Tularemia is a potentially fatal disease and the causative agent, Francisella tularensis (Ft), is one of few bacterial pathogens that can infect both neutrophils (polymorphonuclear leukocytes, PMNs) and macrophages. Notably, macrophages and neutrophils appear to play distinctly different roles in tularemia pathogenesis, with macrophages acting as major vehicles for bacterial growth and dissemination, and PMNs playing a central role in host tissue destruction. Neutrophils 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. In keeping with this, we discovered that Ft inhibits human neutrophil apoptosis and markedly prolongs cell lifespan, and demonstrated that this is achieved via effects on the intrinsic and extrinsic apoptosis pathways, as well as changes in the neutrophil transcriptome that include significant differential expression of 365 unique genes linked to apoptosis and cell fate. Nevertheless, how cell lifespan is prolonged is only partially understood. Herein, we propose groundbreaking studies based on our discovery of neutrophil metabolic reprogramming as a new mechanism for apoptosis inhibition. Our proposed studies are supported by extensive preliminary data, and are highly innovative, as integrated manipulation of glycolysis and organelle function has not been previously documented as a mechanism for regulation of PMN lifespan during infection. Potential effects of these changes on bacterial growth and the influence of PMN metabolites on macrophage polarization will also be determined. In addition, we recently identified bacterial lipoproteins (BLPs) as active factors in Ft conditioned medium (CM) that extend PMN lifespan via a mechanism that is dependent on a common single nucleotide polymorphism (SNP) in human TLR1 (rs5743618, T1805G) that significantly influences the severity and lethality of sepsis as well as the outcomes of many infectious and inflammatory diseases, including but not limited to tuberculosis, pyelonephritis, atherosclerosis, arthritis, lupus, colitis, and cancer. Elucidating the mechanism(s) of BLP and TLR2/1-driven apoptosis inhibition is a second objective of this study. Our experimental design will also utilize drugs that specifically target HIF-1? and TLR2, mitophagy, glycolysis or other relevant signaling intermediates to identify points for therapeutic intervention that are expected to be relevant to many diseases that affect Veterans.
Our specific aims are: 1) To elucidate the mechanisms and functional consequences of neutrophil metabolic reprogramming. 2) To elucidate the mechanisms of BLP and TLR2/1-mediated apoptosis inhibition and potential for theraputic intervention.

Public Health Relevance

Neutrophils are white blood cells that play critical roles in microbe killing and regulation of inflammation, and dysregulation of neutrophil lifespan contributes to tissue destruction in the context of many diseases. The mechanisms that regulate neutrophil apoptosis and lifespan are only partially defined, and we identify a new role for neutrophil metabolic reprogramming as a mechanism to control neutrophil viability during infection that is also influenced by a polymorphism in the human gene that encodes a protein called TLR1. Thus, our studies will provide key new insight into the ability of metabolism and TLR1 signaling to influence neutrophil function and fate. As this TLR1 polymorphism is known to influence the severity of many infectious and inflammatory disorders that affect Veterans, including atherosclerosis, Lyme disease, arthritis, tuberculosis, colitis, bacterial sepsis and cancer, our studies are broadly relevant and our research plan includes testing of drugs that influence these pathways to identify potential targets for therapeutic intervention to improve Veterans? health.

Agency
National Institute of Health (NIH)
Institute
Veterans Affairs (VA)
Type
Non-HHS Research Projects (I01)
Project #
7I01BX002108-07
Application #
10228332
Study Section
Infectious Diseases B (INFB)
Project Start
2013-04-01
Project End
2022-03-31
Budget Start
2020-08-01
Budget End
2021-03-31
Support Year
7
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Harry S. Truman Memorial VA Hospital
Department
Type
DUNS #
082263013
City
Columbia
State
MO
Country
United States
Zip Code
65201
Kinkead, Lauren C; Whitmore, Laura C; McCracken, Jenna M et al. (2018) Bacterial lipoproteins and other factors released by Francisella tularensis modulate human neutrophil lifespan: Effects of a TLR1 SNP on apoptosis inhibition. Cell Microbiol 20:
Kinkead, Lauren C; Fayram, Drew C; Allen, Lee-Ann H (2017) Francisella novicida inhibits spontaneous apoptosis and extends human neutrophil lifespan. J Leukoc Biol 102:815-828
Kinkead, Lauren C; Allen, Lee-Ann H (2016) Multifaceted effects of Francisella tularensis on human neutrophil function and lifespan. Immunol Rev 273:266-81
McCracken, Jenna M; Kinkead, Lauren C; McCaffrey, Ramona L et al. (2016) Francisella tularensis Modulates a Distinct Subset of Regulatory Factors and Sustains Mitochondrial Integrity to Impair Human Neutrophil Apoptosis. J Innate Immun 8:299-313
Allen, Lee-Ann H (2014) Immunofluorescence and confocal microscopy of neutrophils. Methods Mol Biol 1124:251-68
Bandyopadhyay, Sarmistha; Long, Matthew E; Allen, Lee-Ann H (2014) Differential expression of microRNAs in Francisella tularensis-infected human macrophages: miR-155-dependent downregulation of MyD88 inhibits the inflammatory response. PLoS One 9:e109525
McCracken, Jenna M; Allen, Lee-Ann H (2014) Regulation of human neutrophil apoptosis and lifespan in health and disease. J Cell Death 7:15-23
Allen, Lee-Ann H (2013) Neutrophils: potential therapeutic targets in tularemia? Front Cell Infect Microbiol 3:109