The proinflammatory cytokine IL-1β is a critical mediator of host defense against a variety of infectious states. For example, IL-1β is produced in the lung after intratracheal administration of LPS (49
), and its presence is directly associated with lung inflammation in patients with community acquired pneumonia (50
). IL-1 is required for control of a variety of intracellular pathogens, such as Listeria
, and Mycobacterium tuberculosis
). However, little is known about the role of IL-1 in control of Chlamydia pneumoniae
, an obligate intracellular bacterium that is associated with a variety of acute infectious processes, such as atypical pneumonia, bronchitis, and pharyngitis; chronic infection with C. pneumoniae
has also been linked to a variety of chronic inflammatory states, such as asthma and atherosclerosis.
In vitro, C. pneumoniae has been shown to infect and productively replicate within a number of cell types, including macrophages, leading to the upregulation of a variety of proinflammatory cytokines, including TNF-α, IL-1β, IL-6, IL-18, and IFN-γ, and chemokines like KC and MCP-1. IL-1β and IL-18 are unique among this list in that they are regulated at two levels: first, the induction of transcription of a precursor protein, followed by assembly of the inflammasome complex and activation of cysteine protease caspase-1, which cleaves the precursor protein to the biologically active protein that is secreted. Because of its reported role in host defense, specifically pneumonia and ARDS, and chronic inflammatory states, such as asthma and atherosclerosis, we were interested in examining the regulation and function of IL-1β during C. pneumonia infections.
A number of receptors have been implicated in the innate immune response to Chlamydia
, in particular TLR2 and TLR4, which have been shown to activate NF-κB–dependent proinflammatory pathways. Among the NLRs, there have been only a handful of reports suggesting a role for Nod1 and/or Nod2, either in vitro (54
) or in vivo (13
), in the pathogenesis of C. pneumoniae
infection, and except for a recent report that the related species C. trachomatis
can activate caspase-1 in epithelial cells in an NLRP3-dependent manner (55
), activation of the inflammasome has been largely ignored. We also observed inflammasome activation by C. trachomatis
and C. muridarum
; however, C. pneumoniae
induced a far higher degree of caspase-1 processing and production of mature IL-1β in resting macrophages. Our data further suggest the activation of two distinct pathways in the C. pneumoniae
-induced production of mature IL-1β by resting macrophages. First, TLR2 activation leads to the induction of pro–IL-1β (signal 1), followed by activation of the NLRP3/ASC inflammasome, which activates caspase-1 (signal 2) to cleave pro–IL-1β to its mature and biologically active form. Furthermore, C. pneumoniae
-induced caspase-1 activation does not require macrophage priming or any additional signals, such as ATP activation of the P2X7
receptor. Interestingly, caspase-1 activation occurs relatively early in the developmental cycle of C. pneumoniae
, before significant RB replication has occurred, suggesting that it does not require a large intracellular bacterial burden. The fact that only live bacteria are capable of inducing caspase-1 cleavage suggests that it requires uptake and trafficking of bacteria to the inclusion and possibly early gene transcription. Although our in vitro data focused only on the processing and activation of IL-1β and IL-18, recent evidence suggests that caspase-1 is also capable of regulating IL-1α and other proteins involved in cytoprotection and cell survival (56
). Thus, the potential biological effects of caspase-1 activation by C. pneumoniae
are quite broad.
The precise mechanism behind activation of the NLRP3/ASC inflammasome is unknown, although it appears to require potassium flux, lysosomal acidification, and cathepsin B release. This is similar to other particulate activators of the NLRP3 inflammasome, which in some models are hypothesized to trigger lysosomal destabilization (34
). However, in the case of C. pneumoniae
, the connection between replicating bacteria secluded within an intracellular inclusion and lysosomal compartments is unclear. Chlamydiae
do possess a type III secretion system (T3SS), and so one possible mechanism could involve injection of a ligand into the cytosol that leads to effects on neighboring lysosomes, indirectly activating NLRP3. This could be related to the also undefined mechanism by which Chlamydia
actively inhibits lysosomal fusion with the intracellular inclusion. This seems more likely than a cytosolic chlamydial Ag injected via the T3SS acting directly as a NLRP3 ligand. Unfortunately, this hypothesis is difficult to test because the T3SS appears to be essential for chlamydial growth and development. In the study by Baily et al. (57
), small molecule inhibitors of the Yersinia
T3SS were found to inhibit C. pneumoniae
, but not C. trachomatis
, infections in vitro, blocking the developmental cycle and the development of inclusions. Our data demonstrate that productive infection is required for caspase-1 activation; thus, it would be impossible to determine if any changes in caspase-1 activation as a result of the inhibitors were a result of their effect on the T3SS or the lack of intracellular bacterial growth.
Finally, our data are the first to examine a role for IL-1 signaling in the pathogenesis of pneumonia. Studies in patients with ARDS have demonstrated the presence of IL-1β in bronchoalveolar lavage fluid (58
). However, some data suggest that its presence might not be a poor prognostic factor (59
). In fact, several studies have suggested that IL-1β signaling is required for alveolar epithelial repair during acute lung injury (58
). Using an established mouse model for C. pneumoniae
infection, we found a previously unappreciated role for IL-1 signaling in regulating the beneficial host inflammatory response, suggesting that IL-1β, and likely IL-1α, are protective to the host during severe pneumonia. Our data demonstrate that in the absence of IL-1 signaling, lung inflammation is comparable, if not more severe, than that seen in mice with intact IL-1 signaling, and IL-1 does not seem to be required for resolution of the infectious process. However, what we did find was a difference in the cellular infiltrate when IL-1 signaling was absent. We found evidence of increased infiltrating fibroblasts and mesenchymal cells in the lung histology, with increased vimentin-positive cells in the parenchyma. Interestingly, the absolute number of infiltrating neutrophils was actually reduced in the absence of IL-1 signaling, although the chemokine levels, as best we could determine, were comparable.
The role of neutrophils during infection has always been controversial, as it plays an important role in the control of many infectious processes, but the sequestration of activated neutrophils in the lung has long been known to contribute to acute lung injury (62
). However, severe alveolar damage during ARDS can occur even in patients with neutropenia, so there are obviously other mechanisms by which lung damage can occur (64
). The combination of both decreased neutrophils and increased lung fibrosis in the IL-1R–deficient mice initially seemed counterintuitive. However, Tate and colleagues (65
) recently described the outcome of severe influenza pneumonia in neutropenic mice. This group found that depletion of neutrophils in mice infected with influenza virus led to exacerbated pulmonary inflammation, edema, and respiratory dysfunction and that this was not entirely explained by early increased viral replication (65
). Like Tate and colleagues’ data (65
), ours suggests a novel role for neutrophils in ameliorating inflammation and fibrosis during severe infection. The mechanism behind this might be explained by Zhang and colleagues (66
), who recently reported that TLR-stimulated neutrophils are actually poor inducers of proinflammatory signals and, in contrast, produce large amounts of the anti-inflammatory cytokine IL-10. Furthermore, in their model of chronic infection secondary to Mycobacterium bovis
, they found that neutrophil depletion promoted inflammation. Thus, we conclude that both IL-1 signaling and neutrophils are beneficial to the host, at least in the setting of an acute infectious process.
Questions arising from our studies that remain to be answered are exactly how does C. pneumoniae trigger NLRP3/ASC inflammasome activation, and what, if any, role does the T3SS play in the interaction of bacteria with these host cytosolic receptors? Additional studies to determine what cell types are responsible for the beneficial response to IL-1β during lung repair may help us to design better therapies to prevent complications arising from severe pneumonia while preserving the protective immune response that is required for bacterial clearance and the development of lasting immunity. Finally, whereas our data suggest that IL-1 signaling is important during acute C. pneumoniae lung infections, its role in chronic inflammatory states may be the opposite given the suggestion from a number of sources that IL-1 signaling contributes to the development of atherosclerosis and asthma. Thus, the role of IL-1 during C. pneumoniae-induced chronic inflammatory conditions remains to be defined.