Polarized distribution of inducible nitric oxide synthase regulates activity in intestinal epithelial cells

Inducible nitric oxide synthase (iNOS) functions as a homodimer. In cell extracts, iNOS molecules partition both in cytosolic and particulate fractions, indicating that iNOS exists as soluble and membrane associated forms. In this study, iNOS features were investigated in human intestinal epithelial cells stimulated with cytokines and in duodenum from mice exposed to flagellin. Our experiments indicate that iNOS is mainly associated with the particulate fraction of cell extracts. Confocal microscopy showed a preferential localization of iNOS at the apical pole of intestinal epithelial cells. In particulate fractions, iNOS dimers were more abundant than in the cytosolic fraction. Similar observations were seen in mouse duodenum samples. These results suggest that, in epithelial cells, iNOS activity is regulated by localization-dependent processes.Facultad de Ciencias Exacta

Toll-like receptor 5- and lymphotoxin β receptor-dependent epithelial Ccl20 expression involves the same NF-κB binding site but distinct NF-κB pathways and dynamics

Canonical and alternative NF-κB pathways depend on distinct NF-κB members and regulate expression of different gene subset in inflammatory and steady state conditions, respectively. In intestinal epithelial cells, both pathways control the transcription of the gene coding the CCL20 chemokine. Lymphotoxin β receptor (LTβR) mediates long lasting CCL20 expression whereas Toll-like receptor 5 (TLR5) signals promote inducible and transient activation. Here, we investigated whether the regulation of CCL20 expression involves different promoter sites and NF-κB molecules in response to TLR5 and LTβR stimulation. In epithelial cells, both stimulation required the same promoter regions, especially the NF-κB binding site but involved different NF-κB isoforms: p65/p50 and p52/RelB, for TLR5 and LTβR-dependent activation, respectively. The dynamic of activation and interaction with CCL20-specific NF-κB site correlated with gene transcription. Similar Ccl20 expression and NF-κB activation was found in the small intestine of mice stimulated with TLR5 and LTβR agonists. In summary, different NF-κB pathways modulate CCL20 transcription by operating on the same NF-κB binding site in the same cell type.Facultad de Ciencias ExactasLaboratorio de Investigaciones del Sistema Inmun

Polarized distribution of inducible nitric oxide synthase regulates activity in intestinal epithelial cells

Inducible nitric oxide synthase (iNOS) functions as a homodimer. In cell extracts, iNOS molecules partition both in cytosolic and particulate fractions, indicating that iNOS exists as soluble and membrane associated forms. In this study, iNOS features were investigated in human intestinal epithelial cells stimulated with cytokines and in duodenum from mice exposed to flagellin. Our experiments indicate that iNOS is mainly associated with the particulate fraction of cell extracts. Confocal microscopy showed a preferential localization of iNOS at the apical pole of intestinal epithelial cells. In particulate fractions, iNOS dimers were more abundant than in the cytosolic fraction. Similar observations were seen in mouse duodenum samples. These results suggest that, in epithelial cells, iNOS activity is regulated by localization-dependent processes.Facultad de Ciencias Exacta

Toll-like receptor 5- and lymphotoxin beta receptor-dependent epithelial Ccl20 expression involves the same NF-kappaB binding site but distinct NF-kappaB pathways and dynamics.

International audienceCanonical and alternative NF-kappaB pathways depend on distinct NF-kappaB members and regulate expression of different gene subset in inflammatory and steady state conditions, respectively. In intestinal epithelial cells, both pathways control the transcription of the gene coding the CCL20 chemokine. Lymphotoxin beta receptor (LTbetaR) mediates long lasting CCL20 expression whereas Toll-like receptor 5 (TLR5) signals promote inducible and transient activation. Here, we investigated whether the regulation of ccl20 expression involves different promoter sites and NF-kappaB molecules in response to TLR5 and LTbetaR stimulation. In epithelial cells, both stimulation required the same promoter regions, especially the NF-kappaB binding site but involved different NF-kappaB isoforms: p65/p50 and p52/RelB, for TLR5 and LTbetaR-dependent activation, respectively. The dynamic of activation and interaction with CCL20-specific NF-kappaB site correlated with gene transcription. Similar Ccl20 expression and NF-kappaB activation was found in the small intestine of mice stimulated with TLR5 and LTbetaR agonists. In summary, different NF-kappaB pathways modulate CCL20 transcription by operating on the same NF-kappaB binding site in the same cell type

Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediated migration of dendritic cells

Enteropathogenic bacteria elicit mucosal innate and adaptive immune responses. We investigated whether gut epithelial cells played a role in triggering an adaptive immune response by recruiting dendritic cells (DCs). Immature DCs are selectively attracted by the CCL20 chemokine. The expression of the CCL20 gene in human intestinal epithelial cell lines was up-regulated by pathogenic bacteria, including Salmonella species, but not by indigenous bacteria of the intestinal flora. The Salmonella machinery for epithelial cell invasion was not required for CCL20 gene activation. Flagellin but not the lipopolysaccharide was found to be the Salmonella factor responsible for stimulation of epithelial CCL20 production. CCL20 in turn triggered a specific migration of immature DCs. Our data show that crosstalk between bacterial flagellin and epithelial cells is essential for the recruitment of DCs, a mechanism that could be instrumental to initiate adaptive immune responses in the gut

Lymphotoxin beta receptor signaling induces the chemokine CCL20 in intestinal epithelium.

BACKGROUND & AIMS: The follicle-associated epithelium (FAE) that overlies Peyer's patches (PPs) exhibits distinct features compared with the adjacent villus epithelium. Besides the presence of antigen-sampling membranous M cells and the down-regulation of digestive functions, it constitutively expresses the chemokine CCL20. The mechanisms that induce FAE differentiation and CCL20 expression are poorly understood. The aim of this work was to test whether lymphotoxin beta receptor signaling (LTbetaR), which plays a central role in PPs' organogenesis, mediates CCL20 gene expression in intestinal epithelial cells. METHODS: CCL20, lymphotoxin beta (LTbeta) and LTbetaR expression were monitored during embryonic development by in situ hybridization of mouse intestine. The human intestinal epithelial cell line T84 was used to study CCL20 expression following LTalpha(1)/beta(2) stimulation. In vivo CCL20 expression following agonistic anti-LTbetaR antibody treatment was studied by laser microdissection and quantitative RT-PCR. RESULTS: CCL20 was expressed in the FAE before birth at the time when the first hematopoietic CD4(+)CD3(-) appeared in the PP anlage. LTbetaR was expressed in the epithelium during PP organogenesis, making it a putative target for LTalpha(1)beta(2)signals. In vitro, CCL20 was induced in T84 cells upon LTbetaR signaling, either using an agonistic ligand or anti-LTbeta receptor agonistic antibody. LTalpha(1)beta(2)-induced CCL20 expression was found to be NF-kappaB dependent. LTbetaR signaling up-regulated CCL20 expression in the small intestinal epithelium in vivo. CONCLUSIONS: Our results show that LTbetaR signaling induces CCL20 expression in intestinal epithelial cells, suggesting that this pathway triggers constitutive production of CCL20 in the FAE