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T1244 Gap Junction Intercellular Communication Plays a Central Role in the Innate Immune Response of the Intestinal Epithelial Barrier
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Axin-1 and -2, CDX-1, c-jun, Claudin-1, c-myc, CyclD-1, CD44, EphB-3, Ephrin-B1 and -B2, MMP-7, PPAR-δ, APC, CUL-1, DKK-1 and -2, DVL-2 and -3, Frizzled-5, Gastrin, LEF1, LRP-5, PGLYP-1, SFRP-3, Tcf-1) were quantified using real-time PCR with external standards. Target genes exhibiting mRNA expression correlating with Tcf-4 were further investigated. In silico promoter screens for potential Tcf-4 binding sites (WWCAAWG) and gel shift assays for In Vitro confirmation were performed. Total Protein from ileal biopsies of controls and ileal CD patients was extracted and analyzed via Western Blot. Results: In addition to the known decrease of Tcf-4, we found reduced expression and protein of the Wnt pathway transcription factors Tcf-1 in ileal (p=0,0242), but not colonic CD. The mRNA levels of Tcf-1 correlated with Tcf4 (rs=-0,5206; p<0,0001) as well as with HD5 and 6 (p<0.0001). Among the 4 potential Tcf-4 binding sites identified in the Tcf1 promotor, a region between -2492 and -2485bp upstream of the transcription start, exhibited the strongest capability for Tcf4- binding. In contrast to α-defensins, most other Wnt pathway and Tcf4 target genes (26 total) were either unchanged (22) or increased (MMP7: p= 0,0759; Claudin 1: p=0.032;PGLYP-1: p=0,0446). Conclusion: Wnt signaling regulated antimicrobial defense is disturbed in ileal CD. Tcf-4, its target Tcf-1, as well as HD5 and HD6 exhibit decreased gene expression. However, other major pathway targets are unchanged, suggesting a selective function of Tcf-4 in intestinal innate immunity. Since Tcf-1 is an important Wnt pathway transducer in T- cells, its down regulation in ileal CD could also constitute a link to adaptive immunity.
Context-Dependent Splicing of Two Novel 5 Prime Untranslated Exons Contributes to the Complex Regulation of NOD2 Protein Expression in Intestinal Inflammation Philip C. Rosenstiel, Klaus Huse, Andre Franke, Jochen Hampe, Roland Roberts, Christopher G. Mathew, Mathias Platzer, Stefan Schreiber Background: NOD2 is an innate immune receptor for the bacterial cell wall component muramyl-dipeptide. Mutations in the leucine-rich repeat region of NOD2, which lead to an impaired recognition of muramyl-dipeptide, have been associated with Crohn disease, a human chronic inflammatory bowel disease. Tissue specific constitutive and inducible expression patterns of NOD2 have been described that result from complex regulatory events for which the molecular mechanisms are not yet fully understood. Methods & Results: We have identified two novel exons of the NOD2 gene (designated exon 1a and 1b), which are spliced to the canonical exon 2 and constitute the 5 prime untranslated region of two alternative transcript isoforms (i.e. exon 1a/1b/2 and exon 1a/2). The two novel transcripts are abundantly expressed and seem to comprise the majority of NOD2 transcripts in the intestinal tract under physiological conditions. We confirm the expression of the previously known canonical first exon (designated exon 1c) of the gene in unstimulated mononuclear cells. The inclusion of the second alternative exon 1b, which harbours three short upstream open reading frames (uORFs), is downregulated upon stimulation with TNF-α or under proinflammatory conditions in the inflamed intestinal mucosa of Crohn disease patients In Vivo. Using the different 5 prime UTR splice forms fused to a firefly luciferase (LUC) reporter we demonstrate a rapamycin-sensitive inhibitory effect of the uORFs on translation efficacy. Conclusions: The differential usage of two alternative promoters in the NOD2 gene leads to tissue-specific and context-dependent NOD2 transcript isoform patterns. We demonstrate for the first time that context-dependent alternative splicing is linked to uORF-mediated translational repression. The results suggest complex parallel control mechanisms that independently regulate NOD2 expression in the context of inflammatory signaling. The regulatory mechanisms could open up new ways to augment the disturbed barrier function in Crohn disease patients.
T1243 NOD2 Activation By Muramyl Dipeptide Induces Autophagy in Dendritic Cells in An ATG16l1 Dependent Pathway Rachel M. Cooney, John S. Baker, Alison Simmons, Derek P. Jewell Background: Multiple CD susceptibility genes have been identified that function in innate immune processes such as signalling, phagocytosis and autophagy. NOD2 is an intracellular pattern recognition receptor, and mutation of its ligand recognition domain in CD results in defective NF-KB induction following triggering. A major conundrum in understanding the function of NOD2 is how this defective signalling translates into the pro-inflammatory responses in CD. ATG16L1, another susceptibility gene, encodes an autophagy pathway protein. In yeast ATG16 acts early in the autophagy pathway, interacting non-covalently with ATG5 and ATG12, to form a protein complex essential for autophagosome formation. The human ATG16L1 protein has an N-terminal APG16 domain consisting of coiled coils and eight C-terminal WD repeats. In CD, a threonine to alanine substitution at position 300 of the N-terminus of the WD-repeat domain is associated with disease. Autophagy has been implicated in a variety of intracellular processes including facilitation of survival in response to starvation, processing of intracellular bacteria and MHC class II antigen presentation. Dendritic cells (DCs) are prototypic antigen presenting cells responsible for both priming and shaping the nature of the adaptive immune response into either a correctly targeted, tolerant or autoimmune phenotype. Aims: We examine whether NOD2 activation can lead to induction of autophagy, whether this effect requires ATG16L1 and therefore whether NOD2 and ATG16L1 act in the same pathway in DCs. Methods: Immature monocyte derived DCs were isolated from peripheral blood mononuclear cells (PBMC) by CD14+ MACS bead extraction and cultured in IL-4 and GM-CSF. DCs were stimulated with MDP and the degree of autophagosome formation was investigated using confocal microscopy to quantitate number of autophagosome forming cells, assessment of LC3 I/II isoforms by immunoblot and visualisation of autophagosomes in DCs by electron microscopy. siRNAs were used to knockdown NOD2, RIP2 and ATG16L1 in DCs following AMAXA transfection. Results:NOD2 stimulation by MDP induces robust autophagosome formation in DCs that can be inhibited following knockdown of both NOD2 and ATG16L1 together with various components of the NOD2 signalling path. CD patient DCs expressing susceptibility variants associated with CD are also defective in autophagy induction. Conclusion: NOD2 activation by MDP induces autophagy in DCs in a manner dependent on ATG16L1 and components of the NOD2 signalling pathway. CD patients defective in components of the pathway demonstrated reduced levels of autophagy in DCs. Implications of these findings are discussed.
T1241 Microsomal Triglyceride Transfer Protein Regulates CD1d-Restricted Antigen Presentation of Hepatocytes and Controls Natural Killer T Cell Homeostasis Via CD1d-Mediated Apoptosis Sebastian Zeissig, Arthur Kaser, Stephanie K. Dougan, Richard S. Blumberg Background and aims: Natural Killer T (NKT) cells are a subset of unconventional T cells which recognize lipid antigens presented by CD1d. Microsomal triglyceride transfer protein (MTP) is an endoplasmic reticulum-resident protein which can transfer phospholipids onto CD1d and is assumed to assist in loading of endogenous lipids onto CD1d. In this study, we have directly tested whether MTP regulates CD1d function in hepatocytes. Methods: AlbCreMttpflox/flox mice expressing Cre recombinase under control of the albumin promoter were created to generate liver-specific deletion of MTP. Antigen presentation was investigated using a panel of invariant (24.7, 24.8, 24.9, DN32.D3) and non-invariant (14S.6, 14S.7, 14S.15) NKT cell hybridomas or OVA-specific CD8 T cells obtained from OT-I mice which recognize SIINFEKL peptide in the context of H-2Kb. Results: Hepatocytes but not splenocytes from mice with liver-specific MTP deletion showed impaired CD1d-restricted presentation of α-galactosylceramide (αGC) to invariant(i) NKT cells as well as decreased autoreactivity to a panel of non-invariant NKT cells. In contrast, MHC class I-restricted presentation of SIINFEKL peptide to OT-I cells did not differ between hepatocytes of AlbCreMttpflox/ flox mice and their AlbCre-negative littermates confirming a selective defect in CD1drestricted antigen presentation. Analysis of peripheral iNKT cells by CD1d/αGC tetramers revealed an increase in relative and absolute numbers of iNKT cells among liver mononuclear cells but not splenocytes, thymocytes or mesenteric lymph node cells of AlbCreMttpflox/ flox mice. Hepatocyte-specific deletion of MTP specifically affected NKT cells since numbers of CD8+ T cells, B cells and NK cells were not altered. The increase in iNKT cells affected both CD4+ and double negative iNKT cells and was independent of NKT cell TCR Vβ usage. In addition, cell surface expression of maturation, homing and activation/memory markers (NK1.1, CD62L, CD25, CD44, CD69, CXCR3, CXCR6) did not differ between MTP-deficient and wildtype mice. Annexin V staining revealed that the increase in iNKT cells in AlbCreMttpflox/flox was due to decreased apoptosis while NKT cell proliferation measured by 5-Bromo-2′-deoxyuridine labeling or CFSE dilution of transferred NKT cells was normal. Conclusion: MTP regulates CD1d-restricted presentation of endogenous autoantigens and exogenous lipids to liver-resident NKT cells thereby regulating NKT cell homeostasis through CD1d-mediated NKT cell apoptosis. These studies have important implications for a wide variety of diseases that involve CD1d-restricted presentation of lipids in the liver.
T1244 Gap Junction Intercellular Communication Plays a Central Role in the Innate Immune Response of the Intestinal Epithelial Barrier Anna Nierobis, Daniel K. Podolsky, Elke Cario Background: Gap junction intercellular communication (GJIC) is considered to be an important, but not well understood, mechanism for intestinal homeostasis. We have recently demonstrated that Toll-like receptor (TLR) 2 enhances ZO-1-associated barrier integrity of intestinal epithelial cells (IEC). However, the role of GJIC in the innate immune response has not been defined yet. The aim of this study was to delineate the mechanistic and functional relevance of GJIC in the innate immune control of IEC barrier homeostasis via TLR2. Materials & Methods: TLR2 ligand (synthetic Pam3Cys-SK4 (PCSK))-induced modulation of GJ-associated proteins was assessed by western blotting / immunoprecipitation and confocal microscopy using two IEC lines (IEC-6; Caco-2) and a 3D-human intestinal mucosa-like culture model of biopsies (HIMC). The effect of GJIC-selective inhibitors on barrier integrity was assessed by determination of transepithelial electrical resistance (TER) and cytokine profiles analyzed by array. TLR2-mediated GJ function during IEC injury was examined In Vitro using a scrape-wounding assay introducing tracer fluorescent dyes and In Vivo by DSS-colitis (TLR2+/+ vs. TLR2-/- mice). Results: The major GJ protein Connexin (Cx) 43 was expressed at the apicolateral membrane in both IEC lines and primary human and murine TLR2+/+ IEC. Cx43 formed a multiprotein complex, interacting with both TLR2 and ZO-1. Stimulation with the TLR2 ligand PCSK induced serine phosphorylation and cytosolic recruitment of Cx43. In HIMC, PCSK upregulated TLR2 which was associated with apical redistribution and polarization of Cx43 and ZO-1 (3D-reconstruction). PCSKinduced increases of ZO-1-associated TER and secretion of TGF-β2 (known to promote IEC restitution) were abolished by pre-treatment with GJIC-inhibitors. After injury, PCSK treatment especially enhanced GJIC at the wounding edge of IEC and preserved intestinal epithelial Cx43 architecture in DSS colitis, contributing to accelerated mucosal barrier
T1242 Selective Influence of Tcf-4 Mediated Wnt Signaling On Intestinal Innate and Adaptive Immunity of Ileal Crohn's Disease Maureen J. Koslowski, Guoxing Wang, Irmgard E. Kuebler, Michael Gersemann, Klaus Fellermann, Eduard Stange, Jan Wehkamp Background: Ileal Crohn's disease (CD) is characterized by diminished antibacterial activity and a specific decrease of small intestinal Paneth cell α-defensins HD5 and 6. We previously reported a causal link between this decrease and the Wnt pathway transcription factor Tcf4. Wnt signaling has an important function in intestinal epithelium renewal, regulating stem cell maintenance and their transition to Paneth-cells. The involvement of disturbed Wnt signaling, resulting in alleviated innate immunity, reveals a new mechanism for ileal CD pathogenesis. To further investigate the pathways influence in the disease, we aimed to assess expression of Tcf4 target and WNT pathway genes other then HD5 and 6. Methods: RNA was isolated from ileal biopsies of healthy individuals (n=14) and CD patients (ileal CD n=28, colonic CD n=11). The mRNA levels of genes, encoding Wnt/Tcf-4 pathway factors as well as Tcf-4 target genes (β-catenin, CBP, Hic-5, ICAT, NLK, Sox-9, p53, p300
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7/4) or macrophage-specific antigens (CD68, F4-80). Tumor angiogenesis was examined using an antibody against the vascular endothelial cell marker PECAM-1/CD31. Results: Lack of TLR5 or MyD88 expression dramatically enhanced tumor growth and inhibited tumor necrosis in mouse xenograft model of human colon cancer. In contrast, TLR5 activation by peritumoral administration of flagellin substantially increased tumor necrosis, leading to significant tumor regression. Tumors from TLR5- or MyD88-KD cells had reduced production of neutrophil attracting chemokines such as ENA-78, MIP3a, and IL-8. Consequently, neutrophil infiltration was dramatically diminished in TLR5 or MyD88 deficient tumor xenografts, while tumor-associated macrophage infiltration or angiogenesis was not changed. Conclusions: TLR5 engagement by bacterial flagellin mediates innate immune responses and elicits potent anti-tumor activity, indicating that TLR5-dependent signaling could be a potential immunotherapeutic target to modulate tumor development and growth in the gut. Supported by a Research Fellowship Award (S.H.R.) from the “CCFA, Inc.”, Young Clinical Scientific Award from “FAMRI, Inc.” (S.H.R. and E.I.), and NIH RO-1 DK072471 (C.P.).
T1245 The Effect of NOD2 Activation On TLR2 Mediated Cytokine Responses Is Dependent On Activation Dose and NOD2 Genotype Michelle Borm, Ad A. van Bodegraven, Chris J. Mulder, Georg Kraal, Gerd Bouma The mechanism by which mutations in NOD2 predispose to Crohn's disease (CD) is incompletely understood. Previous studies in mice have shown that NOD2 signaling inhibits TLR2 responses. To address whether this also occurs in humans, we tested the effect of NOD2 signaling on TLR responses in monocytes from normal NOD2 expressing individuals and from CD patients with NOD2 mutations. Adding low doses of MDP to TLR2 primed monocytes from normal NOD2 expressing patients and controls resulted in a significant increase in cytokine production as compared to TLR2 primed monocytes, whereas higher doses of MDP led to a striking downregulation of the responses. This downregulation of TLR responses by high dose MDP was not seen in monocytes from NOD2 deficient patients. These findings demonstrate that human monocytes respond to combined TLR2/NOD2 signaling in a biphasic fashion with enhanced cytokine responses at low, but not high concentrations of MDP. The inhibitory role of NOD2 at high concentations of MDP implicates a safety mechanism to prevent exaggerated anti-bacterial immune responses in the gut to high or perpetuating bacterial load. This regulatory mechanism is absent in NOD2 deficient patients and may therefore underlie the onset of CD in this group of patients.
T1248 Regulated Roles of Paneth Cell α-Defensin in Mouse Small Intestine Rie Fukaya, Naoki Sakai, Tokiyoshi Ayabe [Background and Aim] Paneth cells contribute to mucosal innate immunity by sensing bacteria and releasing microbicidal activities mostly from activated α-defensins. In mice, αdefensins, named cryptdins (Crps), consisted of six major isoforms (Crps 1 to 6) are the microbicidal constituents of Paneth cell granules. To clarify topographical distributions of tissue Crp isoforms and roles of Paneth cell secretions in the small intestine, we analyzed expression levels of mRNAs encoding six Crp isoforms, Crps immunoreactivites, as well as secreted Crps by Paneth cells from duodenum, jejunum and ileum. [Materials & Methods] Mucosa from duodenum, proximal jejunum and terminal ileum were obtained from adult CD1 male mice. Intact crypts from duodenum, jejunum and ileum isolated by EDTA dissociation were subjected to quantitative RT-PCR (LightCycler480, Roche) for Crps 16. Isolated crypts and Paneth cells from duodenum, jejunum and ileum were analyzed immunochemically using anti-Crp1, anti-Crp4 antibodies (provided by A.J. Ouellette, UC Irvine) using confocal microscopy (LSM510, Carl Zeiss). Secretions collected from isolated crypts of duodenum, jejunum and ileum exposed to bacteria were assayed for bactericidal activity against a defensin-sensitive S. typhimurium strain. [Results] mRNAs for six Crp isoforms were detected in all isolated crypts tested from duodenum, jejunum and ileum. For each Crp isoform, the expression level in ileum crypts was 5~16 times higher than jejunum or duodenum crypts. The Crp expression showed an increasing proximal to distal gradient in small intestine. Immunochemical analyses on isolated crypts revealed that both Crp1 and Crp4 immunoreactivities were greater in ileal Paneth cells. Bactericidal activities released from ileal crypts exposed to bacteria were significantly higher than those from jejunal crypts. Western blot analyses confirmed that secretions of ileal crypts contained activated Crps. [Conclusion] Our results suggest that Paneth cell antimicrobial peptides appeared to be regulated topographically to control intestinal integrity.
T1246 Toll-Like Receptor 4 Mediates Induction of Inflammatory Pathway By Food Additive Carrageenan in Human Colonic Epithelial Cells Sumit Bhattacharyya, Sangeeta Tyagi, Pradeep K. Dudeja, Joanne K. Tobacman Carrageenan (CGN) is a commonly used food additive in the Western diet, and has been widely used to induce inflammation in models of inflammatory bowel disease. Its effects and mechanisms of action in the human intestine are not well understood. We have previously reported that CGN induces activation of NFκB and IL-8 in human colonic epithelial cells through a pathway of innate immunity mediated by Bcl10 (B-cell CLL/lymphoma 10). This may arise in part due to the unusual α-1,3-galactosidic linkage that is part of the backbone of the sulfated disaccharides that comprise CGN and that is the epitope for the anti-Gal antibody, common to humans and Old World apes that lack an α-1,3-galactosyl transferase enzyme. Experiments were performed that involved silencing of Bcl10 by siRNA and of MyD88 by shRNA, and that utilized fluorescent-tagged CGN, TLR4 blocking antibody, IRAK 1/4 inhibitor, monoclonal antibodies to MALT1, Nemo (IKKγ), ubiquitin, and Bcl10, human colonic epithelial cells, and mouse macrophage cell lines. Our data showed that TLR4 (tolllike receptor 4) is the cell surface receptor for CGN in human colonic epithelial cells. TLR4 is a member of the family of innate immune receptors and is activated by microbial products, including lipopolysaccharide. Experiments with fluorescent-tagged CGN demonstrated marked reduction in CGN binding to human colonic epithelial cells and to RAW 264.7 mouse macrophages, following exposure to TLR4 blocking antibody (HTA-125). Binding to 10ScNCr/23 mouse macrophages, which are deficient in the genetic locus for TLR4, was absent. Additional experiments with TLR4 blocking antibody and TLR4 siRNA showed 80% reductions in CGN-induced increases in Bcl10 and IL-8. Transfection with dominant-negative MyD88 plasmid demonstrated MyD88 dependence of the CGN-TLR4 triggered increases in Bcl10 and IL-8. CGN-induced activation of IL-8 was inhibited by an IRAK 1/4 inhibitor. In addition, our data showed that Bcl10 co-immunoprecipitated with MALT1 and Nemo, and that the Nemo-ubiquitin complex was increased following CGN exposure. Study results indicated that CGN-induced inflammation in human colonocytes proceeds predominantly through a pathway of innate immunity, involving TLR4, Bcl10, Nemo, phospho-IκBα, nuclear translocation of NFκB (p65), and IL-8. Activation of this cascade may arise from the unusual α-1,3-galactosidic linkage characteristic of CGN. These findings suggest how CGN intake may contribute to human intestinal inflammation.
T1249 Tribbles 2 (Trib2), a Novel Regulator in Toll-Like Receptor 5 Signaling, Is Decreased in Inflamed Intestinal Mucosa Shu-Chen Wei, Ian M. Rosenberg, Zhifang Cao, Alan Huett, Ramnik J. Xavier, Daniel K. Podolsky BACKGROUND AND AIMS: Toll-like receptors (TLRs) are expressed by a variety of cell populations, including intestinal epithelia. TLRs activate NF-kB/AP-1 and IRF3/7 pathways to coordinate innate immunity and to shape adaptive immunity. Epithelial-cell-specific NFkB activation or suppression appears to be a nodal point in the regulation of immune responses in inflammatory bowel disease (IBD). Tribbles (Trib), are a recently identified family of three kinase-like proteins that have been highly conserved during evolution. The present study was undertaken to clarify the physiological role of one member of the Trib family, designated human Trib2 in the TLR signaling pathway. METHOD: mRNA expression of Trib2 was analyzed by quantitative PCR and protein expression by Western blot. To determine the involvement of Trib2 in NF-κB pathways, Trib2 as well as NF-κB reporter and renilla constructs were transfected into HEK293/TLR5 cells prior to stimulation with Flagellin. siRNA transfection was used for the transient knockdown of Trib2. Trib2 expression in normal and IBD human tissue was assessed by immunohistochemical staining and quantitative real-time PCR. The subcellular location of Trib2 at baseline and after ligand stimulation was determined by confocal microscopy. RESULTS: Trib2 is expressed in human intestinal epithelium, HEK293, immune cells and is inducible by stimulation with the TLR5 ligand Flagellin. Trib2 inhibits TLR5 mediated activation of NF-kB, abrogating NF-kB signaling downstream of TRAF6. siRNA knockdown of Trib2 expression decreases the TLR5 mediated phosphorylation of p38 and JNK but not p44/p42 (ERK1/2). C/EBP-b increases after Trib2 knockdown and Trib2 binds C/EBP-b, independent of Flagellin stimulation. Trib2 translocalizes from the cytoplasm to the peri-nuclear area following Flagellin stimulation. Trib2 expression was diminished in the epithelium of active inflammatory tissue of IBD patients. Trib2 ratio (inflammatory to non-inflammatory expression) was decreased in active IBD patients. CONCLUSIONS: Trib2 is a novel regulator in the TLR5 signaling pathway. Alterations in its expression may contribute to altered innate responses in IBD.
T1247 Toll-Like Receptor 5 Engagement with Flagellin Mediating the Innate Immunity Elicits Anti-Tumor Activity in Mouse Xenograft Model of Human Colon Cancer Sang Hoon Rhee, Eunok Im, Yoon Jeong Choi, Charalabos Pothoulakis Background / Aims: Toll-like receptors (TLRs)-dependent signaling pathways have been proposed as immunotherapeutic targets against invading pathogens and tumorigenesis. Moreover, host-commensal interactions in the human gut mediated by TLRs play an important role in inflammation, allergy, and host immunity. Here we investigated whether TLR5 engagement with bacterial flagellin mediates innate immunity and elicits anti-tumor activity in a mouse xenograft model of human colon cancer. Methods: The expression of TLR5- or MyD88 was knocked down in DLD-1 colon cancer cells by stably transfecting a construct expressing shRNA against human TLR5 or MyD88, or a control vector (psiRNA-GL3Luc) encoding shRNA targeting luciferase gene. Nude mice were subcutaneously implanted with TLR5-KD, MyD88-KD, or control cells (n=16/group) and tumor growth was monitored for 3 weeks. Tumors were excised and tumor histopathology was examined. Tumor xenografts were also treated with flagellin (5.0 mg/kg, one injection/every 2 days for 3 weeks) and growth and histopathology of these tumors was monitored. Protein micro-array analyses assessed the differential expression of cytokines in these tumors. Tumor leukocytes infiltration was assessed by immunohistochemistry with antibodies against neutrophil markers (Gr-1,
T1250 Luminal Uptake of L-Arginine By C. Parvum Infected Porcine Ileum Is Promoted By Epithelial Induction of Arginase II and Stimulates ProstaglandinDependent Secretory Diarrhea Jody L. Gookin, Derek M. Foster, Stephen H. Stauffer, Maria R. Stone (Coccaro) Cryptosporidium parvum (CP) is a minimally invasive parasite of intestinal epithelium. Synthesis of nitric oxide (NO) by infected epithelium reduces parasite numbers. The exclusive substrate for NO synthesis is L-arginine (ARG). For the neonate, milk is relatively deficient
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restitution during the recovery phase. In contrast, in TLR2-/- IEC, Cx43 was irregularly sequestered in the cytoplasm and DSS-induced damage led to loss of Cx43, suggesting impairment of GJ pore formation in the absence of TLR2. Deregulated GJIC correlated with increased susceptibility to inflammatory injury and compromised wound healing in TLR2-/IEC. Conclusions: GJIC is an important component of the innate immune response of the intestinal epithelium. TLR2-induced GJIC controls IEC barrier function and restitution, enhancing homeostasis between commensals and host.
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Axin-1 and -2, CDX-1, c-jun, Claudin-1, c-myc, CyclD-1, CD44, EphB-3, Ephrin-B1 and -B2, MMP-7, PPAR-δ, APC, CUL-1, DKK-1 and -2, DVL-2 and -3, Frizzled-5, Gastrin, LEF1, LRP-5, PGLYP-1, SFRP-3, Tcf-1) were quantified using real-time PCR with external standards. Target genes exhibiting mRNA expression correlating with Tcf-4 were further investigated. In silico promoter screens for potential Tcf-4 binding sites (WWCAAWG) and gel shift assays for In Vitro confirmation were performed. Total Protein from ileal biopsies of controls and ileal CD patients was extracted and analyzed via Western Blot. Results: In addition to the known decrease of Tcf-4, we found reduced expression and protein of the Wnt pathway transcription factors Tcf-1 in ileal (p=0,0242), but not colonic CD. The mRNA levels of Tcf-1 correlated with Tcf4 (rs=-0,5206; p<0,0001) as well as with HD5 and 6 (p<0.0001). Among the 4 potential Tcf-4 binding sites identified in the Tcf1 promotor, a region between -2492 and -2485bp upstream of the transcription start, exhibited the strongest capability for Tcf4- binding. In contrast to α-defensins, most other Wnt pathway and Tcf4 target genes (26 total) were either unchanged (22) or increased (MMP7: p= 0,0759; Claudin 1: p=0.032;PGLYP-1: p=0,0446). Conclusion: Wnt signaling regulated antimicrobial defense is disturbed in ileal CD. Tcf-4, its target Tcf-1, as well as HD5 and HD6 exhibit decreased gene expression. However, other major pathway targets are unchanged, suggesting a selective function of Tcf-4 in intestinal innate immunity. Since Tcf-1 is an important Wnt pathway transducer in T- cells, its down regulation in ileal CD could also constitute a link to adaptive immunity.
Context-Dependent Splicing of Two Novel 5 Prime Untranslated Exons Contributes to the Complex Regulation of NOD2 Protein Expression in Intestinal Inflammation Philip C. Rosenstiel, Klaus Huse, Andre Franke, Jochen Hampe, Roland Roberts, Christopher G. Mathew, Mathias Platzer, Stefan Schreiber Background: NOD2 is an innate immune receptor for the bacterial cell wall component muramyl-dipeptide. Mutations in the leucine-rich repeat region of NOD2, which lead to an impaired recognition of muramyl-dipeptide, have been associated with Crohn disease, a human chronic inflammatory bowel disease. Tissue specific constitutive and inducible expression patterns of NOD2 have been described that result from complex regulatory events for which the molecular mechanisms are not yet fully understood. Methods & Results: We have identified two novel exons of the NOD2 gene (designated exon 1a and 1b), which are spliced to the canonical exon 2 and constitute the 5 prime untranslated region of two alternative transcript isoforms (i.e. exon 1a/1b/2 and exon 1a/2). The two novel transcripts are abundantly expressed and seem to comprise the majority of NOD2 transcripts in the intestinal tract under physiological conditions. We confirm the expression of the previously known canonical first exon (designated exon 1c) of the gene in unstimulated mononuclear cells. The inclusion of the second alternative exon 1b, which harbours three short upstream open reading frames (uORFs), is downregulated upon stimulation with TNF-α or under proinflammatory conditions in the inflamed intestinal mucosa of Crohn disease patients In Vivo. Using the different 5 prime UTR splice forms fused to a firefly luciferase (LUC) reporter we demonstrate a rapamycin-sensitive inhibitory effect of the uORFs on translation efficacy. Conclusions: The differential usage of two alternative promoters in the NOD2 gene leads to tissue-specific and context-dependent NOD2 transcript isoform patterns. We demonstrate for the first time that context-dependent alternative splicing is linked to uORF-mediated translational repression. The results suggest complex parallel control mechanisms that independently regulate NOD2 expression in the context of inflammatory signaling. The regulatory mechanisms could open up new ways to augment the disturbed barrier function in Crohn disease patients.
T1243 NOD2 Activation By Muramyl Dipeptide Induces Autophagy in Dendritic Cells in An ATG16l1 Dependent Pathway Rachel M. Cooney, John S. Baker, Alison Simmons, Derek P. Jewell Background: Multiple CD susceptibility genes have been identified that function in innate immune processes such as signalling, phagocytosis and autophagy. NOD2 is an intracellular pattern recognition receptor, and mutation of its ligand recognition domain in CD results in defective NF-KB induction following triggering. A major conundrum in understanding the function of NOD2 is how this defective signalling translates into the pro-inflammatory responses in CD. ATG16L1, another susceptibility gene, encodes an autophagy pathway protein. In yeast ATG16 acts early in the autophagy pathway, interacting non-covalently with ATG5 and ATG12, to form a protein complex essential for autophagosome formation. The human ATG16L1 protein has an N-terminal APG16 domain consisting of coiled coils and eight C-terminal WD repeats. In CD, a threonine to alanine substitution at position 300 of the N-terminus of the WD-repeat domain is associated with disease. Autophagy has been implicated in a variety of intracellular processes including facilitation of survival in response to starvation, processing of intracellular bacteria and MHC class II antigen presentation. Dendritic cells (DCs) are prototypic antigen presenting cells responsible for both priming and shaping the nature of the adaptive immune response into either a correctly targeted, tolerant or autoimmune phenotype. Aims: We examine whether NOD2 activation can lead to induction of autophagy, whether this effect requires ATG16L1 and therefore whether NOD2 and ATG16L1 act in the same pathway in DCs. Methods: Immature monocyte derived DCs were isolated from peripheral blood mononuclear cells (PBMC) by CD14+ MACS bead extraction and cultured in IL-4 and GM-CSF. DCs were stimulated with MDP and the degree of autophagosome formation was investigated using confocal microscopy to quantitate number of autophagosome forming cells, assessment of LC3 I/II isoforms by immunoblot and visualisation of autophagosomes in DCs by electron microscopy. siRNAs were used to knockdown NOD2, RIP2 and ATG16L1 in DCs following AMAXA transfection. Results:NOD2 stimulation by MDP induces robust autophagosome formation in DCs that can be inhibited following knockdown of both NOD2 and ATG16L1 together with various components of the NOD2 signalling path. CD patient DCs expressing susceptibility variants associated with CD are also defective in autophagy induction. Conclusion: NOD2 activation by MDP induces autophagy in DCs in a manner dependent on ATG16L1 and components of the NOD2 signalling pathway. CD patients defective in components of the pathway demonstrated reduced levels of autophagy in DCs. Implications of these findings are discussed.
T1241 Microsomal Triglyceride Transfer Protein Regulates CD1d-Restricted Antigen Presentation of Hepatocytes and Controls Natural Killer T Cell Homeostasis Via CD1d-Mediated Apoptosis Sebastian Zeissig, Arthur Kaser, Stephanie K. Dougan, Richard S. Blumberg Background and aims: Natural Killer T (NKT) cells are a subset of unconventional T cells which recognize lipid antigens presented by CD1d. Microsomal triglyceride transfer protein (MTP) is an endoplasmic reticulum-resident protein which can transfer phospholipids onto CD1d and is assumed to assist in loading of endogenous lipids onto CD1d. In this study, we have directly tested whether MTP regulates CD1d function in hepatocytes. Methods: AlbCreMttpflox/flox mice expressing Cre recombinase under control of the albumin promoter were created to generate liver-specific deletion of MTP. Antigen presentation was investigated using a panel of invariant (24.7, 24.8, 24.9, DN32.D3) and non-invariant (14S.6, 14S.7, 14S.15) NKT cell hybridomas or OVA-specific CD8 T cells obtained from OT-I mice which recognize SIINFEKL peptide in the context of H-2Kb. Results: Hepatocytes but not splenocytes from mice with liver-specific MTP deletion showed impaired CD1d-restricted presentation of α-galactosylceramide (αGC) to invariant(i) NKT cells as well as decreased autoreactivity to a panel of non-invariant NKT cells. In contrast, MHC class I-restricted presentation of SIINFEKL peptide to OT-I cells did not differ between hepatocytes of AlbCreMttpflox/ flox mice and their AlbCre-negative littermates confirming a selective defect in CD1drestricted antigen presentation. Analysis of peripheral iNKT cells by CD1d/αGC tetramers revealed an increase in relative and absolute numbers of iNKT cells among liver mononuclear cells but not splenocytes, thymocytes or mesenteric lymph node cells of AlbCreMttpflox/ flox mice. Hepatocyte-specific deletion of MTP specifically affected NKT cells since numbers of CD8+ T cells, B cells and NK cells were not altered. The increase in iNKT cells affected both CD4+ and double negative iNKT cells and was independent of NKT cell TCR Vβ usage. In addition, cell surface expression of maturation, homing and activation/memory markers (NK1.1, CD62L, CD25, CD44, CD69, CXCR3, CXCR6) did not differ between MTP-deficient and wildtype mice. Annexin V staining revealed that the increase in iNKT cells in AlbCreMttpflox/flox was due to decreased apoptosis while NKT cell proliferation measured by 5-Bromo-2′-deoxyuridine labeling or CFSE dilution of transferred NKT cells was normal. Conclusion: MTP regulates CD1d-restricted presentation of endogenous autoantigens and exogenous lipids to liver-resident NKT cells thereby regulating NKT cell homeostasis through CD1d-mediated NKT cell apoptosis. These studies have important implications for a wide variety of diseases that involve CD1d-restricted presentation of lipids in the liver.
T1244 Gap Junction Intercellular Communication Plays a Central Role in the Innate Immune Response of the Intestinal Epithelial Barrier Anna Nierobis, Daniel K. Podolsky, Elke Cario Background: Gap junction intercellular communication (GJIC) is considered to be an important, but not well understood, mechanism for intestinal homeostasis. We have recently demonstrated that Toll-like receptor (TLR) 2 enhances ZO-1-associated barrier integrity of intestinal epithelial cells (IEC). However, the role of GJIC in the innate immune response has not been defined yet. The aim of this study was to delineate the mechanistic and functional relevance of GJIC in the innate immune control of IEC barrier homeostasis via TLR2. Materials & Methods: TLR2 ligand (synthetic Pam3Cys-SK4 (PCSK))-induced modulation of GJ-associated proteins was assessed by western blotting / immunoprecipitation and confocal microscopy using two IEC lines (IEC-6; Caco-2) and a 3D-human intestinal mucosa-like culture model of biopsies (HIMC). The effect of GJIC-selective inhibitors on barrier integrity was assessed by determination of transepithelial electrical resistance (TER) and cytokine profiles analyzed by array. TLR2-mediated GJ function during IEC injury was examined In Vitro using a scrape-wounding assay introducing tracer fluorescent dyes and In Vivo by DSS-colitis (TLR2+/+ vs. TLR2-/- mice). Results: The major GJ protein Connexin (Cx) 43 was expressed at the apicolateral membrane in both IEC lines and primary human and murine TLR2+/+ IEC. Cx43 formed a multiprotein complex, interacting with both TLR2 and ZO-1. Stimulation with the TLR2 ligand PCSK induced serine phosphorylation and cytosolic recruitment of Cx43. In HIMC, PCSK upregulated TLR2 which was associated with apical redistribution and polarization of Cx43 and ZO-1 (3D-reconstruction). PCSKinduced increases of ZO-1-associated TER and secretion of TGF-β2 (known to promote IEC restitution) were abolished by pre-treatment with GJIC-inhibitors. After injury, PCSK treatment especially enhanced GJIC at the wounding edge of IEC and preserved intestinal epithelial Cx43 architecture in DSS colitis, contributing to accelerated mucosal barrier
T1242 Selective Influence of Tcf-4 Mediated Wnt Signaling On Intestinal Innate and Adaptive Immunity of Ileal Crohn's Disease Maureen J. Koslowski, Guoxing Wang, Irmgard E. Kuebler, Michael Gersemann, Klaus Fellermann, Eduard Stange, Jan Wehkamp Background: Ileal Crohn's disease (CD) is characterized by diminished antibacterial activity and a specific decrease of small intestinal Paneth cell α-defensins HD5 and 6. We previously reported a causal link between this decrease and the Wnt pathway transcription factor Tcf4. Wnt signaling has an important function in intestinal epithelium renewal, regulating stem cell maintenance and their transition to Paneth-cells. The involvement of disturbed Wnt signaling, resulting in alleviated innate immunity, reveals a new mechanism for ileal CD pathogenesis. To further investigate the pathways influence in the disease, we aimed to assess expression of Tcf4 target and WNT pathway genes other then HD5 and 6. Methods: RNA was isolated from ileal biopsies of healthy individuals (n=14) and CD patients (ileal CD n=28, colonic CD n=11). The mRNA levels of genes, encoding Wnt/Tcf-4 pathway factors as well as Tcf-4 target genes (β-catenin, CBP, Hic-5, ICAT, NLK, Sox-9, p53, p300
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7/4) or macrophage-specific antigens (CD68, F4-80). Tumor angiogenesis was examined using an antibody against the vascular endothelial cell marker PECAM-1/CD31. Results: Lack of TLR5 or MyD88 expression dramatically enhanced tumor growth and inhibited tumor necrosis in mouse xenograft model of human colon cancer. In contrast, TLR5 activation by peritumoral administration of flagellin substantially increased tumor necrosis, leading to significant tumor regression. Tumors from TLR5- or MyD88-KD cells had reduced production of neutrophil attracting chemokines such as ENA-78, MIP3a, and IL-8. Consequently, neutrophil infiltration was dramatically diminished in TLR5 or MyD88 deficient tumor xenografts, while tumor-associated macrophage infiltration or angiogenesis was not changed. Conclusions: TLR5 engagement by bacterial flagellin mediates innate immune responses and elicits potent anti-tumor activity, indicating that TLR5-dependent signaling could be a potential immunotherapeutic target to modulate tumor development and growth in the gut. Supported by a Research Fellowship Award (S.H.R.) from the “CCFA, Inc.”, Young Clinical Scientific Award from “FAMRI, Inc.” (S.H.R. and E.I.), and NIH RO-1 DK072471 (C.P.).
T1245 The Effect of NOD2 Activation On TLR2 Mediated Cytokine Responses Is Dependent On Activation Dose and NOD2 Genotype Michelle Borm, Ad A. van Bodegraven, Chris J. Mulder, Georg Kraal, Gerd Bouma The mechanism by which mutations in NOD2 predispose to Crohn's disease (CD) is incompletely understood. Previous studies in mice have shown that NOD2 signaling inhibits TLR2 responses. To address whether this also occurs in humans, we tested the effect of NOD2 signaling on TLR responses in monocytes from normal NOD2 expressing individuals and from CD patients with NOD2 mutations. Adding low doses of MDP to TLR2 primed monocytes from normal NOD2 expressing patients and controls resulted in a significant increase in cytokine production as compared to TLR2 primed monocytes, whereas higher doses of MDP led to a striking downregulation of the responses. This downregulation of TLR responses by high dose MDP was not seen in monocytes from NOD2 deficient patients. These findings demonstrate that human monocytes respond to combined TLR2/NOD2 signaling in a biphasic fashion with enhanced cytokine responses at low, but not high concentrations of MDP. The inhibitory role of NOD2 at high concentations of MDP implicates a safety mechanism to prevent exaggerated anti-bacterial immune responses in the gut to high or perpetuating bacterial load. This regulatory mechanism is absent in NOD2 deficient patients and may therefore underlie the onset of CD in this group of patients.
T1248 Regulated Roles of Paneth Cell α-Defensin in Mouse Small Intestine Rie Fukaya, Naoki Sakai, Tokiyoshi Ayabe [Background and Aim] Paneth cells contribute to mucosal innate immunity by sensing bacteria and releasing microbicidal activities mostly from activated α-defensins. In mice, αdefensins, named cryptdins (Crps), consisted of six major isoforms (Crps 1 to 6) are the microbicidal constituents of Paneth cell granules. To clarify topographical distributions of tissue Crp isoforms and roles of Paneth cell secretions in the small intestine, we analyzed expression levels of mRNAs encoding six Crp isoforms, Crps immunoreactivites, as well as secreted Crps by Paneth cells from duodenum, jejunum and ileum. [Materials & Methods] Mucosa from duodenum, proximal jejunum and terminal ileum were obtained from adult CD1 male mice. Intact crypts from duodenum, jejunum and ileum isolated by EDTA dissociation were subjected to quantitative RT-PCR (LightCycler480, Roche) for Crps 16. Isolated crypts and Paneth cells from duodenum, jejunum and ileum were analyzed immunochemically using anti-Crp1, anti-Crp4 antibodies (provided by A.J. Ouellette, UC Irvine) using confocal microscopy (LSM510, Carl Zeiss). Secretions collected from isolated crypts of duodenum, jejunum and ileum exposed to bacteria were assayed for bactericidal activity against a defensin-sensitive S. typhimurium strain. [Results] mRNAs for six Crp isoforms were detected in all isolated crypts tested from duodenum, jejunum and ileum. For each Crp isoform, the expression level in ileum crypts was 5~16 times higher than jejunum or duodenum crypts. The Crp expression showed an increasing proximal to distal gradient in small intestine. Immunochemical analyses on isolated crypts revealed that both Crp1 and Crp4 immunoreactivities were greater in ileal Paneth cells. Bactericidal activities released from ileal crypts exposed to bacteria were significantly higher than those from jejunal crypts. Western blot analyses confirmed that secretions of ileal crypts contained activated Crps. [Conclusion] Our results suggest that Paneth cell antimicrobial peptides appeared to be regulated topographically to control intestinal integrity.
T1246 Toll-Like Receptor 4 Mediates Induction of Inflammatory Pathway By Food Additive Carrageenan in Human Colonic Epithelial Cells Sumit Bhattacharyya, Sangeeta Tyagi, Pradeep K. Dudeja, Joanne K. Tobacman Carrageenan (CGN) is a commonly used food additive in the Western diet, and has been widely used to induce inflammation in models of inflammatory bowel disease. Its effects and mechanisms of action in the human intestine are not well understood. We have previously reported that CGN induces activation of NFκB and IL-8 in human colonic epithelial cells through a pathway of innate immunity mediated by Bcl10 (B-cell CLL/lymphoma 10). This may arise in part due to the unusual α-1,3-galactosidic linkage that is part of the backbone of the sulfated disaccharides that comprise CGN and that is the epitope for the anti-Gal antibody, common to humans and Old World apes that lack an α-1,3-galactosyl transferase enzyme. Experiments were performed that involved silencing of Bcl10 by siRNA and of MyD88 by shRNA, and that utilized fluorescent-tagged CGN, TLR4 blocking antibody, IRAK 1/4 inhibitor, monoclonal antibodies to MALT1, Nemo (IKKγ), ubiquitin, and Bcl10, human colonic epithelial cells, and mouse macrophage cell lines. Our data showed that TLR4 (tolllike receptor 4) is the cell surface receptor for CGN in human colonic epithelial cells. TLR4 is a member of the family of innate immune receptors and is activated by microbial products, including lipopolysaccharide. Experiments with fluorescent-tagged CGN demonstrated marked reduction in CGN binding to human colonic epithelial cells and to RAW 264.7 mouse macrophages, following exposure to TLR4 blocking antibody (HTA-125). Binding to 10ScNCr/23 mouse macrophages, which are deficient in the genetic locus for TLR4, was absent. Additional experiments with TLR4 blocking antibody and TLR4 siRNA showed 80% reductions in CGN-induced increases in Bcl10 and IL-8. Transfection with dominant-negative MyD88 plasmid demonstrated MyD88 dependence of the CGN-TLR4 triggered increases in Bcl10 and IL-8. CGN-induced activation of IL-8 was inhibited by an IRAK 1/4 inhibitor. In addition, our data showed that Bcl10 co-immunoprecipitated with MALT1 and Nemo, and that the Nemo-ubiquitin complex was increased following CGN exposure. Study results indicated that CGN-induced inflammation in human colonocytes proceeds predominantly through a pathway of innate immunity, involving TLR4, Bcl10, Nemo, phospho-IκBα, nuclear translocation of NFκB (p65), and IL-8. Activation of this cascade may arise from the unusual α-1,3-galactosidic linkage characteristic of CGN. These findings suggest how CGN intake may contribute to human intestinal inflammation.
T1249 Tribbles 2 (Trib2), a Novel Regulator in Toll-Like Receptor 5 Signaling, Is Decreased in Inflamed Intestinal Mucosa Shu-Chen Wei, Ian M. Rosenberg, Zhifang Cao, Alan Huett, Ramnik J. Xavier, Daniel K. Podolsky BACKGROUND AND AIMS: Toll-like receptors (TLRs) are expressed by a variety of cell populations, including intestinal epithelia. TLRs activate NF-kB/AP-1 and IRF3/7 pathways to coordinate innate immunity and to shape adaptive immunity. Epithelial-cell-specific NFkB activation or suppression appears to be a nodal point in the regulation of immune responses in inflammatory bowel disease (IBD). Tribbles (Trib), are a recently identified family of three kinase-like proteins that have been highly conserved during evolution. The present study was undertaken to clarify the physiological role of one member of the Trib family, designated human Trib2 in the TLR signaling pathway. METHOD: mRNA expression of Trib2 was analyzed by quantitative PCR and protein expression by Western blot. To determine the involvement of Trib2 in NF-κB pathways, Trib2 as well as NF-κB reporter and renilla constructs were transfected into HEK293/TLR5 cells prior to stimulation with Flagellin. siRNA transfection was used for the transient knockdown of Trib2. Trib2 expression in normal and IBD human tissue was assessed by immunohistochemical staining and quantitative real-time PCR. The subcellular location of Trib2 at baseline and after ligand stimulation was determined by confocal microscopy. RESULTS: Trib2 is expressed in human intestinal epithelium, HEK293, immune cells and is inducible by stimulation with the TLR5 ligand Flagellin. Trib2 inhibits TLR5 mediated activation of NF-kB, abrogating NF-kB signaling downstream of TRAF6. siRNA knockdown of Trib2 expression decreases the TLR5 mediated phosphorylation of p38 and JNK but not p44/p42 (ERK1/2). C/EBP-b increases after Trib2 knockdown and Trib2 binds C/EBP-b, independent of Flagellin stimulation. Trib2 translocalizes from the cytoplasm to the peri-nuclear area following Flagellin stimulation. Trib2 expression was diminished in the epithelium of active inflammatory tissue of IBD patients. Trib2 ratio (inflammatory to non-inflammatory expression) was decreased in active IBD patients. CONCLUSIONS: Trib2 is a novel regulator in the TLR5 signaling pathway. Alterations in its expression may contribute to altered innate responses in IBD.
T1247 Toll-Like Receptor 5 Engagement with Flagellin Mediating the Innate Immunity Elicits Anti-Tumor Activity in Mouse Xenograft Model of Human Colon Cancer Sang Hoon Rhee, Eunok Im, Yoon Jeong Choi, Charalabos Pothoulakis Background / Aims: Toll-like receptors (TLRs)-dependent signaling pathways have been proposed as immunotherapeutic targets against invading pathogens and tumorigenesis. Moreover, host-commensal interactions in the human gut mediated by TLRs play an important role in inflammation, allergy, and host immunity. Here we investigated whether TLR5 engagement with bacterial flagellin mediates innate immunity and elicits anti-tumor activity in a mouse xenograft model of human colon cancer. Methods: The expression of TLR5- or MyD88 was knocked down in DLD-1 colon cancer cells by stably transfecting a construct expressing shRNA against human TLR5 or MyD88, or a control vector (psiRNA-GL3Luc) encoding shRNA targeting luciferase gene. Nude mice were subcutaneously implanted with TLR5-KD, MyD88-KD, or control cells (n=16/group) and tumor growth was monitored for 3 weeks. Tumors were excised and tumor histopathology was examined. Tumor xenografts were also treated with flagellin (5.0 mg/kg, one injection/every 2 days for 3 weeks) and growth and histopathology of these tumors was monitored. Protein micro-array analyses assessed the differential expression of cytokines in these tumors. Tumor leukocytes infiltration was assessed by immunohistochemistry with antibodies against neutrophil markers (Gr-1,
T1250 Luminal Uptake of L-Arginine By C. Parvum Infected Porcine Ileum Is Promoted By Epithelial Induction of Arginase II and Stimulates ProstaglandinDependent Secretory Diarrhea Jody L. Gookin, Derek M. Foster, Stephen H. Stauffer, Maria R. Stone (Coccaro) Cryptosporidium parvum (CP) is a minimally invasive parasite of intestinal epithelium. Synthesis of nitric oxide (NO) by infected epithelium reduces parasite numbers. The exclusive substrate for NO synthesis is L-arginine (ARG). For the neonate, milk is relatively deficient
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restitution during the recovery phase. In contrast, in TLR2-/- IEC, Cx43 was irregularly sequestered in the cytoplasm and DSS-induced damage led to loss of Cx43, suggesting impairment of GJ pore formation in the absence of TLR2. Deregulated GJIC correlated with increased susceptibility to inflammatory injury and compromised wound healing in TLR2-/IEC. Conclusions: GJIC is an important component of the innate immune response of the intestinal epithelium. TLR2-induced GJIC controls IEC barrier function and restitution, enhancing homeostasis between commensals and host.