RESUMEN
In the intestine, epithelial factors condition incoming immune cells including monocytes to adapt their threshold of activation and prevent undesired inflammation. Colonic epithelial cells express Secretory Leukocyte Protease Inhibitor (SLPI), an inhibitor of NF kappa light chain enhancer of activated B cells (NF-κB) that mediates epithelial hyporesponsiveness to microbial stimuli. Uptake of extracellular SLPI by monocytes has been proposed to inhibit monocyte activation. We questioned whether monocytes can produce SLPI and whether endogenous SLPI can inhibit monocyte activation. We demonstrate that human THP-1 monocytic cells produce SLPI and that CD68+ SLPI-producing cells can be detected in human intestinal lamina propria. Knockdown of SLPI in human THP-1 cells significantly increased NF-κB activation and subsequent C-X-C motif chemokine ligand 8 (CXCL8) and TNF-α production in response to microbial stimulation. Reconstitution of SLPI-deficient cells with either full-length SLPI or SLPI lacking its signal peptide rescued inhibition of NF-κB activation and cytokine production, demonstrating that endogenous SLPI inhibits monocytic cell activation. Unexpectedly, exogenous SLPI did not inhibit CXCL8 or TNF-α production, despite efficient uptake. Our data argue that endogenous SLPI can regulate the threshold of activation in monocytes, thereby preventing activation by commensal bacteria in mucosal tissues.
Asunto(s)
FN-kappa B , Inhibidor Secretorio de Peptidasas Leucocitarias , Humanos , FN-kappa B/metabolismo , Monocitos/metabolismo , Factor de Necrosis Tumoral alfa , Transducción de SeñalRESUMEN
OBJECTIVE: Monogenic defects in the interleukin-10 (IL-10) pathway are extremely rare and cause infantile-onset inflammatory bowel disease (IBD)-like pathology. Understanding how immune responses are dysregulated in monogenic IBD-like diseases can provide valuable insight in "classical" IBD pathogenesis. Here, we studied long-term immune cell development and function in an adolescent IL-10 receptor (IL10RA)-deficient patient who presented in infancy with severe colitis and fistulizing perianal disease and is currently treated with immune suppressants. METHODS: Biomaterial was collected from the IL10RA-deficient patient, pediatric patients with IBD, and healthy controls. The frequency and phenotype of immune cells were determined in peripheral blood and intestinal biopsies by flow cytometry and immunohistochemistry. Functional changes in monocyte-derived dendritic cells and T cells were assessed by in vitro activation assays. RESULTS: The IL10RA-deficient immune system developed normally with respect to numbers and phenotype of circulating immune cells. Despite normal co-stimulatory molecule expression, bacterial lipopolysaccharide-stimulated monocyte-derived dendritic cells from the IL10RA-deficient patient released increased amounts of tumor necrosis factor α compared to healthy controls. Upon T-cell receptor ligation, IL10RA-deficient peripheral blood mononuclear cells released increased amounts of T-cell cytokines interferon γ and IL-17 agreeing with high numbers of T-bet and IL-17 cells in intestinal biopsies taken at disease onset. In vitro, the immunosuppressive drug thalidomide used to treat the patient's decreased peripheral blood mononuclear cell-derived tumor necrosis factor production. CONCLUSIONS: With time and during immunosuppressive treatment the IL10RA-deficient immune system develops relatively normally. Upon activation, IL-10 is crucial for controlling excessive inflammatory cytokine release by dendritic cells and preventing interferon γ and IL-17-mediated T-cell responses.
Asunto(s)
Inmunidad Adaptativa/fisiología , Células Dendríticas/metabolismo , Inmunidad Innata/fisiología , Enfermedades Inflamatorias del Intestino/inmunología , Subunidad alfa del Receptor de Interleucina-10/deficiencia , Subgrupos de Linfocitos T/metabolismo , Adolescente , Adulto , Biomarcadores/metabolismo , Estudios de Casos y Controles , Niño , Preescolar , Codón sin Sentido , Femenino , Mutación del Sistema de Lectura , Marcadores Genéticos , Humanos , Lactante , Enfermedades Inflamatorias del Intestino/genética , Subunidad alfa del Receptor de Interleucina-10/genética , Masculino , Persona de Mediana EdadRESUMEN
The intestinal influx oligopeptide transporter peptide transporter 1 (PEPT1) (SLC15A1) is best known for nutrient-derived di- and tripeptide transport. Its role in drug absorption is increasingly recognized. To better understand the disposition of PEPT1 substrate drugs in young infants, we studied intestinal PEPT1 mRNA expression and tissue localization across the pediatric age range. PEPT1 mRNA expression was determined using real-time reverse-transcription polymerase chain reaction in small intestinal tissues collected from surgical procedures (neonates and infants) or biopsies (older children and adolescents). PEPT1 mRNA relative to villin mRNA expression was compared between neonates/infants and older children/adolescents. PEPT1 was visualized in infant tissue using immunohistochemical staining. Other transporters [multidrug resistance protein 1 (MDR1), multidrug resistance-like protein 2 (MRP2), and organic anion transporter polypeptide 2B1 (OATP2B1)] were also stained to describe the localization in relation to PEPT1. Twenty-six intestinal samples (n = 20 neonates/infants, n = 2 pediatric, n = 4 adolescents) were analyzed. The young infant samples were collected at a median (range) gestational age at birth of 29.2 weeks (24.7-40) and postnatal age of 2.4 weeks (0-16.6). The PEPT1 mRNA expression of the neonates/infants was only marginally lower (0.8-fold) than the older children (P < 0.05). Similar and clear apical PEPT1 and MRP2 staining, apical and lateral MDR1 staining, and intraepithelial OATP2B1 staining at the basolateral membrane of the enterocyte were detected in 12 infant and 2 adolescent samples. Although small intestinal PEPT1 expression tended to be lower in neonates than in older children, this difference is small and tissue distribution is similar. This finding suggests similar oral absorption of PEPT1 substrates across the pediatric age range.
Asunto(s)
Recien Nacido Prematuro , Intestino Delgado/metabolismo , Transportador de Péptidos 1/metabolismo , Nacimiento Prematuro , Nacimiento a Término , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Adolescente , Factores de Edad , Niño , Preescolar , Enterocitos/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Humanos , Lactante , Recién Nacido , Masculino , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Transportadores de Anión Orgánico/metabolismo , Transportador de Péptidos 1/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
OBJECTIVE: Repetitive interaction with microbial stimuli renders epithelial cells (ECs) hyporesponsive to microbial stimulation. Previously, we have reported that buccal ECs from a subset of paediatric patients with Crohn's disease are not hyporesponsive and spontaneously released chemokines. We now aimed to identify kinetics and mechanisms of acquisition of hyporesponsiveness to microbial stimulation using primary human buccal epithelium. DESIGN: Buccal ECs collected directly after birth and in later stages of life were investigated. Chemokine release and regulatory signalling pathways were studied using primary buccal ECs and the buccal EC line TR146. Findings were extended to the intestinal mucosa using murine model systems. RESULTS: Directly after birth, primary human buccal ECs spontaneously produced the chemokine CXCL-8 and were responsive to microbial stimuli. Within the first weeks of life, these ECs attained hyporesponsiveness, associated with inactivation of the NF-κB pathway and upregulation of the novel NF-κB inhibitor SLPI but no other known NF-κB inhibitors. SLPI protein was abundant in the cytoplasm and the nucleus of hyporesponsive buccal ECs. Knock-down of SLPI in TR146-buccal ECs induced loss of hyporesponsiveness with increased NF-κB activation and subsequent chemokine release. This regulatory mechanism extended to the intestine, as colonisation of germfree mice elicited SLPI expression in small intestine and colon. Moreover, SLPI-deficient mice had increased chemokine expression in small intestinal and colonic ECs. CONCLUSIONS: We identify SLPI as a new player in acquisition of microbial hyporesponsiveness by buccal and intestinal epithelium in the first weeks after microbial colonisation.
Asunto(s)
Envejecimiento/inmunología , Epitelio/inmunología , Epitelio/microbiología , Mucosa Bucal/citología , Mucosa Bucal/microbiología , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Adulto , Animales , Células Cultivadas , Quimiocina CXCL2/metabolismo , Regulación hacia Abajo , Epitelio/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Tolerancia Inmunológica , Lactante , Recién Nacido , Interleucina-8/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/microbiología , Ratones , Persona de Mediana Edad , FN-kappa B/metabolismo , Peptidoglicano/farmacologíaRESUMEN
BACKGROUND & AIMS: Patients with celiac disease have permanent intolerance to gluten. Because of the high frequency of this disorder (approximately 1 in 100 individuals), we investigated whether oral tolerance to gluten differs from that to other food proteins. METHODS: Using transgenic mice that express human HLA-DQ2 and a gliadin-specific, humanized T-cell receptor, we compared gluten-specific T-cell responses with tolerogenic mucosal T-cell responses to the model food protein ovalbumin. RESULTS: Consistent with previous findings, the ovalbumin-specific response occurred in the mesenteric lymph nodes and induced Foxp3(+) regulatory T cells. In contrast, ingestion of deamidated gliadin induced T-cell proliferation predominantly in the spleen but little in mesenteric lymph nodes. The gliadin-reactive T cells had an effector-like phenotype and secreted large amounts of interferon gamma but also secreted interleukin-10. Despite their effector-like phenotype, gliadin-reactive T cells had regulatory functions, because transfer of the cells suppressed a gliadin-induced, delayed-type hypersensitivity response. CONCLUSIONS: Ingestion of deamidated gliadin induces differentiation of tolerogenic, type 1 regulatory T cells in spleens of HLA-DQ2 transgenic mice. These data indicate that under homeostatic conditions, the T-cell response to deamidated gliadin is tolerance, which is not conditioned by the mucosal immune system but instead requires interleukin-10 induction by antigen presentation in the spleen.
Asunto(s)
Gliadina/inmunología , Tolerancia Inmunológica/inmunología , Bazo/inmunología , Linfocitos T Reguladores/inmunología , Animales , Quimiocina CCL2/metabolismo , Factores de Transcripción Forkhead/inmunología , Gliadina/farmacología , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/inmunología , Antígenos HLA-DQ/metabolismo , Interferón gamma/metabolismo , Interleucina-1/metabolismo , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Ganglios Linfáticos/inmunología , Mesenterio , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Ovalbúmina/inmunología , Bazo/efectos de los fármacos , Linfocitos T Reguladores/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Chronic inflammatory T-cell-mediated diseases such as inflammatory bowel disease (IBD) are often treated with immunosuppressants including corticosteroids. In addition to the intended T-cell suppression, these farmacons give rise to many side effects. Recently, immunosuppressive phospholipids have been proposed as less-toxic alternatives. We aimed to investigate the immunoregulatory capacities of the naturally occurring phospholipid phosphatidylinositol (PI). Systemic PI treatment dramatically reduced disease severity and intestinal inflammation in murine 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis. Moreover, PI treatment inhibited the inflammatory T-cell response in these mice, as T cells derived from colon-draining LN of PI-treated mice secreted less IL-17 and IFN-γ upon polyclonal restimulation when compared to those of saline-treated mice. Further characterization of the suppressive capacity of PI revealed that the phospholipid suppressed Th cell differentiation in vitro irrespective of their cytokine profile by inhibiting proliferation and IL-2 release. In particular, PI diminished IL-2 mRNA expression and inhibited ERK1-, ERK-2-, p38- and JNK-phosphorylation. Crucially, PI did not ablate Treg differentiation or the antigen-presenting capacity of DCs in vitro. These data validate PI as a pluripotent inhibitor that can be applied mucosally as well as systemically. Its compelling functions render PI a promising novel physiological immune suppressant.
Asunto(s)
Antiinflamatorios/uso terapéutico , Colitis/tratamiento farmacológico , Fosfatidilinositoles/uso terapéutico , Animales , Presentación de Antígeno , Movimiento Celular , Proliferación Celular , Células Cultivadas , Colitis/inducido químicamente , Colitis/inmunología , Colitis/patología , Citocinas/inmunología , Citocinas/metabolismo , Células Dendríticas/inmunología , Ratones , Ratones Endogámicos BALB C , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Secretory leukocyte protease inhibitor (SLPI) is a pleiotropic protein produced by healthy intestinal epithelial cells. SLPI regulates NF-κB activation, inhibits neutrophil proteases and has broad antimicrobial activity. Recently, increased SLPI expression was found in various types of carcinomas and was suggested to increase their metastatic potential. Indeed, we demonstrated that SLPI protein expression in colorectal cancer (CRC) liver metastases and matched primary tumors is associated with worse outcome, suggesting that SLPI promotes metastasis in human CRC. However, whether SLPI plays a role in CRC before distant metastases have formed is unclear. Therefore, we examined whether SLPI expression is associated with prognosis in CRC patients with localized disease. Using a cohort of 226 stage II and 160 stage III CRC patients we demonstrate that high SLPI protein expression is associated with reduced disease recurrence in patients with stage III micro-satellite stable tumors treated with adjuvant chemotherapy, independently of established clinical risk factors (hazard rate ratio 0.54, P-value 0.03). SLPI protein expression was not associated with disease-free survival in stage II CRC patients. Our data suggest that the role of SLPI in CRC may be different depending on the stage of disease. In stage III CRC, SLPI expression may be unfavorable for tumors, whereas SLPI expression may be beneficial for tumors once distant metastases have established.
Asunto(s)
Neoplasias Colorrectales , Inhibidor Secretorio de Peptidasas Leucocitarias , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Regulación de la Expresión Génica , Humanos , Recurrencia Local de Neoplasia , Pronóstico , Inhibidor Secretorio de Peptidasas Leucocitarias/genética , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismoRESUMEN
Single genetic mutations predispose to very early onset inflammatory bowel disease (VEO-IBD). Here, we identify a de novo duplication of the 10p15.1 chromosomal region, including the IL2RA locus, in a 2-year-old girl with treatment-resistant pancolitis that was brought into remission by colectomy. Strikingly, after colectomy while the patient was in clinical remission and without medication, the peripheral blood CD4:CD8 ratio was constitutively high and CD25 expression was increased on circulating effector memory, Foxp3+, and Foxp3neg CD4+ T cells compared to healthy controls. This high CD25 expression increased IL-2 signaling, potentiating CD4+ T-cell-derived IFNγ secretion after T-cell receptor (TCR) stimulation. Restoring CD25 expression using the JAK1/3-inhibitor tofacitinib controlled TCR-induced IFNγ secretion in vitro. As diseased colonic tissue, but not the unaffected duodenum, contained mainly CD4+ T cells with a prominent IFNγ-signature, we hypothesize that local microbial stimulation may have initiated colonic disease. Overall, we identify that duplication of the IL2RA locus can associate with VEO-IBD and suggest that increased IL-2 signaling predisposes to colonic intestinal inflammation.
Asunto(s)
Colitis/etiología , Colitis/metabolismo , Duplicación de Gen , Predisposición Genética a la Enfermedad , Subunidad alfa del Receptor de Interleucina-2/genética , Interleucina-2/metabolismo , Transducción de Señal , Edad de Inicio , Alelos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Estudios de Casos y Controles , Cromosomas Humanos Par 10 , Colitis/diagnóstico , Citocinas/metabolismo , Resistencia a Medicamentos , Expresión Génica , Estudios de Asociación Genética , Sitios Genéticos , Humanos , Inmunohistoquímica , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/etiología , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
BACKGROUND & AIMS: The pathogenesis of inflammatory bowel disease involves dysfunctional mucosal immune responses to commensal bacteria in genetically predisposed hosts. Interactions between host cells and bacteria are complicated, making it a challenge to assess their relative contribution to intestinal pathology. We developed a zebrafish model of enterocolitis to study these interactions. METHODS: Enterocolitis was induced by intrarectal administration of the hapten oxazolone in adult wild-type and myeloperoxidase-reporter transgenic zebrafish in the presence or absence of antibiotics. Intestinal inflammation was evaluated by histological and flow cytometry analyses and cytokine profiling with quantitative real-time polymerase chain reaction. Changes in the composition of the intestinal microbiota following antibiotic administration were assessed by 16SrRNA sequencing and bacterial load was quantified by culture on nonselective media (colony-forming units). RESULTS: In zebrafish, the infiltrate and severity of oxazolone-induced enterocolitis are influenced by the composition of the microbiota. Inflammation is characterized by granulocyte influx; epithelial damage; goblet cell depletion; and increased expression of interleukin-1beta, tumor necrosis factor-alpha, and interleukin-10. Zebrafish given vancomycin had bacterial populations dominated by Fusobacteria and reduced enterocolitis scores, intestinal damage, and percentages of infiltrating neutrophils and eosinophils. In contrast, zebrafish given colistin sulphate had a predominance of proteobacteria and reduced eosinophil and lymphocyte infiltration, but enterocolitis scores were not reduced. CONCLUSIONS: In zebrafish with oxazolone-induced enterocolitis, components of the intestinal microbiota affect the severity and composition of the intestinal infiltrate.
Asunto(s)
Adyuvantes Inmunológicos , Enterocolitis/microbiología , Enterocolitis/patología , Intestinos/microbiología , Oxazolona , Adyuvantes Inmunológicos/farmacología , Animales , Antibacterianos/uso terapéutico , Colistina/uso terapéutico , Modelos Animales de Enfermedad , Enterocolitis/tratamiento farmacológico , Intestinos/efectos de los fármacos , Intestinos/patología , Oxazolona/farmacología , Vancomicina/uso terapéutico , Pez CebraRESUMEN
Secretory leukocyte protease inhibitor (SLPI), a pleiotropic protein expressed by healthy intestinal epithelial cells, functions as an inhibitor of NF-κB and neutrophil proteases and exerts antimicrobial activity. We previously showed SLPI suppresses intestinal epithelial chemokine production in response to microbial contact. Increased SLPI expression was recently detected in various types of carcinoma. In addition, accumulating evidence indicates SLPI expression is favorable for tumor cells. In view of these findings and the abundance of SLPI in the colonic epithelium, we hypothesized SLPI promotes colorectal cancer (CRC) growth and metastasis. Here, we aimed to establish whether SLPI expression in CRC is related to clinical outcome. Using a cohort of 507 patients with CRC who underwent resection of liver metastases, we show that high SLPI protein expression in both liver metastases and primary CRC is associated with significantly shorter overall survival after resection of liver metastases. The prognostic value of SLPI in CRC patients with liver metastases implies a role for SLPI in the formation of metastasis of human CRC. Based on the immune regulatory functions of SLPI, we anticipate that expression of SLPI provides tumors with a mechanism to evade infiltration by immune cells.
Asunto(s)
Neoplasias Colorrectales , Neoplasias Hepáticas , Células Epiteliales , Humanos , Pronóstico , Inhibidor Secretorio de Peptidasas Leucocitarias/genéticaRESUMEN
Chemotherapy-induced intestinal damage is a very important dose-limiting side effect for which there is no definitive prophylaxis or treatment. This is in part due to the lack of understanding of its pathophysiology and impact on intestinal differentiation. The objective of this study was to investigate the gene expression of the small intestinal transcription factors HNF-1alpha, Cdx2, GATA-4 in an experimental model of methotrexate (MTX)-induced intestinal damage, and to correlate these alterations with histological damage, epithelial proliferation and differentiation. HNF-1alpha, Cdx2 and GATA-4 are critical transcription factors in epithelial differentiation, and in combination they act as promoting factors of the sucrase-isomaltase (SI) gene, an enterocyte-specific differentiation marker which is distinctly downregulated after MTX treatment. Mice received two doses of MTX i.v. on two consecutive days and were sacrificed 1, 3 and 7 or 9 days after final injection. Segments of the jejunum were taken for morphological, immunohistochemical and quantitative analyses. Intestinal damage was most severe at day 3 and was associated with decreased expression of the transcriptional factors HNF-1alpha, Cdx2 and GATA-4, which correlated well with decreased expression of SI, and seemed inversely correlated with enhanced proliferation of epithelial crypt cells. During severe damage, the epithelium was preferentially concerned with proliferation rather than differentiation, most likely in order to restore the small intestinal barrier function rather than maintaining its absorptive function. Since HNF-1alpha, Cdx2 and GATA-4 are critical for intestine-specific gene expression and therefore crucial in epithelial differentiation, these results may explain, at least in part, why intestinal differentiation is compromised during MTX treatment.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Mucosa Intestinal/efectos de los fármacos , Metotrexato/farmacología , Animales , Factor de Transcripción CDX2 , Factor de Transcripción GATA4/metabolismo , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Proteínas de Homeodominio/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Yeyuno/efectos de los fármacos , Yeyuno/metabolismo , Yeyuno/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Complejo Sacarasa-Isomaltasa/metabolismo , Transactivadores/metabolismoRESUMEN
Although IL-10 promotes a regulatory phenotype of CD11c+ dendritic cells and macrophages in vitro, the role of IL-10 signaling in CD11c+ cells to maintain intestinal tolerance in vivo remains elusive. To this aim, we generated mice with a CD11c-specific deletion of the IL-10 receptor alpha (Cd11ccreIl10rafl/fl). In contrast to the colon, the small intestine of Cd11ccreIl10rafl/fl mice exhibited spontaneous crypt hyperplasia, increased numbers of intraepithelial lymphocytes and lamina propria T cells, associated with elevated levels of T cell-derived IFNγ and IL-17A. Whereas naive mucosal T-cell priming was not affected and oral tolerance to ovalbumin was intact, augmented T-cell function in the lamina propria was associated with elevated numbers of locally dividing T cells, expression of T-cell attracting chemokines and reduced T-cell apoptosis. Upon stimulation, intestinal IL-10Rα deficient CD11c+ cells exhibited increased activation associated with enhanced IL-6 and TNFα production. Following colonization with Helicobacter hepaticus Cd11ccreIl10rafl/fl mice developed severe large intestinal inflammation characterized by infiltrating T cells and increased levels of Il17a, Ifng, and Il12p40. Altogether these findings demonstrate a critical role of IL-10 signaling in CD11c+ cells to control small intestinal immune homeostasis by limiting reactivation of local memory T cells and to protect against Helicobacter hepaticus-induced colitis.
Asunto(s)
Antígeno CD11c/metabolismo , Colitis/prevención & control , Infecciones por Helicobacter/prevención & control , Inmunidad Mucosa , Interleucina-10/metabolismo , Mucosa Intestinal/metabolismo , Intestino Grueso/metabolismo , Intestino Delgado/metabolismo , Linfocitos T/metabolismo , Animales , Antígeno CD11c/deficiencia , Antígeno CD11c/genética , Antígeno CD11c/inmunología , Colitis/inmunología , Colitis/metabolismo , Colitis/microbiología , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/microbiología , Helicobacter hepaticus/inmunología , Helicobacter hepaticus/patogenicidad , Homeostasis , Memoria Inmunológica , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-10/inmunología , Subunidad alfa del Receptor de Interleucina-10/genética , Subunidad alfa del Receptor de Interleucina-10/inmunología , Subunidad alfa del Receptor de Interleucina-10/metabolismo , Interleucina-17/inmunología , Interleucina-17/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Intestino Grueso/inmunología , Intestino Grueso/microbiología , Intestino Delgado/inmunología , Intestino Delgado/microbiología , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/microbiologíaRESUMEN
Disruption of the intestinal epithelial barrier allows bacterial translocation and predisposes to destructive inflammation. To ensure proper barrier composition, crypt-residing stem cells continuously proliferate and replenish all intestinal epithelial cells within days. As a consequence of this high mitotic activity, mucosal surfaces are frequently targeted by anticancer therapies, leading to dose-limiting side effects. The cellular mechanisms that control tissue protection and mucosal healing in response to intestinal damage remain poorly understood. Type 3 innate lymphoid cells (ILC3s) are regulators of homeostasis and tissue responses to infection at mucosal surfaces. We now demonstrate that ILC3s are required for epithelial activation and proliferation in response to small intestinal tissue damage induced by the chemotherapeutic agent methotrexate. Multiple subsets of ILC3s are activated after intestinal tissue damage, and in the absence of ILC3s, epithelial activation is lost, correlating with increased pathology and severe damage to the intestinal crypts. Using ILC3-deficient Lgr5 reporter mice, we show that maintenance of intestinal stem cells after damage is severely impaired in the absence of ILC3s or the ILC3 signature cytokine IL-22. These data unveil a novel function of ILC3s in limiting tissue damage by preserving tissue-specific stem cells.
Asunto(s)
Inmunidad Innata , Mucosa Intestinal/inmunología , Linfocitos/fisiología , Células Madre/fisiología , Animales , Interleucinas/fisiología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Metotrexato/toxicidad , Ratones , Ratones Endogámicos C57BL , Interleucina-22RESUMEN
PURPOSE: The insulin-like growth factor (IGF)-I protein is a growth-promoting polypeptide that can act as an angiogenic agent in the eye. The purpose of the current study was to localize the expression of IGF-I and its receptor (IGF-IR) mRNA and IGF-IR protein in situ in the normal human eye and to examine the presence of expression in eyes with neovascular age-related macular degeneration (AMD). METHODS: Formalin-fixed, paraffin-embedded slides of 4 normal control eyes and 14 eyes with choroidal neovascularization (CNV) secondary to AMD were examined. Three eyes with proliferative diabetic retinopathy were studied as the positive control. IGF-I and IGF-IR mRNA was detected by in situ hybridization with digoxigenin-labeled RNA probes. IGF-IR protein was studied by immunohistochemistry. RESULTS: In the normal retina, IGF-I and IGF-IR mRNA expression was found throughout the neuroretinal layers, in the retinal pigment epithelium (RPE), and in some choriocapillary and retinal capillary endothelial cells. In eyes with CNV we found IGF and IGF-IR mRNA in capillary endothelial cells, some transdifferentiated RPE, and fibroblast-like cells. IGF-IR protein was found in normal eyes in all neuroretinal layers, in the RPE, and in the choroidal vessels. In eyes with CNV, IGF-IR protein was present in the RPE monolayer, in transdifferentiated RPE, and in newly formed vessels. CONCLUSIONS: The colocalization of protein and receptor indicates an autocrine function of IGF-I in the normal human retina. Because IGF-I participates in ocular neovascularization, synthesis of IGF-IR and IGF-I in endothelial cells, RPE cells, and fibroblast-like cells in CNV may point toward a role for this growth factor in the pathogenesis of neovascular AMD.
Asunto(s)
Neovascularización Coroidal/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Degeneración Macular/metabolismo , Receptor IGF Tipo 1/genética , Adulto , Anciano , Anciano de 80 o más Años , Coroides/irrigación sanguínea , Neovascularización Coroidal/etiología , Retinopatía Diabética/metabolismo , Endotelio Vascular/metabolismo , Femenino , Humanos , Técnicas para Inmunoenzimas , Hibridación in Situ , Factor I del Crecimiento Similar a la Insulina/metabolismo , Degeneración Macular/complicaciones , Masculino , Persona de Mediana Edad , Epitelio Pigmentado Ocular/metabolismo , Sondas ARN , ARN Mensajero/metabolismo , Receptor IGF Tipo 1/metabolismo , Células Ganglionares de la Retina/metabolismo , Vasos Retinianos/metabolismoRESUMEN
OBJECTIVE: Hyperthyroidism in rats produces organ hypertrophy and increases in circulating IGF-I and IGF-binding protein (IGFBP)-3. Chronic treatment with thyroxine (T(4)) during pregnancy advances parturition, blocks lactation and changes several hormone receptors in mammary gland and liver. Since IGFs are implicated in mammary and liver growth and in differentiation, we studied the effects of hyperthyroidism, induced by daily injections of T(4) (0.25 mg/kg). DESIGN AND METHODS: Using quantitative RT-PCR and in situ hybridization, the gene expression of IGF-I, IGF-II and the IGFBPs was determined in mammary gland and liver of rats at estrus and days 7, 14 and 21 of pregnancy (G7, G14, G21), day 1 postpartum (L1) and 3 days after removing the litter (L4). Circulating levels of IGF-I, tri-iodothyronine (T(3)), PRL and GH were measured. RESULTS: T(4) treatment (HT) increased circulating T(3) save on G21, did not change serum IGF-I, increased PRL on G21 and decreased GH on L1. PRL decreased on L1 because of the absence of lactation. Hepatic IGF-I mRNA was low during pregnancy and increased on L4. HT advanced this increase to L1. In controls, liver IGFBP-3 mRNA levels decreased from G14 to G21, whereas IGFBP-4 showed an inverse pattern. HT lowered IGFBP-3 mRNA and increased IGFBP-4. Increases in mammary concentrations of IGF-I, IGFBP-3 and IGFBP-4 mRNAs were seen on G21. HT delayed these peaks to L1. Mammary IGF-II and IGFBP-2 mRNA levels were high on G7 and G14, and fell afterwards, with HT having no effects. IGFBP-5 mRNA decreased during pregnancy and increased on L1. HT increased IGFBP-5 levels in early pregnancy and on L1. IGF-I mRNA localized to connective and epithelial mammary tissue, while IGFBP-2 and IGFBP-5 mRNA was only in epithelial cells. CONCLUSION: These results imply a role for IGF-I, IGFBP-3 and IGFBP-4 in terminal mammary development, while IGF-II and IGFBP-2 may be implicated in early growth. IGFBP-5 has been implicated in mammary apoptosis, and the HT-induced increase may play a role in the premature mammary involution of the HT rats.
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Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Lactancia/metabolismo , Preñez/metabolismo , Tiroxina/farmacología , Animales , Femenino , Hormona del Crecimiento/sangre , Hipertiroidismo/inducido químicamente , Hipertiroidismo/metabolismo , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/genética , Hígado/metabolismo , Glándulas Mamarias Animales/metabolismo , Periodo Posparto/metabolismo , Embarazo , Complicaciones del Embarazo/metabolismo , Prolactina/sangre , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Valores de Referencia , Factores de Tiempo , Triyodotironina/sangreAsunto(s)
Biomarcadores de Tumor/metabolismo , Hematopoyesis , Mediadores de Inflamación/metabolismo , Células Madre Mesenquimatosas/patología , Síndromes Mielodisplásicos/patología , FN-kappa B/metabolismo , Biomarcadores de Tumor/genética , Estudios de Casos y Controles , Células Cultivadas , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Células Madre Mesenquimatosas/inmunología , Células Madre Mesenquimatosas/metabolismo , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/inmunología , Síndromes Mielodisplásicos/metabolismo , FN-kappa B/genética , Pronóstico , Factores de RiesgoRESUMEN
BACKGROUND: Celiac disease (CD) is an intestinal inflammation driven by gluten-reactive CD4(+) T cells. Due to lack of selective markers it has not been determined whether defects in inducible regulatory T cell (Treg) differentiation are associated with CD. This is of importance as changes in numbers of induced Treg could be indicative of defects in mucosal tolerance development in CD. Recently, we have shown that, after encounter of retinoic acid during differentiation, circulating gut-imprinted T cells express CD62L(neg)CD38(+). Using this new phenotype, we now determined whether alterations occur in the frequency of natural CD62L(+)Foxp3(+) Treg or mucosally-imprinted CD62L(neg)CD38(+)Foxp3(+) Treg in peripheral blood of CD patients. In particular, we compared pediatric CD, aiming to select for disease at onset, with adult CD. METHODS: Cell surface markers, intracellular Foxp3 and Helios were determined by flow cytometry. Foxp3 expression was also detected by immunohistochemistry in duodenal tissue of CD patients. RESULTS: In children, the percentages of peripheral blood CD4(+)Foxp3(+) Treg were comparable between CD patients and healthy age-matched controls. Differentiation between natural and mucosally-imprinted Treg on the basis of CD62L and CD38 did not uncover differences in Foxp3. In adult patients on gluten-free diet and in refractory CD increased percentages of circulating natural CD62L(+)Foxp3(+) Treg, but normal mucosally-imprinted CD62L(neg)CD38(+)Foxp3(+) Treg frequencies were observed. CONCLUSIONS: Our data exclude that significant numeric deficiency of mucosally-imprinted or natural Foxp3(+) Treg explains exuberant effector responses in CD. Changes in natural Foxp3(+) Treg occur in a subset of adult patients on a gluten-free diet and in refractory CD patients.
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ADP-Ribosil Ciclasa 1/metabolismo , Enfermedad Celíaca/inmunología , Factores de Transcripción Forkhead/metabolismo , Impresión Genómica , Mucosa Intestinal/inmunología , Selectina L/metabolismo , Linfocitos T Reguladores/inmunología , Adolescente , Adulto , Estudios de Casos y Controles , Enfermedad Celíaca/sangre , Enfermedad Celíaca/patología , Movimiento Celular , Niño , Preescolar , Humanos , Lactante , Interleucina-15/sangre , Mucosa Intestinal/patología , Recuento de LinfocitosRESUMEN
During colonization of germfree mice with the total fecal microbial community of their conventionally born and raised siblings (conventionalization), the intestinal mucosal immune system initiates and maintains a balanced immune response. However, the genetic regulation of these balanced, appropriate responses to the microbiota is obscure. Here, combined analysis of germfree and conventionalized mice revealed that the major molecular responses could be detected initiating at day 4 post conventionalization, with a strong induction of innate immune functions followed by stimulation of adaptive immune responses and development and expansion of adaptive immune cells at later stages of conventionalization. This study provides a comprehensive overview of mouse developmental and immune-related cellular pathways and processes that were co-mediated by the commensal microbiota and suggests which mechanisms were involved in this reprogramming. The dynamic, region-dependent mucosal responses to the colonizing microbiota revealed potential transcriptional signatures for the control of intestinal homeostasis in healthy mice, which may help to decipher the genetic basis of pathway dysregulation in human intestinal inflammatory diseases.
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Linfocitos B/inmunología , Infecciones Bacterianas/metabolismo , Intestinos/fisiología , Metagenoma/fisiología , Linfocitos T/inmunología , Inmunidad Adaptativa , Animales , Linfocitos B/microbiología , Infecciones Bacterianas/genética , Infecciones Bacterianas/inmunología , Diferenciación Celular , Células Cultivadas , Biología Computacional , Citocinas/genética , Citocinas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Perfilación de la Expresión Génica , Vida Libre de Gérmenes , Homeostasis , Tolerancia Inmunológica , Inmunidad Innata , Mediadores de Inflamación/metabolismo , Intestinos/microbiología , Ratones , Ratones Endogámicos C57BL , Análisis por Micromatrices , Especificidad de Órganos , Receptores de Reconocimiento de Patrones/genética , Receptores de Reconocimiento de Patrones/metabolismo , Linfocitos T/microbiología , Regulación hacia ArribaRESUMEN
BACKGROUND: Ethanol ('alcohol') is a partly hydrophobic detergent that may affect the accessibility of glycolipids thereby influencing immunological effects of these molecules. METHODS: The study included cellular in vitro tests using α-galactosylceramide (αGalCer), and in vivo NOD mice experiments detecting diabetes incidence and performing behavioural and bacterial analyses. RESULTS: Alcohol in concentrations from 0.6% to 2.5% increased IL-2 production from NKT cells stimulated with αGalCer by 60% (p<0.05). CD1d expressed on HeLa cells contained significantly increasing amounts of αGalCer with increasing concentrations of alcohol, suggesting that alcohol facilitated the passive loading of αGalCer to CD1d. NOD mice were found to tolerate 5% ethanol in their drinking water without signs of impairment in liver function. Giving this treatment, the diabetes incidence declined significantly. Higher numbers of CD3+CD49b+ NKT cells were found in spleen and liver of the alcohol treated compared to the control mice (p<0.05), whereas the amount of CD4+Foxp3+ regulator T cells did not differ. Increased concentrations of IFN-γ were detected in 24-hour blood samples of alcohol treated mice. Behavioural studies showed no change in attitude of the ethanol-consuming mice, and bacterial composition of caecum samples was not affected by alcohol, disqualifying these as protective mechanisms. CONCLUSION: Alcohol facilitates the uptake of glycolipids and the stimulation of NKT cells, which are known to counteract Type 1 diabetes development. We propose that this is the acting mechanism by which treatment with alcohol reduces the incidence of diabetes in NOD mice. This is corroborated by epidemiology showing beneficial effect of alcohol to reduce the severity of atherosclerosis and related diseases.
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Antígenos CD1d/metabolismo , Diabetes Mellitus/prevención & control , Etanol/farmacología , Células T Asesinas Naturales/efectos de los fármacos , Células T Asesinas Naturales/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Electroforesis en Gel de Gradiente Desnaturalizante , Diabetes Mellitus/inmunología , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Células HeLa , Humanos , Ratones , Ratones Endogámicos NOD , Células T Asesinas Naturales/citología , Células T Asesinas Naturales/inmunología , Transporte de Proteínas/efectos de los fármacosRESUMEN
BACKGROUND: T-cells are a main target for antiinflammatory drugs in inflammatory bowel disease. As the innate immune system is also implicated in the pathogenesis of these diseases, T-cell suppressors may not only inhibit T-cell-dependent production of proinflammatory mediators but also affect innate immune cell function. Specifically, these drugs may impair innate immune cell recruitment and activation through inhibition of T-cells or act independent of T-cell modulation. We explored the extent of immune modulation by the T-cell inhibitor tacrolimus in a murine colitis model. METHODS: We assessed the effects of tacrolimus on trinitro-benzene sulphonic acid (TNBS) colitis in wildtype and Rag2-deficient mice. The severity of colitis was assessed by means of histological scores and weight loss. We further characterized the inflammation using immunohistochemistry and by analysis of isolated intestinal leukocytes at various stages of disease. RESULTS: Tacrolimus-treated wildtype mice were less sensitive to colitis and had fewer activated T-cells. Inhibition of T-cell function was associated with strongly diminished recruitment of infiltrating neutrophils in the colon at the early stages of this model. In agreement, immunohistochemistry demonstrated that tacrolimus inhibited production of the neutrophil chemoattractants CXCL1 and CXCL2. Rag2-deficient mice displayed an enhanced baseline level of lamina propria neutrophils that was moderately increased in TNBS colitis and remained unaffected by tacrolimus. CONCLUSIONS: Both the innate and the adaptive mucosal immune system contribute to TNBS colitis. Tacrolimus suppresses colitis directly through inhibition of T-cell activation and by suppression of T-cell-mediated recruitment of neutrophils.