Staphylococcus aureus induces type I IFN signaling in dendritic cells via TLR9

Abstract

The importance of type I IFN signaling in the innate immune response to viral and intracellular pathogens is well established, with an increasing literature implicating extracellular bacterial pathogens, including Staphylococcus aureus, in this signaling pathway. Airway epithelial cells and especially dendritic cells (DCs) contribute to the production of type I IFNs in the lung. We were interested in establishing how S. aureus activates the type I IFN cascade in DCs. In vitro studies confirmed the rapid uptake of S. aureus by DCs followed promptly by STAT1 phosphorylation and expression of IFN-β. Signaling occurred using heat-killed organisms and in the absence of PVL and α-toxin. Consistent with the participation of endosomal and not cytosolic receptors, signaling was predominantly mediated by MyD88, TLR9, and IRF1 and blocked by cytochalasin D, dynasore, and chloroquine. To determine the role of TLR9 signaling in the pathogenesis of S. aureus pneumonia, we infected WT and Tlr9(-/-) mice with MRSA USA300. Tlr9(-/-) mice had significantly improved clearance of S. aureus from the airways and lung tissue. Ifnar(-/-) mice also had improved clearance. This enhanced clearance in Tlr9(-/-) mice was not due to differences in the numbers of recruited neutrophils into the airways, but instead correlated with decreased induction of TNF. Thus, we identified TLR9 as the critical receptor mediating the induction of type I IFN signaling in DCs in response to S. aureus, illustrating an additional mechanism through which S. aureus exploits innate immune signaling to facilitate infection.

Figure 1

Activation of type I interferons by S. aureus in BMDC. A) S. aureus USA300 was incubated with BMDC from WT and IFNAR null mice and levels of gene expression assessed by qRT-PCR. B) Phosphorylation of STAT1 detected using immunoblots from WT BMDC stimulated with S. aureus for 2 h. β-actin was used as a loading control. C) WT BMDC were incubated with WT and mutants strains of S. aureus USA300 and levels of Ifnb assessed by qRT-PCR. D) Assessment of Ifnb induction of live versus heat killed S. aureus USA300 using WT BMDC. Results are representative of two independent experiments. Graphs display means and standard deviation. *P<0.05 (n=3, students t test). UN-unstimulated, Sa-S. aureus.

Figure 2

Ifnar^−/− mice have improved clearance of S. aureus from the lung. Mice were inoculated intranasally with S. aureus USA300 for 20 h before sacrifice. A) Bacteria were enumerated from lung homogenate. B) CD4⁺ cell counts were determined using FACS analysis from BAL samples. FACS data is representative of two independent experiments. Each point represents a mouse. Lines display median values. *P<0.05.

Figure 3

Uptake of S. aureus is required for type I interferon production. WT BMDC were stimulated with S. aureus USA300 in the presence of A) cytochalasin D (cytoD), B) dynasore or C) chloroquine compared to a DMSO control (n=6). D) FITC-labelled S. aureus USA300 (green) visualized inside WT BMDC, cell nuclei are stained with DAPI (blue). Graphs display means and standard deviation and are representative of two independent experiments. *P<0.05.

Figure 4

TLR9 is the receptor for S. aureus DNA induced type I interferon signaling. A) BMDC from WT and knockout mice were cultured with S. aureus for 2 h and levels of Ifnb were detected using qRT-PCR. Data are from two independent experiments (n=6). B) Phosphorylation of IRF3 and 7 was detected using immunoblots from WT BMDC stimulated with S. aureus USA300 for 2 h. β-actin was used as a loading control. BMDC were incubated with S. aureus USA300 for 20 h and supernatants collected for C) IFN-β and D) TNF ELISA. E) BMDC were incubated with S. aureus lysates treated with DNase and RNase and levels of Ifnb quantitated with qRT-PCR. Shown is a representative of two independent experiments (n=3). F) BMDC from WT and Tlr9^−/− mice were stimulated with S. aureus and transcript levels of proinflammatory cytokines measured by qRT-PCR. Shown is a representative of two independent experiments (n=3). *P<0.05. UN-unstimulated.

Figure 5

TLR9 knockout mice have improved clearance of S. aureus from the airways. Mice were inoculated intranasally with S. aureus USA300 and the response to infection assessed 24 h later. Bacteria were enumerated from A) BALF and B) lung homogenate. C) Cell populations were determined using FACS analysis from BAL samples. D) WT and Tlr9^−/− mice were infected with 1-2×10⁸ cfu and assessed for mortality 20 h later (n=8-WT, n=9-Tlr9^−/−). Each point represents a mouse. Lines display median values. *P<0.05.

Figure 6

Cytokine responses of WT and Tlr9^−/− mice to S. aureus. Mice were infected intranasally with S. aureus USA300 for 24 h before BAL was performed. Cytokine levels in BALF were determined using ELISA. Each point represents a mouse. Lines display median values. *P<0.05