Chronic sensing of host-derived lipids is an all-in-one signal that primes and activates NLRP3

Keywords

• OxPAPC (oxidized phospholipids)

• Atherosclerosis, oxLDL, sterile inflammation

• NLRP3 inflammasome, caspase-1, IL-1β

• NRF2 (Nfe2l2), KEAP1

• Mitochondrial ROS, oxidized mtDNA (ox-mtDNA)

Main Findings

Atherosclerosis is a lipid-driven sterile inflammatory disease in which oxidized lipoproteins and phospholipids accumulate in the arterial wall and reprogram infiltrating myeloid cells. A central unresolved question is how lesion-associated macrophages sustain NLRP3 inflammasome activity and IL-1β production in the absence of microbial PAMPs. In this preprint, Dufies et al. test whether chronic exposure to the oxidized phospholipid mixture oxPAPC is sufficient to license macrophages for NLRP3-caspase-1 activation. They further ask whether this licensed state is driven by NRF2 and mechanistically linked to mitochondrial oxidative stress and oxidized mtDNA.

To model chronic lesion-like lipid exposure, the authors establish an oxPAPC-conditioning paradigm in which BMDMs are pre-exposed to oxPAPC for 24 h and then challenged with TLR agonists. Under this sequence, oxPAPC conditioning drives robust, dose-dependent IL-1β secretion after Pam3CSK4, R848, or LPS stimulation, whereas reversing the order (TLR priming followed by oxPAPC) fails to elicit IL-1β. Notably, oxPAPC exposure alone is sufficient to trigger NLRP3-dependent caspase-1 activation, indicating that oxPAPC can provide an activation signal even in the absence of microbial ligands, while downstream IL-1β secretion remains constrained by substrate availability.

The authors next test whether the heightened IL-1β output reflects NLRP3 signaling or requires the non-canonical caspase-11 pathway. IL-1β secretion after oxPAPC conditioning requires NLRP3 and caspase-1 and is blocked by the NLRP3 inhibitor MCC950, whereas caspase-11 is dispensable, arguing against an obligate caspase-11-dependent mechanism.

Immunoblotting then sharpens the conceptual distinction between activation and priming: in primary macrophages, oxPAPC exposure alone does not generate detectable pro-IL-1β, whereas subsequent TLR stimulation supplies the pro-IL-1β substrate that is then processed into mature IL-1β in the conditioned state.

A time-course then supports oxPAPC licensing as a transcriptionally driven process rather than an acute switch. Macrophages require roughly 4-8 h of oxPAPC exposure to acquire IL-1β secretory competence, with maximal effects by 24 h. Consistent with this delayed kinetic profile, bulk RNA-seq at 3 h and 24 h reveals a dominant NRF2-regulated transcriptional program, and biochemical fractionation shows NRF2 stabilization and nuclear translocation within 2 h that persists across the conditioning window.

The authors next argue that NRF2 is both necessary and sufficient for the oxPAPC-licensed inflammasome state. Nfe2l2 deficiency abrogates caspase-1 activation and IL-1β secretion after oxPAPC conditioning, whereas Keap1fl/flLysMcre macrophages, in which NRF2 is constitutively active, recapitulate key features of the licensed response. Mechanistically, they distinguish this pathway from classical K+-efflux-driven NLRP3 activation. Indeed, potassium efflux blockade prevents nigericin-induced IL-1β release, but oxPAPC-conditioned (and KEAP1-deficient) macrophages retain IL-1β output under potassium efflux blockade, consistent with a K+-efflux-independent route to NLRP3 activation.

The work then connects NRF2 licensing to a mitochondria-centered activation signal. oxPAPC-conditioned macrophages exhibit increased mitochondrial ROS, and quenching mitochondrial oxidation with MitoQ suppresses caspase-1 cleavage and IL-1β secretion. Pharmacologic perturbations of mtDNA processing and export (FEN1 inhibition and VDAC1 inhibition with VBIT-4) similarly blunt IL-1β output, consistent with a model in which NRF2-driven mitochondrial oxidative stress promotes an oxidized mtDNA signal that activates NLRP3.

To link this pathway to atherosclerosis, the authors generate Ldlr-/- bone marrow chimeras and show that hematopoietic Nfe2l2 deficiency reduces plaque burden and necrotic core formation without altering plasma cholesterol, implicating NRF2 activity in blood-derived cells in lesion progression. Complementing this, re-analysis of mouse and human plaque scRNA-seq datasets shows enrichment of NRF2 and oxPAPC-conditioning signatures within IL-1β-high plaque myeloid states, providing cross-species cellular context for the proposed mechanism.

Limitations & Suggestions

• Clarify the “all-in-one” framing in the title: In primary macrophages, oxPAPC conditioning does not robustly induce detectable pro-IL-1β, even though it can engage NLRP3-dependent caspase-1 activation and amplify IL-1β maturation once a priming stimulus supplies substrate. Revising the title and/or explicitly defining “all-in-one” as licensing NLRP3 activity rather than priming may better align with the framing of the data.

• NRF2 loss-of-function controls: The reduced IL-1β/caspase-1 outputs in Nfe2l2-/- macrophages would be easier to interpret with matched viability controls under the same oxPAPC conditioning conditions. A simple panel combining cell counts with viability measurements alongside IL-1β and caspase-1 readouts would confirm the phenotype reflects altered licensing rather than reduced cell fitness or survival.

• Validate NRF2 activation in the KEAP1 model: Because the sufficiency argument relies on Keap1fl/flLysMcremacrophages, it would help to show unequivocally that this model produces a robust NRF2 “on” state with NRF2 stabilization/nuclear enrichment. This would help resolve ambiguity surrounding incomplete KEAP1 deletion.

• Directly quantify cytosolic ox-mtDNA: The study presents evidence for oxidized DNA accumulation and shows functional sensitivity to VDAC1 and FEN1 inhibition, but it does not directly quantify cytosolic mtDNA or oxidized mtDNA. A direct readout would better support the proposed mtDNA-export step upstream of NLRP3.

Significance/Novelty

This preprint helps to reframe sterile inflammasome biology in atherosclerosis by proposing that chronic exposure to oxidized phospholipids (oxPAPC) can license macrophages for canonical NLRP3-caspase-1 activation. The work links oxPAPC conditioning to an NRF2-driven transcriptional state coupled to mitochondrial oxidative stress and supports a model consistent with an oxidized mtDNA-linked activation signal, providing a coherent mechanism for how plaque myeloid cells could sustain IL-1β maturation in PAMP-poor environments. Critically, hematopoietic NRF2 deficiency reduces lesion burden in Ldlr-/- bone marrow chimeras, positioning NRF2 as a disease-relevant regulator of lesion progression. Together, these findings are likely to stimulate renewed investigation of how oxidative-stress programs intersect with inflammasome competence across sterile inflammatory settings, and they may inform strategies aimed at upstream control of IL-1β-driven cardiovascular risk.

Credit

Reviewed by John Benjamin W. Duncan as part of a cross-institutional journal club between the Max-Delbrück Center Berlin, the Ragon Institute Boston (Mass General, MIT, Harvard), the University of Virginia, the Medical University of Vienna and other life science institutes in Vienna.

The author declares no conflict of interests in relation to their involvement in the review.