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. 2018 May 15;23(7):2157-2167.
doi: 10.1016/j.celrep.2018.04.044.

mTOR Senses Environmental Cues to Shape the Fibroblast-like Synoviocyte Response to Inflammation

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Free PMC article

mTOR Senses Environmental Cues to Shape the Fibroblast-like Synoviocyte Response to Inflammation

Thomas Karonitsch et al. Cell Rep. .
Free PMC article

Abstract

Accumulating evidence suggests that metabolic master regulators, including mTOR, regulate adaptive and innate immune responses. Resident mesenchymal tissue components are increasingly recognized as key effector cells in inflammation. Whether mTOR also controls the inflammatory response in fibroblasts is insufficiently studied. Here, we show that TNF signaling co-opts the mTOR pathway to shift synovial fibroblast (FLS) inflammation toward an IFN response. mTOR pathway activation is associated with decreased NF-κB-mediated gene expression (e.g., PTGS2, IL-6, and IL-8) but increased STAT1-dependent gene expression (e.g., CXCL11 and TNFSF13B). We further demonstrate how metabolic inputs, such as amino acids, impinge on TNF-mTORC1 signaling to differentially regulate pro-inflammatory signaling circuits. Our results define a critical role for mTOR in the regulation of the pro-inflammatory response in FLSs and unfold its pathogenic involvement in TNF-driven diseases, such as rheumatoid arthritis (RA).

Keywords: SLC38A9; amino acids; fibroblast-like synoviocytes; mechanistic target of rapamycin; nuclear factor ‘kappa-light-chain-enhancer’ of activated B cells; rheumatoid arthritis; signal transducer and activator of transcription 1; tumor necrosis factor.

Figures

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Figure 1
Figure 1
Activation of mTOR in RA (A) Immunohistochemistry showing phosphorylated (p)-mTOR (S2448) and mTOR expression in paraffin-embedded synovial tissue sections from patients suffering from osteoarthritis (OA) or rheumatoid arthritis (RA). Synovial tissue sections were also stained with an isotype-matched control antibody (CTRL). (B) RA (n = 12) and OA (n = 8) synovial tissue sections were evaluated for p-mTOR and mTOR expression using a semiquantitative score (0 = no staining, 3 = high staining). ∗∗∗p = 0.0002 and p < 0.05, unpaired Student’s t test. See also Table S1.
Figure 2
Figure 2
TNF Activates the mTOR Pathway in RA-FLSs (A) Immunoblots of TNF-treated (10 ng/mL) RA fibroblast-like synoviocytes (RA-FLSs). Representative blots for five independent experiments with FLS cell lines from five donors are shown. (p)-S6K: upper band shows P85-S6K, and lower band shows the P70-S6K isoform. (B) RA-FLSs were preincubated with DMSO (Ctrl), rapamycin (250 nM), torin (250 nM), PP242 (1,000 nM), or MK2206 (1,000 nM) for 60 min and then stimulated with TNF (10 ng/mL) for 15 min. Blots are representative for three independent experiments with RA-FLS cell lines from three donors. See also Figure S1.
Figure 3
Figure 3
mTOR Modulates the Gene Expression Response to TNF in FLSs (A) Schematic illustration of the microarray experiment. RA-FLSs from five donors were pretreated with either DMSO (Ctrl) or torin (250 nM) for 1 hr and then stimulated with TNF (10 ng/mL) for 6 hr. Volcano plots show the magnitude of differential expressed transcripts for each treatment. Differentially regulated genes were identified by the significance analysis of microarrays (SAM) (fold induction > 2, FDR < 0.01). The number of differentially regulated transcripts in comparison with DMSO (Ctrl) treatment is depicted. (B) Scatterplot showing the influence of mTOR inhibition (by Torin-1) on TNF-upregulated transcripts. The 25 most significant genes are shown in the upper-right and lower-right quadrant. (C) The top 12 transcription factor binding sites (TFBSs) that are enriched in the TNF-upregulated genes that were positively regulated by the pretreatment with Torin-1 (DAVID) (https://david.ncifcrf.gov/home.jsp). (D) mTOR inhibition decreased the TNF-induced expression of 40 genes. This group was significantly enriched for interferon-regulated genes (IRGs) (92.5% of all genes [37/40]) (http://www.interferome.org/interferome/home.jspx). The bar graph shows the number of genes that were classified as IRGs. See also Figures S2 and S4.
Figure 4
Figure 4
Validation Using a 3D Tissue Culture System (A) Schematic illustration of the 3D tissue culture experiment. RA-FLSs were cultured in micromass organ cultures for 12 days. After serum starvation (overnight), FLSs were treated with DMSO (Ctrl), TNF (10 ng/mL), or TNF (10 ng/mL) + torin (250 nM) for 24 hr. (B) Micromasses were fixed, sectioned, and stained with hematoxylin and specific antibodies for p-mTOR (S2448), mTOR, and TNFSF13B. Representative pictures of three independent experiments performed with RA-FLSs from three donors are shown. For isotype controls, see Figure S3. (C) ELISA of IL-6, IL-8, MMP1 and MMP3 in culture supernatants of micromass organ cultures. Values are the mean ± SEM of three independent experiments that were performed with RA-FLSs from three donors. p < 0.05, paired t test.
Figure 5
Figure 5
mTOR Affects NF-κB Signaling by Influencing IκB-α Dynamics (A and B) Western blots of RA-FLSs that were pretreated with DMSO (Ctrl, TNF), torin (250 nM), or PP242 (1,000 nM) for 60 min and then stimulated with TNF (10 ng/mL). Representatives blots of five (A) or three (B) independent experiments with RA-FLSs from different donors are shown. For quantification of western blots, see Figure S5. (C) NF-κB DNA binding activity by EMSA in nuclear extracts from RA-FLSs, which were treated with DMSO or Torin-1 (250 nM) 1 hr before TNF stimulation (10 ng/mL). Representative blots of four independent experiments performed with RA-FLS cell lines from four donors are shown. For quantification of EMSA, see Figure S5. (D) Boxplots displaying the log2 nuclear-to-perinuclear P65 signal ratio calculated from automatically captured and analyzed images of RA-FLSs. FLSs were treated with either DMSO or 250 nM torin for 60 min before TNF stimulation (10 ng/mL). Pooled data from 6 RA-FLS cell lines are shown. Unpaired Student’s t test has been used to assess the statistical significance of treatment differences. See also Figure S6.
Figure 6
Figure 6
mTOR Promotes TNF-Induced Expression and Activation of STAT1 (A) Immunoblots of S6K1, p-S6K1, Stat1, and p-Stat1 in human RA-FLSs treated with TNF (10 ng/mL). Representative blots of four independent experiments performed with FLSs from four donors are shown. STAT1: upper band shows STAT1α (91 kDa), and lower band shows STAT1β (84 kDa). (B) RA-FLSs were stimulated with TNF (10 ng/mL). qPCR was performed in technical triplicates (error bars, SEM of triplicates). Representative graphs of five independent experiments with different RA-FLS cell lines are shown. mRNA expression is presented relative to untreated cells. (C and D) RA-FLSs were pretreated with JAK inhibitor I (300 nM) for 1 hr and then stimulated with TNF (10 ng/mL). (C) Immunoblots of RA-FLSs. Representative blots of three independent experiments with different RA-FLS cell lines are shown. (D) Gene expression was determined by qPCR. qPCR was performed in technical triplicates (error bars, SEM of triplicates). Representative graphs of two independent experiments with different RA-FLS cell lines are shown. mRNA expression is presented relative to that in untreated cells. (E) Immunoblots of FLSs stimulated with TNF (10 ng/mL) that were pretreated with Torin-1 (250 nM) or DMSO. Representative blots of three independent experiments are shown. (F) Immunoblots of RA-FLSs that were pretreated with DMSO, torin (250 nM), PP242 (1,000 nM), or Jak inhibitor I (300 nM) for 1 hr and then stimulated with TNF (10 ng/mL) for 3 hr. Representative blots of three independent experiments are shown. (G) STAT1 expression in RA-FLSs as determined by qPCR. RA-FLSs from three donors were pretreated with Torin-1 (250 nM) or DMSO for 1 hr and then stimulated with TNF (10 ng/mL). Bars show mean ± SEM. Expression is presented relative to that in DMSO-treated cells. (H) Immunoblots of RA-FLSs pretreated with DMSO or Torin-1 (250 nM) for 1 hr and then stimulated with TNF (10 ng/mL) for 24 hr. Representative blots of at least six independent experiments are shown.
Figure 7
Figure 7
Control of TNF-Induced Gene Expression by Amino Acids (A and B) RA-FLSs cultured in DMEM with or without amino acids (aas) were stimulated with TNF (10 ng/mL) for 6 hr. (A) Levels of IL-6 in the supernatants were measured by ELISA. Values are the mean ± SEM. ∗∗p < 0.01, Student’s paired t test, n = 5. (B) Expression of CXCL11 and TNFSF13B was determined by qPCR. Expression is presented relative to that in untreated cells. Values are the mean ± SEM. p < 0.05, Student’s paired t test, n = 4. (C–E) RA-FLSs were cultured in aa-free DMEM or DMEM that was reconstituted with amino acids (1/3× aa, 2/3× aa, 3/3× amino acid of standard amino acid concentration) and then stimulated with 10 ng/mL of TNF for 6 hr. (C) CXCL11 gene expression was determined by qPCR. Values are the mean ± SD of technical replicates. A representative of three independent experiments is shown. Expression is presented relative to unstimulated cells. (D) IL-6 concentration in supernatants was determined with ELISA. Values represent the mean ± SD of technical replicates. A representative of three independent experiments is shown. (E) Immunoblots of whole-cell lysates using anti-PTGS2 and anti-p-S6K antibodies. Representative blots of three independent experiments are shown. (F) RA-FLSs (n = 7) cultured in DMEM containing or lacking glutamine, leucine, and/or arginine were stimulated with TNF for 6 hr. Gene expression was determined by qPCR. Expression is presented relative to that in untreated cells. Values are the mean ± SEM. p < 0.05, Student’s paired t test. (G) Immunoblots of RA-FLSs after transfection with SLC38A9 or non-targeting control siRNA. (H and I) Transfected FLSs from five donors suffering from RA were treated with TNF (10 ng/mL) for 6 hr (H). TNFSF13B gene expression was determined by qPCR. Expression is presented relative to that in unstimulated cells. (I) Transfected RA-FLSs (n = 5) were treated with TNF (10 ng/mL) for 6 hr. Supernatants were analyzed by ELISA. Values are the mean ± SEM. p < 0.05 and ∗∗p < 0.01, Student’s paired t test.

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