E.coli Induced Larger Neutrophils In The Peritoneal Cavity Of Mice With Severe Septic Peritonitis

Severe trauma and uncontrolled infection can cause sepsis,sepsis were then developed into severe sepsis,septic shock.Sepsis is a public health problem with high morbidity and mortality.When studying the the development of septic peritonitis induced by E.coli,we noticed some of the“giant cells”existed among peritoneal lavage fluid cells(PLCs).Besides the larger size,their nuclei are segmented and flat,squeezed to the marginal zone of the inner membrane.In this study,we verified identity and explore the function of the“giant cells”,as well as their correlation with E.coli induced severe septic peritonitis.The results were as follows:1.Mouse model of E.coli induced septic peritonitisTo establish the mouse model of septic peritonitis,we utilized different dose of E.coli intraperitoneally(i.p.)inject into the mice,recorded survival and monitored their physical conditions.A clinical score was used to evaluate the septic peritonitis severity,based on the clinical manifestations consist of lethargy,piloerection,tremors,periorbital exudates,respiratory distress and diarrhea.The results showed that:1)Low and medium dose of E.coli could induce moderate septic peritonitis(clinical score≤3)with no casualties;2)High dose of E.coli can induce severe septic peritonitis(clinical score>3)leading to 71%of mortality;3)There were excessive or detectable bacterial load in the peritoneal fluid or peripheral blood at 18 hours post-infection.Thus,i.p.injection of E.coli could cause severe septic peritonitis in mice.2.Correlation between the occurrence of“giant cells”and severity of septic peritonitisThe“giant cells”were found in the peritoneal lavage cells(PLCs)in mice with severe septic peritonitis accidentally.Their morphology was characterized by the larger than regular neutrophils in size,segmented and flat nuclei in the marginal zone of the inner membrane.For investigating whether the occurrence of the“giant cells”were related with the severity of septic peritonitis,we injected different dose of E.coli to induce septic peritonitis,collected the PLCs and numerated the“giant cells”.The results showed that:1)High dose of E.coli could elicit“giant cells”whereas low and medium dose of E.coli could not;2)And the numbers were up to 25%after 8 hours post-infection;3)The amount of“giant cells”occupied 13%at 2 hours post-infection,reached a pick at 8 hours post-infection,which were up to 25%of PLCs,and then decreased.These results suggested that the occurrence and the accumulation of“giant cells”were closely related to the severity and mortality of septic peritonitis.3.Identification of the“giant cells”Since the segmented nuclei of“giant cells”,we speculate that they were derived from neutrophils.For identifying the“giant cells”,we purified PLCs of septic peritonitis via density gradient centrifugation,found the layers with“giant cells”,stained the cells with biomarker of the neutrophils(ly6G)and then observed under fluorescence microscope.The results showed that the“giant cells”were mainly in the layers of high density granulocytes(HDG),and could be recognized by ly6G antibody.Thus,the“giant cells”were a kind of ly6G positive neutrophils,therefore designated as E.coli induced larger neutrophils(e-Neus).4.Elements of driving the e-Neus developmentTo investigate the elements of driving the e-Neus development,we tested whether the neutrophils from murine bone marrow neutrophils or human peripheral neutrophils could induce the e-Neus by E.coli or different stimuli(CpG ODN,starch,thioglycollate,H1N1 influenza virus,staphylococcus aureus)could induce the e-Neus in vivo and in vitro.Also,we used E.coli to infect muscles to recruit neutrophils followed by observing the occurrence of the e-Neus.The results showed that:1)Murine bone marrow neutrophils could transform to the e-Neus after E.coli treatment in vitro and in vivo,adoptively transferred bone marrow neutrophils were also could elicit the e-Neus;2)Human peripheral neutrophils but not HL-60 cell line cells could transform to the e-Neus post E.coli-treatment;3)In the CpG ODN,starch,thioglycollate induced SIRS or acute peritonitis in mice,no e-Neus were elicited in the peritoneal cavity;4)In the H1N1 induced acute lung injury in mice,few e-Neus were elicited in the bronchoalveolar lavage fluid(BALF);5)In the staphylococcus aureus(S.aureus)induced septic peritonitis in mice,there were no e-Neus in the PLCs.6)Neutrophils recruited in the muscles after E.coli infection could transfer into the e-Neus.These results suggested that the e-Neus could not be elicited in the non-sterile peritonitis,and be elicited in the bacterial,E.coli but not S.aureus,septic peritonitis.5.Bacterial killing of the e-NeusTo explore the bacterial killing function of e-Neus,we tested the phagocytosis,ROS generation and NETs formation in the e-Neus.The results showed that:1)e-Neus engulfed more bacteria than regular neutrophils in the PLCs of mice with GFP-E.coli induced septic peritonitis;2)Under transmission electron microscope(TEM),the e-Neus were indeed consumed massive E.coli accompanied by“spacious”phagosome-like vacuole;3)The generation of ROS were far lower than that of regular neutrophils;4)e-Neus could not found to release NETs;5)Intracellular bacteria in the e-Neus were thrived on the solid culture medium.Thus,the e-Neus had a powerful phagocytic ability than regular neutrophils,but was ROS~lowow neutrophils.Furthermore,the e-Neus might let the bacteria alive in their spacious vacuole.6.Detection of IL-10 and TNF-αin the e-NeusTo test the inflammatory cytokine level in the e-Neus,we co-cultured bone marrow neutrophils and E.coli for 3,6,12 and 24 hours,and then detect the IL-10 and TNF-αmRNA or protein levels by qRT-PCR,flow cytometry or immunofluorescence assay.The results showed that:1)IL-10 mRNA and TNF-αmRNA level were increased at 6 hours or 12 hours post E.coli-treatment,respectively;2)The ratio of IL-10~+e-Neus were higher than IL-10~+regular neutrophils,however,expression level of IL-10 were significantly lower in the e-Neus than the regular neutrophils;3)The ratio of TNF-α~+e-Neus were obviously lower than that of regular neutrophils.Therefore,the e-Neus were IL-10~lowow neutrophils,indicating that the e-Neus displayed at least less anti-inflammatory function than IL-10~highigh regular neutrophils.7.Detection of surface molecules(CXCR4,CD80,CD11b)on the e-NeusTo detect the“aging”molecules(CXCR4),co-stimulatory molecules(CD80 and CD40),phagocytic receptor(CD11b)on the e-Neus,we utilized the flow cytometry and immunofluorescence assay to test the bone marrow neutrophils co-cultured with E.coli.The results showed that:1)E.coli could drive neutrophils to express CXCR4 at12 and 24 hours post-treatment,but the ratio of CXCR4~+e-Neus were markedly lower than that of regular neutrophils;2)CD80 and CD40 were expressed nearly since the E.coli stimulation,however,the percentage of the CD80~+e-Neus also far lower than that of regular neutrophils;3)The e-Neus were almost the CD11b~+neutrophils which higher than that of regular neutrophils.The results revealed that e-Neus were a type of CXCR4 low expressing,and CD11b~+CD80~lowow neutrophils.Overall,the e-Neus were a kind of neutrophils with distinct morphology,low bacterial killing capacity and less anti-inflammatory function,and their occurrence were positively associated with the severity of septic peritonitis induced by E.coli,which may enrich the understandings on neutrophils transitions in response to various insults,and could be used to diagnose and evaluate the severity of septic peritonitis induced by E.coli.