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Fetal exposure to ethanol has long-term effects on influenza viral immunity and disease severity
Abstracts / Alcohol 43 (2009) 665e671
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IRAK-M plays a central role in differential regulation of acute and chronic alcohol exposure on LPS-induced inflammation in human monocytes Pranoti Mandrekar, Shashi Bala, Donna Catalano, Karen Kodys and Gyongyi Szabo, Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA.
Role of occludin dephosphorylation in ethanol-induced gut barrier disruption R.K. Rao, Department of Physiology & Biophysics, University of Tennessee Health Science Center, Memphis, TN, USA.
Impaired host defense is linked to alcohol abuse due to alterations in inflammatory mediator production. We hypothesized that in human monocytes, acute alcohol induces hyporesponsiveness to LPS leading to decreased production of the proinflammatory cytokine, TNFa whereas chronic alcohol increases TNFa by sensitization to LPS. Human adherence-isolated blood monocytes were exposed to 25 mM alcohol for 7 days (chronic) and then treated with LPS (100ng/ml) for 18 hrs. For acute or short-term treatment, cells were exposed to LPS and 25 mM alcohol for 18 hrs. TNFa levels were analyzed by ELISA and IRAK-M mRNA and protein determined by real-time PCR and western blotting respectively. Our results show that acute alcohol decreases LPS-induced TNFa production (p ! 0.001) compared to LPS treatment alone. Concomitantly, LPS-induced IRAK-M, a negative regulator of inflammation, is increased after acute alcohol exposure. In contrast, chronic alcohol increases LPS-induced TNFa (p ! 0.001) with simultaneously decreased IRAK-M expression in these cells. Signaling molecules downstream to TLR4/ IRAK-M were also investigated to determine their regulation by acute and chronic alcohol exposure in monocytes. Acute alcohol decreases IRAK-1 and IKK activity, NFkB DNA binding and NFkB-driven reporter activity after LPS stimulation but chronic alcohol increases IRAK-1 and IKK kinase activities, NFkB DNA binding and NFkB-reporter activity. Inhibition of IRAK-M in acute-alcohol exposed monocytes using siRNA restores the LPS-induced TNFa whereas over-expression of IRAK-M in chronic alcohol macrophages prevents the increase in TNFa production. Addition of inhibitors of alcohol metabolism did not alter LPS signaling and TNFa production during chronic alcohol exposure. In summary, increased IRAK-M by acute alcohol leads to hyporesponsiveness of monocytes to LPS and reduced TNFa. In contrast, chronic alcohol sensitizes monocytes to LPS through decreased IRAK-M expression and results in increased TNFa production. Our data indicate that IRAK-M plays a central role in the differential regulation of LPS signaling by different lengths of alcohol exposure in monocytes.
A significant body of evidence indicates that gut barrier dysfunction and endotoxemia play an important role in the pathogenesis of alcoholic liver disease. Evidence also indicate that ethanol metabolism into acetaldehyde is required for the disruption of intestinal epithelial barrier. Our recent studies showed that acute administration of ethanol into isolated intestinal loops disrupts tight junctions and increases paracellular permeability in colon, but not in ileum. In Caco-2 cell monolayers, ethanol up to 1.0% failed to affect the tight junctions and paracellular permeability. However, ethanol dose-dependently potentiated the effect of acetaldehyde on the disruption of tight junctions. Inhibition of Src kinase and myosin light chain kinase (MLCK) attenuates the ethanol-mediated sensitization of acetaldehyde-induced tight junction disruption. Disruption of tight junctions by acetaldehyde is associated with a Thrdephosphorylation of occludin and Claudin-4 (tight junction proteins) by a protein phosphatase 2A (PP2A)-dependent mechanism. Our recent study showed that phosphorylation of occludin on Thr-403 and Thr-404 facilitates its assembly into tight junctions. The expression of T403/404D mutant occludin (mimics phosphorylated occludin) significantly attenuates acetaldehyde-induced disruption of tight junctions and barrier dysfunction. In summary, ethanol disrupts the barrier function predominantly in the distal colon in mice. Ethanol and acetaldehyde synergistically disrupts tight junctions. Finally, acetaldehyde-induced disruption of tight junctions involves dephosphorylation of occludin on Thr-403 and Thr-404 by a PP2A-dependent mechanism.
14 Fetal exposure to ethanol has long-term effects on influenza viral immunity and disease severity Jodi McGill1,2, David Meyerholz1, Betty Young1, Ruth A. Coleman1, Annette Schlueter1,2, Thomas J. Waldschmidt1,2, Robert T. Cook1 and Kevin L. Legge1,2, 1 Department of Pathology, University of Iowa, Iowa City, IA, USA, 2 Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City, IA, USA. Alcohol consumption during pregnancy is known to result in well-described cognitive impairment and physical malformations. Additionally, there are data demonstrating that in-utero ethanol exposure results in increased rates of infections in both newborns and young adults. The latter findings suggest fetal alcohol exposure may induce immune lesions that persist for extended periods. In order to determine if fetal alcohol exposure induces long-term immune deficiency, we infected mice that were exposed to ethanol from conception to weaning (fetal alcohol exposed; FAE) and then allowed to mature to adulthood without any additional ethanol intake with influenza A virus (IAV). Our results demonstrate that 14-16 wk old FAE mice exhibit significantly increased IAV associated morbidity and mortality as well as increased and sustained pulmonary viral titers following IAV infection. Additionally these FAE mice mount significantly impaired adaptive immune responses, including decreased numbers of IAV-specific CD8 T cells in the lungs, decreased size and frequency of B cell foci in the lungs, and reduced production of IAV-specific antibody following virus infection. These changes in immunity and disease susceptibility appear long-lived as FAE mice that were exposed to ethanol 6 or 9.5 months previously exhibit similar increases in IAV disease severity as well as reduced levels of IAV-specific antibody and CD8 T cells (6 months of age). These changes are observed on multiple genetic backgrounds and after infection with 2 distinct strains of IAV. Since a proportion of fetal alcohol-exposed individuals go on to abuse alcohol as adults, we further examined FAE mice consuming ethanol as adults. These studies demonstrate that secondary exposure to alcohol as an adult significantly exacerbates both IAV disease severity as well as the reduction in IAV-specific B and T cell immune responses. All together our studies suggest that immune deficiency resulting from fetal alcohol exposure may be long-lived even in the absence of further ethanol consumption, be independent of the host and viral background, and may contribute to the increased disease severity and death observed during IAV infections. Further our results importantly suggest that fetal alcohol exposed individuals could be at increased risk for severe and fatal infections with both seasonal and pandemic IAV. (Supported by NIH AA014405, AA019437, and AA019438).
16 Effects of acute alcohol exposure combined with burn injury on immune cell population in various lymphoid organs Juan Rendon, Xiaoling Li, Shegufta Mahbub, Aleah Brubaker, Elizabeth J. Kovacs and Mashkoor A. Choudhry, Alcohol Research Program, Burn & Shock Trauma Institute, 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 have a measurable ethanol blood level at the time of hospital admission. These patients are at an increased risk of infection and higher mortality when compared to burn patients without alcohol exposure. Our study uses an established mouse model to assess the effects of alcohol and burn injury on immune cell numbers in various lymphoid organs. Preliminary data do not show significant changes in macrophage, dendritic cell or T cell numbers, or expression of MHC class II molecules on macrophages 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 increase in the number of macrophages on day one post alcohol and burn injury as compared to control groups. This increase is accompanied by a 50% decrease in the expression of MHC class II within macrophage populations. While the number of dendritic cells appears to be unaffected within mesenteric lymph nodes, there is a slight increase in their number within Peyer’s patches on day one post alcohol and burn injury. This increase is not accompanied by a decrease in MHC class II expression as was noted with macrophages in the mesenteric lymph nodes. T cell numbers remain unaffected in both mesenteric lymph nodes and Peyer’s patches. These results are based on a 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 injury there are significant changes to antigen presenting cells within intestinal lymphoid organs that are not noted systemically, in the spleen. Therefore, possible early modulation of these changes within the intestinal lymphoid organs may preserve intestinal immunity, barrier function and prevent systemic effects; thus, reducing morbidity and mortality currently seen in patients suffering from burn injuries after acute alcohol exposure (Supported by NIH R01AA015731 (MAC), NIH R01 AA012034 (EJK), R01 AG018859 (EJK), and the Dr. Ralph and Marian C. Falk Medical Research Trust).
669
13
15
IRAK-M plays a central role in differential regulation of acute and chronic alcohol exposure on LPS-induced inflammation in human monocytes Pranoti Mandrekar, Shashi Bala, Donna Catalano, Karen Kodys and Gyongyi Szabo, Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA.
Role of occludin dephosphorylation in ethanol-induced gut barrier disruption R.K. Rao, Department of Physiology & Biophysics, University of Tennessee Health Science Center, Memphis, TN, USA.
Impaired host defense is linked to alcohol abuse due to alterations in inflammatory mediator production. We hypothesized that in human monocytes, acute alcohol induces hyporesponsiveness to LPS leading to decreased production of the proinflammatory cytokine, TNFa whereas chronic alcohol increases TNFa by sensitization to LPS. Human adherence-isolated blood monocytes were exposed to 25 mM alcohol for 7 days (chronic) and then treated with LPS (100ng/ml) for 18 hrs. For acute or short-term treatment, cells were exposed to LPS and 25 mM alcohol for 18 hrs. TNFa levels were analyzed by ELISA and IRAK-M mRNA and protein determined by real-time PCR and western blotting respectively. Our results show that acute alcohol decreases LPS-induced TNFa production (p ! 0.001) compared to LPS treatment alone. Concomitantly, LPS-induced IRAK-M, a negative regulator of inflammation, is increased after acute alcohol exposure. In contrast, chronic alcohol increases LPS-induced TNFa (p ! 0.001) with simultaneously decreased IRAK-M expression in these cells. Signaling molecules downstream to TLR4/ IRAK-M were also investigated to determine their regulation by acute and chronic alcohol exposure in monocytes. Acute alcohol decreases IRAK-1 and IKK activity, NFkB DNA binding and NFkB-driven reporter activity after LPS stimulation but chronic alcohol increases IRAK-1 and IKK kinase activities, NFkB DNA binding and NFkB-reporter activity. Inhibition of IRAK-M in acute-alcohol exposed monocytes using siRNA restores the LPS-induced TNFa whereas over-expression of IRAK-M in chronic alcohol macrophages prevents the increase in TNFa production. Addition of inhibitors of alcohol metabolism did not alter LPS signaling and TNFa production during chronic alcohol exposure. In summary, increased IRAK-M by acute alcohol leads to hyporesponsiveness of monocytes to LPS and reduced TNFa. In contrast, chronic alcohol sensitizes monocytes to LPS through decreased IRAK-M expression and results in increased TNFa production. Our data indicate that IRAK-M plays a central role in the differential regulation of LPS signaling by different lengths of alcohol exposure in monocytes.
A significant body of evidence indicates that gut barrier dysfunction and endotoxemia play an important role in the pathogenesis of alcoholic liver disease. Evidence also indicate that ethanol metabolism into acetaldehyde is required for the disruption of intestinal epithelial barrier. Our recent studies showed that acute administration of ethanol into isolated intestinal loops disrupts tight junctions and increases paracellular permeability in colon, but not in ileum. In Caco-2 cell monolayers, ethanol up to 1.0% failed to affect the tight junctions and paracellular permeability. However, ethanol dose-dependently potentiated the effect of acetaldehyde on the disruption of tight junctions. Inhibition of Src kinase and myosin light chain kinase (MLCK) attenuates the ethanol-mediated sensitization of acetaldehyde-induced tight junction disruption. Disruption of tight junctions by acetaldehyde is associated with a Thrdephosphorylation of occludin and Claudin-4 (tight junction proteins) by a protein phosphatase 2A (PP2A)-dependent mechanism. Our recent study showed that phosphorylation of occludin on Thr-403 and Thr-404 facilitates its assembly into tight junctions. The expression of T403/404D mutant occludin (mimics phosphorylated occludin) significantly attenuates acetaldehyde-induced disruption of tight junctions and barrier dysfunction. In summary, ethanol disrupts the barrier function predominantly in the distal colon in mice. Ethanol and acetaldehyde synergistically disrupts tight junctions. Finally, acetaldehyde-induced disruption of tight junctions involves dephosphorylation of occludin on Thr-403 and Thr-404 by a PP2A-dependent mechanism.
14 Fetal exposure to ethanol has long-term effects on influenza viral immunity and disease severity Jodi McGill1,2, David Meyerholz1, Betty Young1, Ruth A. Coleman1, Annette Schlueter1,2, Thomas J. Waldschmidt1,2, Robert T. Cook1 and Kevin L. Legge1,2, 1 Department of Pathology, University of Iowa, Iowa City, IA, USA, 2 Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City, IA, USA. Alcohol consumption during pregnancy is known to result in well-described cognitive impairment and physical malformations. Additionally, there are data demonstrating that in-utero ethanol exposure results in increased rates of infections in both newborns and young adults. The latter findings suggest fetal alcohol exposure may induce immune lesions that persist for extended periods. In order to determine if fetal alcohol exposure induces long-term immune deficiency, we infected mice that were exposed to ethanol from conception to weaning (fetal alcohol exposed; FAE) and then allowed to mature to adulthood without any additional ethanol intake with influenza A virus (IAV). Our results demonstrate that 14-16 wk old FAE mice exhibit significantly increased IAV associated morbidity and mortality as well as increased and sustained pulmonary viral titers following IAV infection. Additionally these FAE mice mount significantly impaired adaptive immune responses, including decreased numbers of IAV-specific CD8 T cells in the lungs, decreased size and frequency of B cell foci in the lungs, and reduced production of IAV-specific antibody following virus infection. These changes in immunity and disease susceptibility appear long-lived as FAE mice that were exposed to ethanol 6 or 9.5 months previously exhibit similar increases in IAV disease severity as well as reduced levels of IAV-specific antibody and CD8 T cells (6 months of age). These changes are observed on multiple genetic backgrounds and after infection with 2 distinct strains of IAV. Since a proportion of fetal alcohol-exposed individuals go on to abuse alcohol as adults, we further examined FAE mice consuming ethanol as adults. These studies demonstrate that secondary exposure to alcohol as an adult significantly exacerbates both IAV disease severity as well as the reduction in IAV-specific B and T cell immune responses. All together our studies suggest that immune deficiency resulting from fetal alcohol exposure may be long-lived even in the absence of further ethanol consumption, be independent of the host and viral background, and may contribute to the increased disease severity and death observed during IAV infections. Further our results importantly suggest that fetal alcohol exposed individuals could be at increased risk for severe and fatal infections with both seasonal and pandemic IAV. (Supported by NIH AA014405, AA019437, and AA019438).
16 Effects of acute alcohol exposure combined with burn injury on immune cell population in various lymphoid organs Juan Rendon, Xiaoling Li, Shegufta Mahbub, Aleah Brubaker, Elizabeth J. Kovacs and Mashkoor A. Choudhry, Alcohol Research Program, Burn & Shock Trauma Institute, 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 have a measurable ethanol blood level at the time of hospital admission. These patients are at an increased risk of infection and higher mortality when compared to burn patients without alcohol exposure. Our study uses an established mouse model to assess the effects of alcohol and burn injury on immune cell numbers in various lymphoid organs. Preliminary data do not show significant changes in macrophage, dendritic cell or T cell numbers, or expression of MHC class II molecules on macrophages 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 increase in the number of macrophages on day one post alcohol and burn injury as compared to control groups. This increase is accompanied by a 50% decrease in the expression of MHC class II within macrophage populations. While the number of dendritic cells appears to be unaffected within mesenteric lymph nodes, there is a slight increase in their number within Peyer’s patches on day one post alcohol and burn injury. This increase is not accompanied by a decrease in MHC class II expression as was noted with macrophages in the mesenteric lymph nodes. T cell numbers remain unaffected in both mesenteric lymph nodes and Peyer’s patches. These results are based on a 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 injury there are significant changes to antigen presenting cells within intestinal lymphoid organs that are not noted systemically, in the spleen. Therefore, possible early modulation of these changes within the intestinal lymphoid organs may preserve intestinal immunity, barrier function and prevent systemic effects; thus, reducing morbidity and mortality currently seen in patients suffering from burn injuries after acute alcohol exposure (Supported by NIH R01AA015731 (MAC), NIH R01 AA012034 (EJK), R01 AG018859 (EJK), and the Dr. Ralph and Marian C. Falk Medical Research Trust).