The Effects On Macrophage RAW264.7 Infected With GAS

Objective: Group A streptococcus (GAS) is a major human pathogen causing infections ranging from superficial infections of the throat (pharyngitis) or skin (impetigo) to highly invasive and potentially lethal infections such as scarlet fever and streptococcal toxic shock syndrome. It should be noted, however, GAS infections also result in post-infection autoimmunity disease, rheumatic fever, rheumatic heart disease, glomerulonephritis, psoriasis, immunity cerebrosis, and so on. Following invasion to human body by GAS, the primary defence against GAS as well as against many other bacterial pathogens is provided by resident macrophages and recruited neutrophils. Several studies have described the ability of GAS to thwart the antimicrobial activities of neutrophils in various ways, including the release of molecules that interfere with the neutrophils recruitment or the induction of neutrophils cell death,while little is known about how GAS avoid the robust microbicidal effector functions of macrophages.The study aims to detect the state of ultrastructure, generation and molecules of cell surface and excretion of cytokines(CKs)of macrophage RAW264.7 after stimulated by GAS, and to further explore the pathogenic mechanism of GAS.Methods:1 RAW264.7 macrophages were infected with GAS at an MOI (multiphy of infection)ranging from 2:1 to 100:1 (bacteria number per macrophage) for 20,40,60,90,or 120min (PBS and LPS as negative and positive control). After the culture supernatant was abandoned, DMEM supplemented with 10%(v/v)FCS and antibiotics was added to the macrophages, and continued to incubate for 3 days at 37℃5% CO2, at last proliferation of RAW264.7 was determined by the measure of MTT.2 Ultrastructures of the infected RAW264.7 macrophages were observed by transmission electron microscopy, and the apoptosis of RAW264.7 was detected by TUNEL apoptosis detection kit(one-step)and ANNEXIN V-FITC apoptosis detection kit after RAW264.7 cells were infected with GAS at an MOI of 5:1 or 20:1 or 100:1 for 90min.3 The expressions of CD80, CD86 and MHCⅡon RAW264.7 surface were detected by Flow cytometry, the transcriptional levels of IL-1,IL-6,IL-23,TNF-α,TGF-βwere measured by Real-time-PCR and the expression of IL-6 was detected by ELISA after RAW264.7 macrophages were infected with GAS or E.coli or Staphylococcus aureus at an MOI of 5:1 for 90 min, subsequently the culture supernatant was abandoned, and the infected cells were continued to incubate for 3 days at 37℃, 5% CO2. 4 The peritoneal macrophages and the splenocytes were harvested on days 3 and the transcriptional levels of IL-1,IL-6,IL-23,TNF-α,TGF-βof that cells were detected by Real-time-PCR after BALB/c mice were injected GAS at an MOI of 5:1.Results:1 It's showed that small and medium doses of GAS can promote the proliferation of RAW264.7, while the proliferation of RAW264.7 was inhibited by high-dose GAS.2 Following infection by low-dose GAS, organelle function of RAW264.7 was active, mitochondria became edema and the rough endoplasmic reticulum became rougher, by transmission electron microscopy. Infected with the medium-dose GAS, the cell organelle was further active, and there was myeloid lysosomal, suggesting that phagocytosis and digestion were incomplete. While the RAW264.7 stimulated by high-dose GAS, there were vacuolization and incomplete nuclear membrane, suggesting that the cells died or going to die.3 The result of TUNEL and ANNEXIN V-FITC show that, the cells were death rather than apoptosis, following stimulation by high-dose GAS.4The results of semi-quantitative PCR and Real-time-PCR show that small-dose GAS enhanced the expression of IL-1, IL-23,TNF-α, suggesting that small-dose GAS can promote RAW264.7 macrophages to express proinflammatory cytokines, however, the expression of IL-6,TGF-βwere reduced.5 After RAW264.7 cells infected with small-dose E.coli, S. aureus and GAS respectively,the expressions of CD80 on the cell-surface were higher than that of PBS control group. Among these three groups, the expressions of CD80 in E.coli group was the highest. The expression of CD86 was higher in E.coli group than PBS control, and the other groups'were of no significant change. The expressions of MHC-Ⅱon the surface of RAW264.7, stimulated by E.coli and S. aureus, were higher than PBS group, however, that expressed on the RAW264.7 surface was of no significant change.The data of Real-time-PCR showed that the transcriptional levels of IL-6 was increased by stimulation of E.coli and S. aureus, but that was induced by stimulation of GAS group.6 Using Real-time-PCR, it's found that, comparing with PBS control group, the transcriptions of IL-1,TNF-αwere increased in peritoneal macrophages, and the transcriptions of IL-6,IL-23,TGF-βwere of no significant change, while the transcriptions of the above CKs of splenocytes were all increased.Conclusions:1 Low-dose GAS can promote RAW264.7 cells proliferation and enhance it's role, and increase the expression of proinflammatory cytokines, however, the expressions of IL-6,TGF-βwere reduced ,the related mechanisms, such as signal transduction pathway were still to be studied. 2 The expressions of CD80 and MHC-Ⅱis increased after the RAW264.7 cells were stimulated by low-dose GAS, to some extent, they can enhance the role of antigen-presenting, but this effect was not significant in GAS group.3 High-dose GAS can inhibite proliferation of RAW264.7 and the cell is death rather than apoptosis.