Inflammation is recognized as multi-cell network dysregulation with an immunological component. Among the many cell types involved in acute inflammation are macrophages, specialized phagocytes involved in many immune responses. These cells exist in different activation states dependent on their biological stimulus and are unknown to play either a target or anti-target role in the context of inflammation.
Understanding the role that macrophages play in inflammation is critical for the development of novel therapeutics and effective treatment strategies to alleviate the burden that this disease imposes on the Canadian public. Our lab reported in 2014 a striking result in Nature Medicine (Marchant et al 2014) that the extracellular protease matrix metalloproteinase 12 (MMP12) secreted from macrophages traffics to the nucleus of virus-infected cells, binds specific DNA sequences and induces life-saving responses. MMP12 also cleaved intracellular substrates that were regulated at the mRNA level, providing dual regulation.
I hypothesize that MMP12 has autocrine roles in macrophages and a distinct roles according to activation state. The Overall lab has developed effective positional proteomic technologies to identify protease cleavage sites in vivo. Using our mass spectrometric method, Terminal Amine Isotopic Labeling of Substrates (TAILS) to identify MMP12 substrates (at their N terminus) during nuclear translocation in an in vivo murine macrophage model of differentiation (peritoneal macrophages) I will characterize the proteins being cleaved.
Upon stimulation with interferon gamma, macrophages differentiate into inflammatory M1-type, while stimulation with interleukin 4 induces differentiation into M2-like wound healing phenotype. Transcriptional effects of MMP12 will be examined using RNA-seq and Chromatin Immunoprecipitation sequencing (ChIP-seq) with Ilumina sequencing.
Combined with whole proteome characterization by LC-MS/MS and large scale substrate identification, this project will elucidate important molecular mediators of the immunological role of MMP12 in inflammation. These findings will be published in peer-reviewed journals, presented at conference meetings and applied for the development of therapeutics to effectively manage immunological disorders with macrophage-specific components.