MicroRNA-155 regulates host immune response to postviral bacterial pneumonia via IL-23/IL-17 pathway

Abstract

Postinfluenza bacterial pneumonia is associated with significant mortality and morbidity. MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression posttranscriptionally. miR-155 has recently emerged as a crucial regulator of innate immunity and inflammatory responses and is induced in macrophages during infection. We hypothesized upregulation of miR-155 inhibits IL-17 and increases susceptibility to secondary bacterial pneumonia. Mice were challenged with 100 plaque-forming units H1N1 intranasally and were infected with 10(7) colony-forming units of MRSA intratracheally at day 5 postviral challenge. Lungs were harvested 24 h later, and expression of miR-155, IL-17, and IL-23 was measured by real-time RT-PCR. Induction of miR-155 was 3.6-fold higher in dual-infected lungs compared with single infection. miR-155(-/-) mice were protected with significantly lower (4-fold) bacterial burden and no differences in viral load, associated with robust induction of IL-23 and IL-17 (2.2- and 4.8-fold, respectively) postsequential challenge with virus and bacteria, compared with WT mice. Treatment with miR-155 antagomir improved lung bacterial clearance by 4.2-fold compared with control antagomir postsequential infection with virus and bacteria. Moreover, lung macrophages collected from patients with postviral bacterial pneumonia also had upregulation of miR-155 expression compared with healthy controls, consistent with observations in our murine model. This is the first demonstration that cellular miRNAs regulate postinfluenza immune response to subsequent bacterial challenge by suppressing the IL-17 pathway in the lung. Our findings suggest that antagonizing certain microRNA might serve as a potential therapeutic strategy against secondary bacterial infection.

Keywords: IL-17; miR-155; postviral bacterial pneumonia.

Fig. 1.

Mice with viral challenge before bacterial infection have higher bacterial burden in the lung. A: wild-type (WT) mice were challenged with 10⁷ colony-forming units (CFU) methicillin-resistant Staphylococcus aureus (MRSA) either alone or 5 days after infection with 100 PFU H1N1, lungs were harvested at 24 h, and CFU were evaluated; n = 6 mice in each group and experiments were repeated 3 times. *P < 0.05, compared with MRSA infection alone. B: mice were challenged intranasally with 100 PFU H1N1 alone or intratracheally with 10⁷ CFU MRSA alone or at day 5 post-H1N1 infection mice were challenged with 10⁷ CFU MRSA. Survival was assessed for each condition; n = 8 mice in each group and experiments were repeated 3 times. *P < 0.01, compared with the double infection group as measured by log rank test.

Fig. 2.

Dual-infected animals have higher IFNγ in lung compared with either challenge alone. IFNγ^−/− mice and mice treated with anti-IFNγ antibody have lower lung bacterial burden compared with WT mice postsequential infection. Mice were infected with 100 PFU H1N1 or 10⁷ CFU MRSA alone or sequentially, and IFN γ levels in lung digests were measured by real-time RT-PCR (A) ELISA (B); n = 5 mice in each group, experiments repeated twice. *P < 0.05, **P < 0.01, compared with untreated controls. C: WT mice and IFNγ^−/− were infected with 100 PFU H1N1 intranasally and on day 5 challenged with 10⁷ CFU MRSA, lungs were harvested, and bacterial burden was quantitated. WT mice were also treated with 80 μl anti-IFN γ Ab intraperitoneally or control antibody intraperitoneally 6 h before challenge with intratracheally 10⁷ CFU MRSA and on day 5 post-H1N1 intranasally infection, lungs were harvested 24 h postbacterial challenge, and CFU was quantitated; n = 5 mice in each group and experiments were repeated 2 times. **P < 0.01, as compared mice infected WT mice. D: WT mice were also treated with 80 μl anti-IFNγ Ab intraperitoneally or control antibody intraperitoneally 6 h before challenge with intratracheal 10⁷ CFU MRSA and on day 5 post-H1N1 intranasal infection and survival was assessed.

Fig. 3.

Macrophages harvested from mice postdual infection have higher expression of miR-155 RNA compared with viral or bacterial infection alone, which is attenuated by anti-IFNγ Ab treatment. A: WT mice were challenged with 10⁷ CFU MRSA either alone or 5 days after infection with 100 PFU H1N1, lungs were harvested at 24 h, and expression of miR-155 was measured by RT-PCR. ***P < 0.001. B: lung macrophages were harvested 24 h postbacterial challenge by collagenase digest and adherence purification and miR-155 was expression measured by real-time RT-PCR. C: WT mice were infected intranasally with 100 PFU H1N1 and on day 5 mice were inoculated with either control antibody or 80 μl anti-IFNγ antibody intraperitoneally and 6 h later mice were challenged intratracheally with 10⁷ CFU MRSA. Lung macrophages were harvested and expression of miR-155 measured by real-time PCR. *P < 0.05, **P < 0.01; n = 4 in each group, and experiment was repeated 3 times.

Fig. 4.

Decreased expression of IL-23 and IL-17 in lungs postsequential infection. WT mice were challenged with 10⁷ CFU MRSA either alone or 5 days after infection with 100 PFU H1N1, lungs were harvested at 24 h, and IL-17 (A) and IL-23 (B) gene expression was measured by RT-PCR. *P < 0.05; n = 4 in each group, and experiments were repeated 3 times.

Fig. 5.

MiR-155^−/− mice have decreased bacterial burden in the lung and increased expression of IL-23 and IL-17 compared with WT mice. A: WT and miR-155^−/− mice were challenged intranasally with 100 PFU H1N1 and on day 5 post-H1N1 infection challenged with 10⁷ CFU MRSA and survival was assessed. WT and miR-155^−/− mice were challenged intranasally with 100 PFU H1N1 and on day 5 post-H1N1 infection challenged with 10⁷ CFU MRSA. Lungs were harvested at 24 h and viral gene expression was analyzed by RT-PCR (B) and lung bacterial CFU were quantified (C). Lungs were harvested 24 h postbacterial challenge and expression of IL- 23 (D) and IL- 17 (E) levels and measured by real-time PCR. Lung macrophages 24 h postbacterial challenge from H1N1/MRSA-infected mice or uninfected control mice were harvested by collagenase digest and adherence purification and IL-23p19 and expression was measured by real-time PCR (F). *P < 0.05, **P < 0.01; n = 4 in each group, and experiments were repeated 3 times.

Fig. 6.

miR-155^−/− mice treated with anti-IL-17 antibody before bacterial challenge have higher bacterial burden in the lung. A: WT and miR-155^−/− mice were challenged intranasally with 100 PFU H1N1 and on day 5 post-H1N1 infection challenged with 10⁷ CFU MRSA. Four hours before bacterial challenge miR-155^−/− mice were treated with either a control antibody or 100 μg of anti-IL-17 antibody intraperitoneally. Lungs were harvested at 24 h and CFU were quantified. B: WT mice were infected with H1N1 and 4 h before challenge with MRSA given vehicle control or rmIL-23 intratracheally Lungs were harvested at 24 h and CFU quantified. *P < 0.05; n = 5 in each group, and experiments were repeated 2 times.

Fig. 7.

Treatment with miR-155 antagomir decreased bacterial burden in the lung and upregulates expression of IL-23 and IL-17. WT mice were challenged intranasally with 100 PFU H1N1 and on day 5 post-H1N1 infection challenged with 10⁷ CFU MRSA and treated with sham antagomir or miR-155 antagomir intranasally. Lungs were harvested at 24 h and CFU were quantified (A). Cytokine expression of IL-23 and IL-17 were measured in lungs by real-time RT-PCR (B and C). *P < 0.05, **P < 0.01; n = 5 in each group, and experiments were repeated 2 times.

Fig. 8.

Human alveolar macrophages purified from patients with H1N1 and bacterial coinfection had increased expression of miR-155. Alveolar macrophages were purified by adherence purification from bronchoalveolar lavage (BAL) obtained from healthy subjects who underwent bronchoscopy for research purposes or patients admitted to our intensive care unit with respiratory failure from secondary bacterial pneumonia post H1N1, and expression of miR-155 was measured by real-time PCR. *P < 0.05; n = 12 patients and n = 3 controls.