Volume 22, Issue 13, Pages 3468-3479 (March 2018) p53 in Bronchial Club Cells Facilitates Chronic Lung Inflammation by Promoting Senescence  Adi Sagiv, Amir Bar-Shai, Naama Levi, Miki Hatzav, Lior Zada, Yossi Ovadya, Lior Roitman, Gal Manella, Ofer Regev, Julia Majewska, Ezra Vadai, Raya Eilam, Sara W. Feigelson, Michael Tsoory, Michel Tauc, Ronen Alon, Valery Krizhanovsky  Cell Reports  Volume 22, Issue 13, Pages 3468-3479 (March 2018) DOI: 10.1016/j.celrep.2018.03.009 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 22, 3468-3479DOI: (10.1016/j.celrep.2018.03.009) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 Inflammatory Cell Influx into the BAL Fluid and Lung in CCSP-Cre;p53-Loxp (CCSP-p53−/−) Mice following Single LPS Inhalation (A) Schematic presentation of the mouse model used to study the role of p53 in club cells. (B) Lung section from a CCSP-Cre;p53-LoxP;Lox-Stop-Lox-mRFP mouse demonstrating mRFP expression in the bronchial epithelia. Scale bar, 50 μm. (C) Lung sections from CCSP-Cre and CCSP-p53−/− mice (subjected to 8 Gy irradiation) were analyzed for expression of p53. Scale bar, 200 μm. (D–G) CCSP-Cre and CCSP-p53−/− mice were exposed once to either PBS or aerosolized LPS (0.5 mg/mL). 24 hr after exposure to LPS, BAL fluid and single cells extracted from lung homogenates were collected. Flow cytometry was used to determine numbers of (D) Neutrophils in the BAL, (E) alveolar macrophages, (F) neutrophils in the lung tissue, and (G) interstitial macrophages. (H–K) Cytokine levels in the BAL were determined by ELISA: (H) IL-6, (I) CXCL5, (J) CCL2, and (K) KC. (D–K) The data shown are representative of 3 independent experiments; in each, at least 5 animals per group were examined. Statistical analysis was performed by Student’s t test: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.005; (D)−(G), n = 4−6; (H) and (K), n = 5−8; (I) and (J), n = 3−5. Cell Reports 2018 22, 3468-3479DOI: (10.1016/j.celrep.2018.03.009) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 p53 Knockout in Club Cells Protects the Lung Airway Structure from Chronic Bronchitis and Emphysema Triggered by Chronic LPS Exposure CCSP-Cre and CCSP-p53−/− mice were exposed to either PBS or aerosolized LPS (0.5 mg/mL) 3 times a week for 10 weeks. Mouse lungs were then analyzed. Scale bars represent 200 μm. (A) Representative images of H&E-stained lung sections featuring a bronchus of CCSP-Cre and CCSP-p53−/− mice. (B) Airway wall thickness in LPS-treated CCSP-Cre mice and in CCSP-p53−/− mice, measured on the basis of the H&E-stained sections shown in (A). (C) Representative images of H&E-stained lung sections featuring alveoli. (D and E) Morphometric analysis of airway destruction, demonstrated as mean ± SEM of the linear mean intercept (Lm) (D) and the DI (E). Data are representative of 3 independent experiments. In each experiment, at least 5 animals per group were examined. Statistical analysis was performed by Student’s t test: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.005; (B), n = 3 mice per genotype (at least 2 samples per mouse), (D and E), n = 3−5. Cell Reports 2018 22, 3468-3479DOI: (10.1016/j.celrep.2018.03.009) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 p53 Knockout in Club Cells Protects Lung Airways from Chronic Inflammatory Responses Triggered by Chronic LPS Exposure (A–F) CCSP-Cre and CCSP-p53−/− mice were exposed to either PBS or aerosolized LPS (0.5 mg/mL) 3 times a week for 10 weeks. 48 hr following the last LPS exposure, BAL fluid was collected, and flow cytometry was used to determine the numbers of (A) CD3+ T cells, (B) CD4+ T cells, (C) CD8+ T cells, (D) neutrophils, (E) alveolar macrophages, and (F) NK cells. (G–M) Expression levels of the following inflammatory cytokines were assessed in the lung tissue by RT-PCR: (G) TNF-α, (H) IL-6, (I) KC, (J) CXCL2, (K) CXCL5, (L) CCL2, and (M) CXCL10. Data are representative of 3 independent experiments. In each experiment, at least 5 animals per group were examined. Statistical analysis was performed by one-way ANOVA (A−F, n = 8−12) and by Student’s t test (G−M, n = 4−8 for PBS and n = 6−12 for LPS samples). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.005. Cell Reports 2018 22, 3468-3479DOI: (10.1016/j.celrep.2018.03.009) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 p53 in Club Cells Promotes BALT Formation in Response to Chronic LPS Inhalation (A–D) CCSP-Cre and CCSP-p53−/− mice were exposed to either PBS or aerosolized LPS (0.5 mg/mL) 3 times a week for 10 weeks. 48 hr following the last LPS exposure, whole lungs were dissociated into single-cell suspensions and analyzed by flow cytometry to determine (A) numbers of interstitial macrophages, (B) numbers of CD3+ T cells, (C) numbers of CD4+ T cells, and (D) numbers of CD8+ T cells. (E) Representative images of lungs from CCSP-Cre and CCSP-p53−/− mice stained for CD3+ (green) and B220 (red), comprising the BALT. (F and G) Analysis of the numbers (F) and sizes (G) of BALTs in the lungs of CCSP-Cre and CCSP-p53−/− mice. Statistical analysis was performed by one-way ANOVA (A−D, n = 7 for PBS and n = 10−12 for LPS samples) and by Student’s t test (F−G, n = 4). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.005. Scale bars represent 200 μm. Cell Reports 2018 22, 3468-3479DOI: (10.1016/j.celrep.2018.03.009) Copyright © 2018 The Author(s) Terms and Conditions

Figure 5 Accumulation of Senescent Cells Is Decreased in the Lungs of CCSP-P53−/− Mice (A–G) CCSP-Cre and CCSP-p53−/− mice were exposed to either PBS or aerosolized LPS (0.5 mg/mL) 3 times a week for 10 weeks. 48 hr following the last LPS exposure, the lungs were harvested, fixed, and analyzed for markers of apoptosis and senescence. Scale bars represent 200 μm. n = 3−4 mice each. Shown are (A) Immunohistochemistry (IHC) of CCSP, (B) IHC of cleaved caspase-3, (C) IHC of p21, (D) SA-β-gal activity, (E) IHC of HMGB1, (F) IHC of Ki67, and (G) Immunofluorescence of γ-H2AX. Cell Reports 2018 22, 3468-3479DOI: (10.1016/j.celrep.2018.03.009) Copyright © 2018 The Author(s) Terms and Conditions

Figure 6 Accumulation of Senescent Cells Is Decreased in the Lungs of ABT-737-Treated Mice (A–E) C57BL/6 mice were exposed to either PBS or aerosolized LPS (0.5 mg/mL) 3 times a week for 10 weeks. ABT-737 (25 μM) or DMSO was injected intraperitoneally (i.p.) into the LPS-exposed mice on 3 consecutive days every 10 days, starting at week 4 of the inhalations. Lungs were harvested 48 hr following the last LPS exposure, fixed, and analyzed for markers of senescence. Scale bars represent 200 μm. n = 3−4 mice each. Shown are (A) IHC of p21, (B) SA-β-gal activity, (C) IHC of HMGB1, (D) H&E staining, and (E) airway wall thickness. Cell Reports 2018 22, 3468-3479DOI: (10.1016/j.celrep.2018.03.009) Copyright © 2018 The Author(s) Terms and Conditions

Figure 7 ABT-737 Treatment Decreases Chronic Inflammatory Responses Caused by Exposure to Aerosolized LPS C57BL/6 mice were exposed to either PBS or aerosolized LPS (0.5 mg/mL) 3 times a week for 10 weeks. ABT-737 (25 μM) or DMSO solution was injected i.p. into the LPS-exposed mice for 3 consecutive days every 10 days, starting at week 4 of the inhalations. (A−E) 48 hr following the last LPS exposure, BAL fluid was collected. The following were assayed by flow cytometry: (A) numbers of CD3+ T cells, (B) numbers of CD4+ T cells, (C) numbers of CD8+ T cells, (D) numbers of neutrophils, and (E) NK cells. (F−I) Whole lungs were dissociated at the same time point into single-cell suspensions and analyzed by flow cytometry for (F) numbers of interstitial macrophages, (G) numbers of CD3+ T cells, (H) numbers of CD4+ T cells, and (I) numbers of CD8+ T cells. (J) Representative images of lungs stained for CD3+ (green) and B220 (red), depicting accumulation of BALT in DMSO-treated mice. Scale bars represent 200 μm. (K and L) Numbers (K) and sizes (L) of BALTs in the lungs of mice exposed to aerosolized LPS and treated with ABT-737 or DMSO. Statistical analysis was performed by one-way ANOVA (A−E, n = 7−12; F−J, n = 3−5) and by Student’s t test (K and L, n = 4). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.005. Cell Reports 2018 22, 3468-3479DOI: (10.1016/j.celrep.2018.03.009) Copyright © 2018 The Author(s) Terms and Conditions