ETHANOL SUPPRESSES THE UBIQUITIN-PROTEASOME PATHWAY OF ALVEOLAR MACROHPAGES.
Document Type
Presentation
Start Date
22-10-2010 10:45 AM
End Date
22-10-2010 12:00 PM
Description
Alcohol exposure predisposes the drinking host to infection, particularly bacterial pneumonia caused by Klebsiella pneumoniae. A critical pathway in the host inflammatory response to bacterial infection is the ubiquitin-proteasome pathway which is responsible for the degradation of numerous short-lived intracellular proteins. In prior studies, we have shown that ethanol suppresses proteasome activity in macrophages. In this study, we aim to characterize the effect of this proteasomal suppression on downstream cellular functions of the alveolar macrophage, particularly, MHC Class-I antigen processing and presentation.
Murine alveolar macrophage cell line (AMJ2-C11) was exposed to 0, 50 and 1 00mM ethanol concentrations and challenged with heat-killed Klebsiella pneumoniae (MOI: 10:1 ). Cell lysates were analyzed for proteasome activity by release of proteasome-cleaved fluorigenic substrate. We observed that proteasome activity was decreased by as much as 50% in the groups exposed to ethanol, as compared to the control group (p<0.05) in a dose-dependent manner. To study proteasome involvement in MHC Class-I antigen processing and presentation, ethanol treated AMJ2-C11 cells were pulsed with 100Dg/ml ovalbumin for 1 hour. Select groups also received ovalbumin peptide (257-264) (SIINFEKL) as a positive control for antigen presentation. Macrophages were then fixed and co-cultured with CDS+ T-cells obtained from transgenic OT-1 mice (a C57BL/6 background expressing T-cell receptor specific for SIINFEKL peptide of ovalbumin) for 48 hours. Cell supernatants were analyzed for levels of interleukin-2 cytokine as a measure of T-cell activation and stimulation. We conclude that ethanol suppresses proteasome activity in resting and stimulated alveolar macrophages in a dose-dependent manner. We speculate that increased susceptibility of alcoholics to bacterial pneumonia may be due to inhibition of the ubiquitin-proteasome pathway of alveolar macrophages and defective antigen processing.
Recommended Citation
D'Souza, Alain J.; Rudner, X. L.; Desai, S.; and Ruan, S. B., "ETHANOL SUPPRESSES THE UBIQUITIN-PROTEASOME PATHWAY OF ALVEOLAR MACROHPAGES." (2010). Dr. Joseph M. Moerschbaecher, III Graduate Research Day. 9.
https://digitalscholar.lsuhsc.edu/grad_rs/2010/poster1/9
ETHANOL SUPPRESSES THE UBIQUITIN-PROTEASOME PATHWAY OF ALVEOLAR MACROHPAGES.
Alcohol exposure predisposes the drinking host to infection, particularly bacterial pneumonia caused by Klebsiella pneumoniae. A critical pathway in the host inflammatory response to bacterial infection is the ubiquitin-proteasome pathway which is responsible for the degradation of numerous short-lived intracellular proteins. In prior studies, we have shown that ethanol suppresses proteasome activity in macrophages. In this study, we aim to characterize the effect of this proteasomal suppression on downstream cellular functions of the alveolar macrophage, particularly, MHC Class-I antigen processing and presentation.
Murine alveolar macrophage cell line (AMJ2-C11) was exposed to 0, 50 and 1 00mM ethanol concentrations and challenged with heat-killed Klebsiella pneumoniae (MOI: 10:1 ). Cell lysates were analyzed for proteasome activity by release of proteasome-cleaved fluorigenic substrate. We observed that proteasome activity was decreased by as much as 50% in the groups exposed to ethanol, as compared to the control group (p<0.05) in a dose-dependent manner. To study proteasome involvement in MHC Class-I antigen processing and presentation, ethanol treated AMJ2-C11 cells were pulsed with 100Dg/ml ovalbumin for 1 hour. Select groups also received ovalbumin peptide (257-264) (SIINFEKL) as a positive control for antigen presentation. Macrophages were then fixed and co-cultured with CDS+ T-cells obtained from transgenic OT-1 mice (a C57BL/6 background expressing T-cell receptor specific for SIINFEKL peptide of ovalbumin) for 48 hours. Cell supernatants were analyzed for levels of interleukin-2 cytokine as a measure of T-cell activation and stimulation. We conclude that ethanol suppresses proteasome activity in resting and stimulated alveolar macrophages in a dose-dependent manner. We speculate that increased susceptibility of alcoholics to bacterial pneumonia may be due to inhibition of the ubiquitin-proteasome pathway of alveolar macrophages and defective antigen processing.