Phosphodiesterase-4 Inhibitor ROF Suppresses NLRP3 Inflammasome Activation Through Autophagy In Microglial BV-2 Cells

Objective:Previous studies demonstrated that knockdown or inhibition of phosphodiesterase 4(PDE4)suppressed the inflammatory responses in the brain.However,the underlying mechanisms are poorly understood.cAMP induces autoph-agy,and deficiency of autophagy leads to elevated inflammatory factors.Roflupram(ROF)is a novel PDE4 inhibitor that enhances cAMP and suppresses inflammation.However,the contribution of autophagy to the anti-inflammatory effect of ROF in microglial cells is ill-understood.Here,we examined the effects of ROF on autopha-gy against neuroinflammation in microglial BV-2 cells focusing on NLRP3 inflam-masome activation.Methods:MTT assay was used to study the cell viability of microglial BV-2 cells.LysoTracker(LYT)red and acridine orange(AO)-stained the acidic vesicles.Aβ25-35 or LPS with ATP were activated BV-2 cells and NLRP3 inflammasome.ELISA assessed the levels of cytokines.Western blotting detected the levels of pro-inflammatory factors,essential proteins involved in autophagic activities,apop-tosis related protein and synaptic associated protein.(1)To study the effect of ROF on the viability of BV-2 microglia or activated BV-2 microglia,MTT assay was used.The cells were cultured in 96-well flat bottom plates for 12 h before treated.11 μL MTT solution(5 mg/mL in PBS)was added to each well.The cultures were then incubated in the dark at 37 ℃ for living cells to form insoluble violet formazan crystals.After 4 h,15μL of DMSO was added to each well and plates agitated for 5 min to solubilize the crystals.The optical density(OD)was immediately determined at a wavelength of 570 nm with microplate spec-trophotometer.(2)To investigate the effect of ROF on autophagy in BV-2 microglia,essential proteins involved in autophagic activities was dectected by Western blot.Autophagy flux was performed to confirm the effect of ROF on autophagy activity.Acid vesi-cles including autophagolysosomes in the late stage of autophagy were also dectect-ed by AO and LYT stain.Furthermore,Immunofluorescence of microtu-bule-associated protein 1 light chain 3(LC3)was used as specific marker for au-tophagosome.More over,preliminary exploration of the underline mechanism was performed by using RAPA,ROLI and FOSK and 3-MA.(3)To stuy the effect of ROF-induced autophagy on inflammasome in activated BV-2 cells,NLRP3 inflammasome specific activators including LPS plus ATP as well as Aβ25-35 were used to trigger BV-2 cells activation and NLRP3 inflammasome activation.Then ELISA analysis was used to detect the production of IL-1β after the activation of NLRP3 inflammasome pathway.Moreover,protein from culture su-pernatant was precipitated and estimation of IL-1β p17 and caspase-1 p10 were per-formed by Western blot.The protein level of NLRP3,LC3,pro-caspase-1 and pro-IL-1β in the cell lysate were also analysis by Western blot.In addition,3-MA was used as counterevidence for the role of ROF-induced autophagy on its an-ti-inflammation effect.(4)To establish the protective effect of ROF on neuron against micro-glia-mediated neurotoxicity,condition medium(CM)form BV-2 cells was collected and applied to N2a cells culture.Then Hoechst 33342 Stain,phase contrast micro-scope analysis and Western blot were use to detect the the synaptic damage and apoptosis of N2a cells.In addition,3-MA and siRNA ATG7 were used as counterev-idence for the nueronal protection of ROF by microglial autophagy induction.Results:ROF did not affect the cell viability of microglial BV-2 cells while the level of LC3-Ⅱ was increased and p62 was decreased.Enhanced fluorescent signals were observed in BV-2 cells treated with ROF by AO and LYT red staining.In addition,immunofluorescence indicated a significant increase in punctate LC3.Both LPS plus ATP and Aβ25-35 enhanced the conversion of pro-caspase-1 to cleaved-caspase-1 and increased the production of mature IL-1β in BV-2 cells.In-terestingly,these effects were blocked by the treatment of ROF.Moreover,Hoechst staining showed that ROF decreased the apoptosis of neuronal N2a cells in condi-tioned media from BV-2 microglia.These effects were reversed by inhibition of microglial autophagy by 3-MA or siRNA ATG7.(1)Effect of ROF on cell viability in microglial BV-2 cells:ROF(0.625-20 μM)did not exhibit toxicity on BV-2 cells after 24 h incubation.ROF also showed ade-quate compatibility with activated microglial BV-2 cells triggered by LPS plus ATP,as well as,Aβ25-35 exposure for 6 h.(2)ROF induced autophagy in microglial BV-2 cells:ROF increased the protein level of LC3-Ⅱ in BV-2 cells treated with ROF while the that of p62 was signifi-cantly increased in a concentration-and time-dependent manner.Compared to the cells treated with ROF alone,co-treatment with CQ and ROF resulted in increased LC3-Ⅱ accumulation.Compared to the cells in the vehicle control group,the enhanced fluorescence signal was observed in cells treated with ROF.The typical autophagy inducer RAPA also induced fluorescence signal of lysosome The classic PDE4 inhibitor ROLI and an adenosine cyclase activatorFORS could also induce high fluorescence signal of lysosome.Furthermore,immunofluorescence staining showed that the increased LC3 punctuate staining in response to ROF was inhibited by 3-MA.(3)Blocking autophagy enhanced the activation of NLRP3 inflammasome in BV-2 cells:BV-2 cells were in the ’resting’ state in the normal culture medium.After stimulation for 4-6 h,BV-2 cells were in the ’activated’ state with morphological changes.(IL-1β p17)was detectable by Western blot in the supernatant of BV-2 mi-croglial cell 4 h after LPS plus ATP as well as Aβ25-35 simulation.Western blot anal-ysis of caspase-1 showed a gradual increase in cleaved-caspase-1(CASP1 p10)after stimulation for 4-6 h and the changes in CASP1 p10 were similar to that of IL-1βp17.The level of LC3-II was significantly increased in response to LPS plus ATP as well as AP25-35 stimulation as compared to the control.Strikingly,as a response to 3-MA,a significant increase in CASP1 p10 and IL-1β p17 was observed in activated BV-2 cells as compared to cells treated without 3-MA.Moreover,the protein levels of NLRP3 and precursor of interleukin-1β(pro-IL-1β)were increased as a result of 3-MA treatment.(4)ROF-induced autophagy suppressed the activation of NLRP3 inflam-masome in BV-2 cells:ROF significantly increased the level of LC3-Ⅱ in activated BV-2 cells(p<0.05).IL-1β secretion was decreased in a concentration-dependent manner in BV-2 cells stimulated by Aβ25-35 or LPS plus ATP post-ROF treatment.ROF treatment resulted in a significant reduction of matured IL-1β and cleaved CASP1 in activated BV-2 cells.Moreover,the protein levels of NLRP3 and pro-IL-1β were decreased as result of ROF treatment.Interestingly,these effects were abolished in the presence of the autophagy inhibitor,3-MA.(5)ROF protected N2a cells from BV-2 microglia-mediated neurotoxicity in CM:LPS plus ATP-CM or Aβ25-35-CM promoted the apoptosis of N2a cells.Since one of the key events in apoptosis is the activation of caspase-3(CASP3),we evalu-ated the level of CASP3 by Western blotting.Either LPS plus ATP-CM or AP25-35-CM significantly upregulated cleaved-CASP3 in N2a cells(p<0.05).Notably,the alterations in the synaptic structures were observed in some of the surviving cells.After incubation with activated microglial CM for 24 h,the neurites of the surviving N2a cells were shorter than that of the controls.Moreover,the protein level of PSD95 was also reduced in N2a cells incubated with LPS plus ATP-CM or Aβ25-35-CM(p<0.05.Furthermore,the addition of 3-MA led to a significant number of apoptotic cells and shorter neurites in the residual surviving N2a cells.The knockdown of ATG7 exacerbated CM-induced increase of CASP3 in N2a cells while the level of PSD95 was decreased under this condition.Interestingly,CM from acti-vated BV-2 cells treated with ROF appeared to be less acutely toxic to N2a cells.Decreased apoptosis,long neurites,low level of cleaved-CASP3,and increased level of PSD95 were found in N2a cells.However,the protective effect of ROF against neuronal apoptosis and the synaptic injury was abolished by 3-MA or siATG7 treat-ment in BV-2 cells.Conclusions:In this study,for the first time,we show that selective inhibition of PDE4 by ROF enhanced autophagy in microglial BV-2 cells and enhanced au-tophagy contributes to the anti-inflammatory effect of ROF in microglial BV-2 cells.This phenomenon is based on the following observations:(1)ROF induced autoph-agy in microglial BV-2 cells in a time-and concentration-dependent manner.(2)ROF treatment reduced inflammasome activation and the subsequent release of IL-1β via autophagic degradation of NLRP3 and pro-IL-1β in BV-2 cells.(3)ROF protected N2a cells from BV-2 microglia-mediated neurotoxicity in CM.(4)Block-ing autophagy or knocking down ATG7 attenuated the role of ROF.