The Crucial Role of Mitochondria Electron Transport in NLRP3 Inflammasome Activation

Mitochondria, often hailed as the powerhouses of the cell, are central to cellular energy production through the electron transport chain (ETC). Recent research has highlighted their involvement in inflammatory processes, particularly in the activation of the NLRP3 inflammasome. This article delves into the intricate relationship between Mitochondria Electron Transport and NLRP3 inflammasome activation, exploring the roles of specific ETC complexes and metabolic intermediates in this crucial immune pathway.

Mitochondrial Complex II: A Key Player in NLRP3 Inflammasome Priming and Activation

Initial investigations into the link between the ETC and NLRP3 inflammasome activation pointed towards mitochondrial complex II. To examine this connection, researchers utilized Bone Marrow-Derived Macrophages (BMDMs), a model system for studying inflammasome activation. These BMDMs were primed with lipopolysaccharide (LPS), a potent immune stimulant, and then treated with inhibitors of mitochondrial complex II, specifically DMM.

Experiments revealed that inhibiting complex II with DMM led to a decrease in oxygen consumption rates (OCR) in BMDMs, indicating a disruption of mitochondria electron transport. Metabolic analysis further showed that DMM treatment altered LPS-induced metabolic changes and increased succinate levels, a key metabolite in the ETC.

Despite these metabolic shifts, DMM did not affect the LPS-induced expression of pro-inflammatory cytokines Il1b, Tnf, or Il10 mRNA. However, crucially, DMM significantly attenuated the release of secreted IL-1β protein, a hallmark of NLRP3 inflammasome activation, in LPS and ATP-stimulated BMDMs. Furthermore, DMM reduced intracellular cleaved caspase-1 levels, a direct indicator of inflammasome activation. These findings strongly suggest that mitochondrial complex II is essential for caspase-1 activation and subsequent IL-1β production, key steps in NLRP3 inflammasome activation, but not for the initial LPS-induced pro-inflammatory gene expression.

Mitochondrial Complex I: Essential for NLRP3 Inflammasome Activation and Forward Electron Transport

Mitochondrial complexes I and II serve as entry points for electrons into the ETC. Complex I oxidizes NADH, while complex II oxidizes succinate, both feeding electrons into ubiquinone (CoQ). During inflammation, succinate levels rise, potentially leading to a reduced CoQ pool. This can drive reverse electron transport (RET) at complex I, generating superoxide.

To investigate the role of complex I in NLRP3 inflammasome activation, researchers used piericidin A, a specific inhibitor of mitochondrial complex I. Piericidin A treatment, similar to DMM, decreased OCR and altered cellular metabolism. It also abolished LPS-induced metabolite changes, including the increase in succinate. However, piericidin A, like DMM, did not affect the LPS-induced mRNA expression of Il1b, Tnf, or Il10.

Despite not affecting pro-inflammatory mRNA or protein levels, piericidin A significantly decreased secreted IL-1β and intracellular cleaved caspase-1 levels in LPS and ATP-stimulated BMDMs. This inhibitory effect was also observed when using nigericin, another NLRP3 inflammasome activator. Importantly, piericidin A did not reduce TNFα secretion, indicating a specific effect on NLRP3 inflammasome activation. These data indicate that mitochondrial complex I function, like complex II, is crucial for caspase-1 activation and IL-1β production, but not for the early stages of LPS-induced inflammatory gene expression.

Reverse Electron Transport: Not Indispensable for NLRP3 Inflammasome Activation

To further dissect the role of complex I and specifically address the involvement of reverse electron transport (RET), researchers employed BMDMs expressing Saccharomyces cerevisiae NADH dehydrogenase (NDI1). NDI1 is a rotenone-insensitive alternative NADH dehydrogenase that bypasses complex I proton pumping and RET-induced superoxide generation. NDI1 is also resistant to complex I inhibitors like piericidin A.

Using NDI1-expressing BMDMs allowed researchers to inhibit complex I with piericidin A while maintaining NADH oxidation and forward mitochondria electron transport through complexes III and IV. Experiments showed that piericidin A effectively inhibited complex I in wild-type (WT) BMDMs, reducing OCR and the NAD+/NADH ratio, but had minimal effect on NDI1-expressing BMDMs.

Crucially, piericidin A attenuated IL-1β production and caspase-1 activation in WT BMDMs but not in NDI1-expressing BMDMs. This rescue effect in NDI1 cells, where forward mitochondria electron transport is maintained despite complex I inhibition, suggests that it is the disruption of forward electron flow, rather than RET-induced superoxide, that inhibits NLRP3 inflammasome activation. In contrast, DMM, which inhibits complex II and forward electron transport, reduced IL-1β production in both WT and NDI1 BMDMs, further supporting the importance of forward mitochondria electron transport for inflammasome activation.

Mitochondrial Complex III and H2O2: Electron Transport is Key, Not Reactive Oxygen Species

Mitochondrial complex III, downstream of complexes I and II in the ETC, transfers electrons from CoQH2 to cytochrome c and is also a site of superoxide production. To investigate complex III’s role, myxothiazol, a complex III inhibitor, was used. Myxothiazol decreased OCR but, similar to complex I and II inhibitors, did not affect LPS-induced pro-inflammatory mRNA expression. However, it did reduce secreted IL-1β protein levels, suggesting complex III is also necessary for NLRP3 inflammasome activation.

To differentiate between the role of complex III in mitochondria electron transport and its role in ROS production, researchers utilized BMDMs from mice expressing Ciona intestinalis alternative oxidase (AOX) and lacking mitochondrial complex III subunit VII (QPC-KO/AOX BMDMs). AOX bypasses complex III, transferring electrons directly from CoQH2 to oxygen without proton pumping or superoxide production.

QPC-KO/AOX BMDMs, while having reduced superoxide production compared to WT, maintained coupled respiration and ATP production, even with myxothiazol treatment. Importantly, QPC-KO BMDMs showed decreased IL-1β production, while QPC-KO/AOX BMDMs, with restored mitochondria electron transport via AOX, exhibited IL-1β production levels similar to WT BMDMs. Myxothiazol did not further reduce IL-1β production in QPC-KO/AOX BMDMs. These results strongly indicate that it is the forward mitochondria electron transport, and not superoxide production from complex III or RET, that is essential for NLRP3 inflammasome activation.

Mitochondrial Membrane Potential: Not a Direct Link to NLRP3 Inflammasome Activation

Changes in mitochondrial membrane potential (MMP) can influence ETC function and ROS production. To examine if MMP is necessary for NLRP3 activation, BMDMs were treated with oligomycin (complex V inhibitor, increases MMP) or FCCP (protonophore, decreases MMP). Both oligomycin and FCCP attenuated the LPS-dependent increase in secreted IL-1β and cleaved caspase-1 levels, despite having opposite effects on MMP. This suggests that changes in MMP are not directly linked to NLRP3 inflammasome activation. The common effect of both inhibitors is the disruption of normal mitochondria electron transport and ATP production.

Phosphocreatine Shuttle: Bridging Mitochondrial ATP to NLRP3 Inflammasome Activation

To identify a common metabolic link between the effects of ETC inhibitors and MMP disruption on NLRP3 inflammasome activation, researchers analyzed metabolomics data. Phosphocreatine (PCr) emerged as a key metabolite. PCr levels increased during LPS priming but were diminished by all ETC inhibitors and MMP disruptors tested.

PCr, generated in mitochondria from creatine and ATP by creatine kinase, acts as an energy reservoir, shuttling high-energy phosphates to the cytosol to regenerate ATP. Depleting PCr using cyclocreatine (cyCr), a creatine analog, or by knocking down cytosolic creatine kinase (CKB) reduced IL-1β production and caspase-1 activation, mimicking the effects of ETC inhibitors.

Furthermore, inhibiting mitochondria electron transport with piericidin A or depleting PCr with cyCr further decreased ATP levels in LPS-primed and nigericin-stimulated BMDMs. These findings indicate that mitochondrial-derived PCr, generated through mitochondria electron transport and ATP production, is crucial for sustaining cytosolic ATP levels required for NLRP3 inflammasome activation. While glycolysis is important for LPS-stimulated macrophages, it appears insufficient to compensate for the lack of mitochondrial ATP in supporting NLRP3 inflammasome activation.

CL097 and Mitochondrial Complex I: A Unique NLRP3 Inflammasome Activation Pathway

CL097, an imidazoquinoline, activates NLRP3 inflammasome in a K+ efflux-independent manner, unlike ATP or nigericin. It has been proposed to act by inhibiting quinone oxidoreductases, including mitochondrial complex I, leading to ROS production and NLRP3 activation.

Using NDI1-expressing BMDMs, researchers confirmed that CL097 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, indicating that complex I inhibition is necessary for CL097-mediated NLRP3 inflammasome activation.

However, unlike ATP or nigericin, ETC inhibitors like piericidin A or myxothiazol alone were not sufficient to trigger NLRP3 inflammasome activation, suggesting CL097 has additional targets besides complex I. Nevertheless, inhibiting mitochondria electron transport with piericidin A or depleting PCr with cyCr during LPS priming also diminished CL097-induced NLRP3 inflammasome activation, highlighting the consistent requirement for mitochondrial ATP and PCr even in this distinct activation pathway.

Mitochondrial ROS: Not the Driving Force in CL097-Dependent NLRP3 Inflammasome Activation

While CL097 can induce ROS production, experiments using ROS modulators revealed that ROS is not the primary driver of CL097-mediated NLRP3 inflammasome activation. Increasing mitochondrial ROS with antimycin or suppressing superoxide production with MitoTEMPO, S1QEL, or S3QEL did not consistently prevent or rescue CL097-induced NLRP3 inflammasome activation in NDI1-expressing BMDMs. Even increasing ROS production with paraquat failed to induce IL-1β production.

These results suggest that while CL097 inhibits mitochondrial complex I, the subsequent NLRP3 inflammasome activation is not primarily driven by ROS but rather by another mitochondria-dependent mechanism, likely related to the disruption of mitochondria electron transport and ATP supply.

Conclusion: Mitochondria Electron Transport as a Metabolic Gatekeeper of NLRP3 Inflammasome Activation

This research comprehensively demonstrates the critical role of mitochondria electron transport in NLRP3 inflammasome activation. Forward electron flow through mitochondrial complexes I, II, and III is essential for providing mitochondrial-derived ATP, which is then shuttled to the cytosol via phosphocreatine to sustain the ATP hydrolysis required for NLRP3 inflammasome activation. Neither reverse electron transport nor mitochondrial ROS production appears to be necessary for this process. Even in the case of CL097-mediated NLRP3 inflammasome activation, which involves complex I inhibition, the requirement for mitochondrial ATP and mitochondria electron transport remains paramount, independent of ROS.

These findings underscore the intricate metabolic control of immune responses and highlight mitochondria electron transport as a crucial metabolic checkpoint in NLRP3 inflammasome activation, offering potential therapeutic targets for inflammatory diseases.

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