The NLRP3 inflammasome is a multi-protein complex that drives sterile and pathogen-dependent inflammation. Activation of the NLRP3 inflammasome occurs in two steps: priming and activation. Priming occurs in response to an inflammatory stimulus, such as LPS. LPS-primed macrophages are subsequently activated by a second stimuli, most of which require K+ efflux. Upon activation, NLRP3 oligomerization leads to cleavage of pro-caspase-1 to its active form, which then processes pro-IL-1β, pro-IL-18 and gasdermin D. This results in secretion of IL-1β and IL-18. Pharmacologic studies have linked the mitochondria electron transport chain (ETC) to NLRP3 inflammasome activation via reactive oxygen species (ROS). Here we use a variety of pharmacological and genetic perturbations to inhibit ETC function and ROS generation. We ectopically expressed S. cerevisiae NADH dehydrogenase (NDI1) or C. intestinalis alternative oxidase (AOX), which can complement the functional loss of mitochondrial complexes I or II, respectively, and do not generate ROS. We report that mitochondrial ETC function is not required for NLRP3 priming but that forward electron transport by the ETC is required for caspase-1 activation and subsequent secretion of active IL-1β in primary bone marrow-derived macrophages (BMDMs) in response to a second stimuli. Expression of NDI1 or AOX rescued NLRP3 inflammasome activation in BMDMs lacking function of endogenous mitochondrial complex I or III. These results indicate that mitochondrial ROS is not required for NLRP3 inflammasome activation. Metabolomic analysis revealed phosphocreatine (PCr) as a metabolite that is decreased by inhibition of the ETC. PCr can sustain ATP levels during energetic crisis. Depleting PCr from BMDMs during LPS priming decreased ATP levels and inhibited NLRP3 inflammasome activation. The small molecule CL097, which activates the NLRP3 inflammasome independent of K+ efflux, required inhibition of mitochondrial complex I to activate the inflammasome. CL097 also required PCr to support NLRP3 inflammasome activation and did not require mitochondrial generated ROS. Collectively, these findings indicate that mitochondrial-generated ATP, supplied by PCr, sustains NLRP3 inflammasome in a ROS independent mechanism.

Creator

• Billingham, Leah Katherine

DOI

• https://doi.org/10.21985/n2-pr1y-am83

Subject

• Biology

Language

• en

Alternate Identifier

• http://dissertations.umi.com/northwestern:15953
• etdadmin_upload_882213

Keyword

• macrophage
• mitochondria
• immunometabolism
• inflammasome
• metabolism

Date created

• 2022-01-01

Resource type

• Dissertation

Rights statement

• In Copyright