Bacteria have specific metabolite profiles, including volatile organic compounds (VOCs), due to their distinct metabolism (2). Various bacterial metabolites interact with the host nervous and immune systems via receptor activation (4). The interaction between bacterial metabolites and the immune system have only recently started to be investigated, in comparison to the physical components of bacteria which represent pathogen-associated molecular patterns (5, 6). The relationship between bacterial metabolites and host immune responses has expanded as a new research field with a promise to establish strategies against bacterial infection.
Neutrophils are the most abundant leukocytes in the circulation and are the first immune cells that migrate to the sites of infection to kill pathogens through phagocytosis, as well as secretion of granules, reactive oxygen species (ROS), and neutrophil extracellular traps (NETs) (7). Immunocompromised and neutropenic patients are very susceptible to
In this study, we identified 2-undecanone as a major metabolite in the peritoneal fluid of
We attempted to identify metabolic VOCs produced
We identified 2-undecanone as a major VOC during
Many extracellular stimuli that induce neutrophil migration have been reported to use the Gai-type G protein-coupled receptors (GPCRs) (14). Preincubation of neutrophils with pertussis-toxin (PTX), which inhibits ADP-ribosylation of the Gai protein, significantly decreased migration towards 2-undecanone (Fig. 2E). Many extracellular stimulants that bind to PTX-sensitive GPCRs also stimulate PLC activity and increase the level of intracellular calcium (14). 2-Undecanone strongly elicited intracellular calcium increase in neutrophils, which was almost completely blocked by a PLC inhibitor (U-73122) but not by its inactive analog (U-73343) (Fig. 2F). The activation of several kinases such as ERK and Akt accompanies neutrophil activation by extracellular stimuli (15) and it was observed that 2-undecanone induced transient activation of ERK and Akt (Fig. 2G). Collectively, the results suggest that 2-undecanone stimulates neutrophils in a Gai-mediated signaling pathway and PLC-dependent calcium increase.
Since 2-undecanone is a representative VOC produced by
NET is one of the mechanisms of neutrophil-mediated bacterial killing (16).
We found that the phosphorylation of ERK and Akt was increased by 2-undecanone stimulation in neutrophils (Fig. 2G). As both ERK and Akt are important signaling molecules in LPS downstream signaling (19), we checked whether 2-undecanone affects the phosphorylation of ERK and Akt in response to LPS. 2-Undecanone decreased the phosphorylation induced by LPS (Fig. 4A). A previous report demonstrated that Akt acts as a switch between ROS-mediated NETosis and apoptosis. Blocking Akt activity induces cells to enter the apoptosis pathway, instead of entering NET formation, in a caspase-3 dependent manner (20). We found that LPS decreased the level of cleaved caspase-3, and increased an anti-apoptotic marker Bcl-2, which significantly showed the opposite effect in 2-undecanone treatment in neutrophils (Fig. 4B).
Next, we examined whether 2-undecanone affects cell death in the presence of LPS. It was observed that the viability of neutrophils was significantly decreased by 2-undecanone in the presence of LPS at two different time points of 2 h and 12 h after stimulation; however, 2-undecanone alone did not affect the viability of neutrophils (Fig. 4C).
This study provides an example of the immunoregulatory effects of a microbial metabolite on host immune cells. We identified 2-undecanone as a major VOC in systemic infection of
As the research of microbiota actively progresses, the interest in microorganism-derived metabolites has greatly increased. Recently, extensive effort has been expended on the identification of pathogen-derived molecules that affect the host immune system or host physiology. For example, 1-undecene, a VOC from
The growing interest in bacterial VOCs is derived from their roles in intra-kingdom to inter-kingdom and long-distance communication with other organisms (21). 2-Undecanone is a
In conclusion, we demonstrated that 2-undecanone is the major VOC produced from
8- to 10-weeks-old C57BL/6 male mice were purchased from Orient Bio Inc. (Seongnam, Korea). All experiments involving animals were carried out after receiving approval from the Institutional Review Committee for Animal Care and Use at Sungkyunkwan University (Suwon, Korea). Mouse bone marrow neutrophil enrichment was conducted according to a previous report (27). Isolated cells were over 95% Ly6G-positive by flow cytometry (BD FACSCanto II, Franklin Lakes, NJ, USA).
Chemotaxis assays were carried out according to a previous report (27). In the case of PTX treatment, neutrophils were incubated with PTX (100 ng/ml) in 2% FBS containing RPMI 1640 medium (Welgene, Gyeongsan, Korea) at 37°C and 5% CO2 for 4 h before the vehicle or 2-undecanone treatment.
2-Undecanone (4 mg/kg) and vehicle were injected to mice intraperitoneally. After 2 h of injection, mouse peritoneal fluid was collected, and peritoneal cells were stained with antibodies of CD11b and Ly6G (Thermo Fisher Scientific). Stained cells were analyzed by flow cytometry (FACSCanto II).
Degranulation was measured using a β-hexosaminidase assay, as previously reported (27). Briefly, isolated neutrophils were incubated with vehicle or 2-undecanone for 30 min. Both supernatants and lysates were incubated with the substrate solution at 37°C and 5% CO2 for 2 h. O.D. was measured at 405 nm using a spectrophotometer (BioTek, Winooski, VT, USA).
Mouse neutrophils stimulated with 2-undecanone were treated with 5 μM DCF-DA reagent (Thermo Fisher Scientific) in serum-free RPMI 1640 medium at 37°C for 30 min. The levels of cellular ROS were measured with flow cytometry (FACSCanto II).
Intracellular calcium measurement was carried out as previously reported (28). Briefly, fura-2 loaded neutrophils were stimulated with 2-undecanone or vehicle in the absence or presence of U-73122 or U-73343. The changes in fluorescence ratios (340:380 nm) were monitored using a spectrofluorophotometer (RF5301PC, SHIMADZU, Tokyo, Japan).
Mouse neutrophils were stimulated with
Mouse neutrophils (2 × 105 cells/ml) were seeded on 0.01% poly-L-lysine-coated 48-well plates. Neutrophils were incubated with 50 μg/ml of
Mouse neutrophils were resuspended in RPMI 1640 medium (5 × 106 cells/ml) and incubated with GFP-labeled, serum-opsonized
Mouse neutrophils were resuspended in RPMI 1640 medium (5 × 106 cells/ml) and incubated with serum-opsonized
Mouse neutrophils were stimulated with 2-undecanone for the indicated times. Neutrophil apoptosis was determined by the Annexin-V/PI staining kit (BD). The staining procedure was performed according to the manufacturer’s instructions. Co-staining was analyzed on a flow cytometer (FACSCanto II).
Cell lysates were prepared using RIPA sample buffer (iNtRON Biotechnology, Seongnam, Korea). Western blot analysis was conducted according to a previous report (6). Primary antibodies used were anti-phospho-ERK, anti-phospho-Akt, anti-total-Akt, anti-IκB, anti-p-p65, anti-p65, anti-cleaved caspase3, anti-Bcl-2, and anti-β-actin from Cell Signaling Technology (Beverly, MA, USA). The anti-TLR4 and anti-total-ERK were from Santa Cruz Biotechnology (Dallas, TX, USA). Secondary horseradish peroxidase-conjugated antibodies used were anti-mouse IgG and anti-rabbit IgG from Enzo Lifesciences (Farmingdale, NY, USA).
GraphPad Prism software was used to evaluate the results. All results are expressed as the mean ± SEM for the data obtained from the indicated number of experiments. Statistical analysis was performed using the Student’s
This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2020 M3A9D3038435, NRF-2020R1A6A3A13071682, NRF-2021R 1A2C1009258), and by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI20C0026).
The authors have no conflicting interests.