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Caspase-11 interaction with NLRP3 potentiates the noncanonical activation of the NLRP3 inflammasome

Nature Immunology volume 23, pages 705–717 (2022)Cite this article

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Abstract

Caspase-11 detection of intracellular lipopolysaccharide (LPS) from invasive Gram-negative bacteria mediates noncanonical activation of the NLRP3 inflammasome. While avirulent bacteria do not invade the cytosol, their presence in tissues necessitates clearance and immune system mobilization. Despite sharing LPS, only live avirulent Gram-negative bacteria activate the NLRP3 inflammasome. Here, we found that bacterial mRNA, which signals bacterial viability, was required alongside LPS for noncanonical activation of the NLRP3 inflammasome in macrophages. Concurrent detection of bacterial RNA by NLRP3 and binding of LPS by pro-caspase-11 mediated a pro-caspase-11–NLRP3 interaction before caspase-11 activation and inflammasome assembly. LPS binding to pro-caspase-11 augmented bacterial mRNA-dependent assembly of the NLRP3 inflammasome, while bacterial viability and an assembled NLRP3 inflammasome were necessary for activation of LPS-bound pro-caspase-11. Thus, the pro-caspase-11–NLRP3 interaction nucleated a scaffold for their interdependent activation explaining their functional reciprocal exclusivity. Our findings inform new vaccine adjuvant combinations and sepsis therapy.

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Fig. 1: Bacterial mRNA and stimulatory LPS are both required for caspase-11 and noncanonical inflammasome activation.
Fig. 2: Low cytosolic concentration of LPS triggers noncanonical activation of the NLRP3 inflammasome when cytosolic bacterial mRNA is also present.
Fig. 3: NLRP3 inflammasome assembly requires bacterial mRNA with LPS, irrespective of LPS-stimulatory activity.
Fig. 4: Requirement of NLRP3 and ASC for caspase-11 activation in response to live avirulent Gram-negative bacteria.
Fig. 5: Pro-caspase-11 protein is required for assembly of the NLRP3 inflammasome in response to avirulent Gram-negative bacteria.
Fig. 6: Coincident detection of bacterial RNA and LPS triggers a pro-caspase-11–NLRP3 interaction irrespective of LPS-stimulatory activity.
Fig. 7: Pro-caspase-11–NLRP3 interaction is upstream of NLRP3 inflammasome assembly and activation.
Fig. 8: Pro-caspase-11–NLRP3 interaction is mediated by the caspase-11 SCAF domain and NLRP3 LRR and PYD domains.

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Data availability

Datasets generated or analyzed during this study are available on reasonable request. Source data are provided with this paper.

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Acknowledgements

We are grateful to D. Amsen, N. Vabret, T. Kanneganti, K. Fitzgerald, V. Dixit, F. Shao, M. Goldberg, R. Ernst, J. Chipuk, J. Gelles-Hurwitz and R. Flavell for reagents and discussions. We thank D. Filipescu, C. Brou, P. Chastagner, A. Israël, A. Zanin-Zhorov, S. Waksal, M.A. Blander and S.J. Blander for their support. This work was supported by National Institutes of Health (NIH) grants AI127658 to J.M.B., AI139425 to J.C., R35NS111631 to S.R.J. and Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Disease Awards to J.M.B and to J.C. The Blander laboratory was supported by NIH grants DK072201, DK111862, AI095245 and AI123284 to J.M.B. J.M.B. was supported by the Leukemia and Lymphoma Society.

Author information

Author notes

  1. Zachary Hutchins

    Present address: Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA, USA

  2. Soumit Roy

    Present address: 182 E 95th St., New York, NY, USA

  3. Meimei Shan

    Present address: Patch Biosciences, New York, NY, USA

Authors and Affiliations

  1. Jill Roberts Institute for Research in Inflammatory Bowel Disease, Weill Cornell Medicine, Cornell University, New York, NY, USA

    Julien Moretti, Baosen Jia, Zachary Hutchins, Hilary Yip, Meimei Shan & J. Magarian Blander

  2. Joan and Sanford I. Weill Department of Medicine, Weill Cornell Medicine, Cornell University, New York, NY, USA

    Julien Moretti, Baosen Jia, Zachary Hutchins, Hilary Yip, Meimei Shan & J. Magarian Blander

  3. Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Soumit Roy

  4. Department of Pharmacology, Weill Cornell Medicine, Cornell University, New York, NY, USA

    Jiahui Wu & Samie R. Jaffrey

  5. Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, USA

    Jörn Coers

  6. Department of Immunology, Duke University Medical Center, Durham, NC, USA

    Jörn Coers

  7. Department of Microbiology and Immunology, Weill Cornell Medicine, Cornell University, New York, NY, USA

    J. Magarian Blander

  8. Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, Cornell University, New York, NY, USA

    J. Magarian Blander

  9. Immunology and Microbial Pathogenesis Program, Weill Cornell Medicine Graduate School of Medical Sciences, New York, NY, USA

    J. Magarian Blander

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Contributions

J.M. and J.M.B. directed the study, designed experiments and wrote the manuscript. J.M. performed most experiments, data and statistical analyses and all macrophage stimulations. B.J. performed molecular biology, cloning, transfection and ViewRNA ISH related experiments. Z.H. conducted the experiments related to measuring RNAbac in cytosolic extracts and kinetics of macrophage cell death. S.R. performed experiments related to Fig. 1c,f and the dual LPS and RNAbac requirement for noncanonical NLRP3 inflammasome activation during early stages of the work. M.S. conducted confocal microscopy lysosomal localization of bacteria and quantification. H.Y. performed experiments related to Extended Data Fig. 8c. J.W. and S.R.J. provided expertise on Pepper RNA-regulated fluorogenic protein stabilization. J.C. provided conceptual advice.

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Correspondence to J. Magarian Blander.

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Extended data

Extended Data Fig. 1 Bacterial mRNA and stimulatory LPS are both required for caspase-11 and noncanonical inflammasome activation.

a, Immunofluorescence confocal microscopy on bone marrow derived macrophages (hereafter ‘macrophages’) 2 h post stimulation with L or HK E. coli or untreated. b, Bar graph, % cells with LAMP1-LPS colocalization. c, Immunoblots of macrophage concentrated supernatants (20 h) or WCE (6 h), and cytokine concentrations and LDH release in culture supernatants (20 h) post stimulation with L, HK or HK E. coli supplemented with 100 ng ml–1 mRNA isolated from E. coli or eukaryotic cells. LDH measured by cytotoxicity assay; IL-1β, TNF and IL-6 by ELISA. Error bars, mean ± s.e.m. t-test was performed in b (n = 3). ns: nonsignificant. Bacteria:macrophage=20:1. Results represent at least three independent experiments.

Extended Data Fig. 2 Cytosolic levels of LPS and bacterial mRNA after macrophage stimulation with live or heat-killed bacteria.

a, Measurements of LPS in the cytosolic (C) or residual (R) fractions prepared from macrophages 2 hr post-transfection of indicated doses of LPS alone, co-transfection of 2 ng ml–1 LPS with 100 ng ml–1 E. coliLPSmut mRNA, or stimulation with L or HK E. coli or virulent E. coli as indicated. LPS was measured via Limulus Amebocyte Lysate method. b, Immunoblots of cytosolic (C) and residual (R) fractions from macrophages 2 h post stimulation with L E. coli, HK E. coli or L virulent E. coli, and probed with marker antibodies for indicated subcellular compartments. c, RT–qPCR for the bacterial gene groES, groEL, era and dnaE on cytosolic fractions prepared 2 h post macrophage transfection with indicated doses of ultrapure LPS and mRNA prepared from E. coli, or stimulation with L, HK or HK E. coli or virulent E. coli supplemented with mRNA prepared from each bacterium. d,e, mNeonGreen fluorescence measurements on total extracts (d) or cytosolic versus residual fractions (e) of macrophages stably expressing tDeg-tagged mNeonGreen 2 h post stimulation with E. coli expressing or not Pepper RNA, or after treatment with proteasome inhibitor MG132 as indicated. By expressing a Pepper RNA-regulated fluorogenic protein (mNeonGreen-tDeg) in the cytosol of macrophages, we noted 1.5-2-fold increase in mNeonGreen fluorescence following phagocytosis of recombinant Pepper RNA-expressing E. coli compared to the 2-2.5-fold increase with the proteasomal inhibitor MG132, indicating cytosolic access of Pepper RNA derived from phagocytosed E. coli bound to and stabilized the fluorogenic protein in the cytosol of macrophages. f,h, Confocal microscopy of direct fluorescence RNA in situ hybridization (ViewRNA ISH) to detect two RNAbac transcripts encoding for either mCherry (f) or endogenous GroES (h) from recombinant mCherry E. coli showed significantly more probe signal in macrophages at 6 h post-phagocytosis of live (L) compared with killed (K) bacteria. Killed bacteria were visualized by anti-LPS staining due to loss of mCherry fluorescence. RNA probe signal localized with live bacteria in lysosomes labeled with LAMP-1 as expected, but almost half of this signal did not colocalize to these bacteria suggesting cytosolic access. Inserts show magnification of indicated area. g,i, Bar graphs show quantification of probe signal in (f) and (h), respectively. Bacteria:macrophage=20:1. Error bars, mean ± s.e.m. One-way ANOVA followed by multiple comparisons Sidak tests and p values are indicated in bar graphs in g (Total number, Untreated: n = 13, L: n = 19, K: n = 15, K(LPS): n = 17 – Not colocalized, Untreated: n = 11, L, K: n = 14) and i (Total number, Untreated: n = 11, L: n = 14, K, K(LPS): n = 10 – Not colocalized, n = 10). Results represent at least three independent experiments.

Extended Data Fig. 3 Bacterial mRNA and LPS are both required for IL-1β secretion and generation of the active form of caspase-11.

a, Cytokine concentrations and LDH release in culture supernatants (20 h) post-transfection of WT or Nlrp3–/– macrophages as indicated with 2 ng ml–1 ultrapure LPS and/or 10, 30 and 100 ng ml–1 of mRNA prepared from E. coli LPSmut, L. innocua, or in vitro transcribed (IVT). b, Immunoblots of macrophage concentrated supernatants, WCE, mixed concentrated supernatants and WCE, or pulldown of caspases with biotinylated zVAD-FMK from WT, Nlrp3–/– or Casp11–/– macrophages 20 h post stimulation with L, HK or HK E. coli supplemented with E. coli total RNA (RNAtot), or transfection with indicated doses of ultrapure LPS and E. coli RNAtot. c, In vitro zVAD-AMC fluorescence post-incubation with immunoprecipitates of endogenous caspase-11 from macrophages stimulated for 6 h or 12 h as indicated with L, HK, or HK E. coli supplemented with E. coli RNAtot (10 µg ml–1). No IgG served as a control for Protein G-bound proteins alone. Bacteria:macrophage=20:1. Error bars, mean ± s.e.m. Results represent at least three independent experiments.

Extended Data Fig. 4 LPS is required alongside bacterial mRNA for assembly of the NLRP3 inflammasome and regardless of LPS-stimulatory activity.

a–c, Immunofluorescence confocal microscopy on macrophages 16 h post stimulation of indicated macrophage genotypes with L, gentamicin-killed (K) or K red fluorescent protein (RFP) expressing recombinant E. coli supplemented with E. coli RNAtot. Note, the partial nuclear staining pattern has previously been observed (see Methods) and appears to be a specific signal given its absence in Pycard–/– macrophages. In a,b, insets show magnification of indicated areas. White arrowheads point to ASC specks. Phalloidin delineates the macrophage actin cytoskeleton. Scale bar=10 µm. Bar graphs, % cells exhibiting ASC specks. Error bars, mean ± s.e.m. t-test was performed in b (n = 5). One-way ANOVA followed by multiple comparisons Sidak tests were performed in a (n = 5) and c (n = 6). P values are indicated in bar graphs. Bacteria:macrophage=20:1. Results represent at least three independent experiments.

Extended Data Fig. 5 Kinetics of noncanonical activation of the NLPR3 inflammasome in macrophages in response to virulent and avirulent bacteria.

a, LDH released in culture supernatants of macrophages at indicated time points post-stimulation with Live E. coli (avirulent), virulent E. coli or virulent Salmonella. LDH measured by cytotoxicity assay. b,c, Kinetics of SYTOX Red incorporation over 72 h in macrophages stimulated with Live E. coli or virulent E. coli, Salmonella ΔSpi1/2 (avirulent) or wild type (WT) (virulent), or Shigella BS103 (avirulent) or WT (virulent) (b), and representative images of SYTOX Red incorporation at selected time points (c). Peaks of SYTOX incorporation occurred faster (blue boxes) in response to virulent bacteria, before decreasing likely due to destruction of cell structure, while macrophages stimulated with avirulent bacteria were still incorporating SYTOX and peaked later (orange boxes). Scale bar=150 µm. d, Counting of live macrophages at the indicated time points post stimulation with Live E. coli or virulent E. coli. Counts were normalized to the unstimulated macrophages for each time point. e, Immunoblots of macrophage WCE at the indicated time points post stimulation with Live E. coli, virulent E. coli or virulent Salmonella. f, Immunoblots of macrophage concentrated supernatants 6 h post stimulation with Live E. coli or virulent E. coli. Error bars, mean ± s.e.m. Bacteria:macrophage =20:1 for E. coli and virulent E. coli, 5:1 for Salmonella and Shigella strains. Results represent at least three independent experiments.

Extended Data Fig. 6 NLRP3 stimuli specifically couple with LPS in mediating noncanonical activation of the NLRP3 inflammasome.

a,b, Immunoblots of macrophage concentrated supernatants (20 h), WCE (6 h), or cross-linked fractions (16 h), and cytokine concentrations in culture supernatants (20 h) as indicated post stimulation with L, HK or HK E. coli supplemented with indicated doses of RNAtot or Nigericin (a), and post-treatment with Nigericin or transfection of indicated doses of poly(dA:dT) or Flagellin with or without prior priming with 100 ng ml–1 LPS for 12–16 h (b). IL-1β and IL-6 by ELISA. Error bars, mean ± s.e.m.

Extended Data Fig. 7 dTGN colocalization and biochemical pro-caspase-11–NLRP3 interaction.

a,b, Immunofluorescence confocal microscopy 16 h post stimulation of WT macrophages with L or HK virulent E. coli (a), and WT, Casp11–/– and Nlrp3–/– macrophages as indicated with L E coli (b). Scale bar=10 µm. In (a), side micrograph insets in the triple merges show magnification of the indicated areas. c,d, Immunoprecipitation (IP) of endogenous caspase-11 or NLRP3 as indicated, and immunoblotting for co-immunoprecipitating proteins and WCE proteins (labels to left of immunoblot panels) from macrophages stimulated 12 h with L, HK or HK E. coli supplemented with indicated dose of E. coli RNAtot. (c) and L, HK or HK E. coli supplemented with indicated dose of E. coli RNAtot or Nigericin, E. coli RNAtot alone or Nigericin alone (d). Bacteria:macrophage=20:1. Results represent at least three independent experiments.

Extended Data Fig. 8 GSDMD is important for noncanonical activation of the NLRP3 inflammasome in response to Gram-negative bacteria.

a–c, Immunoblots of macrophage concentrated supernatants (20 h) or WCE at 6 h (a,b) and 20 h (c) post stimulation of WT and Gsdmd–/– macrophages with L or HK E. coli (avirulent) or virulent E. coli as indicated. (d) Immunoblots of macrophage concentrated supernatants (20 h) or WCE (6 h) post stimulation of macrophages with L or HK E. coli with or without zVAD-FMK treatment.

Extended Data Fig. 9 Model for noncanonical activation of the NLRP3 inflammasome by live Gram-negative bacteria.

Following phagocytosis of live Gram-negative bacteria, two classes of PAMPs are exposed to cytoplasmic pattern recognition receptors: the classical PAMP LPS, shared by live and killed bacteria, and the vita-PAMP bacterial mRNA (mRNAbac), present only in live bacteria. Coincident detection of mRNAbac and LPS from virulent or avirulent Gram-negative bacteria alike promotes a physical and mutually exclusive interaction between NLRP3 and the intracellular LPS receptor pro-caspase-11. This interaction localizes to the dispersed Trans-Golgi Network (TGN) and is mediated through the pro-caspase-11 SCAF domain and the LRR and PYD domains of NLRP3. The interaction of NLRP3 and pro-caspase-11 is upstream of their activation: It does not require the ability of LPS to activate caspase-11 and can still occur in the absence of GSDMD. It also does not require ASC and caspase-1 which are important for NLRP3 activation. Besides their interaction, NLRP3 and pro-caspase-11 are reciprocally required for their function: LPS binding to but not activation of pro-caspase-11, is necessary for mRNAbac-mediated NLRP3 inflammasome assembly. Reciprocally, NLRP3 and ASC but not caspase-1 are required for pro-caspase-11 activation, indicating the necessity for ‘nascent’ NLRP3 inflammasome assembly upon sensing the viability of Gram-negative bacteria (detection of mRNAbac) and irrespective of bacterial virulence factor expression. Although NLRP3-ASC oligomers can form in the absence of pro-caspase-1, these oligomers are unstable indicating stabilization of the ‘nascent’ assembled NLRP3 inflammasome upon pro-caspase-1 recruitment. Furthermore, higher concentrations of intracellular LPS, likely due to virulence factor activity during infection with virulent cell-invasive Gram-negative bacteria, trigger faster kinetics of plasma membrane permeabilization/pyroptosis compared with avirulent bacteria, and independently of NLRP3 and ASC2,9. Collectively, the model that emerges demonstrates two modes of pro-caspase-11 activation by LPS, a fast NLRP3-independent mode triggered by the concurrent expression of bacterial virulence factors, and a slower NLRP3-dependent mode triggered by coincident detection of the vita-PAMP mRNAbac that signifies bacterial viability. (SCAF, scaffold; PYD, Pyrin; LRR, leucine rich repeat).

Extended Data Fig. 10 Characterization and mapping of the pro-caspase-11 and NLRP3 interaction.

a, Schematic indicating the different truncated mutants of NLRP3 used for co-immunoprecipitation experiments in 293 T cells. All forms were fused to 3xHA tag in N-Terminus. b, Schematic indicating the different truncated mutants of casp11C254A used for co-immunoprecipitation experiments in 293 T cells. All forms were fused to 3xFLAG tag in N-Terminus. c, Immunoprecipitation and immunoblotting for coimmunoprecipitating and WCE proteins of overexpressed FLAG-caspase-11 either casp11C254A FL, Casp-11 C254A ΔCARD or Casp-11 C254A ΔSCAF with or without HA-NLRP3FL in 293 T cells 24 h post-transfection. To equilibrate the levels of FLAG-caspase-11 mutants in the immunoprecipitates, 4 times more protein extracts were submitted to anti-FLAG immunoprecipitation when FLAG-casp11C254A ΔSCAF was expressed (labelled 4X) compared to when either FLAG-casp11C254A FL or FLAG-casp11C254A ΔCARD were expressed (labelled 1X). Therefore, note that all proteins from FLAG-casp11C254A ΔSCAF samples (including HA-NLRP3FL) were 4 times more abundant during anti-FLAG immunoprecipitation, yet HA-NLRP3FL was still much less co-immunoprecipitated with FLAG-casp11C254A ΔSCAF compared to FLAG-casp11C254A FL. Immunoblotted proteins are indicated to left of each immunoblot panel. d, Schematic representation of CARD, DED, CASPASE p20 and CASPASE p10 domains of murine inflammatory and apoptotic caspases. SCAF domain is composed of CASPASE p20 and CASPASE p10 domains. All caspases, with the exception of the short forms of mouse and human caspase-12, have SCAF domains. The alignment E values were calculated using the NCBI alignment tool. e, Similarity coefficients between caspase-11 and other murine caspases. f, Protein sequence alignment for murine caspases. The alignment diagram was generated using the alignment module of SnapGene software. g, Schematic indicating the different caspase-11C254A and caspase-9 chimeras used for co-immunoprecipitation experiments in 293 T cells. All forms were fused to 3xFLAG tag in N-terminus.

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Moretti, J., Jia, B., Hutchins, Z. et al. Caspase-11 interaction with NLRP3 potentiates the noncanonical activation of the NLRP3 inflammasome. Nat Immunol 23, 705–717 (2022). https://doi.org/10.1038/s41590-022-01192-4

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