Elucidation of Eukaryotic Chemorepulsion Signal Transduction Pathways Identify Sex-Based Differences in Human Neutrophils

Abstract

During normal inflammation, neutrophils respond to environmental cues to know when to leave a tissue, but the mechanisms behind neutrophil “repulsion” are not well understood. The ability to selectively repel neutrophils and halt the inflammation feedback loop could lead to novel therapeutics for the neutrophil-based inflammatory diseases. Chemotaxis through amoeboid movement is highly conserved between the eukaryotic amoeba Dictyostelium discoideum and human neutrophils. D. discoideum cells secrete a protein called AprA that inhibits cell proliferation and acts as a signal for cells to move away from the colony center. AprA is structurally similar to the human protein dipeptidyl peptidase IV (DPPIV) and DPPIV repels human and murine neutrophils. However, DPPIV is a prolific serine protease making it a problematic therapeutic. DPPIV induces neutrophil chemorepulsion through activation of protease-activated receptor 2 (PAR2), and PAR2 agonists are sufficient to induce human and murine neutrophil chemorepulsion. However, the signal transduction pathways mediating PAR2 agonist-induced chemorepulsion were unknown. In this dissertation I elucidate the mechanisms involved in AprA-mediated chemorepulsion of D. discoideum cells. Using 29 cell lines with disruptions in proteins necessary for the cyclic adenosine monophosphate (cAMP) chemoattraction pathways, we identified that AprA-induced chemorepulsion requires some, but not all, of the pathways necessary for cAMP-mediated chemoattraction and unlike cAMP-mediated chemoattraction, AprA-mediated chemorepulsion does not induce actin or myosin II polymerization, nor increase pseudopod formation rate. Additionally, in this dissertation I discuss my work investigating PAR2-agonist induced chemorepulsion of human neutrophils. I targeted proteins that were indicated as necessary or unnecessary for AprA-mediated chemorepulsion in D. discoideum cells, and found some similarities in the D. discoideum and human neutrophil chemorepulsion pathways. However, some data showed sex-based differences in neutrophil responses. I found sex-based differences in RNA expression, translation efficacy, protein abundance, and protein localization in unstimulated male and female neutrophils. Together, this data expands our understanding of the eukaryotic chemorepulsion pathways in Dictyostelium and human neutrophils, and furthers our knowledge of sex differences in human neutrophils, an essential component of the innate immunity. This work facilitates the development of new therapeutics for neutrophil-driven diseases, which may require adjustments due to a patient’s biological sex.