Mechanisms of Neutrophil Exhaustion and Resolution

Sepsis is a systemic inflammatory response to infection, which may ultimately lead to multi-organ failure. Sepsis causes millions of deaths each year and creates tremendous financial burdens on the health care system, yet there is no effective cure for sepsis. Even years after the onset of sepsis, patients who have clinically recovered still die from sepsis-related complications due to chronic immune dysfunction. Neutrophils, the most dominant leukocytes in human circulatory systems, play a critical role in not only promoting inflammation to fight against microbe invasion but also facilitating inflammation resolution to restore immune homeostasis. While dysfunctional/exhausted neutrophils have been implicated in the long-term morbidity and mortality of sepsis, the cause of neutrophil exhaustion and the system to rejuvenate the dysregulated immunity are understudied. To fill in the missing piece here, we conducted our trilogy-like projects. First, we established an in vitro culture system to mimic sepsis-like conditions: murine neutrophils prolonged-stimulated with LPS exhibit exhaustion-related phenotype with the elevated expression of both proinflammatory and immunosuppressive makers on the cell surface as well as dysregulated swarming patterns. We found that by knocking out TRAM (TICAM2), an adaptive molecule regulating TLR4 downstream MyD88-independent signaling pathway, neutrophils exhibit attenuated exhaustion on both phenotypic and functional levels. Of note, TRAM contributes to the development of exhausted neutrophils through activating Src family kinases (SFKs)-STAT1 cascade, and deficiency in TRAM provides protective effects on systemic inflammation, reduces tissue injury, and improves survival in a murine colitis-induced sepsis model. Next, in my second project, we reported that neutrophils can be clustered into three subpopulations even at their naïve state based on the single-cell RNA sequencing (scRNAseq) analyses. Of note, neutrophils in one of the clusters are more mature but less apoptotic with the elevated expression of resolving-associated markers Cd86 and Cd200r, hence we termed these neutrophils as 'resolving neutrophils'. We found that the resolving neutrophil population can be expanded via pharmacologically reprogramming with sodium 4-phenylbutyrate (4-PBA) or genetic deletion of TRAM. Resolving neutrophils not only secrete more pro-resolving mediators, such as ResolvinD1 and SerpinB1, but also exert enhanced phagocytic and bactericidal capacities. Mechanistically, we discovered that the development of resolving phenotype in neutrophils is mediated by the PPARγ/LMO4/STAT3 signaling circuitry, which is constitutively suppressed by TRAM. To explore the translational applications of resolving neutrophils, in my third and final project, we conducted adoptive transfer experiments to examine the effects of TRAM-deficient resolving neutrophils in cecal slurry (CS)-induced septic mice. We found that TRAM-deficient mice are more resilient to severe sepsis with reduced tissue injury and less compromised lung integrity as compared to wild-type (WT) mice, and splenic neutrophils from TRAM deficient septic mice better preserve resolving-related features. Moreover, transfusing TRAM deficient neutrophils in WT septic mice renders therapeutic effects with alleviated lung and kidney damage. We also observed TRAM-deficient neutrophil-mediated resolving memory propagation in vitro to promote resolving features of neutrophils, monocytes, and T cells, as well as to strengthen endothelial cell barrier function. In terms of the mechanism, we reported that TRAM is critical for the secretion of neutrophil elastase, a potent protein to compromise endothelium; hence, endothelial cells cocultured with TRAM deficient neutrophils maintain higher levels of adhesion/tight junction markers than cocultured with WT neutrophils. Taken together, our trilogy projects better define exhausted and resolving neutrophils. And most importantly, our works demonstrate that TRAM, an underappreciated molecule, is responsible for inducing neutrophil exhaustion and suppressing resolving neutrophil generation.