Effect of Leptospira interrogans 56606v on apoptosis of murine neutrophil

doi:10.3969/j.issn.1674-8115.2022.11.001

Abstract

Abstract:

Objective ·To investigate the effect of Leptospira interrogans 56606v on apoptosis of murine neutrophils and related mechanisms. Methods ·Murine neutrophils were infected with Leptospira interrogans 56606v at indicated multiplicity of infection (MOI was 100, 300 and 500 respectively) and time points (0.5,6,12 and 24 h), with uninfected neutrophils as control. Cells treated with an equal amount of cell culture medium containing 4 μmol/L astrosporine (STS) were used as positive control for apoptosis. The apoptosis was analyzed by using flow cytometry. The cleavage of caspase-3 and activation of nuclear factor-κB (NF-κB) pathway were detected in murine neutrophils infected with Leptospira interrogans 56606v by Western blotting, and the inhibitory effect of NF-κB inhibitor BAY 11-7082 on NF-κB pathway was also verified by Western blotting. The role of NF-κB pathway in the inhibition of neutrophil apoptosis induced by Leptospira interrogans 56606v was further explored by adding NF-κB inhibitor BAY 11-7082. Macrophages were co-incubated with Leptospira-infected or-uninfected carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled neutrophils, and flow cytometry was used to detect the phagocytosis of neutrophils by macrophages to reflect the effect of the neutrophil apoptosis inhibition induced by Leptospirainterrogans 56606v infection on efferocytosis. Results ·The results of flow cytometry showed that the apoptosis rate of neutrophils in Leptospira infection group was decreased compared with the uninfected control group. The inhibition of Leptospirainterrogans 56606v on neutrophil apoptosis was enhanced with the increase of MOI. Compared with the uninfected control group, the difference was statistically significant under different MOIS (P=0.000). The apoptosis rate was lower than that of the corresponding uninfected control group when Leptospirainterrogans 56606v with MOI=100 infected neutrophils for 0.5, 6 and 12 h (P<0.05), and the apoptosis of neutrophils was enhanced after treatment with STS. The cleavage of caspase-3 was significantly reduced after infection with Leptospira interrogans 56606v and the phosphorylation of NF-κB was significantly increased after infection demonstrated by Western blotting. The activation of NF-κB pathway in both post-infection and non-infection conditions was significantly inhibited by BAY 11-7082. The apoptosis rate of murine neutrophils infected with or without Leptospira interrogans 56606v was significantly increased by BAY 11-7082 (P=0.000). The neutrophils infected with Leptospira interrogans 56606v were significantly less phagocytosed by macrophages than those uninfected (P=0.027). Conclusion ·Leptospira interrogans 56606v inhibits the apoptosis of murine neutrophils by activating the NF-κB pathway, which further reduces efferocytosis of macrophages on neutrophils.

Key words: Leptospirainterrogans, neutrophil, apoptosis, nuclear factor-κB

CLC Number:

DU Lin, HE Ping. Effect of Leptospira interrogans 56606v on apoptosis of murine neutrophil[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(11): 1517-1523.

Figures/Tables 6

Fig 1 Detection of apoptosis of murine neutrophils infected with different concentrations of Leptospira at 6 h post infection by flow cytometry

Fig 2 Detection of apoptosis of murine neutrophils infected with Leptospira (MOI=100) for different periods by flow cytometry

Fig 3 Activation of caspase-3 in murine neutrophils infected with Leptospira

Fig 4 Activation of NF-κB in murine neutrophils by Leptospira infection

Fig 5 Effect of inhibition of NF-κB pathway on apoptosis of murine neutrophils

Fig 6 Efferocytosis of neutrophils by macrophages

TrendMD

References 26

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