral blood monocytes but not at all in the case of U937 and THP1 cell lines. Although in all cases, there was a moderate increase of HLA‐E expressed in the cell surface, lysis by natural killer cells is comparably restored by an anti‐NKG2A antibody in untreated as well as in PMA‐differentiated U937 cells. Instead, the great majority of the HLA‐E is retained in the vesicles of the autophagy‐lysosome network, where they colocalize with the microtubule‐associated protein light chain 3, as well as with the lysosomal‐associated membrane protein 1. We conclude that differently from the classical HLA‐class I molecules, the primary destination of the newly synthesized HLA‐E molecules in macrophages is, rather than the cell membrane, the intracellular autophagy‐lysosomal vesicles where they are stored and where they can encounter the exogenous antigens.

Characterization of a Proteasome and TAP-independent Presentation of Intracellular Epitopes by HLA-B27 Molecules

Background: HLA-B*2705-Asp116 and HLA-B*2709-His-116 associate differently with AS. Both possess an unpaired Cys-67 in the B pocket. Results: The two molecules cross-present antigens through a proteasome-independent route. Cys-67, although not endorsing this alternative pathway, contributes variously to peptide stabilization. Conclusion: B27 molecules can load peptides in post-ER compartments. Asp-116/His-116 influences the need of an intact B-pocket. Significance: Identifying B27 distinctive features might contribute to the understanding of AS pathogenesis. Nascent HLA-class I molecules are stabilized by proteasome-derived peptides in the ER and the new complexes proceed to the cell surface through the post-ER vesicles. It has been shown, however, that less stable complexes can exchange peptides in the Trans Golgi Network (TGN). HLA-B27 are the most studied HLA-class I molecules due to their association with Ankylosing Spondylitis (AS). Chimeric proteins driven by TAT of HIV have been exploited by us to deliver viral epitopes, whose cross-presentation by the HLA-B27 molecules was proteasome and TAP-independent and not restricted to Antigen-Presenting Cells (APC). Here, using these chimeric proteins as epitope suppliers, we compared with each other and with the HLA-A2 molecules, the two HLA-B*2705 and B*2709 alleles differing at residue 116 (D116H) and differentially associated with AS. We found that the antigen presentation by the two HLA-B27 molecules was proteasome-, TAP-, and APC-independent whereas the presentation by the HLA-A2 molecules required proteasome, TAP and professional APC. Assuming that such difference could be due to the unpaired, highly reactive Cys-67 distinguishing the HLA-B27 molecules, C67S mutants in HLA-B*2705 and B*2709 and V67C mutant in HLA-A*0201 were also analyzed. The results showed that this mutation did not influence the HLA-A2-restricted antigen presentation while it drastically affected the HLA-B27-restricted presentation with, however, remarkable differences between B*2705 and B*2709. The data, together with the occurrence on the cell surface of unfolded molecules in the case of C67S-B*2705 mutant but not in that of C67S-B*2709 mutant, indicates that Cys-67 has a more critical role in stabilizing the B*2705 rather than the B*2709 complexes.