Interleukin-4 protects mice against lethal influenza and Streptococcus pneumoniae co-infected pneumonia

Abstract

Streptococcus pneumoniae co-infection post-influenza is a major cause of mortality characterized by uncontrolled bacteria burden and excessive immune response during influenza pandemics. Interleukin (IL)-4 is a canonical type II immune cytokine known for its wide range of biological activities on different cell types. It displays protective roles in numerous infectious diseases and immune-related diseases, but its role in influenza and S. pneumoniae (influenza/S. pneumoniae) co-infected pneumonia has not been reported. In our study, we used C57BL/6 wild-type (WT) and IL-4-deficient (IL-4^-/- ) mice to establish co-infection model with S. pneumoniae after influenza virus infection. Co-infected IL-4^-/- mice showed increased mortality and weight loss compared with WT mice. IL-4 deficiency led to increased bacterial loads in lungs without altering influenza virus replication, suggesting a role of IL-4 in decreasing post-influenza susceptibility to S. pneumoniae co-infection. Loss of IL-4 also resulted in aggravated lung damage together with massive proinflammatory cytokine production and immune cell infiltration during co-infection. Administration of recombinant IL-4 rescued the survival and weight loss of IL-4^-/- mice in lethal co-infection. Additionally, IL-4 deficiency led to more immune cell death in co-infection. Gasdermin D (GSDMD) during co-infection was induced in IL-4^-/- mice that subsequently activated cell pyroptosis. Treatment of recombinant IL-4 or inhibition of GSDMD activity by disulfiram decreased immune cell death and bacterial loads in lungs of IL-4^-/- co-infected mice. These results suggest that IL-4 decreases post-influenza susceptibility to S. pneumoniae co-infection via suppressing GSDMD-induced pyroptosis. Collectively, this study demonstrates the protective role of IL-4 in influenza/S. pneumoniae co-infected pneumonia.

Keywords: Streptococcus pneumoniae; IL-4; co-infection; influenza; pyroptosis.

FIGURE 1

Interleukn (IL)‐4 is required for the survival of mice in influenza/Streptococcus pneumoniae co‐infection. Wild‐type (WT) and IL‐4^−/− mice were infected with PR8 alone, 19F alone or PR8+19F co‐infection. Mice were monitored for 2 weeks after primary PR8 or 19F infection. (a) C57BL/6 WT mice and IL‐4^−/− mice were inoculated intranasally with influenza [PR8; 400 plaque‐forming units (PFU) alone, S. pneumoniae, 19F; 1 × 10⁸ colony‐forming units (CFU)] alone or PR8+19F co‐infection. Secondary 19F super‐challenge was performed 3 days after influenza infection. (b,c) Body weight loss of single infected mice was recorded daily (n = 4–5/group). (d) Kaplan–Meier survival curves were assessed by log‐rank (Mantel–Cox) test (n = 10–12/group). **p < 0.01. (e,f) Co‐infected WT and IL‐4^−/− mice were injected intraperitoneally (i.p.) with 2 μg rIL‐4 at 3 and 5 days after influenza infection (0 and 2 days after 19F co‐infection); the survival and weights were monitored for 2 weeks after influenza (n = 10–13 /group). Kaplan–Meier survival curves were assessed by log‐rank (Mantel–Cox) test for significance. *p < 0.05

FIGURE 2

Interleukin (IL)‐4 deficiency increases post‐influenza susceptibility to Streptococcus pneumoniae co‐infection in mice. Lungs and nasal washes were collected 3 days after PR8 single infection, 1 day after 19F single infection, 1 day after 19F co‐infection (4 days after PR8 infection). (a,b) Viral loads in lungs were determined by PR8 M1 gene copy detected with quantitative polymerase chain reaction (PCR) (n = 3/group). (c–f) Lung homogenates and nasal washes were inoculated on blood agar plates, and bacterial loads were determined by colony‐forming units (CFU) analysis (n = 4–5/group). The labeled CFU/lung or CFU/nasal wash indicates the total number of CFU in each lung homogenate or nasal wash sample. *p < 0.05, **p < 0.01 based on Mann–Whitney U‐test; NS = not significant

FIGURE 3

Interleukin (IL)‐4 deficiency aggravates lung inflammatory damage of mice in influenza/Staphylococcus pneumoniae co‐infection. Lungs and bronchoalveolar lavage fluid (BALF) were collected 3 days after PR8 single infection, 1 day after 19F single infection and 1 and 3 days after 19F co‐infection (4 and 6 days after PR8 infection). (a) Lung sections were stained with hematoxylin and eosin (H&E) for histopathology assay. (b) The lactate dehydrogenase (LDH) activity was tested by LDH assay kits (n = 4–5/group). (c) The total protein levels in BALF were tested by bicinchoninic acid (BCA) protein assay kits (n = 4–5/group). (d) The wet/dry ratios of lungs were determined to evaluate lung oedema of mice in different infection scenarios (n = 4–5/group). (e–h) Levels of proinflammatory cytokines IL‐6, tumour necrosis factor (TNF)‐α, IL‐1β and interferon (IFN)‐γ in lungs were detected by enzyme‐linked immunosorbent assay (ELISA) kits (n = 4–5/group). **p < 0.05, **p < 0.01, ***p < 0.01, based on unpaired Student’s t‐test; NS = not significant

FIGURE 4

Interleukin (IL)‐4 deficiency facilitates macrophage recruitment into lungs of mice in influenza/Staphylococcus pneumoniae co‐infection. Bronchoalveolar lavage fluid (BALF) was collected 3 days after PR8 single infection and 1 day after 19F co‐infection (4 days after PR8 infection). BALF cells were counted, purified and identified by flow cytometry. Neutrophils were determined by gating on CD11b⁺ lymphocyte antigen 6 complex locus G6D (Ly6G⁺) cells, whereas macrophages were determined by gating on CD11b⁺ F4/80⁺ cells. (a,b) Percentages of neutrophils and macrophages. (c–e) Absolute numbers of total cells, macrophages and neutrophils. The absolute number of macrophages and neutrophils was obtained by multiplying the number of cells by the corresponding ratio (n = 5–7/group). **p < 0.01 based on Mann–Whitney U‐test. (f–h) Levels of chemokines chemokine (C‐X‐C motif) ligand 1 (CCL2), CXCL1 and CXCL10 in lungs were measured by ELISA kits (n = 5/group). **p < 0.01 based on unpaired Student’s t‐test; NS = not significant

FIGURE 5

Interleukin (IL)‐4 deficiency increases immune cell death in lungs of mice with influenza/Staphylococcus pneumoniae co‐infection. Bronchoalveolar lavage fluid (BALF) was collected 3 days after PR8 single infection, 1 day after 19F single infection and 1 day after 19F co‐infection (4 days after PR8 infection). (a) The representative images of BALF cell apoptosis analyzed by flow cytometry with the staining of annexin V and propidium iodide (PI). (b,c) Statistical analysis of the percentage of cells positive for annexin V but not PI and cells positive for both annexin V and PI (n = 3/group). **p < 0.01 based on Mann–Whitney U‐test; NS = not significant

FIGURE 6

Recombinant human interleukin (rhIL)‐4 and pyroptosis inhibition by disulfiram decrease immune cell death and post‐influenza susceptibility to Staphylococcus pneumoniae co‐infection. Co‐infected wild‐type (WT) and IL‐4^−/− mice were injected intraperitoneally (i.p) with rIL‐4 (2 μg/mouse) or disulfiram (0.2 mg/mouse). Lungs and bronchoalveolar lavage fluid (BALF) were collected 1 day after 19F co‐infection (4 days after PR8 infection). (a) The expressions of gasdermin D (GSDMD) and IL‐1β in BALF cells were detected by Western blotting. (b) The representative images of BALF cell apoptosis analyzed by flow cytometry with the staining of annexin V and propidium iodide (PI). (c,d) The statistical analysis of percentage of cells positive for annexin V but not PI and cells positive for both annexin V and PI (n = 3/group). (e) The total protein levels in BALF were tested by bicinchoninic acid (BCA) protein assay kits (n = 4–5/group). (f) Lung homogenates inoculated on blood agar plates, and bacterial loads were determined by colony‐forming units (CFU) analysis (n = 4–5/group). (g,h) Levels of proinflammatory cytokines IL‐6, tumor necrosis factor (TNF)‐α and IL‐1β in lungs were detected by enzyme‐linked immunosorbent assay (ELISA) kits (n = 4–5/group). *p < 0.05, **p < 0.01, ***p < 0.001 above all based on Tukey’s multiple comparisons; NS = not significant