Investigations of the role of Semaphorin 5A in innate immunity

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Abstract

Semaphorins are a family of membrane-bound and secreted proteins studied extensively in neuronal development, and more recently explored in airway biology and inflammation and in animal models of immune regulation. Activation of signaling pathways involving these proteins is dependent on the binding of semaphorins to their receptors, plexins. Using a protein biomarker discovery platform, we previously described increases in plasma levels of SEMA5A, (6.4- fold change at 1h) and Semaphorin 6B (SEMA6B, 3.3 fold change at 1h) that decrease over 8h following intravenous (IV) endotoxin (LPS) challenge in healthy volunteers (Am J of Respir Crit Care Med 2018; 197: A4749). This is the first reported association of this SEMA5A with acute inflammatory responses and a detailed examination of semaphorins and their receptors in the context of innate immunity is currently lacking. This project aims to investigate the role of SEMA5A in innate immune responses by addressing the specific aims described below. Specific Aim 1: To determine whether expression and signaling of Semaphorin 5A (SEMA5A) and its receptors, plexin A1 and plexin B3, are increased during acute inflammation. Current status: We have conducted experiments stimulating human and murine macrophage cell lines as well as primary murine and human neutrophils with various TLR stimuli and probing for SEMA5A, and Plexin A1 and B3 expression at the gene and protein level. We are in the process of compiling and confirming these data. Specific Aim 2: To determine whether SEMA5A or SEMA6B loss of function affects the LPS-induced acute inflammatory response. Current Status: SEMA5A and SEMA6B knockout animals are commercially available and have been obtained. Colonies of these animals have been established. These animals, along with appropriate wild type controls, will be stimulated with endotoxin intra-tracheally and lungs, lung lavage fluid, serum and other organs will be obtained and analyzed for inflammatory cell infiltration, architectural changes, and immune cell phenotypes, as well as changes at the gene expression level. In separate experiments, these animals will also be assessed for survival following endotoxin challenge. Specific Aim 3: To determine the role of SEMA5A and SEMA6B in human lung inflammation. Current status: Preliminary experiments suggest that SEMA5A is found in increased levels in lung lavage from patients with acute lung injury. To confirm this finding, we will systematically analyze lavage fluid from healthy volunteers who have received endotoxin endobronchially, patients with various bacterial pneumonias, and patients with ARDS. We will also probe for SEMA6B. No publications are associated with this Annual Report. However, we have published a review on the subject in Frontiers in Immunology.