José Manuel Martín-Villa

Gut immune system and oral tolerance.

Gut mucosal surfaces separate the external environment from the internal sterile environment and so represent a first line of defence system. This barrier faces environments rich in pathogens that have developed effective mechanisms for colonisation of epithelial surfaces and invasion of mucosal tissues, but also harmless antigens such as food, airborne antigens or commensal bacterial flora. The latter represent the vast majority of the encountered antigens and require an appropriate response characterised by either ignorance or active suppression. However, for the former, a robust immune response is needed. Mucosae have developed a complex immune system that is capable of mounting an immune response against pathogenic antigens, while maintaining the required ignorance or active suppression against non-pathogenic antigens. Taking advantage of this knowledge, strategies have been devised to induce oral tolerance to antigens involved in experimental autoimmune disease or human conditions. It is now known that oral tolerance induces the up-regulation and activation of T cells with regulatory properties, a subtype of CD4⁺ T cells whose function is to regulate functions of other T lymphocytes to avoid excessive immune activation. Amongst them, the Th3 cells (cells that express the latency-associated peptide on the surface and secrete transforming growth factor β, a cytokine with immunoregulatory properties) are especially relevant in the induction of oral tolerance. Orally fed antigens seek to generate these types of cells in the treatment of autoimmune diseases in experimental animals or human subjects.

Pro-inflammatory self-reactive T cells are found within murine TCR-αβ(+) CD4(-) CD8(-) PD-1(+) cells.

TCR‐αβ+ double negative (DN) T cells (CD3+TCR‐αβ+CD4−CD8−NK1.1−CD49b−) represent a minor heterogeneous population in healthy humans and mice. These cells have been ascribed pro‐inflammatory and regulatory capacities and are known to expand during the course of several autoimmune diseases. Importantly, previous studies have shown that self‐reactive CD8+ T cells become DN after activation by self‐antigens, suggesting that self‐reactive T cells may exist within the DN T‐cell population. Here, we demonstrate that programmed cell death 1 (PD‐1) expression in unmanipulated mice identifies a subset of DN T cells with expression of activation‐associated markers and a phenotype that strongly suggests they are derived from self‐reactive CD8+ cells. We also found that, within DN T cells, the PD‐1+ subset generates the majority of pro‐inflammatory cytokines. Finally, using a TCR‐activation reporter mouse (Nur77‐GFP), we confirmed that in the steady‐state PD‐1+ DN T cells engage endogenous antigens in healthy mice. In conclusion, we provide evidence that indicates that the PD‐1+ fraction of DN T cells represents self‐reactive cells.

Evaluation of the IL2/IL21, IL2RA and IL2RB genetic variants influence on the endogenous non-anterior uveitis genetic predisposition

BackgroundRecently, different genetic variants located within the IL2/IL21 genetic region as well as within both IL2RA and IL2RB loci have been associated to multiple autoimmune disorders. We aimed to investigate for the first time the potential influence of the IL2/IL21, IL2RA and IL2RB most associated polymorphisms with autoimmunity on the endogenous non-anterior uveitis genetic predisposition.MethodsA total of 196 patients with endogenous non-anterior uveitis and 760 healthy controls, all of them from Caucasian population, were included in the current study. The IL2/IL21 (rs2069762, rs6822844 and rs907715), IL2RA (2104286, rs11594656 and rs12722495) and IL2RB (rs743777) genetic variants were genotyped using TaqMan® allelic discrimination assays.ResultsA statistically significant difference was found for the rs6822844 (IL2/IL21 region) minor allele frequency in the group of uveitis patients compared with controls (P-value=0.02, OR=0.64 CI 95%=0.43-0.94) although the significance was lost after multiple testing correction. Furthermore, no evidence of association with uveitis was detected for the analyzed genetic variants of the IL2RA or IL2RB loci.ConclusionOur results indicate that analyzed IL2/IL21, IL2RA and IL2RB polymorphisms do not seem to play a significant role on the non-anterior uveitis genetic predisposition although further studies are needed in order to clear up the influence of these loci on the non-anterior uveitis susceptibility.

Coeliac- and enteropathy-associated autoantibodies in Spanish insulin-dependent diabetes mellitus patients and their relation to HLA antigens

The frequency of reticulin (ARA), endomysium (EmA), and gut epithelial cell (GECA) autoantibodies, and gliadin antibodies (AGA), was investigated in 86 Spanish diabetic patients by indirect immunofluorescence (IFI) and ELISA, along with their HLA phenotype. Four patients (5%) showed ARA-IgG (R1 pattern), eight (9%) showed AGA-IgG, and eight (9%) showed AGA-IgA. No EmA or GECA-positive patients were found. In diabetic patients, HLA-DR7 is increased in ARA-IgG+ vs. ARA-IgG- (though not significantly), and HLA-DR6 and HLA-DQ1 are significantly increased in the AGA-IgG+ group vs. the AGA-IgG- group. Comparison with a non-diabetic coeliac group showed that HLA-DR4 and HLA-DQ3 are significantly increased in the AGA-IgA+ group, whereas HLA-DQ2 shows a significant decrease in the AGA-IgG+ and AGA-IgA+ patients. Finally, when compared to the healthy group, HLA-DR7 frequency is decreased in the ARA-IgG- group, while HLA-DQ3 is significantly increased and HLA-DR6 and HLA-DQ1 significantly decreased in the AGA-IgG- group.Altogether, these data suggest that the genetic background leading to the appearance of coeliac-specific autoantibodies in Spanish diabetic patients differ depending on the autoantibody produced and is also different to the genetic background leading to diabetes in Spain.

Crk adaptors negatively regulate actin polymerization in pedestals formed by enteropathogenic Escherichia coli (EPEC) by binding to Tir effector.

Infections by enteropathogenic Escherichia coli (EPEC) cause diarrhea linked to high infant mortality in developing countries. EPEC adheres to epithelial cells and induces the formation of actin pedestals. Actin polymerization is driven fundamentally through signaling mediated by Tir bacterial effector protein, which inserts in the plasma membrane of the infected cell. Tir binds Nck adaptor proteins, which in turn recruit and activate N-WASP, a ubiquitous member of the Wiskott-Aldrich syndrome family of proteins. N-WASP activates the Arp2/3 complex to promote actin polymerization. Other proteins aside from components of the Tir-Nck-N-WASP pathway are recruited to the pedestals but their functions are unknown. Here we investigate the function of two alternatively spliced isoforms of Crk adaptors (CrkI/II) and the paralog protein CrkL during pedestal formation by EPEC. We found that the Crk isoforms act as redundant inhibitors of pedestal formation. The SH2 domain of CrkII and CrkL binds to phosphorylated tyrosine 474 of Tir and competes with Nck to bind Tir, preventing its recruitment to pedestals and thereby inhibiting actin polymerization. EPEC infection induces phosphorylation of the major regulatory tyrosine in CrkII and CrkL, possibly preventing the SH2 domain of these proteins from interacting with Tir. Phosphorylated CrkII and CrkL proteins localize specifically to the plasma membrane in contact with EPEC. Our study uncovers a novel role for Crk adaptors at pedestals, opening a new perspective in how these oncoproteins regulate actin polymerization.

Blau syndrome-related CARD15/NOD2 mutations are not linked to idiopathic uveitis in Spanish patients.

Uveitis is a clinical feature of the Blau syndrome, a disease linked to CARD15 (also referred to as NOD2) mutations. Three main mutations in this gene (R334W, R334Q and L469F) have been reported as Blau syndrome risk factors, a disease that manifests uveitis as one of its clinical features. However, little is known on the involvement of this gene in idiopathic uveitis. We thus sought to determine the frequency of these Blau-related CARD15 mutations in a cohort of Spanish patients with idiopathic uveitis. To this aim, 110 patients with idiopathic uveitis, followed at the Department of Ophtalmology of a tertiary hospital (Hospital Universitario Alcalá de Henares, Madrid. Spain) were enrolled. As a control population, 104 healthy subjects were used. DNA was extracted from blood samples and the Blau-related CARD15 mutations were analysed either by PCR-RFLP or direct DNA sequencing. None of the mutations studied was found in any of the individuals tested, whether diseased or healthy. It seems thus that Blau syndrome-related CARD15 mutations are not involved in idiopathic uveitis, a finding which allows us to suggest that the genetic aetiology of the idiopathic uveitis or the Blau-associated uveitis is different.

Increased levels of bovine serum albumin antibodies in patients with type 1 diabetes and celiac disease-related antibodies.

Objectives To detect the presence of antibodies against bovine serum albumin in a cohort of Spanish patients with type 1 insulin-dependent diabetes. Methods Antibodies were measured using an in-house enzyme-linked immunosorbent assay test in 80 patients with type 1 diabetes, subdivided according to the presence or absence in their serum of celiac disease-related antibodies. For comparison, 30 patients with celiac disease (nondiabetic), 13 patients with autoimmune thyroiditis, and 45 healthy volunteers were used. Results Thirty-one percent of patients with diabetes yielded a positive result, with a mean value of 26.1 ± 21.8 arbitrary units (AU). If the group was split into those with celiac disease-related antibodies and those lacking them, the percentages were 53% and 25%, respectively, with a mean value of 39.6 ± 28.4 AU and 22.4 ± 18.3 AU (P = 0.003), respectively. Seventy-three percent of celiac patients showed bovine serum albumin antibodies with a mean level of 38.8 ± 27.7 AU, comparable to that of patients with diabetes with celiac antibodies, but higher than the group lacking them (P = 0.001). Although 46% of patients with autoimmune thyroiditis had positive results, the level detected (22.1 ± 8.7 AU) was significantly lower than that recorded in patients with type 1 diabetes who had celiac disease antibodies (P = 0.04) and celiac patients (P = 0.04). Healthy volunteers showed no antibodies against bovine serum albumin. Conclusions These data suggest that bovine serum albumin antibodies appears in patients with a compromised epithelial permeability, and they reflect a general defect in the process of immunologic tolerance associated with a predisposition to autoimmunity, rather than immunity specific to &bgr; cells.

Characterisation and functional implications of the two new HLA-G alleles found in Amerindian and Caribbean populations.

HLA-G polymorphism has been found to be relatively low in all world populations. In the present paper two new HLA-G molecules are described in ancient American natives. A new HLA-G molecule from a Ecuador Amerindian individual (male) showed four codon changes with respect to HLA-G*01:01:01. Silent changes at α1 domain (residue 57, Pro, CCG→CCA) and α2 domain (residue 93, His, CAC→CAT and residue 100, Gly, GGC→GGT) and one productive change in α3 domain (residue 219 changed from Arg to Trp). This α3 change may dramatically alter HLA-G interactions with beta-2 microglobulin, CD8, ILT-2 and ILT-4 ligands present in subsets of T, B, NK, monocytes, macrophages and dentritic cells. Another HLA-G new molecule was found in a woman from Hispaniola Island, Dominican Republic (Sto Domingo): it presented a silent change at α2 domain residue 107, Gly, GGA→GGT and non-silent change at residue 178, Met→Thr (with respect to HLA-G*01:01:01) which is close to class I molecule/clonotypic T cell receptor interaction sites. Functional implications of these findings are discussed.

New insights into the genetic component of non-infectious uveitis through an Immunochip strategy

Background Large-scale genetic studies have reported several loci associated with specific disorders involving uveitis. Our aim was to identify genetic risk factors that might predispose to uveitis per se, independent of the clinical diagnosis, by performing a dense genotyping of immune-related loci. Methods 613 cases and 3693 unaffected controls from three European case/control sets were genotyped using the Immunochip array. Only patients with non-infectious non-anterior uveitis and without systemic features were selected. To perform a more comprehensive analysis of the human leucocyte antigen (HLA) region, SNPs, classical alleles and polymorphic amino acid variants were obtained via imputation. A meta-analysis combining the three case/control sets was conducted by the inverse variance method. Results The highest peak belonged to the HLA region. A more detailed analysis of this signal evidenced a strong association between the classical allele HLA-A*2902 and birdshot chorioretinopathy (p=3.21E-35, OR=50.95). An omnibus test yielded HLA-A 62 and 63 as relevant amino acid positions for this disease. In patients with intermediate and posterior uveitis, the strongest associations belonged to the rs7197 polymorphism, within HLA-DRA (p=2.07E-11, OR=1.99), and the HLA-DR15 haplotype (DRB1*1501: p=1.16E-10, OR=2.08; DQA1*0102: p=4.37E-09, OR=1.77; DQB1*0602: p=7.26E-10, OR=2.02). Outside the HLA region, the MAP4K4/IL1R2 locus reached statistical significance (rs7608679: p=8.38E-07, OR=1.42). Suggestive associations were found at five other loci. Conclusions We have further interrogated the association between the HLA region and non-infectious non-anterior uveitis. In addition, we have identified a new non-HLA susceptibility factor and proposed additional risk loci with putative roles in this complex condition.

Two Functional Variants of IRF5 Influence the Development of Macular Edema in Patients with Non- Anterior Uveitis

Objective Interferon (IFN) signaling plays a crucial role in autoimmunity. Genetic variation in interferon regulatory factor 5 (IRF5), a major regulator of the type I interferon induction, has been associated with risk of developing several autoimmune diseases. In the current study we aimed to evaluate whether three sets of correlated IRF5 genetic variants, independently associated with SLE and with different functional roles, are involved in uveitis susceptibility and its clinical subphenotypes. Methods Three IRF5 polymorphisms, rs2004640, rs2070197 and rs10954213, representative of each group, were genotyped using TaqMan® allelic discrimination assays in a total of 263 non-anterior uveitis patients and 724 healthy controls of Spanish origin. Results A clear association between two of the three analyzed genetic variants, rs2004640 and rs10954213, and the absence of macular edema was observed in the case/control analysis (P FDR=5.07E-03, OR=1.48, CI 95%=1.14-1.92 and P FDR=3.37E-03, OR=1.54, CI 95%=1.19-2.01, respectively). Consistently, the subphenotype analysis accordingly with the presence/absence of this clinical condition also reached statistical significance (rs2004640: P=0.037, OR=0.69, CI 95%=0.48-0.98; rs10954213: P=0.030, OR=0.67, CI 95%=0.47-0.96), thus suggesting that both IRF5 genetic variants are specifically associated with the lack of macular edema in uveitis patients. Conclusion Our results clearly showed for the first time that two functional genetic variants of IRF5 may play a role in the development of macular edema in non-anterior uveitis patients. Identifying genetic markers for macular edema could lead to the possibility of developing novel treatments or preventive therapies.