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1.
Figure 2

Figure 2. Aging is associated with increased expression of senescence markers in the lungs. From: Cellular senescence increases expression of bacterial ligands in the lungs and is positively correlated with increased susceptibility to pneumococcal pneumonia.

A) Densitometric analyses and representative Western blots for the senescence markers p16, pRb, and mH2A in tissue lysates from lung biopsies obtained from young (43–50 years; n=4), mature (51–64 years; n=5) and aged (65–82 years; n=5) humans. B) The same analyses was performed using whole lung homogenates obtained from young (4–5 months; n=5) and aged (19–22 months; n=5) female Balb/cBy mice. For panels A and B histograms show composite densitometric data (protein/actin) with actin probed from the same lanes and membrane as the tested protein. The representative actin blot shown corresponds to the p16 immunoblot. Statistical significance was determined using a two-tailed Student’s t-test.

Pooja Shivshankar, et al. Aging Cell. ;10(5):798-806.
2.
Figure 4

Figure 4. Aging enhances susceptibility to pneumococcal pneumonia in a K10-dependent manner. From: Cellular senescence increases expression of bacterial ligands in the lungs and is positively correlated with increased susceptibility to pneumococcal pneumonia.

A) Kaplan Meier plot demonstrating percent survival of young and aged Balb/cBy mice intranasally challenged with 107 and 106 CFU of S. pneumoniae (WT: 107 young n=13, 107 aged n=13, 106 young n=6, 106 aged n=6) and an isogenic PsrP-deficient mutant. (ΔpsrP: 107 young n=6, 107 aged n=6, 106 young n=6, 106 aged n=6). Both young and aged mice challenged with 106 ΔpsrP had significantly improved survival versus WT infected mice. Statistical analysis was done using a Kaplan-Meier Log-Rank Test. B) Bacterial burden in the lungs and blood of young and aged mice sacrificed 2 days following intratracheal administration of 105 CFU of WT and ΔpsrP pneumococci (n=6 per group). Note that mice infected with the PsrP deficient strain do not demonstrate an age-dependent increase in bacterial titers versus WT. Asterisks denote a statistical significant difference when using One Way ANOVA (Duncan’s Method; P<0.05).

Pooja Shivshankar, et al. Aging Cell. ;10(5):798-806.
3.
Figure 1

Figure 1. Aged mice experience low-grade lung inflammation. From: Cellular senescence increases expression of bacterial ligands in the lungs and is positively correlated with increased susceptibility to pneumococcal pneumonia.

A) Levels of IL-1α, IL-1β, IL-6, TNFα, and CXCL1 in whole lung homogenates from healthy young (4–5 month, n=5) and aged (19–21 month, n=5) female Balb/cBy mice were assessed by ELISA. B) Representative micrographs of Hematoxylin and Eosin stained lung sections from the same animals. Note the enhanced interstitial and peribronchial inflammation in the aged lung section. C) Scoring of lung inflammation in the same lung sections (1 is no pathology on histological cross-section, 5 is extensive cellular infiltration, edema, and alveolar consolidation). Each diamond indicates the blinded pathological score for an individual mouse. In panels A and C asterisks denote a statistical significant difference when using a two-tailed Student’s t-test.

Pooja Shivshankar, et al. Aging Cell. ;10(5):798-806.
4.
Figure 7

Figure 7. Genotoxic stress enhances susceptibility to pneumonia and is positively correlated with increased bacterial ligand expression. From: Cellular senescence increases expression of bacterial ligands in the lungs and is positively correlated with increased susceptibility to pneumococcal pneumonia.

A) Bacterial titers in the lungs and blood of mice 2 days after challenge with S. pneumoniae (n=6 per cohort). Mice had been intratracheally administered saline (Control), saline with bleomycin (Bleo) at 0.033 mg/kg body weight 3 weeks prior, or had their drinking water supplemented with 0.5% hydrogen peroxide for 3 weeks (H2O2). Asterisks denote a statistical significant difference (P<0.05). Statistical analyses were performed using a two-tailed Student’s t-test. B) Western blot analysis for the senescence markers pRb, p16, and mH2A, as well as pneumococcal ligands K10, LR, and PAFr using whole lung homogenates from control, Bleo and H2O2-administered animals.

Pooja Shivshankar, et al. Aging Cell. ;10(5):798-806.
5.
Figure 3

Figure 3. Aging is associated with increased bacterial ligand expression in the lungs. From: Cellular senescence increases expression of bacterial ligands in the lungs and is positively correlated with increased susceptibility to pneumococcal pneumonia.

A) Densitometric analyses and representative Western blots for the pneumococcal ligands K10, LR, and PAFr in tissue lysates from lung biopsies obtained from young (43–50 years; n=4), mature (51–64 years; n=5) and aged (65–82 years; n=5) humans. B) The same analyses was performed using whole lung homogenates obtained from young (4–5 months; n=5) and aged (19–22 months; n=5) female Balb/cBy mice. For panels A and B histograms show composite densitometric data (protein/actin) with actin probed from the same lanes and membrane as the tested protein. The representative actin blot shown corresponds to the LR immunoblot. Statistical significance was determined using a two-tailed Student’s t-test. C) Representative micrographs of young and aged mouse lung sections immunohistochemically stained for K10. Note, that in aged mice K10 is expressed at high levels in alveolar (alv) and bronchial epithelial cells, but remains low in the vascular endothelium (endo) and fibroblasts (fib) surrounding the bronchi.

Pooja Shivshankar, et al. Aging Cell. ;10(5):798-806.
6.
Figure 6

Figure 6. Normal A549 cells exposed to conditioned media from senescent cells are permissive for bacterial adhesion. From: Cellular senescence increases expression of bacterial ligands in the lungs and is positively correlated with increased susceptibility to pneumococcal pneumonia.

A) Pro-inflammatory cytokine profile of senescent A549 cells (n=3, with duplicate wells per experiment). Intracellular IL-1α as well as secreted IL-1α, IL-1β, TNFα, IL-6, IL-8, IL-10 and IL-12 produced by senescent and normal A549 cells was measured 8 days after a 24 hour pulse with 10 μg/ml bleomycin or mock, respectively, using a cytometric bead array. B) Mean relative adhesion of S. pneumoniae to A549 cells exposed to either conditioned media from normal or senescent A549 cells for 2 hours. Experiments were done in triplicate. For panels A and B, asterisks denote a statistical significant difference when using a two-tailed Student’s t-test. C) Western blots and corresponding densitometric analyses for K10, LR, and PAFr levels in A549 cells exposed to conditioned media after 2 hours. Samples were collected from three independent experiments, from either control (C) or bleomycin-induced senescent cells (S). Histogram shows composite densitometric data (protein/actin) with actin probed from the same lanes and membrane as the tested protein. The representative actin blot corresponds to the PAFr immunoblot.

Pooja Shivshankar, et al. Aging Cell. ;10(5):798-806.
7.
Figure 5

Figure 5. Senescent lung cells express enhanced levels of pneumococcal ligands and are permissive for bacterial adhesion in vitro. From: Cellular senescence increases expression of bacterial ligands in the lungs and is positively correlated with increased susceptibility to pneumococcal pneumonia.

A549 type II alveolar epithelial cells were pulsed with bleomycin to induce cellular senescence. We observed that after 8 days almost all cells had A) characteristic cell flattening and increased senescence associated β-galactosidase activity when treated with 25 μg/ml bleomycin. B) Western blot analysis for the pneumococcal ligands K10, LR, and PAFr as well as the senescence markers p16, pRb, and mH2A in bleomycin pulsed A549 cells. C) Immunofluorescent detection of pRb and K10 showing nuclear localization of pRb with increased expression of K10 in bleomycin pulsed A549 cells. D & E) Adhesion of wild type S. pneumoniae (WT) and the PsrP deficient mutant (ΔpsrP) to bleomycin treated A549 cells. K10 transfected cells were included as a positive control. Given the substantial difference in cell size between normal and senescent cells, the same experimental data is represented in panel D as the percent increase in bacterial adhesion to confluent monolayers with equal surface area (i.e. 2.0 cm2) and in panel E as the number of pneumococci adhered per 100,000 cells. Asterisks denote a statistical significant difference when using a two-tailed Student’s t-test.

Pooja Shivshankar, et al. Aging Cell. ;10(5):798-806.

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