Extracellular matrix sensing in cellular signaling, migration and wound repair

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PROJECT SUMMARY/ABSTRACT Human genetic mutations in integrins and other adhesion proteins cause a range of autoimmune diseases and likely contribute to the chronic inflammation that accompanies many widespread human maladies. However, our knowledge of the molecular mechanisms that link integrin function and immune activation remain incomplete. The long-term goal of this research program is to address this knowledge gap by revealing novel regulatory connections between extracellular matrix (ECM) sensing and macrophage activation. The overall objective of this proposal is to evaluate how ECM sensing (which we refer to as ‘haptosensing’) tunes macrophage activation at the molecular and cellular levels, and to translate this understanding to in vivo wound healing studies. The central hypothesis of this proposal is that haptosensing regulates macrophage activation via integrin-Arp2/3 dependent crosstalk with immune receptors. Strong preliminary data generated by the applicant’s lab supports this hypothesis, as well as demonstrating that the proposed studies are feasible in the applicant’s hands. The rationale for the proposed research plan is that gaining mechanistic insight into haptosensing will allow us to begin understanding its role in chronic human disease states, which may in turn reveal potential pharmacological targets. The hypothesis will be tested further in the context of three specific aims: 1) Define the integrin-initiated signals that activate Arp2/3 during haptosensing; 2) Determine how the haptosensing pathway tunes inflammatory macrophage activation; 3) Determine how macrophage haptosensing is required for wound resolution in vivo. These aims will primarily be pursued by mechanistically dissecting macrophage behavior in custom-designed microfluidic chambers and translating these in vitro findings to in vivo settings using intravital multiphoton microscopy. These studies will employ loss of function approaches (genetic nulls, shRNA knockdown, inhibitor wash-in), which are already established and are effective in the applicant’s hands. Finally, the applicant’s lab will employ careful, rigorous image analysis approaches to study cell migration, actin dynamics and subcellular protein localization in primary macrophages, in vitro and in vivo, responding to numerous extracellular cues, including ECM gradients. The approach is innovative, as it applies mechanistic observations generated in vitro to direct observations of immune cell migration and activity in vivo. The approaches employed to this end by the applicant’s lab represent significant technological advances that make it possible to clarify the connection between cellular adhesion and macrophage activation. The proposed research program is significant as completion of these aims will identify the mechanistic details of macrophage haptosensing and will assign an in vivo function to it. The significant conceptual and technological advances brought to bear in this research study will have significant impact across multiple fields including directional migration, TLR4 signaling and wound repair.