1. TRAIL + monocytes and monocyte ‐ related cells cause lung damage and thereby increase susceptibility to influenza– Streptococcus pneumoniae coinfection by Gregory T Ellis, Sophia Davidson, Stefania Crotta, Nora Branzk, Venizelos Papayannopoulos, and Andreas Wack EMBO Rep. Volume ():e201540473 August 11, 2015 ©2015 by European Molecular Biology Organization

2. Influenza A predisposes mice to Streptococcus pneumoniae coinfection A–CMortality (A), weights (B) and clinical scores (C) following intranasal infection of C57BL/6 mice with 400 TCID50/30 μl IAV X31, 2 × 105 CFU/30 μl S. pneumoniae D39 or mock (PBS) (data... Gregory T Ellis et al. EMBO Rep. doi:10.15252/embr.201540473 ©2015 by European Molecular Biology Organization

3. Mortality in coinfection is linked to outgrowth of live bacteria A–CMortality (A), weights (B) and clinical scores (C) following infection with 8 × 103 TCID50/30 μl IAV X31, 2 × 107 CFU/30 μl S. pneumoniae D39 or mock (PBS) (data shown are pooled from four... Gregory T Ellis et al. EMBO Rep. doi:10.15252/embr.201540473 ©2015 by European Molecular Biology Organization

4. Quantification of cells and cytokines during high ‐ dose coinfection A, BQuantification of inflammatory monocytes (A) and neutrophils (B) during high ‐ dose coinfection at 7 dpi by flow cytometry (data shown are pooled from two independent experiments, n = 2–3... Gregory T Ellis et al. EMBO Rep. doi:10.15252/embr.201540473 ©2015 by European Molecular Biology Organization

5. Mice deficient in inflammatory monocytes and related myeloid populations are resistant to coinfection and have reduced early lung damage AMortality of CCR2−/− and wild ‐ type (WT) mice during low ‐ dose coinfection (data shown are pooled from two independent e... Gregory T Ellis et al. EMBO Rep. doi:10.15252/embr.201540473 ©2015 by European Molecular Biology Organization

6. Myeloid flow cytometry gating strategy Flow cytometry gating strategy used for myeloid cells (neutrophils, alveolar macrophages, inflammatory monocytes and inflammatory monocyte ‐ derived cells); pre ‐ gated for live cells (Death Stain− and exclusion of debris... Gregory T Ellis et al. EMBO Rep. doi:10.15252/embr.201540473 ©2015 by European Molecular Biology Organization

7. Monocytes and monocyte ‐ related cells express TRAIL and are absent in CCR2−/− mice A–FCCR2 and TRAIL expression in different myeloid cell populations as assessed by flow cytometry; pre ‐ gated for live cells (Death Stain− and exclusion of debris by size). Gregory T Ellis et al. EMBO Rep. doi:10.15252/embr.201540473 ©2015 by European Molecular Biology Organization

8. Blockade of TRAIL ameliorates coinfection AQuantification of TRAIL+ lung cells by flow cytometry at 5 dpi in CCR2−/− and wild ‐ type (WT) mice (8 × 103 TCID50); abbreviations as in Fig 2H (n = 4).BQuantification of the DR5+ proportion of lung epithelial cell... Gregory T Ellis et al. EMBO Rep. doi:10.15252/embr.201540473 ©2015 by European Molecular Biology Organization

9. Neutrophils from coinfected mice are functional and contribute to survival and bacterial control ROS production was assessed by luminol assay of PDBu (50 nM) ‐ stimulated lung neutrophils purified by MACS from high ‐ dose ‐ coinfected or S. pneumoniae ‐ infected m... Gregory T Ellis et al. EMBO Rep. doi:10.15252/embr.201540473 ©2015 by European Molecular Biology Organization

10. Neutrophils phagocytose streptococci but do not produce NETs during coinfection Confocal microscopy of lung tissue sections at 8 dpi during high ‐ dose coinfection stained for cell nuclei (DAPI), streptococci (αStrep) and neutrophils (αMPO). Gregory T Ellis et al. EMBO Rep. doi:10.15252/embr.201540473 ©2015 by European Molecular Biology Organization

11. TNF ‐ α contributes to survival and bacterial control in coinfected mice Mortality during low ‐ dose coinfection following treatment with anti ‐ TNF ‐ α or isotype control at 5 and 7 dpi (n = 6– 9).Pneumococcal load in the lung at 8 dpi during low ‐ dose coinfection... Gregory T Ellis et al. EMBO Rep. doi:10.15252/embr.201540473 ©2015 by European Molecular Biology Organization

12. Summary of coinfection pathogenesis Influenza infection leads CCR2 ‐ dependent recruitment of monocytes and other monocyte ‐ related immune cell populations into the lung. Gregory T Ellis et al. EMBO Rep. doi:10.15252/embr.201540473 ©2015 by European Molecular Biology Organization