Variation in Neutrophil Function Among Healthy Subjects Influences Response to Bacteria

Introduction: We have recently shown that healthy subjects have variable neutrophil degranulation responses to antigen/antibody immune complexes (ICs; Duarte ME Blood 2019). With repeated testing over 2 years, we have shown that susceptibility to neutrophil activation represents a fixed phenotype: some healthy subjects have neutrophils which robustly degranulate in response to ICs, while others have neutrophils that are minimally reactive (>30-fold variation in release of MMP9, representative of tertiary granules). Whether this variation in degranulation responses is specific to ICs or is applicable to other stimulants is not known. Because neutrophils are involved in the first steps of host defense against bacterial pathogens, we aimed to determine if these phenotypes are associated with differences in neutrophil effector functions which may contribute to host defense.

Methods/Results: To determine if observed neutrophil phenotypes are relevant to host defense, we compared neutrophil effector responses from "high" and "low" donors after being challenged with bacterial products including the bacterial tripeptide N-formyl-met-leu-phe (fMLP), LPS, and Staphylococcus aureus (USA300) supernatant. As a model IC, we chose to use KKO-platelet factor 4/heparin ICs, as neutrophil responses to this IC have been previously characterized by our lab. As a first step, we examined several healthy subjects and compared degranulation responses to ICs, fMLP, and LPS to determine if susceptibility to degranulation was preserved. As shown in Figure 1, the quantity of MMP9 released in response to ICs was highly correlated with MMP9 released in response to fMLP (Fig 1A) and to LPS (Fig 1B, r = 0.9 for both). We then determined if neutrophil phenotypes were preserved in response to bacteria. As shown in Figure 2, neutrophils from "high" and "low" donors differentially degranulated in response to Staphylococcus aureus supernatant, with continued preservation of the neutrophil phenotype. We next aimed to determine if the neutrophil phenotype was associated with differences in other neutrophil effector functions, besides degranulation, which are relevant to pathogen defense. First, we focused on neutrophil extracellular trap (NET) release, which has an important role in host defense. As shown in Figure 3, "high" and "low" donors differed in NET release in response to ICs, as quantified by visualization using immunofluorescent microscopy. This finding is supported by our previous report that neutrophil phenotypes are correlated with differences in MPO release, a NET component (Duarte ME, et al 2019). We also examined reactive oxygen species (ROS) generation, a primary mechanism by which neutrophils eliminate pathogens. As shown in Figure 4, using DCFH-DA, a fluorescent probe, we show that "high" and "low" donors differ in ROS generation in response to PMA and fMLP.

Conclusions: Taken together, our studies demonstrate that neutrophil responses to a variety of stimuli (ICs, LPS, and fMLP) are highly concordant among healthy donors with "high" or "low" neutrophil phenotypes and that these differences in neutrophil effector functions are likely relevant to host defense. Further studies are needed to determine how differences in innate neutrophil function may affect clinical outcomes during bacterial infection.

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