The Mechanism Underlying Enhancing The Innate Immune Functions Of Macrophages By Piperine Through Regulating MTORC1 Signaling

ObjectiveAs a major bioactive alkaloid isolated from pepper,piperine possesses a vast array of pharmacological activities,including anti-gastroenteritis,anti-depression,anti-epilepsy and neuroprotection effects.But the precise mechanism underlying these effects is largely unclear.This thesis focuses on the effects of piperine on macrophages,especially peritoneal macrophages,and the underlying mechanism.Macrophages are important innate immune cells,and are closely associated with many chronic diseases including atherosclerosis,asthma,inflammatory bowel disease(IBD),rheumatoid arthritis and fibrosis.Macrophages have abundant lysosomes and mitochondria,functioning in phagocytosis and elimination of apoptotic debris,as well as tissue repair.The functions and homeostasis of macrophages are finely regulated by multiple signaling pathways.The mechanistic target of rapamycin(mTOR)is not only the hub regulating metabolism but also a key molecule modulating the immune responses of innate immune cells including monocytes/macrophages,affecting their inflammation related signaling pathways,cytokine expression,activation and differentiation.mTOR binds to other proteins to form two complexes named mTORC1 and mTORC2,each with distinct regulating activities.mTORC1 can be activated by insulin and metabolism of amino acids(including leucine and glutamine).It has been shown that the maintenance of innate immune homeostasis in intestinal mucosa depends on the uptake and metabolism of amino acids by intestinal epithelial cells,which secretes antimicrobial peptides upon the mTORC1 activation.Interestingly,previous studies demonstrated that piperine can increase the absorption of leucine and other amino acids by enterocytes,suggesting that piperine may promote mTORC1 activation,thereby affecting the functions of the innate immune cells including macrophages.To verify this hypothesis,we in this study used the mTOR inhibitor INK128 to explore the effects of mTOR signaling on the activation and functions of macrophages,and we also explored whether piperine regulated the innate functions of peritoneal resident macrophages(pM?s)through mTOR signaling,to elucidate the mechanism by which piperine affected the functions of macrophages.This will provide the experimental basis for potential clinical application of piperine in treating inflammatory diseases.MethodsAt cellular level,WST-1 reagent was used to detect the cytotoxicity of INK128 on murine macrophage RAW 264.7.Western blotting was used to analyze the related proteins of the mTOR and NF-?B pathways.Cytometric bead array(CBA)kit was applied to test the production of IL-6,TNF-? and other cytokines in cell culture supernatants,and the mRNA levels of TNF-? and IL-6 were detected by real-time quantitative PCR(qPCR).HeLa and pM?s cells were treated with serum-and amino acid-free medium,and then the effect of piperine on leucine-and glutamine-induced activation of mTORC1 pathway was detected.The mTORC1 activation was analyzed by western blotting to show the phosphorylation of mTORC1 substrates(p70S6K and 4E-BP1),as well as by immunofluorescence assay to observe the distribution of mTOR on lysosomes(indicted by LAMP2),where mTORC1 activation took place,and the distribution of p-S6(phosphorylated by p70S6K).The effect of piperine on subcellular distribution of amino acid transporter SLC7A5/SLC3A2 was also detected by immunofluorescence observation.Small interfering RNA(siRNA)was used to knockdown SLC7A5 gene in HeLa cells,and then western blotting was applied to detect the phosphorylation of p70S6K(indicative of mTORC1 activity).Besides,CBA kit was applied to detect the effect of piperine on inflammatory cytokines secretion in pM?s cells,and PI3K/AKT/mTOR inhibitors were used to disturb the production of cytokines.Mice were intraperitoneally injected with E.coli and the effect of piperine on the mice survival was explored.Haematoxylin and eosin staining(H&E)was used to observe the inflammatory lesion in the liver and colon.Peritoneal lavage fluids were collected to detect the cytokines by CBA assay,and the corresponding colony-forming units(CFUs)were determined on LB agar plates.Flow cytometry was used to analyze the phagocytosis of fluorescent labeled bacteria by peritoneal macrophages,and the activation of intracellular Caspase-3.ResultsWhen mTOR was inhibited by INK128,the phosphorylation levels of its substrates p70S6 K,4E-BP1 and AKTSer473 were down-regulated.The secretion of IL-6 and TNF-? in LPS-stimulated macrophages(RAW 264.7)was also inhibited,suggesting that mTOR could regulate the expression of inflammatory cytokines in macrophages.Piperine increased the phosphorylation levels of p70S6 K and 4E-BP1,indicating its enhancement of mTORC1 activation.This effect of piperine was depended on the presence of leucine and glutamine.Piperine promoted the redistribution of amino acid transporters SLC7A5/SLC3A2 from the intracellular compartments to the surface of HeLa cells.SLC7A5 knockdown inhibited the phosphorylation level of p70S6 K activated by piperine,leucine and glutamine.Piperine enhanced leucine and glutamine induced colocalization of mTOR and LAMP2(indicative of mTOR activation)in HeLa cells,but this effect could be antagonized by SLC7A5/SLC3A2 inhibitor BCH.These results suggested that piperine could enhance the mTORC1 signal induced by amino acid metabolism.Piperine pretreatment promoted the secretion of IL-6 and TNF-? in LPS-stimulated pM?s,but the enhancement effect was abrogated by PI3K/AKT/mTOR pathway inhibitors.Piperine administration intragastrically could increase the survival of mice infected with E.coli,and reduced the bacterial induced inflammatory lesion of liver and colon.Comparison with vehicle group,piperine significantly increased the intraperitoneal IL-6,TNF-? level at an early stage of bacterial infection(6 h),but the intraperitoneal IL-6,TNF-? levels and the bacterial burden were decreased at the late stage of infection(24 h).Piperine prevented the reduction of F4/80 high pM?s and F4/80highCFSE+ pM?s after the mice were intraperitoneally injected with CFSE-labelled E.coli,indicating that piperine promoted phagocytosis.Piperine also reduced bacterium-induced reduction of CD11bhigh/GATA6 bright pM?s and inhibited cleaved Caspase-3 levels in CD11b+ pM?s and F4/80+ pM?s,while metformin(indirectly inhibiting mTOR activity)could significantly antagonized these protective effects of piperine.These results suggested that piperine enhanced the macrophage function by modulating mTORC1 signaling to exert an anti-infection effect.ConclusionsPiperine increased the distribution of amino acid transporter SLC7A5/SLC3A2 on the cell membrane,thus promoting the uptake and metabolism of leucine and glutamine,and upregulating activation of mTORC1.Piperine also enhanced the secretion of proinflammatory cytokines IL-6 and TNF-? at the early stage of infection,prevented peritoneal resident macrophages from bacterium-induced apoptosis,and increased their bacterial phagocytic ability,thus conferring the mice resistance against bacterial infection and sepsis-induced tissue injury.Collectively,piperine enhanced the innate immune functions of macrophages through promoting the mTORC1 activity by promoting amino acid metabolism,thereby displaying its immune regulation and anti-infectious pharmacological properties.