Pro-inflammatory role of neutrophils populations in trauma patients: monitoring neutrophil populations

Abstract

Background: Trauma is a leading global cause of mortality, and systemic inflammatory response syndrome (SIRS) remains a significant complication, contributing to adverse outcomes. Neutrophils, as first responders to tissue injury, undergo substantial phenotypic and functional changes following trauma. This study investigates neutrophil subpopulations defined by CD16 and CD62L expression in trauma patients, focusing on their correlation with clinical biomarkers, trauma severity, and functional properties.

Methods: We included 50 non-infectious trauma patients, categorized into SIRS and Non-SIRS groups, and 43 elective surgery patients as controls. Neutrophil subsets were analyzed at two time points (TP1 and TP2) using flow cytometry. Functional assays evaluated phagocytosis, oxidative burst, mitochondrial function, and degranulation. Correlations between neutrophil subpopulations and clinical markers, including lactate, creatine kinase, Injury Severity Score, and Trauma and Injury Severity Score, were examined.

Results: Patients with SIRS exhibited higher proportions of banded neutrophils and CD16^lowCD62L^low neutrophils at TP1, alongside reduced levels of mature neutrophils. Elevated lactate and creatine kinase levels positively correlated with banded neutrophils and CD16^lowCD62L^low neutrophils, while negatively correlating with mature neutrophils CD16^highCD62L^high and hypersegmented neutrophils CD16^highCD62L^low. Hypersegmented neutrophils were more prevalent in Non-SIRS patients at TP1 and in SIRS patients at TP2. Banded neutrophils showed a positive correlation with Injury Severity Score and an inverse correlation with Trauma and Injury Severity Score (TRISS), whereas hypersegmented neutrophils were negatively associated with ISS and positively correlated with TRISS. These correlations likely reflect the pro-inflammatory role of banded neutrophils and the inflammation-resolving function of hypersegmented neutrophils. CD16^lowCD62L^low neutrophils displayed impaired phagocytosis, oxidative burst, and degranulation capacity, indicating functional deficiencies.

Conclusion: This study highlights the dynamic changes in neutrophil subpopulations in trauma and their association with systemic inflammation and clinical severity. Increased banded neutrophils correlate with SIRS and metabolic stress, whereas hypersegmented neutrophils may contribute to resolving inflammation. CD16^lowCD62L^low neutrophils exhibit functional impairments, warranting further investigation. Monitoring neutrophil subpopulations could aid in identifying trauma patients at risk for non-infectious SIRS and guide therapeutic interventions.

Keywords: ISS; SIRS; TRISS; creatine kinase; lactate; neutrophils; trauma.

Figure 1

Plots illustrating the gating strategy for neutrophil subpopulations. The initial gate was set on FSC-A and FSC-H to exclude doublets, followed by gating on CD45^high to eliminate debris, and FSC-H and SSC to exclude degranulated cells. Neutrophils were identified as CD45^highCD14^lowCD15^highCD193^low cells and further classified into subpopulations based on surface expression of CD62L and CD16: CD16^highCD62L^high mature neutrophils (MN), CD16^lowCD62L^high banded neutrophils (BN), CD16^highCD62L^low hypersegmented neutrophils, and CD16^lowCD62L^low neutrophils. Representative data from one healthy donor (HD), one Non-SIRS patient, and one SIRS patient were analyzed using the Kaluza software.

Figure 2

The percentages of neutrophil subpopulations for Non-SIRS patients, SIRS patients, and healthy controls. The percentage of (A) banded neutrophils (BN), (B) mature neutrophils (MN), (C) CD16^highCD62L^low hypersegmented neutrophils, and (D) CD16^lowCD62L^low neutrophils. Data were non-normally distributed (Shapiro-Wilk test), and statistical analysis was conducted using the Kruskal-Wallis test with Dunn’s correction for multiple comparisons (all vs. all); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.Sample sizes: N = 39 healthy controls, 33 Non-SIRS patients, and 16 SIRS patients.

Figure 3

Frequency of neutrophil subpopulations at two time points (TP1 and TP2). (A, B) The frequency of mature neutrophils (MN) increases, and the frequency of banded neutrophils (BN) decreases in SIRS patients at TP2. (C) The frequency of hypersegmented neutrophils (HSN) increases in Non-SIRS patients at TP1 and TP2. (D) The frequency of CD16^lowCD62L^low neutrophils increases in Non-SIRS patients at TP2 and remains elevated in SIRS patients. Data were non-normally distributed (Shapiro-Wilk test), and statistical analysis was conducted using the Kruskal-Wallis test with Dunn’s correction for multiple comparisons (all vs. all); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Sample sizes: TP1: N = 39 healthy controls, 33 Non-SIRS patients, 16 SIRS patients; TP2: N = 31 Non-SIRS patients, 13 SIRS patients.

Figure 4

CD marker expression for individual neutrophil subpopulations. Cell surface expression of neutrophil markers on different neutrophil subpopulations at TP1, including (A) CD16, (B) CD62L, (C) CD66b, (D) CD11b, (E) CD10, (F) CD181, and (G) CD182. CD16^lowCD62L^low neutrophils exhibit low expression of all analyzed markers. Decreased expression of CD10 and CD11b on banded neutrophils (BN). Data were non-normally distributed (Shapiro-Wilk test), and statistical analysis was conducted using the Kruskal-Wallis test with Dunn’s correction for multiple comparisons (all vs. all); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Sample size CD16, CD62L CD66b and CD11b: TP1: N = 33 Non-SIRS patients, 16 SIRS patients; CD10: TP1: N = 32 Non-SIRS patients, 9 SIRS patients; CD181: TP1: N = 39 Non-SIRS patients, 11 SIRS patients; CD182: TP1: N = 31 Non-SIRS patients, 12 SIRS patients.

Figure 5

Neutrophil Functional Test: Ingestion capacity in neutrophil subpopulations of FITC-labeled E. coli. Normal ingestion capacity of FITC-labeled E. coli in Non-SIRS and SIRS patients at TP1. (A) CD16^lowCD62L^low neutrophils exhibit reduced ingestion capacity of E. coli (B, C) compared to mature neutrophils (MN). The functional properties of both populations remained unchanged over time (TP1 and TP2) (D). The data are presented as the mean fluorescence intensity of FITC-labeled E. coli. Data were non-normally distributed (Shapiro-Wilk test), and statistical analysis was conducted using the Kruskal-Wallis test with Dunn’s correction for multiple comparisons (all vs. all); ****p ≤ 0.0001. Sample sizes: Ingestion of E. coli (MFI E. coli FITC): TP1, TP2: N = 35 healthy controls, 12 Non-SIRS patients, 6 SIRS patients.

Figure 6

Neutrophil functional test: measurement of oxidative burst in neutrophil subpopulations following Staphylococcus aureus stimulation. Reduced oxidative burst capacity in neutrophils from SIRS patients was observed. (A) The analysis compared the mean fluorescence intensity of rhodamine (MFI DHR) in total neutrophils following Staphylococcus aureus (SA) stimulation. CD16^lowCD62L^low neutrophils demonstrate a reduced oxidative burst response to SA stimulation (B–D) compared to mature neutrophils (MN). The data are presented as the mean fluorescence intensity of rhodamine (MFI DHR) following Staphylococcus aureus stimulation. The functional properties of both populations remained unchanged over time (TP1 and TP2) (B, C). Data were non-normally distributed (Shapiro-Wilk test), and statistical analysis was conducted using the Kruskal-Wallis test with Dunn’s correction for multiple comparisons (all vs. all); *p ≤ 0.05, ****p ≤ 0.0001. oxidative burst (MFI DHR): TP1: N = 29 healthy controls, 24 Non-SIRS patients, 8 SIRS patients; TP2: N = 29 healthy controls, 20 Non-SIRS patients, 6 SIRS patients.

Figure 7

Alterations in CD marker expression among specific neutrophil subpopulations following Staphylococcus aureus stimulation. CD16^lowCD62L^low neutrophils demonstrate significant functional impairments, as evidenced by their reduced degranulation and activation capacity. These neutrophils exhibit a diminished change in CD66b (A), CD11b (B), and CD10 (C) expression compared to mature neutrophils (MN) following Staphylococcus aureus (SA) stimulation, as observed in both patients and healthy controls. In contrast, CD16 expression is comparable between CD16^lowCD62L^low neutrophils and mature neutrophils after SA stimulation (D). Data were non-normally distributed (Shapiro-Wilk test), and statistical analysis was conducted using the Kruskal-Wallis test with Dunn’s correction for multiple comparisons (all vs. all); *p ≤ 0.05, ****p ≤ 0.0001. Sample sizes: TP1: N = 29 healthy controls, 24 Non-SIRS patients, and 8 SIRS patients. S = stimulated; UNS = unstimulated.

Figure 8

Mitochondrial characteristics of neutrophil subpopulations. CD16^lowCD62L^low neutrophils exhibit reduced production of mitochondrial superoxide (MTS) compared to mature neutrophils (MN) (A). (B) Histogram showing mitochondrial superoxide production after Staphylococcus aureus (SA) stimulation in MN neutrophils. (C) Histogram showing mitochondrial superoxide production after SA stimulation in CD16^lowCD62L^low neutrophils. (D) All examined neutrophil subpopulations in Non-SIRS and SIRS patients exhibit comparable mitochondrial content (MFI MTG) and mitochondrial membrane potential (MFI MTP) (E), which differ from those of dead cells. Data were non-normally distributed (Shapiro-Wilk test), and statistical analysis was conducted using the Kruskal-Wallis test with Dunn’s correction for multiple comparisons (all vs. all); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Sample sizes: N = 7 Non-SIRS patients, 5 SIRS patients. S = stimulated; UNS = unstimulated.