Influenza A virus infection predisposes hosts to secondary infection with different

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1 Supplementary information Influenza A virus infection predisposes hosts to secondary infection with different Streptococcus pneumoniae serotypes with similar outcome but serotype-specific manifestation. Niharika Sharma-Chawla 1,2, Vicky Sender 3, Olivia Kershaw 4, Achim D. Gruber 4, Julia Volckmar 1, Birgitta Henriques-Normark 3,5, Sabine Stegemann-Koniszewski 1,2 and Dunja Bruder 1,2 Supplementary 1: a.

2 b. c. Figure S1: Histopathological changes in the lungs of IAV infected animals. (a) Representative example (one out of n=3) of histopathological changes in the lungs of IAV mono-infected or PBS treated mice at day 7, 14 or 21 post IAV infection analyzed after H and E staining at 2x magnification. For day 7 after IAV infection the asterisk indicates acute

3 bronchitis with infiltration of predominantly neutrophils and lymphocytes and the accumulation of suppurative exsudates in the bronchus. Alveolar septae are thickened by lymphocytic infiltration and perivascular edema is present. At days 14 and 21 after IAV infection histopathological changes are similar with widespread lymphocytic infiltration (asterisks). Additionally, there is hyperplasia of pneumocytes type II (arrow heads). (b) Histopathological changes in H and E stained lungs of IAV infected mice 14 days post IAV infection analyzed at 4x magnification. The image depicts the transition zone between unaffected lung parenchyma (left) with only few type II pneumocytes being present (arrows) and affected lung parenchyma (right) with significantly increased numbers of hyperplastic type II pneumocytes type II (double arrows). (c) The image represents the affected lung parenchyma at day 21 after IAV infection with severe lymphocytic, interstitial infiltration and alveoli lined by hyperplastic pneumocytes type II (double arrows). Additionally, high numbers of activated macrophages and fewer neutrophils are present in the alveolar lumina. Supplementary table 1: Compartment Time-point post IAV/PBS treatment Nasopharynx 7 Nasopharynx 14 Nasopharynx 21 IAV + T4 PBS + T4 IAV + 19F PBS + 19F IAV + 7F PBS + 7F 3 ( ) 7 (4-11) 75 ( ) 45 (-1385) 5 (2-115) 17 ( ) 6 (127-1) 12 (45-395) 65 (29-2) 825 ( ) 685 ( ) 5 (16-9) 445 ( ) 25 (16-36) 15 (25-24) 36 (8-665) 48 ( ) 7 (4-12) Table S1: Mice were infected with 1x1 6 CFU of S. pn. strains T4, 19F or 7F on day 7, 14 or 21 post infection with.31tcid 5 of IAV or PBS treatment. CFU counts from the nasopharynx (CFU/ml nasopharyngeal lavage) of the co-infected and mono-infected mice at 18hpi. Data are shown as median with interquartile ranges in parentheses. Data are compiled from a minimum

4 CFU/ml blood of two independent experiments with 3-4 mice per group and were compared by the one-way ANOVA with Bonferroni s multiple comparison test following log-transformation. All groups were compared and no significant changes were detected between the mono-infected, the coinfected or the mono- and co-infected groups. Supplementary 2: IAV + T4 IAV + 19F IAV + 7F Figure S2: Mice were infected with 1x1 6 CFU of S. pn. strains T4, 19F or 7F on day 7 post infection with.31tcid 5 of IAV or PBS treatment. Bacterial burden in the blood for the coinfected animals with lethal co-infection. Lines indicate the median. Data are compiled from a minimum of two independent experiments with 3-4 mice per group. Supplementary table 2: Compartment Time-point post IAV/PBS treatment BALF 21 Lung 21 IAV + T4 PBS + T4 IAV + 19F PBS + 19F IAV + 7F PBS + 7F 2 ( ) 14 (1-56) 6 (-11) 5 (2-3525) 11 (735-38) 27 (375-49) 6 (48-1) 23 (1-48) 1 (1-34) 1 (-525) 1 (-5) (-1) Blood 21 (-25) (-25) (-) (-) (-) (-) Table S2: Mice were infected with 1x1 6 CFU of S. pn. strains T4, 19F or 7F on day 21 post infection with.31tcid 5 of IAV or PBS treatment. CFU counts from the airways, lung tissue

5 and blood of the co-infected and mono-infected mice at 18hpi. Data are shown as median CFU/ml with interquartile ranges in parentheses. Data are compiled from a minimum of two independent experiments with 3-4 mice per group and were compared by the one-way ANOVA with Bonferroni s multiple comparison test following log-transformation. All groups were compared and no significant changes were detected between the mono-infected, the coinfected or the mono- and co-infected groups.