RESUMO
Usually, the massive elimination of cells under steady-state conditions occurs by apoptosis, which is also acknowledged to explain the loss of enterocytes in the small intestine of celiac disease (CD) patients. However, little is known about the role of proinflammatory cell death pathways in CD. Here, we have used confocal microscopy, western blot, and RT-qPCR analysis to assess the presence of regulated cell death pathways in the duodenum of CD patients. We found an increased number of dead (TUNEL+) cells in the lamina propria of small intestine of CD patients, most of them are plasma cells (CD138+). Many dying cells expressed FAS and were in close contact with CD3+ T cells. Caspase-8 and caspase-3 expression was increased in CD, confirming the activation of apoptosis. In parallel, caspase-1, IL-1ß, and GSDMD were increased in CD samples indicating the presence of inflammasome-dependent pyroptosis. Necroptosis was also present, as shown by the increase of RIPK3 and phosphorylate MLKL. Analysis of published databases confirmed that CD has an increased expression of regulated cell death -related genes. Together, these results reveal that CD is characterized by cell death of different kinds. In particular, the presence of proinflammatory cell death pathways may contribute to mucosal damage.
Assuntos
Doença Celíaca , Piroptose , Humanos , Piroptose/genética , Necroptose/genética , Apoptose/genética , Morte CelularRESUMO
Intestinal epithelial cells have a rapid turnover, being rapidly renewed by newly differentiated enterocytes, balanced by massive and constant removal of damaged cells by programmed cell death (PCD). The main forms of PCD are apoptosis, pyroptosis, and necroptosis, with apoptosis being a noninflammatory process, whereas the others drive innate immune responses. Although apoptosis is thought to be the principal means of cell death in the healthy intestine, which mechanisms are responsible for PCD during inflammation are not fully understood. To address this question, we used an in vivo model of enteropathy in wild-type mice induced by a single intragastric administration of the p31-43 gliadin peptide, which is known to elicit transient MyD88, NLRP3, and caspase-1-dependent mucosal damage and inflammation in the small intestine. Here, we found increased numbers of TUNEL+ cells in the mucosa as early as 2 h after p31-43 administration. Western blot and immunofluorescence analysis showed the presence of caspase-3-mediated apoptosis in the epithelium and lamina propria. In addition, the presence of mature forms of caspase-1, IL-1ß, and gasdermin D showed activation of pyroptosis and inhibition of caspase-1 led to decreased enterocyte death in p31-43-treated mice. There was also up-regulation of RIPK3 in crypt epithelium, suggesting that necroptosis was also occurring. Taken together, these results indicate that the inflammatory response induced by p31-43 can drive multiple PCD pathways in the small intestine.
Assuntos
Inflamação/imunologia , Enteropatias/imunologia , Intestino Delgado/imunologia , Morte Celular Regulada/imunologia , Animais , Inflamação/metabolismo , Inflamação/patologia , Enteropatias/metabolismo , Enteropatias/patologia , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Initially described as Th2 promoter cytokine, more recently, IL-33 has been recognized as an alarmin, mainly in epithelial and endothelial cells. While localized in the nucleus acting as a gene regulator, it can be also released after injury, stress or inflammatory cell death. As proinflammatory signal, IL-33 binds to the surface receptor ST2, which enhances mast cell, Th2, regulatory T cell, and innate lymphoid cell type 2 functions. Besides these Th2 roles, free IL-33 can activate CD8+ T cells during ongoing Th1 immune responses to potentiate its cytotoxic function. Celiac Disease (CD) is a chronic inflammatory disorder characterized by a predominant Th1 response leading to multiple pathways of mucosal damage in the proximal small intestine. By immunofluorescence and western blot analysis of duodenal tissues, we found an increased expression of IL-33 in duodenal mucosa of active CD (ACD) patients. Particularly, locally digested IL-33 releases active 18/21kDa fragments which can contribute to expand the proinflammatory signal. Endothelial (CD31+) and mesenchymal, myofibroblast and pericyte cells from microvascular structures in villi and crypts, showed IL-33 nuclear location; while B cells (CD20+) showed a strong cytoplasmic staining. Both ST2 forms, ST2L and sST2, were also upregulated in duodenal mucosa of CD patients. This was accompanied by increased number of CD8+ST2+ T cells and the expression of T-bet in some ST2+ intraepithelial lymphocytes and lamina propria cells. IL-33 and sST2 mRNA levels correlated with IRF1, an IFN induced factor relevant in responses to viral infections and interferon mediated proinflammatory responses highly represented in duodenal tissues in ACD. These findings highlight the potential contribution of IL-33 and its fragments to exacerbate the proinflammatory circuit and potentiate the cytotoxic activity of CD8+ T cells in CD pathology.
Assuntos
Alarminas/imunologia , Doença Celíaca/imunologia , Inflamação/imunologia , Interleucina-33/imunologia , Intestino Delgado/imunologia , Animais , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Citocinas/imunologia , Células HT29 , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1/imunologia , Mucosa Intestinal/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/imunologia , Células Th2/imunologiaRESUMO
Celiac Disease (CD) is a gluten sensitive enteropathy that remains widely undiagnosed and implementation of massive screening tests is needed to reduce the long term complications associated to untreated CD. The main CD autoantigen, human tissue transglutaminase (TG2), is a challenge for the different expression systems available since its cross-linking activity affects cellular processes. Plant-based transient expression systems can be an alternative for the production of this protein. In this work, a transient expression system for the production of human TG2 in Nicotiana benthamiana leaves was optimized and reactivity of plant-produced TG2 in CD screening test was evaluated. First, a subcellular targeting strategy was tested. Cytosolic, secretory, endoplasmic reticulum (C-terminal SEKDEL fusion) and vacuolar (C-terminal KISIA fusion) TG2 versions were transiently expressed in leaves and recombinant protein yields were measured. ER-TG2 and vac-TG2 levels were 9- to 16-fold higher than their cytosolic and secretory counterparts. As second strategy, TG2 variants were co-expressed with a hydrophobic elastin-like polymer (ELP) construct encoding for 36 repeats of the pentapeptide VPGXG in which the guest residue X were V and F in ratio 8:1. Protein bodies (PB) were induced by the ELP, with a consequent two-fold-increase in accumulation of both ER-TG2 and vac-TG2. Subsequently, ER-TG2 and vac-TG2 were produced and purified using immobilized metal ion affinity chromatography. Plant purified ER-TG2 and vac-TG2 were recognized by three anti-TG2 monoclonal antibodies that bind different epitopes proving that plant-produced antigen has immunochemical characteristics similar to those of human TG2. Lastly, an ELISA was performed with sera of CD patients and healthy controls. Both vac-TG2 and ER-TG2 were positively recognized by IgA of CD patients while they were not recognized by serum from non-celiac controls. These results confirmed the usefulness of plant-produced TG2 to develop screening assays. In conclusion, the combination of subcellular sorting strategy with co-expression with a PB inducing construct was sufficient to increase TG2 protein yields. This type of approach could be extended to other problematic proteins, highlighting the advantages of plant based production platforms.
RESUMO
Celiac disease (CD) is an immune-mediated enteropathy that develops in genetically susceptible individuals following exposure to dietary gluten. Severe changes at the intestinal mucosa observed in untreated CD patients are linked to changes in the level and in the pattern of expression of different genes. Fully differentiated epithelial cells express two isoforms of fatty acid binding proteins (FABPs): intestinal and liver, IFABP and LFABP, respectively. These proteins bind and transport long chain fatty acids and also have other important biological roles in signaling pathways, particularly those related to PPARγ and inflammatory processes. Herein, we analyze the serum levels of IFABP and characterize the expression of both FABPs at protein and mRNA level in small intestinal mucosa in severe enteropathy and normal tissue. As a result, we observed higher levels of circulating IFABP in untreated CD patients compared with controls and patients on gluten-free diet. In duodenal mucosa a differential FABPs expression pattern was observed with a reduction in mRNA levels compared to controls explained by the epithelium loss in severe enteropathy. In conclusion, we report changes in FABPs' expression pattern in severe enteropathy. Consequently, there might be alterations in lipid metabolism and the inflammatory process in the small intestinal mucosa.
Assuntos
Doença Celíaca/sangue , Proteínas de Ligação a Ácido Graxo/sangue , Duodeno/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Humanos , Intestino Delgado/metabolismo , PPAR gama/metabolismo , RNA Mensageiro/genéticaRESUMO
Systemic administration of polyinosinic:polycytidylic acid (poly I:C), mimics virally-induced activation of TLR3 signalling causing acute small intestine damage, but whether and how mucosal administration of poly I:C causes enteropathy is less clear. Our aim was to investigate the inflammatory pathways elicited after intraluminal administration of poly I:C and determine acute and delayed consequences of this locally induced immune activation. Intraluminal poly I:C induced rapid mucosal immune activation in C57BL/6 mice involving IFNß and the CXCL10/CXCR3 axis, that may drive inflammation towards a Th1 profile. Intraluminal poly I:C also caused enteropathy and gut dysfunction in gliadin-sensitive NOD-DQ8 mice, and this was prolonged by concomitant oral administration of gliadin. Our results indicate that small intestine pathology can be induced in mice by intraluminal administration of poly I:C and that this is exacerbated by subsequent oral delivery of a relevant dietary antigen.
Assuntos
Progressão da Doença , Gliadina/administração & dosagem , Gliadina/efeitos adversos , Enteropatias/induzido quimicamente , Enteropatias/patologia , Poli I-C/administração & dosagem , Poli I-C/efeitos adversos , Administração Oral , Animais , Citocinas/sangue , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Helicase IFIH1 Induzida por Interferon , Enteropatias/sangue , Mucosa Intestinal/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismoRESUMO
Lymphocytic infiltration in the lamina propria (LP), which is primarily composed of CD4(+) Th1 cells and plasma cells, and increased numbers of intraepithelial lymphocytes (IELs), is a characteristic finding in active celiac disease (CD). Signals for this selective cell recruitment have not been fully established. CXCR3 and its ligands, particularly CXCL10, have been suggested to be one of the most relevant pathways in the attraction of cells into inflamed tissues. In addition, CXCR3 is characteristically expressed by Th1 cells. The aim of this work was to investigate the participation of the chemokine CXCL10/CXCR3 axis in CD pathogenesis. A higher concentration of CXCL10 was found in the serum of untreated CD patients. The mRNA levels of CXCL10 and CXCL11 but not CXCL9 were significantly higher in duodenal biopsies from untreated CD patients compared with non-CD controls or treated patients. The results demonstrate that CXCL10 is abundantly produced in untreated CD and reduced in treated patients, and the expression of CXCL10 was found to be correlated with the IFNγ levels in the tissue. Plasma cells and enterocytes were identified as CXCL10-producing cells. Moreover, the CXCL10 expression in intestinal tissues was upregulated by poly I:C and IL-15. IELs, LP T lymphocytes, and plasma cells, which infiltrate the intestinal mucosa in untreated CD, express CXCR3. The CXCR3/CXCL10 signalling axis is overactivated in the small intestinal mucosa in untreated patients, and this finding explains the specific recruitment of the major cell populations that infiltrate the epithelium and the LP in CD.
Assuntos
Doença Celíaca/imunologia , Doença Celíaca/metabolismo , Quimiocina CXCL10/metabolismo , Intestino Delgado/imunologia , Plasmócitos/imunologia , Receptores CXCR3/metabolismo , Linfócitos T/imunologia , Adulto , Doença Celíaca/sangue , Doença Celíaca/patologia , Quimiocina CXCL10/biossíntese , Quimiocina CXCL10/sangue , Quimiocina CXCL11/metabolismo , Quimiocina CXCL9/metabolismo , Criança , Regulação da Expressão Gênica/imunologia , Humanos , Interferon beta/metabolismo , Interferon gama/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The MICA/B genes (MHC class I chain related genes A and B) encode for non conventional class I HLA molecules which have no role in antigen presentation. MICA/B are up-regulated by different stress conditions such as heat-shock, oxidative stress, neoplasic transformation and viral infection. Particularly, MICA/B are expressed in enterocytes where they can mediate enterocyte apoptosis when recognised by the activating NKG2D receptor present on intraepithelial lymphocytes. This mechanism was suggested to play a major pathogenic role in active celiac disease (CD). Due to the importance of MICA/B in CD pathogenesis we studied their expression in duodenal tissue from CD patients. By immunofluorescence confocal microscopy and flow cytometry we established that MICA/B was mainly intracellularly located in enterocytes. In addition, we identified MICA/B(+) T cells in both the intraepithelial and lamina propria compartments. We also found MICA/B(+) B cells, plasma cells and some macrophages in the lamina propria. The pattern of MICA/B staining in mucosal tissue in severe enteropathy was similar to that found in in vitro models of cellular stress. In such models, MICA/B were located in stress granules that are associated to the oxidative and ER stress response observed in active CD enteropathy. Our results suggest that expression of MICA/B in the intestinal mucosa of CD patients is linked to disregulation of mucosa homeostasis in which the stress response plays an active role.
Assuntos
Doença Celíaca/genética , Duodeno/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Mucosa Intestinal/metabolismo , Estresse Fisiológico/genética , Linfócitos B/metabolismo , Linfócitos B/patologia , Doença Celíaca/metabolismo , Doença Celíaca/patologia , Pré-Escolar , Duodeno/patologia , Enterócitos/metabolismo , Enterócitos/patologia , Feminino , Expressão Gênica , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Mucosa Intestinal/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Plasmócitos/metabolismo , Plasmócitos/patologia , Índice de Gravidade de Doença , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
The composition of human milk is the main base for the development of infant formulas concerning its macronutrients and micronutrients contents and bioactive compounds. Technological advances in the composition of human milk have identified a great number of bioactive compounds such as prebiotics which are responsible for immunological protection and the prevention of different pathologies. In order to achieve similar benefits, they are part of the contents of infant formulas.
Assuntos
Alimentos Infantis , Leite Humano , Prebióticos , Humanos , Alimentos Infantis/análise , Recém-Nascido , Leite Humano/química , Prebióticos/análiseRESUMO
La composición de la leche materna es la base principal para el desarrollo de fórmulas infantiles en cuanto a su contenido de macronutrientes, micronutrientes y compuestos bioactivos. Los avances tecnológicos en el conocimiento de la composición de la leche materna han permitido identificar un gran número de componentes bioactivos, como los prebióticos, responsables de la protección inmunológica y de la prevención de diferentes patologías, lo cual ha llevado a su incorporación en las fórmulas infantiles para lograr beneficios similares.