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sCytokine Signalling (SOCS) 1 and 3 determined by quantitative real-time PCR. Results IFNβwas detected in the lamina propria of both control and IBD tissue and ISGs (MXA and250AS) were expressed by intestinal T cells sorted from tissue. In vitro, IFNβ neutralisationreduced the frequency of pSTAT1+ intestinal T cells (n=6, p=0.05) and, in healthy controls,decreased the proportion of IL10-producing intestinal T cells (n=8, p=0.01). There was atrend for more IFNγ-producers (p=0.059) and IFNγ concentrations in supernatants weresignificantly increased (n=10, p=0.016). In IBD, intestinal T cells were more responsive toIFNβ in vitro, as assessed by ISG induction, (n=10 for patients and controls, p<0.03) andconstitutive pSTAT1 was increased in T-cells isolated from non-inflamed mucosa of IBDpatients compared with controls (n=30 IBD, 16 control, p=0.03). Concordantly, SOCS1expression was decreased in IBD samples compared to controls (n=8 IBD, 6 control), p=0.047). In contrast to control tissue, neutralisation of IFNβ in non-inflamed IBD samplesled to a generalised increase in cytokine production, with a significant increase in thefrequency of T cells producing all cytokines examined (IL-10, IFNγ, IL-17 and TNFα, n=10). Discussion T1IFN is present in the mucosa of the human intestine and in health mayhave a regulatory role by selectively supporting T cell production of IL-10. There is increasedresponsiveness of the T1IFN pathway in T cells from non-inflamed tissue from IBD patients(increased pSTAT1 and ISGs, decreased SOCS1), associated with a generalised suppressionof cytokine production. Thus, immunoregulatory effects of intestinal T1IFN are contextdependent. This may help explain the varied clinical response to T1IFN as a therapeuticagent in IBD.

Sa1779

Vitamin D Modulates T Cell-Mediated Immunity: Results From a RandomizedControlled Trial of Low-Dose and High-Dose Vitamin D3Gauree G. Konijeti, Matthew R. Boylan, Yanna Song, Pankaj Arora, Frank E. Harrell,Christopher Newton-Cheh, Dillon ONeill, Joshua R. Korzenik, Thomas J. Wang, AndrewT. Chan

Background: In vitro and in vivo animal studies have demonstrated that vitamin D affectsdifferentiation of immune cells and modulates immune responses, which may play animportant role in the development and course of chronic immune-mediated disorders, suchas inflammatory bowel disease (IBD). Although studies have linked vitamin D deficiencywith risk of immune disease in humans, data demonstrating a direct effect on T cell functionare sparse. Our objective was to determine whether oral vitamin D3 influences T cellactivation in human subjects with vitamin D deficiency. Methods: The DAYLIGHT trial isa double-blind, multicenter, randomized controlled trial which assessed whether vitamin Dis effective at lowering blood pressure. 534 men and women ages 18 to 50 years old withserum 25-hydroxyvitamin D [25(OH)D] < 20 ng/mL and untreated pre- or early stage Ihypertension were randomized to receive either low (400 IU daily) or high (4000 IU daily)dose oral vitamin D3 for 6 months. In an ancillary study, a subset of 38 patients hadmeasurements of T cell function in whole blood collected at baseline (pre-treatment) andat 4 month exam using the ImmunKnow assay (ViraCor-IBT Laboratories). Following wholeblood stimulation with plant lectin phytohemagglutinin (PHA), CD4+ T cells were selectedusing magnetic particles coated with anti-human CD4 monoclonal antibodies and a magnet.CD4+ T cells were then lysed to release intracellular adenosine triphosphate (ATP), whichwas measured using lucerin/luciferase and a luminometer. A proportional odds model withfollow-up ATP value as the dependent variable, and baseline ATP value and a dose groupindicator as covariates, was fit to test if ATP level changes were significantly different betweentreatment groups. Results: A total of 18 patients were randomized to low dose vitamin D3and 20 patients to high dose vitamin D3. Patients were treated for an average of 117 days(SD 52), resulting in a mean increase in serum 25(OH)D of 6.3 ng/mL and 12.9 ng/mL forthe low and high dose groups, respectively. Treatment with high dose vitamin D3 decreasedintracellular CD4+ ATP release by 95.5 ng/mL (IQR: -219.5 to 105.8). In contrast, treatmentwith low dose vitamin D3 yielded a decrease of only 0.5 ng/mL. (IQR: -69.2 to 148.5). Ina proportional odds model, randomization to high dose vitamin D3 was less likely to increaseATP after antigen stimulation compared to randomization to low dose vitamin D3 (OR,0.29; 95% CI, 0.09-0.94). Conclusions: In this ancillary study of a randomized trial, wedemonstrate treatment with high dose vitamin D3 reduces CD4+ T cell activation, providingdirect human data that vitamin D can influence cell-mediated immunity. These findingsoffer a mechanistic correlate for the potential influence of vitamin D on the course ofimmune-mediated disorders such as IBD.

Sa1780

The Attaching-and-Effacing Pathogen Effector EspF Exploits Tight JunctionRegulatory Activities of N-WASP and SNX9 to Induce Intestinal BarrierDisruptionJohn Garber, Emily Mallick, Karen M. Scanlon, Michael S. Donnenberg, John M. Leong,Scott B. Snapper

Background: The apical junction complex (AJC)—a major determinant of intestinal epithelialpermeability—is linked to dense networks of actin filaments, and the ability to rapidlyremodel the apical cytoskeleton is crucial for allowing the AJC to rapidly adjust its permeabil-ity. A number of bacterial pathogens specifically target normal AJC regulatory processes toenhance their pathogenicity. Attaching and effacing (AE) pathogens express the effectorprotein EspF, which disrupts AJCs and contains non-overlapping binding sites for both N-WASP and SNX9, suggesting that the ability to simultaneously interact with both of thesehost proteins may be important for EspF-induced barrier disruption. Direct binding of EspFto SNX9 alone appears to be dispensable for junction disruption, although whether EspFmust bind N-WASP to induce AJC dysfunction is not known. Methods: N-WASP (NWKD)and SNX9 knockdown (SNX9KD) Caco-2 cell lines were generated using lentiviral constructsexpressing shRNA. Point mutations L31A and R3D were introduced into each proline-richrepeat in EspF, rendering it unable to bind N-WASP and SNX9, respectively. EPEC and C.rodentium (CR) strains lacking EspF were complemented with the N-WASP- or SNX9-binding-defective EspF constructs, generating EPEC or CRΔespF/pEspFNWBD and EPEC orCRΔespF/pEspFSNX9BD. The ability of the strains complemented with N-WASP- or SNX9-binding-defective EspF to disrupt AJCs was assessed by measurement of transepithelial

S-294AGA Abstracts

electrical resistance (TER) and characterization of E-cadherin and ZO-1 morphology byconfocal immunofluorescence during infection of wild type (WT) cells and mice. Results:As expected, EPEC induced a rapid loss of TER and alteration of AJC morphology in WTcells. However, despite the presence of a large number of actin pedestals, EPEC induced alesser degree of TER loss and did not induce relocalization of E-cadherin or ZO-1 from theAJC in either NWKD or SNX9KD cells. In vitro, EPECΔespF/pEspFNWBD and EPECΔespF/pEspFSNX9BD were attenuated in their ability to induce barrier disruption and relocalizationof E-cadherin and ZO-1 during infection of WT cells. Mice infected with WT CR rapidlylost weight and displayed marked disruption in junction localization of ZO-1 during acuteinfection. In contrast, clinical disease was attenuated in mice infected with CRΔespF/pEspFNWBD or CRΔespF/pEspFSNX9BD and, despite the presence of at least 107 CFU/g CRin the stool, N-WASP- and SNX9-binding-defective EspF strains were unable to induce AJCdisruption in vivo. Conclusion: N-WASP is a key regulator of AJC homeostasis, and theA/E pathogen effector EspF must simultaneously bind both N-WASP and SNX9 to disruptintestinal epithelial barrier integrity.

Sa1781

GEF-H1 Is Required for Antiviral Host DefenseJoo Hye Song, Yun Zhao, Hao-Sen Chiang, Song Liu, Ninghai Wang, Cox Terhorst,Megha G. Basavappa, Kate L. Jeffrey, Hans-Christian Reinecker

Background & Aims: The human intestinal microbiome includes viruses that replicate ineukaryotic cells, fungi or bacteria. However, very little is known on how viral nucleicacid recognition shapes mucosal immune responses. We assessed susceptibility of Guaninenucleotide exchange factor H1 (GEF-H1)-deficient mice and macrophages to distinct RNAviruses that activate the innate immune system through RIG-I like receptors (RLRs) andcompared the innate immune responses to those elicited in MAVS-deficient macrophages.Methods: GEF-H1-deficient mice were generated through a gene-trap insertion in Arhgef2.Bone marrow-derived macrophages from Arhgef2-/- mice were generated with M-CSF andinfected with Encephalomyocardititis (EMCV), Influenza A/PR/8/34 or its NS1 deficientmutant. Nuclear and cytoplasmic extracts from wild type, GEF-H1- or MAVS-deficientmacrophages were prepared for analysis of phosphorylation of IRF3, p65, IkBα and viralproteins by immunoprecipitation and immunoblotting. Arhgef2-/- mice and their littermatecontrols were anesthetized and challenged with influenza A intranasally. Alveolar macro-phages were isolated and IFN-β secretion determined after stimulation with HMW poly(I:C).We assessed the susceptibility of Arhgef2-/- mice to influenza A infection to determine whetherGEF-H1 was required for antiviral innate immune responses in vivo. Results: We found thatGEF-H1 was required for the induction of IFN-β by macrophages in response to EMCVand influenza A infection. We similarly observed a reduction of IFN-β in MAVS-deficientmacrophages upon infection with ssRNA viruses. This was due to the lack of phosphorylationand nuclear translocation of IRF3 in the absence of GEF-H1. In contrast IFNα/β receptorsignaling was intact in GEF-H1 deficient macrophages. Remarkably, activation of NF-kB inresponse to infection with ssRNA viruses was independent of GEF-H1 and MAVS in macro-phages. As consequence of reduced IFN-β, virus replication was enhanced in GEF-H1deficient macrophages as indicated by the significantly increased expression of transcriptsencoding for EMCV and influenza A viral proteins. Furthermore, GEF-H1-deficient micewere more susceptible to Influenza A infection compared to wild-type littermates. Four daysafter infection, alveolar macrophages from GEF-H1-deficient mice expressed significantlyless Ifnb1 and Il6 mRNA compared to wildtype controls. Additionally, lungs of GEF-H1-deficient mice demonstrated significantly more signs of severe inflammation, with increasedepithelial damage, mononuclear cell infiltrates, and alveolitis. Conclusions: Generation ofArhgef2-/- mice revealed a pronounced signaling defect that prevented antiviral host responsesagainst EMCV and influenza A. GEF-H1 was required for the restriction of ssRNA virusreplication by MAVS dependent induction of type I interferons.

Sa1782

Opposing Roles of CD14 and TLR4 in Bacterial LPS-Induced ColonocyticApoptosisWei-Ting Kuo, Hsun-Yuan Yang, Chi-Yun Chen, Tsung-Chun Lee, Yen-Zhen Lu, Li-LingWu, Yen-Hsuan Ni, Shu-Chen Wei, Linda C.H. Yu

Bacteria lipopolysaccharide (LPS) receptors play crucial roles in regulating epithelial homeo-stasis; aberrant microbial sensing may lead to enteric dysfunction and tumorigenesis. Diver-gent death and survival/proliferative responses to LPS have been reported in epithelial cells.It remains unclear whether distinct receptor subunit expression may contribute to thephenomenon. The aim was to characterize the roles of epithelial CD14 and TLR4 in pro-and anti-apoptotic responses induced by LPS. Our results showed that human normalcolonocytes and Caco-2 cells were CD14+TLR4-, while cancerous tissues and HT29 cellswere CD14+TLR4+. Enhanced caspase-dependent apoptosis was noted in Caco-2 but notHT29 cells after LPS challenge, and was uncoupled with TLR4 signals. Blockade of CD14by neutralizing antibody and gene silencing, or inhibition to phosphatidylcholine-specificphospholipase C, sphingomyelinase and PKCζ activation, ablated cell apoptosis. Moreover,LPS-induced apoptosis may be rescued by expression of functional TLR4, but not Asp299Gly,Thr399Ile, or Pro714His mutants, in transfected Caco-2 cells. TLR4 knockdown in HT29cells resulted in loss of survival after LPS challenge. Using an early stage colitis-associatedcolorectal cancer model with administration of one or two cycles of azoxymethane/dextransodium sulfate (AOM/DSS), we observed enhanced epithelial apoptosis and PKCζ phosphory-lation associated with elevated mucosal LPS concentration in TLR4-mutant mice. In contrast,the epithelial apoptotic level did not increase in wild type mice with CD14+TLR4+colonocytes,despite high mucosal LPS exposure, after administration of AOM/DSS. Lastly, intracolonicadministration of neutralizing anti-CD14 prevented mucosal apoptosis induced by LPSoverexposure in TLR4-mutant mice. In conclusion, CD14-dependent lipid messengers andPKCζ activation cause epithelial apoptosis following LPS exposure, whereas TLR4 serves anantagonistic signal for anti-apoptosis and may contribute to death/survival imbalances andtumor progression.