Porphyromonas gingivalis persisters induce the immuno-inflammatory responses in macrophages by upregulating the forkhead box1 signaling pathway
Objective To investigate the effects of Porphyromonas gingivalis(Pg)persisters(Ps)on immuno-inflammatory responses in macrophages,and to explore the underlying mechanisms.Methods Pg cells were cultured to the stationary phase(72 h),and subsequently treated by high concentration of metronidazole at 100 mg/L,amoxicillin at 100 mg/L and the combination of them for different time period,named as metronidazole group,amoxicillin group and(metronidazole+amoxicillin)group.Pg cells without treatment were used as Blank control.The survival profile of PgPs cells was measured by colony-forming unit assay.The living state of PgPs was observed by Live/Dead staining.Then,Pg and metronidazole-treated PgPs(M-PgPs)were used to treat macrophages,named as Pg group and M-PgPs group.Transmission electron microscopy(TEM)was used to observe the bacteria in the macrophages.The expression levels of proinflammatory cytokines in macrophages were determined by real-time fluorescence quantitative PCR and enzyme-linked immunosorbent assay.The location of forkhead box transcription factor 1(FOXO1)was detected by confocal immunofluorescence microscopy.After inhibiting or enhancing the FOXO1 expressions using inhibitors(Fi)or activators(Fa)respectively,the macrophages were treated with Pg and M-PgPs,divided as Blank group,Pg group,M-PgPs group,Fi group,(Fi+Pg)group,(Fi+M-PgPs)group,Fa group,(Fa+Pg)group and(Fa+M-PgPs)group.Then,the expression pattens of proinflammatory cytokines were assessed.Results Remarkable number of lived PgPs was observed,both in planktonic culture and Pg biofilms either treated with metronidazole,amoxicillin or both,and those persisters could form new colonies.Pg and M-PgPs were able to enter into the macrophages and the protein expression levels of interleukin(IL)-1β,IL-6,IL-8 and tumor necrosis factor-α(TNF-α)[Pg group:(2 392±188),(162±29),(5 558±661),(789±155)μg/L;M-PgPs group:(2 415±420),(155±3),(5 732±782),(821±176)μg/L]were significantly upregulated than those in Blank group[(485±140),(21±9),(2 332±87),(77±7)μg/L](P＜0.01).Moreover,Pg and M-PgPs could facilitate the nuclear translocation and accumulation of FOXO1.In addition,the relative mRNA expression levels of FOXO1,B-cell lymphoma 6 and Krüppel-like factor 2 were upregulated when compared to Blank group(P＜0.05).Furthermore,the protein expression levels of IL-1β,IL-6,IL-8 and TNF-α in Fi+Pg group[(1 081±168),(70±8),(1 976±544),(420±47)μg/L]were remarkably lower than Pg group[(4 411±137),(179±6),(5 161±929),(934±24)μg/L](P＜0.05).Similarly,the protein expression levels of IL-1β,IL-6,IL-8 and TNF-α in Fi+M-PgPs group[(1 032±237),(74±10),(1 861±614),(405±32)μg/L]were remarkably lower than M-PgPs group[(4 342±314),(164±17),(4 438±1 374),(957±25)μg/L](P＜0.05).On the contrary,the protein expression levels of IL-1β,IL-6,IL-8 and TNF-α in Fa+Pg group[(8 198±1825),(431±28),(8 919±650),(2 186±301)μg/L]and Fa+M-PgPs group[(8 159±2 627),(475±26),(8 995±653),(2 255±387)μg/L]were significantly higher than Pg group and M-PgPs group,respectively(P＜0.05).Conclusions PgPs are highly tolerant to metronidazole and amoxicillin.The M-PgPs could enhance the immuno-inflammatory responses in macrophages by upregulating the FOXO1 signaling pathway,while this effect exhibits no significant difference with Pg.
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