The mechanism of phagocytosis of pathogens remains to be fully characterized. We report a novel phagocytosis pathway for Pseudomonas aeruginosa, which is initiated by cholesterol-rich membrane rafts and is dependent on Lyn, primarily an immune regulator with both positive and negative roles. Blocking of Lyn or blocking of cholesterol synthesis significantly inhibited phagocytosis by alveolar macrophages. We found that Lyn, via Src homology 2 and 3 domains, bound to and then activated PI3K and Akt to regulate intracellular routing of the engulfed P. aeruginosa. Further analysis indicates that Lyn and raft components entered in phagosomes and late lysosomes. Finally, respiratory burst was dependent on Lyn and membrane rafts, as confirmed by small interfering RNA and dominant-negative strategies. Our investigations demonstrate that Lyn along with membrane rafts plays a fundamental role in phagocytosis by alveolar macrophages during infection.

Although alveolar epithelial type II cells (AECII) perform substantial roles in the maintenance of alveolar integrity, the extent of their contributions to immune defense is poorly understood. Here, we demonstrate that AECII activates alveolar macrophages (AM) functions, such as phagocytosis using a conditioned medium from AECII infected by P. aeruginosa. AECII-derived chemokine MCP-1, a monocyte chemoattractant protein, was identified as a main factor in enhancing AM function. We proposed that the enhanced immune potency of AECII may play a critical role in alleviation of bacterial propagation and pneumonia. The ability of phagocytosis and superoxide release by AM was reduced by MCP-1 neutralizing antibodies. Furthermore, MCP-1−/− mice showed an increased bacterial burden under PAO1 and PAK infection vs. wt littermates. AM from MCP-1−/− mice also demonstrated less superoxide and impaired phagocytosis over the controls. In addition, AECII conditioned medium increased the host defense of airway in MCP-1−/− mice through the activation of AM function. Mechanistically, we found that Lyn mediated NFκB activation led to increased gene expression and secretion of MCP-1. Consequently Lyn−/− mice had reduced MCP-1 secretion and resulted in a decrease in superoxide and phagocytosis by AM. Collectively, our data indicate that AECII may serve as an immune booster for fighting bacterial infections, particularly in severe immunocompromised conditions.

Lyn is an important B cell signaling kinase of the Src tyrosine kinase family with a broad range of functions from cytoskeletal changes to induction of apoptosis. However, the role of Lyn in infectious diseases is not clear. Here, we demonstrate that Lyn activation by phosphorylation significantly impacted invasion of an alveolar epithelial cell line, primary lung cells, and rat lungs by Pseudomonas aeruginosa (PA), a common opportunistic lung pathogen affecting individuals with deficient lung immunity. Our results indicate that activation of Lyn and its interaction with rafts and TLR2, played an important role in the initial stages of PA interaction with host cells. The role of Lyn was further evaluated using the pharmacologic Src-specific inhibitor PP2, a dominant negative mutant, and finally confirmed with Lyn-deficient (Lyn -/-) bone marrowderived mast cells. Inhibition of Lyn's function by above approaches prevented PA internalization. Moreover, blocking of Lyn also affected downstream events: induction of inflammatory cytokines and apoptosis. This report brings out a new role of Lyn in infectious diseases and indicates potential new targets for prevention and treatment of infections.

The importance of DNA repair in the pathogenic mechanism of Alzheimer’s Disease (AD) is still poorly understood. Here, we report that a broad range of responses by DNA repair proteins plays a critical role in the regulation of inflammation response in rabbits fed a cholesterol-rich diet, a model system for AD. We found accumulation of oxodG DNA adduct in the brain of cholesterol-enriched diets compared to control diets, which subsequently induced a broad range of DNA repair protein activities. Also, the hippocampus was identified as the primary site of oxidative DNA damage and elevated OGG1 activity. In addition, a physical interaction between XPB and OGG1 may account for a potential mechanism involving these DNA repair responses. DNA repair proteins also impact activation of various signaling cascades, including Src in response to cholesterol oxidation. Furthermore, OGG1 deficient mice showed no IL-6 activation as seen in wt mice but a drastic increase of TNF-α, a pro-inflammatory cytokine. Thus, OGG1 may be associated with cytokine production induced by high cholesterol levels, impacting neurodegeneration. Together, our studies suggest that critical DNA repair proteins are associated with development of AD, and may serve as potential targets for the treatment of AD.

Introduction: Recently, proteomics approach has revealed lipid raft associated proteins such as Flotillin‐1 in phagosome compartments. We have used Pseudomonas aeruginosa (PA) phagocytosis by alveolar macrophage (AM) as a model to study this mechanism. Rationale: Lyn‐ PI3Kinase – Akt pathway is known to be involved in IgG receptor mediated phagosome formation. Since Lyn is also a lipid raft resident protein we propose a role for Lyn in raft mediated phagocytosis of PA by AM. Methods: Phagosome formation and localization of signaling proteins were studied by immunostaining with fluorescent antibodies followed by confocal microscopy. Lyn‐YFP and PH‐Akt‐GFP co‐transfected MHS cells (murine alveolar macrophage cell line, ATCC) were used for studying spatio‐temporal association of Lyn and Akt during PA phagocytosis by live cell confocal microscopy. Lipid rafts were identified by staining with FITC labeled Cholera Toxin B subunit. Phagosomes from PA infected cells were isolated by sucrose density gradient centrifugation for biochemical characterization. Results: Lipid raft marker (green) and Lyn‐YFP (yellow) were found to be co localized with PAO1 (stained red) in phagosome compartment. Both Lyn‐YFP and PH‐Akt‐GFP were found to be co‐localized in the lamellipodium and phagocytic cup. Lyn, PI3Kinase and Akt detected by immunoblotting were found to be actively recruited to phagosome fractions in PA infected cells compared to controls. Conclusions: Our data indicates that Lipid raft mediated Lyn‐PI3Kinase‐Akt pathway is crucial for initiating and regulating phagocytosis of PA by AM. This study will bring out newer insights into PA pathogenesis and help to understand host innate immunity.