Expanded View Figures

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1 Gregory T Ellis et al Lung damage by monocyte TRIL allows coinfection EMO reports Expanded View Figures % survival Clinical score Influenza Matrix /HPRT (log ) CFU/L (log ) 3 irway early h Survival Clinical score Lung viral load.9 + h 7 dpi + h dpi Influenza Matrix /HPRT (log ) s rrn/hprt (log ) 3 Spleen viral load.79 3 <... Control lung CFU/lung (log ) Lung streptococcal RN <. + h % of starting weight 9 Spleen <. + h Lung.79 dpi Weights C D E F I J G CFU/spleen (log ) 7 dpi H Survival: heat treated bacteria % survival Spleen <..79 (HK) Pam3CSK (HK) + Pam3CSK. Figure EV. Mortality in coinfection is linked to outgrowth of live bacteria. C Mortality (), weights () and clinical scores (C) following infection with 3 TCID /3 ll X3, 7 CFU/3 ll S. pneumoniae D39 or mock (PS) (data shown are pooled from four independent experiments, n = 9; this dosing regimen hereafter referred to as high dose ; for clarity, means shown include euthanized mice at endpoint weight or clinical score). D, E Pneumococcal load in the lung (D) from to dpi and in the spleen (E) at dpi during low-dose coinfection (dotted line indicates detection limit, n = ). F Quantitative PCR for influenza matrix RN in the lung during high-dose coinfection from to dpi (n = ). G Quantitative PCR for influenza matrix RN in the spleen at dpi during high-dose coinfection, compared to influenza-infected positive control lung ( dpi 3 TCID ) (n = ). H Mortality following high-dose coinfection with live S. pneumoniae, heat-killed S. pneumoniae or Pam3CSK ( lg) (representative of two independent experiments, n = 9). I Pneumococcal load in the airways early during high-dose coinfection from dpi + hto dpi + h (dotted line indicates detection limit, n = ). J Quantitative PCR for pneumococcal s rrn in the lung during high-dose coinfection from dpi+ hto dpi + h(n = ). Data information: Data are displayed as percentage survival (mortality), geometric means (viral and bacterial loads, bacterial RN) or arithmetic means SEM (weights and clinical scores). Significance was assessed by Mann Whitney U-test (viral and bacterial loads, bacterial RN), two-way NOV (weights and clinical scores) or logrank (Mantel Cox) test (mortality). = not significant. ª Francis Crick Institute EMO reports EV

2 EMO reports Lung damage by monocyte TRIL allows coinfection Gregory T Ellis et al Cells/lung (x ) D Inflammatory Monocytes.3 Cells/lung (x ) Neutrophils.3 C Cells/lung (x ) E pg/ml (log ) CD T CD T NKs Ms TNF-α KC MIP-.9.9 Figure EV. Quantification of cells and cytokines during high-dose coinfection., Quantification of inflammatory monocytes () and neutrophils () during high-dose coinfection at 7 dpi by flow cytometry (data shown are pooled from two independent experiments, n = 3). C Quantification of CD T cells (CD3 + CD + ), CD T cells (CD3 + CD + ), NK cells (CD3 CD CD NKp + ) and alveolar macrophages during high-dose coinfection at 7 dpi (n = ). D H&E staining of lung tissue sections at dpi during high-dose coinfection. Scale bar indicates lm (n = 3). E Multiplex quantification of TNF-a, KC and MIP in the airways at 7 dpi during high-dose coinfection (n = ). Data information: Data are displayed as arithmetic means SEM. Significance was assessed by Mann Whitney U-test (viral and bacterial loads, bacterial RN). = not significant. EV EMO reports ª Francis Crick Institute

3 Gregory T Ellis et al Lung damage by monocyte TRIL allows coinfection EMO reports Figure EV3. 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 by size). Representative dpi wild-type non-lavaged whole lung shown ( 3 TCID )(n = ). ª Francis Crick Institute EMO reports EV3

4 EMO reports Lung damage by monocyte TRIL allows coinfection Gregory T Ellis et al Wild Type (C7L/) C D E F G CCR / H I J K L Figure EV. Monocytes and monocyte-related cells express TRIL and are absent in CCR / mice. F CCR and TRIL expression in different myeloid cell populations as assessed by flow cytometry; pre-gated for live cells (Death Stain and exclusion of debris by size). Representative dpi wild-type non-lavaged whole lung shown ( 3 TCID )(n = ; note this lung is the same as shown in Fig EV3). G L CCR and TRIL expression in different myeloid cell populations as assessed by flow cytometry. Representative dpi non-lavaged whole CCR / lung shown ( 3 TCID )(n = ). Data information: Gating of each myeloid population is shown in the left panels, and histograms of CCR and TRIL expression are shown on the right. Grey histogram represents unstained, and coloured histogram represents stained. EV EMO reports ª Francis Crick Institute

5 Gregory T Ellis et al Lung damage by monocyte TRIL allows coinfection EMO reports DPI α αmpo Z-projection + + C. albicans DPI αmpo αcith3 (NET) αcith3 (NET) Figure EV. Neutrophils phagocytose streptococci but do not produce NETs during coinfection. Confocal microscopy of lung tissue sections at dpi during high-dose coinfection stained for cell nuclei (DPI), streptococci (a) and neutrophils (ampo). lack text indicates infection condition; coloured text indicates staining. Right column shows the Z-projection of individual focal planes; other columns show a single plane. rrows indicate bacteria phagocytosed by neutrophils. Scale bars indicate lm (n = 3). Confocal microscopy of lung tissue sections at dpi during high-dose coinfection (or during C. albicans infection as positive control) stained for cell nuclei (DPI), neutrophils (ampo) or the NET constituent citrullinated histone H3 (acith3). lack text indicates infection condition; coloured text indicates staining. Scale bars indicate lm (n = 3). ª Francis Crick Institute EMO reports EV