mTORC1 Regulates Flagellin-Induced Inflammatory Response in Macrophages

Abstract

Bacterial flagellin triggers inflammatory responses. Phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) regulate the production of pro- and anti-inflammatory cytokines that are induced by extrinsic antigens, but the function of mTORC1 in flagellin-induced inflammatory response is unknown. The purpose of this study was to examine the role and the mechanism of PI3K/Akt/mTOR pathway in flagellin-induced cytokine expression in mouse macrophages. We observed that flagellin upregulated TNF-α time- and dose-dependently. Flagellin stimulated rapid (<15 min) PI3K/Akt/mTOR phosphorylation that was mediated by TLR5. Inhibition of PI3K with LY294002 and wortmannin, and of mTORC1 with rapamycin decreased flagellin-induced TNF-α and IL-6 expression and cell proliferation. The activation of NF-κB p65 and STAT3 was regulated by mTORC1 via degradation of IκBα and phosphorylation of STAT3 in response to flagellin, respectively. Thus, the PI3K/Akt/mTORC1 pathway regulates the innate immune response to bacterial flagellin. Rapamycin is potential therapy that can regulate host defense against pathogenic infections.

Fig 1. Flagellin induces TNF-α expression in a time-dependent and dose-dependent manner.

Ana-1 cells were treated with flagellin, and supernatants were assayed for TNF-α by ELISA. A. flagellin (100 ng/ml) for 0, 4, 8, 12, and 24 h. B. flagellin (0, 1, 10, 100 ng/ml) for 24 h. Experiments were made in triplicate, and data are representative of three separate experiments (mean ± SD).

Fig 2. LY294002, wortmannin and rapamycin dowregulates flagellin-induced TNF-α and IL-6 expression in mouse macrophages.

Ana-1 cells were pretreated with LY294002 (10 μM), wortmannin (10 nM) and rapamycin (100 nM) for 4 h before being stimulated with 100 ng/ml flagellin for 24 h. Supernatants were assayed for TNF-α and IL-6 by ELISA. A. flagellin-induced TNF-α is inhibited by LY294002 and wortmannin. B. flagellin-induced IL-6 expression is inhibited by LY294002 and wortmannin. C. flagellin-induced TNF-α is inhibited by rapamycin. D. flagellin-induced IL-6 expression is inhibited by rapamycin. Experiments were made in triplicate, and data are representative of three separate experiments (mean ± SD). *p< 0.05 compare to untreated controls, # p< 0.05 compared with flagellin group.

Fig 3. LY294002, wortmannin and rapamycin attenuates flagellin-induced cell proliferation.

Ana-1 cells were pretreated with LY294002 (10 μM), wortmannin (10 nM) and rapamycin (100 nM) for 4 h and then stimulated with 100 ng/ml flagellin for 24 h, and cell proliferation was measured by methyl thiazolyl tetrazolium (MTT) assay. A. Proliferation was inhibited by LY294002 and wortmannin in response to flagellin. B. Proliferation was inhibited by rapamycin in response to flagellin. Experiments were made in triplicate, and data are representative of three separate experiments (mean ± SD). *p< 0.05 compare to untreated controls, # p< 0.05 compared with flagellin group.

Fig 4. Flagellin activates PI3K/Akt/mTORC1 signaling via TLR5.

Ana-1 cells were lysed, and total proteins were extracted. A. the cells were stimulated at the indicated times with S. typhimurium flagellin (100 ng/ml). B. the cells were stimulated by flagellin (100 ng/ml) for 24 h in present or absent of TLR5 or TLR4 antibody. Total proteins were subjected to immunoblot with phospho-Akt(Ser473), phospho-mTOR (Ser2448), phospho-S6 (Ser240/244), and phospho-4EBP1 (Thr37/46). Blots were stripped and probed with total Akt, mTOR, S6, and 4EBP1 antibodies. β-actin was used as a loading control. Three separate experiments were performed. *p< 0.05, ns. not significant.

Fig 5. LY294002 decreases flagellin-induced Akt/mTOR activation but does not affect p44/42 MAPK (Erk1/2) activation.

Ana-1 cells were pretreated with LY294002 (10 μM) for 4 h and stimulated with 100 ng/ml flagellin for 24 h. A. Total proteins were subjected to immunoblot with phospho-p44/42 MAPK (Erk1/2)(Thr202/Tyr204), the blot was stripped and probed with p44/42 MAPK (Erk1/2). B. Total proteins were subjected to immunoblot with phospho-Akt (Ser473), phospho-mTOR (Ser2448), phospho-S6 (Ser240/244), and phospho-4EBP1 (Thr37/46) antibodies. The blots were stripped and probed with Akt, mTOR, S6, and 4EBP antibodies. β-actin was used as the loading control. Three separate experiments were performed.

Fig 6. Rapamycin prevents flagellin-induced IκBα degradation and p65 and STAT3 activation.

Ana-1 cells were pretreated with rapamycin (100 nM) for 4 h and stimulated with 100 ng/ml flagellin for 24 h. Total proteins were separated by SDS-PAGE and probed with IκBα, phospho-NF-κB p65 (Ser536), and phospho-STAT3 (Tyr705) antibodies. The blots were stripped and probed with NF-κB p65 and STAT3 antibodies. β-actin was used as the loading control. Three separate experiments were performed.

Fig 7. TLR5 mediates flagellin-induced TNF-α and IL-6 expression and STAT3 activation in macrophages.

Ana-1 cells were pretreated with TLR5-blocking antibody or TLR4-blocking antibody for 24 h and stimulated with 100 ng/ml flagellin for 24 h. Supernatants were assayed for TNF-α and IL-6 by ELISA. A. Flagellin-induced TNF-α expression is inhibited by TLR5 blocking antibody. B. Flagellin-induced IL-6 expression is inhibited by TLR5 blocking antibody. Experiments were made in triplicate, and data are representative of three separate experiments (mean ± SD). *p< 0.05, ns. not significant. C. Total proteins were separated by SDS-PAGE and probed phospho-STAT3 (Tyr705) antibodies. The blots were stripped and probed with STAT3 antibodies. β-actin was used as the loading control. Three separate experiments were performed. *p< 0.05 compare to flagellin group, # p< 0.05 compared with TLR4 antibody group.

Fig 8. Rapamycin downregulates flagellin-induced IL-6 expression in mouse peritoneal macrophages.

Mouse peritoneal macrophages were pretreated with rapamycin (100 nM) for 4 h before being stimulated with 100 ng/ml flagellin for 24 h. Supernatants were assayed for IL-6 by ELISA. flagellin-induced IL-6 expression is inhibited by rapamycin. Experiments were made in triplicate, and data are representative of three separate experiments (mean ± SD). *p< 0.05 compared with flagellin group.

Fig 9. Model of PI3K/Akt/mTORC1 regulation of flagellin-induced TNF-α and IL-6 expression.

Based on our studies and other reports, we propose a model in which flagellin binds toTLR5 and transmits its signal to PI3K via MyD88. Activated PI3K stimulatesAkt/mTOR, and mTOR signaling activates NF-κB and STAT3. These factors translocate to the nucleus and enhance TNF-α and IL-6 expression, promoting cell proliferation and survival.