Roles of N-glycans on neutrophil beta2 integrins in progression of acute lung injury

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PROJECT SUMMARY Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are the common cause of respiratory failure. Despite all medical innovations, treatment of ARDS remains an unsolved problem. Neutrophils are the first immune cells infiltrated into lungs during ALI, where neutrophils release proteinases, cytokines, and oxidants to kill invading microbes, as well as to injure lungs. Clinical data and animal models have proved that neutrophil recruitment and function influences the progression of ALI. Thus, insights into the fine-tuned neutrophil recruitment and function may provide a novel approach for the treatment of ALI. Integrin b2 plays an important role in the regulation of neutrophil recruitment and neutrophil functions. At inflamed sites, the intracellular signaling upregulates the integrin ligand-binding affinity, which allows ligands to bind. Ligand binding to integrin b2 in the high-affinity state arrests neutrophils, a prerequisite step for neutrophil recruitment, as well as causes integrin cluster formation (i.e. integrin valency) to enhance neutrophil arrest. Currently, the intracellular signaling is the only approach to regulate the integrin ligand-binding capability. The extracellular domain of integrin b2 is decorated with N-linked glycans. Yet, whether and how N- glycans regulate the integrin b2-ligand binding has not been studied. In addition, the receptor for advanced glycation end-products (RAGE) on alveolar epithelial cell type I (AT1) is a major mediator for inflammatory responses in lungs. Integrin aMb2 on neutrophils is a ligand for RAGE, however, the role of the interaction of integrin aMb2 with RAGE in the progression of inflammation in ALI is unknown. Our preliminary studies found that removing N-glycans increased integrin aLb2-mediated neutrophil adhesion and ligand binding-induced neutrophil cellular responses. Furthermore, removing sialic acids increased aMb2 binding to RAGE. Thus, we hypothesize that 1) N-glycans on the extracellular domain prevent integrin aLb2 to adopt the high-affinity state and/or limit the integrin cluster formation; and 2) reduced glycosylation of integrin aMb2 facilitates its binding to RAGE that may increase inflammatory responses in lungs. Thus, we propose two Aims to test the hypotheses: Aim 1: Determine if N-glycans on integrin aLb2 negatively regulate its ligand-binding affinity and valency; and Aim 2: Determine if glycans on aMb2 negatively regulate RAGE-mediated inflammatory responses in AT1 cells. Integrin b2 containing mutations on N-glycan bearing sites, a novel tool to measuring integrin affinity states, AT1 cells, and mouse models of ALI will be used to test our hypotheses. Our proposed study will decipher the role of N-glycans on neutrophil b2 integrins in the pathogenesis of ALI, which may lead to the development of a new therapy for ALI.