Sepsis Induces a Dysregulated Neutrophil Phenotype That Is Associated with Increased Mortality

Abstract

Background: Neutrophil dysfunction in sepsis has been implicated in the pathogenesis of multiorgan failure; however, the role of neutrophil extracellular traps (NETs) remains uncertain. We aimed to determine the sequential changes in ex vivo NETosis and its relationship with mortality in patients with sepsis and severe sepsis.

Methods: This was a prospective observational cohort study enrolling 21 healthy age-matched controls and 39 sepsis and 60 severe sepsis patients from acute admissions to two UK hospitals. Patients had sequential bloods for the ex vivo assessment of NETosis in response to phorbol-myristate acetate (PMA) using a fluorometric technique and chemotaxis using time-lapse video microscopy. Continuous data was tested for normality, with appropriate parametric and nonparametric tests, whilst categorical data was analysed using a chi-squared test. Correlations were performed using Spearman's rho.

Results: Ex vivo NETosis was reduced in patients with severe sepsis, compared to patients with sepsis and controls (p = 0.002). PMA NETosis from patients with septic shock was reduced further (p < 0.001) compared to controls. The degree of metabolic acidosis correlated with reduced NETosis (p < 0.001), and this was replicated when neutrophils from healthy donors were incubated in acidotic media. Reduced NETosis at baseline was associated with an increased 30-day (p = 0.002) and 90-day mortality (p = 0.014) in sepsis patients. These findings were accompanied by defects in neutrophil migration and delayed apoptosis. Resolution of sepsis was not associated with the return to baseline levels of NETosis or migration.

Conclusions: Sepsis induces significant changes in neutrophil function with the degree of dysfunction corresponding to the severity of the septic insult which persists beyond physiological recovery from sepsis. The changes induced lead to the failure to effectively contain and eliminate the invading pathogens and contribute to sepsis-induced immunosuppression. For the first time, we demonstrate that reduced ex vivo NETosis is associated with poorer outcomes from sepsis.

Figure 1

Consort diagram of experiments. Modified consort diagram of the number of experiments performed at the various time points during the study. The reducing number of experiments performed between the groups was due to either deaths, discharges from hospital, or refusals of blood sampling from patients.

Figure 2

The ex vivo production of NETs in healthy controls and patients with sepsis and severe sepsis. (a) NETosis from 21 healthy controls, 39 patients with sepsis, and 60 patients with severe sepsis following stimulation with 25 nM PMA for 4 hours ex vivo. An ANOVA between all 3 groups showed a significant difference where p = 0.002, with ∗ representing the significant differences in a post hoc Tukey's test. (b) NETosis from 21 healthy controls, 30 patients with sepsis, and 39 patients with severe sepsis on day 4 following stimulation with 25 nM PMA for 4 hours ex vivo. An ANOVA between all 3 groups showed a significant difference (p = 0.001), with ∗ representing significant differences in a post hoc Tukey's test. On day 7, there are 21 sepsis patients and 29 severe sepsis patients. There was a significant difference between healthy controls and patients with severe sepsis^∗∗.

Figure 3

PMA-induced NET release is lower in nonsurvivors of sepsis at 30 days and 90 days. NET production in recruited patients on enrollment in response to PMA in survivors and nonsurvivors of sepsis at 30 days, 90 days, and 360 days following admission. Bars represent the mean with error bars the SEM, with p values from a Student's t-test.

Figure 4

The impact of acidosis on neutrophil extracellular trap formation. (a) Correlation between standardized base excess and neutrophil extracellular trap formation in 99 patients with sepsis. Spearman's rho suggests that reduction in NET formation is associated with worsening acidosis. (b) Effect of acidosis on the NETs generated from the neutrophils of 5 healthy donors.