Zanoni and colleagues showed in 2016 that oxidized phospholipids (oxPAPC), self-derived lipids generated during tissue injury, bind the inflammatory protease caspase-11 in dendritic cells and trigger inflammasome-dependent interleukin-1 (IL-1) release while the cells stay alive, a state termed hyperactivation. A decade of follow-up work has defined the bacterial-lipopolysaccharide side of caspase-11 activation in structural detail, identified some active and inhibitory oxPAPC components, and extended the hyperactivation concept to macrophages, mouse dendritic-cell subsets, and human type 2 conventional dendritic cells (with non-oxPAPC ligands). However, the central mechanistic questions opened by the 2016 paper remain unanswered: how oxPAPC binding to the caspase-11 catalytic domain produces an enzymatically inactive caspase-11 assembly of undetermined oligomeric state that activates the NLRP3/ASC inflammasome and gasdermin-D-dependent IL-1 secretion without pyroptosis; what the architecture and stoichiometry of the putative oxPAPC-induced caspase-1/caspase-11 complex are; and which lipid determinants and host factors make oxPAPC an agonist in dendritic cells but an antagonist of caspase-11 in macrophages. Subsequent work has additionally raised the related comparative question of whether human antigen-presenting cells, through the caspase-11 orthologs caspase-4 and caspase-5, reproduce this endogenous-lipid-sensing pathway. The problem remains open, with recent field reviews from the original authors' laboratory explicitly listing species-specific lipid-pattern-recognition-receptor interactions as unresolved.

Background

Dendritic cells (DCs) are innate immune sentinels that detect microbes and tissue damage through pattern-recognition receptors and then initiate adaptive T-cell immunity. Interleukin-1 beta (IL-1β), a master inflammatory cytokine that shapes T-helper-cell differentiation, is released in a strictly controlled way: the inactive pro-cytokine must be cleaved by the protease caspase-1, which is activated within a cytosolic multi-protein assembly called an inflammasome. The best-studied inflammasome nucleates around the sensor NLRP3 (nucleotide-binding domain, leucine-rich repeat-containing protein 3) and the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), which recruits and activates caspase-1 through CARD (caspase activation and recruitment domain) interaction motifs. Inflammasome activation is usually coupled to pyroptosis, a lytic inflammatory cell death executed when caspase-1 or the related protease caspase-11 cleave gasdermin D (GSDMD), whose N-terminal fragments perforate the plasma membrane; IL-1β exits the cell through these pores.

Caspase-11 itself is both a sensor and an effector in the so-called non-canonical inflammasome pathway. Shi et al. (2014) discovered that intracellular lipopolysaccharide (LPS), the Gram-negative bacterial cell-wall endotoxin, binds directly to the caspase-11 CARD (and to its human orthologs caspase-4 and caspase-5), inducing caspase-11 oligomerization and protease activation; Kayagaki et al. (2015) showed that activated caspase-11 cleaves GSDMD to cause pyroptosis. Against this background, Zanoni et al. (2016) reported a surprising deviation from this all-or-none scheme. They found that oxPAPC, a heterogeneous mixture of oxidized phospholipids produced when the membrane lipid PAPC (1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) is oxidized in dying or stressed cells (also abundant in atherosclerotic lesions and oxidatively damaged lung), acts as an endogenous caspase-11 ligand. In mouse DCs primed by any of several Toll-like receptor (TLR) ligands, oxPAPC—and purified components including POVPC, PGPC, and KOdiA-PC—elicited IL-1β release through a pathway requiring caspase-11, NLRP3, ASC, and caspase-1, and visible as caspase-11-dependent ASC/caspase-1 specks, yet without pyroptosis: the DCs survived and consequently primed strong adaptive immunity, a state the authors called hyperactivation. Biochemically, the interaction differed from LPS in every tested respect: LPS bound the CARD of caspase-11 with nanomolar affinity, whereas oxPAPC bound the catalytic domain (measured Kd approximately 1.3 micromolar), CARD mutations that abolished LPS binding left oxPAPC binding intact, oxPAPC suppressed both intrinsic and LPS-induced enzymatic activity of caspase-11, and a catalytically inactive caspase-11 mutant (C254A) still supported IL-1β release. Biotinylated oxPAPC, unlike biotinylated LPS, also captured caspase-1 independently of caspase-11, leading to a model of an oxPAPC-induced caspase-1/caspase-11 hetero-complex whose architecture was not determined. The authors closed by stating that the precise mechanisms governing oxPAPC-caspase interactions, and how these interactions promote inflammasome activities, await further investigation.

Subsequent work sharpened rather than settled the puzzle. Zanoni et al. (2017) showed that the LPS receptor CD14 captures extracellular oxPAPC and delivers it to the cytosol, and that defined oxPAPC components can hyperactivate macrophages, not only DCs. Chu et al. (2018) then showed the opposite face of the same interaction: in macrophages, oxPAPC binds caspase-11 and human caspase-4, competes with LPS for binding, and inhibits LPS-induced pyroptosis, IL-1β release, and septic shock. Evavold et al. (2018) established that GSDMD pores can mediate IL-1 secretion from living macrophages, providing a plausible conduit for non-lytic secretion, and Ross et al. (2018) dissected the dimerization and autoprocessing steps of LPS-driven caspase-11 activation. Very recently the LPS side has been resolved structurally: Cao et al. (2025) showed the caspase-11 CARD is a bipartite lipid-binding module with distinct regions engaging charged lipid headgroups and long acyl chains, Wang et al. (2026) used hydrogen-deuterium exchange and native mass spectrometry to show that LPS drives octameric CARD assemblies whereas PGPC occupies the same hydrophobic pocket but holds caspase-11 monomeric (inhibitor mode), and Sever et al. (2026) determined the heterogeneous stoichiometry of the LPS-caspase-11 non-canonical inflammasome. None of these structures captures the Zanoni route—catalytic-domain engagement by oxPAPC species leading to catalysis-independent NLRP3 activation. Reviews from the laboratory that originated the discovery (Zhivaki and Kagan 2022; Choi and Kagan 2025) still describe the oxPAPC-caspase biochemical interaction, the cytosolic delivery route, and species-specific lipid-receptor interactions as open questions.

Problem Statement

Determine the molecular mechanism by which endogenous oxidized phospholipids (oxPAPC and its defined components such as POVPC and PGPC) engage caspase-11 (and caspase-1) to activate the NLRP3/ASC inflammasome and elicit interleukin-1 release from living dendritic cells without caspase-11 catalytic activity and without pyroptosis. A complete answer must specify, as jointly supporting this single objective: (1) the architecture and stoichiometry of the oxPAPC-bound caspase-11 species and of the putative oxPAPC-induced caspase-1/caspase-11 hetero-complex, including which domains and residues contact which lipid species; (2) the lipid molecular determinants that confer caspase engagement, and the structural basis for oxPAPC inhibition of caspase-11 enzymatic activity; and (3) the biochemical steps by which the oxPAPC-bound, enzymatically inactive caspase-11 assembly triggers NLRP3/ASC inflammasome assembly and gasdermin-D-dependent, non-lytic IL-1β secretion, and why this pathway is agonistic in dendritic cells but antagonistic to caspase-11/LPS signaling in macrophages.

References

  1. Zanoni I, Tan Y, Di Gioia M, Broggi A, Ruan J, Shi J, Donado CA, Shao F, Wu H, Springstead JR, Kagan JC. An endogenous caspase-11 ligand elicits interleukin-1 release from living dendritic cells. Science 352, 1232-1236 (2016). DOI: 10.1126/science.aaf3036. https://doi.org/10.1126/science.aaf3036
  2. Shi J, Zhao Y, Wang Y, Gao W, Ding J, Li P, Hu L, Shao F. Inflammatory caspases are innate immune receptors for intracellular LPS. Nature 514, 187-192 (2014). DOI: 10.1038/nature13683. https://doi.org/10.1038/nature13683
  3. Kayagaki N, Stowe IB, Lee BL, O'Rourke K, Anderson K, Warming S, Cuellar T, Haley B, Roose-Girma M, Phung QT, Liu PS, Lill JR, Li H, Wu J, Kummerfeld S, Zhang J, Lee WP, Snipas SJ, Salvesen GS, Morris LX, Fitzgerald L, Zhang Y, Bertram EM, Goodnow CC, Dixit VM. Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling. Nature 526, 666-671 (2015). DOI: 10.1038/nature15541. https://doi.org/10.1038/nature15541
  4. Zanoni I, Tan Y, Di Gioia M, Springstead JR, Kagan JC. By capturing inflammatory lipids released from dying cells, the receptor CD14 induces inflammasome-dependent phagocyte hyperactivation. Immunity 47, 697-709.e3 (2017). DOI: 10.1016/j.immuni.2017.09.010. https://doi.org/10.1016/j.immuni.2017.09.010
  5. Chu LH, Indramohan M, Ratsimandresy RA, Gangopadhyay A, Morris EP, Monack DM, Dorfleutner A, Stehlik C. The oxidized phospholipid oxPAPC protects from septic shock by targeting the non-canonical inflammasome in macrophages. Nature Communications 9, 996 (2018). DOI: 10.1038/s41467-018-03409-3. https://doi.org/10.1038/s41467-018-03409-3
  6. Evavold CL, Ruan J, Tan Y, Xia S, Wu H, Kagan JC. The pore-forming protein gasdermin D regulates interleukin-1 secretion from living macrophages. Immunity 48, 35-44.e6 (2018). DOI: 10.1016/j.immuni.2017.11.013. https://doi.org/10.1016/j.immuni.2017.11.013
  7. Ross C, Chan AH, Von Pein J, Boucher D, Schroder K. Dimerization and auto-processing induce caspase-11 protease activation within the non-canonical inflammasome. Life Science Alliance 1, e201800237 (2018). DOI: 10.26508/lsa.201800237. https://doi.org/10.26508/lsa.201800237
  8. Li W, Zhang W, Deng M, Loughran P, Tang Y, Liao H, Zhang X, Liu J, Billiar TR, Lu B. Stearoyl lysophosphatidylcholine inhibits endotoxin-induced caspase-11 activation. Shock 50, 339-345 (2018). DOI: 10.1097/SHK.0000000000001012. https://doi.org/10.1097/SHK.0000000000001012
  9. Lagrange B, Benaoudia S, Wallet P, Magnotti F, Provost A, Michal F, Martin A, Di Lorenzo F, Py BF, Molinaro A, Henry T. Human caspase-4 detects tetra-acylated LPS and cytosolic Francisella and functions differently from murine caspase-11. Nature Communications 9, 242 (2018). DOI: 10.1038/s41467-017-02682-y. https://doi.org/10.1038/s41467-017-02682-y
  10. Zhivaki D, Borriello F, Chow OA, Doran B, Fleming I, Theisen DJ, Pallis P, Shalek AK, Sokol CL, Zanoni I, Kagan JC. Inflammasomes within hyperactive murine dendritic cells stimulate long-lived T cell-mediated anti-tumor immunity. Cell Reports 33, 108381 (2020). DOI: 10.1016/j.celrep.2020.108381. https://doi.org/10.1016/j.celrep.2020.108381
  11. Di Gioia M, Spreafico R, Springstead JR, Mendelson MM, Joehanes R, Levy D, Zanoni I. Endogenous oxidized phospholipids reprogram cellular metabolism and boost hyperinflammation. Nature Immunology 21, 42-53 (2020). DOI: 10.1038/s41590-019-0539-2. https://doi.org/10.1038/s41590-019-0539-2
  12. Hatscher L, Lehmann CHK, Purbojo A, Onderka C, Liang C, Hartmann A, Cesnjevar R, Bruns H, Gross O, Nimmerjahn F, Ivanovic-Burmazovic I, Kunz M, Heger L, Dudziak D. Select hyperactivating NLRP3 ligands enhance the TH1- and TH17-inducing potential of human type 2 conventional dendritic cells. Science Signaling 14, eabe1757 (2021). DOI: 10.1126/scisignal.abe1757. https://doi.org/10.1126/scisignal.abe1757
  13. Zhivaki D, Kagan JC. Innate immune detection of lipid oxidation as a threat assessment strategy. Nature Reviews Immunology 22, 322-330 (2022). DOI: 10.1038/s41577-021-00618-8. https://doi.org/10.1038/s41577-021-00618-8
  14. Hatscher L, Kaszubowski T, Amon L, Dudziak D, Heger L. Circumventing pyroptosis via hyperactivation shapes superior immune responses of human type 2 dendritic cells compared to type 3 dendritic cells. European Journal of Immunology 53, e2250123 (2023). DOI: 10.1002/eji.202250123. https://doi.org/10.1002/eji.202250123
  15. Eckhart L, Fischer H. Caspase-5: Structure, pro-inflammatory activity and evolution. Biomolecules 14, 520 (2024). DOI: 10.3390/biom14050520. https://doi.org/10.3390/biom14050520
  16. Zhivaki D, Kennedy SN, Park S, Boriello F, Devant P, Cao AB, Bahleda R, Murphy JM, McCabe LR, Evavold C, Chapman HD, Zanoni I, Ashenberg O, Xavier RJ, Kagan JC. Correction of age-associated defects in dendritic cells enables CD4+ T cells to eradicate tumors. Cell 187, 3888-3903.e18 (2024). DOI: 10.1016/j.cell.2024.05.026. https://doi.org/10.1016/j.cell.2024.05.026
  17. Cao AB, Devant P, Wang C, Sun M, Kennedy SN, Ma W, Ruan J, Kagan JC. LPS binding caspase activation and recruitment domains (CARDs) are bipartite lipid binding modules. Science Advances 11, eadt9027 (2025). DOI: 10.1126/sciadv.adt9027. https://doi.org/10.1126/sciadv.adt9027
  18. Choi JH, Kagan JC. Oxidized phospholipid damage signals as modulators of immunity. Open Biology 15, 240391 (2025). DOI: 10.1098/rsob.240391. https://doi.org/10.1098/rsob.240391
  19. Holley CL, Monteleone M, Fisch D, Libert AES, Ju RJ, Choi JH, Condon ND, Emming S, Crawford J, Lawrence GMEP, Coombs JR, Lefevre JG, Bajracharya R, Lahoud MH, Yap AS, Hamilton N, Stehbens SJ, Kagan JC, Ariotti N, Burgener SS, Schroder K. Pyroptotic cell corpses are crowned with F-actin-rich filopodia that engage CLEC9A signaling in incoming dendritic cells. Nature Immunology 26, 42-52 (2025). DOI: 10.1038/s41590-024-02024-3. https://doi.org/10.1038/s41590-024-02024-3
  20. Di Gioia M, Poli V, Tan PJ, Spreafico R, Chu A, Cuenca AG, Wu Z, Benamar M, Pandolfi L, Meloni F, Askarian F, Hsiao J, Borroum E, Nizet V, Gordts PLSM, Witztum JL, Chatila TA, Chou J, Zhou X, Springstead JR, Zanoni I. Epigenetic silencing of interleukin-10 by host-derived oxidized phospholipids supports a lethal inflammatory response to infections. Immunity 58, 2190-2207.e13 (2025). DOI: 10.1016/j.immuni.2025.06.017. https://doi.org/10.1016/j.immuni.2025.06.017
  21. Wang C, Lacey P, Tian T, Teran E, Liaqat M, Shivcharan S, Shumba P, He J, Rathinam VA, Wysocki VH, Ruan J. LPS-induced structural reorganization and polymerization drive noncanonical inflammasome activation. Science Advances 12, eadz4623 (2026). DOI: 10.1126/sciadv.adz4623. https://doi.org/10.1126/sciadv.adz4623
  22. Sever AIM, Aramini JM, Bonin JP, Zhao H, Wang H, Rubinstein JL, Schuck P, Kay LE. The dynamic and heterogeneous structure of the non-canonical inflammasome. Proceedings of the National Academy of Sciences of the United States of America 123, e2613431123 (2026). DOI: 10.1073/pnas.2613431123. https://doi.org/10.1073/pnas.2613431123