Both acute and chronic alcohol consumption has been shown to exacerbate the complications exibited after trauma or burn injury. Alcohol also has been shown to increase the prevalence of pneumonia and other opportunistic infections which normally would be controlled. We propose to elucidate the mechanisms that cause macrophages to exhibit phagocytosis after ethanol exposure. Our model will utilize the murine acute ethanol mode that our lab has previously worked with. We predict that alveolar macrophages isolated from mice that have been treated with ethanol will exhibit a decrease phagocytosis independent on the type of receptors mediated but the pathway of inhibition may be different.
Our first aim will focus on testing the degree of decreased ex vivo phagocytosis in the in vivo ethanol treated macrophages; focusing on targeting the TLR-4, TLR-2, Fcg-R, and CR-3 mediated pathways. We will utilize microscopic techniques to access the degree of inhibition.
The second aim i s to determine whether acute ethanol exposure effects binding and internalization of a pathogen by alveolar macrophages. The first part of this aim will focus on studying the ability of these macrophages to bind to beads coated with specific ligands to TLR-2, FcgR, and CR3. This will be followed by measuring receptor expression on the surface of the macrophage as well as receptor aggregation of the receptor to the coated bead. These data will reveal if decreases of specific ligand binding in ethanol exposed macrophages is due alterations in receptor expression or the inability of the receptor to cluster at the site of the phagolytic particle. The second part of this aim will focus on measuring the rate and degree of focal adhesion protein recruitment to the site of the forming phagosome. We will use time lapse microscopy and immunocytochemistry to measure these levels.
Aim 3 of our study will focus on the downstream signaling, specifically the balance of Rac and Rho activation, as well as the production capability of alveolar macrophages to produce nitric oxide, a molecule involved in the lysis of ingested pathogens. We will utilize western blotting techniques to measure Rho-GTP, Rac-GTP, and total Rho and Rac, and a nitric oxide detection kit for nitric oxide measurements. The studies proposed in this application are necessary to uncover how ethanol exposure modulates the ability of alveolar macrophage to function properly. Understanding how ethanol decreases phagocytosis can lead us to the development of therapeutic targets that can potentially help in the clearance of a pathogen in patients who have consumed alcohol. ? ? ?
