Preliminary Study On The Immune Response Of Macrophages To Enteropathogenic E.coli Infection Under Simulated Microgravity

Background and objectiveMicrogravity is one of the crucial factors that affect the health of astronauts in space.Microgravity or simulated microgravity can disrupt immune homeostasis and increase the risk of infectious diseases in astronauts.Although there have been studies involved infection immunity under microgravity or simulated microgravity,the mechanism of the host becomes less resistant to pathogens under microgravity or simulated microgravity has not been elucidated.The purpose of this study is to investigate the effect of simulated microgravity on the immune response of macrophages to gram-negative bacteria-EPEC,provide experimental basis for elucidating the anti-bacterial infection mechanisms of the host under simulated microgravity,and formulating the prevention and treatment strategies for infectious diseases for astronauts after returning to the ground.Methodsa.Rotary cell culture system(RCCS)was used to simulate microgravity,and the infection model under simulated microgravity was established using macrophages infected with EPEC under simulated microgravity.The production of inflammatory cytokines in macrophages infected with EPEC under simulated microgravity was detected by quantitative real-time PCR.b.RNA sequencing was conducted to analyze and compare differentially expressed genes(DEGs)and the enrichment of signaling pathways between the normal gravity and simulated microgravity.The DEGs related to immunity in enriched signaling pathway were filtered based on InnateDB database.c.The validation in transcription level of screening DEGs was conducted using quantitative real-time PCR,and the activation of enriched signaling pathways related to immunity was conducted using western blotting analysis.Resultsa.Compared with normal gravity,the production of IL-6 and TNF markedly decreased in macrophages infected with EPEC under simulated microgravity,which indicated that simulated microgravity suppressed the immune response of macrophages to EPEC.b.There were 542 up-regulated DEGs in infected macrophages under simulated microgravity(P<0.05),and 720 down-regulated DEGs in infected macrophages under simulated microgravity(P<0.05)as compared to normal gravity.The enrichment signaling pathway of KEGG showed the expression of genes in infected macrophages under simulated microgravity of cytokine-cytokine receptor interaction and MAPK signaling pathway was different(P<0.05).In cytokine-cytokine receptor interaction and MAPK signaling pathway,the DEGs related to immunity were 14(which of 7 were up-regulated,and 7 were down-regulated)and 36(11 of which were up-regulated,and 25 were down-regulated),respectively.In cytokine-cytokine receptor interaction signaling pathway,except for 11 33(P>0.05),the results of mRNA expression of DEGs in macrophages infected with EPEC were consistent with the results of RNA sequencing.In MAPK signaling pathway,the mRNA expression of 11 1?,Cd14,Hspala,Pdgfrb,Il 1a,Fgf7,Igf1r and Pak1 was not consistent with the RNA sequencing;Compared with normal gravity,simulated microgravity did not affect the mRNA expression of Il 1?,Cd14,Hspa1a and Pdgfrb(P>0.05);The Il 1a and Fgf7 down-regulated and Igf1r and Pak1 up-regulated under simulated microgravity at the level of mRNA compared to normal gravity,and the results of the rest of the DEGs were consistent with the results of RNA sequencing.c.Compared with normal gravity,simulated microgravity reduced the phosphorylation of p38 and ERK signaling pathway,but did not affect that of JNK signaling pathway,which suggested that simulated microgravity modulated the immune response of macrophages to EPEC in a p38 and ERK signaling pathway-dependent manner.ConclusionsCompared with the immune response of macrophages to EPEC under normal gravity,simulated microgravity modulates the immune response of macrophages to EPEC in a p38 and ERK signaling pathway-dependent manner and by suppressing the production of IL-6 and TNF in macrophages infected with EPEC.