Synergistic stimulation of type I interferons during influenza virus coinfection promotes Streptococcus pneumoniae colonization in mice

Abstract

Pneumococcal infection of the respiratory tract is often secondary to recent influenza virus infection and accounts for much of the morbidity and mortality during seasonal and pandemic influenza. Here, we show that coinfection of the upper respiratory tract of mice with influenza virus and pneumococcus leads to synergistic stimulation of type I IFNs and that this impairs the recruitment of macrophages, which are required for pneumococcal clearance, due to decreased production of the chemokine CCL2. Type I IFN expression was induced by pneumococcal colonization alone. Colonization followed by influenza coinfection led to a synergistic type I IFN response, resulting in increased density of colonizing bacteria and susceptibility to invasive infection. This enhanced type I IFN response inhibited production of the chemokine CCL2, which promotes the recruitment of macrophages and bacterial clearance. Stimulation of CCL2 by macrophages upon pneumococcal infection alone required the pattern recognition receptor Nod2 and expression of the pore-forming toxin pneumolysin. Indeed, the increased colonization associated with concurrent influenza virus infection was not observed in mice lacking Nod2 or the type I IFN receptor, or in mice challenged with pneumococci lacking pneumolysin. We therefore propose that the synergistic stimulation of type I IFN production during concurrent influenza virus and pneumococcal infection leads to increased bacterial colonization and suggest that this may contribute to the higher rates of disease associated with coinfection in humans.

Figure 1. Type I IFNs are expressed during pneumococcal carriage.

(A) URT lavages were analyzed for quantitative culture at the time points indicated, following intranasal inoculation of C57BL/6 WT mice with strain P1121. Dashed line represents the limit of detection. (B) URT lavages from colonized mice were analyzed by flow cytometry to quantify numbers of neutrophils and macrophages. (C) Levels of Ccl2 mRNA in URT tract lavages were determined using qRT-PCR at each time point and expressed relative to uncolonized mice. (D) Levels of type I IFN mRNA in URT lavages were determined using qRT-PCR at each time point and expressed relative to uncolonized mice. n = 10–15 mice per time point. Values represent mean ± SD.

Figure 2. Stimulation of type I IFNs increased pneumococcal nasopharyngeal colonization.

URT lavages from colonized WT and Ifnar^–/– mice were treated with poly-ICLC (6 μg i.n. daily for 7 days) and compared with PBS vehicle controls for (A) type I IFN mRNA expression in WT mice, (B) density of strain P1121 colonization, (C and D) cell counts in URT lavages analyzed by flow cytometry (F4/80⁺ macrophages and Ly6G⁺CD45⁺ neutrophils), and (E) chemokine KC and Ccl2 mRNA expression in WT mice. n = 9 mice per group. Values represent mean ± SD. Values are expressed relative to the PBS control group. *P < 0.05, **P < 0.01.

Figure 3. Coinfection with influenza virus induces synergistic IFN-β expression and enhances pneumococcal nasopharyngeal colonization.

(A) Experimental protocol for coinfection model. WT mice were given an intranasal inoculation with S. pneumoniae (P1121 strain, 10⁷ CFU) or PBS, followed a day later by an intranasal inoculation with influenza virus (PR8 strain, 1,000 TCID₅₀) or PBS. (B) Mice infected with PR8 strain were monitored for body weight (white squares, P1121+PR8; black diamonds, PR8 alone). (C) Titer of influenza virus in singly infected and coinfected mice was calculated based on viral RNA detected in URT lavages (black bars) and lung homogenates (white bars). (D) H&E staining of representative lung tissue sections from mice infected with S. pneumoniae alone and coinfected with influenza virus. Original magnification, ×200. Horizontal lines indicate mean values. (E) The density of pneumococcal colonization was measured in singly infected and coinfected mice at day 7. (F) Levels of IFN-β mRNA in URT lavages from singly infected and coinfected mice were examined by qRT-PCR. (G) Numbers of F4/80⁺CD45⁺ macrophages and Ly6G⁺CD45⁺ neutrophils recruited to the URT were analyzed by flow cytometry from uninfected, singly infected, and coinfected mice. (H) Levels of Ccl2 and KC mRNA in URT lavages from singly infected and coinfected mice as determined by qRT-PCR. (I) Survival rates of mice infected with serotype 6A pneumococcus alone (squares; n = 20) and serotype 6A pneumococcus coinfected with PR8 (diamonds; n = 20). Values represent mean ± SD. *P < 0.05, **P < 0.01.

Figure 4. The effect of coinfection of influenza strain PR8 on colonization by pneumococcal strain P1121 is dependent on type I IFNs.

(A) Density of pneumococcal colonization in URT lavages from singly infected and coinfected WT and Ifnar^–/– mice at day 7 after challenge with influenza virus. Horizontal lines indicate mean values. (B) Numbers of F4/80⁺ macrophages recruited to URT in coinfected WT and Ifnar^–/– mice. (C) Levels of Ccl2 mRNA expression in URT lavages in coinfected WT and Ifnar^–/– mice were determined by qRT-PCR. Values represent mean ± SD. *P < 0.05, **P < 0.01.

Figure 5. Macrophages and Nod2 contribute to production of type I IFN by S. pneumoniae.

(A) The levels of type I IFN mRNA in URT lavages of WT and Ccr2^–/– mice were analyzed 7 days after intranasal infection with strain P1121 by qRT-PCR. (B) The levels of type I IFN mRNA in URT lavages of WT (black bars) and Tlr2^–/– (white bars) mice were analyzed 3 days after intranasal infection with strain P1121. (C) The levels of IFN-β mRNA in URT lavages of WT (black bars) and Nod2^–/– (white bars) mice were analyzed 3 days after intranasal infection with strain P1121. Levels of IFN-β mRNA in peritoneal macrophages from WT and Nod2^–/– mice were examined 12 hours after stimulation with heat-killed strain P1121. Levels of (D) IFN-β and (E) Ccl2 mRNA in URT lavages of singly infected and coinfected Nod2^–/– mice determined by qRT-PCR. (F) Density of pneumococcal colonization in URT lavages of singly infected and coinfected Nod2^–/– mice day 7 after challenge with influenza virus PR8. Horizontal lines indicate mean values. (G) The levels of type I IFN mRNA in bone marrow–derived macrophages from WT, Irf3^–/–, Irf5^–/–, Irf3^–/–Irf5^–/– mice were examined 12 hours after stimulation with heat-killed strain P1121. Values represent mean ± SD. *P < 0.05, **P < 0.01.

Figure 6. IFN-β inhibits production of CCL2 in peritoneal macrophage stimulated with heat-killed S. pneumoniae.

(A) Effect of treatment of peritoneal macrophages with poly-ICLC or IFN-β prior to stimulation with heat-killed strain P1121 on CCL2 production in culture supernatants as assessed by ELISA. (B) Levels of Ccl2 mRNA expression in P1121-stimulated peritoneal macrophages with the pretreatment indicated as analyzed by qRT-PCR. (C) Luciferase assay comparing NF-κB activity in Nod2-expressing 293T cells treated with MDP with or without pretreatment with poly-ICLC or IFN-β. Values are relative to empty vector controls and expressed as mean ± SD. *P < 0.05, **P < 0.01.

Figure 7. Pore formation by Ply contributes to type I IFN expression.

(A) Levels of IFN-β mRNA determined by qRT-PCR in peritoneal macrophages following stimulation with heat-killed P1121 and isogenic mutants with a deletion of the entire ply (ply^–), with a W433F point mutant defective in pore formation (ply_W433F), or with ply restored in the deletion mutant (ply⁺). (B) The levels of IFN-β mRNA in URT lavages obtained from the mice coinfected with PR8 and the pneumococcal strain indicated were determined by qRT-PCR. (C) Comparison of the colonization density in URT lavages of WT mice singly infected or coinfected with the pneumolysin mutant indicated and PR8 at day 7 after influenza virus infection. Values represent mean ± SD. Horizontal lines indicate mean values. *P < 0.05.