DOCK8 inhibits the immune function of neutrophils in sepsis by regulating aerobic glycolysis

Abstract

Introduction: This study endeavored to investigate the role of DOCK8 in modulating the immune function triggered by sepsis.

Methods: Expression of DOCK8 in the whole blood of sepsis patients and its enrichment pathways were assayed by bioinformatics. Pearson analysis was used to predict the relationship between glycolytic signaling pathway and its relevance to neutrophil function in sepsis. A sepsis mouse model was then built by performing cecal ligation and puncture treatment on male mice. Neutrophils were isolated, and their purity was tested by flow cytometry. Neutrophils were then stimulated by lipopolysaccharide to build a sepsis cell model. Next, quantitative reverse transcription polymerase chain reaction and CCK-8 were applied to test the expression of DOCK8 and cell viability, western blot to assay the expression of HK-2, PKM2, and LDHA proteins, ELISA to measure the concentrations of TNF-α, IL-1β, and IL-6, Transwell to detect the chemotaxis of neutrophils and flow cytometry to detect the phagocytic activity of neutrophils. Finally, in different treatment groups, we used Seahorse XF 96 to analyze the extracellular acidification rate (ECAR) of sepsis cells and used enzyme-linked immunosorbent assay to detect the contents of pyruvic acid, lactic acid, and ATP in sepsis cells.

Results: DOCK8 was downregulated in sepsis blood and activated neutrophils. Aerobic glycolysis was positively correlated with sepsis. Activated neutrophils promoted the expression of inflammatory factors TNF-α, IL-1β, and IL-6. Low expression of DOCK8 facilitated the proliferation, chemotaxis, and phagocytic activity of sepsis cells and promoted the expression of inflammatory factors. Bioinformatics analysis revealed that DOCK8 was enriched in the glycolytic signaling pathway. Low expression of DOCK8 induced ECAR, promoted the protein expression of HK-2, PKM2 and LDHA, and favored the increase of pyruvate, lactate, and ATP contents. While 2-DG treatment could restore these effects.

Conclusion: DOCK8 may inhibit sepsis-induced neutrophil immune function by regulating aerobic glycolysis and causing excessive inflammation, which helps to explore potential therapeutic targets.

Keywords: DOCK8; aerobic glycolysis; immune function; neutrophils; sepsis.

Figure 1

Low expression of DOCK8 in Sepsis. (A) Bioinformatics analysis of DOCK8 expression in sepsis blood; (B) DOCK8 expression in sepsis mouse blood. All experiments were repeated three times. ***p < .001.

Figure 2

Reduced expression of DOCK8 in neutrophils during sepsis. (A) Flow cytometry analysis of neutrophil purity; (B–D) ELISA analysis of TNF‐α, IL‐1β, and IL‐6 concentrations in cells; (E) qRT‐PCR analysis of DOCK8 expression. All experiments were repeated three times. ***p < .001. ELISA, enzyme‐linked immunosorbent assay; IL, interleukin; qRT‐PCR, quantitative reverse transcription polymerase chain reaction; TNF‐α, tumor necrosis factor α.

Figure 3

DOCK8 inhibition of neutrophil immune function in sepsis. (A) qRT‐PCR detection of DOCK8 expression; (B–D) ELISA detection of TNF‐α, IL‐1β, and IL‐6 concentrations in cells; (E) qRT‐PCR detection of transfection efficiency; (F–G) CCK‐8 detection of cell viability; (H–J) ELISA detection of TNF‐α, IL‐1β, and IL‐6 concentrations in cells; (K) transwell assay for neutrophil chemotaxis; (L) elow cytometry detection of phagocytosis; All experiments were repeated three times. **p < .01, ***p < .001. CCK‐8, Counting Kit‐8; ELISA, enzyme‐linked immunosorbent assay; IL, interleukin; qRT‐PCR, quantitative reverse transcription polymerase chain reaction; TNF‐α, tumor necrosis factor α.

Figure 4

DOCK8 suppresses the immune function of neutrophils in sepsis through aerobic glycolysis inhibition. (A) Enrichment analysis of DOCK8 pathway; (B) Pearson analysis was used to predict the relationship between glycolytic signaling pathway and its relevance to neutrophil function in sepsis; (C) CCK‐8 assay for cell viability; (D) western blot analysis of the expression levels of specific genes (HK2, PKM2, and LDHA) related to glycolytic metabolism pathway; (E) seahorse XF 96 analyses of ECAR; (F) detection of the levels of pyruvate, lactate, and ATP in sepsis cells of each treatment group; (G) ELISA detection of TNF‐α, IL‐1β, and IL‐6 concentrations in cells; (H) transwell assay was utilized to measure the chemotactic effect of Neutrophils; and (I) flow cytometry analysis of phagocytic function. All experiments were repeated three times. **p < .01, ***p < .001. CCK‐8, Counting Kit‐8; ELISA, enzyme‐linked immunosorbent assay; IL, interleukin; qRT‐PCR, quantitative reverse transcription polymerase chain reaction; TNF‐α, tumor necrosis factor α.