Role of mechanical signal in regulating neutrophil function

It is not understood, how mechanical signals influence the behavior and action of circulating cells in our body. Previous studies showed that the process of migration through narrow slits might enhance lifespan of polymorpho-nuclear cells (PMN / neutrophil). But it is unknown, how the mechanical signal is received, relayed, and translated into specific phenotype shift in PMN. Here we investigated whether compressional force affects defense system of circulating lymphocytes by increasing their killing ability during migration through narrow endothelial junctions. To decipher the receiving and transducing components of this mechanical signal relay process, we performed gene expression analysis between transmigrated and non-transmigrated neutrophils. Comparison of early gene-expression pattern of transmigrated and non-transmigrated neutrophils revealed a huge change in gene expression profile. Molecular and genetic analysis of the proteins revealed from gene-expression analysis led us to understand the mechanism of mechanical signal relay from the neutrophil membrane to bacterial-killing machinery. As neutrophils are most abundant and immediate-responding circulating immune cells in our body, our study advances the path for understanding the general process of mechano-transduction, and mechano-modulatory applications with mechanically enhanced neutrophils. Overall design: Neutrophils were mobilized to lung of 8-10 weeks old male Catchup (heterozygous) or non-labelled C57BL/6-background mice by exposure to 1mg/ml of nebulized LPS (Sigma, Escherichia coli O55:B5) inside the fumigation chamber. 20 ug of anti-Ly6G-BV421 antibody (Biolegend, #127628) was i.v. injected 2 minutes before sacrifice. Lungs were perfused with 10 ml of PBS through right ventricle of heart and collected for PMN isolation, biochemical, and histopathological analyses. For PMN isolation, a single cell suspension was prepared by digestion of minced lung lobes with collagenase A (1 mg/ml; Millipore-Sigma # SCR103) at a 370C shaking water bath for 45 min. Digested lung samples were passed through 16-17G needle and filtered through 40 um cell strainer (Thermo Fisher, #08-771-1). Red blood cells were removed by using RBC lysis buffer (Thermo Fisher, #00-4333-57). A single cell suspension prepared from Catchup (heterozygous) mice were directly subjected to flow sorting for separation of td-Tomato-positive or double td-Tomato and anti-Ly6G-BV421-positive PMN. PMN, labelled with td-Tomato (or anti-Ly6G-AF-594) and td-Tomato and anti-Ly6G-BV-421 (or anti-Ly6G-AF-594 and anti-Ly6G-BV-421), were sorted in a sterile environment by MoFlo Astrios and MoFlo Astrios EQ (Beckman Coulter) cell sorter equipped with 405, 488, 561 and 635 nm lasers, and collected in RPMI-1640 medium (Thermo Fisher #72400047). Both the sorters are Gates were initially established on forward and side scatter, based on granularity and relative size. Aggregates were gated out accordingly. Selective gates were drawn to sort the single positive and dual positive PMN. Software SUMMIT, 6.1 (Beckman Coulter) was used for sorting and analysis. Cpp is control dual stained vascular LPSpp is LPS treated dual stained vascular LPSp is LPS treated single stained transmigrated to lung