[Phenotypic characteristics and functional alterations of lipopolysaccharide-induced low-density neutrophil]

To observe the generation of low-density neutrophil (LDN) induced by lipopolysaccharide (LPS) stimulation in vitro to simulate a septic environment, and to analyze their generation mechanism, phenotype, and functional characteristics.

1) Analysis of LDN proportion in septic patients: A case-control study was conducted. Peripheral blood samples were collected from 45 septic patients admitted to the intensive care unit (ICU) of Suzhou Municipal Hospital from June 2022 to January 2024, and from 32 age- and sex-matched healthy volunteers as the control group. The proportion of LDN in the peripheral blood mononuclear cell (PBMC) layer and among total neutrophils was analyzed. The expression of CD10, a marker of mature neutrophils, was detected by flow cytometry to analyze the proportion of immature LDN. 2) LPS-induced LDN generation and mechanism analysis: Neutrophils were isolated from the peripheral blood of healthy volunteers. They were stimulated with different concentrations of LPS (10, 100, 1 000 μg/L) for 4 hours. High-density neutrophil (HDN) and LDN were separated by Percoll density gradient centrifugation, and the proportion of LDN was calculated. Neutrophil extracellular trap (NET) levels in the culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Neutrophils from healthy volunteers were divided into HDN control, LDN control, LPS-stimulated HDN, and LPS-stimulated LDN groups. The LPS-stimulated groups were treated with LPS (1 000 μg/L) for 4 hours, while the control groups received an equal volume of culture medium. Morphological changes were observed under transmission electron microscope. Additionally, neutrophils from healthy volunteers were divided into control group (an equal volume of culture medium), LPS group (stimulated with 1 000 μg/L LPS for 4 hours), and phorbol 12-myristate 13-acetate (PMA) group (stimulated with 100 nmol/L PMA for 4 hours). NET formation was observed under scanning electron microscope. In another experiment, neutrophils from healthy volunteers were divided into LPS group (stimulated with 1 000 μg/L LPS for 4 hours) and GSK484 pretreatment group (pretreated with 50 nmol/L GSK484, a NET release inhibitor, for 30 minutes before LPS stimulation). LDN generation was detected by flow cytometry, and NET levels in the supernatant were detected by ELISA. 3) Phenotypic and functional analysis of neutrophils: Neutrophils from healthy volunteers were divided into the same four groups as described above (HDN control, LDN control, LPS-stimulated HDN, LPS-stimulated LDN groups). The expression of granulocyte maturation markers (CD66b, CD15, CD16), chemokine receptors (CXCR2, CXCR4), and cell activation markers (CD11b, CD62L) was detected by flow cytometry. Chemotactic function was assessed using an agarose chemotaxis model, and the chemotactic distance (CD), chemotactic cell ratio (CCR), and chemotactic index (CI) were analyzed.

1) Analysis results of LDN proportion in septic patients: The proportion of neutrophils in the PBMC layer (0.362±0.125), the proportion of LDN among total neutrophils (0.439±0.162), and the proportion of CD10^- LDN (0.222±0.093) in septic patients were higher than those in healthy controls (0.040±0.013, 0.014±0.004, and 0.005±0.002, respectively, all P<0.05). 2) LPS-induced LDN generation and mechanism analysis results: Transmission electron microscope showed increased surface protrusions and intracellular vacuoles in both HDN and LDN after LPS stimulation, particularly in the LPS-stimulated LDN group. After LPS stimulation, both the proportion of LDN and NET levels in the supernatant increased in a concentration-dependent manner (all P<0.05). Scanning electron microscope revealed that LPS stimulation induced NET formation in neutrophils, but to a lesser extent than PMA stimulation. GSK484 intervention inhibited LPS-induced LDN generation and NET release, with statistically significant differences compared with the LPS group [LDN count (×10⁵): 3.75±0.52 vs. 7.07±1.14, NET (A value): 0.96±0.14 vs. 1.36±0.27, both P<0.05]. 3) Results of phenotypic and functional analysis of neutrophils: Cell phenotype analysis revealed that compared with the control groups, after LPS stimulation, both HDN and LDN showed increased expression of CD66b and CD11b, and decreased expression of CD16, CXCR2, and CD62L. Compared with the LPS-stimulated HDN group, the LPS-stimulated LDN group exhibited higher expression of CD66b [mean fluorescence intensity (MFI): 16 424±2 074 vs. 9 470±1 201, P<0.05] and lower expression of CD16 (MFI: 3 647±1 458 vs. 7 815±1 143, P<0.05), while CD15 expression showed no significant change. Regarding cell chemokine receptors, compared with the LPS-stimulated HDN group, the LPS-stimulated LDN group showed decreased CXCR2 expression and increased CXCR4 expression. Regarding activation markers, compared with the LPS-stimulated HDN group, the LPS-stimulated LDN group showed increased CD11b expression (MFI: 11 684±2 131 vs. 7 782±373, P<0.05) and decreased CD62L expression [(59.75±7.42)% vs. (82.18±14.06)%, P<0.05]. Chemotaxis assays showed that after LPS stimulation, CD, CCR, and CI of HDN and LDN were all decreased, with more pronounced decreases in the LPS-stimulated LDN group than in the LPS-stimulated HDN group [CD (μm): 886.5±342.7 vs. 1 633.0±295.0, CCR: (0.69±0.25)% vs. (2.44±1.40)%, CI: (9.63±8.94)% vs. (34.62±8.31)%, all P<0.05].

LPS induces LDN generation through a NET formation-dependent pathway. This LDN subpopulation displays a unique phenotype characterized by high expression of CD66b, CXCR4, CD11b and low expression of CD16, CXCR2, CD62L, accompanied by chemotactic dysfunction.