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
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
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
Human renal membrane transporters play key roles in the disposition of renally cleared drugs and endogenous substrates, but their ontogeny is largely unknown. Using 184 human postmortem frozen renal cortical tissues (preterm newborns to adults) and a subset of 62 tissue samples, we measured the mRNA levels of 11 renal transporters and the transcription factor pregnane X receptor (PXR) with quantitative real-time polymerase chain reaction, and protein abundance of nine transporters using liquid chromatography tandem mass spectrometry selective reaction monitoring, respectively. Expression levels of p-glycoprotein, urate transporter 1, organic anion transporter 1, organic anion transporter 3, and organic cation transporter 2 increased with age. Protein levels of multidrug and toxin extrusion transporter 2-K and breast cancer resistance protein showed no difference from newborns to adults, despite age-related changes in mRNA expression. Multidrug and toxin extrusion transporter 1, glucose transporter 2, multidrug resistance-associated protein 2, multidrug resistance-associated protein 4 (MRP4), and PXR expression levels were stable. Using immunohistochemistry, we found that MRP4 localization in pediatric samples was similar to that in adult samples. Collectively, our study revealed that renal drug transporters exhibited different rates and patterns of maturation, suggesting that renal handling of substrates may change with age.
Asunto(s)
Corteza Renal/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Receptor X de Pregnano/metabolismo , Proteómica/métodos , ARN Mensajero/biosíntesis , Adolescente , Adulto , Factores de Edad , Anciano , Niño , Preescolar , Cromatografía Liquida , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masas en Tándem , Adulto JovenRESUMEN
Disease heterogeneity hampers achieving long-term disease remission in inflammatory bowel disease (IBD). Monitoring ongoing tissue-localized regulatory and inflammatory T-cell responses in peripheral blood would empower disease classification. We determined whether regulatory and inflammatory phenotypes of circulating CD38+ effector (CD62LnegCD4+) T cells, a population enriched for cells with mucosal antigen specificity, classify disease course in pediatric IBD patients. In healthy individuals, circulating CD38+ effector T cells had a predominant regulatory component with lower frequencies of IFNγ-secreting T cells, higher frequencies of IL-10-secreting T cells and higher frequencies of inhibitory molecule T-cell immunoglobulin and ITIM domain+ (TIGIT) cells than CD38neg effector T cells. TIGIT expression was stable upon stimulation and marked CD38+ T cells with inhibitory properties. In IBD patients with active intestinal inflammation this predominant regulatory component was lost: circulating CD38+ effector T cells had increased activated CD25+CD45RAneg and decreased TIGIT+ cell frequencies. TIGIT percentages below 25% before treatment associated with shorter duration of clinical remission. In conclusion, phenotypic changes in circulating CD38+ effector T cells, in particular the frequency of TIGIT+ cells, classify pediatric IBD patients and predict severity of disease course. These findings have relevance for IBD and can be exploited in graft-versus-host-disease and checkpoint inhibitor-induced inflammation in cancer.
Asunto(s)
Células Dendríticas/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Mucosa Intestinal/inmunología , Linfocitos T Reguladores/inmunología , ADP-Ribosil Ciclasa 1/metabolismo , Adulto , Circulación Sanguínea , Estudios de Casos y Controles , Células Cultivadas , Técnicas de Cocultivo , Estudios de Cohortes , Progresión de la Enfermedad , Humanos , Interleucina-10/metabolismo , Receptores Inmunológicos/metabolismoRESUMEN
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.
Asunto(s)
Inmunidad Adaptativa/efectos de los fármacos , Colitis/tratamiento farmacológico , Colitis/inmunología , Neutrófilos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Inmunidad Adaptativa/inmunología , Animales , Colitis/inducido químicamente , Proteínas de Unión al ADN/genética , Células Dendríticas/efectos de los fármacos , Células Dendríticas/patología , Toxina Diftérica/toxicidad , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Inmunosupresores/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neutrófilos/inmunología , Neutrófilos/patología , Índice de Severidad de la Enfermedad , Linfocitos T/inmunología , Linfocitos T/patología , Tacrolimus/farmacologíaRESUMEN
The accumulation of certain species of bacteria in the intestine is involved in both tissue homeostasis and immune-mediated pathologies. The host mechanisms involved in controlling intestinal colonization with commensal bacteria are poorly understood. We observed that under specific pathogen-free or germ-free conditions, intragastric administration of Pseudomonas aeruginosa, E. coli, Staphylococcus aureus, or Lactobacillus gasseri resulted in increased colonization of the small intestine and bacterial translocation in mice lacking Cd1d, an MHC class I-like molecule, compared with WT mice. In contrast, activation of Cd1d-restricted T cells (NKT cells) with alpha-galactosylceramide caused diminished intestinal colonization with the same bacterial strains. We also found prominent differences in the composition of intestinal microbiota, including increased adherent bacteria, in Cd1d-/- mice in comparison to WT mice under specific pathogen-free conditions. Germ-free Cd1d-/- mice exhibited a defect in Paneth cell granule ultrastructure and ability to degranulate after bacterial colonization. In vitro, NKT cells were shown to induce the release of lysozyme from intestinal crypts. Together, these data support a role for Cd1d in regulating intestinal colonization through mechanisms that include the control of Paneth cell function.
Asunto(s)
Antígenos CD1d/fisiología , Bacterias/inmunología , Intestinos/inmunología , Intestinos/microbiología , Animales , Degranulación de la Célula/fisiología , Escherichia coli/inmunología , Heces/microbiología , Galactosilceramidas/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Intestino Delgado/inmunología , Intestino Delgado/microbiología , Lactobacillus/inmunología , Ganglios Linfáticos/microbiología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Células T Asesinas Naturales/inmunología , Células de Paneth/fisiología , Células de Paneth/ultraestructura , Pseudomonas aeruginosa/inmunología , ARN Ribosómico 16S/análisis , Vesículas Secretoras/química , Vesículas Secretoras/ultraestructura , Organismos Libres de Patógenos Específicos/inmunología , Staphylococcus aureus/inmunologíaRESUMEN
The notion that the mucosal immune system maintains a tolerogenic response to harmless Ags while continually being challenged with microbial products seems an enigma. The aim of this study was to unravel mechanisms that are involved in regulating the development of tolerance under constant microbial pressure. The tolerogenic response to Ags administered via the nasal mucosa is dependent on the organized lymphoid tissue of the cervical lymph nodes (LN). We show that cervical LN differentially express secretory leukoprotease inhibitor (SLPI) compared with peripheral LN. SLPI was expressed by dendritic cells (DCs) and because SLPI is known to suppress LPS responsiveness, it was hypothesized that its expression in mucosal DCs may be required to regulate cellular activation to microbial products. Indeed, compared with wild-type controls, bone marrow-derived DCs from SLPI(-/-) mice released more inflammatory cytokines and enhanced T cell proliferation after stimulation with low dose LPS. This increased sensitivity to LPS was accompanied by increased NF-kappaB p65 activation in SLPI(-/-) DCs. In vivo, nasal application of OVA with LPS to SLPI(-/-) mice resulted in enhanced DC activation in the cervical LN reflected by increased costimulatory molecule expression and release of inflammatory cytokines. This led to failure to maintain tolerance to nasal OVA application in the presence of low doses of LPS. We propose that expression of SLPI functions as a rheostat by controlling the level of bacterial stimuli that induce mucosal DC activation. As such, it regulates the quality of the ensuing Ag-specific immune response in the mucosa draining LN.