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Chronic ethanol exposure selectively inhibits the influenza-specific CD8 T cell response during influenza A virus infection
Abstracts / Alcohol 47 (2013) 567–576
571
13. Chronic ethanol exposure selectively inhibits the influenza-specific CD8 T cell response during influenza A virus infection
15. Binge ethanol exposure alters CD206 expression on alveolar macrophages from burn injured mice
E.A. Hemann, J.L. McGill, T.J. Waldschmidt, K.L. Legge, Immunology Graduate Program and Department of Pathology, University of Iowa, Iowa City, IA, USA
J.A. Ippolito, B.J. Curtis, E.J. Kovacs, Alcohol Research Program, Burn & Shock Trauma Research Institute, Departments of Surgery and Microbiology & Immunology, Program in Integrative Cell Biology, Loyola University Chicago Health Sciences Campus, Maywood, IL, USA
Chronic ethanol exposure increases disease severity during influenza A virus (IAV) infection. Mice chronically consuming ethanol fail to mount an optimal IAV-specific CD8 T cell response within the lungs during IAV challenge. Further, these IAV-specific CD8 T cells produce significantly less IFNg compared to IAV-infected, water-consuming controls. Given the key role of IAV-specific CD8 T cells in clearance of IAV infection, we evaluated the extent of the lesion within the IAV-specific CD8 T cell response of mice chronically consuming ethanol. Our results demonstrate that while there is a defect in IFNg production by IAV-specific CD8 T cells in mice chronically consuming ethanol, the ability of these cells to produce both TNFa and IL-2 remains unaltered on day 8 post infection (p.i.). Further, although we detected no significant differences in number (or amount) of granzyme B-expressing pulmonary CD8 T cells between water and ethanol consuming mice, there was a significant reduction in degranulation. We also observed a reduction in the number of TRAIL- and FasL-expressing, IAV-specific CD8 T cells within the lungs of ethanol consuming mice compared to water controls. Together, these results suggest that the EtOH induced lesion in the IAV-specific CD8 T cell response extends beyond IFNg into other effector functions. Finally, in order to identify when this lesion in the IAV-specific CD8 T cell response first appears, we analyzed the IAV-specific T cell response on day 4 p.i. in the draining lymph nodes and found an early defect in proliferation and IFNg production in ethanol consuming mice. Together, these findings highlight the previously unrecognized depth of the lesion in the IAV-specific CD8 T cell response of mice chronically consuming ethanol. Given the important role CD8 T cell immunity plays in control of IAV, further examination of these defects may help fashion therapies to decrease the serious outcomes associated with IAV infection during chronic ethanol exposure (Supported by AA018671 [KLL], AA019438 [TJW], T32 AI007533 [EAH], and the University of Iowa Department of Pathology).
14. Macrophages – Matrix interactions in alcoholic liver injury and hepatic fibrogenesis I.N. Hines, G. Son, A.M. Norris, D.C. Gillis, E.J. Sanderlin, III, S.M. Moore, East Carolina University, Greenville, NC 27514, USA Chronic alcohol consumption is a known risk factor for the development of acute and chronic liver pathologies from hepatitis to fibrosis and cirrhosis. Macrophage production of tumor necrosis factor alpha (TNFa) and transforming growth factor beta (TGFb) are critical for the observed early tissue pathology and fibrosis induction. Intriguingly, significant heterogeneity exists among macrophage populations, particularly in their cytokine production profiles and matrix remodeling repertoire, key factors regulating the dynamic process of inflammation, fibrosis and resolution. Thus, an understanding of the mechanisms of macrophage recruitment and activation are critical to treat or prevent early as well as late stage alcoholic liver disease as well as to potentially resolve tissue damage. Integrins are an important component of cell adhesion, migration, and infiltration in normal and damaged tissues. The function of integrins and their signaling through integrin linked kinase (ILK) during the dynamic process of alcoholic liver disease have not been explored. Chronic exposure to ethanol enhances hepatic ILK activation and b integrin expression both in parenchymal and non-parenchymal cell populations. Selective deletion of ILK in macrophages (Mɸ-ILK mice) limits chronic ethanol-induced tissue injury in conjunction with reduced hepatic TNFa and interleukin 6 (IL6) production in the absence of significant changes in hepatic F4/80+ cell numbers. Similarly, TNFa production following in vitro stimulation of primary KCs with lipopolysaccharide (LPS) or in vivo administration of LPS is reduced in Mɸ-ILK mice. To define the role of ILK in macrophages during fibrogenesis, wild type or Mɸ-ILK mice administered CCl4 for 4 weeks to induce fibrosis. Loss of ILK in macrophages did not reduce the progression of hepatic fibrosis however Mɸ-ILK-deficient mice showed a significantly greater pace of resolution 5 and 7 days post-CCl4 exposure. This enhancement in resolution was correlated with a shifted, CD11b+ and Gr-1lo macrophage population and reduced tissue inhibitor of matrix metalloprotease expression. Taken together, these data demonstrate the importance of ILK in the regulation of macrophage function and differentiation within the alcohol exposed liver and highlight its contribution to fibrosis resolution. Therapies directed at inhibiting ILK function in macrophages may prove useful to treat multiple phases of alcoholic liver disease in humans.
Alcohol intoxication is involved in 50% of all burn injuries that require hospitalization, resulting in increased mortality and an increased risk of pulmonary infections, relative to burn injury alone. Our work has also demonstrated that in a mouse model of episodic binge ethanol exposure and burn injury there is significant, prolonged pulmonary inflammation, relative to either insult alone. The magnitude of pulmonary inflammation is mediated in part by alveolar macrophage (AM) function. The ability of these multifaceted cells to shift their phenotype from a pro-inflammatory, M1, macrophage, to an anti-inflammatory, M2, state is essential to resolving inflammation, yet an excess or shortage of either subset can result in adverse pulmonary complications. Here we sought to identify the effect of alcohol on AM expression of M2 marker, CD206. Mice were subjected to episodic binge alcohol exposure (3 days of ethanol, 4 days off, and 3 days of ethanol). Thirty minutes following the final ethanol exposure, mice were anesthetized and given a 15% total body surface area dorsal scald injury. At 24 and 72 h postinjury bronchoalveolar lavage cells were collected and AMs were identified by flow cytometry as CD45+Cd11c+CD11b . M2 phenotype was assessed using the mean fluorescent intensity (MFI) of CD206. At 24 h, there was no change in the MFI of CD206 on AMs. However, at 72 h AMs from burn injury alone had a 25% increase in CD206, relative to sham groups. Interestingly, combined injured mice exhibited a 23% increase relative to burn injury alone and an overall 42% increase, relative to sham groups (p < 0.05). These data indicate that alcohol exposure prior to burn injury does not alter the activation of M2 macrophages until 72 h after injury. This upregulation of CD206 at the latter time point suggests that the earlier pro-inflammatory response seen after combined injury may trigger a heightened anti-inflammatory response. The marked elevation in M2 macrophages may represent a possible mechanism behind the increased susceptibility to pulmonary infections seen in intoxicated burn patients (Supported by R01 AA012034 [EJK], T32 AA013527 [EJK], F31 AA022566 [JAI], F32 AA021636 [BJC], and Ralph and Marian C. Falk Medical Research Trust [EJK]). 16. Alcohol negatively regulates human TGF-b production and regulatory T cell induction in a human umbilical cord blood model K. Jaeger, E.J. Kovacs, M. Iwashima, Alcohol Research Program, Departments of Microbiology & Immunology and Surgery, Loyola University Chicago Health Sciences Campus, Maywood, IL 60153, USA To determine the effect of alcohol (EtOH) on human regulatory T cell (Treg) biology, we utilized our model of ex vivo human Treg induction from umbilical cord blood (UCB). While this approach does not exactly mimic the pathology of fetal alcohol syndrome (FAS), we believe this is a relevant model to study the neonatal immune system and better understand the increased risk of infection associated with intrauterine alcohol exposure. This research is essential as up to 30% of women report consuming alcohol at some time during their pregnancy and the prevalence of FAS in the United States remains between 0.2 and 1.5 in every 1000 live births. To determine the effect of EtOH on human Treg induction, human UCB culture was treated with increasing concentrations of EtOH and Treg induction was evaluated via flow cytometry at day 14. These data demonstrated a significant decrease in Treg frequency of total CD4+ T cells and absolute Treg cell number with exposure to 50 mM EtOH as compared to untreated UCB cultures. TGF-b inhibition assays have demonstrated that TGF-b plays an essential role in promoting human Treg induction, so we were interested in whether EtOH altered TGFb production by freshly isolated UCB mononuclear cells. To determine the influence of EtOH on TGF-b production, we cultured freshly isolated mononuclear cells from human UCB in the presence or absence of EtOH and performed a TGF-b bioassay on the culture supernatants after 24 h. EtOH exposure resulted in significantly decreased TGF-b production by UCB mononuclear cells6. These data suggest that not only does EtOH negatively regulate human Treg induction, but also that EtOH may exert its influence on human Tregs via the TGF-b pathway, which is known to play a critical role in human Treg differentiation as well as in the maintenance of maternal fetal tolerance. Human UCB is a physiological model of an in utero or neonatal environment and we believe that our data support that EtOH exposure via maternal consumption can have a significant effect on the immune system of a developing fetus. The significant decrease in both human Treg induction and TGF-b production from human UCB mononuclear cells after EtOH exposure incites a number of future directions. Additionally, understanding the effect of EtOH on the human immune system, particularly Treg biology, is critical to comprehending the intricate maintenance of maternal fetal tolerance and development of the fetal immune system (Supported by NIH T32 AA013527 [EJK] and F30 AA021323 [KEJ]).
571
13. Chronic ethanol exposure selectively inhibits the influenza-specific CD8 T cell response during influenza A virus infection
15. Binge ethanol exposure alters CD206 expression on alveolar macrophages from burn injured mice
E.A. Hemann, J.L. McGill, T.J. Waldschmidt, K.L. Legge, Immunology Graduate Program and Department of Pathology, University of Iowa, Iowa City, IA, USA
J.A. Ippolito, B.J. Curtis, E.J. Kovacs, Alcohol Research Program, Burn & Shock Trauma Research Institute, Departments of Surgery and Microbiology & Immunology, Program in Integrative Cell Biology, Loyola University Chicago Health Sciences Campus, Maywood, IL, USA
Chronic ethanol exposure increases disease severity during influenza A virus (IAV) infection. Mice chronically consuming ethanol fail to mount an optimal IAV-specific CD8 T cell response within the lungs during IAV challenge. Further, these IAV-specific CD8 T cells produce significantly less IFNg compared to IAV-infected, water-consuming controls. Given the key role of IAV-specific CD8 T cells in clearance of IAV infection, we evaluated the extent of the lesion within the IAV-specific CD8 T cell response of mice chronically consuming ethanol. Our results demonstrate that while there is a defect in IFNg production by IAV-specific CD8 T cells in mice chronically consuming ethanol, the ability of these cells to produce both TNFa and IL-2 remains unaltered on day 8 post infection (p.i.). Further, although we detected no significant differences in number (or amount) of granzyme B-expressing pulmonary CD8 T cells between water and ethanol consuming mice, there was a significant reduction in degranulation. We also observed a reduction in the number of TRAIL- and FasL-expressing, IAV-specific CD8 T cells within the lungs of ethanol consuming mice compared to water controls. Together, these results suggest that the EtOH induced lesion in the IAV-specific CD8 T cell response extends beyond IFNg into other effector functions. Finally, in order to identify when this lesion in the IAV-specific CD8 T cell response first appears, we analyzed the IAV-specific T cell response on day 4 p.i. in the draining lymph nodes and found an early defect in proliferation and IFNg production in ethanol consuming mice. Together, these findings highlight the previously unrecognized depth of the lesion in the IAV-specific CD8 T cell response of mice chronically consuming ethanol. Given the important role CD8 T cell immunity plays in control of IAV, further examination of these defects may help fashion therapies to decrease the serious outcomes associated with IAV infection during chronic ethanol exposure (Supported by AA018671 [KLL], AA019438 [TJW], T32 AI007533 [EAH], and the University of Iowa Department of Pathology).
14. Macrophages – Matrix interactions in alcoholic liver injury and hepatic fibrogenesis I.N. Hines, G. Son, A.M. Norris, D.C. Gillis, E.J. Sanderlin, III, S.M. Moore, East Carolina University, Greenville, NC 27514, USA Chronic alcohol consumption is a known risk factor for the development of acute and chronic liver pathologies from hepatitis to fibrosis and cirrhosis. Macrophage production of tumor necrosis factor alpha (TNFa) and transforming growth factor beta (TGFb) are critical for the observed early tissue pathology and fibrosis induction. Intriguingly, significant heterogeneity exists among macrophage populations, particularly in their cytokine production profiles and matrix remodeling repertoire, key factors regulating the dynamic process of inflammation, fibrosis and resolution. Thus, an understanding of the mechanisms of macrophage recruitment and activation are critical to treat or prevent early as well as late stage alcoholic liver disease as well as to potentially resolve tissue damage. Integrins are an important component of cell adhesion, migration, and infiltration in normal and damaged tissues. The function of integrins and their signaling through integrin linked kinase (ILK) during the dynamic process of alcoholic liver disease have not been explored. Chronic exposure to ethanol enhances hepatic ILK activation and b integrin expression both in parenchymal and non-parenchymal cell populations. Selective deletion of ILK in macrophages (Mɸ-ILK mice) limits chronic ethanol-induced tissue injury in conjunction with reduced hepatic TNFa and interleukin 6 (IL6) production in the absence of significant changes in hepatic F4/80+ cell numbers. Similarly, TNFa production following in vitro stimulation of primary KCs with lipopolysaccharide (LPS) or in vivo administration of LPS is reduced in Mɸ-ILK mice. To define the role of ILK in macrophages during fibrogenesis, wild type or Mɸ-ILK mice administered CCl4 for 4 weeks to induce fibrosis. Loss of ILK in macrophages did not reduce the progression of hepatic fibrosis however Mɸ-ILK-deficient mice showed a significantly greater pace of resolution 5 and 7 days post-CCl4 exposure. This enhancement in resolution was correlated with a shifted, CD11b+ and Gr-1lo macrophage population and reduced tissue inhibitor of matrix metalloprotease expression. Taken together, these data demonstrate the importance of ILK in the regulation of macrophage function and differentiation within the alcohol exposed liver and highlight its contribution to fibrosis resolution. Therapies directed at inhibiting ILK function in macrophages may prove useful to treat multiple phases of alcoholic liver disease in humans.
Alcohol intoxication is involved in 50% of all burn injuries that require hospitalization, resulting in increased mortality and an increased risk of pulmonary infections, relative to burn injury alone. Our work has also demonstrated that in a mouse model of episodic binge ethanol exposure and burn injury there is significant, prolonged pulmonary inflammation, relative to either insult alone. The magnitude of pulmonary inflammation is mediated in part by alveolar macrophage (AM) function. The ability of these multifaceted cells to shift their phenotype from a pro-inflammatory, M1, macrophage, to an anti-inflammatory, M2, state is essential to resolving inflammation, yet an excess or shortage of either subset can result in adverse pulmonary complications. Here we sought to identify the effect of alcohol on AM expression of M2 marker, CD206. Mice were subjected to episodic binge alcohol exposure (3 days of ethanol, 4 days off, and 3 days of ethanol). Thirty minutes following the final ethanol exposure, mice were anesthetized and given a 15% total body surface area dorsal scald injury. At 24 and 72 h postinjury bronchoalveolar lavage cells were collected and AMs were identified by flow cytometry as CD45+Cd11c+CD11b . M2 phenotype was assessed using the mean fluorescent intensity (MFI) of CD206. At 24 h, there was no change in the MFI of CD206 on AMs. However, at 72 h AMs from burn injury alone had a 25% increase in CD206, relative to sham groups. Interestingly, combined injured mice exhibited a 23% increase relative to burn injury alone and an overall 42% increase, relative to sham groups (p < 0.05). These data indicate that alcohol exposure prior to burn injury does not alter the activation of M2 macrophages until 72 h after injury. This upregulation of CD206 at the latter time point suggests that the earlier pro-inflammatory response seen after combined injury may trigger a heightened anti-inflammatory response. The marked elevation in M2 macrophages may represent a possible mechanism behind the increased susceptibility to pulmonary infections seen in intoxicated burn patients (Supported by R01 AA012034 [EJK], T32 AA013527 [EJK], F31 AA022566 [JAI], F32 AA021636 [BJC], and Ralph and Marian C. Falk Medical Research Trust [EJK]). 16. Alcohol negatively regulates human TGF-b production and regulatory T cell induction in a human umbilical cord blood model K. Jaeger, E.J. Kovacs, M. Iwashima, Alcohol Research Program, Departments of Microbiology & Immunology and Surgery, Loyola University Chicago Health Sciences Campus, Maywood, IL 60153, USA To determine the effect of alcohol (EtOH) on human regulatory T cell (Treg) biology, we utilized our model of ex vivo human Treg induction from umbilical cord blood (UCB). While this approach does not exactly mimic the pathology of fetal alcohol syndrome (FAS), we believe this is a relevant model to study the neonatal immune system and better understand the increased risk of infection associated with intrauterine alcohol exposure. This research is essential as up to 30% of women report consuming alcohol at some time during their pregnancy and the prevalence of FAS in the United States remains between 0.2 and 1.5 in every 1000 live births. To determine the effect of EtOH on human Treg induction, human UCB culture was treated with increasing concentrations of EtOH and Treg induction was evaluated via flow cytometry at day 14. These data demonstrated a significant decrease in Treg frequency of total CD4+ T cells and absolute Treg cell number with exposure to 50 mM EtOH as compared to untreated UCB cultures. TGF-b inhibition assays have demonstrated that TGF-b plays an essential role in promoting human Treg induction, so we were interested in whether EtOH altered TGFb production by freshly isolated UCB mononuclear cells. To determine the influence of EtOH on TGF-b production, we cultured freshly isolated mononuclear cells from human UCB in the presence or absence of EtOH and performed a TGF-b bioassay on the culture supernatants after 24 h. EtOH exposure resulted in significantly decreased TGF-b production by UCB mononuclear cells6. These data suggest that not only does EtOH negatively regulate human Treg induction, but also that EtOH may exert its influence on human Tregs via the TGF-b pathway, which is known to play a critical role in human Treg differentiation as well as in the maintenance of maternal fetal tolerance. Human UCB is a physiological model of an in utero or neonatal environment and we believe that our data support that EtOH exposure via maternal consumption can have a significant effect on the immune system of a developing fetus. The significant decrease in both human Treg induction and TGF-b production from human UCB mononuclear cells after EtOH exposure incites a number of future directions. Additionally, understanding the effect of EtOH on the human immune system, particularly Treg biology, is critical to comprehending the intricate maintenance of maternal fetal tolerance and development of the fetal immune system (Supported by NIH T32 AA013527 [EJK] and F30 AA021323 [KEJ]).