We show here that Casp1-dependent IL-1β secretion is critically required for host defense against CP
lung infection. Casp1−/−
mice displayed delayed pulmonary bacterial clearance leading to increased mortality compared to WT mice. Macrophages play a key role in this process, as they respond to CP
via the NLRP3 inflammasome. Casp1−/−
mice showed delayed IFN-γ production and defective iNOS activation in Casp1−/−
AM, consistent with reports demonstrating a critical role of IFN-γ and iNOS in clearing CP
In our model of CP lung infection, IL-1β plays a critical role in orchestrating a successful host defense against infection. In addition to Casp-1−/− mice, blockade of IL-1β signaling using the IL-1RA resulted in increased mortality to a CP infection. Indeed, early IL-1β signaling proved to be critical as rIL-1β given to caspase-1−/− was able to rescue these mice from a lethal CP infection, but only when given at the earliest time points. These data also highlight that it is unlikely that IL-18 plays a significant role during CP infection.
IL-1β has been known to be an important initiator of acute phase inflammatory responses to infections 
and more recently, found to play an critical role in establishing a Th17 response 
. In our model, we found that in caspase-1−/−
mice IFN-γ production was significantly delayed, resulting in poor bacterial clearance. It has been well established that IFN-γ is required for proper clearance of CP
in mice 
. We also found that iNOS was not induced in alveolar macrophages in caspase-1−/−
mice at day 2. While alveolar macrophages are a major site of CP
replication, they also play a critical role in bacterial clearance. Importantly, iNOS is critically important for CP
clearance and can be induced by both IFN-γ and IL-1β 
. Therefore, the defective iNOS induction in alveolar macrophages early during infection (day 2) most likely plays a significant role in the defective bacterial clearance.
IL-1β was found to be critically important for several other bacterial infections, including S. aureus
, B. anthracis
, and M. tuberculosis
. Miller et al. found that mice lacking IL-1β developed large skin lesions due to a reduction in neutrophil recruitment during a cutaneous S. aureus
. In another study, Moayeri et al. found similar results indicating the requirement of IL-1β for proper neutrophil recruitment against B. anthracis
. Finally, IL-1β−/−
mice showed greatly increased mortality to M tuberculosis
. Interestingly, these mice did not have any defects in nitrite production or IFN-γ or cellular recruitment, thus the mechanism by which IL-1β acts is unknown in this model. Taken together and including our data, it is clear that IL-1β can play a critical role in the host defense against a bacterial infection.
In our study we found that CP
infection induced IL-1β processing through TLR2/MyD88 signaling and activation of the NLRP3 inflammasome. This process required live bacteria, as UVCP did not induce IL-1β secretion without additional stimuli such as ATP. Additionally, entry into the cells was required for inflammasome activation as was active protein synthesis in the bacteria. CP
does possess a type III secretion system and it is possible that it might be involved in NALP3 inflammasome activation. However, as there is no genetic manipulation available yet for CP
and the type III secretion inhibitors proposed to be specific against CP 
have many off target and non-specific inhibitory effects, the direct role of type III secretion in NALP3 activation can not be assessed currently.
Similar to our findings, He et al.
recently reported that CP
required TLR2 and the NLRP3/ASC inflammasome for IL-1β production 
. However, unlike our study, they were unable to determine a role for IL-1β in the model they used. These investigators used IL-1R deficient mice, and these mice showed little difference if any on the course of infection. We demonstrate for the first time the critical role of IL-1β in host defenses against CP
lung infection. While we used caspase-1−/−
mice, which would affect both IL-1β and IL-18 production, our reconstitution experiment with IL-1β in caspase-1−/−
mice, plus the use of the IL-1RA in WT mice, clearly showed that IL-18 is dispensable in the host response to CP
infection. A source of the differences between our results and those by He et al.
could be due to different models used, including different CP
strains used in these two studies (A03 strain by He et al.
as opposed to CM-1 strain used in our study), and a much higher dose of CP
) used by He et al
. versus (1×106
) used in our study. CP
strain-specific differences likely led to the much milder lung infection seen in the study by He et al
. and perhaps this accounts for the large differences found between our study and theirs regarding infection course and mortality following murine CP
It is currently not clear how CP
infection activates the NLRP3 inflammasome. A wide range of cytosolic danger signals have been shown to lead to activation of the NLRP3 inflammasome. It is believed that three broad physiological changes—reactive oxygen species (ROS) generation, potassium cation (K+) efflux, or lysosomal leakage—activate the NLRP3 inflammasome 
, while direct mechanistic studies as to how they activate NLRP3 are yet to be provided. Furthermore, these three proposed models of NLRP3 activation are not even reconciled with one another and no model that offers a unifying paradigm exists. Our data indicates that ROS is not involved in activating the NLRP3 inflammasome during CP
infection. Even though UVCP and CP
induced similar amounts of ROS in macrophages, UVCP does not elicit IL-1β secretion while live CP
does. Moreover, our results call in to question results from studies that use the antioxidant N-acetyl cysteine (NAC). Though this agent was found to reduce IL-1β secretion, it also caused a concomitant reduction in TNF-α production, indicating that NAC likely affects pro-IL-1β production via NF-κB. Lastly, in agreement with previous studies 
, we report here that macrophages deficient in NADPH oxidase activity, and thus in phagocytic ROS production (gp91phox−/−
), exhibit normal IL-1β production in response to NLRP3 stimuli, refuting the role of ROS in NLRP3 activation. However these data only determined the role for cellular derived ROS, not mitochondrial derived ROS. Recent publications have found an important role for mitochondrial ROS in NLRP3 activation, indicating an important role for this organelle 
The lysosome rupture model also does not seem to be mechanism of CP
infection induced NLRP3 inflammasome activation, as the cathepsin inhibitor that we used had no effect on CP
-induced IL-1β production. This observation is different than those reported by He et al.
who observed that cathepsin activity and lysosomal acidification both play a role in CP-
induced IL-1β secretion. However, the inhibitors used in that study, CA-074Me (a cathepsin B and L inhibitor) and bafilomycin A (a lysosomal acidification inhibitor), both have off-target effects (as do most pharmacological inhibitors) 
. Additionally, CP
is a small infectious elementary body (EB); 300–600 nm diameter compared to other intracellular bacteria, and so internalization of the EB is unlikely to exceed the capacity of the phagolysosome. As part of the CP
life cycle, infectious EB converts to the vegetative reticulate body (RB), which forms inclusion bodies in the host cell phagosome (6.0–7.4 µm diameter). Though these inclusions might be large enough to cause vesicle rupture, Chlamydia
are known to actively inhibit the process of phagolysosomal fusion 
. So even if CP
-containing phagosomes ruptured, lysosomal enzymes would ostensibly not be present. Also, we found that CP
is able to induce IL-1β secretion in the presence of a specific cathepsin B inhibitor, Ac-LLM, further arguing against lysosomal degradation as the means by which CP
activates the NLRP3 inflammasome.
Two recent papers have identified mitochondrial dysfunction as being involved in the activation of the NLPR3 inflammasome, especially in relation to autophagy. Zhou et al. found that both ROS generation and inflammasome activation are suppressed when mitochondrial activity is dysregulated by inhibition of the voltage-dependent anion channel 
. Nakahira et al. also found that mitochondrial dysfunction played a role in inflammasome activation, and that mitochondrial DNA might play a role in this 
. To this end we investigated the effect of CP
infection on mitochondrial function. Our results indicated that both CP
infection and the commonly used inflammasome activator LPS plus ATP resulted in mitochondrial dysfunction as measured by a reduction in mitochondrial membrane potential and reduced O2
consumption. Identifying mitochondria as a player in NLRP3 inflammasome induction could help explain the many differing pathways that result in NLRP3 activation. Both ROS and cathepsins released from the lysosome can affect mitochondrial membrane potential, as can K+
levels in the cell 
,  
. With the addition of a bacterial infection to this mix, the role that mitochondria might play in inflammasome activation remains an important subject and may hold the key to allow us to understand the mechanism of NLRP3 activation.