Tularemia is a potentially fatal zoonosis of humans and the causative agent, Francisella tularensis (Ft), is a facultative intracellular bacterium, and is one of few pathogens that can infect both macrophages and neutrophils (polymorphonuclear leukocytes, PMNs). In this regard, it is noteworthy that macrophages and neutrophils appear to play distinctly different roles in pathogenesis, with macrophages acting as major vehicles for bacterial growth and dissemination, and PMNs playing a central role in host tissue destruction. PMNs are short lived, and unlike other leukocytes are preprogrammed to undergo apoptosis ~24 h after release into the circulation. Tight spatial and temporal control of this process is critical for elimination of infection and resolution of inflammation, and for this reason defects in PMN turnover exemplify a dysregulated and ineffective inflammatory response that promotes tissue destruction and disease. Consistent with this, we discovered that Ft inhibits human neutrophil apoptosis and markedly prolongs cell lifespan, and demonstrated previously that this is achieved, in part, by effects on the intrinsic and extrinsic apoptosis pathways, as well as changes in the neutrophil transcriptome that include significant differential expression of genes linked to apoptosis and cell fate. Nevertheless, how PMN lifespan is prolonged is only partially understood. Herein, we present compelling preliminary data which suggest that apoptosis inhibition also requires global metabolic reprogramming Ft- infected PMNs that is mediated by HIF-1? and p38 MAP kinase, which act at multiple levels to upregulate glycolysis and induce selective mitophagy. The studies we propose are innovative, as integrated regulation of glycolysis and organelle dynamics has not been documented previously as a mechanism for regulation of PMN lifespan during infection. Notably, the metabolic reprogramming we describe differs markedly from metabolic changes recently reported to occur in Ft-infected murine macrophages Thus, we predict that completion of this study will reveal additional differences between these phagocyte types, and thereby reinforce the established cell type-specificity of crosstalk between metabolism and inflammation and immunity, while also identifying potential new targets for therapeutic intervention. Specifically, we hypothesize that HIF-1? and p38 MAPK act at multiple levels for coordinated induction of glycolysis and selective mitophagy in the first hours after Ft infection, and that enhanced ATP production and mitochondrial depletion act in concert to sustain PMN viability, while also providing additional nutrients for bacterial growth. To interrogate this hypothesis, we propose the following Specific Aims: 1) To elucidate the mechanisms and functional consequences of glycolysis induction by F. tularensis Schu S4 with a focus on HIF-1? and p38 MAPK. 2) To elucidate how crosstalk between glycolysis and mitochondria contributes to apoptosis inhibition, with a focus on hexokinase- and VDAC-dependent mitochondrial outer membrane stabilization and NIX- and BNIP3-dependent mitophagy.
Francisella tularnesis is a bacterium that can be inhaled into the lungs, causing a serious and potentially lethal infection that is associated with pneumonia and spreading of bacteria to other ogans, including the liver and spleen. White blood cells, including cells called neutrophils, are an essential part of the immune system, and under normal circumstances these cells rapidly kill bacteria. However, F. tularensis manipulates neutrophil function to persist in the body and cause disease; thus, understanding how neutrophil function is altered is an important first step in the development of new vaccines or therapeutics that control this infection by manipulation of the inflammatory response.
