RESUMEN
BACKGROUND & AIMS: Loss-of-function variants in the PTPN2 gene are associated with increased risk of inflammatory bowel disease. We recently showed that Ptpn2 is critical for intestinal epithelial cell (IEC) barrier maintenance, IEC-macrophage communication, and modulation of the gut microbiome in mice, restricting expansion of a small intestinal pathobiont associated with inflammatory bowel disease. Here, we aimed to identify how Ptpn2 loss affects ileal IEC subtypes and their function in vivo. METHODS: Constitutive Ptpn2 wild-type, heterozygous, and knockout (KO) mice, as well as mice with inducible deletion of Ptpn2 in IECs, were used in the study. Investigation was performed using imaging techniques, flow cytometry, enteroid culture, and analysis of gene and protein levels of IEC markers. RESULTS: Partial transcriptome analysis showed that expression of Paneth cell-associated antimicrobial peptides Lyz1, Pla2g2a, and Defa6 was down-regulated markedly in Ptpn2-KO mice compared with wild-type and heterozygous. In parallel, Paneth cell numbers were reduced, their endoplasmic reticulum architecture was disrupted, and the endoplasmic reticulum stress protein, C/EBP-homologous protein (CHOP), was increased in Ptpn2-KO mice. Despite reduced Paneth cell number, flow cytometry showed increased expression of the Paneth cell-stimulatory cytokines interleukin 22 and interferon γ+ in CD4+ T cells isolated from Ptpn2-KO ileum. Key findings in constitutive Ptpn2-KO mice were confirmed in epithelium-specific Ptpn2ΔIEC mice, which also showed impaired lysozyme protein levels in Paneth cells compared with Ptpn2fl/fl control mice. CONCLUSIONS: Constitutive Ptpn2 deficiency affects Paneth cell viability and compromises Paneth cell-specific antimicrobial peptide production. The observed effects may contribute to the increased susceptibility to intestinal infection and dysbiosis in these mice.
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Enfermedades Inflamatorias del Intestino , Células de Paneth , Ratones , Animales , Células de Paneth/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 2/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 2/metabolismo , Supervivencia Celular , Enfermedades Inflamatorias del Intestino/genética , Íleon/metabolismo , Ratones NoqueadosRESUMEN
Giardia lamblia is a leading protozoal cause of diarrheal disease worldwide. Infection is associated with abdominal pain, malabsorption and weight loss, and protracted post-infectious syndromes. A human vaccine is not available against G. lamblia. Prior studies with human and murine immune sera have identified several parasite antigens, including surface proteins and metabolic enzymes with intracellular functions. While surface proteins have demonstrated vaccine potential, they can exhibit significant variation between G. lamblia strains. By comparison, metabolic enzymes show greater conservation but their vaccine potential has not been established. To determine whether such proteins can serve as vaccine candidates, we focused on two enzymes, α-enolase (ENO) and ornithine carbamoyl transferase (OCT), which are involved in glycolysis and arginine metabolism, respectively. We show in a cohort of patients with confirmed giardiasis that both enzymes are immunogenic. Intranasal immunization with either enzyme antigen in mice induced strong systemic IgG1 and IgG2b responses and modest mucosal IgA responses, and a marked 100- to 1,000-fold reduction in peak trophozoite load upon oral G. lamblia challenge. ENO immunization also reduced the extent and duration of cyst excretion. Examination of 44 cytokines showed only minimal intestinal changes in immunized mice, although a modest increase of CCL22 was observed in ENO-immunized mice. Spectral flow cytometry revealed increased numbers and activation state of CD4 T cells in the small intestine and an increase in α4ß7-expressing CD4 T cells in mesenteric lymph nodes of ENO-immunized mice. Consistent with a key role of CD4 T cells, immunization of CD4-deficient and Rag-2 deficient mice failed to induce protection, whereas mice lacking IgA were fully protected by immunization, indicating that immunity was CD4 T cell-dependent but IgA-independent. These results demonstrate that conserved metabolic enzymes can be effective vaccine antigens for protection against G. lamblia infection, thereby expanding the repertoire of candidate antigens beyond primary surface proteins.
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Giardia lamblia , Giardiasis , Animales , Antígenos de Protozoos , Giardia , Giardiasis/parasitología , Humanos , Inmunoglobulina A , Inmunoglobulina G , Proteínas de la Membrana , RatonesRESUMEN
The protozoan pathogen Giardia lamblia is an important worldwide cause of diarrheal disease and malabsorption. Infection is managed with antimicrobials, although drug resistance and treatment failures are a clinical challenge. Prior infection provides significant protection, yet a human vaccine has not been realized. Individual antigens can elicit partial protection in experimental models, but protection is weaker than after prior infection. Here, we developed a multivalent nanovaccine by coating membranes derived from the parasite onto uniform and stable polymeric nanoparticles loaded with a mucosal adjuvant. Intranasal immunization with the nanovaccine induced adaptive immunity and effectively protected mice from G. lamblia infection.
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Giardia lamblia , Giardiasis , Nanopartículas , Parásitos , Adyuvantes Inmunológicos , Animales , Giardiasis/parasitología , Giardiasis/prevención & control , Humanos , Inmunidad Mucosa , RatonesRESUMEN
Genome-wide association studies revealed that loss-of-function mutations in protein tyrosine phosphatase non-receptor type 2 (PTPN2) increase the risk of developing chronic immune diseases, such as inflammatory bowel disease (IBD) and celiac disease. These conditions are associated with increased intestinal permeability as an early etiological event. The aim of this study was to examine the consequences of deficient activity of the PTPN2 gene product, T cell protein tyrosine phosphatase (TCPTP), on intestinal barrier function and tight junction organization in vivo and in vitro. Here, we demonstrate that TCPTP protected against intestinal barrier dysfunction induced by the inflammatory cytokine IFN-γ by 2 mechanisms: it maintained localization of zonula occludens 1 and occludin at apical tight junctions and restricted both expression and insertion of the cation pore-forming transmembrane protein, claudin-2, at tight junctions through upregulation of the inhibitory cysteine protease, matriptase. We also confirmed that the loss-of-function PTPN2 rs1893217 SNP was associated with increased intestinal claudin-2 expression in patients with IBD. Moreover, elevated claudin-2 levels and paracellular electrolyte flux in TCPTP-deficient intestinal epithelial cells were normalized by recombinant matriptase. Our findings uncover distinct and critical roles for epithelial TCPTP in preserving intestinal barrier integrity, thereby proposing a mechanism by which PTPN2 mutations contribute to IBD.
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Mucosa Intestinal/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 2/metabolismo , Uniones Estrechas/metabolismo , Adolescente , Adulto , Anciano , Animales , Claudinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Técnicas In Vitro , Enfermedades Inflamatorias del Intestino/etiología , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Permeabilidad , Polimorfismo de Nucleótido Simple , Proteína Tirosina Fosfatasa no Receptora Tipo 2/deficiencia , Proteína Tirosina Fosfatasa no Receptora Tipo 2/genética , Uniones Estrechas/patología , Adulto JovenRESUMEN
Coronavirus disease (COVID-19), caused by SARS-CoV-2, has affected over 65 million individuals and killed over 1.5 million persons (December 8, 2020; www.who.int)1. While fatality rates are higher among the elderly and those with underlying comorbidities2, host factors that promote susceptibility to SARS-CoV-2 infection and severe disease are poorly understood. Although individuals with certain autoimmune/inflammatory disorders show increased susceptibility to viral infections, there is incomplete knowledge of SARS-CoV-2 susceptibility in these diseases.3-7 We report that the autoimmune PTPN2 risk variant rs1893217 promotes expression of the SARS-CoV-2 receptor, ACE2, and increases cellular entry mediated by SARS-CoV-2 spike protein. Elevated ACE2 expression and viral entry were mediated by increased JAK-STAT signalling, and were reversed by the JAK inhibitor, tofacitinib. Collectively, our findings uncover a novel risk biomarker for increased expression of the SARS-CoV-2 receptor and viral entry, and identify a clinically approved therapeutic agent to mitigate this risk.
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Giardia lamblia, one of the most common protozoal infections of the human intestine, is an important worldwide cause of diarrheal disease, malabsorption, malnutrition, delayed cognitive development in children, and protracted postinfectious syndromes. Despite its medical importance, no human vaccine is available against giardiasis. A crude veterinary vaccine has been developed, and experimental vaccines based on expression of multiple variant-specific surface proteins have been reported, but poorly defined vaccine components and excessive antigen variability are problematic for pharmaceutical vaccine production. To expand the repertoire of antigen candidates for vaccines, we reasoned that surface proteins may provide an enriched source of such antigens since key host effectors, such as secretory IgA, can directly bind to such antigens in the intestinal lumen and interfere with epithelial attachment. Here, we have applied a proteomics approach to identify 23 novel surface antigens of G. lamblia that show >90% amino acid sequence identity between the two human-pathogenic genetic assemblages (A and B) of the parasite. Surface localization of a representative subset of these proteins was confirmed by immunostaining. Four selected proteins, uridine phosphorylase-like protein-1, protein 21.1 (GL50803_27925), α1-giardin, and α11-giardin, were subsequently produced in recombinant form and shown to be immunogenic in mice and G. lamblia-infected humans and confer protection against G. lamblia infection upon intranasal immunization in rodent models of giardiasis. These results demonstrate that identification of conserved surface antigens provides a powerful approach for overcoming a key rate-limiting step in the design and construction of an effective vaccine against giardiasis.
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Antígenos de Protozoos/inmunología , Giardia lamblia/inmunología , Giardiasis/parasitología , Proteoma/inmunología , Proteínas Protozoarias/inmunología , Vacunas Antiprotozoos/inmunología , Adulto , Animales , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Reacciones Cruzadas , Femenino , Giardia lamblia/química , Giardia lamblia/genética , Giardiasis/inmunología , Giardiasis/prevención & control , Humanos , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Proteoma/química , Proteoma/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Vacunas Antiprotozoos/química , Vacunas Antiprotozoos/genética , Adulto JovenRESUMEN
The lumen-dwelling protozoan Giardia is an important parasitic cause of diarrheal disease worldwide. Infection can persist over extended periods with minimal intestinal inflammation, suggesting that Giardia may attenuate host responses to ensure its survival, although clearance eventually occurs in most cases. IL-10 is an anti-inflammatory regulator critical for intestinal homeostasis and controlling host responses to bacterial exposure, yet its potential role in coordinating antiprotozoal host defense in the intestine is not known. In this study, we found that murine infection with the natural enteric pathogen Giardia muris induced a transient IL-10 response after 2-4 wk at the primary site of infection in the upper small intestine, but parasite colonization and eradication were not affected by the absence of the cytokine in gene-targeted mice. However, IL-10 was critical for controlling infection-associated immunological sequelae in the colon because severe and persistent diarrhea and colitis were observed in IL-10-deficient mice within 1-2 wk postinfection but not in uninfected littermate controls. Inflammation was characterized by epithelial hyperplasia, neutrophil and macrophage expansion, and Th1 induction and could be prevented by blockade of IL-12/IL-23 p40 but not depletion of CD11c+ dendritic cells. Furthermore, the intestinal microbiota underwent characteristic shifts in composition and was required for disease because antibiotics and loss of TLR signaling in MyD88-deficient mice protected against colitis. Together, our data suggest that transient infection by a luminal and seemingly noninflammatory pathogen can trigger sustained colitis in genetically susceptible hosts, which has broader implications for understanding postinfectious syndromes and other chronic intestinal inflammatory conditions.
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Colitis/inmunología , Giardia/fisiología , Giardiasis/inmunología , Interleucina-10/metabolismo , Mucosa Intestinal/inmunología , Intestino Delgado/fisiología , Células TH1/inmunología , Animales , Células Cultivadas , Enfermedad Crónica , Predisposición Genética a la Enfermedad , Humanos , Interleucina-10/genética , Interleucina-12/metabolismo , Mucosa Intestinal/parasitología , Intestino Delgado/parasitología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Receptores Toll-Like/metabolismoRESUMEN
BACKGROUND AND AIMS: The enteric nervous system (ENS) plays a crucial role in the control of gastrointestinal motility, secretion and absorption functions. Immunohistochemistry has been widely used to visualize neurons of the ENS for more than two decades. Genetically engineered mice that report specific proteins can also be used to visualize neurons of the ENS. The goal of our study was to develop a mouse that expresses fluorescent neuronal nitric oxide synthase (nNOS) and choline acetyltransferase (ChAT), the two proteins expressed in 95% of the ENS neurons. We compared ENS neurons visualized in the reporter mouse with the wild type mouse stained using classical immunostaining techniques. METHODS: Mice hemizygous for ChAT-ChR2-YFP BAC transgene with expression of the mhChR2:YFP fusion protein directed by ChAT promoter/enhancer regions on the BAC transgene were purchased commercially. The Cre/LoxP technique of somatic recombination was used to construct mice with nNOS positive neurons. The two mice were crossbred and tissues were harvested and examined using fluorescent microscopy. Immunostaining was performed in the wild type mice, using antibodies to nNOS, ChAT, Hu and PGP 9.5. RESULTS: Greater than 95% of the ENS neurons were positive for either nNOS or ChAT or both. The nNOS and ChAT neurons and their processes in the ENS were well visualized in all the regions of the GI tract, i.e., esophagus, small intestine and colon. The number of nNOS and ChAT neurons was approximately same in the reporter mouse and immunostaining method in the wild type mouse. The nNOS fluorescence in the reporter mouse was seen in both cytoplasm as well as nucleus but in the immunostained specimens it was seen only in the cytoplasm. CONCLUSION: We propose that the genetically engineered double reporter mouse for ChAT and nNOS proteins is a powerful tool to study of the effects of various diseases on the ENS without the need for immunostaining.
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Sistema Nervioso Entérico/metabolismo , Expresión Génica , Genes Reporteros , Imagen Molecular , Animales , Colina O-Acetiltransferasa/genética , Colina O-Acetiltransferasa/metabolismo , Neuronas Colinérgicas/metabolismo , Técnica del Anticuerpo Fluorescente , Tracto Gastrointestinal/metabolismo , Inmunohistoquímica , Ratones , Ratones Transgénicos , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa de Tipo I/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Transporte de ProteínasRESUMEN
BACKGROUND & AIMS: Intestinal microfold (M) cells are specialized epithelial cells that act as gatekeepers of luminal antigens in the intestinal tract. They play a critical role in the intestinal mucosal immune response through transport of viruses, bacteria and other particles and antigens across the epithelium to immune cells within Peyer's patch regions and other mucosal sites. Recent studies in mice have demonstrated that M cells are generated from Lgr5+ intestinal stem cells (ISCs), and that infection with Salmonella enterica serovar Typhimurium increases M cell formation. However, it is not known whether and how these findings apply to primary human small intestinal epithelium propagated in an in vitro setting. METHODS: Human intestinal crypts were grown as monolayers with growth factors and treated with recombinant RANKL, and assessed for mRNA transcripts, immunofluorescence and uptake of microparticles and S. Typhimurium. RESULTS: Functional M cells were generated by short-term culture of freshly isolated human intestinal crypts in a dose- and time-dependent fashion. RANKL stimulation of the monolayer cultures caused dramatic induction of the M cell-specific markers, SPIB, and Glycoprotein-2 (GP2) in a process primed by canonical WNT signaling. Confocal microscopy demonstrated a pseudopod phenotype of GP2-positive M cells that preferentially take up microparticles. Furthermore, infection of the M cell-enriched cultures with the M cell-tropic enteric pathogen, S. Typhimurium, led to preferential association of the bacteria with M cells, particularly at lower inoculum sizes. Larger inocula caused rapid induction of M cells. CONCLUSIONS: Human intestinal crypts containing ISCs can be cultured and differentiate into an epithelial layer with functional M cells with characteristic morphological and functional properties. This study is the first to demonstrate that M cells can be induced to form from primary human intestinal epithelium, and that S. Typhimurium preferentially infect these cells in an in vitro setting. We anticipate that this model can be used to generate large numbers of M cells for further functional studies of these key cells of intestinal immune induction and their impact on controlling enteric pathogens and the intestinal microbiome.
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Mucosa Intestinal/citología , Intestino Delgado/citología , Ganglios Linfáticos Agregados/citología , Células Madre/citología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Cultivadas , Humanos , Inmunidad Mucosa , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Intestino Delgado/inmunología , Intestino Delgado/microbiología , Ganglios Linfáticos Agregados/inmunología , Ganglios Linfáticos Agregados/microbiología , Ligando RANK/inmunología , Salmonella typhimurium/inmunología , Células Madre/inmunologíaRESUMEN
Mesenchymal stem cell (MSC) therapy is an emerging field of regenerative medicine; however, it is often unclear how these cells mediate repair. Here, we investigated the use of MSCs in the treatment of intestinal disease and modeled abnormal repair by creating focal wounds in the colonic mucosa of prostaglandin-deficient mice. These wounds developed into ulcers that infiltrated the outer intestinal wall. We determined that penetrating ulcer formation in this model resulted from increased hypoxia and smooth muscle wall necrosis. Prostaglandin I2 (PGI2) stimulated VEGF-dependent angiogenesis to prevent penetrating ulcers. Treatment of mucosally injured WT mice with a VEGFR inhibitor resulted in the development of penetrating ulcers, further demonstrating that VEGF is critical for mucosal repair. We next used this model to address the role of transplanted colonic MSCs (cMSCs) in intestinal repair. Compared with intravenously injected cMSCs, mucosally injected cMSCs more effectively prevented the development of penetrating ulcers, as they were more efficiently recruited to colonic wounds. Importantly, mucosally injected cMSCs stimulated angiogenesis in a VEGF-dependent manner. Together, our results reveal that penetrating ulcer formation results from a reduction of local angiogenesis and targeted injection of MSCs can optimize transplantation therapy. Moreover, local MSC injection has potential for treating diseases with features of abnormal angiogenesis and repair.
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Colon , Mucosa Intestinal , Trasplante de Células Madre Mesenquimatosas , Neovascularización Fisiológica , Factor A de Crecimiento Endotelial Vascular/metabolismo , Cicatrización de Heridas , Heridas y Lesiones , Aloinjertos , Animales , Colon/lesiones , Colon/metabolismo , Colon/patología , Epoprostenol/genética , Epoprostenol/metabolismo , Mucosa Intestinal/lesiones , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Ratones Noqueados , Factor A de Crecimiento Endotelial Vascular/genética , Heridas y Lesiones/genética , Heridas y Lesiones/metabolismo , Heridas y Lesiones/patología , Heridas y Lesiones/terapiaRESUMEN
Giardia lamblia is a leading protozoan cause of diarrheal disease worldwide. It colonizes the lumen and epithelial surface of the small intestine, but does not invade the mucosa. Acute infection causes only minimal mucosal inflammation. Effective immune defenses exist, yet their identity and mechanisms remain incompletely understood. Interleukin (IL)-17A has emerged as an important cytokine involved in inflammation and antimicrobial defense against bacterial pathogens at mucosal surfaces. In this study, we demonstrate that IL-17A has a crucial function in host defense against Giardia infection. Using murine infection models with G. muris and G. lamblia, we observed marked and selective induction of intestinal IL-17A with peak expression after 2 weeks. Th17 cells in the lamina propria and innate immune cells in the epithelial compartment of the small intestine were responsible for the IL-17A response. Experiments in gene-targeted mice revealed that the cytokine, and its cognate receptor IL-17RA, were required for eradication of the parasite. The actions of the cytokine were mediated by hematopoietic cells, and were required for the transport of IgA into the intestinal lumen, since IL-17A deficiency led to marked reduction of fecal IgA levels, as well as for increased intestinal expression of several other potential effectors, including ß-defensin 1 and resistin-like molecule ß. In contrast, intestinal hypermotility, another major antigiardial defense mechanism, was not impacted by IL-17A loss. Taken together, these findings demonstrate that IL-17A and IL-17 receptor signaling are essential for intestinal defense against the important lumen-dwelling intestinal parasite Giardia.
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Anticuerpos Antiprotozoarios/biosíntesis , Giardia/inmunología , Giardiasis/inmunología , Inmunoglobulina A/biosíntesis , Interleucina-17/metabolismo , Animales , Anticuerpos Antiprotozoarios/inmunología , Linfocitos T CD4-Positivos/inmunología , Quimera , Giardia lamblia/inmunología , Células Madre Hematopoyéticas/inmunología , Inmunoglobulina A/inmunología , Interleucina-17/genética , Mucosa Intestinal/inmunología , Mucosa Intestinal/parasitología , Intestino Delgado/inmunología , Intestino Delgado/parasitología , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Transducción de Señal/inmunología , Organismos Libres de Patógenos Específicos , Células Th17/inmunologíaRESUMEN
Enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium are attaching-and-effacing (A/E) pathogens that cause intestinal inflammation and diarrhea. The bacteria adhere to the intestinal epithelium, destroy microvilli, and induce actin-filled membranous pedestals but do not invade the mucosa. Adherence leads to activation of several host cell kinases, including FYN, n-SRC, YES, ABL, and ARG, phosphorylation of the bacterial translocated intimin receptor, and actin polymerization and pedestal formation in cultured cells. However, marked functional redundancy appears to exist between kinases, and their physiological importance in A/E pathogen infections has remained unclear. To address this question, we employed a novel dynamic in vitro infection model that mimics transient and short-term interactions in the intestinal tract. Screening of a kinase inhibitor library and RNA interference experiments in vitro revealed that ABL and platelet-derived growth factor (PDGF) receptor (PDGFR) kinases, as well as p38 MAP kinase, have unique, indispensable roles in early attachment of EPEC to epithelial cells under dynamic infection conditions. Studies with mutant EPEC showed that the attachment functions of ABL and PDGFR were independent of the intimin receptor but required bacterial bundle-forming pili. Furthermore, inhibition of ABL and PDGFR with imatinib protected against infection of mice with modest loads of C. rodentium, whereas the kinases were dispensable for high inocula or late after infection. These results indicate that ABL and PDGFR have indispensable roles in early A/E pathogen attachment to intestinal epithelial cells and for in vivo infection with limiting inocula but are not required for late intimate bacterial attachment or high inoculum infections.
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Adhesión Bacteriana/fisiología , Escherichia coli Enteropatógena/metabolismo , Células Epiteliales/fisiología , Proteínas Oncogénicas v-abl/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Línea Celular , Escherichia coli Enteropatógena/citología , Escherichia coli Enteropatógena/fisiología , Infecciones por Escherichia coli/microbiología , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Oncogénicas v-abl/genética , Inhibidores de Proteínas Quinasas/farmacología , Receptores del Factor de Crecimiento Derivado de Plaquetas/genéticaRESUMEN
Interleukin-10 (IL-10) curtails immune responses to microbial infection and autoantigens and contributes to intestinal immune homeostasis, yet administration of IL-10 has not been effective at attenuating chronic intestinal inflammatory conditions, suggesting that its immune functions may be context dependent. To gain a broader understanding of the importance of IL-10 in controlling mucosal immune responses to infectious challenges, we employed the murine attaching and effacing pathogen Citrobacter rodentium, which colonizes primarily the surfaces of the cecum and colon and causes transient mucosal inflammation driven by Th17 and Th1 T helper cells. Infection induced macrophage and dendritic cell production of IL-10, which diminished antibacterial host defenses, because IL-10-deficient mice cleared infection faster than wild-type controls. In parallel, the mice had less acute infection-associated colitis and resolved it more rapidly than controls. Importantly, transient C. rodentium infection protected IL-10-deficient mice against the later development of spontaneous colitis that normally occurs with aging in these mice. Genome-wide expression studies revealed that IL-10 deficiency was associated with downregulation of proinflammatory pathways but increased expression of the anti-inflammatory cytokine IL-27 in response to infection. IL-27 was found to suppress in vitro Th17 and, to a lesser degree, Th1 differentiation independent of IL-10. Furthermore, neutralization of IL-27 resulted in more severe colitis in infected IL-10-deficient mice. Together, these findings indicate that IL-10 is dispensable for resolving C. rodentium-associated colitis and further suggest that IL-27 may be a critical factor for controlling intestinal inflammation and Th17 and Th1 development by IL-10-independent mechanisms.
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Citrobacter rodentium , Infecciones por Enterobacteriaceae/microbiología , Inflamación/microbiología , Interleucina-10/metabolismo , Envejecimiento , Animales , Infecciones por Enterobacteriaceae/metabolismo , Infecciones por Enterobacteriaceae/patología , Femenino , Regulación de la Expresión Génica/inmunología , Interleucina-10/genética , Interleucinas/genética , Interleucinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
The p53 protein has not only important tumor suppressor activity but also additional immunological and other functions, whose nature and extent are just beginning to be recognized. In this article, we show that p53 has a novel inflammation-promoting action in the intestinal tract, because loss of p53 or the upstream activating kinase, ATM, protects against acute intestinal inflammation in murine models. Mechanistically, deficiency in p53 leads to increased survival of epithelial cells and lamina propria macrophages, higher IL-6 expression owing to enhanced glucose-dependent NF-κB activation, and increased mucosal STAT3 activation. Blockade or loss of IL-6 signaling reverses the protective effects of p53 deficiency. Conversely, IL-6 treatment protects against acute colitis in a manner dependent on STAT3 signaling and induction of cytoprotective factors in epithelial cells. Together, these results indicate that p53 promotes inflammation in the intestinal tract through suppression of epithelium-protective factors, thus significantly expanding the spectrum of physiological and immunological p53 activities unrelated to cancer formation.
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Colitis/inmunología , Colitis/prevención & control , Inflamación/inmunología , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada , Células de la Médula Ósea/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Colitis/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Dendríticas/metabolismo , Activación Enzimática , Células Epiteliales/metabolismo , Inflamación/prevención & control , Interleucina-6/biosíntesis , Interleucina-6/farmacología , Interleucinas/biosíntesis , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Estrés Oxidativo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Interleucina-22RESUMEN
The intestinal epithelium is dynamic, with proliferation of undifferentiated crypt cells balanced by terminal differentiation and cell death at the colon surface or small intestinal villus tips. Cyclic AMP, induced by agonists such as prostaglandin E(2) and vasoactive intestinal polypeptide, promotes proliferation and ion secretion and suppresses apoptosis in intestinal epithelial cells. Here, we show that cell differentiation in a model intestinal epithelium leads to attenuation of cAMP production in response to G protein-coupled receptor and receptor-independent agonists. Concomitantly, key components of the cAMP cascade, the alpha subunit of the stimulatory G protein, G(s), and adenylyl cyclase (AC) isoforms 3, 4, 6, and 7 are down-regulated. By contrast, AC1, AC2, AC8, and AC9, and the receptors for prostaglandin E(2) and vasoactive intestinal polypeptide, are not expressed or not affected by differentiation. We confirmed key findings in normal murine colon epithelium, in which the major AC isoforms and G(s)alpha are markedly down-regulated in differentiated surface cells. Suppression of AC isoforms and G(s)alpha is functionally important, because their constitutive expression completely reverses differentiation-induced cAMP attenuation. Thus, down-regulation of AC isoforms and G(s)alpha is an integral part of the intestinal epithelial differentiation program, perhaps serving to release cells from cAMP-promoted anti-apoptosis as a prerequisite for cell death upon terminal differentiation.
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Adenilil Ciclasas/biosíntesis , Diferenciación Celular/fisiología , Colon/enzimología , Células Epiteliales/enzimología , Proteínas de Unión al GTP/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Mucosa Intestinal/enzimología , Animales , Línea Celular , Dinoprostona/metabolismo , Regulación hacia Abajo/fisiología , Humanos , Isoenzimas/biosíntesisRESUMEN
Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrheal disease in young children, yet symptoms and duration are highly variable for unknown reasons. Citrobacter rodentium, a murine model pathogen that shares important functional features with EPEC, colonizes mice in colon and cecum and causes inflammation, but typically little or no diarrhea. We conducted genome-wide microarray studies to define mechanisms of host defense and disease in C. rodentium infection. A significant fraction of the genes most highly induced in the colon by infection encoded CXC chemokines, particularly CXCL1/2/5 and CXCL9/10, which are ligands for the chemokine receptors CXCR2 and CXCR3, respectively. CD11b(+) dendritic cells were the major producers of CXCL1, CXCL5, and CXCL9, while CXCL2 was mainly induced in macrophages. Infection of gene-targeted mice revealed that CXCR3 had a significant but modest role in defense against C. rodentium, whereas CXCR2 had a major and indispensable function. CXCR2 was required for normal mucosal influx of neutrophils, which act as direct antibacterial effectors. Moreover, CXCR2 loss led to severe diarrhea and failure to express critical components of normal ion and fluid transport, including ATPase beta(2)-subunit, CFTR, and DRA. The antidiarrheal functions were unique to CXCR2, since other immune defects leading to increased bacterial load and inflammation did not cause diarrhea. Thus, CXCR2-dependent processes, particularly mucosal neutrophil influx, not only contribute to host defense against C. rodentium, but provide protection against infection-associated diarrhea.
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Adhesión Bacteriana/inmunología , Citrobacter rodentium/patogenicidad , Colitis/inmunología , Diarrea/inmunología , Diarrea/prevención & control , Mucosa Intestinal/inmunología , Infiltración Neutrófila/inmunología , Receptores de Interleucina-8B/fisiología , Animales , Citrobacter rodentium/inmunología , Colitis/microbiología , Colitis/prevención & control , Diarrea/microbiología , Infecciones por Enterobacteriaceae/genética , Infecciones por Enterobacteriaceae/inmunología , Infecciones por Enterobacteriaceae/microbiología , Femenino , Perfilación de la Expresión Génica , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infiltración Neutrófila/genética , Receptores CXCR3/deficiencia , Receptores CXCR3/genética , Receptores de Interleucina-8B/biosíntesis , Receptores de Interleucina-8B/deficiencia , Receptores de Interleucina-8B/genéticaRESUMEN
Enteric infections with attaching/effacing lesion-inducing bacterial pathogens are a worldwide health problem. A murine infection model with one such pathogen, Citrobacter rodentium, was used to elucidate the importance of the pleiotropic immune regulator, IL-6, in the pathogenesis of infection. IL-6 was strongly induced in colonic epithelial cells and macrophages upon C. rodentium infection and was required for effective host defense, because mice lacking IL-6 failed to control bacterial numbers 2-3 wk after infection and exhibited increased mortality. IL-6 was not needed for mounting effective T and B cell responses to the pathogens, nor was it important for induction of IFN-gamma or TNF-alpha, cytokines involved in host defense against the bacteria, or the antibacterial effector, NO. Instead, IL-6 played a key role in mucosal protection, since its absence was associated with marked infection-induced apoptosis in the colonic epithelium and subsequent ulcerations. Cell culture studies confirmed that IL-6 protected colon epithelial cells directly against inducible apoptosis, which was accompanied by increased expression of an array of genes encoding antiapoptotic proteins, including Bcl-x(L), Mcl-1, cIAP-2, and Bcl-3. Ulcerations appeared to be pathogenetically important, because bacteria localized preferentially to those regions, and chemically induced colonic ulcerations promoted bacterial colonization. Furthermore, blood components likely present in ulcer exudates, particularly alanine, asparagine, and glycine, promoted bacterial growth. Thus, IL-6 is an important regulator of host defense against C. rodentium by protecting the mucosa against ulcerations which can act as a microbial niche for the bacteria.
Asunto(s)
Citrobacter rodentium/patogenicidad , Infecciones por Enterobacteriaceae/inmunología , Interleucina-6/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Úlcera/microbiología , Animales , Apoptosis/fisiología , Citrobacter rodentium/inmunología , Infecciones por Enterobacteriaceae/metabolismo , Inmunohistoquímica , Interleucina-6/inmunología , Mucosa Intestinal/patología , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Úlcera/patologíaRESUMEN
MicroRNAs (miRNAs) are endogenously expressed 20-24 nucleotide RNAs thought to repress protein translation through binding to a target mRNA (1-3). Only a few of the more than 250 predicted human miRNAs have been assigned any biological function. In an effort to uncover miRNAs important during adipocyte differentiation, antisense oligonucleotides (ASOs) targeting 86 human miRNAs were transfected into cultured human pre-adipocytes, and their ability to modulate adipocyte differentiation was evaluated. Expression of 254 miRNAs in differentiating adipocytes was also examined on a miRNA microarray. Here we report that the combination of expression data and functional assay results identified a role for miR-143 in adipocyte differentiation. miR-143 levels increased in differentiating adipocytes, and inhibition of miR-143 effectively inhibited adipocyte differentiation. In addition, protein levels of the proposed miR-143 target ERK5 (4) were higher in ASO-treated adipocytes. These results demonstrate that miR-143 is involved in adipocyte differentiation and may act through target gene ERK5.
Asunto(s)
Adipocitos/citología , Adipocitos/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Adipocitos/efectos de los fármacos , Secuencia de Bases , Diferenciación Celular , Células Cultivadas , Sondas de ADN/genética , Humanos , MicroARNs/antagonistas & inhibidores , Oligodesoxirribonucleótidos Antisentido/genética , Oligodesoxirribonucleótidos Antisentido/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , TransfecciónRESUMEN
The mechanism of cytokine secretion is not well understood, but cytokines appear to be synthesized and released in a polarized fashion toward an Ag-specific target cell. In this study, we demonstrate that the Wiskott-Aldrich syndrome protein (WASp) is an essential component of the cytokine secretory pathway in CD4(+) T cells. Murine WASp-deficient CD4(+) T cells fail to polarize cytokines toward a target and show an unexpected and striking block in cytokine secretion. In contrast, chemokine secretion and trafficking of plasma membrane proteins, transported via the constitutive secretory pathway, are unaffected by the lack of WASp. These results suggest that CD4(+) T cell cytokines require a specialized, WASp-dependent pathway for cellular traffic and/or vesicle release that is distinct from that required for chemokine release. We propose that the use of different secretory pathways for cytokines and chemokines enables CD4(+) T cell activity to be further fine-tuned to serve specialized effector functions.