The tuberous sclerosis complex 1 (TSC1) is a tumor suppressor that inhibits the mammalian target of rapamycin (mTOR), which serves as a key regulator of inflammatory responses after bacterial stimulation in monocytes, macrophages, and primary dendritic cells. Previous studies have shown that TSC1 knockout (KO) macrophages produced increased inflammatory responses including tumor necrosis factor-α (TNF-α) and IL-12 to pro-inflammatory stimuli, but whether and how TSC1 regulates pro-IL-1β expression remains unclear. Here using a mouse model in which myeloid lineage-specific deletion of TSC1 leads to constitutive mTORC1 activation, we found that TSC1 deficiency resulted in impaired expression of pro-IL-1β in macrophages following lipopolysaccharide stimulation. Such decreased pro-IL-1β expression in TSC1 KO macrophages was rescued by reducing mTORC1 activity with rapamycin or deletion of mTOR. Rictor deficiency has no detectable effect on pro-IL-1β synthesis, suggesting that TSC1 positively controls pro-IL-1β expression through mTORC1 pathway. Moreover, mechanism studies suggest that mTORC1-mediated downregulation of the CCAAT enhancer-binding protein (C/EBPβ) critically contributes to the defective pro-IL-1β expression. Overall, these findings highlight a critical role of TSC1 in regulating innate immunity by control of the mTOR1-C/EBPβ pathway.
Macrophages are migratory phagocytic cells and play a key role in the innate immune response and adaptive immune response1. Macrophages are polarized to the classic M1 macrophages by exposure to IFN-γ and GM-CSF, or in the presence of bacterial products such as lipopolysaccharide (LPS). M1 macrophages produce high levels of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), IL-6, IL-12, and IL-23, and increased levels of reactive oxygen species2,3,4,5,6. IL-1β, an important pro-inflammatory cytokine, belongs to the IL-1 family of cytokines. The significant pro-inflammatory function of cytokine IL-1β is to recruit inflammatory and immunocompetent cells infiltrating from the circulation into the extravascular space and then into the site of injury or infection7,8,9. IL-1β is also an angiogenic factor and functions in tumor angiogenesis, invasiveness, and metastasis10,11. Moreover, IL-1β has long been linked to the pathogenesis of atherosclerosis and rheumatoid arthritis diseases12,13,14. The cytokine IL-1β is mostly produced by activated monocytes, macrophages, and primary dendritic cells (DCs) mainly through a NF–κB-dependent pathway15,16. The expression of IL-1 is regulated at the levels of transcription, mRNA stabilization, and post-translational proteolytic processing14. IL-1β is first translated as an inactive precursor protein, pro-IL-1β, which is then proteolytically cleaved at Asp116 into its mature form by an inflammasome17.
The mammalian target of rapamycin (mTOR) is a conserved serine–threonine kinase that is essential in broad aspects of cellular activity, including cell growth, survival and aging. mTORC1 can be activated by growth factors through the tuberous sclerosis complex (TSC), which consists of TSC tumor suppressors, TSC1 and TSC2, and Tre2-TBC1D718. TSC1/2 complex negatively regulates mTORC1 through the GTPase activation property of TSC2 to RheB, a small GTPase protein that promotes mTORC1 activation19. Some studies showed that activation of mTOR downregulated IL-12p70 and IL-23 production in human macrophages20,21. Moreover, IL-12 was enhanced, whereas IL-10 was blocked after mTOR inhibition in mouse bone marrow-derived macrophages (BMDMs) and mDCs22. These findings indicate that activated mTOR may limit pro-inflammatory responses. On the contrary, other studies indicated that TSC1-deficient macrophages produced elevated pro-inflammatory cytokines including TNF-α, IL-12p40, and IL-6 in response to multiple TLR ligands19,23. Moreover, TSC1-deficient BMDMs treated by LPS secreted more of the pro-inflammatory cytokines such as IL-6 and TNF-α but less of the anti-inflammatory cytokine IL-1024.
The involvement of mTOR pathway in the regulation of and IL-1β expression has been studied in mouse and human macrophages22,25,26. In the present study, using TSC1-deficient macrophages and macrophage cell lines, we studied the role of TSC1 on pro-inflammatory cytokine IL-1β expression and explored the associated molecular mechanism. We found that LPS-induced pro-IL-1β synthesis was significantly downregulated at both the mRNA and protein levels in TSC1-deficient macrophages, and prolonged rapamycin (Rapa) treatment or mTOR deletion significantly rescued this phenotype. mTORC1, but not mTORC2, was involved in the suppression of pro-IL-1β transcription by selectively inhibiting the CCAAT enhancer-binding protein (C/EBPβ) expression. Moreover, overexpression of C/EBPβ significantly reversed the defect of pro-IL-1β expression in TSC1 knockout (KO) BMDMs.
MATERIALS AND METHODS
Myeloid-specific TSC1, mTOR, and Rictor conditional KO mice were obtained by crossing TSC1loxp/loxp , mTORloxp/loxp , or Rictorloxp/loxp mice with mice-expressing Cre recombinase under the control of the Lysozyme promoter (LysMCre)23. LysMCre+/−TSC1loxp/loxp , LysMCre+/−mTORloxp/loxp , and LysMCre+/−Rictorloxp/loxp were referred to as TSC1 KO, mTOR KO, and Rictor KO, respectively. LysMCre-negative littermates served as the control (referred to as wild-type (WT)). About 4- to 5-week-old mice were usually used for the in vitro experiments. TSC1loxp/loxp mice were the generous gifts of Dr Hongbing Zhang (Institute of Basic Medical Sciences and School of Basic Medicine, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China)27,28. mTORloxp/loxp mice were kindly provided by Dr Zhongzhou Yang from the Center of Model Animal Research at Nanjing University, China27. LysMCre mice were kindly offered by Dr Lianfeng Zhang from the Key Laboratory of Human Diseases Comparative Medicine, Ministry of Health; Institute of Laboratory Animal Science, CAMS & PUMC. All mice were maintained in a specific pathogen-free facility. All experimental manipulations were undertaken in accordance with the Institutional Guidelines for the Care and Use of Laboratory Animals, Institute of Zoology (Beijing, China).
Rapa (LC laboratory) was reconstituted in ethanol at 60 mg/mL and then diluted with DMSO (Sigma-Aldrich) to 60 μg/mL. Bacterial LPSs (E. coli 055:B5) were purchased from Sigma-Aldrich (St Louis, MO, USA). The antibodies against TSC1, TSC2, CREB, H3, C/EBPβ, Rictor, IL-1β, ASC, p-S6 (Ser240/244), p-JNK (Thr183/Tyr185), p-P38 (Thr180/Tyr182), p-IKKα/β (Ser176/180), p-IκBα (Ser32/36), and p-P65 (Ser536) were obtained from Cell Signaling Technology. Anti-c-Fos and anti-c-Jun antibodies were purchased from Enogene. Anti-NLRP3 and anti-caspase-1 (mouse) antibodies were from Enzo Life Sciences. The above-mentioned antibodies were diluted at 1:1000 in 5% bovine serum albumin (BSA), except for anti-p-S6 (Thr421/Ser424; 1:4000) and anti-H3 (1:3000). Anti-β-Actin mAb (diluted at 1:50 000) was purchased from Sigma-Aldrich. PD98059 and SP600125 were from Selleck Chemicals. Mouse TLR1-9 agonist kit was obtained from InvivoGen.
Bone marrow cells were cultured with DMEM containing 10% (V/V) FBS and 10 ng/ml of mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) for 7–12 days to obtain BMDMs29. The non-adherent cells and loosely attached cells were washed by swirling the plates on day 3, 5, 7, and 930,31. The purity of BMDMs was more than 95% (Supplementary Figure 1A). Primary mouse peritoneal macrophages were obtained from the peritoneal exudates of 4-week-old mice. The peritoneal exudate cells were washed twice with PBS solution, adjusted to 1 × 106 cells/mL in DMEM, and then cultured for 3–4 h at 37 °C and 5% CO2. The non-adherent cells were removed by washing with warm PBS. The purification rate of macrophage was analyzed by FCM (Beckman, CA) using the mouse macrophage marker F4/80. F4/80+ macrophages constituted more than 90% of the adherent cells.
Quantitative PCR analysis
Total RNA was isolated with TRIzol (Invitrogen), and reverse transcription was performed with M-MLV superscript reverse transcriptase according to the manufacturer’s instructions32. Real-time PCR kit (SYBR Premix Ex Taq, DRR041A) was purchased from Takara Bio Inc. PCR was performed on CFX96 (Bio-Rad). All mRNA expression levels were normalized to that of HPRT. Each sample was run at least in triplicate. Primers used for the amplification are summarized in Table 1.
Western blot assay
Cells were cultured in DMEM medium with 10% FCS in 12-well plate. Cells were then treated with LPS (1 μg/mL) for different durations. After stimulation, the cells were washed once in cold PBS, lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA) with protease and phosphatase inhibitor cocktails (Sigma) for 10 min on a rocker at 4 °C. Cells were scraped, centrifuged at 12 000 rpm for 10 min at 4 °C and the supernatants were mixed with ×5 protein loading buffer. Protein concentration was determined using a BCA assay. Protein samples were analyzed on SDS-PAGE and transferred onto PVDF membrane (Millipore). PVDF membrane was blocked with TBST (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween 20) with 5% non-fat dried milk for 1 h, then incubated with primary antibodies overnight on a shaker at 4 °C. The appropriate HRP-coupled secondary antibody was then added and was detected through chemiluminescence (Millipore). β-Actin was used as a protein-loading control.
shRNA sequences, lentiviral preparation, and cell infection
Constitutively active Rheb (Rheb QL64) was amplified by PCR method and cloned into lentiviral overexpression vector pWPXLd-puro. The scrambled shRNA (non-target shRNA vector) and shRNA for TSC1 and mTOR vectors were purchased from Santa Cruz Biotechnology. TSC2 and Rictor shRNA were generated by cloning the target oligonucleotides into the BamHI and EcoRI sites of pShRNA lentiviral plasmid. The target sequences of shRNAs used in this study were: TSC2, 5′-CCTCTCTCTTAAACTTGATAT; and Rictor, 5′-CGGTTCATACAAGAGTTATTT. Lentiviral plasmids were co-transfected with the lentiviral-packaging plasmids psPAX2 and pMD2.G into HEK293T cells for virus production. RAW264.7 cells were infected with lentivirus supernatant with 8 μg/mL polybrene (Invitrogen). Stable shRNA or overexpressing cells were selected with puromycin (3 μg/mL) for two weeks.
Overexpression of C/EBPβ in BMDMs using adenoviral vectors
An adenoviral construct encoding C/EBPβ was prepared with a ViraPower adenovirus expression system (Invitrogen) according to the manufacturer’s instructions. cDNA that encodes for the C/EBPβ gene and Ubic promoter-GFP fragment were subcloned into the pENTR vector. As a control, only Ubic promoter-GFP fragment was subcloned into the pENTR vector. These constructs were then recombined with pAd/CMV/V5-DEST, using LR Clonase II (Invitrogen) according to the manufacturer’s protocol, to generate the pAd plasmids. The plasmids were linearized using PacI and then transfected into 293A cells with Lipofectamine 2000. After 10 days, cells from wells showing 100% cytopathic effect were collected by scraping, lysed with three rounds of freeze/thaw and centrifuged to collect the supernatant. One more round of infection with the supernatant was performed to enhance the viral titer. BMDMs were prepared as described above and infected with the freshly prepared viral supernatant (50% viral supernatant containing empty vector or C/EBPβ, supplied with 50% DMEM complete media) for 24 h. The cells were then washed and stimulated with LPS for an additional 6 h.
In vivo LPS challenge
WT and TSC1 KO mice were intraperitoneally injected with LPS (5 mg/kg body weight). The serum was collected after 2 h and IL-1β was measured by ELISA according to the manufacturer’s instructions (Invitrogen).
Detection of IL-1β by ELISA assay
Mature IL-1β protein levels in cultured supernatant samples or sera were measured using an IL-1β Mouse ELISA Kit according to the manufacturer’s instructions (Invitrogen).
All data are presented as the mean ± SD. Student’s unpaired t-test for comparison of means was used to compare groups. A P-value less than 0.05 was considered to be statistically significant.
Decreased pro-IL-1β expression in TSC1 KO macrophages
First we measured the efficiency of TSC1 deletion in macrophages. Immunoblotting assays demonstrated that TSC1 was absent in the TSC1 KO peritoneal macrophages and BMDMs, in which phosphorylation of the mTORC1 downstream targets ribosomal S6 was enhanced (Figure 1A and B). Following LPS stimulation for different durations, freshly isolated TSC1 KO peritoneal macrophages produced less pro-inflammatory cytokines pro-IL-1β at the mRNA level than WT cells (P < 0.01, Figure 1C). Moreover, the mRNA expression level of pro-IL-1β was noticeably impaired in TSC1 KO BMDMs relative to WT BMDMs (P < 0.001, Figure 1D). To test the protein level of pro-IL-1β, the WT and TSC1 KO peritoneal macrophages were stimulated with LPS for 6 h. We found that the expression level of pro-IL-1β protein significantly decreased in TSC1 KO peritoneal macrophages and BMDMs (Figure 1E and F).
To further investigate the role of TSC1 on pro-inflammatory cytokine IL-1β expression, we generated a cell line that stably expressed short hairpin RNA against TSC1 (TSC1 shRNA) in RAW264.7 cells, which was a mouse macrophage cell line. As shown in Figure 1G, TSC1 protein was severely decreased in TSC1 shRNA cells compared with scramble cells. TSC1 knockdown also reduced TSC2 protein level (Figure 1G), which is consistent with previous reports that TSC1 is required for TSC2 stability19. In addition, LPS stimulation induced rapid phosphorylation of S6, and p-S6 level was higher in TSC1 shRNA cells than in scramble cells, indicating that mTORC1 activation was enhanced (Figure 1G). Next, quantitative real-time PCR analysis was performed to assess mature mRNA expression of pro-IL-1β in scramble and TSC1 shRNA cells after time-varying exposure to LPS (0, 1, 3, 6, 12, 18, and 24 h). We found that mRNA expression of pro-IL-1β increased rapidly after time-varying exposure to LPS and began to decrease after 3 h. Less mRNA synthesis of pro-IL-1β was observed in TSC1 shRNA cells compared with scramble cells at each time point (Figure 1H). Notably, suppression of pro-IL-1β transcription in TSC1 shRNA cells had already occurred as early as 1 h after LPS stimulation (P < 0.01, Figure 1H). Immunoblotting analysis demonstrated that pro-IL-1β protein expression peaked after 6 h exposure to LPS and TSC1 shRNA cells produced less pro-IL-1β protein levels than scramble cells at each time point (Figure 1I). Moreover, concentration gradient experiment showed that pro-IL-1β protein expressions were decreased at each concentration of LPS in TSC1 shRNA cells compared with scramble cells, although pro-IL-1β protein was barely detected in TSC1 shRNA cells treated with low concentration of LPS (0.01 or 0.1 μg/mL) (Figure 1J). In addition, TLR1-9 ligands were used to induce IL-1β synthesis in scramble and TSC1 shRNA cells. Although TLR1-3 ligand only induced low level of pro-IL-1β protein comparing with LPS, TSC1 shRNA also inhibited pro-IL-1β expression after stimulation with these ligands. We did not detect pro-IL-1β protein expression in both scramble and TSC1 shRNA RAW264.7 cells treated with TLR5, 6, 7, or 9 ligand (Supplementary Figure 2).
Pro-IL-1β protein is cleaved into the active form by an inflammasome and then secrets into the supernatant. WT and TSC1 KO peritoneal macrophages were stimulated with LPS for 6 h followed by ATP treatment. We found dramatically decreased mature IL-1β secretion in the supernatant of TSC1 KO macrophages (P < 0.001, Figure 1K). Similarly, IL-1β level in the sera of TSC1KO mice were also decreased compared with WT mice after treatment with LPS for 2 h (P < 0.001, Figure 1L). Further research showed that TSC1 KO in macrophages also dramatically suppressed caspase-1 activation (Supplementary Figure 3A) but did not affect the expression of inflammasome components at both the mRNA and protein level (Supplementary Figure 3B and C). Thus, pro-IL-1β expression was inhibited at both the mRNA and post-translational levels in TSC1 KO macrophages.
The decreased pro-IL-1β expression in TSC1 KO macrophages is due to prolonged mTORC1 hyperactivity
It is well known that the TSC complex serves as a negative regulator of mTORC1 and enhanced mTORC1 activity is detected in TSC1 KO macrophages. It is possible that the effect of TSC1 deficiency on pro-IL-1β production is due to over-activated mTOR signaling pathway. To test our hypothesis, we generated a cell line that stably expressed short hairpin RNA against TSC2 (TSC2 shRNA) in RAW264.7. The basal and LPS-induced p-S6 increased in TSC2 shRNA cells, suggesting enhanced mTORC1 activity (Figure 2A). As expected, the pro-IL-1β expression was also decreased in the TSC2 shRNA cells compared with the scramble cells at both mRNA and protein levels after stimulation with LPS (P < 0.001, Figure 2A and B). Moreover, overexpression of constitutively active Rheb-Q64L, a direct activator of mTORC1, inhibited pro-IL-1β expression in RAW264.7 cells similarly (Figure 2C). Thus, our data suggest that active mTORC1 signaling pathway may contribute to the low pro-IL-1β expression in TSC1/2-deficient macrophages.
To further demonstrate the role of over activated mTORC1 on pro-IL-1β expression, we pretreated scramble and TSC1 shRNA cells with the well-known mTOR-specific inhibitor Rapa followed by LPS stimulation. Though pre-treatment with Rapa drastically decreased basal and LPS-induced p-S6 as determined by immunoblotting in RAW264.7 cells (Figure 2D), Rapa pre-treatment for a short period of time (1 h) failed to rescue the poor pro-IL-1β expression at both mRNA and protein levels in TSC1 shRNA cells (Figure 2E and F). The same phenomenon was also observed in freshly isolated TSC1 KO peritoneal macrophages (Figure 2G). Our results are consistent with the previous study showing that intracellular pro-IL-1β production after LPS stimulation are not influenced in BMDMs pretreated with Rapa for 1 h22. Next, we prolonged Rapa pre-treatment (48 h) for chronic mTOR inhibition followed by stimulation with LPS for 6 h. The results showed that pro-IL-1β mRNA expression was significantly rescued by Rapa treatment for long periods of time in TSC1 KO BMDMs (P < 0.001, Figure 2H). The pro-IL-1β protein level was similar between WT and TSC1 KO peritoneal macrophages after Rapa treatment for 48 h (Figure 2I). Moreover, extended (48 h) Rapa treatment also rescued pro-IL-1β expression at both mRNA and protein levels in TSC1 shRNA RAW264.7 cells compared with scramble cells (Figure 2J, Supplementary Figure 4). To further confirm the ability of mTOR to suppress pro-IL-1β expression, we knocked down mTOR in WT and TSC1 shRNA RAW264.7 cells. Consistent with the results mentioned above, the decreased pro-IL-1β levels in TSC1 shRNA RWA264.7 cells was significantly rescued in TSC1/mTOR double knockdown cells (Figure 2K). The studies using mTOR KO and TSC1/mTOR KO BMDMs also demonstrated that deletion of mTOR recovered pro-IL-1β expression in TSC1 KO macrophages (Figure 2L). In summary, our results demonstrate that mTORC1 hyperactivity inhibited pro-IL-1β expression.
mTORC2 has no detectable effect on pro-IL-1β expression in macrophages
mTOR forms two functionally distinct complexes in mammalian cells: mTORC1 and mTORC2. mTORC1 is highly sensitive to Rapa, but mTORC2 is relatively insensitive to Rapa. Rapa acutely and specifically inhibits mTORC1, whereas prolonged Rapa treatment leads to disruption of mTORC2 assembly33,34. Based on our data that prolonged Rapa treatment or mTOR KO can rescue pro-IL-1β expression in TSC1 KO macrophages and TSC1 shRNA RAW264.7 cells, we wondered whether mTORC2 complex also participated in regulating pro-IL-1β synthesis. To test this hypothesis, the short hairpin RNA against Rictor, the unique component in mTORC2, was used to dissect mTORC2 involvement in pro-IL-1β synthesis. As shown in Figure 3, Rictor knockdown in TSC1 shRNA cells failed to reverse decreased pro-IL-1β synthesis in TSC1 shRNA cells (Figure 3A and B). Moreover, the mRNA and protein levels of pro-IL-1β were similar between WT and Rictor KO peritoneal macrophages after LPS stimulation in vitro (Figure 3C and D). Therefore, these data suggest that decreased pro-IL-1β synthesis in TSC1 KO macrophages was mainly mediated by mTORC1 pathway but not mTORC2.
TSC1 deficiency does not inhibit activation of MAPKs and NF-κB in macrophages
We have demonstrated that the mRNA expression of pro-IL-1β was impaired in TSC1-deficient macrophages. Next, we investigated whether the synthesis of unspliced IL-1β mRNA, pre-mRNA, was also inhibited in TSC1 KO macrophages. As shown in Figure 4, pro-IL-1β pre-mRNA, was evidently inhibited in TSC1 KO peritoneal macrophages and TSC1 shRNA cells (P < 0.001, Figure 4A and B), indicating that the regulation of TSC1 on IL-1β mRNA expression occurred at the level of transcription.
LPS is known to signal through the TLR4 signaling pathway, which plays a role in the production of inflammatory cytokines including pro-IL-1β through activating the NF-κB and MAPK pathway35,36,37. We therefore investigated the activation of the TLR4 signaling pathway in LPS-stimulated macrophages. Scramble and TSC1 shRNA cells were stimulated with LPS for different durations (0, 0.5, 1, 3, 6, 12, and 24 h) and cell lysates were immunoblotted with antibodies recognizing activated forms of the proteins involved in the NF-κB and MAPK pathways. LPS induced rapid IKKα/β, IκBα, p65, p38, ERK, and JNK activation by phosphorylation in both scramble and TSC1 shRNA cells. However, identical phosphorylation levels of these proteins were found after LPS stimulation between scramble and TSC1 shRNA cells except p-ERK (Figure 4C).
We have demonstrated that increased p-ERK in TSC1 KO macrophages was dependent on the loss of TSC2 GTPase-activating protein (GAP) activity23. However, blocking ERK and JNK activity (PD98059 and SP600125, respectively) significantly inhibited pro-IL-1β expression in WT and TSC1 KO macrophages (Supplementary Figure 5). Thus, increased p-ERK was not responsible for the impaired pro-IL-1β expression in TSC1 KO macrophages.
Given that only prolonged Rapa treatment or mTOR deletion can rescue pro-IL-1β expression in TSC1 KO macrophages and TSC1 shRNA RAW264.7 cells, this raised the possibility that enhanced mTORC1 activity inhibited certain related transcriptional factor expression, which regulates pro-IL-1β synthesis in macrophages. To test this, we assayed the mRNA and protein levels of SP1, PU.1, CREB, c-Jun, c-fos by quantitative real-time PCR, but no obvious difference was found between scramble and TSC1 shRNA cells (Figure 4C and D). These data were further confirmed in WT and TSC1 KO BMDMs (Figure 4E). To further explore whether the decreased pro-IL-1β expression in TSC1 shRNA cells was due to altered transcriptional factor nuclear translocation, including P65, c-fos, and CREB, scramble and TSC1 shRNA RAW264.7 cells were stimulated with LPS for 1 h and then nuclear extracts were prepared. The results showed that LPS-stimulated rapid nuclear translocation of P65, c-fos, and CREB. However, TSC1 deficiency did not impair the nuclear translocation of these transcriptional factors (Figure 4F). Moreover, the same profile of p65 nuclear translocation was observed in WT and TSC1 KO peritoneal macrophage (Figure 4G). These results indicate that TSC1 deficiency did not significantly affect the activity of P65, c-fos, and CREB in macrophages during LPS stimulation.
TSC1 controls pro-IL-1β expression via mTORC1-dependent C/EBPβ
Previous studies showed that C/EBPβ protein, which could be induced by LPS (Supplementary Figure 6)14,38, is required for the activation of mouse and human IL-1β gene expression39,40,41,42,43. We found that TSC1 KO peritoneal macrophages had markedly reduced C/EBPβ protein expression compared with WT macrophages (Figure 5A), whereas its expression was not affected in Rictor KO macrophages (Figure 5B). In addition, prolonged Rapa treatment for 48 h, but not short period Rapa treatment (1 h), markedly reversed the impaired C/EBPβ expression in TSC1 KO macrophages (Figure 5C). Similarly, reduced C/EBPβ expression was observed in TSC1 shRNA cells and its expression increased after treatment with Rapa for 24 h and was rescued completely after 48 h in TSC1 shRNA cells compared with scramble cells (Supplementary Figure 7). Notably, deletion of mTOR significantly reversed the decreased C/EBPβ expression in TSC1 KO peritoneal macrophages as determined by immunoblotting (Figure 5D). To test whether the decreased C/EBPβ expression was responsible for the decreased pro-IL-1β expression in TSC1 KO macrophages, we overexpressed C/EBPβ in WT and TSC1 KO BMDMs using adenovirus (Figure 5E, Supplementary Figure 8). Overexpression of C/EBPβ significantly rescued the decreased pro-IL-1β expression in TSC1 KO macrophages as determined by immunoblotting (Figure 5E). The present results collectively reveal that the decreased C/EBPβ expression was induced by the over-activated mTOR activity and was involved in impaired pro-IL-1β expression in TSC1 KO macrophages.
The role of the TSC–mTOR pathway in TLR-induced pro-inflammatory cytokine production was controversial. In TSC2-deficient MEFs, pro-inflammatory responses are decreased due to impaired IKK activation and NF-κB translation to the nuclei44. However, other studies reported that Rapa treatment enhances IL-12 production in myeloid DCs by promoting NF-κB activation but inhibits IL-12 production in monocyte-derived DCs and bone marrow-derived DCs20,45. In addition, TSC1-deficient macrophages in response to LPS stimulation produce elevated pro-inflammatory cytokines, such as TNF-α, IL-12, and IL-619,23,24. The reasons for this inconsistency are unclear, possibly due to the different cell types and duration of mTOR inhibition with Rapa or mTOR deletion used in these studies. So far, how TSC1 regulates pro-inflammatory cytokine IL-1β expression in macrophages remains unclear. Our results show that pro-IL-1β production was significantly inhibited at mRNA and protein levels in TSC1 KO primary macrophages and TSC1 shRNA RAW264.7 cells (Figure 1). In addition, our results are similar with previous report that over-activated mTOR suppressed caspase-1 activation then active IL-1β production (Supplementary Figure 3)22. Thus, TSC1 is required for the synthesis and maturation of IL-1β in macrophages19,20,24,44,45.
It is well known that the NF-κB and MAPK signaling pathway are important in the activation of inflammatory cytokines including pro-IL-1β expression46. Previous research showed that JNK activity19, but not P38 or ERK, was enhanced in TSC1 KO peritoneal macrophages. However, TSC1 shRNA cells showed similar phosphorylation levels of P38 and JNK relative to scramble cells after stimulation with LPS (Figure 4C). Interestingly, increased p-ERK was observed in TSC1 KO macrophages due to loss of TSC2 GTPase-activating protein (GAP) activity23. However, pro-IL-1β expression was upregulated by ERK and JNK in macrophages (Supplementary Figure 5). NF-κB signaling is also critical for IL-1β transcription and is involved in activation of mTOR pathway47,48,49,50,51. In addition, mTOR pathway is also relative to NF-κB pathway activation. Ghosh et al. reported that NF-κB signaling is attenuated in TSC2-deficient MEFs and several human tumor cell lines stimulated by DNA damage52. It was also reported that inhibition of mTORC2 signaling abrogates NF-κB activity and also decreases the NF-κB DNA-binding activity in U87/EGFRvIII cells53. In our experiments, we have not observed obvious defect of IKKα/β and IκBα as well as NF-κB p65 nuclear translocation in TSC1-deficient macrophages compared with scramble cells (Figure 4C, F, and G). But our data is generally in agreement with previous studies in macrophage cells19. Our results are different from those observed in TSC2-deficient MEFs or tumor cells lines and such differences could be attributed to the different cells examined53.
Molecular mechanism studies showed that the length and timing of Rapa treatment might have different effects on LPS-stimulated IL-1β expression. Our data showed that pre-treatment with Rapa for 1 h followed by stimulation with LPS for 6 h did not rescue pro-IL-1β expression in TSC1 KO macrophages (Figure 2F and G). This is consistent with a previous report that Rapa treatment (1 h) has no effect on intracellular pro-IL-1β expression in macrophages22. However, it was reported that activation of autophagy with Rapa promotes the degradation of pro-IL-1β after its translation and blocks secretion of the mature cytokine in immortalized BMDMs and bone marrow-derived DCs26. The reason for this inconsistency is possibly due to the different cell types used. Previous study showed that long-term treatment with Rapa (1 h then followed by LPS stimulation for another 24 h) promotes peripheral blood mononuclear cells to produce more IL-1β in culture supernatants25. We also observed the inhibition of mTORC1 by Rapa (48 h) or deletion of mTOR almost completely rescued the pro-IL-1β expression at the mRNA and protein levels in TSC1 KO macrophages, inferring that mTORC1 inhibited pro-IL-1β production indirectly and blocking mTORC1 pathway facilitated its synthesis. Given that mTOR is a conserved serine/threonine kinase that controls diverse processes including protein synthesis, ribogenesis, and mitochondrial capacity54, we asked whether over-activated mTORC1 in TSC1 KO macrophages altered specific transcription factor expression important for pro-IL-1β production. Unfortunately, all of the detected transcription factors were unimpaired in TSC1 KO macrophages relative to WT cells except for the C/EBPβ (Figure 4D and E and Figure 5A), which were essential for LPS-induced pro-IL-1β expression39,40,41,42. The C/EBPβ gene can produce several N-terminally truncated isoforms, LAP*, LAP, and LIP, by alternative translation initiation at downstream AUG codons55. It was reported that C/EBPβ isoform production is regulated by mTORC1 pathway: activated mTORC1 enhances generation of LIP while inhibition of mTOR promotes LAP expression56,57. In our studies, we found that TSC1 KO macrophages displayed enhanced mTOR activity and decreased C/EBPβ protein level. We have treated the scramble and TSC1 shRNA cells with Rapa for different durations. Brief treatment with Rapa had no effect on C/EBPβ expression (treated for 6 h and 12 h), or slightly rescued its expression in TSC1 shRNA cells (treated for 24 h). Only prolonged treatment (48 h) with Rapa or deletion of the mTOR gene could completely rescue C/EBPβ expression in TSC1-deficient cells (Figure 5C and Supplementary Figure 7). Moreover, overexpression of C/EBPβ significantly reversed the defect of pro-IL-1β expression in TSC1 KO macrophages. These results indicate that the pro-IL-1β expression deficiency in TSC1 KO macrophages is, at least partially, mediated by the mTOR-C/EBPβ pathway.
In summary, pro-IL-1β expression is suppressed in TSC1-deficient macrophages at both the mRNA and protein levels, indicating that TSC1 is essential for pro-IL-1β expression in macrophages. TSC1 regulates pro-IL-1β expression through downregulation of mTOR and subsequently upregulation of C/EBPβ pathway (Figure 6).
The authors wish to thank Dr Chenming Sun, Dr Lina Sun, and Mr Steven Shao for their kind review of the manuscript. This work was supported by grants from the National Basic Research Program of China (2011CB710903, 2010CB945301, Yong Zhao), the National Natural Science Foundation of China for General and Key Programs (C81130055, C81072396, Yong Zhao), and the CAS/SAFEA International Partnership Program for Creative Research Teams (Yong Zhao).