Memory Th17 cell-mediated protection against lethal secondary pneumococcal pneumonia following influenza infection

Abstract

Streptococcus pneumoniae (Sp) frequently causes secondary pneumonia after influenza A virus (IAV) infection, leading to high morbidity and mortality worldwide. Concomitant pneumococcal and influenza vaccination improves protection against coinfection but does not always yield complete protection. Impaired innate and adaptive immune responses have been associated with attenuated bacterial clearance in influenza virus-infected hosts. In this study, we showed that preceding low-dose IAV infection caused persistent Sp infection and suppression of bacteria-specific T-helper type 17 (Th17) responses in mice. Prior Sp infection protected against subsequent IAV/Sp coinfection by improving bacterial clearance and rescuing bacteria-specific Th17 responses in the lungs. Furthermore, blockade of IL-17A by anti-IL-17A antibodies abrogated the protective effect of Sp preinfection. Importantly, memory Th17 responses induced by Sp preinfection overcame viral-driven Th17 inhibition and provided cross-protection against different Sp serotypes following coinfection with IAV. These results indicate that bacteria-specific Th17 memory cells play a key role in providing protection against IAV/Sp coinfection in a serotype-independent manner and suggest that a Th17-based vaccine would have excellent potential to mitigate disease caused by coinfection. IMPORTANCE Streptococcus pneumoniae (Sp) frequently causes secondary bacterial pneumonia after influenza A virus (IAV) infection, leading to increased morbidity and mortality worldwide. Current pneumococcal vaccines induce highly strain-specific antibody responses and provide limited protection against IAV/Sp coinfection. Th17 responses are broadly protective against Sp single infection, but whether the Th17 response, which is dramatically impaired by IAV infection in naïve mice, might be effective in immunization-induced protection against pneumonia caused by coinfection is not known. In this study, we have revealed that Sp-specific memory Th17 cells rescue IAV-driven inhibition and provide cross-protection against subsequent lethal coinfection with IAV and different Sp serotypes. These results indicate that a Th17-based vaccine would have excellent potential to mitigate disease caused by IAV/Sp coinfection.

Keywords: Streptococcus pneumoniae; Th17 responses; coinfection; cross-protection; influenza A virus.

Fig 1

Low-dose flu infection increases susceptibility and mortality during secondary bacterial infection. Mice were infected with a sublethal dose of PR8 (50 TCID₅₀), and 5 days later infected with a sublethal dose of T4 (10⁶ CFU/mouse). Heart rate (A), breath rate (B), arterial oxygen saturation (SpO₂, C), body weight loss (D), and survival rate (E, >30% body weight loss as an endpoint) were measured and recorded on different days after PR8 (PR8, n = 12), T4 single infection (T4, n = 13), or PR8 and T4 coinfection (PR8/T4, n = 12). Bacterial (F) and viral (G) loads in lung homogenate on the indicated days after PR8/T4 infection. dpi=day post-infection. Data are mean ± s.e.m. from 5 to 13 mice in each group. ****P < 0.0001; ***P < 0.001; ^NSP > 0.05.

Fig 2

Bacteria-specific T-helper type 17 (Th17) responses are inhibited in the lung during virus and bacterial coinfection. Cytokines interleukin-17A (IL-17A, A) and interferon-γ (IFN-γ, B) in bronchoalveolar lavage fluid (BALF) on days 2 and 6 after T4 infection in T4 and PR8/T4 infected mice. IL-17A (C, D) and IFN-γ (E, F) production by CD4⁺T cells after stimulation with heat-killed T4 as (C, E) visualized by FACS and (D, F) calculated as the number of IL-17A⁺CD44⁺ and IFN-γ⁺CD44⁺ producing CD4⁺T cells per lung on 6 days after T4 infection. Data are mean ± s.e.m. from 3 to 13 mice in each group. ****P < 0.0001; **P < 0.01; *P < 0.05; ^NSP > 0.05. FACS, fluorescence-activated cell sorting.

Fig 3

Prior Sp infection protects against virus and bacterial coinfection. Mice were inoculated with T4, and 21 days later, were first infected with PR8 and secondarily infected with T4 (A). Body weight loss (B), survival rates (C, >30% body weight loss as an endpoint), arterial oxygen saturation (SpO₂, D), heart rate (E), and breath rate (F) on different days after PR8/T4 infection in PBS (n = 12) or T4 preinfected mice (n = 14). Bacterial (G, in lung and blood) and viral loads (H) in lung homogenates of PBS or T4 preinfected mice on the indicated days after PR8/T4 infection. Data are mean ± s.e.m. from 5 to 10 mice in each group. ****P < 0.0001; **P < 0.01; *P < 0.05; ^NSP > 0.05. PBS, phosphate-buffered saline.

Fig 4

Prior Sp infection increases Sp-specific Th17 responses in virus and bacterial coinfection. IL-17A (A, B) and IFN-γ (C, D) production by CD4⁺T cells after stimulation with heat-killed T4 as (A, C) visualized by FACS and (B, D) calculated as the number of IL-17A⁺CD44⁺ and IFN-γ⁺CD44⁺ producing CD4⁺T cells per lung on the indicated days of PR8/T4 infection in PBS or T4 preinfected mice. Data are mean ± s.e.m. from 5 to 11 mice in each group. **P < 0.01; *P < 0.05; ^NSP > 0.05. FACS, fluorescence-activated cell sorting; PBS, phosphate-buffered saline.

Fig 5

Protection from coinfection induced by prior Sp infection is dependent on IL-17A. At 21 days postinoculation with T4, B6 mice were first infected with PR8 and secondarily infected with T4 (A). Preinfected mice also treated with IL-17-neutralizing antibody (anti-IL-17, clone 17F3) or isotype control antibody (IgG) intraperitoneally on days −1, 0, and 1 and intranasally on day 0 during T4 challenge following PR8 infection. Virus loads (B) and bacterial loads (C) in lung homogenate of naïve or T4 preinfected mice treated with IL-17-neutralizing antibody (anti-IL-17) or isotype control antibody on 7/2 days after PR8/T4 infection. Data are mean±s.e.m. from 7 to 14 mice in each group. ****P < 0.0001; **P < 0.01; *P < 0.05; ^NSP > 0.05.

Fig 6

Prior Sp infection induces a cross-specific Th17 response and provides heterologous protection against coinfection. At 21 days postexposure with P1121, mice were first infected with PR8 and secondarily infected with T4 (A). Body weight loss (B) and survival rates (C, >30% body weight loss as an endpoint) on the indicated days after PR8/T4 infection in PBS (n = 10) or T4 preinfected mice (n = 10). Bacterial loads (D) in lung homogenate and blood of PBS or T4 preinfected mice on 7/2 days after PR8/T4 infection. IL-17A production by CD4⁺ T cells after stimulation with heat-killed T4 as visualized by FACS (E) and (F) calculated as the number of IL-17A⁺CD44⁺ and IFN-γ⁺CD44⁺ producing CD4⁺ T cells per lung on the indicated days after PR8/T4 infection in PBS or T4 preinfected mice. Data are mean±s.e.m. from 4 to 10 mice in each group. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ^NSP > 0.05. FACS, fluorescence-activated cell sorting; PBS, phosphate-buffered saline.