RESUMEN
The inflammatory state associated with Crohn's disease (CD) and ulcerative colitis (UC) remains incompletely defined. To understand more clearly the extracellular milieu associated with inflammatory bowel disease (IBD), we employed a bioassay whereby plasma of treatment naive paediatric IBD patients (n = 22 CD, n = 15 UC) and unrelated healthy controls (uHC, n = 10) were used to induce transcriptional responses in a healthy leucocyte population. After culture, gene expression was measured comprehensively with microarrays and analysed. Relative to uHC, plasma of CD and UC patients induced distinct responses consisting, respectively, of 985 and 895 regulated transcripts [|log2 ratio| ≥ 0·5 (1·4-fold); false discovery rates (FDR) ≤ 0·01]. The CD:uHC and UC:uHC signatures shared a non-random, commonly regulated, intersection of 656 transcripts (χ(2) = P < 0·001) and were highly correlative [Pearson's correlation coefficient = 0·96, 95% confidence interval (CI) = 0.96, 0.97]. Despite sharing common genetic susceptibility loci, the IBD signature correlated negatively with that driven by plasma of type 1 diabetes (T1D) patients (Pearson's correlation coefficient = -0·51). Ontological analyses revealed the presence of an immunoregulatory plasma milieu in IBD, as transcripts for cytokines/chemokines, receptors and signalling molecules consistent with immune activation were under-expressed relative to uHC and T1D plasma. Multiplex enzyme-linked immunosorbent assay (ELISA) and receptor blockade studies confirmed transforming growth factor (TGF)-ß and interleukin (IL)-10 as contributors to the IBD signature. Analysis of CD patient signatures detected a subset of transcripts associated with responsiveness to 6-mercaptopurine treatment. Through plasma-induced signature analysis, we have defined a unique, partially TGF-ß/IL-10-dependent immunoregulatory signature associated with IBD that may prove useful in predicting therapeutic responsiveness.
Asunto(s)
Proteínas Sanguíneas/farmacología , Colitis Ulcerosa/sangre , Enfermedad de Crohn/sangre , Factores Inmunológicos/farmacología , Leucocitos Mononucleares/efectos de los fármacos , ARN Mensajero/genética , Transcriptoma , Adolescente , Niño , Preescolar , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/patología , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/patología , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/inmunología , Femenino , Voluntarios Sanos , Humanos , Interleucina-10/farmacología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Masculino , Cultivo Primario de Células , Análisis por Matrices de Proteínas , ARN Mensajero/inmunología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
The innate and adaptive immune systems in the intestine cooperate to maintain the integrity of the intestinal barrier and to regulate the composition of the resident microbiota. However, little is known about the crosstalk between the innate and adaptive immune systems that contribute to this homeostasis. We find that CD4+ T cells regulate the number and function of barrier-protective innate lymphoid cells (ILCs), as well as production of antimicrobial peptides (AMPs), Reg3γ and Reg3ß. RAG1-/- mice lacking T and B cells had elevated ILC numbers, interleukin-22 (IL-22) production, and AMP expression, which were corrected by replacement of CD4+ T cells. Major histocompatibility class II-/- (MHCII-/-) mice lacking CD4+ T cells also had increased ILCs, IL-22, and AMPs, suggesting that negative regulation by CD4+ T cells occurs at steady state. We utilized transfers and genetically modified mice to show that reduction of IL-22 is mediated by conventional CD4+ T cells and is T-cell receptor dependent. The IL-22-AMP axis responds to commensal bacteria; however, neither the bacterial repertoire nor the gross localization of commensal bacteria differed between MHCII+/- and MHCII-/- littermates. These data define a novel ability of CD4+ T cells to regulate intestinal IL-22-producing ILCs and AMPs.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Inmunidad Innata , Interleucinas/metabolismo , Intestinos/inmunología , Linfocitos/inmunología , Inmunidad Adaptativa , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Genes RAG-1/genética , Linfocitos/citología , Ratones , Ratones Noqueados , Interleucina-22RESUMEN
Innate immune recognition of the bacterial cell wall constituent peptidoglycan by the cytosolic nucleotide-binding oligomerization domain 2 (Nod2) receptor has a pivotal role in the maintenance of intestinal mucosal homeostasis. Whereas peptidoglycan cleavage by gut-derived lysozyme preserves the recognition motif, the N-acetylmuramoyl-L-alanine amidase activity of the peptidoglycan recognition protein 2 (PGLYRP-2) destroys the Nod2-detected muramyl dipeptide structure. PGLYRP-2 green fluorescent protein (GFP) reporter and wild-type mice were studied by flow cytometry and quantitative RT-PCR to identify Pglyrp-2 expression in cells of the intestinal mucosa and reveal a potential regulatory function on epithelial peptidoglycan recognition. CD3(+)/CD11c(+) T lymphocytes revealed significant Pglyrp-2 expression, whereas epithelial cells and intestinal myeloid cells were negative. The mucosal Pglyrp-2-expressing lymphocyte population demonstrated a mixed T-cell receptor (TCR) αß or γδ phenotype with predominant CD8α and less so CD8ß expression, as well as significant staining for the activation markers B220 and CD69, presenting a typical intraepithelial lymphocyte phenotype. Importantly, exposure of peptidoglycan to PGLYRP-2 significantly reduced Nod2/Rip2-mediated epithelial activation. Also, moderate but significant alterations of the intestinal microbiota composition were noted in Pglyrp-2-deficient animals. PGLYRP-2 might thus have a significant role in regulation of the enteric host-microbe homeostasis.
Asunto(s)
Mucosa Intestinal/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Proteínas/metabolismo , Linfocitos T/metabolismo , Animales , Antígenos CD/metabolismo , Células Cultivadas , Ingeniería Genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Interacciones Huésped-Patógeno , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Activación de Linfocitos , Metagenoma , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Adaptadora de Señalización NOD2/genética , Proteína Adaptadora de Señalización NOD2/inmunología , Peptidoglicano/inmunología , Proteínas/genética , Proteínas/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
Immaturity of local innate defenses has been suggested as a factor involved in the pathophysiology of necrotizing enterocolitis (NEC). The mRNA of enteric human defensins 5 (HD5) and 6 (HD6), antibiotic peptides expressed in Paneth cells of the small intestine, have significantly lower levels of expression in fetal life compared with the term newborn and adult. In the current study, intracellular HD5 was demonstrated by immunohistochemistry at 24 wk of gestation, but at low levels, consistent with findings at the mRNA level. These data suggest that the low level enteric defensin expression, characteristic of normal intestinal development, may contribute to the immaturity of local defense, which predisposes the premature infant to NEC. To test if levels of defensin expression are altered in NEC, specimens from six cases of patients with NEC and five control subjects (four patients with atresia and one with meconium ileus) were analyzed to determine HD5 and HD6 mRNA levels by in situ hybridization. Compared with the control group, the level of enteric defensin expression per Paneth cell assessed by image analysis was increased 3-fold in cases of NEC (p = 0.02, analysis of variance and covariance). In addition, the number of Paneth cells was increased 2-fold in the small intestinal crypts of NEC specimens compared with those of control subjects (p < 0.01, covariance analysis). In healthy tissue, peptide levels within Paneth cells paralleled mRNA levels through development. In tissue from infants with NEC, the steady state level of intracellular peptide was not increased in conjunction with the observed rise in defensin mRNA. A straightforward interpretation of this finding is that HD5 is actively secreted in this setting and the Paneth cells maintain a constant steady state level of intracellular peptide, but the possibility of translational regulation of peptide expression is also consistent with these data. The associations between NEC and enteric defensin expression reported here offer support for future studies to address the role of these endogenous host defense factors in the pathophysiology of this disease.
Asunto(s)
Proteínas Sanguíneas/genética , Enterocolitis Seudomembranosa/fisiopatología , Células de Paneth/metabolismo , Adulto , Análisis de Varianza , Actividad Bactericida de la Sangre , Proteínas Sanguíneas/biosíntesis , Defensinas , Enterocolitis Seudomembranosa/cirugía , Feto , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Humanos , Hibridación in Situ , Lactante , Recién Nacido , Intestino Delgado/crecimiento & desarrollo , Intestino Delgado/metabolismo , Intestino Delgado/patología , Células de Paneth/patología , ARN Mensajero/biosíntesis , Valores de Referencia , Transcripción GenéticaAsunto(s)
Células/metabolismo , Endocitosis , Animales , Células CHO , Membrana Celular/metabolismo , Cricetinae , Citometría de Flujo/métodos , Microscopía Fluorescente/métodos , Modelos Biológicos , Pinocitosis , Receptores de Superficie Celular/metabolismo , Espectrometría de Fluorescencia/métodosRESUMEN
A fluorescence assay developed for the quantitation of intracellular fusion of sequentially formed endocytic compartments (Salzman, N. H., and F. R. Maxfield. 1988 J. Cell Biol. 106:1083-1091) has been used to measure the time course of endosome fusion accessibility along the recycling and degradative endocytic pathways. Transferrin (Tf) was used to label the recycling pathway, and alpha2-macroglobulin (alpha 2 M) was used to label the lysosomal degradative pathway. Along the degradative pathway, accessibility of vesicles containing alpha 2M to fusion with subsequently formed endocytic vesicles decreased with apparent first order kinetics. The t12 for the loss of fusion accessibility was approximately 8 min. The behavior of Tf is more complex. Initially the fusion accessibility of Tf decayed rapidly (t1/2 less than 3 min), but a constant level of fusion accessibility was then observed for 10 min. This suggests that Tf moves through one fusion accessible endosome rapidly and then enters a second fusion accessible compartment on the recycling pathway. At 18 degrees C, fusion of antifluorescein antibodies (AFA) containing vesicles with F-alpha 2M was observed when the interval between additions was 10 min. However, if the interval was increased to 1 h, no fusion with incoming vesicles was observed. These results identify the site of F-alpha 2M accumulation at 18 degrees C as a prelysosomal late endosome that no longer fuses with newly formed endosomes since no delivery to lysosomes is observed at this temperature.
Asunto(s)
Endocitosis , Lisosomas/fisiología , Animales , Línea Celular , Fluoresceínas , Técnica del Anticuerpo Fluorescente , Humanos , Lisosomas/ultraestructura , Modelos Biológicos , Mutación , Receptores de Transferrina/genética , Receptores de Transferrina/metabolismo , Espectrometría de Fluorescencia , Transfección , Transferrina/metabolismoRESUMEN
A polyclonal anti-fluorescein antibody (AFA) which quenches fluorescein fluorescence has been used to distinguish between two models of intracellular vesicle traffic. These models address the question of whether sequentially endocytosed probes will mix intracellularly or whether they are carried through the cell in a sequential, isolated manner. Using transferrin (Tf) as a recycling receptor marker, we incubated Chinese hamster ovary (CHO) cells with fluorescein-Tf (F-Tf) which is rapidly endocytosed. After the F-Tf was completely cleared from the surface, AFA was added to the incubation medium and entered endocytic compartments by fluid phase endocytosis. Fusion of a vesicle containing AFA with the compartment containing F-Tf results in binding of AFA to fluorescein and the quenching of fluorescein fluorescence. When AFA was added to the culture medium 2 min after clearance of F-Tf from the surface, time dependent fluorescence quenching occurred. After 20 min, 67% saturation of F-Tf with AFA was observed. When the interval between F-Tf clearance and AFA addition was increased to 5 min only 41% saturation of F-Tf was found. These data indicate that there are some compartments which are accessible for mixing with subsequently endocytosed molecules, but the efficiency of mixing falls off rapidly as the interval between pulses is increased. In CHO cells Tf swiftly segregates to a collection of vesicles or tubules in the para-Golgi region, and at steady state most of the F-Tf is in this compartment. Using digital image analysis to quantify quenching in this region, we have found that F-Tf/AFA mixing is occurring either within this compartment or before transferrin enters it.