Gut Microbiota-Derived Small Extracellular Vesicles Endorse Memory-like Inflammatory Responses in Murine Neutrophils

Abstract

Neutrophils are classically characterized as merely reactive innate effector cells. However, the microbiome is known to shape the education and maturation process of neutrophils, improving their function and immune-plasticity. Recent reports demonstrate that murine neutrophils possess the ability to exert adaptive responses after exposure to bacterial components such as LPS (Gram-negative bacteria) or LTA (Gram-positive bacteria). We now ask whether small extracellular vesicles (EVs) from the gut may directly mediate adaptive responses in neutrophils in vitro. Murine bone marrow-derived neutrophils were primed in vitro by small EVs of high purity collected from colon stool samples, followed by a second hit with LPS. We found that low-dose priming with gut microbiota-derived small EVs enhanced pro-inflammatory sensitivity as indicated by elevated levels of TNF-α, IL-6, ROS and MCP-1 and increased migratory and phagocytic activity. In contrast, high-dose priming resulted in a tolerant phenotype, marked by increased IL-10 and decreased transmigration and phagocytosis. Alterations in TLR2/MyD88 as well as TLR4/MyD88 signaling were correlated with the induction of adaptive cues in neutrophils in vitro. Taken together, our study shows that small EVs from stools can drive adaptive responses in neutrophils in vitro and may represent a missing link in the gut-immune axis.

Keywords: inflammation; memory-like; microbiota; neutrophils; phagocytosis; priming; small EVs; transmigration.

Figure 1

Schematic illustration of the isolation and purification process of small EVs from murine stool and their characterization. (A) Small EVs (sEV) isolated from the stool of mice from cecum and colon using qEV columns. Analysis of sEV concentration and their size distribution were determined by TRPS and TEM (B,C). Total protein concentration (D) was determined using the Pierce™ 660 nm Protein Assay Kit. Determination of endotoxin amount (E) was performed by Pierce LAL Chromogenic Endotoxin Quantification kit assay, whereas the amount of LTA (F) was measured by Mouse lipoteichoic acids (LTA) ELISA Kit. Protein expression of β-actin (G) was analyzed by Western blotting. Data are shown as scatter dot plots, mean + SEM (repeated measurements).

Figure 2

Cytokine responses with increasing concentrations of small EVs. (A) Bone marrow neutrophils were primed in a protein concentration-dependent manner with microbiota-derived small EVs ((B,C); n = 4) for 45 min and later after resting using the two-step protocol, re-challenged by 100 ng/mL LPS for 4 h expressing adaptive manners ((D,E); n = 5; normalized to total protein concentration). The cytokine production of TNF-α and IL-6 was measured using ELISA. Data are presented as scatter dot plots, mean + SEM, # p < 0.05, # versus unstimulated state (US) (B,C); # p < 0.05, # versus unprimed state (UP, gray bar) (D,E).

Figure 3

Role of microbiota-derived small EVs altering pro- and anti-inflammatory mediators in bone marrow neutrophils. Murine bone marrow neutrophils were primed for 45 min by microbiota-derived small EVs (low concentration: 1 ng/mL; high concentration: 28,1 µg/mL) and re-challenged with 100 ng/mL LPS for 4 h on day 2. Production of (A) ROS (n = 7) was determined by DCFDA assay, whereas (B) MCP-1 (n = 7) and (C) IL-10 (n = 7) were measured by ELISA (normalized to total protein concentration). Real-time qPCR was used to analyze the gene expression of (D) IL-10 (n = 3; unprimed state assigned as 1.0). Data are presented as scatter dot plots, mean + SEM, # p < 0.05, # versus unprimed condition (UP, gray bar).

Figure 4

Signaling events behind gut microbiota-derived small EVs in murine neutrophils. Murine bone marrow neutrophils were primed for 45 min by microbiota-derived small EVs (low concentration: 1 ng/mL; high concentration: 28.1 µg/mL) and later, on day 2, were re-challenged by 100 ng/mL LPS for 4 h as described above. Protein expression of TLR2 (A, n = 5), TLR4 (B, n = 5) and MyD88 (C, n = 5) were assayed by Western blotting and quantified (unprimed cells assigned as 1.0). Data are presented as scatter dot plots, mean + SEM, # p < 0.05, # versus unprimed condition (UP, gray bar).

Figure 5

Microbiota-derived small EVs reshape migratory effects and phagocytic activity in murine neutrophils. Neutrophils were primed for 45 min by microbiota-derived small EVs (low concentration: 1 ng/mL; high concentration: 28.1 µg/mL) and re-challenged by 100 ng/mL LPS for 4 h on day 2. Migratory activities of neutrophils (A, n = 6) were analyzed using commercially available kits as described above and data were shown as relative fluorescence units (RFU), whereas real-time qPCR was used to analyze the gene expression of (B) CD11a (n = 4) and (D) CD32 (n = 4) (unprimed state assigned as 1.0). Phagocytic activity (C, n = 7) of murine neutrophils was determined by commercially available kits as described above and data were expressed as optical density (OD) values. Data are presented as scatter dot plots, mean + SEM, # p < 0.05, # versus unprimed condition (UP, gray bar).