Neutrophil-derived trail is a proinflammatory subtype of neutrophil-derived extracellular vesicles

Abstract

Aims: Extracellular vesicles (EVs) are membrane-derived vesicles that mediate intercellular communications. Neutrophils produce different subtypes of EVs during inflammatory responses. Neutrophil-derived trails (NDTRs) are generated by neutrophils migrating toward inflammatory foci, whereas neutrophil-derived microvesicles (NDMVs) are thought to be generated by neutrophils that have arrived at the inflammatory foci. However, the physical and functional characteristics of neutrophil-derived EVs are incompletely understood. In this study, we aimed to investigate the differences between NDTRs and NDMVs. Methods: The generation of neutrophil-derived EVs were visualized by live-cell fluorescence images and the physical characteristics were further analyzed using nanotracking analysis assay, scanning electron microscopic analysis, and marker expressions. Functional characteristics of neutrophil-derived EVs were analyzed using assays for bactericidal activity, monocyte chemotaxis, phenotype polarization of macrophages, and miRNA sequencing. Finally, the effects of neutrophil-derived EVs on the acute and chronic inflammation were examined in vivo. Results: Both EVs share similar characteristics including stimulators, surface marker expression, bactericidal activity, and chemoattractive effect on monocytes via MCP-1. However, the integrin-mediated physical interaction was required for generation of NDTRs whereas NDMV generation was dependent on PI3K pathway. Interestingly, NDTRs contained proinflammatory miRNAs such as miR-1260, miR-1285, miR-4454, and miR-7975, while NDMVs contained anti-inflammatory miRNAs such as miR-126, miR-150, and miR-451a. Although both EVs were easily uptaken by monocytes, NDTRs enhanced proinflammatory macrophage polarization whereas NDMVs induced anti-inflammatory macrophage polarization. Moreover, NDTRs showed protective effects against lethality in a murine sepsis model and pathological changes in a murine chronic colitis model. Conclusion: These results suggest that NDTR is a proinflammatory subtype of neutrophil-derived EVs distinguished from NDMV.

Keywords: EV, extracellular vesicle; NDMV, neutrophil-derived microvesicle; NDTR, neutrophil-derived trail.

Figure 1

Characterization of NDTRs. (A-B) Various stimulators induce formation of both NDTRs and NDMVs (A) Representative time-lapse images of EVs released from neutrophils. Neutrophils were stained with cell-tracker, stimulated with the indicated stimulators, and visualized using immunofluorescence microscopy for 1 h. The time denotes minutes after stimulation. The arrows indicate elongated uropods. The arrowheads indicate deposited neutrophil-derived EVs. All data are representative of more than three independent experiments with n = 3 per each group. (B) Quantification of relative amounts of generated neutrophil-derived EVs. Neutrophils were stained with calcein-AM and stimulated with the indicated stimulators. n = 4-7 per group. Relative fluorescence normalized to the fluorescence of EVs isolated from unstimulated neutrophils. (C) Representative density plots and bar graph for sizes and concentrations of neutrophil-derived EVs. The size distribution and concentration of neutrophil-derived EVs was measured using Nanosight tracking analysis (NTA). n = 3 per group. (B-C) The data shown are the mean ± SEM. (B-C) ***P < 0.001 vs control. (D) *P < 0.05; **P < 0.01; ***P < 0.001. (D-E) Scanning electron microscopic analysis of neutrophil-derived EVs. Neutrophils were stimulated with fMLP (1 µM) and neutrophil-derived EVs were separated. Neutrophil-derived EVs were coated on filter membrane and visualized with scanning microscopy. (D) Representative picture of NDTRs. The size of particle ranged up to 502 nm. (E) Representative picture of NDMVs. The size of particle ranged up to 570 nm.

Figure 2

NDTRs exert bactericidal activity via ROS- and granule-dependent pathway. (A) Schematic depiction of experimental design. (B) Bactericidal activity of neutrophil-derived EVs. Opsonized E. coli or S. aureus were exposed to neutrophil-derived EVs separated from neutrophils stimulated with respective bacteria. (C) Bactericidal activity of remnant neutrophils (RNs) after EV formations. RNs were recovered after generation of neutrophil-derived EVs. (D-E) The effects of inhibitors on the specific pathways for bactericidal activity by neutrophil-derived EVs. Bacteria were exposed to neutrophil-derived EVs in presence of absence of indicated inhibitors. Veh, DDW; DPI, an inhibitor of NDAPH oxidase, 10 µM; ProI, protease inhibitor cocktail, 10 µM; DNase, DNase I, 10 µM. The data are pooled from three independent experiments (n = 4-7 per each group) and shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

NDTRs induce monocyte chemotaxis via an MCP-1-dependent pathway. (A-C) Neutrophils were stimulated with indicated stimulators and EVs were isolated. The lane was coated with isolated neutrophil-derived EVs and monocytes were allowed to migrate toward isolated neutrophil-derived EVs. The distances traveled by migrating cells were tracked on every minute for 45 min. Representative tracking results of thirty cells per each group are presented. (A) Monocyte migration tracking analysis. (B) The effects of MCP-1 inhibitor on chemotaxis of monocytes against neutrophil-derived EVs. Upper panels, monocytes were allowed to migrate toward MCP-1 (100 ng/mL) in the presence or absence of CCR1 antagonist. Middle and bottom panels, monocytes were allowed to migrate toward neutrophil-derived EVs in the presence of CCR2 antagonist (+MCP-1 inhibitor, 1 µg/mL). (C) Mean distance and velocity of monocytes traveled towards neutrophil-derived EVs. The data are shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. All data are representative of three independent experiments (n = 3-4 per group).

Figure 4

NDTRs induce proinflammatory phenotype polarization of macrophages. (A-B) Phagocytosis of neutrophil-derived EVs by M0-differentiated THP-1 cells. (A) Time-lapse images of M0-differentiated THP-1 cells acquiring neutrophil-derived EVs. Microfluidic chamber was coated with neutrophil-derived EVs and M0-differentiated THP-1 cells were allowed to phagocytose. Green, neutrophil-derived EVs stained with Cell Tracker Green; Arrowheads, neutrophil-derived EVs attached to the plates; Arrows, neutrophil-derived EVs phagocytosed by M0-differentiated THP-1 cells. Representative images of three independent experiments. (B) Flow cytometric analysis showing the uptake of neutrophil-derived EVs by M0-differentiated THP-1 cells. n = 3 per group. (C) Schematic depiction of experimental design for macrophage phenotype polarization. (D-E) The expressions of markers for M1 and M2 in M0-differentiated THP-1 cells exposed to neutrophil-derived EVs (n = 3-4 per each group). (D) Fold changes in the expression levels of cytokines in M0-differentiated THP-1 cells exposed to neutrophil-derived EVs using qPCR (E) The expression levels of surface markers in M0-differentiated THP-1 cells exposed to neutrophil-derived EVs using flow cytometry. The data are shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 compared to control.

Figure 5

Proinflammatory miRNA profiles in NDTRs. (A) Hierarchical clustering of differentially expressed miRNAs in NDTRs and NDMVs. The miRNA profiles of NDTRs and NDMVs (n = 6 per group) clustered. Cluster analysis based on log10-transformed data. A red color represents relatively higher expression and a green color represents relatively lower expression. (B) Principal component analysis (PCA) plot of the miRNA expression profiles of NDTRs and NDMVs. (C) Summary of selected miRNAs differentially expressed in NDTRs and NDMVs. (D) Validation of selected miRNAs in NDTRs and NDMVs using RT-PCR (n = 10 per each group). (E) The effects of miRNA mimics (miR-1260a, miR-1285-5p, miR-4454, miR-7975, miR-126-3p, miR-150p, and miR-451a) on the expressions of iNOS and arginase in M0-differentiated THP-1 cells (n = 3 per each group). The data are shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6

Protective effects of NDTRs on murine models of acute and chronic inflammation. (A) Effects of NDTRs and NDMVs in a murine model of sepsis. Experimental sepsis was induced by CLP. Survival rates of septic mice after the administration of neutrophil-derived EVs. BALB/c mice were administered an i.p. injection of either NDTRs (n = 8), NDMVs (n = 12) or vehicle (saline, n = 12) 1 h before surgery and on days 1 and 3 after surgery. *P < 0.05 compared to vehicle. (B) The expressions of markers for M1 and M2 in peritoneal macrophages isolated from septic mice injected with neutrophil-derived EVs. (C) The cytokine levels of peritoneal fluid isolated from septic mice injected with neutrophil-derived EVs. (D-F) The effects of NDTRs and NDMVs in a murine model of chronic colitis. Mice were administered with two cycles of 2% DSS for 5 days. At the beginning of the second cycle, mice were treated with NDTRs and NDMVs on every 2 days. n = 4 per each group. (D) Percentage of initial weight. (E-F) Mice were sacrificed on day 22 and subjected to evaluation. (E) Left panel, colon length. Right panel, representative photographs of the colon and cecum. (F) Macroscopic colonic damage. Left panel, histological score. Right panel, representative images of hematoxylin and eosin (H&E) staining. The arrows indicate crypt damage and inflammatory cell infiltration. The data are shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.