Lisa Holt

Transforming Growth Factor-β1 Inhibits Non-pathogenic Gramnegative Bacteria-induced NF-κB Recruitment to the Interleukin-6 Gene Promoter in Intestinal Epithelial Cells through Modulation of Histone Acetylation

We have shown that non-pathogenic enteric Gramnegative Bacteroides vulgatus induces RelA phosphorylation, NF-κB activation, and proinflammatory gene expression in primary and intestinal epithelial cell (IEC) lines. We now demonstrate the transient induction of nuclear phospho-RelA (day 3) followed by persistent activation of phospho-Smad2 (days 3 and 7) in IEC from mucosal tissue sections of B. vulgatus-monoassociated rats, indicating that both NF-κB and transforming growth factor-β1 (TGF-β1) signaling are induced in vivo following bacterial colonization. Interestingly, TGF-β1 inhibited B. vulgatus- and lipopolysaccharide (LPS)-induced NF-κB transcriptional activity as well as interleukin-6 (IL-6) mRNA accumulation and protein secretion in IEC. The inhibitory effect of TGF-β1 is mediated independently of B. vulgatus/LPS-induced IκBα, Akt, and RelA phosphorylation as well as NF-κB DNA binding activity. Moreover, the specific histone deacetylase inhibitor trichostatin A blocked B. vulgatus/LPS-induced histone acetylation/phosphorylation (Lys-9/Ser-10) and reversed TGF-β1-mediated inhibition of IL-6 gene expression. Chromatin immunoprecipitation analysis revealed that B. vulgatus/LPS-induced RelA recruitment to the IL-6 promoter is inhibited by TGF-β1 treatment. Adenoviral delivery of Smad7 and dominant negative Smad3 (SmadΔ3) reversed the TGF-β1-mediated inhibition of NF-κB transcriptional activity and NF-κB recruitment to the IL-6 promoter. In addition, TGF-β1 and Ad5Smad3/4 prevent B. vulgatus/LPS-induced CBP/p300 and p65 nuclear co-association. We concluded that the TGF-β1/Smad signaling pathway helps maintain normal intestinal homeostasis to commensal luminal enteric bacteria by regulating NF-κB signaling in IEC through altered histone acetylation.

Bacterial cell wall polymers promote intestinal fibrosis by direct stimulation of myofibroblasts

Normal luminal bacteria and bacterial cell wall polymers are implicated in the pathogenesis of chronic intestinal inflammation. To determine the direct involvement of bacteria and their products on intestinal fibrogenesis, the effects of purified bacterial cell wall polymers on collagen and cytokine synthesis were evaluated in intestinal myofibroblast cultures established from normal fetal and chronically inflamed cecal tissues. In this study, the intestines of Lewis rats were intramurally injected with peptidoglycan-polysaccharide polymers. Collagen and transforming growth factor (TGF)-beta1 mRNA levels were measured and correlated with mesenchymal cell accumulation by immunohistochemistry. The direct effects of cell wall polymers on fibrogenic cytokine and collagen alpha1 (type I) expression were evaluated in intestinal myofibroblast cultures. We found that intramural injections of bacterial cell wall polymers induced chronic granulomatous enterocolitis with markedly increased collagen synthesis and concomitant increased TGF-beta1 and interleukin (IL)-6 expression. Intestinal myofibroblast cultures were established, which both phenotypically and functionally resemble the mesenchymal cells that are involved in fibrosis in vivo. Bacterial cell wall polymers directly stimulated collagen alpha1 (I), TGF-beta1, IL-1beta, and IL-6 mRNA expression in the intestinal myofibroblasts derived from both normal and inflamed cecum. Neutralization of endogenous TGF-beta1 inhibited in vitro collagen gene expression. From our results, we conclude that increased exposure to luminal bacterial products can directly activate intestinal mesenchymal cells, which accumulate in areas of chronic intestinal inflammation, thus stimulating intestinal fibrosis in genetically susceptible hosts.

Butyrate modulates intestinal epithelial cell‐mediated neutrophil migration

Butyrate, a short‐chain fatty acid released by colonic bacteria and administered therapeutically in inflammatory bowel diseases, exerts immunomodulatory properties. The aim of the study was to determine the functional consequences of butyrate exposure on the proinflammatory responsiveness of human intestinal epithelial cells (IEC). IL‐8 promoter activity in IEC pretreated with butyrate then exposed to proinflammatory stimuli was assayed by transfection of luciferase constructs. IL‐8 secretion was determined by ELISA and neutrophil migration by flow cytometry. Receptor mRNA was assessed by reverse transcriptase–polymerase chain reaction (RT‐PCR). Butyrate modulated proinflammatory IL‐8 secretion differentially in Caco‐2 and HT‐29 cells on the transcriptional level. Pointing to the potentially underlying mechanism of increased IL‐1β‐stimulated IL‐8 secretion in HT‐29 cells, butyrate up‐regulated IL‐1RI mRNA but not IL‐1RII. Butyrate pretreatment of IEC lines stimulated by IL‐1β modulated neutrophil migration significantly: reduction towards Caco‐2 and enhancement towards HT‐29/p cells. Pharmacological inhibition of protein tyrosine phosphatases or treatment with mesalamine or sulphasalazine diminished IL‐1β‐stimulated IL‐8 secretion by butyrate‐exposed HT‐29 cells substantially. Immunomodulatory effects of butyrate on IEC are functionally relevant for neutrophil migration. Pharmacological inhibition of enhanced IL‐1β‐mediated IL‐8 secretion in a subpopulation of IEC may improve the clinical efficacy of butyrate.

Gene expression patterns in experimental colitis in IL‐10‐deficient mice

Background: While others have described gene expression patterns in humans with inflammatory bowel diseases and animals with chemically induced colitis, a genome‐wide comparison of gene expression in genetically susceptible animals that develop spontaneous colitis has not been reported. Methods: We used microarray technology to compare gene expression profiles in cecal specimens from specific pathogen‐free IL10‐deficient (IL10−/−) mice with colitis and normal wildtype (WT) mice. RNA isolated from ceca of IL10−/− and WT mice was subjected to microarray analysis. The results were confirmed by real‐time polymerase chain reaction (PCR) and immunofluorescence microscopy of selected molecules. Expression of the selected genes in dextran sodium sulfate (DSS)‐treated mice with colitis and epithelial cell lines activated with pathophysiologic stimuli was measured by real‐time PCR. Results: Histological inflammation of the colon and IL‐12/23p40 secretion from intestinal explants were greater in IL10−/− and DSS‐treated mice versus WT and untreated mice. Microarray analysis demonstrated >10‐fold induction of the following molecules in the ceca of IL10−/− mice: mitochondrial ribosomal protein‐L33, aquaporin‐4, indoleamine‐pyrrole‐2,3‐dioxygenase, and MHC class II with 63, 25, 20, and 12‐fold increases, respectively. Cytochrome‐P450, pancreatic lipase‐related protein‐2, and transthyretin were downregulated in IL10−/− mice. MHC II was increased throughout the colon, and aquaporin‐4 was increased in the basolateral aspect of cecal epithelial cells. MHC II mRNA was increased in epithelial cells treated with IFN‐&ggr;, but not TNF or Toll‐like receptor ligands. Conclusions: Although most upregulated genes in experimental colitis are immune‐related, aquaporin‐4 and mitochondrial ribosomal protein‐L33, which have not been previously associated with inflammation, were most highly upregulated.

Differential expression of interleukin 1 receptor antagonist isoforms in human intestinal epithelial cells.

BACKGROUND & AIMS Regulatory cytokines mediate intestinal epithelial cell (IEC) participation in mucosal immune responses. The aim of this study was to investigate the expression of secretory and intracellular isoforms of interleukin 1 receptor antagonist (IL-1Ra) in human primary IECs and carcinoma-derived cell lines. METHODS Primary IECs were isolated from patients with Crohns disease or ulcerative colitis and from normal controls. Isoform-specific IL-1Ra messenger RNA (mRNA) and protein were assessed by reverse-transcription polymerase chain reaction and Western blot analysis. Expression during cellular differentiation was determined by in situ immunohistochemistry on sequentially released, native IECs and in vitro differentiated cell lines. Intracellular IL-1Ra I function was analyzed by permanent transfection of Caco-2 cells. RESULTS Intracellular IL-1Ra I protein accumulated in surface IECs with extension to the crypts during inflammation. Secretory IL-1Ra and intracellular IL-1Ra II mRNA, but not the corresponding protein, was detected. Transcription of intracellular IL-1Ra I mRNA was significantly up-regulated with inflammation and in vitro by phorbol myristate acetate and interleukin 1beta. In vitro differentiated cells had higher constitutive intracellular IL-1Ra I protein content. Intracellular IL-1Ra I expression in Caco-2 cells decreased IL-1beta-stimulated interleukin 8 secretion. CONCLUSIONS Native human IECs and certain cell lines constitutively express intracellular IL-1Ra type I, which is up-regulated by inflammation, inflammatory stimuli, and cellular differentiation. Constitutive expression of this anti-inflammatory cytokine may contribute to mucosal protection.

Identification and characterization of rat intestinal lamina propria cells: consequences of microbial colonization

Germ-free (GF) animals exhibit an abnormally diminished, cell-mediated immune response which can be rapidly normalized by bacterial colonization of the intestine. This conventionalization suggests that the development and/or regulation of the immune system is dependent upon intestinal bacteria or their products. Here we consider the ontogeny of gut-associated lymphoid tissue (GALT) immunocytes by isolating and characterizing the intestinal lamina propria cells (LPC) of GF rats responding to bacterial colonization or an irrelevant protein antigen, and compared to LPC of specific pathogen-free (SPF) rats which were conventionalized (CV) from birth. Isolation of cells was accomplished by successive EDTA washings of small intestine to remove the epithelium, and enzymatic digestion of the tissue generating single-cell suspensions. Resulting cell suspensions were characterized by monoclonal antibodies directed against leukocyte epitopes using flow cytometry. Functional characterization was measured by the tritiated thymidine proliferation assay with concanavalin A (Con A) and lipopolysaccharide (LPS) as co-stimulators. Germ-free and SPF rats had fewer total LPC than CV rats. Antibody staining revealed that GF rats had fewer total leukocytes than CV and SPF rats, and that CV rats had a greater percentage of T-cells and cells positive for the C3 receptor than GF rats. Co-stimulation of LPC with mitogens only increased proliferation of cells from CV rats compared to GF and SPF rats. In addition, spleen cells from CV rats demonstrated significantly enhanced proliferative responses compared to spleen cells from GF rat and were more analogous to spleen cells from SPF rats in their ability to proliferate in vitro, with and without mitogens. We conclude that T-cells and CD35-positive (C3BR+) cells are recruited and/or proliferate in response to intestinal bacteria and/or their products, and that this results in the induction of immune competency.

Oral peptidoglycan-polysaccharide stimulates systemic immunocompetency in germ-free mice

Germ-free and conventional mice were fed sterile peptidoglycan-polysaccharide polymers to determine whether intestinal bacterial cell wall products influence systemic immunity. Germ-free mice fed saline had significantly less footpad swelling after challenge with sheep erythrocytes than conventional mice. Feeding peptidoglycan-polysaccharide polymers to germ-free mice restored their deficient cellular immune response to conventional levels. Feeding peptidoglycan-polysaccharide did not alter anti-erythrocyte antibody production in germ-free mice. Spontaneous in vitr o proliferation of unfractionated splenocytes taken from germ-free mice fed peptidoglycan-polysaccharide polymers was enhanced compared with proliferation of splenocytes from germ-free mice fed human serum albumin. In vitro conconavalin A stimulation of unfractionated splenocytes obtained from germ-free mice fed cell wall polymers resulted in a significant decrease in proliferation relative to that seen for germ-free mice fed human serum albumin. Respective proliferation results were abrogated when splenocytes were fractionated by removal of plastic-adherent cells. These results suggest that luminal peptidoglycan-polysaccharide polymers are important in the functional development of systemic cellular immunity, and that plastic-adherent splenocytes regulate this response. Keywords - Germ free, Peptidoglycan, Delayed-type hypersensitivity, Splenocyte proliferation, Immunoregulation.

IRGM1 Deficiency Selectively Affects Intestinal Paneth Cell Morphology and Function

G A A b st ra ct s features irregular thickening of epithelium and variable width of capillaries, the % of agreement was 41.5% and 44.5%, respectively, while estimates of kappa were 0.12 and 0.11, respectively. Discussion: In this study we attempted to create classification criteria for advanced adenomas of the colon using selected features during a preliminary review of randomized pCLE videos. Overall sensitivity was low, albeit improving when diagnoses were changed to advanced when confidence level was low. Despite this, NPV is high due to the low prevalence of advanced neoplasia. Further refinement in the classification scheme is required before a diagnose and discard strategy can be implemented in routine clinical practice. The moderate inter observer agreement for certain feature does have promising implications. However, further studies are needed to confirm these findings.