Lipopolysaccharide and outer membrane proteins of Leptospira interrogans induce macrophage apop-tosis via Fas/FasL pathway

Abstract

Objective To investigate the role of pathogenic Leptospira interrogans lipopolysaccha-ride （L-LPS） and outer membrane proteins （L-OMP） in the apoptosis of mouse macrophages （J774A.1） and their association with Fas/FasL pathway .Methods Phenol-water extraction and Triton X-114 phase separation were used to extract L-LPS and L-OMP from L.interrogans serogroup icterohaemorrhagiae serovar Lai strain Lai, respectively.Polymyxin B （ PMB） and protease K （ PK） were used to treat L-LPS and L-OMP, respectively.J774A.1 cells were stimulated by L.interrogans strain Lai with or without ultraviolet inactivation.In parallel, the cells were stimulated by extracted L-LPS and L-OMP with or without PMB and PK treatments .The apoptosis and necrosis of J 774 A.1 cells before and after treatment were detected by flow cytometry.The siRNAs were used to silence the expression of Fas or FasL gene in J 774A.1 cells and their inhibitory effects were further validated by using real-time fluorescent quantitative RT-PCR.Flow cytometry was used to detect the effects of L-LPS or L-OMP on the apoptosis of J774A.1 cells with Fas or FasL gene-knockdown .Results L.interrogans strain Lai with or without ultraviolet inactivation could cause similar early apoptosis rates （47.1%and 55.6%） and late apoptosis/necrosis rates （7.6%and 7.9%）.The ear-ly apoptosis rates of 1×10^5 J774A.1 cells were 40.4%and 34.0%after the treatment with 100 ng of L-LPS and 100 μg of L-OMP for 4 h.The late apoptosis/necrosis rates of the cells were 7.5%and 6.9%upon the treatments with L-LPS and L-OMP, respectively.However, the apoptosis or necrosis of the cells was not ob-served when using L-LPS and L-OMP pre-treated by PMB and PK, respectively.Silenced expression of Fas or FasL gene reduced the L-LPS-induced J774A.1 cells apoptosis （P〈0.05）, while decreased early apopto-sis rate of J774A.1 cells mediated by L-OMP was only observed in Fas gene-knockdown cells （P〈0.05）. Conclusion Both L-LPS and L-OMP can cause the Fas/FasL-associated apoptosis of macrophages , which is beneficial for L.interrogans to establish the productive infection in hosts . Key words: Fas/FasL; Leptospira interrogans; Lipopolysaccharide/outer membrane proteins; Macrophage/apoptosis; Fas/FasL