Pirfenidone Treatment Improves Mitochondrial Respiration and ATP Production by Aged Macrophages in Response to S. pneumoniae. Aged and/or young macrophages were cultured with media alone or media containing pirfenidone (500 μg/ml) for 24 hours prior to culture with S. pneumoniae (MOI = 25). (A) mRNA was isolated from untreated or pirfenidone treated aged macrophages at select time points of S. pneumoniae infection and gene expression was analyzed by real time PCR using RT2 Profiler Assays (Mouse Mitochondrial Energy Metabolism PAMM-008ZA, fold regulation values are listed in Table 1). (B) Mitochondrial respiration was assessed using the MITO-ID Extracellular O2 Sensor Kit. Pirfenidone treated cells were cultured with S. pneumoniae (2 hours) prior to addition of the MITO-ID Extracellular O2 probe. Results were calculated as % effect from age-matched media treated samples (t-test: ****P < 0.0001 and one-way ANOVA ****P < 0.0001). (C) ATP concentration in untreated and pirfenidone treated aged macrophages was evaluated in the presence or absence of CCCP (25μM) (t-test: **P < 0.01, ***P < 0.001, and ****P < 0.0001). (D) ATP concentration in response to pirfenidone young and aged macrophages was examined at baseline and at 2 hours of infection (t-test: ***P < 0.001 and ****P < 0.0001 and one-way ANOVA ****P < 0.0001). (E) MPTP activation was measured in young and aged macrophages after 2 hours post S. pneumoniae infection (t-test: ***P < 0.001 and ****P < 0.0001 and one-way ANOVA ****P < 0.0001). Similar results were obtained from at least three independent experiments. Data are expressed as the mean ± SD.