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 ():e August 11, 2015 ©2015 by European Molecular Biology Organization

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: /embr ©2015 by European Molecular Biology Organization

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: /embr ©2015 by European Molecular Biology Organization

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: /embr ©2015 by European Molecular Biology Organization

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: /embr ©2015 by European Molecular Biology Organization

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: /embr ©2015 by European Molecular Biology Organization

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: /embr ©2015 by European Molecular Biology Organization

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: /embr ©2015 by European Molecular Biology Organization

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: /embr ©2015 by European Molecular Biology Organization

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: /embr ©2015 by European Molecular Biology Organization

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: /embr ©2015 by European Molecular Biology Organization

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: /embr ©2015 by European Molecular Biology Organization