Mesenchymal Stromal Cells, Autophagy, and the Host Response to Systemic Bacterial Infection - PROJECT ABSTRACT: Sepsis is a disease process that causes significant morbidity and mortality by inciting a systemic inflammatory response. The invading organism(s) activate immune cells of the host, in an effort to eradicate the infection. However, this pro-inflammatory response often persists, leading to organ dysfunction and subsequent death. Mesenchymal stromal cells (MSCs), and the autophagy process, are key mediators of the response to sepsis. Autophagy represents a pathway for the turnover of damaged proteins and organelles (such as mitochondria), which can promote survival under various disease conditions. Genes critical for the regulation of autophagy include Beclin 1. MSCs are a population of cells, originally isolated from the bone marrow, that are a promising platform for cell-based therapy. We have shown that MSCs improve survival in murine sepsis by decreasing organ injury, improving bacterial clearance, interacting with immune cells and promoting resolution of inflammation. Low-dose CO exposure induces autophagy in vivo, however the impact of CO conditioning on the autophagy response in MSCs, and its effect on MSC function during sepsis, requires investigation. We hypothesize that autophagy is a key mediator in regulating the ability of CO (ex vivo) to enhance MSC function in experimental models of sepsis, both by cell-to-cell and paracrine actions of MSCs. To test our hypothesis, we propose three aims. In Aim 1, we will decipher the importance of the autophagy pathway, for enhanced MSC function during sepsis, after CO pre-conditioning of MSCs ex vivo. MSCs will be harvested from Beclin1+/– and wild-type (WT) mice, or Beclin1 will be silenced (shBeclin1) in WT MSCs, and then administered to WT mice after cecal ligation and puncture (CLP), an experimental model of sepsis. We will determine whether the enhanced function of CO pre-conditioning is lost in autophagy protein deficient MSCs. We will also study the effect of CO on WT and autophagy protein deficient MSCs to promote bacterial clearance, resolution of inflammation, preservation of organ function, and survival. In Aim 2 we will determine the role of extracellular vesicles (EVs) in the paracrine actions of MSCs pre-conditioned with CO, and investigate the importance of autophagy in this response. EVs will be isolated from MSCs derived from WT and Beclin1+/– mice, or from shBeclin1 MSCs, and given to WT mice after the onset of sepsis. In Aim 3, we will determine whether ex vivo pre-conditioning of MSCs with CO improves the outcome of autophagy protein deficient mice during sepsis. WT MSCs pre-conditioned with CO, will be administered to Beclin1+/– mice, and mice in which autophagy is pharmacologically inhibited, after CLP-induced sepsis. In addition, we will assess EVs derived from MSCs exposed to CO, on sepsis outcome in autophagy protein deficient mice. If successful, this application will elucidate mechanisms by which MSCs improve the host response to sepsis. Elucidating the importance of autophagy in MSCs, and the enhancement of MSC function by CO will also advance our understanding of MSC biology and optimize their potential therapeutic use.
