Selective Modulation of Neutrophils in Critical Illness

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Project Summary Caring for the critically ill accounts for 5-10% of all U.S. healthcare spending, while in-hospital mortality remains high (20-40%). Yet, there are currently no effective therapeutic interventions beyond supportive care and antibiotics. The development of new therapeutic strategies requires filling important gaps in our knowledge of the pathophysiological mechanisms underlying immune dysfunction in the critically ill. Hemorrhagic shock induces a systemic response that results in immune dysfunction, rendering the host susceptible to severe secondary infections that are a significant cause of morbidity and mortality in hospital ICUs. The focus of the research program outlined in this MIRA renewal is in understanding how hemorrhagic shock alters the response of neutrophils, the essential mediators of acute host defense against bacteria/fungi. When dysregulated, neutrophils prematurely deploy their antimicrobial arsenal of cytotoxic proteases and reactive oxidants, directly injuring host tissue. Following hemorrhagic shock, neutrophils become “primed” and subsequently induce acute lung injury, coupled with a significant deficit in their capacity to find and fight infection. Our long-term goals are to understand the mechanisms of neutrophil dysfunction that occurs as a result of hemorrhagic shock and to identify targets for correcting aberrant neutrophil function. Our studies will interrogate three aspects of the neutrophil response: priming, trafficking, and antimicrobial function. We have established an innovative approach for the rapid generation of neutrophil-specific chimeric mice by transplantation of conditionally-immortalized neutrophil progenitor cell lines. Importantly, this technique does not require animal irradiation and yields robust chimerism of donor-derived neutrophils (>40%) that are functionally equivalent to endogenous neutrophils. This system is genetically tractable, enabling in vivo studies into the mechanistic aspects of neutrophil dysfunction following hemorrhagic shock. In Project 1, we will continue using our clinically relevant “two-hit” mouse model of critical illness through hemorrhagic shock and secondary respiratory infection. We will determine the mechanisms underlying the dysregulation of specific subset of neutrophil effector functions (e.g., reactive oxidant generation, degranulation) in response to hemorrhagic shock, evaluating the role of novel genes in acute lung injury and bacterial clearance. In Project 2, we will employ our microfluidic platform to investigate the mechanisms of neutrophil sequestration in model pulmonary capillaries. As the lungs are particularly susceptible to neutrophil-mediated injury, this model provides a means to evaluate new strategies for modulating aberrant neutrophil trafficking that contributes to acute lung injury. In Project 3, we will pursue several targets for enhancing the antimicrobial function of neutrophils without exacerbating injury to host tissue, focusing on methicillin-resistant Staphylococcus aureus. Together, these integrated approaches will address major knowledge gaps in neutrophil pathobiology by spanning the molecular, cellular, and organism/disease levels of experimentation.