669Abstracts / Alcohol 43 (2009) 665e671

13IRAK-M plays a central role in differential regulation of acute and chronicalcohol exposure on LPS-induced inflammation in human monocytesPranoti Mandrekar, Shashi Bala, Donna Catalano, Karen Kodys and Gyongyi Szabo,Department of Medicine, University of Massachusetts Medical School, Worcester,MA, USA.

Impaired host defense is linked to alcohol abuse due to alterations in inflammatorymediator production. We hypothesized that in human monocytes, acute alcoholinduces hyporesponsiveness to LPS leading to decreased production of the pro-inflammatory cytokine, TNFa whereas chronic alcohol increases TNFa by sensitiza-tion to LPS. Human adherence-isolated blood monocytes were exposed to 25 mMalcohol for 7 days (chronic) and then treated with LPS (100ng/ml) for 18 hrs. Foracute or short-term treatment, cells were exposed to LPS and 25 mM alcohol for18 hrs. TNFa levels were analyzed by ELISA and IRAK-M mRNA and protein deter-mined by real-time PCR and western blotting respectively. Our results show thatacute alcohol decreases LPS-induced TNFa production (p ! 0.001) compared toLPS treatment alone. Concomitantly, LPS-induced IRAK-M, a negative regulatorof inflammation, is increased after acute alcohol exposure. In contrast, chronicalcohol increases LPS-induced TNFa (p ! 0.001) with simultaneously decreasedIRAK-M expression in these cells. Signaling molecules downstream to TLR4/IRAK-M were also investigated to determine their regulation by acute and chronicalcohol exposure in monocytes. Acute alcohol decreases IRAK-1 and IKK activity,NFkB DNA binding and NFkB-driven reporter activity after LPS stimulation butchronic alcohol increases IRAK-1 and IKK kinase activities, NFkB DNA bindingand NFkB-reporter activity. Inhibition of IRAK-M in acute-alcohol exposed mono-cytes using siRNA restores the LPS-induced TNFa whereas over-expression ofIRAK-M in chronic alcohol macrophages prevents the increase in TNFa production.Addition of inhibitors of alcohol metabolism did not alter LPS signaling and TNFaproduction during chronic alcohol exposure. In summary, increased IRAK-M byacute alcohol leads to hyporesponsiveness of monocytes to LPS and reduced TNFa.In contrast, chronic alcohol sensitizes monocytes to LPS through decreased IRAK-Mexpression and results in increased TNFa production. Our data indicate that IRAK-Mplays a central role in the differential regulation of LPS signaling by different lengthsof alcohol exposure in monocytes.

14Fetal exposure to ethanol has long-term effects on influenza viral immunity anddisease severityJodi McGill1,2, David Meyerholz1, Betty Young1, Ruth A. Coleman1, AnnetteSchlueter1,2, Thomas J. Waldschmidt1,2, Robert T. Cook1 and Kevin L. Legge1,2,1Department of Pathology, University of Iowa, Iowa City, IA, USA,2Interdisciplinary Graduate Program in Immunology, University of Iowa, IowaCity, IA, USA.

Alcohol consumption during pregnancy is known to result in well-described cogni-tive impairment and physical malformations. Additionally, there are data demon-strating that in-utero ethanol exposure results in increased rates of infections inboth newborns and young adults. The latter findings suggest fetal alcohol exposuremay induce immune lesions that persist for extended periods. In order to determineif fetal alcohol exposure induces long-term immune deficiency, we infected mice thatwere exposed to ethanol from conception to weaning (fetal alcohol exposed; FAE)and then allowed to mature to adulthood without any additional ethanol intake withinfluenza A virus (IAV). Our results demonstrate that 14-16 wk old FAE mice exhibitsignificantly increased IAV associated morbidity and mortality as well as increasedand sustained pulmonary viral titers following IAV infection. Additionally theseFAE mice mount significantly impaired adaptive immune responses, includingdecreased numbers of IAV-specific CD8 T cells in the lungs, decreased size andfrequency of B cell foci in the lungs, and reduced production of IAV-specific antibodyfollowing virus infection. These changes in immunity and disease susceptibilityappear long-lived as FAE mice that were exposed to ethanol 6 or 9.5 months previ-ously exhibit similar increases in IAV disease severity as well as reduced levels ofIAV-specific antibody and CD8 T cells (6 months of age). These changes areobserved on multiple genetic backgrounds and after infection with 2 distinct strainsof IAV. Since a proportion of fetal alcohol-exposed individuals go on to abuse alcoholas adults, we further examined FAE mice consuming ethanol as adults. These studiesdemonstrate that secondary exposure to alcohol as an adult significantly exacerbatesboth IAV disease severity as well as the reduction in IAV-specific B and T cellimmune responses. All together our studies suggest that immune deficiency resultingfrom fetal alcohol exposure may be long-lived even in the absence of further ethanolconsumption, be independent of the host and viral background, and may contribute tothe increased disease severity and death observed during IAV infections. Further ourresults importantly suggest that fetal alcohol exposed individuals could be atincreased risk for severe and fatal infections with both seasonal and pandemic IAV.(Supported by NIH AA014405, AA019437, and AA019438).

15Role of occludin dephosphorylation in ethanol-induced gut barrier disruptionR.K. Rao, Department of Physiology & Biophysics, University of Tennessee HealthScience Center, Memphis, TN, USA.

A significant body of evidence indicates that gut barrier dysfunction and endotoxemiaplay an important role in the pathogenesis of alcoholic liver disease. Evidence alsoindicate that ethanol metabolism into acetaldehyde is required for the disruption ofintestinal epithelial barrier. Our recent studies showed that acute administration ofethanol into isolated intestinal loops disrupts tight junctions and increases paracellu-lar permeability in colon, but not in ileum. In Caco-2 cell monolayers, ethanol up to1.0% failed to affect the tight junctions and paracellular permeability. However,ethanol dose-dependently potentiated the effect of acetaldehyde on the disruptionof tight junctions. Inhibition of Src kinase and myosin light chain kinase (MLCK)attenuates the ethanol-mediated sensitization of acetaldehyde-induced tight junctiondisruption. Disruption of tight junctions by acetaldehyde is associated with a Thr-dephosphorylation of occludin and Claudin-4 (tight junction proteins) by a proteinphosphatase 2A (PP2A)-dependent mechanism. Our recent study showed that phos-phorylation of occludin on Thr-403 and Thr-404 facilitates its assembly into tightjunctions. The expression of T403/404D mutant occludin (mimics phosphorylatedoccludin) significantly attenuates acetaldehyde-induced disruption of tight junctionsand barrier dysfunction. In summary, ethanol disrupts the barrier function predomi-nantly in the distal colon in mice. Ethanol and acetaldehyde synergistically disruptstight junctions. Finally, acetaldehyde-induced disruption of tight junctions involvesdephosphorylation of occludin on Thr-403 and Thr-404 by a PP2A-dependentmechanism.

16Effects of acute alcohol exposure combined with burn injury on immune cellpopulation in various lymphoid organsJuan Rendon, Xiaoling Li, Shegufta Mahbub, Aleah Brubaker, Elizabeth J. Kovacsand Mashkoor A. Choudhry, Alcohol Research Program, Burn & Shock TraumaInstitute, Department of Surgery, Program in Cell Biology, Neurobiology, &Anatomy, Loyola University Medical Center, Maywood, IL, USA.

Multiple studies have established that greater than 50% of adult burn patients havea measurable ethanol blood level at the time of hospital admission. These patientsare at an increased risk of infection and higher mortality when compared to burnpatients without alcohol exposure. Our study uses an established mouse model toassess the effects of alcohol and burn injury on immune cell numbers in variouslymphoid organs. Preliminary data do not show significant changes in macrophage,dendritic cell or T cell numbers, or expression of MHC class II molecules on macro-phages and dendritic cells in the spleen on day one post alcohol and burn injury.However, within mesenteric lymph nodes, there appears to be a three-fold increasein the number of macrophages on day one post alcohol and burn injury as comparedto control groups. This increase is accompanied by a 50% decrease in the expressionof MHC class II within macrophage populations. While the number of dendritic cellsappears to be unaffected within mesenteric lymph nodes, there is a slight increase intheir number within Peyer’s patches on day one post alcohol and burn injury. Thisincrease is not accompanied by a decrease in MHC class II expression as was notedwith macrophages in the mesenteric lymph nodes. T cell numbers remain unaffectedin both mesenteric lymph nodes and Peyer’s patches. These results are based ona small number of animals and will need to be confirmed within a larger sample size.However, data demonstrate that on day one post alcohol exposure and burn injurythere are significant changes to antigen presenting cells within intestinal lymphoidorgans that are not noted systemically, in the spleen. Therefore, possible early modu-lation of these changes within the intestinal lymphoid organs may preserve intestinalimmunity, barrier function and prevent systemic effects; thus, reducing morbidity andmortality currently seen in patients suffering from burn injuries after acute alcoholexposure (Supported by NIH R01AA015731 (MAC), NIH R01 AA012034(EJK), R01 AG018859 (EJK), and the Dr. Ralph and Marian C. Falk MedicalResearch Trust).