Elucidation of a Eukaryotic Chemorepulsion Mechanism

The directed movement of eukaryotic cells away from a source of a chemorepellent appears to play a major role in development and the resolution of inflammation, but very little is known about eukaryotic chemorepellents and how they direct cell motility. Chemorepulsion of Dictyostelium cells from a secreted protein called AprA is a model where one can combine biochemistry and genetics to elucidate chemorepulsion. Using available mutants and a preliminary genetic screen, we identified the AprA receptor, identified several components of the AprA signal transduction pathway, and found that AprA chemorepulsion involves a fundamentally different mechanism from chemoattraction. AprA has predicted structural similarity to the human secreted protein dipeptidyl peptidase IV (DPPIV), and shares functional properties with DPPIV. We found that DPPIV is a chemorepellent for human and mouse neutrophils, found that the G protein-coupled receptor PAR2 mediates DPPIV chemorepulsion, and found that small molecule PAR2 agonists act as neutrophil chemorepellents. Preliminary studies indicate that there are strong similarities between the AprA and PAR2 agonist chemorepulsion mechanisms. Acute respiratory distress syndrome (ARDS) is an untreatable disease involving damage to the lungs causing neutrophils to enter the lungs. The neutrophils further damage the lungs, causing more neutrophil entry in a positive feedback loop, and this results in the death of ~40% of the ~200,000 ARDS patients per year in the US. An exciting insight into a possible therapeutic approach is that when DPPIV or PAR2 agonists are aspirated into the lungs, they induce neutrophils to leave the lungs in two mouse models of ARDS. The key roadblock to moving this into the clinic is that we need to know what the PAR2 agonist chemorepulsion mechanism is to anticipate potential drug interactions and side effects. To gain insights into a fundamental mechanism, and ways to induce neutrophils to leave a tissue, we propose to elucidate the AprA and PAR2 agonist chemorepulsion mechanisms using the power of Dictyostelium to lead the work. We will rigorously test newly identified Dictyostelium chemorepulsion pathway components, and determine where they function in the pathway. We will complete the Dictyostelium genetic screens to gain further insight into chemorepulsion, and then use this information to guide an examination of possibly similar mechanisms in neutrophils. While examining human neutrophil chemorepulsion, we observed a significant difference between male and female neutrophils, and we will delineate the extent and molecular mechanism underlying this difference. Together, the proposed work will elucidate a poorly understood fundamental mechanism, elucidate an unexpected sex difference in the innate immune system, and help elucidate how PAR2 agonists could be used clinically to drive excessive neutrophils out of a tissue, with possibly different treatments for men and women.