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1 Supporting Information Aldridge et al /pnas Fig. S1. Flow diagram of sublethal (a) and lethal (b) influenza virus infections. (a) Infection of lung epithelial cells by influenza virus induces, among other things, secretion of MCP-1 and MCP-3 two chemokines capable of signaling through the CCR2 receptor. These chemokines recruit TNF- /inducible nitric oxide synthase-producing dendritic cells (tipdcs) to the site of infection, where they acquire, process, and present influenza antigen in the context of MHC class I antigen to CD8 T cells emigrating from the draining mediastinal lymph node (MLN). Antigen presentation by tipdcs results in CD8 T-cell expansion, which ultimately aids in viral clearance. (b) During lethal influenza infections, the chemokines MCP-1 and MCP-3 accumulate to significantly higher levels in the airways, resulting in increased recruitment of tipdcs. Lethal infection results in decreased levels of CD8 T cells through a mechanism that has not been identified. Together, decreased numbers of CD8 T cells and increased numbers of tipdcs result in impaired viral clearance and elevated host pathology, respectively. 1of7

2 Fig. S2. Innate cell recruitment to the lungs. (a d) Graphs represent the mean SEM. of the number of macrophages (a), neutrophils (b), conventional dendritic cells (DCs) (c), or natural killer cells (d) recruited to the airways on days 3, 5, and 7 after infection with either 10 3 pfu of PR8 (lethal) or 10 5 pfu of x-31 (sublethal). Infection caused a change in cell counts (virus effect, P 0.003), but this was not influenced by the type of virus (interaction effect, P 0.05). Results are representative of 2 independent experiments (n 5). 2of7

3 Fig. S3. Cytokine and chemokine expression in lungs of influenza virus-infected animals. (a i) Graphs represent the mean SEM of the level of expression of MCP-1 (CCL2) (a), MIP-1 (b), MIP-1 (c), IL-1 (d), IL-6 (e), TNF- (f), KC (g), G-CSF (h), and GM-CSF (i) in the cell-free bronchoalveolar lavage was on days 3, 5, and 7 after infection with either 10 3 pfu of PR8 (lethal) or 10 5 pfu of x-31 (sublethal). MCP-1, IL-1, KC, and GM-CSF exhibit a significant virus time interaction, so simple effects were investigated (asterisks, P 0.05). The quantity of MIP-1 increased over time (P ), and the amount was higher in PR8-infected mice (P ). Of the remaining cytokines and chemokines, model simplification revealed a significant effect for virus alone (P 0.03). Results are representative of 2 independent experiments (n 5). 3of7

4 Fig. S4. Innate cell recruitment to the lungs. (a d) Graphs represent the mean SEM of the number of macrophages (a), neutrophils (b), conventional dendritic cells (DCs) (c), or natural killer cells (NKs) (d) recruited to the airways on days 3, 5, and 7 after infection with either 10 2 pfu of VN1203 (lethal) or 10 2 pfu of HK213 (sublethal). Lethal infections exhibited higher cell counts for macrophages (P 0.005) and NKs (P 0.01) and fewer cdcs (P ). No differences in neutrophil cell counts were observed (P 0.12). The number of cells for each cell type changed over time for both lethal and sublethal infections (P ). Results are representative of 2 independent experiments (n 5). 4of7

5 Fig. S5. Cytokine expression in lungs of CCR2 / and CCR2 / animals. Graphs represent the mean SEM of the level of expression of IL-2 or IL-4 in the cell-free bronchoalveolar lavage wash from CCR2 / or CCR2 / animals on day 7 after infection with 10 5 pfu of x-31. Results are representative of 2 independent experiments (n 5). Results of the two-tailed Student s t test showed no significant differences (P 0.05). 5of7

6 Fig. S6 Pioglitazone does not inhibit virus replication. (A) Virus titer (plaque-forming units) of the BAL fluid was determined on days 3, 6, and 9 after infection from animals treated with either PBS or pioglitazone. Results of the two-tailed Student s t test showed no significant differences (P 0.05) in virus titer between treatment groups. (B) Confluent MDCK cells in 6-well plates were infected with PR8 by using a multiplicity of infection of either 0.25, 0.5, or 1.0. Five hours after infection, the cells were stained for the influenza NP via intracellular staining and read on a FACSCalibur. There was no significant difference in NP accumulation between treatment groups. 6of7

7 Table S1. Cellular characterization Cell CD11b CD11c Ly6c Ly6g MHC class II NK1.1 T-cell receptor inos TNF- Macrophage Neutrophil NK cdc tipdc Cell surface expression levels:, high level of expression;, intermediate expression;, low expression;, negative. NK, natural killer cell; cdc, conventional dendritic cell; tipdc, TNF- /inducible nitric oxide synthase (inos)-producing dendritic cell. 7of7

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