Acute alcohol intoxication increases lymphatic permeability and promotes a perilymphatic adipose tissue inflammatory milieu

s / Alcohol 47 (2013) 567–576 575 29. Alcohol-induced tight junction dysfunction primes airways for RSV infection S.M. Simet, T.A. Wyatt, J.A. Pavlik, J.H. Sisson, Division of Pulmonary, Critical Care, Sleep & Allergy, Department of Internal Medicine, Omaha, NE, USA Tight junctions form a belt-like structure between adjacent cells whose degree of permeability changes according to external stimuli, physiological and pathological conditions. We have previously shown that alcohol increases tight junction permeability and disrupts tight junction proteins (ZO-1 and claudin) in cultured airway epithelial cells through PKCa. It is well established that individuals who consume heavy and prolonged amounts of alcohol are more likely to suffer from pulmonary diseases and infections. Alcohol triggers tight junctions in airway epithelial cells to “leak”, which likely primes the airways for pulmonary infections. Thus, a second injury, such as viral or bacterial infection, is likely intensified by alcohol consumption. Not only does alcohol increase tight junction permeability, but it also perturbs wound healing. Since RSV infection (alone) is known to cause shedding of airway epithelium resulting in an injury wound, we suspect that alcohol exacerbates airway epithelial RSV-induced wounds and increases the time it takes for migrating airway epithelia cells to heal the wound. To date, very few studies have focused on the combination of alcohol and RSV infection. While both alcohol and RSV have been separately shown to disrupt tight junctions, little research has examined the combination of alcohol and RSV infection on tight junction function. We hypothesize that alcohol exacerbates RSV infection significantly increasing tight junction permeability, airway epithelial injury and tight junction mediated wound-healing time via a PKCa/ROCK pathway. Preliminary data demonstrates that Go6976, a PKCa inhibitor, and Y27632, a Rho-associated protein kinase (ROCK) inhibitor, prevent alcohol-induced tight junction “leak”. In addition, the combination of alcohol exposure and RSV infection increase tight junction permeability considerably more than RSV or alcohol alone. Wound healing assays demonstrate that alcohol increases wound healing time. Pretreatment with PKC inhibitors both increases and decreases wound-healing time compared to controls, isoform dependent. Together our preliminary data suggest that alcohol-induced tight junction disruption primes the airways for RSV infection and increases wound healing time (Supported by NIH F32 AA019859 [SMS] and R01 AA008769 [JHS]). 30. Chronic binge alcohol (CBA) consumption increases cell death and alters myogenic gene expression in skeletal muscle satellite cells L. Simon, N. LeCapitaine, P. Berner, T. Dodd, C. Vande Stouwe, P. Molina, Department of Physiology, Louisiana State University Health Sciences Center, New Orleans, LA, USA Chronic alcohol abuse is associated with skeletal muscle myopathy. This can be attributed to increased protein degradation and inability to rebuild muscle mass, which might be due to an inability to form new myofibers. Satellite cells (SCs) are resident stem cells in skeletal muscle that upon stimulation, proliferate, self-renew, and differentiate into multinucleated myofibers. Previously we have shown that CBA produces a chronic proinflammatory and pro-oxidative environment in skeletal muscle; which would be expected to activate quiescent SCs. However, we hypothesize that the increased magnitude and duration of tissue inflammation affects the ability of SCs to repair and/or regenerate myofibers. In this study, we have examined the effects of CBA on satellite cell proliferative and differentiation potential. SC were isolated from CBA rhesus macaques and seeded on collagen plates for 14 days. The cells were cultured for the first 7 days in proliferation media (Ham’s F12 with 20% fetal bovine serum and 10 ng/ml of epidermal growth factor) and the next 7 days in differentiation media (Ham’s F12 with 2% horse serum). SC isolated from skeletal muscle of CBA macaques showed significantly increased apoptosis and necrosis, and no change in autophagy during ex vivo proliferation. In addition, SCs isolated from skeletal muscle of CBA macaques showed significantly reduced Pax7 and MyoD gene expression during ex vivo proliferation and differentiation and significantly decreased expression of myogenic genes such as Myf5 and Mef2C during differentiation. These changes in gene expression translated into a marked decrease in the ability of satellite cells to differentiate as evidenced by JennerGiemsa staining andmyonuclei fusion index. Moreover, a significant increase in SC Smad 2 gene expression, the downstream signaling intermediate of TGF-b1 signaling, was observed during proliferation; suggesting a role for this pathway in inhibiting myogenesis. These results provide evidence of dysregulation of SC function which may contribute to the inability of these cells to regenerate new myofibers in CBA macaques (Supported by NIH P60 AA09803). 31. Acute alcohol intoxication increases lymphatic permeability and promotes a perilymphatic adipose tissue inflammatory milieu F. Souza-Smith, P. Berner, P. Molina, Department of Physiology, Alcohol and Drug Abuse Center of Excellence, LSUHSC, New Orleans, LA, USA The lymphatics of the digestive tract transport around two thirds of all lymph produced in the body and have key role in mucosal immunity through their contribution to antigen transport and immune cell trafficking. We previously showed that acute alcohol intoxication (AAI) regulates mesenteric collecting lymphatic intrinsic contractile cycle, modulating the driving force for lymph flow. Specifically, AAI reduces lymphatic myogenic constriction in response to a step increase in luminal pressure. Whether AAI alters collecting lymphatic vessel permeability is not known. We hypothesized that AAI increases lymphatic permeability, impacting the perilymphatic adipose tissue (PLAT) milieu. Male Sprague–Dawley rats received an intragastric infusion of 2.5 g/kg of alcohol. Isovolumic administration of water (vehicle) served as control. Lymphatic permeability was examined by extravasation of Evans Blue to mesenteric PLAT isolated 30 min after alcohol administration. PLAT inflammatory milieu was examined at 30 min and 24 h post-alcohol administration. In a separate group of animals treated as described above, mesenteric PLAT was isolated and fixed in paraformaldehyde for chemical staining and immunohistochemistry examination for mast cells and neutrophils. The results showed that PLAT from alcohol-treated animals had greater concentration of Evans Blue compared to controls. PLAT cytokine (IL6, TNF-a, and GM-CSF) expression was not different between control and alcohol-treated animals at 30 min. However cytokine expression was greater in PLAT from alcoholtreated animals compared to controls at 24 h post alcohol administration. PLAT mast cell and neutrophil density was significantly greater in alcoholtreated animals than in controls. These data indicate that AAI induces mesenteric lymphatic hyperpermeability and promotes PLAT inflammatory milieu. We speculate that crosstalk between PLAT and mesenteric lymphatics may participate in alcohol-induced disruption of lymphatic contractility (Supported by NIH F32 AA021049 and Departmental Funds). 32. Alcohol exposure dysregulates hematopoietic progenitor cell notchmediated dendropoiesis G.M. Upchurch, R.W. Siggins, LSU Health Sciences Center, Department of Physiology, Alcohol and Drug Abuse Center of Excellence, and the Comprehensive Alcohol Research Center, New Orleans, LA 70112, USA Chronic alcohol abuse is known to cause immunosuppression resulting in increased risk and severity of infection. Myeloid dendritic cells (mDCs) are the principal bridge between the innate and adaptive immune systems. Chronic alcohol decreases the bone marrow and circulating pools of mDCs. We hypothesize that alcohol inhibits hematopoietic progenitor cell (HPC) dendropoiesis through a NOTCH-mediated pathway. This study examined the effects of 50 mM alcohol on human-derived MUTZ-3 CD34+ HPC NOTCHmediated dendropoiesis. Cell culture dishes were coated with 0–2 mM of the NOTCH ligand Delta-like ligand 4 (DLL4) before plating HPCs in order to determine the effect of alcohol on CD34+ HPC differentiation. Cultures were differentiated for 7 d to produce immature mDCs and were assayed by FACS. FACS analysis of 7 d mDCs revealed a DLL4 dose-dependent increase in mDC1 phenotype (HLA-DR+CD11c+CD1c+; P 0.05) compared to 0mM alcohol cells. Interestingly, alcohol treatment of cultures during differentiation resulted in promotion of a highly mature mDC2 phenotype (HLA-DR++; P < 0.05) in the absence of DLL4. These results support the hypothesis that NOTCH promotes dendropoiesis and that alcohol exposure attenuates this effect. Additional studies are needed to assess the effect of alcohol on mDC2 differentiation because of this phenotype’s ability to polarize T cells to a TH2 phenotype, a cell type correlated to enhanced human immunodeficiency virus (HIV) disease progression (Supported by NIH AA020312, AA009803, AA21097).