RESUMO
Celiac disease (CD) is an immune-driven disease characterized by tissue damage in the small intestine of genetically-susceptible individuals. We evaluated here a crucial immune regulatory pathway involving TYRO3, AXL, and MERTK (TAM) receptors and their ligands PROS1 and GAS6 in duodenal biopsies of controls and CD patients. We found increased GAS6 expression associated with downregulation of PROS1 and variable TAM receptors levels in duodenum tissue of CD patients. Interestingly, CD3+ lymphocytes, CD68+, CD11c+ myeloid and epithelial cells, showed differential expressions of TAM components comparing CD vs controls. Principal component analysis revealed a clear segregation of two groups of CD patients based on TAM components and IFN signaling. In vitro validation demonstrated that monocytes, T lymphocytes and epithelial cells upregulated TAM components in response to IFN stimulation. Our findings highlight a dysregulated TAM axis in CD related to IFN signaling and contribute to a deeper understanding of the pathophysiology of CD.
Assuntos
Receptor Tirosina Quinase Axl , Doença Celíaca , Duodeno , Peptídeos e Proteínas de Sinalização Intercelular , Mucosa Intestinal , Proteína S , Receptores Proteína Tirosina Quinases , c-Mer Tirosina Quinase , Feminino , Humanos , Masculino , c-Mer Tirosina Quinase/genética , c-Mer Tirosina Quinase/metabolismo , Doença Celíaca/imunologia , Doença Celíaca/metabolismo , Doença Celíaca/genética , Duodeno/metabolismo , Duodeno/imunologia , Duodeno/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interferons/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/imunologia , Proteína S/metabolismo , Proteína S/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/imunologia , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
The intestinal mucosa is covered by a layer of epithelial cells that is constantly challenged by commensal, opportunistic, and pathogenic microorganisms, their components, and harmful compounds. Any inflammatory response to these materials must be tightly controlled to limit tissue damage and restore the integrity of the mucosal barrier. We have shown previously that production of IL-1ß via activation of the inflammasome can lead to mucosal damage in the small intestinal pathology that occurs after intragastric administration of a gluten derived peptide, p31-43. Here we show that specific inhibition of caspase-1 or NLRP3 abolishes the damage induced by p31-43, and that antibody-mediated blocking of IL-1ß inhibits the both the histological changes and the induction of apoptosis and caspase-3 activation driven by p31-43. Understanding the role of IL-1ß in sterile inflammation may help to understand chronic inflammatory pathological processes, and design new intervention strategies.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Caspase 1/metabolismo , Inflamação/patologia , Intestino Delgado/patologia , ApoptoseRESUMO
Celiac disease (CD) is a chronic inflammatory disease caused by a genetic predisposition to an abnormal T cell-mediated immune response to the gluten in the diet. Different environmental proinflammatory factors can influence and amplify the T cell-mediated response to gluten. The aim of this manuscript was to study the role of enterocytes in CD intestinal inflammation and their response to different proinflammatory factors, such as gliadin and viruses. Intestinal biopsies from CD patients on a gluten-containing (GCD-CD) or a gluten-free diet (GFD-CD) as well as biopsies from potential CD patients (Pot-CD) before the onset of intestinal lesions and controls (CTR) were used to investigate IL-1ß and IL-6 mRNA levels in situ. Organoids from CD patients were used to test the levels of NF-κB, ERK, IL-6, and IL-1ß by Western blot (WB), ELISA, and quantitative PCR. The Toll-like receptor ligand loxoribine (Lox) and gliadin peptide P31-43 were used as proinflammatory stimuli. In CD biopsies inflammation markers IL-1ß and IL-6 were increased in the enterocytes, and also in Pot-CD before the onset of the intestinal lesion and in GFD-CD. The inflammatory markers pNF-κB, pERK, IL-1ß, and IL-6 were increased and persistent in CD organoids; these organoids were more sensitive to P31-43 and Lox stimuli compared with CTR organoids. Taken together, these observations point to constitutive inflammation in CD enterocytes, which are more sensitive to inflammatory stimuli such as food components and viruses.
Assuntos
Doença Celíaca/metabolismo , Doença Celíaca/patologia , Enterócitos/metabolismo , Enterócitos/patologia , Inflamação/metabolismo , Inflamação/patologia , Adolescente , Biomarcadores/metabolismo , Criança , Pré-Escolar , Dieta Livre de Glúten , Feminino , Glutens/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Transdução de Sinais/fisiologiaRESUMO
The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.
Assuntos
Doenças Autoimunes/imunologia , Citometria de Fluxo , Infecções/imunologia , Neoplasias/imunologia , Animais , Doença Crônica , Humanos , Camundongos , Guias de Prática Clínica como AssuntoRESUMO
The small intestine has a high rate of cell turnover under homeostatic conditions, and this increases further in response to infection or damage. Epithelial cells mostly die by apoptosis, but recent studies indicate that this may also involve pro-inflammatory pathways of programmed cell death, such as pyroptosis and necroptosis. Celiac disease (CD), the most prevalent immune-based enteropathy, is caused by loss of oral tolerance to peptides derived from wheat, rye, and barley in genetically predisposed individuals. Although cytotoxic cells and gluten-specific CD4+ Th1 cells are the central players in the pathology, inflammatory pathways induced by cell death may participate in driving and sustaining the disease through the release of alarmins. In this review, we summarize the recent literature addressing the role of programmed cell death pathways in the small intestine, describing how these mechanisms may contribute to CD and discussing their potential implications.
Assuntos
Apoptose , Doença Celíaca/patologia , Intestino Delgado/patologia , Animais , Doença Celíaca/etiologia , HumanosRESUMO
Coeliac disease (CD) is the prototype of an inflammatory chronic disease induced by food. In this context, gliadin p31-43 peptide comes into the spotlight as an important player of the inflammatory/innate immune response to gliadin in CD. The p31-43 peptide is part of the p31-55 peptide from α-gliadins that remains undigested for a long time, and can be present in the small intestine after ingestion of a gluten-containing diet. Different biophysical methods and molecular dynamic simulations have shown that p31-43 spontaneously forms oligomeric nanostructures, whereas experimental approaches using in vitro assays, mouse models, and human duodenal tissues have shown that p31-43 is able to induce different forms of cellular stress by driving multiple inflammatory pathways. Increased proliferative activity of the epithelial cells in the crypts, enterocyte stress, activation of TG2, induction of Ca2+, IL-15, and NFκB signaling, inhibition of CFTR, alteration of vesicular trafficking, and activation of the inflammasome platform are some of the biological effects of p31-43, which, in the presence of appropriate genetic susceptibility and environmental factors, may act together to drive CD.
Assuntos
Gliadina/efeitos adversos , Inflamação/etiologia , Fragmentos de Peptídeos/efeitos adversos , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Gliadina/química , Humanos , Inflamassomos/metabolismo , Inflamação/patologia , Fragmentos de Peptídeos/química , Transdução de Sinais , Triticum/químicaRESUMO
Celiac disease (CD) is a chronic enteropathy elicited by a Th1 response to gluten peptides in the small intestine of genetically susceptible individuals. However, it remains unclear what drives the induction of inflammatory responses of this kind against harmless antigens in food. In a recent work, we have shown that the p31-43 peptide (p31-43) from α-gliadin can induce an innate immune response in the intestine and that this may initiate pathological adaptive immunity. The receptors and mechanisms responsible for the induction of innate immunity by p31-43 are unknown and here we present evidence that this may reflect conformational changes in the peptide that allow it to activate the NLRP3 inflammasome. Administration of p31-43, but not scrambled or inverted peptides, to normal mice induced enteropathy in the proximal small intestine, associated with increased production of type I interferon and mature IL-1ß. P31-43 showed a sequence-specific spontaneous ability to form structured oligomers and aggregates in vitro and induced activation of the ASC speck complex. In parallel, the enteropathy induced by p31-43 in vivo did not occur in the absence of NLRP3 or caspase 1 and was inhibited by administration of the caspase 1 inhibitor Ac-YVAD-cmk. Collectively, these findings show that p31-43 gliadin has an intrinsic propensity to form oligomers which trigger the NLRP3 inflammasome and that this pathway is required for intestinal inflammation and pathology when p31-43 is administered orally to mice. This innate activation of the inflammasome may have important implications in the initial stages of CD pathogenesis.
Assuntos
Caspase 1/metabolismo , Gliadina/metabolismo , Inflamassomos/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fragmentos de Peptídeos/metabolismo , Multimerização Proteica , Sequência de Aminoácidos , Animais , Apoptose , Doença Celíaca/etiologia , Doença Celíaca/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Gliadina/química , Gliadina/ultraestrutura , Mucosa Intestinal/ultraestrutura , Intestino Delgado , Masculino , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/ultraestrutura , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Gliadin peptide presentation and T-cell activation are critical events in the pathogenesis of celiac disease. Several studies have been performed to identify the toxic gliadin peptides but the complexity of the antigenic fraction makes this analysis difficult. In this work, an in vitro model for the analysis of gliadin peptide presentation is studied. METHODS: The human cell lines U937 and THP-1 (monocytic), DUCAF and VAVY (immortalised B cells) and HT-29 and Caco-2 (intestinal epithelial cells) were incubated with biotin-labelled gliadin (bG). FITC-labelled streptavidin was used to detect biotinylated peptides at the cell surface by flow cytometry. RESULTS: All cell lines tested showed a fluorescence signal derived from bG, that was highest when cells were stimulated with IFN-gamma for 48 h. Time course experiments performed using THP-1 cells showed that after 4-h incubation, almost a maximal signal can be reached. THP-1 cells incubated at 4 degrees C or after paraformaldehyde fixation showed a substantial signal reduction, suggesting that metabolic activity was necessary for the detection of the maximal fluorescence signal at the cell surface. The presence of HLA class II-bound biotinylated peptides was observed in cell lysates of THP-1 cells incubated with bG. CONCLUSIONS: In all cell lines tested, a specific biotin-peptide-derived signal was observed. This was increased after IFN-gamma treatment and decreased after fixation or incubation at low temperature. The signal was higher in monocytic and B-cell lines than in the epithelial cell lines. The use of this procedure could be a useful tool to study the in vitro processing and presentation of naturally gliadin-derived peptides.
Assuntos
Gliadina/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Fragmentos de Peptídeos/metabolismo , Biotina/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática/métodos , Gliadina/química , Gliadina/farmacologia , Antígenos de Histocompatibilidade Classe II/efeitos dos fármacos , Humanos , Interferon gama/farmacologia , Mucosa Intestinal/metabolismo , Intestinos/citologia , Fragmentos de Peptídeos/análise , Fatores de Tempo , Células Tumorais CultivadasRESUMO
La enfermedad celíaca (EC) es una enfermedad gastrointestinal crónica de muy alta incidencia en nuestro país. Se estima que puede afectar 1 de cada 300 habitantes. En individuos susceptibles, la patología es provocada por la ingestión de mínimas cantidades de prolaminas de los cereales: trigo, triticale, cebada y centeno. Su único tratamiento es una estricta dieta libre de dichas proteínas. La Organización Mundial de la Salud (OMS) establece que un alimento puede ser considerado como apto para consumo por enfermos celíacos sólo si su contenido de gluten es inferior a 1 mg/100 g de producto seco. La detección precisa de estas proteínas requiere entonces de métodos de alta detectabilidad y especificidad para discriminar entre las proteínas nocivas y las de otros vegetales frecuentemente usados como reemplazo en la formulación de los alimentos para estos pacientes. En este trabajo, se muestra el desarrollo de un ELISA con anticuerpos policlonales, para la cuantificación de gliadinas en alimentos destinados a enfermos celíacos. Se empleó un diseño de ELISA competitivo secuencial con un antisuero obtenido en conejos que detecta selectivamente las prolaminas tóxicas. Este inmunoensayo presenta un nivel de detección de 0,1 mg de gluten/100 g de producto y cumple con los niveles de detectabilidad aconsejados por la OMS. Mediante el ELISA descripto se han analizado una gran variedad de muestras comerciales, pudiendo cuantificar las prolaminas incluso en alimentos que sufrieron tratamientos térmicos durante su fabricación