An efficient urine peptidomics workflow identifies chemically defined dietary gluten peptides from patients with celiac disease

Celiac disease (CeD) is an autoimmune disorder induced by consuming gluten proteins from wheat, barley, and rye. Glutens resist gastrointestinal proteolysis, resulting in peptides that elicit inflammation in patients with CeD. Despite well-established connections between glutens and CeD, chemically defined, bioavailable peptides produced from dietary proteins have never been identified from humans in an unbiased manner. This is largely attributable to technical challenges, impeding our knowledge of potentially diverse peptide species that encounter the immune system. Here, we develop a liquid chromatographic-mass spectrometric workflow for untargeted sequence analysis of the urinary peptidome. We detect over 600 distinct dietary peptides, of which ~35% have a CeD-relevant T cell epitope and ~5% are known to stimulate innate immune responses. Remarkably, gluten peptides from patients with CeD qualitatively and quantitatively differ from controls. Our results provide a new foundation for understanding gluten immunogenicity, improving CeD management, and characterizing the dietary and urinary peptidomes.Ministerio de Ciencia e Innovación SAF2017-83700-

Cystamine and Disulfiram Inhibit Human Transglutaminase 2 via an Oxidative Mechanism

The catalytic activity of transglutaminase 2 (TG2), a ubiquitously expressed mammalian enzyme, is regulated by multiple post-translational mechanisms. Because elevated activity of TG2 in the extracellular matrix is associated with organ-specific diseases such as celiac disease and renal fibrosis, there is growing therapeutic interest in inhibitors of this enzyme. Cystamine, a symmetric disulfide compound, is one of the earliest reported TG2 inhibitors. Despite its widespread use as a tool compound to block TG2 activity <i>in vitro</i> and <i>in vivo</i>, its mechanism of action has remained unclear. Here, we demonstrate that cystamine irreversibly inhibits human TG2 (<i>k</i>_inh/<i>K</i>_i = 1.2 mM^–1 min^–1) via a mechanism fundamentally distinct from those proposed previously. Through mass spectrometric disulfide mapping and site-directed mutagenesis, we show that cystamine promotes the formation of a physiologically relevant disulfide bond between Cys370 and Cys371 that allosterically abrogates the catalytic activity of human TG2. This discovery led us to evaluate clinically useful thiol → disulfide oxidants for TG2 inhibitory activity. It is demonstrated that disulfiram, a relatively safe oral thiuram disulfide, is a fairly potent TG2 inhibitor (<i>k</i>_inh/<i>K</i>_i = 8.3 mM^–1 min^–1) and may therefore provide a practical tool for clinically validating this emerging therapeutic target in intestinal disorders such as celiac disease

Generation of food-grade recombinant Lactobacillus casei delivering Myxococcus xanthus prolyl endopeptidase

Prolyl endopeptidases (PEP) (EC 3.4.21.26), a family of serine proteases with the ability to hydrolyze the peptide bond on the carboxyl side of an internal proline residue, are able to degrade immunotoxic peptides responsible for celiac disease (CD), such as a 33-residue gluten peptide (33-mer). Oral administration of PEP has been suggested as a potential therapeutic approach for CD, although delivery of the enzyme to the small intestine requires intrinsic gastric stability or advanced formulation technologies. We have engineered two food-grade Lactobacillus casei strains to deliver PEP in an in vitro model of small intestine environment. One strain secretes PEP into the extracellular medium, whereas the other retains PEP in the intracellular environment. The strain that secretes PEP into the extracellular medium is the most effective to degrade the 33-mer and is resistant to simulated gastrointestinal stress. Our results suggest that in the future, after more studies and clinical trials, an engineered food-grade Lactobacillus strain may be useful as a vector for in situ production of PEP in the upper small intestine of CD patients. © 2014 Springer-Verlag.This research was supported by project 201370E094 from the CSIC. P.A. is the recipient of a fellowship from FICYT (BP09093) and B.D.R. is a beneficiary of a JAE-DOC contract (CSIC). C.K. is supported by a grant from the NIH (R01 DK 063158).Peer Reviewe

KIR+CD8+ T cells suppress pathogenic T cells and are active in autoimmune diseases and COVID-19

In this work, we find that CD8+ T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49+CD8+ regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8+ T cells efficiently eliminated pathogenic gliadin-specific CD4+ T cells from the leukocytes of celiac disease patients in vitro. We also find elevated levels of KIR+CD8+ T cells, but not CD4+ regulatory T cells, in COVID-19 patients, correlating with disease severity and vasculitis. Selective ablation of Ly49+CD8+ T cells in virus-infected mice led to autoimmunity after infection. Our results indicate that in both species, these regulatory CD8+ T cells act specifically to suppress pathogenic T cells in autoimmune and infectious diseases

KIR + CD8 + T cells suppress pathogenic T cells and are active in autoimmune diseases and COVID-19

In this work, we find that CD8+ T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49+CD8+ regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8+ T cells efficiently eliminated pathogenic gliadin-specific CD4+ T cells from the leukocytes of celiac disease patients in vitro. We also find elevated levels of KIR+CD8+ T cells, but not CD4+ regulatory T cells, in COVID-19 patients, correlating with disease severity and vasculitis. Selective ablation of Ly49+CD8+ T cells in virus-infected mice led to autoimmunity after infection. Our results indicate that in both species, these regulatory CD8+ T cells act specifically to suppress pathogenic T cells in autoimmune and infectious diseases

KIR+CD8+ T cells suppress pathogenic T cells and are active in autoimmune diseases and COVID-19.

In this work, we find that CD8+ T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49+CD8+ regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8+ T cells efficiently eliminated pathogenic gliadin-specific CD4+ T cells from the leukocytes of celiac disease patients in vitro. We also find elevated levels of KIR+CD8+ T cells, but not CD4+ regulatory T cells, in COVID-19 patients, correlating with disease severity and vasculitis. Selective ablation of Ly49+CD8+ T cells in virus-infected mice led to autoimmunity after infection. Our results indicate that in both species, these regulatory CD8+ T cells act specifically to suppress pathogenic T cells in autoimmune and infectious diseases

Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease

Viral infections have been proposed to elicit pathological processes leading to the initiation of T helper 1 (TH1) immunity against dietary gluten and celiac disease (CeD). To test this hypothesis and gain insights into mechanisms underlying virus-induced loss of tolerance to dietary antigens, we developed a viral infection model that makes use of two reovirus strains that infect the intestine but differ in their immunopathological outcomes. Reovirus is an avirulent pathogen that elicits protective immunity, but we discovered that it can nonetheless disrupt intestinal immune homeostasis at inductive and effector sites of oral tolerance by suppressing peripheral regulatory T cell (pTreg) conversion and promoting TH1 immunity to dietary antigen. Initiation of TH1 immunity to dietary antigen was dependent on interferon regulatory factor 1 and dissociated from suppression of pTreg conversion, which was mediated by type-1 interferon. Last, our study in humans supports a role for infection with reovirus, a seemingly innocuous virus, in triggering the development of CeD