Study On The Role And Its Mechanism Of The Development Of Neutrophil Extracellular Traps Induced By Leishmania Infantum

Objective:Neutrophils play an immunomodulatory role through the releasing of neutrophil extracellular traps(NETs).Neutrophil in response to infection could release NETS,which claimed correlation with disease severity.At present,the endemic desert sub-type zoonotic visceral leishmaniasis is the most pernicious leishmaniasis without effective control measures in China.A better understanding of the immunological mechanism of Leishmania-induced NETs provide an important research basis for better illustration of the immune regulation mechanism and exploring potential therapeutic targets for Leishmania infection.In this study,we constructed an experimental model of L.infantum(desert sub-type)(MHOM/CN/2008/JIASHI-5,L.infantum for short)infected BALB/c and analyzed the L.infantum-induced effect on the ability of NET releasing.The mechanism of NET formation induced by L.infantum promastigotes was further investigated.Methods:Establish an experimental mouse model of L.infantum through intraperitoneal inoculation.Neutrophils were purified from peripheral blood which obtained from Leishmania-infected mice and control mice,respectively.Neutrophils were stimulated by PMA to release NETs in vitiro.The efficiency of PMA-induced NET releasing by neutrophils sourced from Leishmania-infected mice and control mice was compared to analyzed the L.infantum-induced immunological effect on the NET formation during infection through confocal and flow cytometry.Neutrophils were purified from peripheral blood of control mice.Neutrophils pretreated with antagonist(Boc)or agonist(LXA4)to block or activate the lipoxin A4 receptor followed by promastigotes stimulation.Comparing the variation of NET formation among different experimental groups to explore the possible role of lipoxin A4 receptor during L.infantum-induced NET formation through confocal,dsDNA quantification and flow cytometry.HL-60 cells were induced to differentiate into neutrophil-like cells by 1.25%dimethyl sulfoxide(DMSO).Neutrophil-like cells model with down-regulated lipoxin A4 receptor based on RNA interference(RNAi)was constructed.Then the Leishmania-induced H3Cit(a marker of NETs)expressing between the silenced and the non-silenced cells were compared to further demonstrated the role of Lipoxin A4 receptor in L.infantum-induced NET formation.Results:1.Laser confocal microscopic imaging analysis showed that the proportion of cells produced filamentous or circular DNA skeleton structure,and the expressing of H3Cit and MPO particles were increased of the neutrophils sourced from Leishmania-infected mice stimulated by PMA than that of uninfected mice.The proportion of H3Cit+MPO+neutrophils in the neutrophils sourced from Leishmania-infected mice was(1.880±0.251)%and that of uninfected mice was(0.623±0.087)%,which showed statistical difference(P-0.0014).2.Promastigotes of L.infantum can effectively induce neutrophils to form NETs(P<0.01).There was a significant decrease in the Boc-primed neutrophil group as compared to the unprimed group stimulated by promastigotes(P<0.01),while the antagonistic effect is partially enforced on L.in/antum-induced NETs extrusion.The agonist LXA4 can increase NET formation significantly(P<0.01),while the antagonist Boc can significantly reverse the agonistic effects of LXA4(P<0.01).3.The percentage of H3Cit+neutrophil-like cells in differentiated HL-60 cells stimulated by promastigotes was significantly increased(P<0.001).There was a significant decrease in the proportion of H3Cit-positive neutrophils in the Lipoxin A4 receptor-silenced neutrophil-like cells as compared to the non-silenced neutrophil samples stimulated by promastigotes(P<0.001).Conclusion:1.L.infantum infection immunologically change the neutrophil and significantly enhance the ability of neutrophils to form NETs,which may cause deterioration of host immunity.2.Lipoxin A4 receptor as one of the mediators conducting the formation of NETs induced by L.infantum promastigotes.3.The lipoxin A4 receptor,which anchored on the neutrophils plays a crucial role duing Leishmania infection,might be an alternative target for visceral leishmaniasis control.