In this study we found that Beclin 1 and LC3B, autophagic proteins required in the initial and late phases of autophagosome formation, act as critical regulators of caspase-1-mediated immune responses in vitro
and in vivo
. The mechanism by which these autophagic proteins regulate caspase-1 activation is related to their positive contributions to mitochondrial quality control. We observed that autophagic protein depleted macrophages accumulate physiologically abnormal mitochondria, which produce elevated amounts of ROS at baseline. These abnormal mitochondria are prone to undergo more severe structural derangement and dysfunction after LPS and ATP treatment. The changes to mitochondria after LPS and ATP treatment are important because we observed that the loss of mitochondrial integrity was critical for the enhanced activation of caspase-1, both in wild-type macrophages and in autophagy protein depleted (Map1lc3b−/−
) macrophages. The increased mitochondrial ROS generation produced by Map1lc3b−/−
macrophages was sufficient to explain their increased efficiency of caspase-1 activation in our model. As a consequence of mitochondrial ROS and subsequent MPT, we found that LPS and ATP treatment released mtDNA into the cytosolic compartment. Translocation of mtDNA into the cytosol required NALP3 inflammsome activation, and directly contributed to downstream activation of caspase-1 in response to LPS and ATP treatment. Importantly, we found that Map1lc3b−/−
macrophages released mtDNA in excess quantities into the cytosol in response to LPS and ATP treatment, further explaining the increased caspase-1 activation and IL-1β and IL-18 secretion observed in these cells. While prior reports have described the important role for autophagy in mitochondrial homeostasis13,32
, our results clarify the molecular link between mitochondrial quality control, as regulated by autophagy, and NALP3-dependent activation of caspase-1. We also identified endogenous cytosolic mtDNA as a novel intracellular second-messenger molecule that connects mitochondrial dysfunction to caspase-1 activation.
Mitochondrial ROS generation was a relatively upstream mechanistic step in our system, as treatment with Mito-TEMPO abolished subsequent caspase-1 activation and mtDNA translocation in response to LPS and ATP. The production of mitochondrial ROS was likely a consequence of ATP-driven activation of the P2X7 receptor33
, since this was previously shown to induce mitochondrial damage34,35
, and also to contribute to NALP3 inflammasome assembly9
. Interestingly, we found that Mito-TEMPO did not inhibit IL-1β secretion induced by LPS and MSU. Crystalline particles such as asbestos, silica and MSU have previously shown to activate the NALP3 inflammasome, through ROS generation by phagocytic NADPH oxidase28,36
. Thus, while there appears to be a general requirement for ROS in NALP3 inflammasome activation, the subcellular source ROS may differ between stimuli.
Based on previous studies6,9
, we expected that NALP3 would be situated downstream of both mitochondrial ROS and cytosolic mtDNA release in the caspase-1 activation pathway. However, our results indicate that NALP3 is required at a step upstream of MPT and mtDNA release but downstream of mitochondrial ROS production in response to LPS and ATP treatment. Since deletion of the NALP3 binding partner ASC produced a similar attenuation of the response, it is likely that the NALP3 inflammasome complex is responsible for mediating MPT in our model. At present, it is unclear whether the NALP3 inflammasome is additionally responsible for more distal steps in the caspase-1 activation pathway after LPS and ATP treatment. Further research will be needed to clarify this point.
mtDNA stimulates inflammatory responses through TLR9 when released into plasma in human as a consequence of mechanical trauma27
. We showed that mtDNA also mediates inflammatory responses in the intracellular space through the activation of caspase-1 in response to LPS and ATP, a NALP3 inflammsome model. Multiple groups have shown that introduction of exogenous or foreign DNA into the cytosol activates caspase-1 via
the AIM2 inflammsome23,24
. However, the contribution of endogenous mtDNA to caspase-1 regulation has not previously been described. Our conclusion that mtDNA is important for caspase-1 activation, in response to LPS and ATP, is based on several results. First, respiration-deficient ρ0
cells, which are also depleted for mtDNA content, produced markedly less IL-1β and IL-18 secretion when challenged with LPS and ATP. Second, transfection of DNase1 into the cytoplasm, which reduced mtDNA copy number in the cytosol, attenuated IL-1β secretion after LPS and ATP treatment in macrophages. Since nuclear DNA does not translocate to the cytoplasm after LPS and ATP treatment, the inhibitory effect of DNase1 on caspase-1 dependent cytokine secretion correlates with its ability to degrade mtDNA in the cytosol.
Third, direct transfection of mtDNA into aim2−/−
macrophages, where the dominant AIM2-dependent inflammasome response to exogenous DNA in cytosol has been removed, nevertheless enhanced caspase-1 dependent cytokine secretion in response to LPS and ATP. Cytosolic mtDNA was insufficient to stimulate IL-1β and IL-18 secretion on its own but required at a minimum the presence of LPS6,7
. This fact may explain why Map1lc3b−/−
macrophages do not constitutively secrete elevated amounts of IL-1β and IL-18 on the basal condition, in spite of the fact that they have mildly elevated amounts of mtDNA in the cytoplasm at baseline. While the AIM2 inflammasome clearly plays a dominant role in response to transfected DNA and particular intracellular pathogens23,24
, it is worth noting that other cytosolic DNA sensors potentially could serve as the dominant receptors for cytosolic mtDNA in our model37,38
. Further research will be needed to clarify the downstream mechanism by which cytosolic mtDNA contributes to caspase-1 activation during LPS and ATP stimulation.
In this study, we showed the physiological relevance of autophagic proteins in regulating caspase-1-mediated inflammatory responses in two animal models of sepsis: the endotoxic shock model and the CLP model of polymicrobial sepsis. In addition, our clinical data confirmed that circulating plasma levels of IL-18 were increased in patients with sepsis. Marked autophagic vacuolization has been observed in liver biopsies obtained from patients who died of sepsis39
. However, it is unclear whether this observation represented increased autophagic activity in septic patients or downstream inhibition of autophagy that led to the inappropriate accumulation of autophagosomes. Since sepsis has long been recognized to produce mitochondrial dysfunction in humans40,41
, further research is needed to investigate whether autophagy is dysregulated by septic conditions and as such contributes to sepsis-induced mitochondrial dysfunction. In conclusion, the present study provides a mechanistic explanation for how autophagy, a core cellular housekeeping process, modulates caspase-1 mediated immune responses. Agents that stimulate autophagic activity may confer beneficial anti-inflammatory effects through the improvement of mitochondrial quality control and the removal of molecules such as mitochondrial ROS and cytosolic mtDNA. It might be useful to investigate the potential for therapeutic targeting of autophagic processes to mitigate inflammatory diseases.