The inflammasome potentiates influenza/Staphylococcus aureus superinfection in mice

Abstract

Secondary bacterial respiratory infections are commonly associated with both acute and chronic lung injury. Influenza complicated by bacterial pneumonia is an effective model to study host defense during pulmonary superinfection due to its clinical relevance. Multiprotein inflammasomes are responsible for IL-1β production in response to infection and drive tissue inflammation. In this study, we examined the role of the inflammasome during viral/bacterial superinfection. We demonstrate that ASC-/- mice are protected from bacterial superinfection and produce sufficient quantities of IL-1β through an apoptosis-associated speck-like protein containing CARD (ASC) inflammasome-independent mechanism. Despite the production of IL-1β by ASC-/- mice in response to bacterial superinfection, these mice display decreased lung inflammation. A neutrophil elastase inhibitor blocked ASC inflammasome-independent production of IL-1β and the IL-1 receptor antagonist, anakinra, confirmed that IL-1 remains crucial to the clearance of bacteria during superinfection. Delayed inhibition of NLRP3 during influenza infection by MCC950 decreases bacterial burden during superinfection and leads to decreased inflammatory cytokine production. Collectively, our results demonstrate that ASC augments the clearance of bacteria, but can also contribute to inflammation and mortality. ASC should be considered as a therapeutic target to decrease morbidity and mortality during bacterial superinfection.

Keywords: Bacterial infections; Immunology; Influenza; Pulmonology.

Figure 1. The ASC inflammasome promotes decreased bacterial clearance and increased mortality during influenza and bacterial superinfection.

C57BL/6 mice were infected with 100 PFU of influenza A/PR/8/34 or vehicle for 6 days, and then challenged with 10⁸ CFU of methicillin-sensitive Staphylococcus aureus (MSSA) or vehicle for 24 hours. (A–C) Gene expression of inflammasome components in lung (n = 3–12). C57BL/6 and ASC^–/– mice were infected with 100 PFU of influenza A/PR/8/34 or vehicle for 6 days, and then challenged with 10⁸ CFU of MSSA or vehicle for 24 hours. (D) Bacterial colony counts in lung homogenate (n = 10–12). (E) Gene expression of influenza matrix protein in lung (n = 11–12). (F) Survival curve for WT and ASC^–/– mice challenged with influenza followed by MSSA (n = 8). (G) C57BL/6 mice were infected with 100 PFU of influenza A/PR/8/34 or vehicle for 6 days, and then challenged with 5 × 10⁷ CFU of methicillin-resistant S. aureus (MRSA) or vehicle for 24 hours (n = 12). *P < 0.05 versus naive WT by 1-way ANOVA; ^#P < 0.05 comparing SA with Flu/SA by unpaired t test. Data points reflect individual values ± SEM. Each experiment was independently performed at least twice and data are shown from combined experiments with the exception of naive mouse data, which was performed once.

Figure 2. The ASC inflammasome drives increased inflammation during influenza and bacterial superinfection.

C57BL/6 and ASC^–/– mice were infected with 100 PFU of influenza A/PR/8/34 or vehicle for 6 days, and then challenged with 10⁸ CFU of methicillin-sensitive Staphylococcus aureus (MSSA) or vehicle for 24 hours. (A) Bronchoalveolar lavage (BAL) cell differential counts (n = 7–8). (B–E) Cytokine concentrations in lung homogenate as measured by Bio-Plex immunoassay (n = 11–12). *P < 0.05 versus Flu/MSSA by unpaired t test. Data points reflect individual values ± SEM. Each experiment was independently performed at least twice and data are shown from combined experiments. (F) Oxygen consumption rate (OCR) of CD11c⁺ cells isolated from WT and ASC^–/– measured at the basal level and then after sequential treatment with 1 μM oligomycin, 1.5 μM FCCP, and 0.1 μM rotenone plus 1 μM antimycin-A using the XFe-96 extracellular flux assay system. The assay is representative of 3 independent experiments, run in triplicate, and the mean values ± SEM are shown. NS, not significant.

Figure 3. Mice lacking the ASC inflammasome produce IL-1β through inflammasome-independent mechanisms and IL-1β is crucial to the effective clearance of bacteria during superinfection.

C57BL/6 and ASC^–/– mice were infected with influenza A/PR/8/34 or vehicle for 6 days, and then challenged with 10⁸ CFU of methicillin-sensitive Staphylococcus aureus (MSSA) or vehicle for 24 hours. (A) IL-1β concentrations in bronchoalveolar lavage fluid as measured by Bio-Plex immunoassay (n = 11–12). (B) IL-1β concentrations in lung homogenate as measured by ELISA (n = 7–8). C57BL/6 and ASC^–/– mice were infected with influenza for 6 days, and then challenged with methicillin-resistant S. aureus (MRSA) for 24 hours. Mice received silvestat, 10 μg/g i.p. or vehicle control, on days 4–6 after influenza. (C) Bacterial colony counts in lung homogenate (n = 9). (D) IL-1β concentrations in lung homogenate as measured by Bio-Plex immunoassay (n = 6). (E) Gene expression of type 17 immunity–related antimicrobial peptides (n = 7–8). C57BL/6 and ASC^–/– mice were infected with influenza for 6 days, and then challenged with MSSA for 24 hours. Mice received anakinra, 600 μg in 200 μl of PBS i.p., on days 0–6 after influenza. (F) Bacterial colony counts in lung homogenate (n = 8). *P < 0.05 versus Flu/SA by 1-way ANOVA or unpaired t test. Data points reflect individual values ± SEM. Each experiment was independently performed at least twice and data are shown from combined experiments. NS, not significant.

Figure 4. IL-18 does not alter bacterial clearance during superinfection.

C57BL/6 and ASC^–/– mice were infected with influenza A/PR/8/34 or vehicle for 6 days, and then challenged with 10⁸ CFU of methicillin-sensitive Staphylococcus aureus (MSSA) or vehicle for 24 hours. (A) Gene expression of caspase-1 (n = 7–8). (B and C) Protein measurement of caspase-1 p20 and caspase-1 p45 products measured by Western blot (n = 8). (D) Gene expression of IL-18 (n = 7–8). (E) IL-18 concentrations measured by ELISA (n = 6). C57BL/6 mice were infected with influenza A/PR/8/34 for 6 days, received 200 μg of IL-18 inhibitor or IgG control, and then challenged with 10⁸ CFU of MSSA 4 hours later and harvested at 24 hours. (F) CFU in lung homogenate (n = 6). *P < 0.05 versus Flu/SA by 1-way ANOVA or unpaired t test. Data points reflect individual values ± SEM. Each experiment was independently performed at least twice and data are shown from combined experiments with the exception of the IL-18 inhibition experiment that was performed once. NS, not significant.

Figure 5. Temporal NLRP3 inhibition enhances bacterial clearance but does not affect mortality during influenza and bacterial superinfection.

C57BL/6 and AIM^–/– mice were infected with influenza A/PR/8/34 for 6 days, and then challenged with 10⁸ CFU of methicillin-sensitive Staphylococcus aureus (MSSA) for 24 hours. (A) Bacterial colony counts in lung homogenate (n = 6). (B) Survival curve for WT and AIM^–/– mice challenged with influenza followed by MSSA (n = 7–8). C57BL/6 mice were infected with 100 PFU of influenza A/PR/8/34 or vehicle for 6 days, and then challenged with 10⁸ CFU of MSSA or vehicle for 24 hours. Mice received MCC950 5 mg/kg by oropharyngeal aspiration or PBS control on days 4 and 6 after influenza. (C) Bacterial colony counts in lung homogenate (n = 4 in MSSA-alone groups and 9–10 in Flu/MSSA groups). (D–F) Cytokine concentrations in lung homogenate as measured by Bio-Plex immunoassay (n = 9–10). (G) IL-1β concentrations in lung homogenate as measured by ELISA (n = 9–10). (H) Survival curve for WT and MCC950-treated mice challenged with influenza followed by MSSA (n = 8). *P < 0.05 versus Flu/MSSA by 1-way ANOVA or unpaired t test. Data points reflect individual values ± SEM. Each experiment was independently performed at least twice and data are shown from combined experiments with the exception of AIM^–/– mouse data displayed in panel A and survival curves, which were performed once.