In this report, we demonstrate the participation of airway epithelial cells in the initial recognition of an important bacterial pathogen, S. pneumoniae, in the respiratory tract. This immune response is mediated by activation of the epithelial type I IFN cascade by the intracellular accumulation of pneumococcal DNA. As this pathway is readily activated by viral nucleic acids that accumulate in airway epithelial cells, it is not unexpected that bacterial nucleic acids could have a similar effect. The upper respiratory tract is continually exposed to commensal flora, but the bacterial components, including DNA released from lysed organisms, do not appear to activate mucosal signaling. S. pneumoniae, a pathogen, is distinct from commensal flora by virtue of its expression of pneumolysin, the pore-forming toxin that enables ligands to gain access to the epithelial cytosol and initiate type I IFN signaling. As the induction of proinflammatory responses in the lung is so potentially deleterious to respiration, it seems logical that mucosal signaling is activated only by PAMPs that are sensed intracellularly, by organisms that have breached the barrier function of the airway mucosa. Our findings suggest that type I IFN signaling may be a general host response to perceived cellular invasion and does not discriminate among types of pathogens, whether viral or bacterial, but instead responds to the nature of the specific PAMPS within the cell.
In a model of nasopharyngeal colonization, the usual route of pneumococcal infection, the type I IFN response analogous to that induced by influenza virus contributed to bacterial clearance. Production of pneumolysin seems to be critical in triggering immune recognition of S. pneumoniae
as a pathogen. The role of type I IFN signaling in the host response to pneumococcal infection has been previously reported by Weigent et al. (40), who demonstrated increased susceptibility to infection in mice treated with anti-IFN-α/β antibodies. Mancuso et al. (41) similarly investigated the importance of type I IFN signaling in S. pneumoniae
sepsis and meningitis models. The significance of nasopharyngeal colonization in the initiation of host defenses against S. pneumoniae
was recently highlighted in a microarray study that identified the induction of a number of type I IFN-related genes in murine nasal lymphoid tissue early in the course of a 6-week colonization (42
). Less clear is how necessary type I IFN induction is in the context of active pneumococcal pneumonia, as models of pneumonia using direct tracheal inoculation of the organisms fail to demonstrate a notable phenotype (39
). However, as upper airway colonization precedes the development of pneumonia, this is a critical stage in the pathogenesis of systemic pneumococcal infection.
It is increasingly apparent that the mucosal epithelium of the lung provides much more than a physical barrier to infection but actively participates in sensing and initiating immune signaling in response to inhaled pathogens, both viral and bacterial. This signaling includes not only the proinflammatory cascades set off by the apically exposed TLRs but also intracellular sensing systems. For at least two important pulmonary pathogens, S. aureus
and S. pneumoniae
, shed components that are either endocytosed (S. aureus
protein A [36
]) or taken up via pore formation (pneumolysin) trigger IFN-β production in both epithelial and immune cells. The ability of these PAMPs from Gram-positive extracellular bacteria to access intracellular receptors suggests a central role for the type I IFN cascade in mucosal defenses against respiratory pathogens of all types.
The expression of pneumolysin is critical in the activation of type I IFN signaling by S. pneumoniae
. Pneumolysin has been shown previously to facilitate the introduction of peptidoglycan into host cells, as well as induce host signaling from osmotic changes (28
). DNA from lysed commensal flora in the upper respiratory tract could potentially be immunostimulatory, if the organisms were able to deliver it into mucosal cells. The data suggest that pneumolysin facilitates entry of DNA via its pore formation. We observed the requirement for pneumococcal lysis and release of pneumococcal DNA as an autolysin null strain was unable to induce type I IFN signaling. We could obviate this requirement artificially by transfection of DNA or exposing naturally phagocytic DCs to cell lysates. This suggests that the ability to access cytosolic receptors may also contribute significantly to virulence. However, the addition of purified pneumolysin and DNA did not activate signaling (data not shown). The inability to artificially recapitulate the system in vitro
indicates a missing component, alternative ligand, or stoichiometric effects necessary to activate signaling.
Several discrete receptors in both the cytosol and endosomes of the cells that make up the respiratory mucosa participate in type I IFN signaling. Our data suggest that DAI, whose role in immune signaling has been debated (15
), may be important in the response to pneumococcal DNA and other bacterial DNA. There was a significant induction of DAI expression by S. pneumoniae
and a significant requirement for DAI to induce Ifnb
in response to pneumococcal DNA. In addition, the contribution of other potential DNA sensors and signaling components (MAVS, RIG-I, and TLRs) was specifically excluded (although there was a trend to reduced induction in the MAVS null background, suggesting a limited role for RNA polymerase III [12
]). The expected downstream components of this pathway, including the recently identified STING (stimulator of IFN genes), TBK1, and IRF3 proteins, were also shown to be involved in the induction of Ifnb
in the contexts of both DNA and the whole organism.
There is a growing literature detailing how bacterial pathogens induce the type I IFN cascade in macrophages (6
). This report provides a second example of how an extracellular pathogen can activate type I IFN signaling, an intracellular response (36
). We now show that epithelial cells similarly participate in the induction of type I IFN signaling. Exactly how the epithelial cells, which are not actively phagocytic, are stimulated to express IFN-β in response to other types of airway pathogens remains to be established. However, based upon our observations with the pneumococcus, it seems likely that a general induction of type I IFN signaling in the lung, including the airway mucosal cells, is an important consequence of both bacterial and viral infections of the lung.