Extracellular matrix stiffness control of NLRP3 inflammasome activity - PROJECT SUMMARY Fibrotic interstitial lung disease (ILD) is a debilitating illness whose course is worsened by paroxysms of infection- induced acute exacerbations (AE-ILD) with mortality rates greater than 50%. Fibrotic ILD is associated with (a) increased extracellular matrix (ECM) deposition that increases lung stiffness and (b) decreased NLRP3 inflammasome activities that leads to ineffective host defense to bacterial and viral infections. However, whether ECM stiffness alters NLRP3 inflammasome activities, and its relevance to inflammatory disease, is a largely unexplored area of biology. The study of NLRP3 inflammasome activity often excludes the variable of ECM stiffness in experimental design. Most experiments in vitro are conducted on plastic plates, which are 1,000,000- fold stiffer than tissue. Thus, an exploration of the interaction between ECM stiffness and NLRP3 inflammasome activities will inform our understanding of AE-ILD while simultaneously illuminating basic principles of inflammasome biology. The objective of this proposal is to define the mechanisms by which ECM stiffness regulates the NLRP3 inflammasome in macrophages. To explore the impact of ECM stiffness on macrophage inflammasome activities, I developed an in vitro system where macrophages are cultured on material where the stiffness is varied in a biologically relevant way. Using this system, I have identified NRF2 as a mechanosensitive transcription factor that regulates NLRP3 inflammasome activities. I have found that macrophage NRF2 activities are also controlled by ECM stiffness in human ILD. Based on these preliminary data, I hypothesize that ECM stiffness controls NRF2 function, which regulates the NLRP3 inflammasome in macrophages with functional consequences for control of pneumonia and development of fibrosis. To test this hypothesis, I will use innovative experimental systems to define the mechanisms of ECM stiffness control of NRF2 activity and novel mouse models whereby NRF2 activity is genetically controlled in macrophages. Specifically, I propose to (Aim 1) define how ECM stiffness regulates NRF2 activity in vitro, and (Aim 2) determine the role of NRF2 regulation of the NLRP3 inflammasome in disease. This research will identify novel mechanisms of ECM- mediated control of NLRP3 inflammasome activities and inform discussions of new therapies for patients with AE-ILD while also answering fundamental questions of biology. I will perform the work in this K08 proposal under the mentorship of Dr. Jonathan Kagan at Boston Children’s Hospital (BCH). Massachusetts General Hospital and BCH are ideal environments for me given their strong focus and track record of training the next generation of physician-scientists. I have developed a career development plan consisting of coursework in the biology of the ECM and bioinformatics as well as assembled a Training Advisory Committee to provide expertise and assistance in these areas. The goal of this K08 award is to provide me with the intellectual and technical training to become an independent, R01-funded physician-scientist with expertise in the mechanisms by which the ECM regulates innate immunity.
