Diversity In Ability And Mechanism Of Mononuclear-macrophages And Neutrophils Anti-leptospira Interrogans During Infection

Background:Leptospirosis is a worldwide prevalence zoonotic infectious disease caused by pathogenic Leptospira species,which is one of the major infectious diseases in China’s key prevention and control.Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai is the most dominant pathogenic Leptospira in China.Mononuclear-macrophages and neutrophils,the major phagocytes in innate immunity,play a decisive role in defending against invaded pathogens.However,little is known about these phagocytes to phagocytose and kill L.interrogans during infection.In this paper,we explore the difference between mononuclear-macrophages and neutrophils in elimination of L.interrogans during infection.Methods:The infiltration of peripheral blood derived mononuclear-macrophages and neutrophils in lung,liver and kidney of infected C3H/HeJ mice were assayed by immunohistochemistry.The histological examination and the morphological analysis of L.interrogans in lung,liver and kidney from infected mouse were detected by HE staining and silver staining,respectively.Chemokines in sera of Leptospira-infected mice and leptospirosis patients were detected by microarray,and the vascular endothelial cell adhesion molecules(VECAMs)(VCAM-1,E-selectin,P-selectin and ICAM-1)in lung,liver and kidney of infected mice were determined by immunohistochemistry.A L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lain infected human HL-60 cells,human peripheral blood neutrophils,human THP-1 cells,human peripheral blood monocytes,murine peripheral blood neutrophils,murine J774A.1 cells,or murine peripheral blood monocytes models were established for subsequent experiments.The phagocytosis of leptospires was determined by confocal microscopy and transmission electron microscopy,the number of leptospires in different phagocytes was determined by bacterial counting chamber.The viability and dead percentages of phagocytosed leptospires were assayed by confocal microscopy and spectrofluorimetry,respectively.The survival and growth ability of intracellular leptospires were tested by colony forming unit and enumeration,respectively.Confocal microscopy was used to measure the colocalization of leptospires with lysosomes in different phagocytes.The expressions and the role of total ROS,NO,and[Ca2+]i in different phagocytes were tetected by confocal microscopy and spectrofluorimetry.Results:A large number of peripheral blood-derived mononuclear-macrophages infiltrated into the lungs,liver,and kidneys of leptospire-infected C3H/HeJ mice,but a low number of neutrophils were detected in injury tissues.Mononuclear-macrophage-but not neutrophil-specific chemokines were significantly increased in the sera from the infected mice and patients(p<0.05).Meanwhile,the mononuclear-macrophage-but not neutrophil-specific VECAMs in lungs,liver and kidneys of infected mice were significantly elevated(p<0.05).The primary and passage mononuclear-macrophages from human or murine origin(human THP-1 cells,human peripheral blood mononuclear-macrophages,murine J774A.1 cells,or murine peripheral blood mononuclear-macrophages)presented more powerful ability to phagocytose and kill the leptospires compared to the neutrophils(human HL-60 cells,human peripheral blood neutrophils,murine peripheral blood neutrophils)(p<0.05).The higher total ROS and NO levels and[Ca2+]i in the mononuclear-macrophages were related to the Leptospira-killing mechanism.The human microphages presented lower phagolysosome-forming ratios and Leptospira-killing ability than the mouse macrophages(p<0.05).Conclusion:The present study revealed that mononuclear-macrophages but not neutrophils act as the major infiltrating and Leptospira-killing phagocyte during leptospirosis.Less phagolysosome-formation may be responsible for lower Leptospira-killing ability of human mononuclear-macrophages.