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Natural killer T cells play an important role in chronic plus binge ethanol-induced liver injury
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Abstracts / Alcohol 47 (2013) 567–576
17. Toll-like receptor 2 modulates the expression of T cell transcription factors following alcohol and burn injury
19. Natural killer T cells play an important role in chronic plus binge ethanol-induced liver injury
X. Li, J.L. Rendon, M.A. Choudhry, Alcohol Research Program, Burn & Shock Trauma Research Institute, Department of Surgery, Loyola University Chicago Health Sciences Division, Maywood, IL 60153, USA
S.A. Mathews, D. Feng, B. Gao, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD 20892, USA
Previous studies from our laboratory have shown that acute alcohol (ethanol) intoxication combined with burn injury suppresses T cell proliferation, IL-2 and IFN-g production. We further found that T cell stimulation with Toll-like receptor 2 (TLR2) agonist (HKLM) prevents the decrease in T cell IFN-g release following ethanol and burn injury. This study examined the effect of ethanol and burn injury on T cell transcription factors (e.g., NFAT, Tbx21, Jun and Fos) and determined whether TLR2 agonist modulates the expression of these factors following ethanol and burn injury. To test this, male C57/BL6 mice (w25 g) were gavaged with ethanol (w2.9 mg/kg) to achieve a blood ethanol level of w100 mg/dl prior to receiving w12.5% total body surface area sham or burn injury. One day after injury, mice were sacrificed and splenic T cells were isolated. T cells were cultured with platebound anti-CD3 antibody in the presence or absence of TLR2 agonist (HKLM 108 cells/ml) for 48 h. Supernatants were collected for the measurement of IFN-g by ELISA and cells were lysed. The cells lysates were analyzed for NFAT, Tbx21, Jun and Fos mRNA expression by RT-PCR. As reported earlier, there was a significant decrease in IFN-g release following ethanol and burn injury compared to shams. T cells stimulated with anti-CD3 combined with TLR2 agonist did not show a decrease in IFN-g release following ethanol and burn injury. This was accompanied with a significant decrease in NFAT, Tbx21, Jun and Fos expression in T cells stimulated with anti-CD3 following ethanol and burn injury. However T cells stimulated with anti-CD3 combined with TLR2 agonist did not exhibit a decrease in expression of these transcription factors. Together, these findings suggest that TLR2 stimulation of T cells normalizes IFN-g by modulating NFAT, Tbx21, Jun and Fos expression following ethanol intoxication combined with burn injury (Supported by NIH R01 AA015731 [MAC], F30 AA020167 [JR] and the Dr. Ralph and Marian C. Falk Medical Research Trust).
Despite years of ongoing research, the molecular mechanisms contributing to progression of alcoholic liver disease (ALD) are only partially understood. The liver is enriched in natural killer T (NKT) cells that play an important role in host defense against hepatic viral infection and tumor transformation; however, the role of NKT cells in pathogenesis of ALD remains unknown. We used a mouse model of chronic plus binge ethanol (EtOH) feeding to determine how NKT cells affect EtOH-induced liver injury and inflammation. NKT deficient (Jalpha18 KO or CD1d KO) mice and their wild-type controls were fed a 5% EtOH liquid diet for 10 days, followed by single gavage of EtOH (5 g/kg). Sera and liver tissue were collected 9 h postgavage and analyzed for markers of liver injury. Neutrophils and lymphocytes were isolated 3 h post-gavage and subjected to flow cytometry. Histological and Oil Red O staining revealed that EtOH feeding-induced hepatic steatosis was lower in Jalpha18 KO and CD1d KO mice compared to WT mice; which correlated with a lower liver/body weight ratio and less hepatic triglyceride in EtOH-fed NKT deficient mice. Sera from EtOH-fed NKT deficient mice had significantly less ALT compared to WT mice. Immunological staining for cytochrome P450 2E1 was comparable in all EtOH-fed mice, suggesting that the resistance of NKT-deficient mice to ALD was not due to changes in EtOH metabolism. Importantly, flow cytometric analyses revealed an increased number of activated NKT cells in the livers of WT mice after chronic plus binge EtOH feeding, which correlated to increased neutrophil accumulation, thus supporting a role for NKT cells in alcohol-induced liver injury. Real-time PCR analysis showed that induction of the pro-inflammatory cytokines TNFalpha and IL-1beta was significantly blunted in EtOH-fed NKT deficient mice compared to that in WT. Finally, hepatic expression of the inflammationassociated genes for e-selectin and MCP-1 were drastically reduced in EtOHfed NKT deficient mice compared to WT. Taken together, our data suggest NKT cells play a critical role in the development of early alcoholic liver injury, neutrophil recruitment, and inflammation. Future studies will be aimed at elucidating the mechanism(s) by which ethanol activates NKT cells and further investigating how activated NKT cells modify the critical inflammatory pathways involved in progression of ALD.
18. Chronic ethanol consumption impacts post-AMI cardiac function and modulates gene expression in cardiac cell types through alteration of histone 3 lysine 79 methylation A.R. Mackie, E.E. Vaughan, S. Verma, P. Krishnamurthy, V. Ramirez, A. Ito, T. Abramova, S. Misener, R. Kishore, Feinberg Cardiovascular Research Institute, Northwestern University, Chicago, IL, USA The beliefs surrounding the cardiac effects of chronic ethanol (EtOH) consumption in humans are known to be dictated by the frequency of ingestion. Studies have established that moderate consumption (i.e., 1–2 drinks/day) imparts a cardiac benefit to patients by reducing the frequency of adverse cardiovascular events (ACE). EtOH consumption beyond moderate levels (i.e., >2 drinks per day) is associated with a significant increase in ACEs. Despite this knowledge, little is known regarding the functional impact of chronic EtOH consumption on post-myocardial infarct repair or the cellular mechanisms involved in this process. Therefore, we investigated the post-AMI functional consequences of chronic ethanol consumption in mice. Mice received chronic ethanol via the Lieber-DeCarli paradigm and each group of mice received either: 0%, 1% (moderate) or 5% (high) ethanol v/v in an isocaloric fashion for 8 weeks. After 8 weeks, mice then underwent a 60 min ischemic/reperfusion injury and the subsequent assessment of their cardiac function at 1, 2 and 4 weeks after AMI. As early as two weeks postAMI, mice fed the 1% EtOH displayed significant improvements in ejection fraction, systolic ventricular volumes and infarct size as compared to control mice. Conversely, mice that consumed the 5% EtOH diet displayed diminished ejection fraction and increased systolic chamber volume and infarct size. To investigate the mechanistic basis behind these changes, isolated murine cardiac fibroblasts (CFBs) were exposed to EtOH using a “chronic” 5 day treatment paradigm in which media EtOH was replaced daily. CFB lysates were then harvested and several epigenetic histone marks were assessed via western blotting. Specifically, methylation of histone 3 lysine 79 (H3K79) was significantly increased at the moderate dose (0.1% v/v) and severely diminished at the high dose (0.5% v/v). H3K79 is exclusively methylate by the methyl-transferase Dot1, suggesting that chronic ethanol may have biphasic dose effects on the regulation of genes influenced by H3K79 methylation. This data represents the first indication that chronic EtOH may affect cells of the post-ischemic heart by influencing their gene expression profiles through manipulation of their epigenetic fingerprint.
20. Moderate alcohol consumption enhances vaccine-induced responses in rhesus macaques I. Messaoudi, University of California, Riverside, Riverside, CA 92521, USA We have recently shown that chronic alcohol consumption in a rhesus macaque model of ethanol self-administration significantly modulates the serum cytokine profile. In this study, we extended these observations by investigating the impact of chronic ethanol exposure on the immune response to modified vaccinia Ankara (MVA). All animals were vaccinated with MVA before ethanol exposure and then again after 7 months of 22 h/day of “open-access” drinking of 4% (w/v) ethanol. Our results indicate that animals whose blood ethanol concentration (BEC) chronically exceeded 80 mg/dl had lower CD4 and CD8 T cell proliferation as well as IgG responses following MVA booster than control animals. In contrast, relatively moderate drinkers whose BEC remained below 80 mg/ml exhibited more robust MVAspecific IgG and CD8 T cell responses than controls. To begin to uncover mechanisms underlying the differences in MVA-specific responses between the three groups, we analyzed plasma cytokine levels and microRNA expression in peripheral blood mononuclear cells following MVA booster. Our findings suggest that moderate ethanol consumption results in higher levels of antiviral cytokines and an expression profile of microRNAs linked to CD8 T cell differentiation. In summary, moderate alcohol consumption enhances recall vaccine responses, whereas chronic alcohol intoxication suppresses this response.
Abstracts / Alcohol 47 (2013) 567–576
17. Toll-like receptor 2 modulates the expression of T cell transcription factors following alcohol and burn injury
19. Natural killer T cells play an important role in chronic plus binge ethanol-induced liver injury
X. Li, J.L. Rendon, M.A. Choudhry, Alcohol Research Program, Burn & Shock Trauma Research Institute, Department of Surgery, Loyola University Chicago Health Sciences Division, Maywood, IL 60153, USA
S.A. Mathews, D. Feng, B. Gao, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD 20892, USA
Previous studies from our laboratory have shown that acute alcohol (ethanol) intoxication combined with burn injury suppresses T cell proliferation, IL-2 and IFN-g production. We further found that T cell stimulation with Toll-like receptor 2 (TLR2) agonist (HKLM) prevents the decrease in T cell IFN-g release following ethanol and burn injury. This study examined the effect of ethanol and burn injury on T cell transcription factors (e.g., NFAT, Tbx21, Jun and Fos) and determined whether TLR2 agonist modulates the expression of these factors following ethanol and burn injury. To test this, male C57/BL6 mice (w25 g) were gavaged with ethanol (w2.9 mg/kg) to achieve a blood ethanol level of w100 mg/dl prior to receiving w12.5% total body surface area sham or burn injury. One day after injury, mice were sacrificed and splenic T cells were isolated. T cells were cultured with platebound anti-CD3 antibody in the presence or absence of TLR2 agonist (HKLM 108 cells/ml) for 48 h. Supernatants were collected for the measurement of IFN-g by ELISA and cells were lysed. The cells lysates were analyzed for NFAT, Tbx21, Jun and Fos mRNA expression by RT-PCR. As reported earlier, there was a significant decrease in IFN-g release following ethanol and burn injury compared to shams. T cells stimulated with anti-CD3 combined with TLR2 agonist did not show a decrease in IFN-g release following ethanol and burn injury. This was accompanied with a significant decrease in NFAT, Tbx21, Jun and Fos expression in T cells stimulated with anti-CD3 following ethanol and burn injury. However T cells stimulated with anti-CD3 combined with TLR2 agonist did not exhibit a decrease in expression of these transcription factors. Together, these findings suggest that TLR2 stimulation of T cells normalizes IFN-g by modulating NFAT, Tbx21, Jun and Fos expression following ethanol intoxication combined with burn injury (Supported by NIH R01 AA015731 [MAC], F30 AA020167 [JR] and the Dr. Ralph and Marian C. Falk Medical Research Trust).
Despite years of ongoing research, the molecular mechanisms contributing to progression of alcoholic liver disease (ALD) are only partially understood. The liver is enriched in natural killer T (NKT) cells that play an important role in host defense against hepatic viral infection and tumor transformation; however, the role of NKT cells in pathogenesis of ALD remains unknown. We used a mouse model of chronic plus binge ethanol (EtOH) feeding to determine how NKT cells affect EtOH-induced liver injury and inflammation. NKT deficient (Jalpha18 KO or CD1d KO) mice and their wild-type controls were fed a 5% EtOH liquid diet for 10 days, followed by single gavage of EtOH (5 g/kg). Sera and liver tissue were collected 9 h postgavage and analyzed for markers of liver injury. Neutrophils and lymphocytes were isolated 3 h post-gavage and subjected to flow cytometry. Histological and Oil Red O staining revealed that EtOH feeding-induced hepatic steatosis was lower in Jalpha18 KO and CD1d KO mice compared to WT mice; which correlated with a lower liver/body weight ratio and less hepatic triglyceride in EtOH-fed NKT deficient mice. Sera from EtOH-fed NKT deficient mice had significantly less ALT compared to WT mice. Immunological staining for cytochrome P450 2E1 was comparable in all EtOH-fed mice, suggesting that the resistance of NKT-deficient mice to ALD was not due to changes in EtOH metabolism. Importantly, flow cytometric analyses revealed an increased number of activated NKT cells in the livers of WT mice after chronic plus binge EtOH feeding, which correlated to increased neutrophil accumulation, thus supporting a role for NKT cells in alcohol-induced liver injury. Real-time PCR analysis showed that induction of the pro-inflammatory cytokines TNFalpha and IL-1beta was significantly blunted in EtOH-fed NKT deficient mice compared to that in WT. Finally, hepatic expression of the inflammationassociated genes for e-selectin and MCP-1 were drastically reduced in EtOHfed NKT deficient mice compared to WT. Taken together, our data suggest NKT cells play a critical role in the development of early alcoholic liver injury, neutrophil recruitment, and inflammation. Future studies will be aimed at elucidating the mechanism(s) by which ethanol activates NKT cells and further investigating how activated NKT cells modify the critical inflammatory pathways involved in progression of ALD.
18. Chronic ethanol consumption impacts post-AMI cardiac function and modulates gene expression in cardiac cell types through alteration of histone 3 lysine 79 methylation A.R. Mackie, E.E. Vaughan, S. Verma, P. Krishnamurthy, V. Ramirez, A. Ito, T. Abramova, S. Misener, R. Kishore, Feinberg Cardiovascular Research Institute, Northwestern University, Chicago, IL, USA The beliefs surrounding the cardiac effects of chronic ethanol (EtOH) consumption in humans are known to be dictated by the frequency of ingestion. Studies have established that moderate consumption (i.e., 1–2 drinks/day) imparts a cardiac benefit to patients by reducing the frequency of adverse cardiovascular events (ACE). EtOH consumption beyond moderate levels (i.e., >2 drinks per day) is associated with a significant increase in ACEs. Despite this knowledge, little is known regarding the functional impact of chronic EtOH consumption on post-myocardial infarct repair or the cellular mechanisms involved in this process. Therefore, we investigated the post-AMI functional consequences of chronic ethanol consumption in mice. Mice received chronic ethanol via the Lieber-DeCarli paradigm and each group of mice received either: 0%, 1% (moderate) or 5% (high) ethanol v/v in an isocaloric fashion for 8 weeks. After 8 weeks, mice then underwent a 60 min ischemic/reperfusion injury and the subsequent assessment of their cardiac function at 1, 2 and 4 weeks after AMI. As early as two weeks postAMI, mice fed the 1% EtOH displayed significant improvements in ejection fraction, systolic ventricular volumes and infarct size as compared to control mice. Conversely, mice that consumed the 5% EtOH diet displayed diminished ejection fraction and increased systolic chamber volume and infarct size. To investigate the mechanistic basis behind these changes, isolated murine cardiac fibroblasts (CFBs) were exposed to EtOH using a “chronic” 5 day treatment paradigm in which media EtOH was replaced daily. CFB lysates were then harvested and several epigenetic histone marks were assessed via western blotting. Specifically, methylation of histone 3 lysine 79 (H3K79) was significantly increased at the moderate dose (0.1% v/v) and severely diminished at the high dose (0.5% v/v). H3K79 is exclusively methylate by the methyl-transferase Dot1, suggesting that chronic ethanol may have biphasic dose effects on the regulation of genes influenced by H3K79 methylation. This data represents the first indication that chronic EtOH may affect cells of the post-ischemic heart by influencing their gene expression profiles through manipulation of their epigenetic fingerprint.
20. Moderate alcohol consumption enhances vaccine-induced responses in rhesus macaques I. Messaoudi, University of California, Riverside, Riverside, CA 92521, USA We have recently shown that chronic alcohol consumption in a rhesus macaque model of ethanol self-administration significantly modulates the serum cytokine profile. In this study, we extended these observations by investigating the impact of chronic ethanol exposure on the immune response to modified vaccinia Ankara (MVA). All animals were vaccinated with MVA before ethanol exposure and then again after 7 months of 22 h/day of “open-access” drinking of 4% (w/v) ethanol. Our results indicate that animals whose blood ethanol concentration (BEC) chronically exceeded 80 mg/dl had lower CD4 and CD8 T cell proliferation as well as IgG responses following MVA booster than control animals. In contrast, relatively moderate drinkers whose BEC remained below 80 mg/ml exhibited more robust MVAspecific IgG and CD8 T cell responses than controls. To begin to uncover mechanisms underlying the differences in MVA-specific responses between the three groups, we analyzed plasma cytokine levels and microRNA expression in peripheral blood mononuclear cells following MVA booster. Our findings suggest that moderate ethanol consumption results in higher levels of antiviral cytokines and an expression profile of microRNAs linked to CD8 T cell differentiation. In summary, moderate alcohol consumption enhances recall vaccine responses, whereas chronic alcohol intoxication suppresses this response.