176 Abstracts / Alcohol 46 (2012) 173e179

9Alcohol-induced gut leakiness as a potential trigger for activation of theimmuno-inflammatory cascade in alcoholics: role of circadian machineryC.B. Forsyth1, M. Shaikh1, R.M. Voigt1, Y. Tang1, F.W. Turek2 and A. Keshavarzian1,1Department of Internal Medicine, Section of Gastroenterology, Rush UniversityMedical Center, Chicago, IL USA, 2Center for Sleep and Circadian Biology,Department of Neurobiology and Physiology, Northwestern University, Evanston, IL,USA.

We and others have shown that gut derived endotoxin is the required co-factor to activatethe immune-inflammatory cascade in a subset of alcoholicswithorgan damage especiallyliver disease.We showed that gut leakiness is a primary cause of endotoxemia in a subsetof alcoholics.However, it is not knownwhyonlya subset of alcoholics ismore susceptibleto alcohol-induced gut leakiness. We also recently showed that disruption of circadianrhythm makes the colon susceptible to injury. Several recent studies have demonstratedthe effects of alcohol on central circadian rhythms regulated by the suprachiasmaticnucleus in the brain. Accordingly, we hypothesized that alcohol might also affect circa-dian clock gene expression in the intestinal epithelium in a subset of alcoholics and thisin turn couldmake their intestinemore susceptible to injurious effects of alcohol resultingin hyperpermeability [leaky gut].WeusedCaco-2monolayers grownon culture inserts asan invitromodel of intestinal permeability and performedwestern blotting, permeability,and siRNA inhibition studies to examine the role of Clock and Per2 circadian genes inalcohol-induced hyperpermeability and a possible role for CYP2E1 in alcohol-induced,circadian mediated, disruption of intestinal barrier integrity. We also measured PER2protein levels in intestinal mucosa of alcohol fed rats with intestinal hyperpermeabilityand endotoxemia. Alcohol, as low as 0.2%, induced time dependent increases in bothCaco-2 cell monolayer permeability and in CLOCK and PER2 proteins and thesealcohol-induced changes are CYP2E1 dependent. SiRNA specific inhibition of eitherClock or Per2 significantly inhibited alcohol-induced monolayer hyperpermeability.SiRNA specific for CYP2E1 significantly inhibited the alcohol-induced effects on clockgene expression. Alcohol-fed rats with increased total gut permeability, assessed byurinary sucralose, also had significantly higher levels of PER2 protein in their duodenumand proximal colon than control rats. Our studies: (1) demonstrate a novelmechanism foralcohol-induced intestinal hyperpermeability through stimulation of intestinal circadianclock gene expression, and (2) provide direct evidence for a role for CYP2E1alcohol-induced effects on intestinal circadian genes in the regulation of intestinal permeabilityin response to alcohol that result in intestinal hyperpermeability, endotoxemiaandhepaticinflammation in alcoholics. (Supported byNIHAA013745, AA020216, RC2AA019405(AK), and AA018729 (YT) and a gift from Mr. and Mrs. Larry Field (AK)).

10Fetal/neonatal exposure to ethanol alters pulmonary immunity and increases theseverity of respiratory infectionsK. Kulhankova, K.J. Fan, L.T. Tygrett, B.M. Young, R.A. Coleman, D.K. Meyerholz,A. Schlueter, T.J. Waldschmidt, R.T. Cook and K.L. Legge, The University of IowaCarver College of Medicine, Iowa City, IA, USA.

In addition to the well-described developmental and neurological defects in childrenwith fetal alcohol syndrome (FAS), lesions in immunity have also been reported.Several studies have documented fetal alcohol syndrome FAS children to haveincreased rates of bacterial infection not only during the newborn period, but wellinto the first decade. In order to model the effects of in utero alcohol exposure inthe mouse, we utilized the ethanol-in-drinking water model. Specifically, femaleC57BL/6 mice were maintained on 5% w/v ethanol for at least 2 wks prior to mating.During and after mating, pregnant mice were provided 10% w/v until parturition.During the nursing period, the ethanol dose was elevated to 12% w/v. After weaning,neonatal mice were provided normal drinking water until tested at 12+ wks. Whenexamining total spleen cell recovery, mice exposed to ethanol during the fetal/neonatal period exhibited significantly lower cell numbers in the B cell compartment.Moreover, alcohol decreased the ability of dendritic cells isolated from the spleen tostimulate allogeneic T cells. In addition our findings suggest that fetal/neonatal expo-sure to ethanol leads to an altered splenic immune architecture. These findingssuggest that exposure to ethanol during the fetal/neonatal period may compromisedevelopment of the peripheral immune system and lead to long-term deficits. To testthis possibility, 12 wk old mice exposed to alcohol during the in utero and nursingperiod, as well as water controls, were infected i.n. with mouse-adapted H1N1 influ-enza virus or group A streptococcus (GAS) and followed for mortality and morbidity,as well as pulmonary CD8+ T cell responses. Ethanol-exposed mice had increasedmorbidity and mortality to both pathogens. Further our results demonstrate reducednumbers of total and virus-specific CD8+ T cells in the lungs. These results indicatethat mice derived from alcohol-consuming mothers harbor immune abnormalitieswell into adulthood, thereby providing a model to explore the immune lesionsobserved in FAS children. (Supported by NIH AA014405, AA019437, AA019438and the Department of Pathology).

11Role of p38/ERK in TLR2-mediated increase in T cell IFN-g release followingalcohol and burn injuryX. Li, J. Rendon, S. Akhtar and M.A. Choudhry, Alcohol Research Program, Burn &Shock Trauma Institute, Dept. of Surgery, Loyola University Chicago MedicalCenter, Maywood, IL, USA.

Toll-like receptors (TLRs) play a critical role in host immunity. Recent findings fromour laboratory have shown that TLR2 agonist directly modulates T cell release ofIFN-g, via the MYD88/TIRAP-dependent pathway, following alcohol/ethanol(EtOH) and burn injury. Furthermore, we observed that acute EtOH combined withburn injury suppresses the activation of T cell p38 and ERK members of mitogenactivated protein kinase (MAPK) family. We determined whether TLR2 utilizesthe p38/ERK pathways in mediating its effect on T cell effector function followingEtOH and burn injury. To test this, male mice (|25g) were gavaged with 0.4 ml of25% EtOH (|3g/kg) or the same volume of H2O, prior to receiving 12-15% totalbody surface area sham or full thickness burn injury. One day after injury, mice weresacrificed and splenic T cells were isolated. T cells (5x106 cells/ml) were culturedwith or without plate-bound anti-CD3 (2 mg/ml) in the presence or absence ofTLR2 agonist (HKLM 108 cell/ml), p38 inhibitor (SB203580 100mM) and/ orERK inhibitor (PD98059 50mM) for 48 hours, supernatants were collected to deter-mine IFN-g production by ELISA. T cells were lysed and p38 and ERK phosphor-ylation and protein levels were determined by western blot. A significant decreasein T cell IFN-g was observed following EtOH and burn injury compared to shams.Stimulation of T cells directly with TLR2 agonist also causes IFN-g release but thiswas not found to be different between sham and EtOH plus burn injured mice.Furthermore, TLR2 stimulation potentiated anti-CD3-depdendent release of IFN-gby T cells in both sham (|2.5 folds) and EtOH plus burn injured (|5 folds) mice.We also observed a significant increase in the phosphorylation of p38 and ERK inT cells stimulated with TLR2 agonist in the presence of anti-CD3 following EtOHand burn injury. The increase in IFN-g was abolished in T cells cultured in presenceof p38 or ERK inhibitor. Taken together, these data suggest that MAPK pathwayplays an important role in TLR2-mediated modulation of T cell IFN-g releasefollowing EtOH and burn injury. (Supported by NIH R01AA015731(MAC),F30AA020167 (JR) and the Dr. Ralph and Marian C. Falk Medical ResearchTrust).

12Alcohol exposure and burn injury drive acute adipose inflammation andalterations in glucose transporter GLUT1Y. Qin1, M.D. Bird2, M. Chen2, E.J. Kovacs2 and L. Makowski1, 1Nutrition ObesityResearch Center, Department of Nutrition, Gillings School of Global Public Health,UNC Chapel Hill, NC, USA, 2Alcohol Research Program, Burn & Shock TraumaInstitute, Department of Surgery, Department of Cell Biology, Neurobiology, &Anatomy, Loyola University Medical Center, Maywood, IL, USA.

Macrophages (MF) are central to the formation of obesity. Alcohol is a dietarymodulator of the inflammatory response, exacerbating inflammation acutely aftertrauma. Serum fatty acids are increased during stress to allow for a fuel substrateswitch systemically to increase availability of glucose needed for the pro-inflamma-tory response. We hypothesize that adipose tissue is a likely source of fatty acidsthrough MF-derived cytokines that act to induce lipolysis. While chronic alcoholexposure has been shown to drive macrophage infiltration into adipose tissue, littleis known about acute effects. To examine the role of inflammation in adipose duringtrauma after an acute alcohol exposure, mice were first exposed to saline or intraper-itoneal ethanol (1.12 g/Kg) and then subjected to a 15% total body surface area scaldor sham injury 30 minutes later. Alcohol alone caused little induction of inflammationin adipose over sham-vehicle. Burn induced elevations in the level of pro-inflamma-tory mediator interleukin-6 (IL-6) in adipose by 2 to 5 fold by protein (p ! 0.03) ormRNA levels (p 5 ns). Dramatic elevations in IL-6 were detected in adipose tissueafter acute alcohol and burn injury at the protein (10-fold, p 5 0.03) and mRNA(20-fold, p 5 0.02) levels compared to either exposure alone or control. Our previouswork has demonstrated a critical role for the glucose transporter GLUT1 and subse-quent glucose metabolism in the pro-inflammatory MF response. Similar to IL-6,adipose tissue GLUT1 was not affected by alcohol, increased 3-fold (p 5 0.01) uponburn trauma, and the exposure to ethanol and burn trauma together synergisticallyincreased GLUT1 expression 4-fold (p 5 0.02). Taken together, these data suggestthat the pro-inflammatory response in adipose may play a role in systemic responseto injury which is exacerbated upon alcohol exposure. (Supported by NIH-R00AA017376 (LM), Nutrition Obesity Research Center (LM), and sanofi aventis(YQ), R01AA012034 (EJK), T32AA013527 (EJK)).