Gut microbiota profiles and the role of anti-CdtB and anti-vinculin antibodies in patients with functional gastrointestinal disorders (FGID).

BACKGROUND: Distinct faecal microbiota profiles are reported to be associated with various subtypes of IBS. Circulating antibodies to cytolethal distending toxin B (CdtB) and vinculin are proposed as biomarkers to identify post-infectious IBS. The aim of our study was to analyse serum levels of anti-CdtB and anti-vinculin antibodies in patients with different functional gastrointestinal disorders (FGID) and their correlation with the composition of faecal microbiome. METHODS: The study cohort comprised 65 prospectively recruited individuals: 15 with diarrhoea-type-IBS (IBS-D), 13 with constipation-type-IBS (IBS-C), 15 with functional dyspepsia (FD) and 22 healthy controls. FGID subgroups were defined according to Rome III criteria. Serum levels of anti-CdtB and anti-vinculin antibodies were measured by ELISA. Faecal microbiome composition analysis and assessment of dysbiosis were performed by GA-map® Dysbiosis Test. RESULTS: Positivity rate either for anti-CdtB or anti-vinculin antibodies was higher in the IBS-C group (76.9%) compared to IBS-D (40.0%), FD (60%) and healthy (63.6%) groups. Dysbiosis was more frequent in subjects positive for anti-CdtB antibodies and in IBS-C patients, who showed an increased amount of opportunistic/pro-inflammatory bacteria and reduced gut protective bacteria. IBS-C patients showed a high inter-individual variation of bacterial communities compared to other FGID subgroups and healthy individuals, whereas microbial profiles of patients with IBS-D and FD were overlapping with those of healthy controls. No bacteria markers showed significant differences between FGID subgroups and healthy controls. CONCLUSION: Neither anti-CdtB/anti-vinculin antibodies nor faecal microbial profiles allowed to discriminate between specific FGID subgroups. Dysbiosis was more frequent in patients presenting with anti-CdtB antibodies and in IBS-C patients.

Combining Calcium Phosphates with Polysaccharides: A Bone-Inspired Material Modulating Monocyte/Macrophage Early Inflammatory Response.

The use of inorganic calcium/phosphate supplemented with biopolymers has drawn lots of attention in bone regenerative medicine. While inflammation is required for bone healing, its exacerbation alters tissue regeneration/implants integration. Inspired by bone composition, a friendly automated spray-assisted system was used to build bioactive and osteoinductive calcium phosphate/chitosan/hyaluronic acid substrate (CaP-CHI-HA). Exposing monocytes to CaP-CHI-HA resulted in a secretion of pro-healing VEGF and TGF-ß growth factors, TNF-α, MCP-1, IL-6 and IL-8 pro-inflammatory mediators but also IL-10 anti-inflammatory cytokine along with an inflammatory index below 1.5 (versus 2.5 and 7.5 following CaP and LPS stimulation, respectively). Although CD44 hyaluronic acid receptor seems not to be involved in the inflammatory regulation, results suggest a potential role of chemical composition and calcium release from build-up substrates, in affecting the intracellular expression of a calcium-sensing receptor. Herein, our findings indicate a great potential of CaP-CHI-HA in providing required inflammation-healing balance, favorable for bone healing/regeneration.

Assessment of Anti-vinculin and Anti-cytolethal Distending Toxin B Antibodies in Subtypes of Irritable Bowel Syndrome.

BACKGROUND: Antibodies to cytolethal distending toxin B (CdtB) and vinculin are novel biomarkers that rule-in and differentiate irritable bowel syndrome with diarrhea (IBS-D) from other causes of diarrhea and healthy controls. AIM: To determine whether these antibodies can also diagnose and differentiate other IBS subtypes. METHODS: Subjects with IBS-D based on Rome III criteria (n = 2375) were recruited from a large-scale multicenter clinical trial (TARGET 3). Healthy subjects without gastrointestinal (GI) diseases or symptoms (n = 43) and subjects with mixed IBS (IBS-M) (n = 25) or IBS with constipation (IBS-C) (n = 30) were recruited from two major medical centers. Plasma levels of anti-CdtB and anti-vinculin antibodies in all subjects were determined by enzyme-linked immunosorbent assay. Optical densities of ≥1.68 and ≥2.80 were considered positive for anti-vinculin and anti-CdtB, respectively. Plasma levels of anti-CdtB and anti-vinculin antibodies were highest in IBS-D and lowest in IBS-C and healthy controls (P < 0.001). Levels in IBS-C subjects were not statistically different from controls (P > 0.1). Positivity for anti-CdtB or anti-vinculin resulted in a statistically significant negative gradient from IBS-D (58.1%) to IBS-M (44.0%), IBS-C (26.7%), and controls (16.3%) (P < 0.001). CONCLUSIONS: Anti-CdtB and anti-vinculin titers and positivity rates differ in IBS subtypes, with higher antibody levels and positivity rates in IBS-D and IBS-M, and lower levels in IBS-C subjects that are similar to those in healthy controls. These antibodies appear useful in the diagnosis of IBS-M and IBS-D, but not IBS-C. Furthermore, these findings suggest that IBS-C is pathophysiologically distinct from subtypes with diarrheal components (i.e., IBS-M and IBS-D).

Vinculin arrests motile B cells by stabilizing integrin clustering at the immune synapse.

Lymphocytes use integrin-based platforms to move and adhere firmly to the surface of other cells. The molecular mechanisms governing lymphocyte adhesion dynamics are however poorly understood. In this study, we show that in mouse B lymphocytes, the actin binding protein vinculin localizes to the ring-shaped integrin-rich domain of the immune synapse (IS); the assembly of this platform, triggered by cognate immune interactions, is needed for chemokine-mediated B cell motility arrest and leads to firm, long-lasting B cell adhesion to the APC. Vinculin is recruited early in IS formation, in parallel to a local phosphatidylinositol (4,5)-bisphosphate wave, and requires spleen tyrosine kinase activity. Lack of vinculin at the IS impairs firm adhesion, promoting, in turn, cell migration with Ag clustered at the uropod. Vinculin localization to the B cell contact area depends on actomyosin. These results identify vinculin as a major controller of integrin-mediated adhesion dynamics in B cells.

Autoimmunity Links Vinculin to the Pathophysiology of Chronic Functional Bowel Changes Following Campylobacter jejuni Infection in a Rat Model.

BACKGROUND: Acute gastroenteritis can precipitate irritable bowel syndrome (IBS) in humans. Cytolethal distending toxin is common to all pathogens causing gastroenteritis. Its active subunit, CdtB, is associated with post-infectious bowel changes in a rat model of Campylobacter jejuni infection, including small intestinal bacterial overgrowth (SIBO). AIM: To evaluate the role of host antibodies to CdtB in contributing to post-infectious functional sequelae in this rat model. METHODS: Ileal tissues from non-IBS human subjects, C. jejuni-infected and control rats were immunostained with antibodies to CdtB, c-Kit, S-100, PGP 9.5 and vinculin. Cytosolic and membrane proteins from mouse enteric neuronal cell lysates were immunoprecipitated with anti-CdtB and analyzed by mass spectrometry. ELISAs were performed on rat cardiac serum using CdtB or vinculin as antigens. RESULTS: Anti-CdtB antibodies bound to a cytosolic protein in interstitial cells of Cajal (ICC) and myenteric ganglia in C. jejuni-infected and naïve rats and human subjects. Mass spectrometry identified vinculin, confirmed by co-localization and ELISAs. Anti-CdtB antibodies were higher in C. jejuni-infected rats (1.27 ± 0.15) than controls (1.76 ± 0.12) (P < 0.05), and rats that developed SIBO (2.01 ± 0.18) vs. rats that did not (1.44 ± 0.11) (P = 0.019). Vinculin expression levels were reduced in C. jejuni-infected rats (0.058 ± 0.053) versus controls (0.087 ± 0.023) (P = 0.0001), with greater reductions in rats with two C. jejuni infections (P = 0.0001) and rats that developed SIBO (P = 0.001). CONCLUSIONS: Host anti-CdtB antibodies cross-react with vinculin in ICC and myenteric ganglia, required for normal gut motility. Circulating antibody levels and loss of vinculin expression correlate with number of C. jejuni exposures and SIBO, suggesting that effects on vinculin are important in the effects of C. jejuni infection on the host gut.

Targets of anti-endothelial cell antibodies in pulmonary hypertension and scleroderma.

Anti-endothelial cell antibodies (AECAs) have been identified in patients with systemic sclerosis (SSc) with and without pulmonary arterial hypertension (PAH) and in patients with idiopathic pulmonary arterial hypertension (iPAH). However, their target antigens remain poorly identified. Sera from 24 patients with SSc without PAH, 20 patients with SSc with PAH, 30 with iPAH and 12 healthy controls were collected. Target antigens were identified by two-dimensional electrophoresis and immunoblotting in protein extracts of human umbilical vein endothelial cells. Targeted antigens were identified by mass spectrometry. Serum immunoglobulin G from patients with SSc with or without PAH and patients with iPAH specifically recognised 110, 82 and 37 protein spots, respectively. Among others, target antigens of AECAs included lamin A/C, tubulin ß-chain and vinculin. One-dimension immunoblotting experiments confirmed the identification of lamin A/C and tubulin ß-chain. In conclusion, our results confirm the presence of AECA in patients with systemic sclerosis with and without pulmonary arterial hypertension and in those with idiopathic pulmonary arterial hypertension, and provide evidence for the identification of target antigens of these autoantibodies including lamin A/C and tubulin ß-chain.

Protein kinase G signaling disrupts Rac1-dependent focal adhesion assembly in liver specific pericytes.

Nitric oxide (NO) regulates the function of perivascular cells (pericytes), including hepatic stellate cells (HSC), mainly by activating cGMP and cGMP-dependent kinase (PKG) via NO/cGMP paracrine signaling. Although PKG is implicated in integrin-mediated cell adhesion to extracellular matrix, whether or how PKG signaling regulates the assembly of focal adhesion complexes (FA) and migration of HSC is not known. With the help of complementary molecular and cell biological approaches, we demonstrate here that activation of PKG signaling in HSC inhibits vascular tubulogenesis, migration/chemotaxis, and assembly of mature FA plaques, as assessed by vascular tubulogenesis assays and immunofluorescence localization of FA markers such as vinculin and vasodilator-stimulated phosphoprotein (VASP). To determine whether PKG inhibits FA assembly by phosphorylation of VASP at Ser-157, Ser-239, and Thr-278, we mutated these putative phosphorylation sites to alanine (VASP3A, phosphoresistant mutant) or aspartic acid (VASP3D, phosphomimetic), respectively. Data generated from these two mutants suggest that the effect of PKG on FA is independent of these three phosphorylation sites. In contrast, activation of PKG inhibits the activity of small GTPase Rac1 and its association with the effector protein IQGAP1. Moreover, PKG activation inhibits the formation of a trimeric protein complex containing Rac1, IQGAP1, and VASP. Finally, we found that expression of a constitutively active Rac1 mutant abolishes the inhibitory effects of PKG on FA formation. In summary, our data suggest that activation of PKG signaling in pericytes inhibits FA formation by inhibiting Rac1.

Autoreactive cytotoxic T lymphocytes in human immunodeficiency virus type 1-infected subjects.

A subtractive analysis of peptides eluted from major histocompatibility complex (MHC) class I human histocompatibility leukocyte antigen (HLA)-A2.1 molecules purified from either human immunodeficiency virus type-1 (HIV-1)-infected or uninfected cells was performed using micro high-performance liquid chromatography and mass spectrometry. Three peptides unique to infected cells were identified and found to derive from a single protein, human vinculin, a structural protein not known to be involved in viral pathogenesis. Molecular and cytofluorometric analyses revealed vinculin mRNA and vinculin protein overexpression in B and T lymphocytes from HIV-1-infected individuals. Vinculin peptide-specific CTL activity was readily elicited from peripheral blood lymphocytes of the majority of HLA-A2.1+, HIV+ patients tested. Our observations suggest that atypical vinculin expression and MHC class I-mediated presentation of vinculin-derived peptides accompany HIV infection of lymphoid cells in vivo, with a resultant induction of antivinculin CTL in a significant portion of HIV+ (HLA-A2.1+) individuals.

Immunization with cytolethal distending toxin B produces autoantibodies to vinculin and small bowel bacterial changes in a rat model of postinfectious irritable bowel syndrome.

BACKGROUND: Recent data substantiate the importance of acute gastroenteritis in the development of irritable bowel syndrome (IBS). An animal model of postinfectious IBS determined the importance of cytolethal distending toxin B (CdtB) during live Campylobacter jejuni infection and its development of autoimmunity to vinculin. In this study, we examine whether subcutaneous exposure to CdtB alone is sufficient to produce the postinfectious IBS effect and autoimmunity. METHODS: Sixty adult Sprague Dawley rats were randomized into 2 groups to receive subcutaneous injection of either CdtB or vehicle and administered a booster injection of the same product 3 weeks later. Serum was collected for anti-CdtB and anti-vinculin titers. Duodenal and ileal luminal contents for total eubacterial qPCR, and ileal bowel segments were harvested for vinculin and ileal expression. In a second experiment, 4 adult, Sprague Dawley rats were injected with either Cy7-labeled anti-CdtB and anti-vinculin antibodies were injected into the tail vein and imaged to determine organ localization of the antibodies. KEY RESULTS: Rats that received CdtB increased in serum anti-CdtB after injection. CdtB exposure also precipitated significant elevation in anti-vinculin antibodies (P < .001). This was associated with a reduction in intestinal vinculin expression (P < .001) that negatively correlated with serum anti-CdtB levels. CdtB exposure was also associated with greater levels of duodenal (P < .001) and ileal (P < .01) bacteria by qPCR that positively correlated with anti-CdtB levels. CONCLUSIONS AND INFERENCES: Rats injected with CdtB developed a postinfectious IBS-like phenotype and autoimmunity to vinculin with corresponding reduction in intestinal vinculin expression.

Circulating Anti-cytolethal Distending Toxin B and Anti-vinculin Antibodies as Biomarkers in Community and Healthcare Populations With Functional Dyspepsia and Irritable Bowel Syndrome.

OBJECTIVES: Anti-cytolethal distending toxin B (CdtB) and anti-vinculin antibodies have been proposed as biomarkers that discriminate irritable bowel syndrome (IBS) diarrhea from inflammatory bowel disease; however, it is unknown whether they can also discriminate patients with IBS and IBS subtypes and functional dyspepsia (FD) from healthy individuals in the general population. We aimed to determine whether anti-CdtB and anti-vinculin can discriminate IBS and FD from health and from organic gastrointestinal (GI) disease. METHODS: Adults were enrolled from 2 Australian studies: (i) a random, population-based study (n = 331) with subjects diagnosed with IBS (n = 63) or FD (n = 61) by modified Rome III criteria or healthy control subjects (n = 246) who did not meet criteria for IBS and/or FD and (ii) an outpatient-based study with subjects diagnosed with IBS (n = 256) and/or FD (n = 55) or organic GI disease (n = 182) by an independent clinician. Serum levels of anti-CdtB/anti-vinculin antibodies were determined by enzyme-linked immunosorbent assay. RESULTS: There was a significantly higher mean value of anti-CdtB in FD vs healthy controls (mean = 2.46 [SD = 0.72] vs mean = 2.14 [SD = 0.77]; P = 0.005) and IBS/FD overlap vs healthy controls (mean = 2.47 [SD = 0.78] vs mean = 2.14 [SD = 0.77]; P = 0.02). There were no significant differences in anti-CdtB in IBS and FD outpatients or IBS/FD subgroups compared with patients with organic GI disease. In terms of anti-vinculin, there were no significant differences between IBS and FD and healthy controls or between IBS and FD and organic GI disease controls. DISCUSSION: We did not confirm that anti-CdtB/anti-vinculin discriminated IBS diarrhea from organic GI disease in Australian subjects. However, we did find higher anti-CdtB in FD and IBS/FD overlap vs healthy controls. Postinfectious FD may be more common than currently recognized.

Coupling of ß2 integrins to actin by a mechanosensitive molecular clutch drives complement receptor-mediated phagocytosis.

αMß2 integrin (complement receptor 3) is a major receptor for phagocytosis in macrophages. In other contexts, integrins' activities and functions are mechanically linked to actin dynamics through focal adhesions. We asked whether mechanical coupling of αMß2 integrin to the actin cytoskeleton mediates phagocytosis. We found that particle internalization was driven by formation of Arp2/3 and formin-dependent actin protrusions that wrapped around the particle. Focal complex-like adhesions formed in the phagocytic cup that contained ß2 integrins, focal adhesion proteins and tyrosine kinases. Perturbation of talin and Syk demonstrated that a talin-dependent link between integrin and actin and Syk-mediated recruitment of vinculin enable force transmission to target particles and promote phagocytosis. Altering target mechanical properties demonstrated more efficient phagocytosis of stiffer targets. Thus, macrophages use tyrosine kinase signalling to build a mechanosensitive, talin- and vinculin-mediated, focal adhesion-like molecular clutch, which couples integrins to cytoskeletal forces to drive particle engulfment.

The novel Rho-family GTPase rif regulates coordinated actin-based membrane rearrangements.

Small GTPases of the Rho family have a critical role in controlling cell morphology, motility and adhesion through dynamic regulation of the actin cytoskeleton [1,2]. Individual Rho GTPases have been shown to regulate distinct components of the cytoskeletal architecture; RhoA stimulates the bundling of actin filaments into stress fibres [3], Rac reorganises actin to produce membrane sheets or lamellipodia [4] and Cdc42 causes the formation of thin, actin-rich surface projections called filopodia [5]. We have isolated a new Rho-family GTPase, Rif (Rho in filopodia), and shown that it represents an alternative signalling route to the generation of filopodial structures. Coordinated regulation of Rho-family GTPases can be used to generate more complicated actin rearrangements, such as those underlying cell migration [6]. In addition to inducing filopodia, Rif functions cooperatively with Cdc42 and Rac to generate additional structures, increasing the diversity of actin-based morphology.

Fluctuation of zonulin levels in blood vs stability of antibodies.

AIM: To evaluate the measurement of zonulin level and antibodies of zonulin and other tight junction proteins in the blood of controls and celiac disease patients. METHODS: This study was conducted to assess the variability or stability of zonulin levels vs IgA and IgG antibodies against zonulin in blood samples from 18 controls at 0, 6, 24 and 30 h after blood draw. We also measured zonulin level as well as zonulin, occludin, vinculin, aquaporin 4 and glial fibrillary acidic protein antibodies in the sera of 30 patients with celiac disease and 30 controls using enzyme-linked immunosorbent assay methodology. RESULTS: The serum zonulin level in 6 out of 18 subjects was low or < 2.8 ng/mL and was very close to the detection limit of the assay. The other 12 subjects had zonulin levels of > 2.8 ng/mL and showed significant fluctuation from sample to sample. Comparatively, zonulin antibody measured in all samples was highly stable and reproducible from sample to sample. Celiac disease patients showed zonulin levels with a mean of 8.5 ng/mL compared to 3.7 ng/mL in controls (P < 0.0001). Elevation of zonulin level at 2SD above the mean was demonstrated in 37% of celiac disease patients, while antibodies against zonulin, occludin and other tight junction proteins was detected in up to 86% of patients with celiac disease. CONCLUSION: Due to its fluctuation, a single measurement of zonulin level is not recommended for assessment of intestinal barrier integrity. Measurement of IgG and IgA antibodies against zonulin, occludin, and other tight junction proteins is proposed for the evaluation of the loss of intestinal barrier integrity.

PREL1 provides a link from Ras signalling to the actin cytoskeleton via Ena/VASP proteins.

Ena/VASP family proteins are important modulators of cell migration and localize to focal adhesions, stress fibres and the very tips of lamellipodia and filopodia. Proline-rich proteins like vinculin and zyxin are well established interaction partners, which mediate Ena/VASP-recruitment via their EVH1-domains to focal adhesions and stress fibres. However, it is still unclear, which binding partners Ena/VASP proteins may have at lamellipodia tips and how their recruitment to these cellular protrusions is regulated. Here, we report the identification of a novel protein with high similarity to the C. elegans MIG-10 protein, which we termed PREL1 (Proline Rich EVH1 Ligand). PREL1 is a 74 kDa protein and shares homology with the Grb7-family of signalling adaptors. We show that PREL1 directly binds to Ena/VASP proteins and co-localizes with them at lamellipodia tips and at focal adhesions in response to Ras activation. Moreover, PREL1 directly binds to activated Ras in a phosphoinositide-dependent manner. Thus, our data pinpoint PREL1 as the first direct link between Ras signalling and cytoskeletal remodelling via Ena/VASP proteins during cell migration and spreading.

Distinct patterns of dystrophin organization in myocyte sarcolemma and transverse tubules of normal and diseased human myocardium.

BACKGROUND: Genetic mutations of dystrophin and associated glycoproteins underlie cell degeneration in several inherited cardiomyopathies, although the precise physiological role of these proteins remains under discussion. We studied the distribution of dystrophin in relation to the force-transducing vinculin-rich costameres in left ventricular cardiomyocytes from normal and failing human hearts to further elucidate the function of this protein complex. METHODS AND RESULTS: Single- and double-label immunoconfocal microscopy and parallel high-resolution immunogold fracture-label electron microscopy were used to localize dystrophin and vinculin in human left ventricular myocytes from normal (n=6) and failing hearts (idiopathic dilated cardiomyopathy, n=7, or ischemic heart disease, n=5). In control cardiomyocytes, dystrophin had a continuous distribution at the peripheral sarcolemma, with concentrated bands corresponding to the vinculin-rich costameres. Intracellular labeling extended along transverse (T) tubule membranes. Fracture-label confirmed this distribution, showing significantly greater label on plasma membrane fractures overlying I-bands (I-band 4.1+/-0.3 gold particles/micrometer A-band 3.3+/-0.2 gold particles/micrometer mean+/-SE, P=0.02). Hypertrophied myocytes from failing hearts showed maintenance of this surface distribution except in degenerating cells; there was a clear increase in intracellular dystrophin label reflecting T-tubule hypertrophy. CONCLUSIONS: Dystrophin partially colocalizes with costameric vinculin in normal and hypertrophied myocytes, a distribution lost in degenerating cells. This suggests a primarily mechanical role for dystrophin in maintenance of cell membrane integrity in normal and hypertrophied myocytes. The presence of dystrophin in the cardiac T-tubule membrane, in contrast to its known absence in skeletal muscle T-tubules, implies additional roles for dystrophin in membrane domain organization.

PREL1 provides a link from Ras signalling to the actin cytoskeleton via Ena/VASP proteins.

Ena/VASP family proteins are important modulators of cell migration and localize to focal adhesions, stress fibres and the very tips of lamellipodia and filopodia. Proline-rich proteins like vinculin and zyxin are well established interaction partners, which mediate Ena/VASP-recruitment via their EVH1-domains to focal adhesions and stress fibres. However, it is still unclear, which binding partners Ena/VASP proteins may have at lamellipodia tips and how their recruitment to these cellular protrusions is regulated. Here, we report the identification of a novel protein with high similarity to the C. elegans MIG-10 protein, which we termed PREL1 (Proline Rich EVH1 Ligand). PREL1 is a 74 kDa protein and shares homology with the Grb7-family of signalling adaptors. We show that PREL1 directly binds to Ena/VASP proteins and co-localizes with them at lamellipodia tips and at focal adhesions in response to Ras activation. Moreover, PREL1 directly binds to activated Ras in a phosphoinositide-dependent manner. Thus, our data pinpoint PREL1 as the first direct link between Ras signalling and cytoskeletal remodelling via Ena/VASP proteins during cell migration and spreading.

Regulation and localization of CAS substrate domain tyrosine phosphorylation.

Crk-associated substrate (CAS) is a tyrosine kinase substrate implicated in integrin control of cell behavior. Phosphorylation, by Src family kinases, of multiple tyrosine residues in the CAS substrate domain (SD) is a major integrin signaling event that promotes cell motility. In this study, novel phosphospecific antibodies directed against CAS SD phosphotyrosine sites ("pCAS" antibodies) were characterized and employed to investigate the cellular regulation and localization of CAS SD tyrosine phosphorylation. An analysis of CAS and focal adhesion kinase (FAK) variants expressed in CAS- and FAK-deficient cell lines, respectively, indicated that CAS SD tyrosine phosphorylation is substantially achieved by Src family kinases brought into association with CAS through two distinct mechanisms: direct binding to the CAS Src-binding domain and indirect association through a FAK bridge. Cell immunostaining with pCAS antibodies revealed that CAS SD tyrosine phosphorylation occurs exclusively at sites of integrin adhesion including both nascent focal complexes formed at the edges of extending lamellipodia as well as mature focal adhesions underlying the cell body. These findings further document a role for FAK as an important upstream regulator of CAS SD tyrosine phosphorylation and implicate CAS-mediated signaling events in promoting membrane protrusion/lamellipodium extension during cell motility.

Crossreactivity to vinculin and microbes provides a molecular basis for HLA-based protection against rheumatoid arthritis.

The HLA locus is the strongest risk factor for anti-citrullinated protein antibody (ACPA)(+) rheumatoid arthritis (RA). Despite considerable efforts in the last 35 years, this association is poorly understood. Here we identify (citrullinated) vinculin, present in the joints of ACPA(+) RA patients, as an autoantigen targeted by ACPA and CD4(+) T cells. These T cells recognize an epitope with the core sequence DERAA, which is also found in many microbes and in protective HLA-DRB1*13 molecules, presented by predisposing HLA-DQ molecules. Moreover, these T cells crossreact with vinculin-derived and microbial-derived DERAA epitopes. Intriguingly, DERAA-directed T cells are not detected in HLA-DRB1*13(+) donors, indicating that the DERAA epitope from HLA-DRB1*13 mediates (thymic) tolerance in these donors and explaining the protective effects associated with HLA-DRB1*13. Together our data indicate the involvement of pathogen-induced DERAA-directed T cells in the HLA-RA association and provide a molecular basis for the contribution of protective/predisposing HLA alleles.