Streptococcus pneumoniae DNA initiates type I interferon signaling in the respiratory tract

Abstract

The mucosal epithelium is the initial target for respiratory pathogens of all types. While type I interferon (IFN) signaling is traditionally associated with antiviral immunity, we demonstrate that the extracellular bacterial pathogen Streptococcus pneumoniae activates the type I IFN cascade in airway epithelial and dendritic cells. This response is dependent upon the pore-forming toxin pneumolysin. Pneumococcal DNA activates IFN-β expression through a DAI/STING/TBK1/IRF3 cascade. Tlr4(-/-), Myd88(-/-), Trif(-/-), and Nod2(-/-) mutant mice had no impairment of type I IFN signaling. Induction of type I IFN signaling contributes to the eradication of pneumococcal carriage, as IFN-α/β receptor null mice had significantly increased nasal colonization with S. pneumoniae compared with that of wild-type mice. These studies suggest that the type I IFN cascade is a central component of the mucosal response to airway bacterial pathogens and is responsive to bacterial pathogen-associated molecular patterns that are capable of accessing intracellular receptors.

Importance: The bacterium Streptococcus pneumoniae is a leading cause of bacterial pneumonia, leading to upwards of one million deaths a year worldwide and significant economic burden. Although it is known that antibody is critical for efficient phagocytosis, it is not known how this pathogen is sensed by the mucosal epithelium. We demonstrate that this extracellular pathogen activates mucosal signaling typically activated by viral pathogens via the pneumolysin pore to activate intracellular receptors and the type I interferon (IFN) cascade. Mice lacking the receptor to type I IFNs have a reduced ability to clear S. pneumoniae, suggesting that the type I IFN cascade is central to the mucosal clearance of this important pathogen.

FIG 1

S. pneumoniae activates the type I IFN response in vivo. Mice were infected with 2 × 10⁷ CFU of S. pneumoniae and analyzed 24 h later. (A) qRT-PCR of type I IFN and inflammatory cytokine genes from RNA extracted from infected mouse lungs. Graphs display means with standard deviations (n = 3). (B) Immunoblots of transcription factors P-STAT1 and P-STAT3 from mouse lungs. β-Actin was used as a loading control. Each lane represents an individual mouse. Data are representative of two experiments. LPS from E. coli (50 µg per mouse) was used as a positive control.

FIG 2

Pneumolysin is involved in the induction of type I IFN. C57BL/6J mice were infected with 2 × 10⁷ CFU of S. pneumoniae (S. p.) intranasally and studied at 4 h (A to D). (A) WT-infected lung data are shown in black, and ply mutant-infected lung data are shaded grey. *, P < 0.05 (Student’s t test; n = 8). Lung homogenates were analyzed for bacterial counts (B), neutrophils (percentage of Ly6G Ly6C⁺ CD45⁺ cells) (C), and CD11c⁺ cells (D) by FACS. Lines represent median values. *, P < 0.05 (WT versus ply mutant; Mann-Whitney test; n = 8, except for the PBS controls). (E) Murine nasal epithelial cells in primary culture were stimulated for the times indicated with WT or ply mutant S. pneumoniae D39 in the presence or absence of cytochalasin D (CytoD) (F). DMSO, dimethyl sulfoxide. *, P < 0.05 (Student’s t test; n = 3). (G) Mice were intranasally administered S. pneumoniae strains (P1121 background) expressing WT pneumolysin, no pneumolysin, or a nonhemolytic toxoid variant and examined 4 h later. Lung homogenates were immunoblotted for P-STAT1. Each lane represents an individual mouse. (H) AF488-labeled pneumolysin is visualized in murine nasal epithelial cells at 1 h poststimulation. Murine epithelial cells in primary culture following stimulation with S. pneumoniae D39 or purified pneumolysin (Ply) (I) or with ply lysates with and without purified pneumolysin or PdB (J). *, P < 0.05 compared to unstimulated cells (Student’s t test; n = 3). RNA was extracted and analyzed for levels of IFN-β by qRT-PCR. All graphs display means with standard deviations.

FIG 3

Induction of IFN-β production by S. pneumoniae in mice with mutations in TLR4, Nod2, and innate adapters. WT and Tlr4^−/−, Trif^−/−, MyD88^−/−, and Nod2^−/− mutant C57BL/6J mice were infected intranasally with 10⁷ CFU of S. pneumoniae and examined 24 h later. (A) Lung homogenates were assayed for STAT1 phosphorylation. Each lane represents an individual mouse. Shown are representative experiments. (B) Lung tissue was analyzed for induction of IFN-β by qRT-PCR (n = 4, except for Nod2^−/− mutant mice [n = 6]).

FIG 4

S. pneumoniae DNA activates type I IFN production. (A) Murine nasal epithelial cells were stimulated for 4 h with pneumococcal lysate that had been left untreated or treated with DNase. (B) Murine nasal epithelial cells were stimulated for 4 h with the WT or lytA mutant strain (n = 3). IFN-β induction was assessed by qRT-PCR and is displayed as fold induction over that in PBS-only controls. (C) Murine nasal epithelial cells were stimulated for 4 h with the WT and ply mutant strains (n = 3). DNA was extracted from the epithelial cells, and levels of S. pneumoniae 16S rRNA were measured by qRT-PCR. Un, untreated lysate. *, P < 0.05 compared to WT or untreated samples (Student’s t test). All results shown are representative of at least two independent experiments.

FIG 5

Pneumococcal DNA is sensed by the cytosolic DAI sensor. (A) Mice were infected with 10⁷ CFU of S. pneumoniae D39 (S.p), and lung tissue was analyzed for induction of DAI 24 h later (n = 4). (B) Bone marrow-derived cells were stimulated with enzymatically treated S. pneumoniae lysates and then analyzed for Ifnb production by qRT-PCR (n = 3). (C) Bone marrow-derived cells from DAI^−/− (n = 6) and STING^−/− (n = 6) mice were transfected with pneumococcal DNA, and Ifnb induction was analyzed by qRT-PCR. Ifnb induction of pneumococcal DNA-stimulated knockout bone marrow-derived cells and their respective controls downstream of DAI (D and E) and other type I signaling components (F to H) (n = 3). Bone marrow-derived DCs and macrophages from DAI^−/− (I) and STING^−/− (J) mice (n = 6) and TBK1^−/− (K) and IRF3^−/− (L) mice (n = 3) were stimulated with S. pneumoniae for 2 h, and Ifnb levels were quantitated by qRT-PCR. Graphs display means plus standard deviations and are representative of two experiments (excluding STING data), and data are expressed as fold induction compared to that of the control cell line stimulated with PBS alone. *, P < 0.05 compared to unstimulated or WT samples (Student’s t test).

FIG 6

Type I IFN production contributes to pneumococcal clearance. WT and Ifnar^−/− mutant mice (129/SvEV background for both) were infected with 10⁷ CFU of S. pneumoniae intranasally and examined 7 days later. Shown are bacterial counts recovered from nasal lavage fluid samples (A) and percentages of monocytic cells (percentage of CD11b⁺ CD45⁺ cells) (B), DCs (percentage of CD11b⁺ CD11c⁺ CD45⁺ cells) (C), and neutrophils (percentage of Ly6G Ly6C⁺ CD45⁺ cells) (D) from lung homogenates analyzed by FACS. Each data point represents an individual mouse (n = 12). Data are combined from two independent experiments. Lines represent median values. *, P < 0.05 compared to WT mice (Mann-Whitney test).