T12 - Exploring the molecular mechanism underlying neutrophil upstream migration

Introduction: : Neutrophil migration during inflammation is a key feature of the immune system and is regulated by the leukocyte adhesion cascade in the endothelial cell-lined vasculature (Fig. 1A). Neutrophils use surface integrins to crawl along the apical endothelium, adhering to cell adhesion molecules before transmigrating through the basal endothelium to infection sites. Their ability to move against fluid shear flow, known as upstream migration, is vital for immune and inflammatory responses, though its molecular mechanisms remain unclear. During this process, chemokine-activated integrins like Mac-1 and LFA-1 enable neutrophil adhesion to endothelial molecules such as ICAM-1 and VCAM-1. Blocking LFA-1 has also been shown to promote upstream migration in T cell migration. Upstream migration may speed extravasation by enabling cells to tunnel beneath the endothelial monolayer, reducing transit time to inflammatory sites. This is supported by the observation that T cells moving on vascular endothelial cell surfaces exhibit a twofold increase in transmigration speed when crawling upstream compared to downstream. Building on these discoveries, our central hypothesis is to determine the molecular cues or signals downstream of Mac-1 that will allow neutrophils to transmigrate against shear flow more quickly and arrive at the injury site.

Materials and

Methods: : Previous research from our laboratory highlights the crucial contributions of LFA-1 and Mac-1 in the upstream migration of neutrophils. In upstream migration, the actin structure is essential for generating the force to push the cell upstream. We also employ analogous screening strategies like RNA-Seq to pinpoint signaling molecules downstream of Mac-1 that hinder the process of upstream migration. To investigate whether ICAM-1 and VCAM-1 differentially activate these downstream signaling pathways, we will initially serum-starve differentiated HL-60 cells or primary neutrophils for 3 hours in IMDM without supplements. Subsequently, the following experiments will be carried out on both ICAM-1/VCAM-1 using appropriate controls, like our previous work (Fig.1B, C). We will examine the differences in signaling downstream of LFA-1 and Mac-1 upon interacting with ICAM-1. Various structural inhibitors for the differential pathways identified will also be screened to identify signals downstream of MAC-1. Knockouts of HL-60 cells for the identified signaling genes will be created using specific synthetic single guide RNAs (sgRNAs) from Synthego (Redwood City, CA). We will then differentiate the HL-60 knockout (KO) cells into neutrophil-like cells using DMSO (1.25% v/v for 5 days) and assess their directional migratory preferences under shear flow. Once the directional migration preferences on various surfaces and under different blocking conditions have been verified in differentiated HL-60 KO cells, we will replicate the results using CRISPR knockouts in primary neutrophils following a similar strategy. Similar experiments will be carried out to compare the transmigration time between the KOs and WTs on HUVEC surfaces.

Results, Conclusions, and Discussions:: We will observe the different signaling pathways downstream of LFA-1 and Mac-1 in these knockouts; this methodology follows previously published methods we have demonstrated in our previous work on T cells. Furthermore, we will test the preferences of directional migration of edited primary neutrophils and HL-60 cells in a microfluidic chamber where the types and densities of ligands can be carefully controlled, to accurately identify the critical downstream signals, which, when blocked, would allow neutrophils to migrate upstream. It is conceivable that differential signals between ICAM-1 and VCAM-1 or between LFA-1 and Mac-1 on ICAM-1 may not be identified, so as an alternative strategy, specific pharmacological inhibitors targeting signaling molecules such as ROCK, RhoA, Cdc42, or myosin will be employed. Alternatively, a CRISPR screening approach may be utilized to identify new downstream signals if a specific molecule downstream of Mac-1 is not identified via western blotting. The feasibility of these alternatives is supported by previous work, where upstream migration in T-cells and HSPCs was successfully achieved through antibody blocking of integrin chains on HL-60 and neutrophils. We expect to see differential responses of WTs and KOs in their directional migration patterns since KOs without their integrins can transmigrate upstream quicker than their WTs. We also expect to observe differences in their transmigration movement on gels of varying stiffness coated with the different integrin (ICAM-1 and LFA-1) ligands. We also expect to measure the different traction stresses of the WTs and KOs using TFM and understand the biophysical aspect of upstream migration in HL60 cells.

Acknowledgements and/or References (Optional):: This work was funded by R01GM143357 to ABJ