RESUMEN
The Na+/H+ exchanger transporters (NHE) play an important role in various biologic processes including Na+ absorption, intracellular pH homeostasis, cell volume regulation, proliferation, and apoptosis. The wide expression pattern and cellular localization of NHEs make these proteins pivotal players in virtually all human tissues and organs. In addition, recent studies suggest that NHEs may be one of the primeval transport protein forms in the history of life. Among the different isoforms, the most well-characterized NHEs are the Na+/H+ exchanger isoform 1 (NHE1) and Na+/H+ exchanger isoform 3 (NHE3). However, Na+/H+ exchanger isoform 8 (NHE8) has been receiving attention based on its recent discoveries in the gastrointestinal tract. In this review, we will discuss what is known about the physiological function and potential role of NHE8 in the main organ systems, including useful overviews that could inspire new studies on this multifaceted protein.
Asunto(s)
Productos Biológicos , Intercambiadores de Sodio-Hidrógeno , Equilibrio Ácido-Base , Humanos , Concentración de Iones de Hidrógeno , Isoformas de Proteínas/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
BACKGROUND & AIMS: Intestinal epithelial cells (IECs) provide a barrier that separates the mucosal immune system from the luminal microbiota. IECs constitutively express low levels of major histocompatibility complex (MHC) class II proteins, which are upregulated upon exposure to interferon gamma. We investigated the effects of deleting MHCII proteins specifically in mice with infectious, dextran sodium sulfate (DSS)-, and T-cell-induced colitis. METHODS: We disrupted the histocompatibility 2, class II antigen A, beta 1 gene (H2-Ab1) in IECs of C57BL/6 mice (I-AbΔIEC) or Rag1-/- mice (Rag1-/-I-AbΔIEC); we used I-AbWT mice as controls. Colitis was induced by administration of DSS, transfer of CD4+CD45RBhi T cells, or infection with Citrobacter rodentium. Colon tissues were collected and analyzed by histology, immunofluorescence, xMAP, and reverse-transcription polymerase chain reaction and organoids were generated. Microbiota (total and immunoglobulin [Ig]A-coated) in intestinal samples were analyzed by16S amplicon profiling. IgA+CD138+ plasma cells from Peyer's patches and lamina propria were analyzed by flow cytometry and IgA repertoire was determined by next-generation sequencing. RESULTS: Mice with IEC-specific loss of MHCII (I-AbΔIEC mice) developed less severe DSS- or T-cell transfer-induced colitis than control mice. Intestinal tissues from I-AbΔIEC mice had a lower proportion of IgA-coated bacteria compared with control mice, and a reduced luminal concentration of secretory IgA (SIgA) following infection with C rodentium. There was no significant difference in the mucosal IgA repertoire of I-AbΔIEC vs control mice, but opsonization of cultured C rodentium by SIgA isolated from I-AbΔIEC mice was 50% lower than that of SIgA from mAbWT mice. Fifty percent of I-AbΔIEC mice died after infection with C rodentium, compared with none of the control mice. We observed a transient but significant expansion of the pathogen in the feces of I-AbΔIEC mice compared with I-AbWT mice. CONCLUSIONS: In mice with DSS or T-cell-induced colitis, loss of MHCII from IECs reduces but does not eliminate mucosal inflammation. However, in mice with C rodentium-induced colitis, loss of MHCII reduces bacterial clearance by decreasing binding of IgA to commensal and pathogenic bacteria.
Asunto(s)
Colitis/etiología , Colitis/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Antígenos de Histocompatibilidad Clase II/metabolismo , Mucosa Intestinal/patología , Animales , Colitis/metabolismo , Modelos Animales de Enfermedad , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
The loss of the intestinal Na+/H+ exchanger isoform 8 (NHE8) results in an ulcerative colitis-like condition with reduction of mucin production and dysbiosis, indicating that NHE8 plays an important role in intestinal mucosal protection. The aim of this study was to investigate the potential rebalance of the altered microbiota community of NHE8-deficient mice via fecal microbiota transplantation (FMT) and feeding probiotic VSL#3. We also aimed to stimulate mucin production by sodium butyrate administration via enema. Data from 16S rRNA sequencing showed that loss of NHE8 contributes to colonic microbial dysbiosis with reduction of butyrate-producing bacteria. FMT increased bacterial adhesion in the colon in NHE8 knockout (NHE8KO) mice. Periodic-acid Schiff reagent (PAS) stain and quantitative PCR showed no changes in mucin production during FMT. In mice treated with the probiotic VSL#3, a reduction of Lactobacillus and segmented filamentous bacteria (SFB) in NHE8KO mouse colon was detected and an increase in goblet cell theca was observed. In NHE8KO mice receiving sodium butyrate (NaB), 1 mM NaB stimulated Muc2 expression without changing goblet cell theca, but 10 mM NaB induced a significant reduction of goblet cell theca without altering Muc2 expression. Furthermore, 5 mM and 10 mM NaB-treated HT29-MTX cells displayed increased apoptosis, while 0.5 mM NaB stimulated Muc2 gene expression. These data showed that loss of NHE8 leads to dysbiosis with reduction of butyrate-producing bacteria and FMT and VSL#3 failed to rebalance the microbiota in NHE8KO mice. Therefore, FMT, VSL#3, and NaB are not able to restore mucin production in the absence of NHE8 in the intestine.NEW & NOTEWORTHY Loss of Na+/H+ exchanger isoform 8 (NHE8), a Slc9 family of exchanger that contributes to sodium uptake, cell volume regulation, and intracellular pH homeostasis, resulted in dysbiosis with reduction of butyrate-producing bacteria and decrease of Muc2 production in the intestine in mice. Introducing fecal microbiota transplantation (FMT) and VSL#3 in NHE8 knockout (NHE8KO) mice failed to rebalance the microbiota in these mice. Furthermore, administration of FMT, VSL#3, and sodium butyrate was unable to restore mucin production in the absence of NHE8 in the intestine.
Asunto(s)
Mucosa Intestinal/fisiología , Intercambiadores de Sodio-Hidrógeno/fisiología , Animales , Butiratos/metabolismo , Ácido Butírico/administración & dosificación , Colon/microbiología , Disbiosis/etiología , Disbiosis/microbiología , Disbiosis/terapia , Trasplante de Microbiota Fecal , Microbioma Gastrointestinal/fisiología , Células Caliciformes/efectos de los fármacos , Células Caliciformes/fisiología , Células HT29 , Humanos , Lactobacillus/fisiología , Ratones , Ratones Noqueados , Mucinas/biosíntesis , Probióticos/administración & dosificación , Intercambiadores de Sodio-Hidrógeno/deficienciaRESUMEN
Poly(ADP-ribose) polymerases (PARPs) synthesize and bind branched polymers of ADP-ribose to acceptor proteins using NAD as a substrate and participate in the control of gene transcription and DNA repair. PARP1, the most abundant isoform, regulates the expression of proinflammatory mediator cytokines, chemokines, and adhesion molecules, and inhibition of PARP1 enzymatic activity reduced or ameliorated autoimmune diseases in several experimental models, including colitis. However, the mechanism(s) underlying the protective effects of PARP1 inhibition in colitis and the cell types in which Parp1 deletion has the most significant impact are unknown. The objective of the current study was to determine the impact of Parp1 deletion on the innate immune response to mucosal injury and on the gut microbiome composition. Parp1 deficiency was evaluated in DSS-induced colitis in WT, Parp1(-/-), Rag2(-/-), and Rag2(-/-)×Parp1(-/-) double knock-out mice. Genome-wide analysis of the colonic transcriptome and fecal 16S amplicon profiling was performed. Compared with WT, we demonstrated that Parp1(-/-) were protected from dextran-sulfate sodium-induced colitis and that this protection was associated with a dramatic transcriptional reprogramming in the colon. PARP1 deficiency was also associated with a modulation of the colonic microbiota (increases relative abundance of Clostridia clusters IV and XIVa) and a concomitant increase in the frequency of mucosal CD4(+)CD25(+) Foxp3(+) regulatory T cells. The protective effects conferred by Parp1 deletion were lost in Rag2(-/-) × Parp1(-/-) mice, highlighting the role of the adaptive immune system for full protection.
Asunto(s)
Inmunidad Adaptativa , Colitis/inmunología , Colon/inmunología , Inmunidad Innata , Inmunidad Mucosa , Mucosa Intestinal/inmunología , Poli(ADP-Ribosa) Polimerasas/deficiencia , Animales , Colitis/inducido químicamente , Colitis/genética , Colitis/patología , Colon/lesiones , Colon/patología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Sulfato de Dextran/toxicidad , Mucosa Intestinal/lesiones , Mucosa Intestinal/patología , Ratones , Ratones Noqueados , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
It has been hypothesized that apically expressed L-type Ca2+ channel Cav1.3 (encoded by CACNA1D gene) contributes toward an alternative TRPV6-independent route of intestinal epithelial Ca2+ absorption, especially during digestion when high luminal concentration of Ca2+ and other nutrients limit TRPV6 contribution. We and others have implicated altered expression and activity of key mediators of intestinal and renal Ca2+ (re)absorption as contributors to negative systemic Ca2+ balance and bone loss in intestinal inflammation. Here, we investigated the effects of experimental colitis and related inflammatory mediators on colonic Cav1.3 expression. We confirmed Cav1.3 expression within the segments of the mouse and human gastrointestinal tract. Consistent with available microarray data (GEO database) from inflammatory bowel disease (IBD) patients, mouse colonic expression of Cav1.3 was significantly reduced in trinitrobenzene sulfonic acid (TNBS) colitis. In vitro, IFNγ most potently reduced Cav1.3 expression. We reproduced these findings in vivo with wild-type and Stat1-/- mice injected with IFNγ. The observed effect in Stat1-/- suggested a noncanonical transcriptional repression or a posttranscriptional mechanism. In support of the latter, we observed no effect on the cloned Cav1.3 gene promoter activity and accelerated Cav1.3 mRNA decay rate in IFNγ-treated HCT116 cells. While the relative contribution of Cav1.3 to intestinal Ca2+ absorption and its value as a therapeutic target remain to be established, we postulate that Cav1.3 downregulation in IBD may contribute to the negative systemic Ca2+ balance, to increased bone resorption, and to reduced bone mineral density in IBD patients.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Colitis/metabolismo , Colon/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Interferón gamma/farmacología , Interferencia de ARN/efectos de los fármacos , Animales , Calcio/metabolismo , Canales de Calcio Tipo L/genética , Línea Celular , Colon/efectos de los fármacos , Bases de Datos Factuales , Regulación hacia Abajo , Humanos , Ratones , Regiones Promotoras GenéticasRESUMEN
NCX1 is a Na(+)/Ca(2+) exchanger, which is believed to provide a key route for basolateral Ca(2+) efflux in the renal epithelia, thus contributing to renal Ca(2+) reabsorption. Altered mineral homeostasis, including intestinal and renal Ca(2+) transport may represent a significant component of the pathophysiology of the bone mineral density loss associated with Inflammatory Bowel Diseases (IBD). The objective of our research was to investigate the effects of TNBS and DSS colitis and related inflammatory mediators on renal Ncx1 expression. Colitis was associated with decreased renal Ncx1 expression, as examined by real-time RT-PCR, Western blotting, and immunofluorescence. In mIMCD3 cells, IFNγ significantly reduced Ncx1 mRNA and protein expression. Similar effects were observed in cells transiently transfected with a reporter construct bearing the promoter region of the kidney-specific Ncx1 gene. This inhibitory effect of IFNγ is mediated by STAT1 recruitment to the proximal promoter region of Ncx1. Further in vivo study with Stat1(-/-) mice confirmed that STAT1 is indeed required for the IFNγ mediated Ncx1 gene regulation. These results strongly support the hypothesis that impaired renal Ca(2+) handling occurs in experimental colitis. Negative regulation of NCX1- mediated renal Ca(2+) absorption by IFNγ may significantly contribute to the altered Ca(2+) homeostasis in IBD patients and to IBD-associated loss of bone mineral density.
Asunto(s)
Colitis/genética , Interferón gamma/metabolismo , Túbulos Renales Distales/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Cartilla de ADN , Ratones , Ratones Transgénicos , Regiones Promotoras GenéticasRESUMEN
While the intestine plays an important role in digestion and absorption, the mucus lining the epithelium represents a pivotal function in mucosal protection. Goblet cells are scattered in both the crypts and among enterocytes, and they secrete an important component of mucus, mucin. We have reported that sodium/hydrogen exchanger (NHE) 8 is a novel player in mucosal protection, since loss of NHE8 function resulted in reduced mucin production and increased bacterial adhesion. While NHE8 has been shown to be expressed in enterocytes and its expression is reduced during intestinal inflammation, nothing is known about the role of NHE8 in goblet cells. This current study is designed to define the expression of NHE8 and the role of TNF-α in the regulation of NHE8 in goblet cells. Using HT29-MTX cells as an in vitro model, we detected abundant NHE8 mRNA in goblet cells. Immunohistochemical staining localized NHE8 protein on the plasma membrane and in the intracellular compartments in goblet cells. Furthermore, NHE8 expression in goblet cells is regulated by the proinflammatory cytokine TNF-α. The expression of NHE8 in HT29-MTX cells was significantly reduced at both mRNA and protein levels in the presence of TNF-α. This inhibition of NHE8 mRNA expression could be blocked by the transcriptional inhibitor actinomycin D. Promoter reporter assay showed that NHE8 promoter activity was indeed reduced by TNF-α. Mechanistically, TNF-α reduced Sp3 protein binding to the human NHE8 basal promoter region. Therefore, NHE8 is expressed in goblet cells, and the inflammatory cytokine TNF-α downregulates NHE8 expression by a transcriptional mechanism.
Asunto(s)
Células Caliciformes/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Línea Celular , Dactinomicina/farmacología , Células Caliciformes/efectos de los fármacos , Humanos , Mucosa Intestinal/metabolismo , Regiones Promotoras Genéticas , Inhibidores de la Síntesis de la Proteína/farmacología , Factor de Transcripción Sp3/metabolismoRESUMEN
Previous studies reported that administration of somatostatin (SST) to human patients mitigated their diarrheal symptoms. Octreotide (an analog of SST) treatment in animals resulted in upregulation of sodium/hydrogen exchanger 8 (NHE8). NHE8 is important for water/sodium absorption in the intestine, and loss of NHE8 function results in mucosal injury. Thus we hypothesized that NHE8 expression is inhibited during colitis and that SST treatment during pathological conditions can restore NHE8 expression. Our data showed for the first time that NHE8 is expressed in the human colonic tissue and that NHE8 expression is decreased in ulcerative colitis (UC) patients. We also found that octreotide could stimulate colonic NHE8 expression in colitic mice. Furthermore, the somatostatin receptor 2 (SSTR2) agonist seglitide and the somatostatin receptor 5 (SSTR5) agonist L-817,818 could restore NHE8 expression via its role in suppressing ERK1/2 phosphorylation. Our study uncovered a novel mechanism of SST stimulation of NHE8 expression in colitis.
Asunto(s)
Colitis/metabolismo , Colon/metabolismo , Fármacos Gastrointestinales/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Recto/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Somatostatina/farmacología , Adulto , Animales , Células CACO-2 , Colitis/inducido químicamente , Femenino , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Persona de Mediana Edad , Octreótido/farmacología , Receptores de Somatostatina/metabolismoRESUMEN
Multiple sodium/hydrogen exchanger (NHE) isoforms are expressed in the testes, and they play various roles in cell volume regulation, intracellular pH regulation, and fluid absorption. NHE8, the most recently characterized NHE family member, is detected in the Leydig cells in humans and mice in great abundance by immunohistochemistry in the current study. Male mice lacking NHE8 expression were infertile. Despite having intact male reproductive organs, male NHE8-/- mice have smaller testes and lacked spermatozoon in the seminiferous tubules and the epididymis. At the age of 39 wk, few spermogonia were seen in the testis in NHE8-/- mice. Although male NHE8-/- mice have normal serum levels of luteinizing hormone and follicle-stimulating hormone, serum testosterone level was significantly reduced. These mice have decreased expression of luteinizing hormone receptor in the testes. In NHE8 small-interfering RNA-transfected mouse Leydig cells (MLTC-1), silencing of NHE8 decreased the expression of luteinizing hormone receptor by â¼70%. Moreover, loss of NHE8 function in Leydig cells resulted in disorganized luteinizing hormone receptor membrane distribution. Therefore, male infertility in NHE8-/- mice is at least partially due to the disruption of luteinizing hormone receptor distribution and consequent low testosterone production, which leads to Sertoli cell dysfunction. Our work identified a novel role of NHE8 in male fertility through its effect on modifying luteinizing hormone receptor function.
Asunto(s)
Fertilidad , Infertilidad Masculina/metabolismo , Células Intersticiales del Testículo/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Factores de Edad , Animales , Células Cultivadas , Epidídimo/metabolismo , Epidídimo/patología , Hormona Folículo Estimulante/sangre , Genotipo , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Infertilidad Masculina/fisiopatología , Células Intersticiales del Testículo/patología , Hormona Luteinizante/sangre , Masculino , Ratones de la Cepa 129 , Ratones Noqueados , Fenotipo , Interferencia de ARN , Receptores de HL/metabolismo , Túbulos Seminíferos/metabolismo , Túbulos Seminíferos/patología , Intercambiadores de Sodio-Hidrógeno/genética , Espermatozoides/metabolismo , Espermatozoides/patología , Testosterona/sangre , TransfecciónRESUMEN
Sodium/hydrogen exchanger (NHE) 8 is expressed at the apical membrane of the epithelial cells and plays important roles in neutral sodium absorption in the gastrointestinal tract and the kidney. It also has an important role in epithelial mucosal protection in the gastric gland and the intestine. Although NHE8 has broad tissue distribution, the precise location and the physiological role of NHE8 in the eye remain unknown. In the present study, we successfully detected the expression of NHE8 in the ocular surface by PCR and Western blot in human and mouse eyes. Immunohistochemistry staining located NHE8 protein at the plasma membrane of the epithelial cells in the conjunctiva, the cornea, and the lacrimal gland both in human and mouse. We also detected the expression of downregulated-in-adenoma (DRA, a Cl(-)/HCO3 (-) transporter) in the ocular surface epithelial cells. Using NHE8-/- mouse model, we found that loss of NHE8 function resulted in reduced tear production and increased corneal staining. These NHE8-/- mice also showed increased expression of TNF-α and matrix metalloproteinase 9 (MMP9) genes. The expression of epithelial keratinization marker genes, small proline-rich protein 2h (Sprr2h) and transglutaminase 1 (Tgm1), were also increased in NHE8-/- eyes. Furthermore, DRA expression in NHE8-/- mice was reduced in the conjunctiva, the cornea, and the lacrimal glands in association with a reduction in conjunctival mucosal pH. Altered ocular surface function and reduced epithelial DRA expression in NHE8-/- mice suggest that the role of NHE8 in ocular surface tissue involve in tear production and ocular epithelial protection. This study reveals a potential novel mechanism of dry eye condition involving abnormal NHE8 function.
Asunto(s)
Células Epiteliales/metabolismo , Ojo/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Antiportadores/genética , Antiportadores/metabolismo , Conjuntiva/metabolismo , Conjuntiva/patología , Córnea/metabolismo , Córnea/patología , Proteínas Ricas en Prolina del Estrato Córneo/genética , Proteínas Ricas en Prolina del Estrato Córneo/metabolismo , Regulación hacia Abajo , Células Epiteliales/patología , Ojo/patología , Femenino , Genotipo , Humanos , Concentración de Iones de Hidrógeno , Aparato Lagrimal/metabolismo , Aparato Lagrimal/patología , Masculino , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones Noqueados , Fenotipo , ARN Mensajero/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Transportadores de Sulfato , Lágrimas/metabolismo , Transglutaminasas/genética , Transglutaminasas/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Butyrate is a major metabolite in colonic lumen. It is produced from bacterial fermentation of dietary fiber. Butyrate has been shown to stimulate electroneutral sodium absorption through its regulation on sodium/hydrogen exchanger 3 (NHE3). Although NHE8, the newest addition of intestinal NHE family, is involved in sodium absorption in the intestinal tract, whether butyrate modulates NHE8 expression in the intestinal epithelial cells is not known. In the current study, we showed that butyrate treatment strongly induced NHE8 protein and NHE8 mRNA expression in human intestinal epithelial cells. Transfection with the human NHE8 promoter reporter constructs showed that butyrate treatment stimulated reporter gene expression at an amount comparable with its stimulation of NHE8 mRNA expression. Interestingly, a similar result was also observed in human NHE8 promoter transfected cells after trichostatin (TSA) treatment. Gel mobility shift assay identified an enhanced Sp3 protein binding on the human NHE8 basal promoter region upon butyrate stimulation. Furthermore, Sp3 acetylation modification is involved in butyrate-mediated NHE8 activation in Caco-2 cells. Our findings suggest that the mechanism of butyrate action on NHE8 expression involves enhanced Sp3 interaction at the basal promoter region of the human NHE8 gene promoter to activate NHE8 gene transcription. Thus butyrate is involved in intestinal regulation of NHE8 resulting enhanced sodium absorption.
Asunto(s)
Ácido Butírico/farmacología , Intercambiadores de Sodio-Hidrógeno/biosíntesis , Intercambiadores de Sodio-Hidrógeno/genética , Acetilación , Células CACO-2 , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros/efectos de los fármacos , Genes Reporteros/genética , Humanos , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Factor de Transcripción Sp3/metabolismoRESUMEN
The newest member of the Na(+)/H(+) exchanger (NHE) family, NHE8, is abundantly expressed at the apical membrane of the intestinal epithelia. We previously reported that mucin 2 expression was significantly decreased in the colon in NHE8(-/-) mice, suggesting that NHE8 is involved in intestinal mucosal protection. In this study, we further evaluated the role of NHE8 in intestinal epithelial protection after dextran sodium sulfate (DSS) challenge. Compared with wild-type mice, NHE8(-/-) mice have increased bacterial adhesion and inflammation, especially in the distal colon. NHE8(-/-) mice are also susceptible to DSS treatment. Real-time PCR detected a remarkable increase in the expression of IL-1ß, IL-6, TNF-α, and IL-4 in DSS-treated NHE8(-/-) mice compared with DSS-treated wild-type littermates. Immunohistochemistry showed a disorganized epithelial layer in the colon of NHE8(-/-) mice. Periodic acid-Schiff staining showed a reduction in the number of mature goblet cells and the area of the goblet cell theca in NHE8(-/-) mice. Phyloxine/tartrazine staining revealed a decrease in functional Paneth cell population in the NHE8(-/-) small intestinal crypt. The expression of enteric defensins was also decreased in NHE8(-/-) mice. The reduced mucin production in goblet cells and antimicrobial peptides production in Paneth cells lead to disruption of the intestinal mucosa protection. Therefore, NHE8 may be involved in the establishment of intestinal mucosal integrity by regulating the functions of goblet and Paneth cells.
Asunto(s)
Colitis/metabolismo , Colon/metabolismo , Mucosa Intestinal/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Adhesión Bacteriana , Traslocación Bacteriana , Colitis/inducido químicamente , Colitis/genética , Colitis/microbiología , Colitis/patología , Colon/microbiología , Colon/patología , Citocinas/genética , Citocinas/metabolismo , Defensinas/metabolismo , Sulfato de Dextran , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Células Caliciformes/metabolismo , Células Caliciformes/patología , Mediadores de Inflamación/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Masculino , Ratones Noqueados , Mucinas/metabolismo , Células de Paneth/metabolismo , Células de Paneth/patología , ARN Mensajero/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Factores de TiempoRESUMEN
OBJECTIVE: Children with congenital heart disease have loss of intestinal epithelial barrier function, which increases their risk for postoperative sepsis and organ dysfunction. We do not understand how postoperative cardiopulmonary support or the inflammatory response to cardiopulmonary bypass might alter intestinal epithelial barrier function. We examined variation in a panel of plasma biomarkers to reflect intestinal epithelial barrier function (cellular and paracellular) after cardiopulmonary bypass and in response to routine ICU care. DESIGN: Prospective cohort. SETTING: University medical center cardiac ICU. PATIENTS: Twenty children aged between newborn and 18 years undergoing repair or palliation of congenital heart disease with cardiopulmonary bypass. INTERVENTIONS: We measured baseline and repeated plasma intestinal fatty acid-binding protein, citrulline, claudin 3, and dual sugar permeability testing to reflect intestinal epithelial integrity, epithelial function, paracellular integrity, and paracellular function, respectively. We measured baseline and repeated plasma proinflammatory (interleukin-6, tumor necrosis factor-α, and interferon-γ) and anti-inflammatory (interleukin-4 and interleukin-10) cytokines, known to modulate intestinal epithelial barrier function in murine models of cardiopulmonary bypass. MEASUREMENTS AND MAIN RESULTS: All patients had abnormal baseline intestinal fatty acid-binding protein concentrations (mean, 3,815.5 pg/mL; normal, 41-336 pg/mL). Cytokine response to cardiopulmonary bypass was associated with early, but not late, changes in plasma concentrations of intestinal fatty acid-binding protein 2 and citrulline. Variation in biomarker concentrations over time was associated with aspects of ICU care indicating greater severity of illness: claudin 3, intestinal fatty acid-binding protein 2, and dual sugar permeability test ratio were associated with symptoms of feeding intolerance (p < 0.05), whereas intestinal fatty acid-binding protein was positively associated with vasoactive-inotrope score (p = 0.04). Citrulline was associated with larger arteriovenous oxygen saturation difference (p = 0.04) and had a complex relationship with vasoactive-inotrope score. CONCLUSIONS: Children undergoing cardiopulmonary bypass for repair or palliation of congenital heart disease are at risk for intestinal injury and often present with evidence for loss of intestinal epithelial integrity preoperatively. Greater severity of illness requiring increased cardiopulmonary support rather than the inflammatory response to cardiopulmonary bypass seems to mediate late postoperative intestinal epithelial barrier function.
Asunto(s)
Biomarcadores/sangre , Citocinas/sangre , Cardiopatías Congénitas/cirugía , Enfermedades Intestinales/etiología , Mucosa Intestinal/patología , Niño , Preescolar , Estudios de Cohortes , Femenino , Humanos , Lactante , Inflamación , Unidades de Cuidado Intensivo Pediátrico , Enfermedades Intestinales/sangre , Masculino , Periodo Posoperatorio , Estudios ProspectivosRESUMEN
OBJECTIVE: The inner mucus layer in mouse colon normally separates bacteria from the epithelium. Do humans have a similar inner mucus layer and are defects in this mucus layer a common denominator for spontaneous colitis in mice models and ulcerative colitis (UC)? METHODS AND RESULTS: The colon mucus layer from mice deficient in Muc2 mucin, Core 1 O-glycans, Tlr5, interleukin 10 (IL-10) and Slc9a3 (Nhe3) together with that from dextran sodium sulfate-treated mice was immunostained for Muc2, and bacterial localisation in the mucus was analysed. All murine colitis models revealed bacteria in contact with the epithelium. Additional analysis of the less inflamed IL-10(-/-) mice revealed a thicker mucus layer than wild-type, but the properties were different, as the inner mucus layer could be penetrated both by bacteria in vivo and by fluorescent beads the size of bacteria ex vivo. Clear separation between bacteria or fluorescent beads and the epithelium mediated by the inner mucus layer was also evident in normal human sigmoid colon biopsy samples. In contrast, mucus on colon biopsy specimens from patients with UC with acute inflammation was highly penetrable. Most patients with UC in remission had an impenetrable mucus layer similar to that of controls. CONCLUSIONS: Normal human sigmoid colon has an inner mucus layer that is impenetrable to bacteria. The colon mucus in animal models that spontaneously develop colitis and in patients with active UC allows bacteria to penetrate and reach the epithelium. Thus colon mucus properties can be modulated, and this suggests a novel model of UC pathophysiology.
Asunto(s)
Colitis Ulcerosa/microbiología , Colitis/microbiología , Colon/microbiología , Mucosa Intestinal/microbiología , Mucina 2/metabolismo , Moco/microbiología , Adolescente , Adulto , Anciano , Animales , Colitis/metabolismo , Colitis/patología , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Colon/metabolismo , Colon/patología , Femenino , Humanos , Hibridación Fluorescente in Situ , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Ratones , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND & AIMS: Dysregulated Ca(2+) homeostasis likely contributes to the etiology of inflammatory bowel disease-associated loss of bone mineral density. Experimental colitis leads to decreased expression of Klotho, a protein that supports renal Ca(2+) reabsorption by stabilizing the transient receptor potential vanilloid 5 (TRPV5) channel on the apical membrane of distal tubule epithelial cells. METHODS: Colitis was induced in mice via administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) or transfer of CD4(+)interleukin-10(-/-) and CD4(+), CD45RB(hi) T cells. We investigated changes in bone metabolism, renal processing of Ca(2+), and expression of TRPV5. RESULTS: Mice with colitis had normal serum levels of Ca(2+) and parathormone. Computed tomography analysis showed a decreased density of cortical and trabecular bone, and there was biochemical evidence for reduced bone formation and increased bone resorption. Increased fractional urinary excretion of Ca(2+) was accompanied by reduced levels of TRPV5 protein in distal convoluted tubules, with a concomitant increase in TRPV5 sialylation. In mouse renal intermedullary collecting duct epithelial (mIMCD3) cells transduced with TRPV5 adenovirus, the inflammatory cytokines tumor necrosis factor, interferon-γ, and interleukin-1ß reduced levels of TRPV5 on the cell surface, leading to its degradation. Cytomix induced interaction between TRPV5 and UBR4 (Ubiquitin recoginition 4), an E3 ubiquitin ligase; knockdown of UBR4 with small interfering RNAs prevented cytomix-induced degradation of TRPV5. The effects of cytokines on TRPV5 were not observed in cells stably transfected with membrane-bound Klotho; TRPV5 expression was preserved when colitis was induced with TNBS in transgenic mice that overexpressed Klotho or in mice with T-cell transfer colitis injected with soluble recombinant Klotho. CONCLUSIONS: After induction of colitis in mice via TNBS administration or T-cell transfer, tumor necrosis factor and interferon-γ reduced the expression and activity of Klotho, which otherwise would protect TRPV5 from hypersialylation and cytokine-induced TRPV5 endocytosis, UBR4-dependent ubiquitination, degradation, and urinary wasting of Ca(2+).
Asunto(s)
Densidad Ósea , Canales de Calcio/metabolismo , Calcio/metabolismo , Colitis/metabolismo , Riñón/metabolismo , Procesamiento Proteico-Postraduccional , Canales Catiónicos TRPV/metabolismo , Animales , Biomarcadores/metabolismo , Linfocitos T CD4-Positivos/trasplante , Colitis/inducido químicamente , Colitis/inmunología , Glucuronidasa/metabolismo , Interferón gamma/metabolismo , Proteínas Klotho , Ratones , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tomografía Computarizada por Rayos X , Ácido Trinitrobencenosulfónico , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In vitro data and transgenic mouse models suggest a role for TGF-ß signaling in dendritic cells (DCs) to prevent autoimmunity primarily through maintenance of DCs in their immature and tolerogenic state characterized by low expression of MHC class II (MHCII) and costimulatory molecules and increased expression of IDO, among others. To test whether a complete lack of TGF-ß signaling in DCs predisposes mice to spontaneous autoimmunity and to verify the mechanisms implicated previously in vitro, we generated conditional knockout (KO) mice with Cre-mediated DC-specific deletion of Tgfbr2 (DC-Tgfbr2 KO). DC-Tgfbr2 KO mice die before 15 wk of age with multiorgan autoimmune inflammation and spontaneous activation of T and B cells. Interestingly, there were no significant differences in the expression of MHCII, costimulatory molecules, or IDO in secondary lymphoid organ DCs, although Tgfbr2-deficient DCs were more proinflammatory in vitro and in vivo. DC-Tgfbr2 KO showed attenuated Foxp3 expression in regulatory T cells (Tregs) and abnormal expansion of CD25(-)Foxp3(+) Tregs in vivo. Tgfbr2-deficient DCs secreted elevated levels of IFN-γ and were not capable of directing Ag-specific Treg conversion unless in the presence of anti-IFN-γ blocking Ab. Adoptive transfer of induced Tregs into DC-Tgfbr2 KO mice partially rescued the phenotype. Therefore, in vivo, TGF-ß signaling in DCs is critical in the control of autoimmunity through both Treg-dependent and -independent mechanisms, but it does not affect MHCII and costimulatory molecule expression.
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Enfermedades Autoinmunes/prevención & control , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Proteínas Serina-Treonina Quinasas/deficiencia , Receptores de Factores de Crecimiento Transformadores beta/deficiencia , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/patología , Colitis/genética , Colitis/inmunología , Colitis/prevención & control , Células Dendríticas/patología , Modelos Animales de Enfermedad , Tolerancia Inmunológica/genética , Inmunofenotipificación , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Proteínas Serina-Treonina Quinasas/fisiología , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Linfocitos T Reguladores/patologíaRESUMEN
Matrix vesicles (MVs) provide the initial site for amorphous hydroxyapatite (HA) formation within mineralizing osteoblasts. Although Na+/Ca2+ exchanger isoform-3 (NCX3, SLC8A3) was presumed to function as major Ca2+ transporter responsible for Ca2+ extrusion out of osteoblast into the calcifying bone matrix, its presence and functional role in MVs have not been investigated. In this study, we investigated the involvement of NCX3 in MV-mediated mineralization process and its impact on bone formation. Using differentiated MC3T3-E1 cells, we demonstrated that NCX3 knockout in these cells resulted in a significant reduction of Ca2+ deposition due to reduced Ca2+ entry within the MVs, leading to impaired mineralization. Consequently, the capacity of MVs to promote extracellular HA formation was diminished. Moreover, primary osteoblast isolated from NCX3 deficient mice (NCX3-/-) exhibits reduced mineralization efficacy without any effect on osteoclast activity. To validate this in vitro finding, µCT analysis revealed a substantial decrease in trabecular bone mineral density in both genders of NCX3-/- mice, thus supporting the critical role of NCX3 in facilitating Ca2+ uptake into the MVs to initiate osteoblast-mediated mineralization. NCX3 expression was also found to be the target of downregulation by inflammatory mediators in vitro and in vivo. This newfound understanding of NCX3's functional role in MVs opens new avenues for therapeutic interventions aimed at enhancing bone mineralization and treating mineralization-related disorders.
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Calcificación Fisiológica , Calcio , Ratones Noqueados , Osteoblastos , Intercambiador de Sodio-Calcio , Animales , Osteoblastos/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Ratones , Calcio/metabolismo , Masculino , Osteogénesis , Diferenciación Celular , Femenino , Vesículas Extracelulares/metabolismo , Línea CelularRESUMEN
Although the role of the intestinal microbiota in the pathogenesis of inflammatory bowel disease (IBD) is beyond debate, attempts to verify the causative role of IBD-associated dysbiosis have been limited to reports of promoting the disease in genetically susceptible mice or in chemically induced colitis. We aimed to further test the host response to fecal microbiome transplantation (FMT) from Crohn's disease patients on mucosal homeostasis in ex-germ-free (xGF) mice. We characterized and transferred fecal microbiota from healthy patients and patients with defined Crohn's ileocolitis (CD_L3) to germ-free mice and analyzed the resulting microbial and mucosal homeostasis by 16S profiling, shotgun metagenomics, histology, immunofluorescence (IF) and RNAseq analysis. We observed a markedly reduced engraftment of CD_L3 microbiome compared to healthy control microbiota. FMT from CD_L3 patients did not lead to ileitis but resulted in colitis with features consistent with CD: a discontinued pattern of colitis, more proximal colonic localization, enlarged isolated lymphoid follicles and/or tertiary lymphoid organ neogenesis, and a transcriptomic pattern consistent with epithelial reprograming and promotion of the Paneth cell-like signature in the proximal colon and immune dysregulation characteristic of CD. The observed inflammatory response was associated with persistently increased abundance of Ruminococcus gnavus, Erysipelatoclostridium ramosum, Faecalimonas umbilicate, Blautia hominis, Clostridium butyricum, and C. paraputrificum and unexpected growth of toxigenic C. difficile, which was below the detection level in the community used for inoculation. Our study provides the first evidence that the transfer of a dysbiotic community from CD patients can lead to spontaneous inflammatory changes in the colon of xGF mice and identifies a signature microbial community capable of promoting colonization of pathogenic and conditionally pathogenic bacteria.
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Clostridioides difficile , Colitis , Enfermedad de Crohn , Microbioma Gastrointestinal , Microbiota , Humanos , Ratones , Animales , Enfermedad de Crohn/microbiología , Trasplante de Microbiota Fecal , Disbiosis/microbiologíaRESUMEN
The Naâº/H⺠exchanger NHE8 is expressed on the apical membrane of intestinal epithelial cells and is particularly abundant in the colon. Our previous study showed that Muc2 expression was significantly reduced in NHE8-knockout (NHE8-/-) mice, suggesting that NHE8 plays a role in mucosal protection in the colon. The current study confirms and extends our studies on the role of NHE8 in mucosal protection. The number of bacteria attached on the distal colon was significantly increased in NHE8-/- mice compared with their wild-type littermates. As expected, IL-4 expression was markedly increased in NHE8-/- mice compared with wild-type mice. Immunohistochemistry showed disorganization in the mucin layer of NHE8-/- mice, suggesting a possible direct bacteria-epithelia interaction. Furthermore, NHE8-/- mice were susceptible to dextran sodium sulfate-induced mucosal injury. In wild-type mice, dextran sodium sulfate treatment inhibited colonic NHE8 expression. In Caco-2 cells, the absence of NHE8 expression resulted in higher adhesion rates of Salmonella typhimurium but not Lactobacillus plantarum. Similarly, in vivo, S. typhimurium adhesion rate was increased in NHE8-/- mice compared with wild-type mice. Our study suggests that NHE8 plays important roles in protecting intestinal epithelia from infectious bacterial adherence.
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Adhesión Bacteriana/fisiología , Inmunidad Mucosa/fisiología , Mucosa Intestinal/metabolismo , Salmonella typhimurium/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Bacterias/clasificación , Células CACO-2 , Colitis/inducido químicamente , Colitis/metabolismo , Colon/metabolismo , Colon/microbiología , Citocinas/metabolismo , Sulfato de Dextran , Regulación de la Expresión Génica/inmunología , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Mucosa Intestinal/microbiología , Masculino , Ratones , Ratones Noqueados , Salmonelosis Animal/inmunología , Salmonelosis Animal/microbiología , Intercambiadores de Sodio-Hidrógeno/genética , Factores de Tiempo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Sodium/hydrogen exchanger (NHE) 8 is an apically expressed membrane protein in the intestinal epithelial cells. It plays important roles in sodium absorption and bicarbonate secretion in the intestine. Although NHE8 mRNA has been detected in the stomach, the precise location and physiological role of NHE8 in the gastric glands remain unclear. In the current study, we successfully detected the expression of NHE8 in the glandular region of the stomach by Western blotting and located NHE8 protein at the apical membrane in the surface mucous cells by a confocal microscopic method. We also identified the expression of downregulated-in-adenoma (DRA) in the surface mucous cells in the stomach. Using NHE8(-/-) mice, we found that NHE8 plays little or no role in basal gastric acid production, yet NHE8(-/-) mice have reduced gastric mucosal surface pH and higher incidence of developing gastric ulcer. DRA expression was reduced significantly in the stomach in NHE8(-/-) mice. The propensity for gastric ulcer, reduced mucosal surface pH, and low DRA expression suggest that NHE8 is indirectly involved in gastric bicarbonate secretion and gastric mucosal protection.