Supplementary Material
Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.
Supplementary material ERJ-01953-2016_Supplement
Figure S1. Localisation of influenza A virus Pan/99(H3N2) (IAV) and Streptococcus pneumoniae D39 (S. pneumoniae) in single and co-infected human lung tissue and IAV induced interferon (IFN) expression in single and co-infected human lung tissue. (A) Human lung tissue was either mock infected, (B) challenged with the seasonal IAV for 24 h, (C) S. pneumoniae for 16 h, or (D) co-infected with the IAV subsequently followed by S. pneumoniae. IAV (green, white arrowheads) typically replicated in alveolar epithelial type II cells indicated by pro-SP-C (blue, asterisk, B, D). S. pneumoniae closely attached to alveolar epithelial cells (open arrowheads, C, D) and alveolar macrophages (white arrow, D). Lung structure was visualised by differential interference contrast (grey) and nuclei were counterstained using DAPI (orange). Scale bar 10 µm. ERJ-01953-2016_Supplementary_Figure_S1
Figure S2. Co-infection with influenza A virus Pan/99(H3N2) (IAV) and Streptococcus pneumoniae (S. pneumoniae) shows differential cytokine regulation in human lungs. Lung explants were ex vivo mock infected or challenged with IAV (1 × 106 PFU/ml) for 24 h. (A-F) Afterwards, dedicated specimen were either infected with the S. pneumoniae (1 × 106 CFU/ml) (A - D) or (E, F) the clinical isolate of serotype 3 S. pneumoniae (1 × 106 CFU/ml), respectively. Supernatants were collected 16 h after pneumococcal infection and assayed for release of (A) IL-6, (B) IL-8, (C) IL-10, (D) TNFα, (E) IL-1β, (F) GM-CSF as indicated. Data are presented as mean ± SEM of six donors within independent experiments. **p≤0.01. ERJ-01953-2016_Supplementary_Figure_S2
Figure S3. Influenza A virus Pan/99(H3N2) (IAV) infection or interferon (IFN) treatment of human lung tissue fails to suppress Streptococcus pneumoniae D39 (S. pneumoniae) induced tumour necrosis factor α (TNFα) release. Lung explants were ex vivo mock infected or challenged for 24 h with influenza A virus Pan/99(H3N2) (IAV) (1 × 106 PFU/ml) or 16 h pre-treated with a combination of IFNβ and IFNγ (100 U/ml each). Mock or S. pneumoniae (1 × 106 CFU/ml) infection followed and supernatants of lung specimen were analysed after additional 16 h for release of TNFα. Data are presented as mean ± SEM of six donors within independent experiments. **p≤0.01. ERJ-01953-2016_Supplementary_Figure_S3
Figure S4. TNFα stimulation induces cyclooxygenase-2 (COX-2) expression and granulocyte macrophage - colony stimulating factor (GM-CSF) expression is time shifted to IL-1β in human lungs. (A) Stimulation with TNFα (100 ng/ml) of human lung specimen for 16 h induces pro-inflammatory COX-2. (B) Human lung explants were either mock infected or challenged with S. pneumoniae (1 × 106 CFU/ml) for 2, 4, 6, 8, and 16 h to demonstrate time shifted release of GM-CSF in relation to earlier IL-1β liberation. Data are presented as fold of control and mean ± SEM of three/four donors within independent experiments. ERJ-01953-2016_Supplementary_Figure_S4
Figure S5. Alveolar macrophages (AM) and alveolar epithelial type II cells (AEC II) were isolated from fresh human lung tissue and seeded on glass coverslips, fixed and phenotyped with different cell markers by immunofluorescence and confocal microscopy. (A) AM, cultured for 2 days, were positive for CD-68 (red). AEC II were cultured for 4 days after isolation. (B) Cells were positive for pan-Cytokeratin (red) and pro-SP-C (green). For nuclear counterstaining DAPI (blue) was used. Microscope: Carl Zeiss LSM780, Plan Apo-Chromat 63× oil/NA 1.4. Scale bar 5 µm ERJ-01953-2016_Supplementary_Figure_S5
Figure S6. Tumour necrosis factor α (TNFα) expression was not suppressed by interferons (IFN) in isolated human alveolar macrophages (AM). Isolated AM were cultured for 2 days and either mock challenged or stimulated with a combination of interferon β and γ (100 U/ml each) for 16 h before pneumococcal infection. Afterwards, supernatants were assayed for release of TNFα. Data are represented as mean ± SEM of four donors within independent experiments. *p≤0.05. ERJ-01953-2016_Supplementary_Figure_S6
Figure S7. IL-1β induced granulocyte macrophage - colony stimulating factor (GM-CSF) release in human lung tissue is suppressed by interferons (IFN). Human lungs were pre-treated with a combination of IFNβ and IFNγ (100 U/ml each) for 16 h and subsequent IL-1β stimulation (10 ng/ml) for 16 h. Supernatants of lung specimen were analysed for release of GM-CSF. Data are presented as mean ± SEM of three donors within independent experiments. *p≤0.05. ERJ-01953-2016_Supplementary_Figure_S7