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Epithelial Nlrp10 inflammasome mediates protection against intestinal autoinflammation

Nature Immunology volume 24, pages 585–594 (2023)Cite this article

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Abstract

Unlike other nucleotide oligomerization domain-like receptors, Nlrp10 lacks a canonical leucine-rich repeat domain, suggesting that it is incapable of signal sensing and inflammasome formation. Here we show that mouse Nlrp10 is expressed in distal colonic intestinal epithelial cells (IECs) and modulated by the intestinal microbiome. In vitro, Nlrp10 forms an Apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-dependent, m-3M3FBS-activated, polyinosinic:polycytidylic acid-modulated inflammasome driving interleukin-1β and interleukin-18 secretion. In vivo, Nlrp10 signaling is dispensable during steady state but becomes functional during autoinflammation in antagonizing mucosal damage. Importantly, whole-body or conditional IEC Nlrp10 depletion leads to reduced IEC caspase-1 activation, coupled with enhanced susceptibility to dextran sodium sulfate-induced colitis, mediated by altered inflammatory and healing programs. Collectively, understanding Nlrp10 inflammasome-dependent and independent activity, regulation and possible human relevance might facilitate the development of new innate immune anti-inflammatory interventions.

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Fig. 1: Nlrp10 expressed in mouse distal colon IECs forms an in vitro and ex vivo inflammasome.
Fig. 2: Nlrp10 inflammasome is inactivated at steady state.
Fig. 3: Nlrp10 inflammasome forms during the induction of DSS colitis.
Fig. 4: Nlrp10-deficient mice have greater DSS-induced intestinal inflammation.
Fig. 5: Nlrp10 activates protective mucosal pathways during the induction of autoinflammation.

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Data availability

RNA sequences are available at the European Nucleotide Archive under accession no. PRJEB50590. Metagenomics sequences are available at the European Nucleotide Archive under accession no. PRJEB50590. The single-cell data analysis was performed using count data for WT mice downloaded from Gene Expression Omnibus accession no. GSE148794. Source data are provided with this paper.

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Acknowledgements

We thank the members of the Elinav laboratory, Weizmann Institute of Science and members of the German Cancer Research Center Microbiome & Cancer division for insightful discussions; C. Bar-Nathan for dedicated GF mouse husbandry. D.Z. is the recipient of the European Crohn’s and Colitis Organization Fellowship and is supported by the Ke Lin Program of the First Affiliated Hospital, Sun Yat‐sen University. L.K. is funded by the postdoctoral Walter Benjamin fellowship from the German Research Foundation (no. 447836288). Y.H. is supported by the Ke Lin Program of the First Affiliated Hospital, Sun Yat‐sen University. M.S.D. is supported by the Marie Skłodowska-Curie Individual Fellowship (Horizon 2020 grant no. GAP-845066). R.V.-M. is the recipient of the Weizmann Institute ‘La Caixa’ Foundation Postdoctoral Fellowship. H.S. is an incumbent of the Vera Rosenberg Schwartz Research Fellow Chair. S.K.A. is supported by the Israeli Ministry of Science and Technology Zvi Yanai Fellowship. M.H. is funded by the German Research Foundation (no. 438122637). E.E. is supported by the Adelis Foundation, Pearl Welinsky Merlo Scientific Progress Research Fund, Park Avenue Charitable Fund, Hanna and Dr. Ludwik Wallach Cancer Research Fund, Daniel Morris Trust, Wolfson Family Charitable Trust and Wolfson Foundation, Ben B. and Joyce E. Eisenberg Foundation, White Rose International Foundation, Estate of Bernard Bishin for the WIS-Clalit Program, Else Kröener-Fresenius Foundation, Jeanne and Joseph Nissim Center for Life Sciences Research and by grants funded by the European Research Council, Israel Science Foundation, Israel Ministry of Science and Technology, Israel Ministry of Health, Helmholtz Foundation, Garvan Institute of Medical Research, European Crohn’s and Colitis Organization, Deutsch-Israelische Projektkooperation, Infectious Diseases Society of America Foundation and Wellcome Trust. E.E. is the incumbent of the Sir Marc and Lady Tania Feldmann Professorial Chair, a senior fellow of the Canadian Institute of Advanced Research and an international scholar of the Bill & Melinda Gates Foundation and Howard Hughes Medical Institute.

Author information

Author notes

  1. These authors contributed equally: Danping Zheng, Gayatree Mohapatra, Lara Kern.

Authors and Affiliations

  1. Systems Immunology Department, Weizmann Institute of Science, Rehovot, Israel

    Danping Zheng, Gayatree Mohapatra, Lara Kern, Yiming He, Merav D. Shmueli, Rafael Valdés-Mas, Aleksandra A. Kolodziejczyk, Miguel Camacho Rufino, Emmanuelle Ceddaha, Sandy Shimshy, Ryan James Hodgetts, Mally Dori-Bachash, Christian Kleimeyer, Kim Goldenberg, Melina Heinemann, Hagit Shapiro, Yifat Merbl, Suhaib K. Abdeen & Eran Elinav

  2. Department of Gastroenterology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China

    Danping Zheng, Yiming He & Minhu Chen

  3. Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany

    Tomasz Próchnicki, Matilde B. Vasconcelos & Eicke Latz

  4. Division of Cancer-Microbiome Research, German Cancer Research Center, Heidelberg, Germany

    Lena Schorr, Franziska Hertel, Ye Seul Lee, Jens Puschhof & Eran Elinav

  5. Faculty of Biosciences, Heidelberg University, Heidelberg, Germany

    Lena Schorr

  6. Lydia Becker Institute of Immunology and Inflammation, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK

    Ryan James Hodgetts

  7. Department of Veterinary Resources, Weizmann Institute of Science, Rehovot, Israel

    Noa Stettner & Alon Harmelin

  8. Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA

    Richard A. Flavell

  9. Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA

    Richard A. Flavell

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Contributions

D.Z., G.M. and L.K. conceived the study, designed, performed, analyzed and interpreted the experiments, and wrote the manuscript. Y.H., M.D.S., T.P., M.B.V., J.P., L.S., F.H., Y.S.L., M.C.R., E.C., S.S., R.J.H., S.K.A., C.K., K.G. and M.H. performed and assisted with the experiments. R.V-M. and A.K. performed the computational analyses and provided essential tools and insights. M.D-B. performed sample processing and next-generation DNA sequencing. N.S. supervised mouse experimentation and provided critical insights. A.H. analyzed the pathology specimens. H.S., M.C., R.A.F., E.L. and Y.M. provided essential tools and key insights. S.K.A. jointly supervised the study and wrote the manuscript. E.E. conceived and supervised the study and wrote the manuscript.

Corresponding authors

Correspondence to Suhaib K. Abdeen or Eran Elinav.

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Competing interests

E.L. is cofounder and consultant of IFM Therapeutics and Odyssey Therapeutics as well as cofounder and board member of Dioscure Therapeutics and Stealth Biotech. E.E. is a scientific founder of DayTwo and BiomX, and a paid consultant to Hello Inside, Igen and Aposense, in topics unrelated to this manuscript. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Generation strategy of Nlrp10-deficient mice.

(a) Nlrp10, Epcam and Ptprc mRNA expression in colonic epithelial and lamina propria fractions (n = 5). IEC: intestinal epithelial cells; IEL: intraepithelial lymphocytes; LP: lamina propria (Unpaired t-test, ****P = 0.00001, **P = 0.008149, ****P = 0.000089). (b) Generation strategy of Nlrp10D/D mice. (c, d) Distal colon mRNA expression of (c) Nlrp10 (Nlrp10loxP/loxP n = 5; Nlrp10D/D n = 11) (Unpaired t-test, ****P < 0.0001) and (d) Dock8 (Unpaired t-test) (Nlrp10loxP/loxP n = 3; Nlrp10D/D n = 4) in Nlrp10loxP/loxP and Nlrp10D/D littermate mice. Mean ± SEM. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. Statistical test used in (a), (c) and (d) was two-sided. Created with BioRender.com.

Source data

Extended Data Fig. 2 Nlrp10 forms an in vitro inflammasome complex.

(a) ASC speck formation in human ASCTagBFP HEK cells (HEKASC) and human ASCTagBFP HEK cells expressing human NLRP10mCitrine (HEKASC_Nlrp10) untreated or treated with m-3M3FBS (n = 2, in each sample 5 independent random fields were quantified) (Unpaired t-test, ****P = 0.000002). (b) IL-18 ELISA of supernatants of human ASCTagBFP HEK cells (HEKASC) and human ASCTagBFP HEK cells expressing human Nlrp10mCitrine (HEKASC_Nlrp10) transfected with human myc-caspase-1, and pro- IL-18, and treated with m-3M3FBS (n = 3) (Unpaired t-test, **P = 0.00789). (c) Representative confocal images of ASC speck formation in human ASCTagBFP HEK cells (HEKASC) and human ASCTagBFP HEK cells expressing human Nlrp10mCitrine (HEKASC_Nlrp10) untreated, or treated with poly(I:C), m-3M3FBS either alone or in combination (corresponding to Fig. 1e), scale- 50 µm. ASC-tag BFP is pseudocolored as red, nucleus is stained with DRAQ5 and pseudocolored as blue. Mean ± SEM. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. Statistical test used in (a-b) was two-sided.

Source data

Extended Data Fig. 3 In vitro activation and modulation of the Nlrp10 inflammasome.

(a) IL-18 ELISA of supernatants of human ASCTagBFP HEK cells (HEKASC) and human ASCTagBFP HEK cells expressing human Nlrp10mCitrine (HEKASC_Nlrp10) transfected with human myc- caspase-1, and pro-IL-18, and treated with m-3M3FBS and a panel of toll-like receptor (TLR) agonists, including the TLR2 agonist HKLM, TLR2/6 agonist FSL1, TLR4 agonist LPSEK, TLR5 agonist FLAST, and TLR3 agonist poly(I:C) (LMW: low molecular weight, HMW: high molecular weight) (n = 3) (Unpaired t-test, P values are *P = 0.018948, **P = 0.005017, ***P = 0.000883). (b) Human ASCTagBFP HEK cells (HEKASC) and human ASCTagBFP HEK cells expressing human Nlrp10mCitrine (HEKASC_Nlrp10) and HEK cells expressing human Nlrp10mCitrine (HEKNlrp10) immunoprecipitated using anti-GFP beads to pull down Nlrp10 and immunoblotted with anti-GFP and anti-ASC. (c) IL-18 (Unpaired t-test, ****P < 0.0001) and (d) IL-1β (Unpaired t-test, ***P = 0.0001, **P = 0.005, ****P < 0.0001) ELISA of supernatants of Aim2−/−CRISPR immortalized mouse macrophages stably expressing full-length human Nlrp10- mCitrine (MacsAim2−/−_hNlrp10), mouse Nlrp10-mCitrine (MacsAim2−/−_mNlrp10), human Nlrp10-PYD (only)-mCitrine (MacsAim2−/−_PYD), or human Nlrp10-NACHT (only)-mCitrine (MacsAim2−/−_NACHT), treated with HMW poly(I:C), m-3M3FBS either alone or in combination (n = 3). (e) IL-1β ELISA of supernatants of differentiated organoids derived from the distal colons of Nlrp10loxP/loxPCMV-Cre+ and Nlrp10loxP/loxP littermates treated with vehicle, HMW poly(I:C), or both HMW Poly(I:C) and m-3M3FBS (n = 6, results pooled from 2 independent experiments; n = 3 in the positive control group). CMV: Cytomegalovirus. Recombinant IL-1β was used as the positive control. Mean ± SEM. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. Statistical test used in (a), (c-d) was two-sided.

Source data

Extended Data Fig. 4 Generation of IEC- specific Nlrp10- deficient mice.

(a) Generation strategy of Nlrp10loxP/loxPVil1+ mice. (b) Expression of Nlrp10 mRNA in sorted EpCAM+ cells of Nlrp10loxP/loxPVil1+ and Nlrp10loxP/loxP littermate mice (Nlrp10loxP/loxP n = 4; Nlrp10loxP/loxPVil1+ n = 8) (Unpaired t-test, ****P < 0.0001). (c) Expression of Nlrp10 mRNA in spleens of Nlrp10loxP/loxPVil1+ and Nlrp10loxP/loxP littermate mice (Nlrp10loxP/loxP n = 6; Nlrp10loxP/loxPVil1+ n = 7) (Unpaired t-test). Mean ± SEM. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. Statistical test used in (b-c) was two-sided.

Source data

Extended Data Fig. 5 Nlrp10 inflammasome is activated upon induction of DSS colitis.

(a) A representative Immunoblot of pro-caspase-1 (p45), cleaved caspase-1 (p20), pro-IL-18 and cleaved IL-18 (n = 6), (b) IL-18 ELISA of supernatants of distal colon explants (n = 6) in Nlrp10D/D and Nlrp10loxP/loxP littermate mice at day 4 after DSS initiation (Unpaired t-test, P = 0.073). (c) Densitometry quantification of cleaved IL-18/pro-IL-18 (Mann-Whitney U test, *P = 0.0286) (d) and cleaved IL-18/actin (Unpaired t-test. *P = 0.0303) in distal colons of Nlrp10D/D and Nlrp10loxP/loxP littermate mice at day 10 after DSS initiation (corresponding to Fig. 3d) (n = 4). (e) A representative immunoblot and (f) quantification (Mann-Whitney U test) (f) of cleaved IL-1β in distal colons of Nlrp10D/D and Nlrp10loxP/loxP littermate mice at day 10 after DSS initiation (n = 4). (g) A representative immunoblot and (h) quantification (Unpaired t-test) of cleaved GSDMD in distal colons of Nlrp10D/D and Nlrp10loxP/loxP littermate mice at day 10 after DSS initiation (n = 4). GSDMD: gasdermin D. Numbers labeled in (a), (e) and (g) represent individual mice in each group. Mean ± SEM. *: p < 0.05. Statistical test used in (b- d), (f) and (h) was two-sided.

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Extended Data Fig. 6 Nlrp10D/D mice feature an exacerbated DSS- induced intestinal inflammation.

(a-d) Quantification of the severity of DSS colitis in Nlrp10D/D and Nlrp10loxP/loxP littermate mice. (a) Representative images of colonoscopy and (b) colon lengths at day 7 after DSS initiation, (c) colon length (cm) (Nlrp10loxP/loxP n = 19; Nlrp10D/D n = 18) and (Unpaired t-test, ***P = 0.0007) (d) representative images of H&E- stained colonic sections at day 12 after DSS initiation (scale bar-100 µm). (e) IL-6 ELISA (n = 4) (Unpaired t-test, ***P = 0.0002), (f) and TGF-β (Nlrp10loxP/loxP n = 4; Nlrp10D/D n = 6) in distal colon homogenates of Nlrp10D/D and Nlrp10loxP/loxP littermate mice at day 10 after DSS initiation (Unpaired t-test). Mean ± SEM. *: p < 0.05, **: p < 0.01, ***: p < 0.001. Statistical test used in (c), (e-f) was two-sided.

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Extended Data Fig. 7 Induction of DSS colitis in 4-Way Bone marrow chimeric mice.

a) Schematic of bone marrow transplantation generating 4 types of bone marrow chimeric mice, using both wild-type (WT) and Nlrp10D/D mice as donors and recipients. (b-h) Quantification of colitis severity (for (b-d), WT to WT, KO to WT, WT to KO n = 7; KO to KO n = 9; for (f and h), WT to WT, KO to WT n = 7; WT to KO n = 6; KO to KO n = 8). (b) Body weight, (c) area under the curve (AUC) of the weight change (unpaired t test), (d) colitis severity score (Unpaired t-test, *P = 0.0171, Mann-Whitney U test, ***P = 0.0009), and (e) representative colonoscopy images at day 7 after DSS initiation, (f) colon length (Mann-Whitney U test, unpaired t-test, *P = 0.0472, *P = 0.0482), (g) representative images of the colon length, (h) IL-18 ELISA of supernatants of distal colon explants at day 12 after DSS initiation (unpaired t test, Mann-Whitney U test *P = 0.0196, **P = 0.002). Mean ± SEM. *: p < 0.05, **: p < 0.01, ***: p < 0.001. Statistical test used in (c-d), (f) and (h) was two- sided. Created with BioRender.com.

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Extended Data Fig. 8 Exacerbated DSS- induced colitis in intestinal epithelial Nlrp10-deficient mice.

(a, b) Quantification of DSS colitis severity in Nlrp10loxP/loxPVil1+ and Nlrp10loxP/loxP littermate mice. (a) Representative colonoscopy images at day 7 after DSS initiation, (b) representative images of the colon length at day 12 after DSS initiation. (c–e) ELISA of (c) IL-6 (Mann-Whitney U test), (d) TNF-α (Mann-Whitney U test) and (e) IL-10 (Mann-Whitney U test) in supernatants of distal colon explants in Nlrp10loxP/loxPVil1+ vs. Nlrp10loxP/loxP littermate mice at day 12 after DSS initiation (Nlrp10loxP/loxP n = 8; Nlrp10loxP/loxPVil1+ n = 12) Mean ± SEM. *: p < 0.05. Statistical test used in (c-e) was two-sided.

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Extended Data Fig. 9 Flow cytometry assessment of colonic lamina propria immune cells.

(a, b) Flow cytometry comparison of colonic lamina propria (a) myeloid (Mann-Whitney U test, unpaired t test, *P = 0.0481, unpaired t test, *P = 0.0109) and (b) lymphocyte cell populations between Nlrp10D/D and Nlrp10loxP/loxP littermate mice at day 4 after DSS initiation (n = 5) (unpaired t test, Mann- Whitney U test). (c, d) Flow cytometry Comparison of colonic lamina propria (c) myeloid cell (unpaired t test, *P = 0.0135, **P = 0.0096) and (d) lymphocyte (unpaired t test, *P = 0.0184, **P = 0.0095, **P = 0.005, Mann-Whitney U test, *P = 0.0159) populations between Nlrp10D/D and Nlrp10loxP/loxP littermate mice at day 7 after DSS initiation (Nlrp10loxP/loxP n = 5; Nlrp10D/D n = 4). Mean ± SEM. *: p < 0.05, **: p < 0.01. Statistical test used in (a- d) was two-sided.

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Extended Data Fig. 10 Severe colitis in Nlrp10D/D is associated with altered phospho NF-κB.

(a) A representative immunoblot and (b, c) quantification of phospho-NF-κB in the distal colons of Nlrp10D/D and Nlrp10loxP/loxP littermate mice at day 10 after DSS initiation (unpaired t test **P = 0.0022; Mann-Whitney U test *P = 0.0238) (Nlrp10loxP/loxP n = 3; Nlrp10D/D n = 6). (d) A representative immunoblot (e, f) and quantification of phospho-Stat1 in distal colons of Nlrp10D/D and Nlrp10loxP/loxP littermate mice at day 10 after DSS initiation (unpaired t test) (n = 4). (g) A representative immunoblot and (h-i) quantification of phospho-Stat3 in distal colons of Nlrp10D/D and Nlrp10loxP/loxP littermate mice at day 10 after DSS initiation (Mann-Whitney U test) (n = 4). (j) Systemic fluorescein isothiocyanate-dextran (FITC-Dextran) levels 3 hours following FITC-Dextran gavage to Nlrp10D/D and Nlrp10loxP/loxP littermate mice at day 4 after DSS initiation (unpaired t test) (Nlrp10loxP/loxP n = 5; Nlrp10D/D n = 6). Numbers labeled in (a), (d) and (g) represent individual mice in each group. Mean ± SEM. *: p < 0.05, **: p < 0.01. Exact p values and statistical tests are presented in Supplementary Table 1.

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Supplementary information

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Zheng, D., Mohapatra, G., Kern, L. et al. Epithelial Nlrp10 inflammasome mediates protection against intestinal autoinflammation. Nat Immunol 24, 585–594 (2023). https://doi.org/10.1038/s41590-023-01450-z

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