Volume 22, Issue 13, Pages 3493-3506 (March 2018) Telomere Dysfunction Disturbs Macrophage Mitochondrial Metabolism and the NLRP3 Inflammasome through the PGC-1α/TNFAIP3 Axis  Yanhua Kang, Hang Zhang, Yufang Zhao, Yan Wang, Wei Wang, Yan He, Wei Zhang, Weiwei Zhang, Xudong Zhu, Yong Zhou, Lingling Zhang, Zhenyu Ju, Liyun Shi  Cell Reports  Volume 22, Issue 13, Pages 3493-3506 (March 2018) DOI: 10.1016/j.celrep.2018.02.071 Copyright © 2018 The Authors Terms and Conditions

Cell Reports 2018 22, 3493-3506DOI: (10.1016/j.celrep.2018.02.071) Copyright © 2018 The Authors Terms and Conditions

Figure 1 Terc−/− Mice Are More Susceptible to Bacterial Infection than Their WT Littermates (A) Age- and sex-matched WT and Terc−/− mice (n = 10) were instilled with 108 colony-forming units (CFUs) of S. aureus, and their survival rates were analyzed. (B–G) WT and Terc−/− mice (n = 5) were inoculated intratracheally (i.t.) with S. aureus (107 CFU/mice) and sacrificed at the indicated time. Shown are total cell and neutrophil counts (B); BALF levels of IL-6, TNF-α, IL-1β, and KC (C); H&E staining of murine lung tissues 6 hr post-infection (D); protein centration in BALF (E); TUNEL staining of lung tissues 6 hr post-infection (F); and bacterial loads in BALF (G). All results are expressed as the mean ± SD (B–G). ∗p < 0.05, ∗∗p < 0.01 by Student’s t test. See also Figure S1. Cell Reports 2018 22, 3493-3506DOI: (10.1016/j.celrep.2018.02.071) Copyright © 2018 The Authors Terms and Conditions

Figure 2 Terc−/− Macrophages Exhibit Enhanced Inflammatory and Antibacterial Responses (A–D) Terc−/− or WT BMDMs were stimulated with S. aureus at the indicated time points. Shown are mRNA (A) and protein (B) levels of TNF-α, IL-6, and IL-1β; levels of total and the phosphorylated NF-κB-related proteins (C) and MAPKs (D) as indicated. Shown are representative images and quantification of band densities. (E) Uptake of carboxyfluorescein succinimidyl ester (CFSE)-labeled S. aureus by Terc−/− and WT macrophages. Shown are representative histogram 30 min post-infection (left) and the quantification of macrophages phagocytosing fluorescein isothiocyanate (FITC)-labeled bacteria at the indicated time periods (right). (F and G) Fluorescence microscopy of intracellular bacteria (F) and enumeration of bacterial loads in WT and Terc−/− macrophages at the indicated time periods (G). All results are from three independent experiments and presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01 by Student’s t test. See also Figure S2. Cell Reports 2018 22, 3493-3506DOI: (10.1016/j.celrep.2018.02.071) Copyright © 2018 The Authors Terms and Conditions

Figure 3 Telomere Dysfunction Leads to Hyperactivation of the NLRP3 Inflammasome (A) Terc−/− or WT BMDMs were infected with S. aureus for the indicated time periods. Shown are levels of NLRP3, caspase-1, and IL-1β in cell lysates (Lys) and culture supernatants. (B) Immunostaining of anti-IL-1β p17 in BMDMs 3 hr post-infection. (C) Immunostaining of lung tissues with anti-caspase-1 p20 6 hr post-infection. (D and E) IL-1β secretion by Terc−/− or WT BMDMs that were pretreated with Z-WEHD-FMK or DMSO (D) or transfected with non-specific control (NC) or NLRP3-targeted siRNA (E), followed by staphylococcal infection for 12 hr. (F and G) PBMCs from young or old healthy donors (n = 15) were uninfected or infected with S. aureus for 8 hr. Representative images show the cleavage of pro-IL-1β and pro-caspase-1 (F) and ELISA analysis of IL-1β secretion (G). Results are from three independent experiments (B–E) and are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01 by Student’s t test. See also Figure S3. Cell Reports 2018 22, 3493-3506DOI: (10.1016/j.celrep.2018.02.071) Copyright © 2018 The Authors Terms and Conditions

Figure 4 IL-1R Signaling Contributes to Telomere-Related Immunopathology (A–F) Terc−/− and WT mice (n = 5) were intraperitoneally injected with IL-1Ra or vehicle, followed by staphylococcal infection for 12 hr. Shown are total cell counts (A), TNF-α and IL-6 levels (B), protein concentrations (C) and bacterial loads in BALF (D), H&E staining of lung sections (E), and MPO activity in lung tissues (F). (G) Survival rates of Terc−/− mice (n = 10) instilled with IL-1Ra (25 mg/kg, i.p.) or vehicle prior to infection. Results are expressed as the mean ± SD (A–F). ∗p < 0.05, ∗∗p < 0.01 by Student’s t test. ns, not significant. Cell Reports 2018 22, 3493-3506DOI: (10.1016/j.celrep.2018.02.071) Copyright © 2018 The Authors Terms and Conditions

Figure 5 Dysfunctional Telomeres Cause Mitochondrial Abnormity and Oxidative Stress and Hence the Exaggerated Inflammasome (A–F) Terc−/− and WT BMDMs were infected with S. aureus for 12 hr. (A) Electron microscopy of BMDMs. Black arrow, mitochondria. Flow cytometry of BMDMs stained with MitoTracker Green and MitoTracker. (B) Representative histograms and relative fluorescence quantification. (C) Levels of cellular ATP. Flow cytometry of BMDMs labeled with Mito-SOX. (D) Representative histograms and relative fluorescence quantification. (E) Immunofluorescence microscopy of BMDMs labeled with Mito-SOX. (F) mRNA levels of the indicated genes. (G) PGC-1α levels in Terc−/− and WT BMDMs infected with S. aureus for the indicated time periods. (H and I) Cleaved caspase-1 and IL-1β (H) and secretion of IL-1β (I) in Terc−/− and WT BMDMs pretreated with DMSO or Mito-tempo prior to infection. (J–L) BMDMs from young or aged mice were stimulated with S. aureus for the indicated time periods. (J) mRNA levels of NLRP3, caspase-1, and IL-1β. (K) Secretion of IL-1β. (L) Flow cytometry of BMDMs labeled with Mito-SOX. Data are presented as the means ± SD from three independent experiments. ∗p < 0.05; ∗∗p < 0.01 by Student’s t test. See also Figure S4. Cell Reports 2018 22, 3493-3506DOI: (10.1016/j.celrep.2018.02.071) Copyright © 2018 The Authors Terms and Conditions

Figure 6 TNFAIP3 Is Involved in Telomere-Associated Inflammapathology (A and B) mRNA (A) and protein (B) levels of TNFAIP3 in Terc−/− and WT BMDMs infected with S. aureus for the indicated time periods. (C) Immunohistochemical staining of TNFAIP3 in lung tissues 12 hr post-infection. (D–F) Terc−/− and WT BMDMs were infected with TNFAIP3-exprssing adenovirus (ad-T) or control adenovirus (ad) (MOI = 100) and then challenged with S. aureus for 12 hr. Cleaved caspase-1 and IL-1β (D), IL-1β secretion (E), and bacterial loads (F). (G–L) Terc−/− mice were instilled (i.t.) with ad-TNFAIP3-expressing or control adenovirus (5 × 108 plaque-forming units (PFUs)/mouse, n = 5) and infected with S. aureus for 6 hr. Shown are total cell counts (G); protein concentrations (H); IL-1β, TNF-α, and IL-6 levels (I) in BALF; MPO activity in lung tissues (J); H&E staining of lung sections (K); and bacterial enumeration in BALF (L). All results are expressed as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01 by Student’s t test. See also Figure S5. Cell Reports 2018 22, 3493-3506DOI: (10.1016/j.celrep.2018.02.071) Copyright © 2018 The Authors Terms and Conditions

Figure 7 The PGC-1α/TNFAIP3 Axis Links Metabolic and Inflammatory Pathways under the Control of Telomeres (A) TNFAIP3 promoter activity in Terc−/− and WT BMDMs 6 hr post-infection. (B) TNFAIP3 mRNA levels in RAW264.7 cells transfected with PGC-1α- and/or ERRα-expressing plasmids or control plasmids. (C) 5′ UTR of TNFAIP3 containing the intact or mutant ERRα-binding sites (top). Luciferase activity was measured in 293T cells transfected with the TNFAIP3 reporter construct carrying the intact or mutant 5′ UTR sequence, along with PGC-1α- and/or ERRα-expressing plasmids (lower). (D) ChIP assays in RAW 264.7 cells transfected with PGC-1α- and/or ERRα-expressing plasmids or control plasmids. Immunoprecipitation was performed using anti-FLAG and/or anti-RFP antibodies, and qPCR analysis was conducted using Tnfaip3-specific primers. (E) TNFAIP3 protein levels in RAW264.7 cells transfected with PGC-1α- and/or ERRα-expressing plasmids. (F) Release of IL-1β by Terc−/− or WT BMDMs co-transfected with PGC-1α- and ERRα-expressing plasmids 12 hr post-infection. (G and H) BMDMs from young or aged mice were stimulated with S. aureus for the indicated time periods. Shown are relative mRNA (G) or protein (H) levels of TNFAIP3 and PGC-1α. (I) Young or aged mice (n = 3) were inoculated with S. aureus for 12 hr, and the BALF level of IL-1β was measured. (J) The action mode whereby dysfunctional telomeres disturb immune and metabolic homeostasis during the host defensive response. All results are expressed as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01 by Student’s t test. See also Figure S6. Cell Reports 2018 22, 3493-3506DOI: (10.1016/j.celrep.2018.02.071) Copyright © 2018 The Authors Terms and Conditions