RESUMO
The integrin α4ß7 selectively regulates lymphocyte trafficking and adhesion in the gut and gut-associated lymphoid tissue (GALT). Here, we describe unexpected involvement of the tyrosine phosphatase Shp1 and the B cell lectin CD22 (Siglec-2) in the regulation of α4ß7 surface expression and gut immunity. Shp1 selectively inhibited ß7 endocytosis, enhancing surface α4ß7 display and lymphocyte homing to GALT. In B cells, CD22 associated in a sialic acid-dependent manner with integrin ß7 on the cell surface to target intracellular Shp1 to ß7. Shp1 restrained plasma membrane ß7 phosphorylation and inhibited ß7 endocytosis without affecting ß1 integrin. B cells with reduced Shp1 activity, lacking CD22 or expressing CD22 with mutated Shp1-binding or carbohydrate-binding domains displayed parallel reductions in surface α4ß7 and in homing to GALT. Consistent with the specialized role of α4ß7 in intestinal immunity, CD22 deficiency selectively inhibited intestinal antibody and pathogen responses.
Assuntos
Linfócitos B/enzimologia , Imunidade nas Mucosas , Cadeias beta de Integrinas/metabolismo , Integrinas/metabolismo , Mucosa Intestinal/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/virologia , Quimiotaxia de Leucócito , Modelos Animais de Doenças , Endocitose , Feminino , Cadeias beta de Integrinas/imunologia , Integrinas/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/virologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 6/deficiência , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Rotavirus/imunologia , Rotavirus/patogenicidade , Infecções por Rotavirus/genética , Infecções por Rotavirus/imunologia , Infecções por Rotavirus/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/deficiência , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Transdução de Sinais , Técnicas de Cultura de TecidosRESUMO
Intestinal epithelial cell (IEC) damage by T cells contributes to graft-versus-host disease, inflammatory bowel disease and immune checkpoint blockade-mediated colitis. But little is known about the target cell-intrinsic features that affect disease severity. Here we identified disruption of oxidative phosphorylation and an increase in succinate levels in the IECs from several distinct in vivo models of T cell-mediated colitis. Metabolic flux studies, complemented by imaging and protein analyses, identified disruption of IEC-intrinsic succinate dehydrogenase A (SDHA), a component of mitochondrial complex II, in causing these metabolic alterations. The relevance of IEC-intrinsic SDHA in mediating disease severity was confirmed by complementary chemical and genetic experimental approaches and validated in human clinical samples. These data identify a critical role for the alteration of the IEC-specific mitochondrial complex II component SDHA in the regulation of the severity of T cell-mediated intestinal diseases.
Assuntos
Colite/enzimologia , Colo/enzimologia , Citotoxicidade Imunológica , Complexo II de Transporte de Elétrons/metabolismo , Células Epiteliais/enzimologia , Doença Enxerto-Hospedeiro/enzimologia , Mucosa Intestinal/enzimologia , Mitocôndrias/enzimologia , Linfócitos T/imunologia , Animais , Estudos de Casos e Controles , Comunicação Celular , Células Cultivadas , Colite/genética , Colite/imunologia , Colite/patologia , Colo/imunologia , Colo/ultraestrutura , Modelos Animais de Doenças , Complexo II de Transporte de Elétrons/genética , Células Epiteliais/imunologia , Células Epiteliais/ultraestrutura , Feminino , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Humanos , Imunidade nas Mucosas , Mucosa Intestinal/imunologia , Mucosa Intestinal/ultraestrutura , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/imunologia , Mitocôndrias/ultraestrutura , Fosforilação Oxidativa , Ácido Succínico/metabolismo , Linfócitos T/metabolismoRESUMO
Serrated adenocarcinoma, an alternative pathway for colorectal cancer (CRC) development, accounts for 15%-30% of all CRCs and is aggressive and treatment resistant. We show that the expression of atypical protein kinase C ζ (PKCζ) and PKCλ/ι was reduced in human serrated tumors. Simultaneous inactivation of the encoding genes in the mouse intestinal epithelium resulted in spontaneous serrated tumorigenesis that progressed to advanced cancer with a strongly reactive and immunosuppressive stroma. Whereas epithelial PKCλ/ι deficiency led to immunogenic cell death and the infiltration of CD8+ T cells, which repressed tumor initiation, PKCζ loss impaired interferon and CD8+ T cell responses, which resulted in tumorigenesis. Combined treatment with a TGF-ß receptor inhibitor plus anti-PD-L1 checkpoint blockade showed synergistic curative activity. Analysis of human samples supported the relevance of these kinases in the immunosurveillance defects of human serrated CRC. These findings provide insight into avenues for the detection and treatment of this poor-prognosis subtype of CRC.
Assuntos
Mucosa Intestinal/imunologia , Neoplasias Intestinais/imunologia , Isoenzimas/imunologia , Proteína Quinase C/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/imunologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Feminino , Humanos , Vigilância Imunológica/genética , Vigilância Imunológica/imunologia , Mucosa Intestinal/enzimologia , Mucosa Intestinal/patologia , Neoplasias Intestinais/enzimologia , Neoplasias Intestinais/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismoRESUMO
The coevolution of mammalian hosts and their beneficial commensal microbes has led to development of symbiotic host-microbiota relationships1. Epigenetic machinery permits mammalian cells to integrate environmental signals2; however, how these pathways are fine-tuned by diverse cues from commensal bacteria is not well understood. Here we reveal a highly selective pathway through which microbiota-derived inositol phosphate regulates histone deacetylase 3 (HDAC3) activity in the intestine. Despite the abundant presence of HDAC inhibitors such as butyrate in the intestine, we found that HDAC3 activity was sharply increased in intestinal epithelial cells of microbiota-replete mice compared with germ-free mice. This divergence was reconciled by the finding that commensal bacteria, including Escherichia coli, stimulated HDAC activity through metabolism of phytate and production of inositol-1,4,5-trisphosphate (InsP3). Both intestinal exposure to InsP3 and phytate ingestion promoted recovery following intestinal damage. Of note, InsP3 also induced growth of intestinal organoids derived from human tissue, stimulated HDAC3-dependent proliferation and countered butyrate inhibition of colonic growth. Collectively, these results show that InsP3 is a microbiota-derived metabolite that activates a mammalian histone deacetylase to promote epithelial repair. Thus, HDAC3 represents a convergent epigenetic sensor of distinct metabolites that calibrates host responses to diverse microbial signals.
Assuntos
Microbioma Gastrointestinal/fisiologia , Histona Desacetilases/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Intestinos/enzimologia , Intestinos/microbiologia , Ácido Fítico/metabolismo , Animais , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/enzimologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Intestinos/citologia , Intestinos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Organoides/enzimologia , Organoides/metabolismo , Organoides/patologia , SimbioseRESUMO
Caspase-8 is a protease with both pro-death and pro-survival functions: it mediates apoptosis induced by death receptors such as TNFR11, and suppresses necroptosis mediated by the kinase RIPK3 and the pseudokinase MLKL2-4. Mice that lack caspase-8 display MLKL-dependent embryonic lethality4, as do mice that express catalytically inactive CASP8(C362A)5. Casp8C362A/C362AMlkl-/- mice die during the perinatal period5, whereas Casp8-/-Mlkl-/- mice are viable4, which indicates that inactive caspase-8 also has a pro-death scaffolding function. Here we show that mutant CASP8(C362A) induces the formation of ASC (also known as PYCARD) specks, and caspase-1-dependent cleavage of GSDMD and caspases 3 and 7 in MLKL-deficient mouse intestines around embryonic day 18. Caspase-1 and its adaptor ASC contributed to the perinatal lethal phenotype because a number of Casp8C362A/C362AMlkl-/-Casp1-/- and Casp8C362A/C362AMlkl-/-Asc-/- mice survived beyond weaning. Transfection studies suggest that inactive caspase-8 adopts a distinct conformation to active caspase-8, enabling its prodomain to engage ASC. Upregulation of the lipopolysaccharide sensor caspase-11 in the intestines of both Casp8C362A/C362AMlkl-/- and Casp8C362A/C362AMlkl-/-Casp1-/- mice also contributed to lethality because Casp8C362A/C362AMlkl-/-Casp1-/-Casp11-/- (Casp11 is also known as Casp4) neonates survived more often than Casp8C362A/C362AMlkl-/-Casp1-/- neonates. Finally, Casp8C362A/C362ARipk3-/-Casp1-/-Casp11-/- mice survived longer than Casp8C362A/C362AMlkl-/-Casp1-/-Casp11-/- mice, indicating that a necroptosis-independent function of RIPK3 also contributes to lethality. Thus, unanticipated plasticity in death pathways is revealed when caspase-8-dependent apoptosis and MLKL-dependent necroptosis are inhibited.
Assuntos
Caspase 8/metabolismo , Morte Celular/genética , Mucosa Intestinal/citologia , Animais , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 8/genética , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Mucosa Intestinal/enzimologia , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
Caspase-8 is the initiator caspase of extrinsic apoptosis1,2 and inhibits necroptosis mediated by RIPK3 and MLKL. Accordingly, caspase-8 deficiency in mice causes embryonic lethality3, which can be rescued by deletion of either Ripk3 or Mlkl4-6. Here we show that the expression of enzymatically inactive CASP8(C362S) causes embryonic lethality in mice by inducing necroptosis and pyroptosis. Similar to Casp8-/- mice3,7, Casp8C362S/C362S mouse embryos died after endothelial cell necroptosis leading to cardiovascular defects. MLKL deficiency rescued the cardiovascular phenotype but unexpectedly caused perinatal lethality in Casp8C362S/C362S mice, indicating that CASP8(C362S) causes necroptosis-independent death at later stages of embryonic development. Specific loss of the catalytic activity of caspase-8 in intestinal epithelial cells induced intestinal inflammation similar to intestinal epithelial cell-specific Casp8 knockout mice8. Inhibition of necroptosis by additional deletion of Mlkl severely aggravated intestinal inflammation and caused premature lethality in Mlkl knockout mice with specific loss of caspase-8 catalytic activity in intestinal epithelial cells. Expression of CASP8(C362S) triggered the formation of ASC specks, activation of caspase-1 and secretion of IL-1ß. Both embryonic lethality and premature death were completely rescued in Casp8C362S/C362SMlkl-/-Asc-/- or Casp8C362S/C362SMlkl-/-Casp1-/- mice, indicating that the activation of the inflammasome promotes CASP8(C362S)-mediated tissue pathology when necroptosis is blocked. Therefore, caspase-8 represents the molecular switch that controls apoptosis, necroptosis and pyroptosis, and prevents tissue damage during embryonic development and adulthood.
Assuntos
Apoptose/genética , Caspase 8/genética , Caspase 8/metabolismo , Necroptose/genética , Piroptose/genética , Animais , Linhagem Celular , Células Cultivadas , Ativação Enzimática/genética , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Inflamassomos/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/enzimologia , Queratinócitos/citologia , Queratinócitos/patologia , Camundongos , Mutação , Receptor TIE-2/genética , Receptor TIE-2/metabolismoRESUMO
Apoptosis is widely believed to be crucial for epithelial cell death and shedding in the intestine, thereby shaping the overall architecture of the gastrointestinal tract, but also regulating tolerance induction, pinpointing a role of apoptosis intestinal epithelial cell (IEC) turnover and maintenance of barrier function, and in maintaining immune homeostasis. To experimentally address this concept, we generated IEC-specific knockout mice that lack both executioner caspase-3 and caspase-7 (Casp3/7ΔIEC), which are the converging point of the extrinsic and intrinsic apoptotic pathway. Surprisingly, the overall architecture, cellular landscape, and proliferation rate remained unchanged in these mice. However, nonapoptotic cell extrusion was increased in Casp3/7ΔIEC mice, compensating apoptosis deficiency, maintaining the same physiological level of IEC shedding. Microbiome richness and composition stayed unaffected, bearing no sign of dysbiosis. Transcriptome and single-cell RNA sequencing analyses of IECs and immune cells revealed no differences in signaling pathways of differentiation and inflammation. These findings demonstrate that during homeostasis, apoptosis per se is dispensable for IEC turnover at the top of intestinal villi intestinal tissue dynamics, microbiome, and immune cell composition.
Assuntos
Apoptose , Caspase 3/metabolismo , Caspase 7/metabolismo , Células Epiteliais/enzimologia , Homeostase , Mucosa Intestinal/enzimologia , Transdução de Sinais , Animais , Caspase 3/genética , Caspase 7/genética , Camundongos , Camundongos TransgênicosRESUMO
Lgr5+ intestinal stem cells (ISCs) exhibit self-renewal and differentiation features under homeostatic conditions, but the mechanisms controlling Lgr5 + ISC self-renewal remain elusive. Here, we show that the chromatin remodeler SRCAP is highly expressed in mouse intestinal epithelium and ISCs. Srcap deletion impairs both self-renewal of ISCs and intestinal epithelial regeneration. Mechanistically, SRCAP recruits the transcriptional regulator REST to the Prdm16 promoter and induces expression of this transcription factor. By activating PPARδ expression, Prdm16 in turn initiates PPARδ signaling, which sustains ISC stemness. Rest or Prdm16 deficiency abrogates the self-renewal capacity of ISCs as well as intestinal epithelial regeneration. Collectively, these data show that the SRCAP-REST-Prdm16-PPARδ axis is required for self-renewal maintenance of Lgr5 + ISCs.
Assuntos
Adenosina Trifosfatases/metabolismo , Mucosa Intestinal/enzimologia , Transdução de Sinais , Células-Tronco/enzimologia , Adenosina Trifosfatases/genética , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Mucosa Intestinal/citologia , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Células-Tronco/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The DNA-sensing enzyme cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) regulates inflammation and immune defense against pathogens and malignant cells. Although cGAS has been shown to exert antitumor effects in several mouse models harboring transplanted tumor cell lines, its role in tumors arising from endogenous tissues remains unknown. Here, we show that deletion of cGAS in mice exacerbated chemical-induced colitis and colitis-associated colon cancer (CAC). Interestingly, mice lacking cGAS were more susceptible to CAC than those lacking stimulator of interferon genes (STING) or type I interferon receptor under the same conditions. cGAS but not STING is highly expressed in intestinal stem cells. cGAS deficiency led to intestinal stem cell loss and compromised intestinal barrier integrity upon dextran sodium sulfate-induced acute injury. Loss of cGAS exacerbated inflammation, led to activation of STAT3, and accelerated proliferation of intestinal epithelial cells during CAC development. Mice lacking cGAS also accumulated myeloid-derived suppressive cells within the tumor, displayed enhanced Th17 differentiation, but reduced interleukin (IL)-10 production. These results indicate that cGAS plays an important role in controlling CAC development by defending the integrity of the intestinal mucosa.
Assuntos
Neoplasias do Colo/enzimologia , Mucosa Intestinal/enzimologia , Proteínas de Neoplasias/metabolismo , Nucleotidiltransferases/metabolismo , Animais , Neoplasias do Colo/genética , Camundongos , Camundongos Knockout , Células Supressoras Mieloides/enzimologia , Proteínas de Neoplasias/genética , Nucleotidiltransferases/genética , Células-Tronco/enzimologia , Células Th17/enzimologiaRESUMO
OBJECTIVES: We investigated potential relationships among initial lesions of the intestinal mucosa, fecal enzymatic activities and microbiota profiles. METHODS: Fecal samples from 54 volunteers were collected after recruitment among individuals participating in a colorectal cancer (CRC) screening program in our region (Northern Spain) or attending for consultation due to clinical symptoms; intestinal mucosa samples were resected during colonoscopy. Enzymatic activities were determined in fecal supernatants by a semi-quantitative method. The fecal microbiota composition was determined by 16S rRNA gene-based sequencing. The results were compared between samples from clinical diagnosis groups (controls and polyps), according with the type of polyp (hyperplastic polyps or conventional adenomas) and considering the grade of dysplasia for conventional adenomas (low and high grade dysplasia). RESULTS: High levels of α-glucosidase activity were more frequent among samples from individuals diagnosed with intestinal polyps, reaching statistical significance for conventional adenomas and for low grade dysplasia adenomas when compared to controls. Regarding the microbiota profiles, higher abundance of Christensenellaceae_R-7 group and Oscillospiraceae_UCG-002 were found in fecal samples displaying low α-glucosidase activity as compared with those with higher activity as well as in controls with respect to conventional adenomas. A relationship was evidenced among intestinal mucosal lesions, gut glucosidase activities and intestinal microbiota profiles. CONCLUSIONS: Our findings suggest a relationship among altered fecal α-glucosidase levels, the presence of intestinal mucosal lesions, which can be precursors of CRC, and shifts in defined microbial groups of the fecal microbiota.
Assuntos
Fezes , Microbioma Gastrointestinal , Mucosa Intestinal , alfa-Glucosidases , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , alfa-Glucosidases/metabolismo , alfa-Glucosidases/genética , Neoplasias Colorretais/microbiologia , Neoplasias Colorretais/patologia , Fezes/microbiologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Mucosa Intestinal/enzimologia , RNA Ribossômico 16S/genética , EspanhaRESUMO
BACKGROUND & AIMS: Because inflammatory bowel disease is increasing worldwide and can lead to colitis-associated carcinoma (CAC), new interventions are needed. We have shown that spermine oxidase (SMOX), which generates spermidine (Spd), regulates colitis. Here we determined whether Spd treatment reduces colitis and carcinogenesis. METHODS: SMOX was quantified in human colitis and associated dysplasia using quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. We used wild-type (WT) and Smox-/- C57BL/6 mice treated with dextran sulfate sodium (DSS) or azoxymethane (AOM)-DSS as models of colitis and CAC, respectively. Mice with epithelial-specific deletion of Apc were used as a model of sporadic colon cancer. Animals were supplemented or not with Spd in the drinking water. Colonic polyamines, inflammation, tumorigenesis, transcriptomes, and microbiomes were assessed. RESULTS: SMOX messenger RNA levels were decreased in human ulcerative colitis tissues and inversely correlated with disease activity, and SMOX protein was reduced in colitis-associated dysplasia. DSS colitis and AOM-DSS-induced dysplasia and tumorigenesis were worsened in Smox-/- vs WT mice and improved in both genotypes with Spd. Tumor development caused by Apc deletion was also reduced by Spd. Smox deletion and AOM-DSS treatment were both strongly associated with increased expression of α-defensins, which was reduced by Spd. A shift in the microbiome, with reduced abundance of Prevotella and increased Proteobacteria and Deferribacteres, occurred in Smox-/- mice and was reversed with Spd. CONCLUSIONS: Loss of SMOX is associated with exacerbated colitis and CAC, increased α-defensin expression, and dysbiosis of the microbiome. Spd supplementation reverses these phenotypes, indicating that it has potential as an adjunctive treatment for colitis and chemopreventive for colon carcinogenesis.
Assuntos
Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Colite/genética , Neoplasias do Colo/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Espermidina/uso terapêutico , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Azoximetano , Colite/induzido quimicamente , Colite/enzimologia , Colite/prevenção & controle , Colite Ulcerativa/enzimologia , Colite Ulcerativa/genética , Colo/enzimologia , Colo/patologia , Neoplasias do Colo/prevenção & controle , Sulfato de Dextrana , Microbioma Gastrointestinal/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mucosa Intestinal/enzimologia , Mucosa Intestinal/patologia , Masculino , Camundongos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Lesões Pré-Cancerosas/enzimologia , Fatores de Proteção , RNA Mensageiro/metabolismo , Índice de Gravidade de Doença , Espermidina/metabolismo , Espermidina/farmacologia , Redução de Peso/efeitos dos fármacos , alfa-Defensinas/genética , alfa-Defensinas/metabolismo , Poliamina OxidaseRESUMO
BACKGROUND & AIMS: Sulfation is a conjugation reaction essential for numerous biochemical and cellular functions in mammals. The 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase 2 (PAPSS2) is the key enzyme to generate PAPS, which is the universal sulfonate donor for all sulfation reactions. The goal of this study was to determine whether and how PAPSS2 plays a role in colitis and colonic carcinogenesis. METHODS: Tissue arrays of human colon cancer specimens, gene expression data, and clinical features of cancer patients were analyzed. Intestinal-specific Papss2 knockout mice (Papss2ΔIE) were created and subjected to dextran sodium sulfate-induced colitis and colonic carcinogenesis induced by a combined treatment of azoxymethane and dextran sodium sulfate or azoxymethane alone. RESULTS: The expression of PAPSS2 is decreased in the colon cancers of mice and humans. The lower expression of PAPSS2 in colon cancer patients is correlated with worse survival. Papss2ΔIE mice showed heightened sensitivity to colitis and colon cancer by damaging the intestinal mucosal barrier, increasing intestinal permeability and bacteria infiltration, and worsening the intestinal tumor microenvironment. Mechanistically, the Papss2ΔIE mice exhibited reduced intestinal sulfomucin content. Metabolomic analyses revealed the accumulation of bile acids, including the Farnesoid X receptor antagonist bile acid tauro-ß-muricholic acid, and deficiency in the formation of bile acid sulfates in the colon of Papss2ΔIE mice. CONCLUSIONS: We have uncovered an important role of PAPSS2-mediated sulfation in colitis and colonic carcinogenesis. Intestinal sulfation may represent a potential diagnostic marker and PAPSS2 may serve as a potential therapeutic target for inflammatory bowel disease and colon cancer.
Assuntos
Neoplasias Associadas a Colite/prevenção & controle , Colite/prevenção & controle , Colo/enzimologia , Mucosa Intestinal/enzimologia , Mucinas/metabolismo , Complexos Multienzimáticos/metabolismo , Sulfato Adenililtransferase/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Colite/enzimologia , Colite/genética , Colite/patologia , Neoplasias Associadas a Colite/enzimologia , Neoplasias Associadas a Colite/genética , Neoplasias Associadas a Colite/patologia , Colo/patologia , Bases de Dados Genéticas , Modelos Animais de Doenças , Humanos , Mucosa Intestinal/patologia , Metaboloma , Metabolômica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multienzimáticos/genética , Prognóstico , Receptores Citoplasmáticos e Nucleares/metabolismo , Sulfato Adenililtransferase/genéticaRESUMO
BACKGROUND & AIMS: The molecular checkpoints driving T cell activation and cytokine responses in ulcerative colitis (UC) are incompletely understood. Here, we studied the Tec kinase ITK in UC. METHODS: We analyzed patients with inflammatory bowel disease (n = 223) and evaluated ITK activity as well as the functional effects of cyclosporine-A (CsA). In addition, 3 independent murine colitis models were used to investigate the functional role of ITK. Finally, the activity of ITK was blocked via pharmacological inhibitors and genetically engineered mice. Readout parameters were mini-endoscopy, histopathology, mucosal T cell apoptosis, and cytokine production. RESULTS: We found an expansion of pITK-expressing mucosal CD4+ T cells in UC rather than Crohn's disease that correlated with disease severity. CsA suppressed activation of ITK in cultured CD4+ T cells and calcineurin-containing microclusters adjacent to the T cell receptor signaling complex. Functionally, the capacity of CsA to suppress activity of experimental colitis was critically dependent on ITK. Genetic inactivation of Itk via gene targeting or induction of allele-sensitive Itk mutants prevented experimental colitis in 3 colitis models, and treatment with pharmacological ITK blockers suppressed established colitis. In addition, ITK controlled apoptosis and activation of mucosal Th2 and Th17 lymphocytes via NFATc2 signaling pathways. CONCLUSIONS: ITK activation was detected in UC and could be down-regulated in cultured T cells by CsA administration. Selective targeting of ITK emerges as an attractive approach for treatment of chronic intestinal inflammation and potentially UC by driving resolution of mucosal inflammation.
Assuntos
Anti-Inflamatórios/farmacologia , Colite Ulcerativa/prevenção & controle , Colo/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Linfócitos Intraepiteliais/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Colite Ulcerativa/enzimologia , Colite Ulcerativa/imunologia , Colite Ulcerativa/patologia , Colo/enzimologia , Colo/imunologia , Colo/patologia , Ciclosporina/farmacologia , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Mucosa Intestinal/enzimologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Linfócitos Intraepiteliais/enzimologia , Linfócitos Intraepiteliais/imunologia , Linfócitos Intraepiteliais/patologia , Camundongos Knockout , Terapia de Alvo Molecular , Fosforilação , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Transdução de SinaisRESUMO
The role of angiotensin converting enzyme 2 has expanded from regulating the renin angiotensin system to regulating intestinal amino acid homeostasis and the gut microbiome. Recently, angiotensin converting enzyme 2 was identified as a primary receptor for severe acute respiratory syndrome coronaviruses 1 and 2 being expressed in multiple tissues including the luminal surface of the gut. In this brief perspective, we examine the role of angiotensin converting enzyme 2 as the receptor for severe acute respiratory syndrome coronavirus 2 and the impact of coronavirus disease 19 infection on the gut microbiome and on the gut epithelium.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/enzimologia , Gastroenterite/enzimologia , Microbioma Gastrointestinal , Mucosa Intestinal/enzimologia , Receptores Virais/metabolismo , SARS-CoV-2/patogenicidade , Enzima de Conversão de Angiotensina 2/uso terapêutico , Animais , Anti-Inflamatórios/uso terapêutico , Antivirais/uso terapêutico , COVID-19/microbiologia , COVID-19/virologia , Fezes/microbiologia , Fezes/virologia , Gastroenterite/tratamento farmacológico , Gastroenterite/microbiologia , Gastroenterite/virologia , Microbioma Gastrointestinal/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/virologia , Sistema Renina-Angiotensina , SARS-CoV-2/efeitos dos fármacos , Internalização do Vírus , Tratamento Farmacológico da COVID-19RESUMO
BACKGROUND AND AIMS: The presence of gastrointestinal symptoms and high levels of viral RNA in the stool suggest active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication within enterocytes. METHODS: Here, in multiple, large cohorts of patients with inflammatory bowel disease (IBD), we have studied the intersections between Coronavirus Disease 2019 (COVID-19), intestinal inflammation, and IBD treatment. RESULTS: A striking expression of ACE2 on the small bowel enterocyte brush border supports intestinal infectivity by SARS-CoV-2. Commonly used IBD medications, both biologic and nonbiologic, do not significantly impact ACE2 and TMPRSS2 receptor expression in the uninflamed intestines. In addition, we have defined molecular responses to COVID-19 infection that are also enriched in IBD, pointing to shared molecular networks between COVID-19 and IBD. CONCLUSIONS: These data generate a novel appreciation of the confluence of COVID-19- and IBD-associated inflammation and provide mechanistic insights supporting further investigation of specific IBD drugs in the treatment of COVID-19. Preprint doi: https://doi.org/10.1101/2020.05.21.109124.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/enzimologia , Doenças Inflamatórias Intestinais/enzimologia , Mucosa Intestinal/enzimologia , SARS-CoV-2/patogenicidade , Serina Endopeptidases/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Animais , Anti-Inflamatórios/uso terapêutico , Antivirais/uso terapêutico , COVID-19/genética , COVID-19/virologia , Estudos de Casos e Controles , Ensaios Clínicos como Assunto , Estudos Transversais , Modelos Animais de Doenças , Feminino , Redes Reguladoras de Genes , Interações Hospedeiro-Patógeno , Humanos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/genética , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/virologia , Estudos Longitudinais , Masculino , Camundongos , SARS-CoV-2/efeitos dos fármacos , Serina Endopeptidases/genética , Transdução de Sinais , Tratamento Farmacológico da COVID-19RESUMO
We investigated the role of the inflammasome effector caspases-1 and -11 during Salmonella enterica serovar Typhimurium infection of murine intestinal epithelial cells (IECs). Salmonella burdens were significantly greater in the intestines of caspase-1/11 deficient (Casp1/11-/-), Casp1-/- and Casp11-/- mice, as compared to wildtype mice. To determine if this reflected IEC-intrinsic inflammasomes, enteroid monolayers were derived and infected with Salmonella. Casp11-/- and wildtype monolayers responded similarly, whereas Casp1-/- and Casp1/11-/- monolayers carried significantly increased intracellular burdens, concomitant with marked decreases in IEC shedding and death. Pretreatment with IFN-γ to mimic inflammation increased caspase-11 levels and IEC death, and reduced Salmonella burdens in Casp1-/- monolayers, while high intracellular burdens and limited cell shedding persisted in Casp1/11-/- monolayers. Thus caspase-1 regulates inflammasome responses in IECs at baseline, while proinflammatory activation of IECs reveals a compensatory role for caspase-11. These results demonstrate the importance of IEC-intrinsic canonical and non-canonical inflammasomes in host defense against Salmonella.
Assuntos
Caspase 1/imunologia , Caspases Iniciadoras/imunologia , Inflamassomos/imunologia , Intestinos/enzimologia , Intestinos/imunologia , Infecções por Salmonella/enzimologia , Salmonella typhimurium/imunologia , Animais , Células Epiteliais/enzimologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Feminino , Imunidade nas Mucosas , Inflamassomos/metabolismo , Interferon gama/imunologia , Mucosa Intestinal/enzimologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Intestinos/microbiologia , Lipopolissacarídeos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Salmonella/imunologia , Salmonella typhimurium/patogenicidadeRESUMO
Changes in the gut microbiota may underpin many human diseases, but the mechanisms that are responsible for altering microbial communities remain poorly understood. Antibiotic usage elevates the risk of contracting gastroenteritis caused by Salmonella enterica serovars, increases the duration for which patients shed the pathogen in their faeces, and may on occasion produce a bacteriologic and symptomatic relapse. These antibiotic-induced changes in the gut microbiota can be studied in mice, in which the disruption of a balanced microbial community by treatment with the antibiotic streptomycin leads to an expansion of S. enterica serovars in the large bowel. However, the mechanisms by which streptomycin treatment drives an expansion of S. enterica serovars are not fully resolved. Here we show that host-mediated oxidation of galactose and glucose promotes post-antibiotic expansion of S. enterica serovar Typhimurium (S. Typhimurium). By elevating expression of the gene encoding inducible nitric oxide synthase (iNOS) in the caecal mucosa, streptomycin treatment increased post-antibiotic availability of the oxidation products galactarate and glucarate in the murine caecum. S. Typhimurium used galactarate and glucarate within the gut lumen of streptomycin pre-treated mice, and genetic ablation of the respective catabolic pathways reduced S. Typhimurium competitiveness. Our results identify host-mediated oxidation of carbohydrates in the gut as a mechanism for post-antibiotic pathogen expansion.
Assuntos
Antibacterianos/farmacologia , Metabolismo dos Carboidratos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/crescimento & desenvolvimento , Estreptomicina/farmacologia , Animais , Metabolismo dos Carboidratos/efeitos dos fármacos , Metabolismo dos Carboidratos/genética , Ceco/efeitos dos fármacos , Ceco/enzimologia , Ceco/microbiologia , Feminino , Galactose/metabolismo , Gastroenterite/microbiologia , Ácido Glucárico/metabolismo , Glucose/metabolismo , Mucosa Intestinal/enzimologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Masculino , Camundongos , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Óperon/genética , Oxirredução/efeitos dos fármacos , Espécies Reativas de Nitrogênio/metabolismo , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Açúcares Ácidos/metabolismoRESUMO
The small intestine plays a critical role in the absorption and metabolism of orally administered drugs. Therefore, a model capable of evaluating drug absorption and metabolism in the small intestine would be useful for drug discovery. Patients with genotype UGT1A1*6 (exon 1, 211G > A) treated with the antineoplastic drug SN-38 have been reported to exhibit decreased glucuronide conjugation and increased incidence of intestinal toxicity and its severe side effects, including severe diarrhea. To ensure the safety of drugs, we must develop a drug metabolism and toxicity evaluation model which considers UGT1A1*6. In this study, we generated CYP3A4·POR·UGT1A1 KI- and CYP3A4·POR·UGT1A1*6 KI-Caco-2 cells for pharmaceutical research using a PITCh system. The CYP3A4·POR·UGT1A1 KI-Caco-2 cells were shown to express functional CYP3A4 and UGT1A1. The CYP3A4·POR·UGT1A1*6 KI-Caco-2 cells were sensitive to SN-38-induced intestinal toxicity. We thus succeeded in generating CYP3A4·POR·UGT1A1 KI- and CYP3A4·POR·UGT1A1*6 KI-Caco-2 cells, which can be used in pharmaceutical research. We also developed an intestinal epithelial cell model of patients with UGT1A1*6 and showed that it was useful as a tool for drug discovery.
Assuntos
Citocromo P-450 CYP3A/genética , Glucuronosiltransferase/genética , Mucosa Intestinal/enzimologia , Intestino Delgado/enzimologia , Antineoplásicos/toxicidade , Células CACO-2/enzimologia , Descoberta de Drogas/métodos , Genótipo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Intestino Delgado/citologia , Intestino Delgado/efeitos dos fármacos , Irinotecano/toxicidadeRESUMO
The stem/progenitor cells of the developing intestine are biologically distinct from their adult counterparts. Here, we examine the microenvironmental cues that regulate the embryonic stem/progenitor population, focusing on the role of Notch pathway factor delta-like protein-1 (DLK1). mRNA-seq analyses of intestinal mesenchymal cells (IMCs) collected from embryonic day 14.5 (E14.5) or adult IMCs and a novel coculture system with E14.5 intestinal epithelial organoids were used. Following addition of recombinant DLK1 (rDLK) or Dlk1 siRNA (siDlk1), epithelial characteristics were compared using imaging, replating efficiency assays, qPCR, and immunocytochemistry. The intestinal phenotypes of littermate Dlk1+/+ and Dlk1-/- mice were compared using immunohistochemistry. Using transcriptomic analyses, we identified morphogens derived from the embryonic mesenchyme that potentially regulate the developing epithelial cells, to focus on Notch family candidate DLK1. Immunohistochemistry indicated that DLK1 was expressed exclusively in the intestinal stroma at E14.5 at the top of emerging villi, decreased after birth, and shifted to the intestinal epithelium in adulthood. In coculture experiments, addition of rDLK1 to adult IMCs inhibited organoid differentiation, whereas Dlk1 knockdown in embryonic IMCs increased epithelial differentiation to secretory lineage cells. Dlk1-/- mice had restricted Ki67+ cells in the villi base and increased secretory lineage cells compared with Dlk1+/+ embryos. Mesenchyme-derived DLK1 plays an important role in the promotion of epithelial stem/precursor expansion and prevention of differentiation to secretory lineages in the developing intestine.NEW & NOTEWORTHY Using a novel coculture system, transcriptomics, and transgenic mice, we investigated differential molecular signaling between the intestinal epithelium and mesenchyme during development and in the adult. We show that the Notch pathway factor delta-like protein-1 (DLK1) is stromally produced during development and uncover a new role for DLK1 in the regulation of intestinal epithelial stem/precursor expansion and differentiation to secretory lineages.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Comunicação Celular , Diferenciação Celular , Proliferação de Células , Células-Tronco Embrionárias/enzimologia , Células Epiteliais/enzimologia , Mucosa Intestinal/enzimologia , Células Estromais/enzimologia , Animais , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Linhagem da Célula , Células Cultivadas , Técnicas de Cocultura , Regulação da Expressão Gênica no Desenvolvimento , Mucosa Intestinal/embriologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Organoides , Via Secretória , Transdução de Sinais , Nicho de Células-Tronco , TranscriptomaRESUMO
BACKGROUND & AIMS: The mechanisms by which macrophages regulate intestinal epithelial cell (IEC) barrier properties are poorly understood. Protein tyrosine phosphatase non-receptor type 2 (PTPN2) protects the IEC barrier from inflammation-induced disruption and regulates macrophage functions. We investigated whether PTPN2 controls interactions between IECs and macrophages to maintain intestinal barrier function. METHODS: Human IEC (Caco-2BBe/HT-29.cl19a cells) and mouse enteroid monolayers were cocultured with human macrophages (THP-1, U937, primary monocyte-derived macrophages from patients with inflammatory bowel disease [IBD]) or mouse macrophages, respectively. We assessed barrier function (transepithelial electrical resistance [TEER] and permeability to 4-kDa fluorescently labeled dextran or 70-kDa rhodamine B-dextran) and macrophage polarization. We analyzed intestinal tissues from mice with myeloid cell-specific deletion of PTPN2 (Ptpn2-LysMCre mice) and mice without disruption of Ptpn2 (controls); some mice were given injections of a neutralizing antibody against interleukin (IL) 6. Proteins were knocked down in macrophages and/or IECs with small hairpin RNAs. RESULTS: Knockdown of PTPN2 in either macrophages and/or IECs increased the permeability of IEC monolayers, had a synergistic effect when knocked down from both cell types, and increased the development of inflammatory macrophages in macrophage-IEC cocultures. Colon lamina propria from Ptpn2-LysMCre mice had significant increases in inflammatory macrophages; these mice had increased in vivo and ex vivo colon permeability to 4-kDa fluorescently labeled dextran and reduced ex vivo colon TEER. Nanostring analysis showed significant increases in the expression of IL6 in colon macrophages from Ptpn2-LysMCre mice. An IL6-blocking antibody reversed the effects of PTPN2-deficient macrophages, reducing the permeability of IEC monolayers in culture and in Ptpn2-LysMCre mice. Macrophages from patients with IBD carrying a single-nucleotide polymorphism associated with the disease (PTPN2 rs1893217) had the same features of PTPN2-deficient macrophages from mice, including reduced TEER and increased permeability in cocultures with human IEC or mouse enteroid monolayers, which were restored by anti-IL6. CONCLUSIONS: PTPN2 is required for interactions between macrophages and IECs; loss of PTPN2 from either cell type results in intestinal barrier defects, and loss from both cell types has a synergistic effect. We provide a mechanism by which the PTPN2 gene variants compromise intestinal epithelial barrier function and increase the risk of inflammatory disorders such as IBD.