The electron transport chain (ETC) in mitochondria is pivotal in cellular energy production, but emerging research highlights its crucial role in inflammation. This article delves into the intricate relationship between the mitochondrial ETC and the NLRP3 inflammasome, a key component of the innate immune system. Building upon recent studies, we explore how different complexes within the Electron Transport Chain In Mitochondria influence NLRP3 inflammasome activation, offering a comprehensive understanding for researchers and those interested in immunometabolism.
Mitochondrial Complex II: A Key Player in NLRP3 Inflammasome Activation
Initial investigations into the link between the electron transport chain in mitochondria and inflammation often pointed to mitochondrial complex II. To examine this connection, experiments were conducted using bone marrow-derived macrophages (BMDMs) primed with lipopolysaccharide (LPS). The impact of inhibiting the electron transport chain in mitochondria at complex II was assessed by measuring caspase-1 activation and IL-1β production, both critical markers of NLRP3 inflammasome activation.
Treating BMDMs with dimethyloxaloylglycine (DMM), a complex II inhibitor, resulted in decreased oxygen consumption rates, confirming ETC inhibition. Metabolic analysis revealed that DMM also altered LPS-induced metabolite changes and increased succinate levels, a substrate for complex II, without affecting the NAD+/NADH ratio. Interestingly, while DMM did not reduce LPS-induced mRNA expression of pro-inflammatory cytokines like Il1b and Tnf, it significantly attenuated the release of secreted IL-1β protein. Furthermore, DMM diminished intracellular cleaved caspase-1 levels, indicating a crucial role for mitochondrial complex II in caspase-1 activation and subsequent IL-1β production, downstream of initial inflammatory gene expression. These findings underscore the importance of complex II of the electron transport chain in mitochondria for NLRP3 inflammasome activation.
The Necessity of Mitochondrial Complex I for NLRP3 Inflammasome Activation
Mitochondrial complexes I and II are entry points to the electron transport chain in mitochondria, transferring electrons to ubiquinone. During inflammation, increased succinate levels can lead to reverse electron transport (RET) at complex I, generating superoxide. While RET-derived superoxide has been implicated in later stages of inflammation, the immediate role of complex I in NLRP3 inflammasome activation remained under investigation.
Utilizing piericidin A, a mitochondrial complex I inhibitor, experiments mirrored those with complex II inhibition. Piericidin A decreased oxygen consumption and altered cellular metabolism, abolishing LPS-induced metabolite changes. Similar to complex II inhibition, complex I inhibition did not affect the initial LPS-induced mRNA expression of Il1b, Tnf, or Il10, nor did it reduce pro-IL-1β or pro-caspase-1 protein levels. However, piericidin A effectively decreased secreted IL-1β and intracellular cleaved caspase-1 upon NLRP3 inflammasome stimulation. This data reveals that mitochondrial complex I, part of the electron transport chain in mitochondria, is essential for caspase-1 activation and IL-1β protein production, but not for the early inflammatory gene induction by LPS.
Fig. 1: NDI1 expression counteracts mitochondrial complex I inhibitor piericidin A.
Schematic representation of the electron transport chain in mitochondria in wild-type (WT) and NDI1-expressing BMDMs under LPS stimulation. Piericidin A inhibition of complex I is bypassed by NDI1 expression. The figure also includes data panels (b-h) illustrating the effects of NDI1 expression and piericidin A on mRNA levels, oxygen consumption rates (OCR), NAD+/NADH ratio, H2O2 production, and metabolite profiles in BMDMs. These data support the role of complex I in electron flow and metabolic changes during inflammation.
Reverse Electron Transport: Not Indispensable for NLRP3 Inflammasome Activation
To specifically address the role of complex I inhibition, and to differentiate it from potential off-target effects of inhibitors, researchers employed BMDMs expressing Saccharomyces cerevisiae NADH dehydrogenase (NDI1). NDI1 bypasses complex I, restoring NADH oxidation without proton pumping or RET-induced superoxide production. NDI1 is also resistant to piericidin A, allowing for targeted investigation of complex I’s role in the electron transport chain in mitochondria.
NDI1-expressing BMDMs, when treated with piericidin A, maintained NADH oxidation and downstream electron flow in the electron transport chain in mitochondria, bypassing complex I inhibition. Experiments showed that while piericidin A still reduced OCR and the NAD+/NADH ratio in wild-type cells, it had minimal impact on NDI1-expressing cells. Crucially, piericidin A failed to attenuate IL-1β production and caspase-1 activation in NDI1-expressing BMDMs, unlike in wild-type cells. This critical finding demonstrates that the requirement of mitochondrial complex I for NLRP3 inflammasome activation is specifically linked to NADH oxidation and forward electron flow within the electron transport chain in mitochondria, rather than RET-induced superoxide production.
Fig. 2: Reverse electron transport is dispensable for NLRP3 inflammasome activation.
This figure presents data on IL-1β and caspase-1 protein levels in wild-type (WT) and NDI1-expressing mice and BMDMs. Panels (a-h) illustrate the effects of piericidin A and DMM treatments on IL-1β secretion and caspase-1 cleavage in LPS and ATP-stimulated cells. The data demonstrates that NDI1 expression rescues the inhibitory effects of piericidin A on NLRP3 inflammasome activation, confirming that reverse electron transport is not essential for this process, but forward electron flow through the electron transport chain in mitochondria is.
Mitochondrial H2O2 Production: Not Essential for NLRP3 Inflammasome Activation
Mitochondrial complex III, another vital component of the electron transport chain in mitochondria, is a major site of reactive oxygen species (ROS) production, specifically superoxide and hydrogen peroxide (H2O2). To investigate the role of complex III and mitochondrial ROS in NLRP3 inflammasome activation, myxothiazol, a complex III inhibitor, was used.
Myxothiazol treatment effectively decreased oxygen consumption. Similar to complexes I and II inhibitors, myxothiazol did not affect LPS-induced Il1b, Tnf, or Il10 mRNA expression, but it did reduce secreted IL-1β protein levels and caspase-1 activation. Importantly, NDI1 expression did not rescue the inhibitory effects of myxothiazol, indicating that complex III, like complexes I and II of the electron transport chain in mitochondria, is required for NLRP3 inflammasome activation through a mechanism independent of complex I and RET.
To further dissect the role of complex III-derived ROS, researchers used BMDMs from mice expressing Ciona intestinalis alternative oxidase (AOX) in a mitochondrial complex III deficient background (QPC-KO/AOX). AOX bypasses complex III, allowing electron flow to oxygen without proton pumping or ROS generation at complex III, while still enabling ATP production via complex I.
Fig. 3: Mitochondrial-derived H2O2 production is not necessary for NLRP3 inflammasome activation.
Schematic illustrating the electron transport chain in mitochondria in wild-type (WT) and QPC-KO/AOX BMDMs, highlighting the bypass of complex III by AOX. The figure includes data panels (b-h) showing the effects of myxothiazol and genetic manipulation of complex III and AOX expression on OCR, H2O2 production, IL-1β secretion, and caspase-1 processing. The results demonstrate that while complex III is necessary for inflammasome activation, mitochondrial-generated H2O2 is not.
QPC-KO/AOX BMDMs exhibited reduced H2O2 production compared to wild-type cells, but maintained coupled respiration and ATP production. Notably, QPC-KO/AOX BMDMs showed no significant difference in IL-1β production or caspase-1 activation compared to wild-type BMDMs, and these responses were not affected by myxothiazol. This compelling evidence indicates that neither complex III-generated ROS nor RET-derived ROS are required for NLRP3 inflammasome activation. Instead, forward electron flow through the electron transport chain in mitochondria itself is crucial.
Mitochondrial Membrane Potential: Not Directly Linked to NLRP3 Inflammasome Activation
Mitochondrial membrane potential (MMP) is intimately linked to the electron transport chain in mitochondria and ROS production. To investigate whether changes in MMP are necessary for NLRP3 activation, oligomycin (complex V inhibitor, increasing MMP) and FCCP (protonophore, decreasing MMP) were used.
Both oligomycin and FCCP, despite having opposite effects on MMP, attenuated LPS-dependent increases in secreted IL-1β and caspase-1 activation. These findings demonstrate that alterations in MMP, whether increased or decreased, do not directly drive NLRP3 inflammasome activation. This suggests that the crucial role of the electron transport chain in mitochondria lies elsewhere, beyond simply modulating MMP.
Mitochondrial-Generated Phosphocreatine Supports NLRP3 Inflammasome Activation
To identify a common metabolic link across different ETC inhibitors, metabolomic data was analyzed. Phosphocreatine (PCr) emerged as a key metabolite. PCr levels increased during LPS priming and were consistently diminished by inhibitors of complexes I, II, III, and V, as well as by MMP disruption.
PCr, generated in mitochondria and shuttled to the cytosol, provides readily available ATP. Depleting PCr using cyclocreatine (cyCr), a creatine analog, reduced ATP levels and decreased IL-1β production and caspase-1 activation. Similarly, inhibiting cytosolic creatine kinase (CKB), which utilizes PCr to regenerate cytosolic ATP, also reduced inflammasome activation. Furthermore, in vivo administration of cyCr reduced LPS-induced IL-1β production.
Fig. 4: Mitochondrial-generated PCr during priming is essential for NLRP3 inflammasome activation.
This figure presents data on phosphocreatine (PCr) and ATP levels in BMDMs and the effects of cyclocreatine (cyCr) and CKB knockdown on NLRP3 inflammasome activation. Panels (a-i) show the impact of cyCr on PCr levels, ATP levels under different stimulations, IL-1β secretion, and caspase-1 processing. The data strongly suggests that mitochondrial-derived PCr, and consequently, ATP supply, is critical for NLRP3 inflammasome activation.
These results strongly indicate that mitochondrial-generated PCr, facilitating cytosolic ATP availability, is a critical metabolic support for NLRP3 inflammasome activation. This highlights a key function of the electron transport chain in mitochondria beyond just bulk ATP production.
Glycolysis is Insufficient, Mitochondrial ATP is Essential for NLRP3 Inflammasome Activation
While LPS stimulation is known to enhance glycolysis for ATP supply, experiments using nigericin, which triggers NLRP3 activation and reduces ATP levels, revealed that glycolysis-derived ATP is not sufficient for inflammasome activation in the absence of mitochondrial ATP. Inhibiting the electron transport chain in mitochondria with piericidin A during nigericin stimulation, despite maintaining high glycolytic flux, still prevented NLRP3 activation. This emphasizes the unique and essential contribution of mitochondrial ATP, generated through the electron transport chain in mitochondria, for NLRP3 inflammasome activation.
Fig. 5: Nigericin reduces OCR in a caspase-1-dependent manner.
This figure displays metabolic flux analysis data, including oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), in BMDMs treated with LPS, Nigericin, VX-765 (caspase-1 inhibitor), piericidin A, oligomycin, and 2-deoxy-d-glucose (2DG). Panels (a-g) illustrate the dynamic changes in OCR and ECAR under different treatment conditions, highlighting the interplay between mitochondrial respiration, glycolysis, and caspase-1 activity during NLRP3 inflammasome activation. The data supports the conclusion that mitochondrial ATP, derived from the electron transport chain in mitochondria, is essential, and glycolysis alone is insufficient for NLRP3 inflammasome activation.
CL097-Induced NLRP3 Inflammasome Activation Requires Mitochondrial Complex I Inhibition
CL097, an NLRP3 inflammasome activator known to act independently of K+ efflux, has been proposed to function by inhibiting quinone oxidoreductases, including mitochondrial complex I, leading to ROS production. Using NDI1-expressing BMDMs, researchers confirmed that CL097 indeed inhibits mitochondrial complex I, as NDI1 expression prevented CL097-induced OCR reduction.
Importantly, NDI1 expression also abolished CL097-dependent IL-1β production and caspase-1 activation, demonstrating that mitochondrial complex I inhibition is necessary for CL097-mediated NLRP3 inflammasome activation. Furthermore, similar to ATP and nigericin-induced activation, piericidin A and cyCr administration during LPS priming also diminished CL097-induced inflammasome activation, reinforcing the requirement for mitochondrial ATP and PCr support even in K+ efflux-independent NLRP3 activation pathways.
Fig. 6: Mitochondrial complex I inhibition is required for CL097-induced NLRP3 inflammasome activation.
This figure presents data on the effects of CL097 and piericidin A on oxygen consumption rate (OCR) and NLRP3 inflammasome activation in wild-type (WT) and NDI1-expressing BMDMs. Panels (a-n) illustrate the impact of CL097 on LDH release, OCR dynamics, IL-1β secretion, and caspase-1 processing, and how NDI1 expression and PCr depletion affect these responses. The data confirms that mitochondrial complex I inhibition is a crucial step in CL097-mediated NLRP3 inflammasome activation, and mitochondrial ATP support remains essential.
Mitochondrial ROS: Not the Driving Force in CL097-Dependent NLRP3 Inflammasome Activation
While CL097 and complex I inhibition can induce ROS production, experiments using antimycin (ROS generator at complex III) and myxothiazol (inhibitor of ROS production at complex III) to manipulate ROS levels in NDI1-expressing BMDMs revealed a surprising outcome. Both antimycin and myxothiazol, as well as oligomycin and FCCP, rescued CL097-inhibited inflammasome activation in NDI1-expressing cells. This rescue effect, observed with agents having diverse impacts on ROS production and MMP, suggests that ROS is not the primary driver of CL097-dependent NLRP3 inflammasome activation.
Furthermore, directly scavenging mitochondrial superoxide using MitoTEMPO or site-specific superoxide suppressors (S1 and S3) failed to prevent CL097 or ATP-induced NLRP3 inflammasome activation. Similarly, increasing ROS production with paraquat did not rescue inflammasome activation in NDI1-expressing BMDMs treated with CL097. Collectively, these findings strongly argue against mitochondrial ROS as the essential mediator of CL097-induced NLRP3 inflammasome activation.
Fig. 7: Mitochondrial ROS is not required for CLO97-dependent NLRP3 inflammasome activation.
This figure presents data on the role of mitochondrial reactive oxygen species (ROS) in CL097-dependent NLRP3 inflammasome activation. Panels (a-h) show the effects of antimycin, myxothiazol, FCCP, oligomycin, MitoTEMPO, S1QEL, S3QEL, and paraquat on caspase-1 processing and IL-1β secretion in WT and NDI1-expressing BMDMs. The data indicates that mitochondrial ROS is not essential for CL097-mediated NLRP3 inflammasome activation, and the rescue effects observed with ETC modulators are independent of ROS.
Conclusion: Forward Electron Flow and Mitochondrial ATP via the Electron Transport Chain are Key for NLRP3 Inflammasome Activation
This comprehensive study demonstrates that forward electron flow through the electron transport chain in mitochondria, specifically involving complexes I, II, and III, is crucial for NLRP3 inflammasome activation. Mitochondrial-derived ATP, supplied via the PCr shuttle, provides essential metabolic support for this process. Importantly, neither mitochondrial ROS production nor changes in mitochondrial membrane potential appear to be the primary triggers for NLRP3 inflammasome activation in this context. These findings advance our understanding of the intricate link between mitochondrial metabolism, specifically the electron transport chain in mitochondria, and innate immunity, opening new avenues for therapeutic targeting of inflammatory diseases.
Fig. 8: Schematic summary of NLRP3 inflammasome activation pathways.
Schematic representation summarizing the K+ efflux-dependent and -independent NLRP3 inflammasome activation pathways, highlighting the essential role of the electron transport chain in mitochondria and mitochondrial ATP generation in both pathways.
