IRF7 Triggers Macrophage Autophagy To Kill Bacteria In Sepsis

BackgroundAutophagy is emerging as an defence against intracellular pathogens in sepsis.It is cleared that transcription factors determinate the autophagy-related gene repertoire in infected host.IRF7,a endogenous transcription factor that is resistant against virus by triggering genes esseential for innate immunity,such as type I interferons.However,whether and how IRF7 defend against pathogens in sepsis is poorly understood.MethodsSix-to eight-week-old male C57BL/6 mice were used to construct cecal ligation and puncture modle or were administrated with LPS(20mg/kg).Firstly,we performed RT-QPCR assay to determine Irf7,Ifnα,Ifnβ mRNA and protein level in sepsis modle of mice and macrophages.According to the trend of IRF7 level in sepsis,we knockout Irf7 of mice and investigate effects of IRF7 on cleaning bacteria in sepsis mice.Furthermore,we interfere and overexpress Irf7 to clarify the role of IRF7 in vitro.Immunofluorescence and transmission electron microscope assay were performed to detect bacteria blocked by autophagosomes.Intracellular killing rate of macrophage on bacteria were analyzed by clone formation unit assay.To further clarify the role of IRF7 on transcription of autophagy-related genes,we test 53 Atgs mRNA level after rescue Irf7 in Irf7-/-bone-marrow derived macrophages(BMDMs),and analyzed the correlation coefficient among Irf7 and Atgs.At last,dual luciferase reporter assay was applied to identify whether IRF7 directly regulate transcription of Lc3b.ResultTo investigate whether IRF7 is associated with sepsis pathogenesis,we first monitored 7-day survival rate of Irf7-/-and wild type mice performed CLP.All Irf7 deficient mice died within 2 days,but 50%of WT mice survived until the seventh day.To explore a role of IRF7 in sepsis,we determined levels of Irf7 in mice administrated with LPS(20 mg/kg)for 0,2,4,8,16,24 hours.Irf7 mRNA increased at 2 hour and reach top level at 16 hour.Data similar to that found from Raw264.7 cells treated with LPS(100 ng/mL)were observed.These results suggested that IRF7 is required for anti-infection in the late phase of sepsis.We next sought to identify the function of IRF7 in infection.Because studies reveal that IRF7,a member of interferon regulate factor family,stimulates type I interferon transcription.We performed RT-QPCR assay to detect Ifnα and Ifnβ mRNA level in LPS treated Raw264.7 cells.Unexpectedly,Ifnα and Ifnβ mRNA expression reach maximal level at 2 hour but not 16 hour.These results suggested that IRF7 have another role in sepsis besides of regulating type I interferon transcription.We next investigated whether IRF7 is required for killing bacteria.BMDMs were transfected with pcDNA3-HA-Irf7 to overexpress Irf7.Western Blot assay revealed that IRF7 was efficiently overexpressed.Compare with null vector group,overexpressing Irf7 promoted bone-marrow derived macrophages to kill E.coli.To block clearance of bacteria pathway,we treated Irf7-overexpressing BMDMs with Bafilomycin A1(Baf A1),a known inhibitor of lysosome acidification,and found that Baf A1 treatment markedly inhibited clearance of bacteria.To further confirm the relevance of IRF7 as a critical regulator with killing bacteria.Small interference RNA was applied to silence Irf7.Obviously,killing bacteria rate was reduced in Irf7 knockdown BMDMs.Similar data were detected when using pathogenic S.typhimurium and V.vulnificus.Taken together,the results support a role of IRF7 in clearance of bacteria.Mounts of studies demonstrated that autophagy,an intracellular degradation process,maintained cellular homeostasis by transporting autophagic cargo to lysosome.Given that killing intracellular bacteria required of cell autonomous clearance mechanism,we hypothesized that IRF7 was associated with autophagy in bacteria infection disease,such as sepsis.To test this idea,we monitored level of LC3B and p62 after LPS treatment in vitro.It is noteworthy that LC3II,a component of autophagosome membrane reach top level at 16 hour,whereas p62,degradated in autophagy process,was reduced from 4 to 24 hour.These results suggested that IRF7 was associated with autophagy.To further confirm whether IRF7 regulate autophagy in sepsis,we determined LC3B expression in Irf7-overexpressing BMDMs.Compared with null vector group,LC3II was increased after administrated Baf A1.Underexpected,LC3II level reduced after knockdown Irf7 in LPS-treated BMDMs.We also performed immunofluorescence and TEM assay to monitor the effect of Irf7 on autophagy.The assay revealed that LC3 positive puncta were present in BMDMs treated with LPS,whereas decreased in Irf7-/-group.Ultrastructure analysis showed that to interfere Irf7 expression decreased the number of dual/multi-layer membrane autophagosome in infected macrophages.So far,our studies have independently revealed the effects of IRF7 on clearance of bacteria and autophagy in sepsis.Given that autophagy is an defence against intracellular pathogens.Next,we performed immunofluorescence and TEM assay to monitor the intracellular bacteria.The results showed that the rate of autophagosome-contained bacteria was decreased afer knockdown Irf7.Underexpected,overexpressing Irf7 promoted bone-marrow derived macrophages to kill E.coli and S.typhimurium,but decreased in BMDMs administrated with autophagy inhibitor Wortmaine.These results suggested that IRF7 promoted macrophages to kill bacteria via autophagy.How did IRF7 regulate autophagy to promote macrophage to kill bacteria?Given that IRF7 is a endogenous transcription factor,we monitored the location of IRF7 in BMDMs.Obviously,IRF7 translocated to the cell nuclear after LPS treatment.Rescue experiment showed that transfer Irf7 to Irf7-/-BMDMs restored the expression of 10 Atgs.Further,dual-luciferase reporter assay revealed that the rescue of Irf7 restored relative luciferase activity of Lc3b.Taken together,IRF7 promote autophagy relative genes transcription in sepsis.ConclusionsIn this study,we identified that IRF7 triggered transcription of many autophagy-related genes.As a result,IRF7 facilitated autophagy to clear bacteria,thus inhibited organ injury and inflammatory response,decreased death of septic mice.