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Environmental allergens trigger type 2 inflammation through ripoptosome activation

Nature Immunology volume 22, pages 1316–1326 (2021)Cite this article

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Abstract

Environmental allergens, including fungi, insects and mites, trigger type 2 immunity; however, the innate sensing mechanisms and initial signaling events remain unclear. Herein, we demonstrate that allergens trigger RIPK1–caspase 8 ripoptosome activation in epithelial cells. The active caspase 8 subsequently engages caspases 3 and 7, which directly mediate intracellular maturation and release of IL-33, a pro-atopy, innate immunity, alarmin cytokine. Mature IL-33 maintained functional interaction with the cognate ST2 receptor and elicited potent pro-atopy inflammatory activity in vitro and in vivo. Inhibiting caspase 8 pharmacologically and deleting murine Il33 and Casp8 each attenuated allergic inflammation in vivo. Clinical data substantiated ripoptosome activation and IL-33 maturation as likely contributors to human allergic inflammation. Our findings reveal an epithelial barrier, allergen-sensing mechanism that converges on the ripoptosome as an intracellular molecular signaling platform, triggering type 2 innate immune responses. These findings have significant implications for understanding and treating human allergic diseases.

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Fig. 1: Effect of environmental allergens on ripoptosome-dependent, intracellular pIL-33 maturation and release.
Fig. 2: Effect of caspase 3 and 7 on proteolytic maturation of pIL-33 and ST2-interacting mIL-33 forms.
Fig. 3: IL-33 bioactivity as a function of G176-270T C-terminal domain.
Fig. 4: Effect of ripoptosome inhibition on IL-33-dependent airway inflammation.
Fig. 5: Effect of caspase 8 on allergic airway inflammation.
Fig. 6: Ripoptosome activation relationship with mIL-33 expression and eosinophilia in EoE.

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Data availability

No accession codes, unique identifiers or web links for publicly available datasets were generated or required for this publication. Source data including the associated immunoblot scans for the main and extended figures are provided with this paper. All figures have associated raw data. The additional data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

This work was supported in part by NIH grant no. R37 AI045898; the Campaign Urging Research for Eosinophilic Disease (CURED); the Sunshine Charitable Foundation and its supporters, Denise and David Bunning (M.E.R.); and by grant nos. R01 AI123176, R01 AI113125 and R01 CA231303 (C.P.). We thank S. Hottinger for medical writing assistance.

Author information

Authors and Affiliations

  1. Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    Michael Brusilovsky, Mark Rochman, Yrina Rochman, Julie M. Caldwell, Lydia E. Mack, Jennifer M. Felton, Jeff E. Habel & Marc E. Rothenberg

  2. Center for Autoimmune Genomics and Etiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    Aleksey Porollo

  3. Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    Aleksey Porollo

  4. Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA

    Aleksey Porollo, Chandrashekhar Pasare & Marc E. Rothenberg

  5. Division of Immunobiology, Center for Inflammation and Tolerance, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    Chandrashekhar Pasare

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  1. Michael Brusilovsky
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Contributions

M.B. and M.E.R. designed the study. M.B., M.E.R. and C.P. designed the experiments. M.B., M.R., Y.R., J.M.C., L.E.M., J.M.F. and J.E.H. performed the experiments and data analysis. A.P. performed the modeling and protein structure analysis. M.B., M.E.R. and C.P. interpreted the results and wrote the manuscript. All authors read and commented on the manuscript.

Corresponding author

Correspondence to Marc E. Rothenberg.

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Competing interests

M.E.R. is a consultant for Pulm One, Spoon Guru, ClostraBio, Serpin Pharm, Allakos, Celgene, Astra Zeneca, Adare/Ellodi Pharma, Glaxo Smith Kline, Regeneron/Sanofi, Revolo Biotherapeutics and Guidepoint, and has an equity interest in the first five listed, and royalties from reslizumab (Teva Pharmaceuticals), PEESSv2 (Mapi Research Trust) and UpToDate. M.E.R. is an inventor of patents owned by Cincinnati Children’s Hospital. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Immunology thanks Hirohito Kita and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Zoltan Fehervari was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Ripoptosome-dependent intracellular pIL-33 maturation.

A–E. Immunoblot analysis of intracellular IL-33 and cellular components from total cell lysates of cells expressing endogenous pIL-33 and exposed to various stimuli. EPC2 cells were treated for 8 hours with control medium alone (Mock), 10 nM of Poly (I:C) or LPS, or 25 µg/mL of A. alternata (A.Alt), house dust mite (HDM), or A. fumigatus (A.Fum) allergen extracts as indicated. EPC2 cells were treated in the presence of control medium (Mock) or in the presence of 20 µM pan-caspase inhibitor (Q-VD-OPH), caspase 8 inhibitor (Z-IETD-FMK), caspase 3 and 7 inhibitor (Z-DEVD-FMK), or caspase 1 inhibitor (Ac-YVAD-CHO); 50 µM inactive necrostatin 1 (Inact Ctr); and/or 20 µM or 50 µM of necrostatin 1 (NEC-1), necrostatin 5 (NEC-5), necrostatin 7 (NEC-7), or necrostatin 1 s (NEC-1s) as indicated. E. Immunoblot analysis of EPC2 cells treated for 8 hours with control medium (Mock), 10 nM of Poly (I:C) or 25 μg/mL of A. alternata (A.Alt), house dust mite (HDM), or A. fumigatus (A.Fum) allergen extracts in medium alone or pre-mixed with complete protease inhibitor cocktail (Prot Inhib; Roche see Methods). F-G. LDH (F) and IL-33 (G) release analysis in cell supernatants of EPC2 cells expressing endogenous pIL-33 and exposed to various stimuli. Cells were treated as above (A-E) for 2, 4 and 8 hours as indicated. Lysis control are cells treated with Triton 100 lysis buffer for 45 minutes to determine maximum LDH and IL-33 release (CyQUANT LDH cytotoxicity assay - see Methods). Each data point is a mean of a technical duplicate ±s.d. of in vitro assays. Statistics were performed by two-way ANOVA with Tukey’s multiple comparisons test: p value ≤ 0.0003 (***). H-I. Immunoblot analysis of intracellular IL-33 and cellular components from total cell lysates of cells expressing endogenous pIL-33. H. Immunoblot analysis of control (TLR3 +) and CRISPR/Cas9 TLR3 knockout (TLR3 -) EPC2 cells treated as above (A-E). I. Immunoblot analysis of total cell lysates of human esophageal epithelial cells (EPC2), skin epithelial cells (HaCaT), bronchial epithelial cells (HBEC3-KT) expressing endogenous IL-33, and fibroblasts (FEF3) cells; IL-33 expression in FEF3 cells was induced with 100 pg/mL TNF-α for 16 hours. Then all the cells were incubated with either control medium or 10 nM Poly (I:C).

Source data

Extended Data Fig. 2 Peptide profiles of intact and cleaved precursor IL-33.

A. Precursor IL-33 reference sequence (UniProtKB O95760). Peptides covered by analysis are underlined. Bold letter Ds indicate residues 175 (left) and 178 (right). B-F. Summary table and peptide profiles of recombinant human IL-33 peptides identified by MALDI-TOF Mass Spectrometry and Tandem Liquid Chromatography MALDI-TOF Mass Spectrometry (LCMS) before and after cleavage by recombinant human caspases. C-F. Exact profiles corresponding to identified peptides sequences of precursor (full-length control; C, E) and cleaved (D, F) GST–IL-33 are labeled with colors in the inserts, and the first letter of each color in the histogram peptide profiles as indicated: green (G), blue (B), and purple (P). Bold red indicates extra sequence identified by nano-LCMS. G-H. The 159-VLLSYYESQHPSNESGD-175 peptide profiles were generated by caspase 3 and caspase 7 cleavage, respectively. I-J. 159-VLLSYYESQHPSNESGDGVD-178 peptide profiles generated by caspase 3 and caspase 7 cleavage, respectively. A-J. Data are a summary of n = 4 independent experiments. m/z, mass-to-charge ratio.

Extended Data Fig. 3 Flow cytometry gating strategy for Fig. 3 E-G.

A-D. IL-33 knock out (KO) mice were intraperitoneally injected with equimolar quantities (10 nM) of recombinant pIL-33 and mIL-33 forms in PBS or PBS alone (Mock). Single-cell dot plot data and gating strategy for live, mouse, intraperitoneal cells (A) where P0 are neutrophils (Neut; GR1/Ly6ChighCD11b+c-KIT-) and inflammatory macrophages (iMɸ; GR1/Ly6CmediumCD11b+c-KIT-). A-B. Flow cytometry analysis of single-cell dot plot data with corresponding gates (B) for neutrophils (P1; GR1high CD11b+) and inflammatory macrophages (P2; GR1medium CD11b+). Summary plots show neutrophil (C) and inflammatory macrophages (D) influx in peritoneal cavity. Data are representative of n = 3 independent experiments. C-D. Data are summary of n = 3 independent experiments. Each data point is a mean of a technical duplicate ± SD of in vivo (individual mouse) assays. Statistics were performed by one-way ANOVA with Tukey’s multiple comparisons test: p value ≤ 0.0001 (****), p value ≤ 0.0002 (***), and p value ≤ 0.008 (**).

Extended Data Fig. 4 Chromatin-binding domain containing mIL-33 forms are released in complex with histones, which potentiate mIL-33 biological activity.

A. Quantification analysis of released (cell medium) IL-33 from TE-7 cells overexpressing pIL-33 (1-270). Cells were treated with control medium or Poly (I:C) (10 nM) for 0-24 hours, and medium was collected for each time point. B. Immunoblot analysis of released (concentrated cell medium) and intracellular (cellular; total cell lysates) IL-33 from the corresponding TE-7 cells. GAPDH was used as a loading control. C. UV absorption plot of size exclusion column fractions 1-95. D. Immunoblot analysis of TE-7 cells overexpressing pIL-33 (1-270). Cells were treated with TLR3 agonist (Poly (I:C)) for 8 hours in serum-free medium (Opti-MEM). Medium containing secreted IL-33 was supplemented with complete protease inhibitors, filtered through 45- µM pores, DNase treated, and concentrated 10 fold using a 10-kDa cutoff membrane filter. Samples were run on the size exclusion column. Fractions 1-95 were collected, concentrated 20 fold using a 10-kDa cutoff membrane filter, and analyzed via immunoblotting in the following order: protein standard ladder (L), secreted IL-33 medium loading control (LC), and fractions 1-95. E. Size exclusion column standard curve by fraction number as a function of molecular weight (MW). F, G. IL-33 bioactivity assay as function of IL-8 secretion by HMC-I human mast cells. Cells were treated for 8 h with 2.5-10 nM of wheat germ extract–produced IL-33 forms in medium alone (Mock) or medium supplemented with 500 ng of acetone-purified histones in the presence of IgG control (IgG) or anti-ST2 blocking antibody (aST2). A-G. Data are representative of n = 3 independent experiments. Immunoblot left margin (throughout): protein molecular weight (kDa). Right margin (throughout): protein names (m, mature; p, precursor); # are non-specific bands. A, F, G. Each data point is a mean of a technical duplicate ± SD. Statistics were performed by 2-way ANOVA with Tukey’s multiple comparisons test: p value ≤ 0.0001 (****) and p value ≤ 0.015 (*). NS, not significant.

Source data

Extended Data Fig. 5 Flow cytometry gating strategy and BALF cytokines for Fig. 4 D-I.

A. Single-cell dot plot data and gating strategy for the live, mouse bronchoalveolar fluid (BALF) cells: the P1 CD45+CD11c- population is derived from total single cells; the P2 population was identified on the basis of total single cells and applied on the P1 population. Finally, the P2-derived ST2+ cells are neutrophils (Neut; SiglecF-GR1/Ly6Chigh) and eosinophils (Eos; SiglecF+GR1/Ly6Cmedium/low). Data are representative of n = 3 independent experiments. B-C. BALF cytokines in WT (B) and IL-33 KO (C) mice were measured by ELISA with and without treatment with a specific inhibitor of caspase 8 (Z-IETD-FMK). Data are summary of n = 3 independent experiments. Each data point is a mean ± SD of in vivo (individual mouse) assays. Statistics were performed by unpaired t-test: p value ≤ 0.0001 (****), p value ≤ 0.0067 (***), p value ≤ 0.0099 (**), p value ≤ 0.0431 (*). N/S is not significant. Arrowheads are comparison of A. alternata (A.Alt) challenges alone between WT and IL-33 KO mice. Statistics were performed by unpaired one-sided t-test: p value ≤ 0.0001 (****), p value ≤ 0.0006 (***), p value ≤ 0.0034 (**), p value ≤ 0.028 (*).

Extended Data Fig. 6 Flow cytometry gating strategy and BALF cytokines for Fig. 5 D-E.

A. Single-cell dot plot data and gating strategy for live BALF cells (D, E): the P1 CD45+CD11c- population is derived from total single cells; the P2 population was identified on the basis of total single cells and applied on the P1 population. Finally, the P2-derived ST2+ cells are neutrophils (Neut; SiglecF-GR1/Ly6Chigh) and eosinophils (Eos; SiglecF+GR1/Ly6Cmedium/low). Data are representative of n = 3 independent experiments. B. BALF cytokines in WT and caspase 8 KO mice were measured by ELISA. Data are summary of n = 3 independent experiments. Each data point is a mean ± SD of in vivo (individual mouse) assays. Statistics were performed by unpaired one-sided t-test: p value ≤ 0.0003 (***), p value ≤ 0.0058 (**), p value ≤ 0.0442 (*). N/S is not significant.

Extended Data Fig. 7 Active caspase 8 and IL-33 interaction in airway inflammation.

A-E. Wildtype (WT; +) and caspase 8 knock out (KO; -) mice were treated intratracheally with A. alternata (A.Alt) extract in PBS or PBS alone (Mock) - see Fig. 5. A. Representative images of active caspase 8 and IL-33 staining in murine lungs bronchi epithelial cells in mice treated with PBS (Mock) and A. alternata (A.Alt). Positive (grey) and negative (white) staining is indicated with arrowheads. The 100-µm scale bars are included in all images. B. Active caspase 8 quantification. C. IL-33 quantification. D-E. Correlation of IL-33 with active caspase 8 in WT (D) and caspase 8 KO (E) mice. Statistics are by Pearson correlation (D-E): R2 (r) and p values are as indicated. Data are representative (A) or a summary (B-E) of n = 3 independent experiments. Each data point is a mean ± SD of multiple sections measurement in an individual mouse. Statistics were performed by one-way ANOVA with Tukey’s multiple comparisons test: p-value ≤ 0.0001 (****), p value ≤ 0.0025 (**), p value ≤ 0.0139 (*). N/S is not significant.

Extended Data Fig. 8 RipIL-33 pathway for environmental allergen sensing.

Allergen exposure triggers RIP phosphorylation and ripoptosome assembly: RIP (RIP) in complex with cFLIPL, FADD, TRADD, and pro-caspase 8. Following RIP phosphorylation (pRIP), FADD-bound pro-caspase 8 is self-cleaved and activated. Active caspase 8 cleaves and deactivates pRIP and activates effector pro-caspases 3 and 7. Active effector caspases in turn target and cleave histone-bound pIL-33 at amino acids D175 and D178. mIL-33 is released to initiate type 2 innate immune responses.

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Brusilovsky, M., Rochman, M., Rochman, Y. et al. Environmental allergens trigger type 2 inflammation through ripoptosome activation. Nat Immunol 22, 1316–1326 (2021). https://doi.org/10.1038/s41590-021-01011-2

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