Supplementary Figures

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1 Inhibition of Pulmonary Anti Bacterial Defense by IFN γ During Recovery from Influenza Infection By Keer Sun and Dennis W. Metzger Supplementary Figures d a Ly6G Percentage survival f Time (d) after pneumo infection b c CFU (% of inoculum) h 2 h PMN PMN. 9.3 CD11b hr Unit distribution 3min 2hr e g scid Rag2 / CFU (% of inoculum) 2 Time (hr) after pneumo infection CFU/Lung (log 1 ) PMN+ PMN 8 C3-/- 6 CFU (% of inoculum) * Pneumococci 2 2 Time (hr) after pneumo infection Supplementary Figure 1. Bacterial clearance in naive mice (a) Survival of C57BL/6 mice after respiratory infection with the indicated doses of S. pneumoniae (5 mice/group). (b, c) Bar graphs showing the relative numbers of bacteria in C57BL/6 wild-type lungs and BALF, (b) scid lungs, and (c) Rag2 / BALF hr after infection with 1 5 CFU of

2 A66.1 S. pneumoniae (5 mice/group). (d) Flow cytometry analysis of C57BL/6 BALF cells and 2 hr after infection with 1 CFU S. pneumoniae. (-PMN), mice depleted of neutrophils using anti-ly6g mab treatment; (+PMN), neutrophil-competent mice (-5 mice/group). (e) Relative numbers of bacteria remaining in the lungs 2 and hr after infection of C57BL/6 mice with 1 5 CFU S. pneumoniae. (-PMN), mice depleted of neutrophils; (+PMN), neutrophil-competent mice. In (b, c, e), the mean CFU ± s.d. (5 mice/group) are represented as percentages of the inoculation dose. (f) Flow cytometry analysis of CD11c + alveolar macrophages at different time points after infection of C57BL/6 mice with 1 5 CFU of PKH26 red fluorescent-labeled S. pneumoniae. The results are representative of three mice per group. (g) Numbers of bacteria (mean CFU ± s.d) in the lungs after infection of C57BL/6 wild-type and C3 / mice with 1 5 CFU of S. pneumoniae ( mice/time point). The data are representative of two independent experiments. *, P<.5, compared to wild-type mice.

3 a Ly6G Ifngr1-/ b +PMN CFU/BALF ( 1 ) PMN PMN *** ** PMN +FLU Ifngr1 / CD11b Supplementary Figure 2. Neutrophil depletion in influenza infected mice. (a) Flow cytometry analysis of CD11b + Ly6G + neutrophils in BALF hr after superinfection of C57BL/6 wild-type and Ifngr1 / mice with 1 5 CFU S. pneumoniae on day 9 of viral infection. (-PMN), mice depleted of neutrophils using anti-ly6g mab treatment; (+PMN), neutrophil-competent mice. Pooled BALF cells from three mice per group are shown. (b) Numbers of bacteria in BALF hr after pneumococcal infection of day 9 postinfluenza mice (+FLU). (-PMN), neutrophil-depleted mice. **, P <.1, ***, P <.1, relative to influenza infected wild-type mice.

4 % Binding and Uptake ** Ifngr1 / IFN-γ + IFN-γ Supplementary Figure 3. Specific role of IFN-γ on alveolar macrophage bacterial uptake. Flow cytometric analysis of bacterial uptake by CD11c + BALF cells from C57BL/6 wild-type and Ifngr1 / mice after overnight incubation with 1 ng/ml of IFN-γ. The results are expressed as the relative percentages of bacterial uptake normalized to values observed in the absence of IFN-γ. The data represent the mean + s.d of three independent experiments. **, P<.1, compared to IFN-γ-treated CD11c + BALF cells from Ifngr1 / mice (paired t-test).

5 a IFN-γ CD.3.6 CD FLU + FLU b IFN-γ (ng/ml) FLU +FLU *** ** ** Cd / CFU(% of inoculum) ** ** Cd / Supplementary Figure. Influenza-induced T cell IFN-γ expression in the lung. (a) Lung lymphocytes were isolated from C57BL/6 mice on day 7 after influenza infection and intracellular IFN-γ expression was determined by flow cytometry. Results from the lung lymphocyte gate are shown. (b) C57BL/6 wild-type and Cd / mice were infected with influenza virus and challenged with 1 5 CFU of A66.1 S. pneumoniae on day 8 after viral infection. The levels of IFN-γ (left) and (c) bacterial CFU (right) in BALF hr after pneumococcal infection show that in the absence of CD + T cells, there is still sufficient IFN-γ production in influenza-infected mice to inhibit hr clearance of pneumococci. **, P <.1, ***, P <.1, relative to influenza infected wild-type mice.

6 c a Weight (g/mouse) TNF-α (ng/ml) Rat IgG XMG Time (d) after influenza infection *** *** PFU/BALF (log 1 ) d IL-1β (ng/ml) b Rat IgG XMG1.2 *** **. FLU Rat IgG XMG1.2 +FLU FLU Rat IgG XMG1.2 +FLU Supplementary Figure 5. IFN-γ neutralization following viral infection. (a) Mean weight change of BALB/c mice after infection with 1 PFU of influenza virus, IFN-γ neutralization, and inoculation of 1 5 CFU S. pneumoniae D39 on day 9 postinfluenza infection (8 mice/group). Mean levels ± s.d of (b) viral PFU, (c) TNF-α and (d) IL-1β in BALF hr after i.n. inoculation of 1 5 CFU S. pneumoniae into either naïve C57BL/6 mice or mice infected 8 days earlier with 1 PFU of influenza virus (5-7 mice/group). Influenza infected mice were also treated with rat IgG or XMG1.2 anti-ifn-γ mab i.n. on day 5 after viral inoculation. The data are representative of two independent experiments. **, P <.1, ***, P <.1, compared to influenza infected wild-type mice.

7 CD11b + Unit distribution FLU + FLU+PBS + FLU+XMG1.2 CD11c + MHCII Supplementary Figure 6. Flow cytometry of BALF cells for MHCII expression after influenza infection. Analysis of MHCII expression on CD11b + (top) and CD11c + (bottom) BALF cells from C57BL/6 mice following i.p. treatment with XMG1.2 anti- IFN-γ mab or PBS after viral infection. The data are representative of four mice per group.

8 Supplementary Methods Murine model of viral and bacterial infection Titers of virus stocks and viral levels in the BALF and lungs of infected mice were determined by plaque assays on MDCK cell monolayers. Respiratory bacterial burdens were measured by sacrificing infected mice at various time points as indicated, and incubating serial 1-fold dilutions of BALF and lung homogenates on blood agar plates at 37 C overnight. Studies examining superinfection with pneumococci were all performed at the time that the mice began to regain weight following primary viral infection, which varied among individual experiments from days 7 to 1 after sublethal influenza infection. An infection dose of 1 5 CFU was chosen for bacterial clearance studies and a dose of 1 CFU was chosen for survival studies in superinfected mice when the A66.1 pneumococcus strain was used. Binding of pneumococci by BALF cells S. pneumoniae were labeled with a PKH26 Red Fluorescent General Cell Linker Kit (Sigma). 1 7 CFU of S. pneumoniae were incubated with 2 µm PKH26 at 25 C for 5 min, followed by addition of 1% BSA in PBS, and additional incubation for 1 min. The bacterial cell suspension was washed twice with PBS containing 1% BSA and once with PBS, and then resuspended in PBS to a concentration of CFU/ml. Anesthetized mice were inoculated i.n. with 5 μl of the labeled S. pneumoniae. BALF cells were collected at the indicated time points and stained for CD11b and CD11c surface markers. In vivo labeling of resident alveolar macrophages PKH26-PLC (Sigma) is a phagocytespecific, lipophilic dye that stably integrates into cell membranes for more than 21 days in vivo and is ideal for long-term staining of resting cells such as alveolar macrophages. Neutrophil depletion For viral infected mice, RB6-8C5 anti-ly6g mab was given on day 7 and day 8 after influenza infection. The efficiency of neutrophil depletion in BALF of pneumococcal-infected mice was confirmed by using Diff-Quick stained cytospin preparations and by flow cytometry (Supplementary Figs. 1d and 2a). Intracellular cytokine staining Single-cell suspensions were obtained from lungs by collagenase D and DNase I digestion, passage through a cell strainer (BD Falcon, Bedford, MA), filtering through a nylon/cotton wool column and then density gradient centrifugation on Lympholyte M (Cedarlane Laboratories Limited, Ontario, Canada).

9 The lung lymphocytes were cultured in DMEM medium containing 1% FBS for hr at 37 C in the presence of 5 ng/ml PMA, 5 ng/ml ionomycin, and 1 μg/ml Brefeldin A. The cells were then fixed with 2% paraformaldehyde and stained with Alexa Fluorconjugated anti-ifn-γ (BD Biosciences), FITC-conjugated anti-cd (BD Biosciences) and PE-conjugated anti-cd8 mab (Caltag). The stained cells were stored in the dark at o C and analyzed within 2 hr on a FACSCanto using FACSDiva software.