Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression

Abstract

The aggregation of hypertrophic macrophages constitutes the basis of all granulomatous diseases, such as tuberculosis or sarcoidosis, and is decisive for disease pathogenesis. However, macrophage-intrinsic pathways driving granuloma initiation and maintenance remain elusive. We found that activation of the metabolic checkpoint kinase mTORC1 in macrophages by deletion of the gene encoding tuberous sclerosis 2 (Tsc2) was sufficient to induce hypertrophy and proliferation, resulting in excessive granuloma formation in vivo. TSC2-deficient macrophages formed mTORC1-dependent granulomatous structures in vitro and showed constitutive proliferation that was mediated by the neo-expression of cyclin-dependent kinase 4 (CDK4). Moreover, mTORC1 promoted metabolic reprogramming via CDK4 toward increased glycolysis while simultaneously inhibiting NF-κB signaling and apoptosis. Inhibition of mTORC1 induced apoptosis and completely resolved granulomas in myeloid TSC2-deficient mice. In human sarcoidosis patients, mTORC1 activation, macrophage proliferation and glycolysis were identified as hallmarks that correlated with clinical disease progression. Collectively, TSC2 maintains macrophage quiescence and prevents mTORC1-dependent granulomatous disease with clinical implications for sarcoidosis.

Figure 1. Hypertrophic M2-like macrophages spontaneously form granulomas in Tsc2^fl/flLyz2-Cre mice.

(a,c) Lung sections evaluated by H&E staining of Tsc2^fl/fl and Tsc2^fl/flLyz2-Cre mice at the age of (a) 3 and (c) 6 months. (b) Immunofluorescence for Mac-2 and p-S6 in lung sections of 3 month-old mice. Boxed region shows p-S6 staining only. (d) Image of a Tsc2^fl/flLyz2-Cre mouse at the age of 6 months (left) and immunohistochemistry of Mac-2 of a paw (right). (e) Immunofluorescence for Mac-2 and F4/80 in paw sections of 6 month-old Tsc2^fl/flLyz2-Cre mice. (f) Flow cytometric scatterplot of the G1 population of Tsc2^fl/fl and Tsc2^fl/flLyz2-Cre peritoneal macrophages. (g) Tsc2^fl/fl and Tsc2^fl/flLyz2-Cre bone marrow was injected into irradiated wild-type recipient mice and evaluated after 3 months by histology. Left panel: H&E staining of lung sections. Right panel: IHC of Mac-2 in liver. (h) Flow cytometry analysis of hypertrophic macrophages in the lungs of Tsc2^fl/fl (n=5) and Tsc2^fl/flLyz2-Cre (n= 4) mice. Shown are means. (i) Expression of the indicated mRNAs in the lungs of 6 month-old mice. Shown are means ± SE (n= 5). *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant (Student's t test). Representative histological images or plots are from one mouse; each analysis was performed on three (b,d,e,g) or five (a,c,f) mice per genotype. Numerical data are representative of two independent (h) or cumulative of two independent (i) experiments. Scale bars, 100 μm (a,c,e,g), 10 μm (b), 1 mm (d).

Figure 2. TSC2 inhibits granulomatous aggregation, hypertrophy and macrophage proliferation in vitro.

(a) Immunoblots of CSF1-deprived Tsc2^fl/fl and Tsc2^fl/flLyz2-Cre bone marrow-derived macrophages (BMDM), treated for 18 hours with 100 nM rapamycin or solvent control, hybridized with the indicated antibodies. (b) Surface expression of the indicated proteins on Tsc2^fl/fl (n=6) and Tsc2^fl/flLyz2-Cre (n=4) BMDM after differentiation. (c) Left: Images of IL-4 stimulated (10 ng/ml) Tsc2^fl/fl and Tsc2^fl/flLyz2-Cre BMDM treated with solvent or 100 nM rapamycin for four days. Right: Quantification of cluster formation. Clusters in three pictures per condition (4x magnification) were counted (n= 3). (d) Flow cytometry scatterplot of CSF1-deprived BMDM on day 7. (e,f) Cell cycle analysis of (e) BMDM by 7-AAD and EdU staining and of (f) peritoneal macrophages by PI staining from Tsc2^fl/fl and Tsc2^fl/flLyz2-Cre mice (n= 5). (g) IHC of Ki-67 in lung sections of 3 month-old Tsc2^fl/fl and Tsc2^fl/flLyz2-Cre mice. (h) Proliferation analysis of BMDM stimulated with 40 ng/ml CSF1 and treated with solvent or 100 nM rapamycin (n=4). Shown are means ± SE (b,f,h) or SD (c). *p < 0.001 (Student's t test) (c,f). Data are representative of three (a,d,g,c) or two (e) or cumulative from two independent experiments (b,f,h) Scale bar, 100 µm (c), 50 μm (g).

Figure 3. TSC2 globally regulates macrophage proliferation, apoptosis and inflammation.

(a) Scatterplot of global gene-expression profiles of CSF1-deprived Tsc2^fl/fl and Tsc2^fl/flLyz2-Cre BMDM. Blue dots, 426 genes significantly higher expressed in Tsc2^fl/fl BMDM; red dots, 401 genes significantly higher expressed in Tsc2^fl/flLyz2-Cre BMDM. (b) Gene-set enrichment analysis (GSEA) of hallmark gene sets (H.all) from the Molecular Signatures Database of the Broad Institute, showing the most significantly enriched gene sets in Tsc2^fl/fl and Tsc2^fl/flLyz2-Cre BMDM and their normalized enrichment scores (NES) as well as their false discovery rates (FDR). (c,d) GSEA plot of the E2F targets (c) and Tnfa signaling via NFKB (d) gene signature in Tsc2^fl/flLyz2-Cre BMDM relative to Tsc2^fl/fl BMDM from the analysis in (b) with members of the gene set presented in the ranked list of genes ('bar code' below) and the signal-to-noise ranking metric (bar at bottom). (e) GSEA of the 'KEGG_apoptosis' gene signature in Tsc2^fl/flLyz2-Cre BMDM relative to Tsc2^fl/fl BMDM. (f) The most significantly enriched transcription factor target gene sets (C3.TFT) from the Molecular Signatures Database in unstimulated Tsc2^fl/fl and Tsc2^fl/flLyz2-Cre BMDM derived by GSEA. NES, normalized enrichment score; FDR, false discovery rate. Data are from one experiment with four biological replicates per genotype obtained from two independent experiments.

Figure 4. CSF1 induces CDK4 expression via TSC2/mTORC1 in macrophages.

(a,b,c) BMDM were treated with 100 nM rapamycin, solvent control and 10 ng/ml CSF1 as indicated for 18 h. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. (d) IHC of CDK4 in lung sections of 3 month-old mice. (e) Left, analysis of CSF1-induced (40 ng/ml) proliferation of BMDM treated with solvent or the indicated amounts of the CKD4 inhibitor PD-0332991 (n= 4). Right, cell cycle analysis of CSF1-stimulated BMDM treated with solvent or 1 µM PD-0332991 for 18 h (n= 3). (f,g) Tsc2^fl/flLyz2-Cre bone marrow was injected into irradiated wild-type recipient mice. After ten days mice were treated daily with PD-0332991 (n=10) or solvent control (n=10) daily for two month. (f) Lung (left panel) and liver (right panel) sections were evaluated by IHC of Mac-2. (g) Area of Mac-2 positive cells compared to total lung area of the treated mice. Two random images per animal were evaluated. (h) BMDM were treated with 1 µM PD-0332991 or solvent control and stimulated with 10 ng/ml CSF1 as indicated for 18 h. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. Shown are means ± SE (e) or SD (g). *p < 0.01, **p < 0.001 (Student's t test). Data are representative of three (a,b,c,d), one (f,g) or two (h) independent, or cumulative of two (e) experiments. Scale bar, 40 μm (d), 100 μm (f).

Figure 5. TSC2 regulates the cellular metabolism in macrophages via CDK4 to promote proliferation.

(a) Basal extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of BMDM reported in units of mpH/min and pmol/min, respectively (n=8). (b) Uptake of 2-NBDG in BMDM treated with solvent control or 100 nM rapamycin for 18 h; analyzed by flow cytometry (n=4). (c) Uptake of 2-NBDG in peritoneal macrophages from Tsc2^fl/fl (n=4) and Tsc2^fl/flLyz2-Cre (n=5) mice. (d) Total amount of glucose in lung tissue of mice (n=10). (e) Mitotracker Green staining analyzed by flow cytometry of BMDM treated with solvent control or 100 nM rapamycin for 18 h (n=4). (f) Immunofluorescence for F-ATPase β of BMDM deprived of CSF1. (g) ECAR of Tsc2^fl/flLyz2-Cre BMDM that were treated for 30 min with solvent or 1 µM PD-0332991 (n=8). (h) ECAR of Tsc2^fl/fl that were treated overnight with solvent or 10 ng/ml CSF1 and at the indicated time point with 1 µM PD-0332991 (n=8). (i) Images of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity on frozen lung sections of mice in situ. Sections were additionally stained for Mac-2, p-S6, and DAPI. (j) Fraction (in %) of p-S6-positive Mac-2 macrophages in lungs of mice with high activities of GAPDH, lactate dehydrogenase (LDH), isocitrate dehydrogenase (IDH) and succinate dehydrogenase (SDH) (n=3-4). (k) Analysis of CSF1-induced (40 ng/ml) proliferation of BMDM treated with solvent or the indicated amounts of 2-DG (n= 4). (l) Cell cycle analysis of CSF1-stimulated BMDM treated with solvent or 1 mM 2-DG for 18 h (n= 3). (m) BMDM were treated with 1 mM 2-DG or solvent control and then stimulated with 10 ng/ml CSF1 for 18 h. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. Shown are means ± SD (a,g,h,j) or SE (b,c,e,k,l) or boxplots with means, 25^th to 75^th percentile and minimum to maximum bars (d). *p<0.05, **p<0.001, ***p<0.001 (Student's t test). Data are representative of one (d) or two (a,f-j,m) independent experiments or cumulative of 2 experiments (b,c,e,k,l). Scale bar, 10 μm (f), 100 μm (i).

Figure 6. Disease progression in human sarcoidosis is associated with mTORC1 signaling and proliferation.

(a) Sections of three sarcoidosis patients stained with p-S6 by IHC. (b) Immunofluorescence for Mac-2 and p-S6 in a sarcoidosis granuloma. (c) GSEA analysis of the ‘mTORC1 signaling’, ‘E2F targets’, and ´Glycolysis´ gene signatures in progressive relative to self-limiting sarcoidosis from the data of Lockstone et al. (d) An unsupervised cluster analysis of the microarray data of Tsc2^fl/fl and Tsc2^fl/flLyz2-Cre BMDM (Fig. 3a) performed with the genes that were differentially expressed in progressive relative to self-limiting sarcoidosis patients. (e) IHC for p-S6 and Ki-67 of granulomas from sarcoidosis patients. (f) Immunofluorescence for Mac-2 and Ki-67 in a sarcoidosis granuloma. (g) Relationship between p-S6 and Ki-67 expression in granulomas of 27 human sarcoidosis biopsies. Ki-67 high, > 5 % Ki-67 positive cells in the granuloma; Ki-67 low, < 5 % Ki-67 positive cells in the granulomas. The relationship was investigated using fisher’s exact test. Data are representative of 27 human sarcoidosis patients (a,b,e,f) or from 8 self-limiting and 7 progressive sarcoidosis patients (c,d). Scale bar, 200 μm (a), 20 μm (b), 100 μm (e,f).

Figure 7. Inhibition of mTORC1 restores homeostasis in Tsc2^fl/flLyz2-Cre mice.

(a) 2 month-old and (b) 6 month-old Tsc2^fl/flLyz2-Cre mice were treated with placebo or everolimus for three weeks and lung sections were evaluated by H&E staining. (c) Paw and tail images of a 6 month-old Tsc2^fl/flLyz2-Cre mouse at day 0 and 14 of everolimus treatment. (d) IHC for Mac-2 of paw sections of 6 month-old Tsc2^fl/flLyz2-Cre mice treated with placebo or everolimus. (e) IHC for p-S6 and cleaved caspase 3 of paw sections of 6 month-old Tsc2^fl/flLyz2-Cre mice treated with placebo or everolimus for two days. Representative histological images are from one mouse; each analysis was performed on four (a,b,c) or three (d,e) mice per genotype. Scale bar, 40 μm (a), 100 μm (b), 1 mm (d), 50 μm (e).