Novel protective mechanism for interleukin-33 at the mucosal barrier during influenza-associated bacterial superinfection

Abstract

Influenza A is a highly contagious respiratory virus that causes seasonal epidemics and occasional worldwide pandemics. The primary cause of influenza-related mortality is bacterial superinfection. There are numerous mechanisms by which preceding influenza infection attenuates host defense, allowing for increased susceptibility to bacterial pneumonia. Herein, we demonstrate that influenza inhibits Staphylococcus aureus-induced production of interleukin-33 (IL-33). Restoration of IL-33 during influenza A and methicillin-resistant S. aureus superinfection enhanced bacterial clearance and improved mortality. Innate lymphoid Type 2 cells and alternatively activated macrophages are not required for IL-33-mediated protection during superinfection. We show that IL-33 treatment resulted in neutrophil recruitment to the lung, associated with improved bacterial clearance. These findings identify a novel role for IL-33 in antibacterial host defense at the mucosal barrier.

Figure 1. Influenza attenuates the IL-33 response to secondary bacterial challenge

C57BL/6 mice were infected with 100 PFU of influenza A PR/8/34 for 6 days, then challenged with 5×10⁷ CFU of MRSA for 24 hours. A - IL-33 expression in lung tissue measured by RT-PCR (n=3–7). C57BL/6 mice were infected with influenza A and harvested at different time points following influenza infection. B – IL-33 expression in lung tissue measured by RT-PCR (n=3–4). Mice were infected with influenza and MRSA. C – IL-33 protein in lung tissue measured by Western Blot (n=8). C57BL/6 mice were infected with 100 PFU of influenza A PR/8/34 for 6 days, then challenged with 1×10⁸ CFU of MSSA for 24 hours. D - IL-33 expression in lung tissue measured by RT-PCR (n=3–7). E – IL-33 protein in lung tissue measured by Lincoplex (n=7). C57BL/6 mice were infected with 100 PFU of influenza A PR/8/34 for 6 days, then challenged with PA01 for 24 hours. F – IL-33 expression in lung tissue measured by RT-PCR (n=3–8). * p < 0.05. Significance was tested by unpaired t test or one-way ANOVA. Each experiment was independently performed twice and data are shown from combined experiments with the exception of Panel A data from naïve mice and singular infection with influenza and Panel B, these experiments were performed once. Panel F represents two independently performed experiments with representative data shown.

Figure 2. Restoration of IL-33 improves S. aureus clearance in the lung during super-infection

Mice were infected with influenza or control and MRSA and received 2 μg recombinant mouse IL-33 at 24 and 2 hrs prior to MRSA challenge. A – Bacterial colony counts in the lung (n=6 in MRSA groups and n=12 in Flu/MRSA groups). B – Survival curve (n=5–6). C, E, F – Type 2 immune-related gene expression in lung tissue measured by RT-PCR (n=12). D, G–I, – Protein production in lung tissue measured by Lincoplex (n=12). J – CXCR2 gene expression in lung tissue measured by RT-PCR. * p < 0.05 Significance was tested by unpaired t test or one-way ANOVA. Each experiment was independently performed twice and data are shown from combined experiments with the exception of the singular MRSA groups in panel A and the survival curve in panel B, these experiments were performed once.

Figure 3. IL-33 mediated protection during super-infection does not require ILC2 or alternatively activated macrophages

C57BL/6 WT mice and B6-Rag2/ll2rg double knockout mice were infected with influenza and MRSA. Mice received IL-33 prior to MRSA challenge. A - Bacterial colony counts in the lung. B - Protein production of IL-5 in lung homogenate as measured by Lincoplex (n=12). C – Type 2 cytokine/protein-related gene expression in lung tissue measured by RT-PCR (n=12). D – M2a macrophage-related gene expression in lung tissue measured by RT-PCR (n=12). E - Protein production in lung homogenate as measured by Lincoplex (n=12). * p < 0.05 Significance was tested by unpaired t test or one-way ANOVA. Each experiment was independently performed twice and data are shown from combined experiments with the exception of panel A, which was performed twice with representative data presented.

Figure 4. IL-33 recruits neutrophils to the lung during super-infection, which are required for MRSA clearance

C57BL/6 WT mice were infected with influenza and MRSA. Mice received IL-33 prior to MRSA challenge. A – Total F480⁺CD11c⁺SiglecF⁺ cells in the lung by flow cytometry (n=6). B – Total F480⁺CD11c⁺SiglecF⁻ cells in the lung by flow cytometry (n=6). C – Macrophage scavenger receptor gene expression in lung tissue measured by RT-PCR (n=12). D – Total CD11b⁺Ly6G⁺ cells in the lung by flow cytometry (n=12). E–F – Serine protease gene expression in lung tissue measured by RT-PCR (n=12). C57BL/6 WT mice were infected with influenza and MRSA. Mice received IL-33 prior to MRSA challenge Mice received 250 μg anti-Ly-6G antibody at 48, 24, and 2 hrs prior to MRSA challenge. G - Bacterial colony counts in the lung (n=8–16). H - Total CD11b⁺Ly6G⁺ cells in the lung by flow cytometry (n=16). * p < 0.05 versus control group. Each experiment was independently performed twice and data are shown from combined experiments with the exception of panels A and B, these experiments were performed once.