P55PIK Promotes LPS-induced Inflammation By Inhibiting Autophagy

Autophagy is a catabolic process in which cells undergo inflammatory stimuli or stress responses,and can degrade endogenous or exogenous components such as damaged organelles,denatured proteins,and pathogenic microorganisms.Autophagy can mediate the metabolism and renewal of cells,and participate in various pathophysiological processes such as infection,inflammation and tumor.Inflammation and autophagy promote and restrict each other to maintain immune homeostasis: First,infection-mediated inflammation can initiate autophagy through PAMP stimulation and binding to PRR,and autophagy pathways to clear intracellular pathogens.In turn,autophagy initiated by inflammatory stimulation(PAMP)can also play a role in negative regulation/inhibition of inflammation by inhibiting the inflammatory corpuscle pathway and other mechanisms,thereby controlling the inflammatory response.Phosphoinositide 3-kinases(PI3Ks)is an important molecule in the growth factor regulatory signaling pathway and plays a very important role in inflammation,proliferation,differentiation,apoptosis,and glucose metabolism.PI3 K is classified into Class I(IA and IB),II,and III,and Class IA PI3 K is composed of a catalytic subunit(p110?/?/?)and a regulatory subunit(p85?/?,p55?/?,p50?).Heterodimer.It is reported in the literature that the classical Class IA PI3 K pathway can inhibit the production of autophagosomes by mTORC1,and the effect of non-canonical PI3 K regulatory subunit p55?(ie p55PIK)on autophagy has not been reported.The preliminary results of our laboratory show that in the mouse AOM-DSS enteritis model,p55PIK-specific inhibitor(TAT-N15)can alleviate the inflammatory basic traits in acute and chronic inflammatory phase,and reduce the acute and chronic inflammatory phase.Expression of cytokines(TNF-?,IL-1? and IL-6)suggests that p55 PIK promotes acute and chronic inflammatory responses in the AOM-DSS enteritis model.From this we raise the scientific question,can p55 PIK exert its proinflammatory effect by affecting autophagy? In this study,macrophages(bone marrowderived macrophages BMDM and THP-1 cell lines)with moderate expression of p55 PIK were selected as subjects.The aim was to explore the effects of p55 PIK on LPS-induced inflammatory effects by inhibiting autophagy.The main results are as follows: ?.Selection of subjects: BMDM and human THP-1 cell line-derived macrophagesThe protein expression of p55 PIK in different subgroups of mouse Th1,Th2,Th17 and Treg and human monocyte cell lines was detected by western blot using BIOGPS and GeneCards.Bone marrow-derived macrophages(BMDM)and THP-1 cell lines were selected as subjects for this study.?.p55 PIK promotes LPS-induced inflammatory response 2.1 P55PIK-specific inhibitor TAT-N15 inhibits LPS-induced inflammationIn the mature bone marrow-derived macrophage BMDM and THP-1 cell lines,the p55 PIK specific inhibitor TAT-N15 was pre-treated for 12 h,and then stimulated with LPS for 4h to collect the total RNA and cell culture supernatant,Real.Time-PCR and ELISA were used to detect mRNA and protein levels of pro-inflammatory cytokines(TNF-?,IL-1?,IL-6)and anti-inflammatory cytokines(TGF-? and IL-10),respectively.The results showed that in BMDM and THP-1,TAT-N15 significantly inhibited LPSinduced proinflammatory cytokine(TNF-?,IL-1?,IL-6)mRNA and protein levels.There was no effect on the expression of mRNA and protein levels of anti-inflammatory cytokines(TGF-? and IL-10).This result suggests that the p55PIK-specific inhibitor TAT-N15 can inhibit LPS-induced inflammation.2.2 Low expression of p55 PIK inhibits LPS-induced inflammation in the THP-1 cell lineFirst,sgRNA was designed by CRISPR-Cas9 technology,and 293 T was transfected with lenti-viral packaging plasmid.The lenti-virus infected THP-1 cell line and successfully screened the p55 PIK stably transfected cell line.LPS-1 was used to stimulate the low expression of p55 PIK stably transfected cell line(p55PIK-KO)for 4h.Total RNA and cell culture supernatant were collected.The pro-inflammatory cytokines(TNF-?,IL-1?,IL-6)were detected by Real Time-PCR and ELISA.The results showed that LPS-induced proinflammatory cytokine(TNF-?,IL-1?,IL-6)mRNA levels And protein expression was significantly inhibited in the THP-1 cell line stably expressing p55PIK(p55PIK-KO)compared to the control cell line.But the mRNA and protein expression of anti-inflammatory cytokines(TGF-? and IL-10)did not change significantly.This result suggests that low expression of p55 PIK can inhibit LPS-induced inflammation.2.3 High expression of p55 PIK promotes LPS-induced inflammation in the THP-1 cell lineThe THP-1 cell line was highly transfected with a high expression p55 PIK lenti-virus,and a highly expressed p55 PIK stably transfected cell line(p55PIK-OE)was successfully screened.The cells were also stimulated with LPS for 4h,and the total RNA and cell culture supernatant were collected.Real Time-PCR and ELISA were used to detect pro-inflammatory cytokines(TNF-?,IL-1?,IL-6)and anti-inflammatory cytokines(TGF-? and IL-10)mRNA and protein levels.The results showed that in the THP-1 cell line,high expression of p55 PIK significantly promoted the mRNA and protein levels of LPS-induced proinflammatory cytokines(TNF-?,IL-1?,IL-6)against anti-inflammatory cytokines(TGF-? and IL-10).There was no significant inhibition of mRNA and protein levels of TGF-? and IL-10.This result suggests that high expression of p55 PIK promotes LPS-induced inflammation.3.In macrophages,p55 PIK promotes p65 activation in the LPS-NF-?B signaling pathway 3.1 In LPS-stimulated THP-1 cells,p55 PIK expression is up-regulated and p65 is activatedIn THP-1,different time points(0h,2h,4h)were stimulated with LPS,and p55 PIK and p-p65 were detected by western blot.The results showed that p55 PIK protein expression and p65 phosphorylation gradually increased with the increase of LPS treat time compared with the control group.This result suggests that p55 PIK activates the NF-?B signaling pathway in LPS-stimulated THP-1 cells.3.2.TAT-N15 inhibits p65 activation in LPS-NF-?B signaling pathway in LPSstimulated BMDM cellsBMDM was pretreated with TAT-N15 for 12 h,and then stimulated with LPS for different time points(0h,2h,4h,6h),and p-Akt(S473)and p-p65 were detected by western blot.The results showed that TAT-N15 significantly inhibited p-p65 at different stimulation time points of LPS,but had no significant effect on p-Akt(S473)expression.This result suggests that TAT-N15 inhibits the activation of p65 in an Akt-independent manner in LPS-stimulated BMDM.?.p55 PIK inhibits autophagy in LPS-stimulated macrophages 4.1.In BMDM,p55PIK-specific inhibitor TAT-N15 promotes autophagyBMDM was treated with autophagy inhibitors BafA and TAT-N15 alone or simultaneously,and protein expression of autophagy important markers LC3 and p62 were detected by Western blot.The results showed that the expression of autophagyassociated marker proteins LC3 and p62 increased simultaneously with the increase in the time of TAT-N15 treatment compared with the BafA group.The RFP-GFP-LC3 double-fluorescence confocal results showed that compared with CP and the control group,the red-green-fused dual-fluorescence-yellow-fluorescent aggregated particles of LC3 in the TAT-N15 group increased significantly,suggesting that TAT-N15 may promote BMDM.Intracellular autophagy bodies are formed.In addition,compared with CP and control group,the red fluorescent aggregated particles of LC3 were also significantly increased in TAT-N15 group,suggesting that TAT-N15 may promote autophagic flow in BMDM.4.2.Low expression of p55 PIK promotes autophagy in LPS-stimulated THP-1 cell lineIn the control cell line and THP-1 cell line with low expression of p55 PIK,prestimulation with LPS for 8h,followed by treatment with BafA for 4h,and LC3 expression was detected by Western blot.The results showed that compared with the control cell line,the expression of LC3 was significantly increased in the p55 PIK low expression stable cell line,suggesting that low expression of p55 PIK may promote the formation of autophagosomes.4.3.High expression of p55 PIK inhibits autophagy in LPS-stimulated THP-1 cell lineIn the control cell line and high expression p55 PIK THP-1 cell line,LPS was prestimulated for 8h,and then treated with Rapamycin for 4 h.Western blot was used to detect LC3 protein expression.The results showed that the expression level of LC3 was significantly down-regulated in the p55 PIK high expression group compared with the control cell line.The change of autophagy was detected by RFP-GFP-LC3 double fluorescence confocal microscopy.After high expression of p55 PIK,the red single fluorescent aggregated particles of LC3 were significantly decreased.This result suggests that autophagy flow is observed when THP-1 cells express p55 PIK under the treatment of LPS.The occurrence is suppressed.The above results suggest that high expression of p55 PIK inhibits autophagy in the LPS-stimulated THP-1 cell line.?.In macrophages,p55 PIK promotes LPS-induced inflammation by inhibiting autophagy 5.1.In THP-1,autophagy inhibits LPS-mediated inflammationTHP-1 cells were pretreated with autophagy inhibitor 3-MA and autophagy inducer Rapamycin for 1h,and then stimulated with LPS for 4h to collect total RNA and medium supernatant.Real Time-PCR and ELISA were used to detect mRNA and protein levels of pro-inflammatory cytokines(TNF-?,IL-1?,IL-6),respectively.The results showed that the mRNA and protein levels of cytokine IL-1? were significantly increased in the inhibition of autophagy.The mRNA and protein levels of cytokine IL-1? were significantly reduced in the promotion of autophagy.This result suggests that autophagy can negatively regulate inflammation in THP-1.5.2.In the THP-1 stably transfected cell line with low expression of p55 PIK,inhibition of autophagy partially reversed the expression of IL-1? down-regulated by low expression of p55 PIK.In the THP-1 stable cell line with low expression of p55 PIK,autophagy inhibitor 3-MA was added for 1h,and then stimulated with LPS for 4h to collect total RNA and medium supernatant.Real Time-PCR and ELISA were used to detect mRNA and protein levels of pro-inflammatory cytokines(TNF-?,IL-1?,IL-6),respectively.The results showed that under the action of LPS,low expression of p55 PIK promoted autophagy and inhibited the mRNA level and protein expression of pro-inflammatory cytokines,but the low-expression p55 PIK stably transfected cell line was treated with autophagy inhibitor 3-MA.That is,after blocking the effect of promoting autophagy,its inhibition of IL-1? secretion is partially reversed,and the inhibition of TNF-? and IL-6 has no reversal effect.This result suggests that inhibition of autophagy partially reverses the effect of low expression of p55 PIK to down-regulate IL-1? expression in THP-1 stable cell lines with low expression of p55 PIK.5.3 Activation of autophagy partially reverses the up-regulation of IL-1? expression by p55 PIK in a THP-1 stable cell line with high expression of p55PIKIn the THP-1 stable cell line with high expression of p55 PIK,the autophagy inducer Rapamycin was added for 1h,and then stimulated with LPS for 4h to collect total RNA and medium supernatant.Real Time-PCR and ELISA were used to detect mRNA and protein levels of pro-inflammatory cytokines(TNF-?,IL-1?,IL-6),respectively.The results showed that under the action of LPS,high expression of p55 PIK inhibited autophagy and promoted the mRNA level and protein expression of pro-inflammatory cytokines,but the highly expressed p55PIK-stable cell line was treated with autophagy inducer Rapa,ie,After the effect of inhibiting autophagy,its effect of promoting the secretion of IL-1? and IL-6 was partially reversed,and the promotion of TNF-? secretion was not reversed.This result suggests that activation of autophagy partially reverses the effect of p55 PIK on up-regulating IL-1? expression in a THP-1 stable cell line with high expression of p55 PIK.This study verified the pro-inflammatory effect of p55 PIK on macrophages,an innate immune cell,and elucidated the promotion/inhibition effect of p55 PIK and p55PIK-specific inhibitor TAT-N15 on the innate immune response and its mechanism.The biological effect of p55 PIK promoting LPS-induced inflammation by inhibiting autophagy was first revealed.This study not only enriched the inhibition of autophagy by Class IA PI3 K non-canonical regulatory subunit p55 PIK,but also the p55 PIK specific inhibitor TAT-N15 provides a theoretical basis for the therapeutic effect in the induction of inflammatory diseases,and provides new targets and treatments for the prevention and treatment of infectious diseases such as endotoxin shock caused by LPS.