Immune suppressive activities of low-density neutrophils in sepsis and potential use as a novel biomarker of sepsis-induced immune suppression

Data of low-density neutrophils (LDN), the neutrophils in the peripheral blood mononuclear cells (PBMC) fraction, in sepsis is still less. As such, LDN (CD66b-positive cells in PBMC) was highest in intensive care unit (ICU) patients with sepsis (n=24) compared with non-sepsis (n=10) and healthy control (n=20), with a negative correlation with lymphocyte count and could predict secondary infection and mortality with the area under the curve (AUC) at 0.79 and 0.84, respectively. Compared with sepsis normal-density neutrophils (NDN), sepsis-LDN demonstrated higher expression of CD66b, CD63, CD11b, and CD184, but lower expression of CD62L and CD182 and defects of effector functions, including phagocytosis and apoptosis. The t-distributed stochastic neighbor embedding (t-SNEs) demonstrated high program cell death ligand-1 (PD-L1) in sepsis-LDN. In sepsis samples, the T cell proliferation in PBMC (T cells with LDNs) was lower than that in the isolated T cells (T cells alone) and incubation of anti-PD-L1 neutralizing antibody, but not a reactive oxygen species (ROS) scavenger (N-acetyl cysteine), improved the T cell suppression. Additionally, 30 min lipopolysaccharide (LPS) activation altered healthy control NDN into LPS-LDN (reduced density) and LPS-NDN (maintain density) with similarly elevated CD66b, CD11B, and CD62L. However, LPS-LDN (in vitro LDN) showed lower expression of CD63, CD184, and PD-L1 compared with LDN from patients (sepsis-LDN), suggesting a partial LPS impact on LDN generation. From the microscopic-based method (Wright's staining in PBMC), sepsis-LDN demonstrated a mixed population of mature and immature cells with a good correlation with the flow-based analysis (Bland-Altman analysis and AUC). In conclusion, LDN in sepsis, partly generated by LPS activation, was associated with secondary infection and T cell suppression, mainly through the expression of PD-L1, which might be an immune suppression biomarker, especially with a less expensive microscopic-based method.