Exogenous Interleukin-33 Contributes to Protective Immunity via Cytotoxic T-Cell Priming against Mucosal Influenza Viral Infection

Abstract

Influenza is an infectious respiratory illness caused by the influenza virus. Though vaccines against influenza exist, they have limited efficacy. To additionally develop effective treatments, there is a need to study the mechanisms of host defenses from influenza viral infections. To date, the mechanism by which interleukin (IL)-33 modulates the antiviral immune response post-influenza infection is unclear. In this study, we demonstrate that exogenous IL-33 enhanced antiviral protection against influenza virus infection. Exogenous IL-33 induced the recruitment of dendritic cells, increased the secretion of pro-inflammatory cytokine IL-12, and promoted cytotoxic T-cell responses in the local microenvironment. Thus, our findings suggest a role of exogenous IL-33 in the antiviral immune response against influenza infection.

Keywords: IL-33; antiviral immunity; influenza virus.

Figure 1

Exogenous interleukin (IL)-33, but not endogenous IL-33, improved the survival of mice infected with PR8 influenza virus. (A) C57BL/6 mice were injected intranasally with 0.5 μg of rIL-33 or phosphate-buffered saline (PBS) daily for five days and infected with 50 PFU of PR8 influenza virus. (B) Survival was monitored for 20 days post-infection. (C) At the indicated days post-infection, PR8 viral titers in BAL fluids were measured on Madin–Darby canine kidney (MDCK) cells. (D,E) IL-33^+/− and IL-33^−/− mice were intranasally infected with 50 PFU of PR8 influenza virus, and (D) survival was monitored for 10 days post-infection. (E) At eight days post-infection, PR8 viral titers in BAL fluids were measured in MDCK cells. * p < 0.05; ** p < 0.01; ns, not significant. BAL: Bronchoalveolar lavage.

Figure 2

Exogenous IL-33 affected immune cells in the lung. (A) C57BL/6 mice were injected intranasally with 0.5 μg rIL-33 or PBS daily for five days, and lungs were collected for analyses of immune cell populations. (B) The numbers of cells isolated from the lungs were counted. The frequency and number of lineage-specific (C) Sca-1⁺ ST2⁺ type 2 innate lymphoid cells (ILC2s), (D) CD11c⁺ MHCII⁺ dendritic cell (DCs), (E) Siglec-F⁺ CD11c^hi CD11b^int alveolar macrophages, and Siglec-F⁺ CD11c^int CD11b^hi eosinophils were analyzed by flow cytometry. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

Figure 3

Exogenous IL-33 enhanced IL-12p40 secretion in BAL fluids, bone marrow-derived dendritic cell (BMDC) culture media, and BMDC maturation. (A) C57BL/6 mice were injected intranasally with 0.5 μg of rIL-33 or PBS daily for five days and then infected with 50 PFU of PR8 influenza virus. At the indicated days post-infection, BAL fluids were collected, and IL12p40 levels were measured by an enzyme-linked immunosorbent assay (ELISA). (B) BMDCs were stimulated with the indicated concentrations of rIL-33. At 24 h post-stimulation, IL-12p40 levels in culture media were measured by ELISA. (C) BMDCs were infected with 1, 10, and 50 multiplicity of infection (MOI) PR8 virus with 10 ng/mL of rIL-33 or PBS. At 24 h post-infection, IL-12p40 levels in culture media were measured by ELISA. (D) BMDCs were stimulated with 10 ng/ml of rIL-33, and the expression of CD86 and MHCII in BMDCs was assessed by flow cytometry at 24 h post-stimulation. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 4

Exogenous IL-33, but not endogenous IL-33, enhanced CD8 T-cell responses against influenza infection. (A–C) C57BL/6 mice were injected intranasally with 0.5 μg of rIL-33 or PBS daily for five days and infected with 50 PFU of PR8 influenza virus. (A) On day seven post-infection, the recruitment of CD3ε⁺ CD8⁺ T-cells to lungs was assessed by flow cytometry. (B) IFN-γ production by CD3ε⁺ CD8⁺ CD44^hi T-cells post-stimulation with NP_366–374 peptide was measured by intracellular staining. (C) NP_366-374 antigen-specific CD3ε⁺ CD8⁺ CD44⁺ T-cells were measured by pentamer staining. (D) IL-33^+/− and IL-33^−/− mice were infected with 50 PFU of PR8 influenza virus. On day seven post-infection, IFN-γ production by CD3ε⁺ CD8⁺ CD44^hi T-cells post-stimulation with NP_366-374 peptide was measured by intracellular staining. ** p < 0.01; ns, not significant.