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IL-1R3 blockade broadly attenuates the functions of six members of the IL-1 family, revealing their contribution to models of disease

Nature Immunology volume 20, pages 1138–1149 (2019)Cite this article

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Abstract

Interleukin (IL)-1R3 is the co-receptor in three signaling pathways that involve six cytokines of the IL-1 family (IL-1α, IL-1β, IL-33, IL-36α, IL-36β and IL-36γ). In many diseases, multiple cytokines contribute to disease pathogenesis. For example, in asthma, both IL-33 and IL-1 are of major importance, as are IL-36 and IL-1 in psoriasis. We developed a blocking monoclonal antibody (mAb) to human IL-1R3 (MAB-hR3) and demonstrate here that this antibody specifically inhibits signaling via IL-1, IL-33 and IL-36 in vitro. Also, in three distinct in vivo models of disease (crystal-induced peritonitis, allergic airway inflammation and psoriasis), we found that targeting IL-1R3 with a single mAb to mouse IL-1R3 (MAB-mR3) significantly attenuated heterogeneous cytokine-driven inflammation and disease severity. We conclude that in diseases driven by multiple cytokines, a single antagonistic agent such as a mAb to IL-1R3 is a therapeutic option with considerable translational benefit.

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Fig. 1: MAB-hR3 binds dose-dependently and with high affinity to IL-1R3.
Fig. 2: MAB-hR3 inhibits IL-1R1, IL-1R4 and IL-1R6 signaling in vitro and is functional ex vivo.
Fig. 3: MAB-mR3 reduces in vivo MSU crystal-mediated peritonitis.
Fig. 4: IL-1R3 blockade results in a broader anti-inflammatory phenotype compared to primary pathway inhibition.
Fig. 5: MAB-mR3 decreases OVA-induced allergic airway inflammation.
Fig. 6: Improvement of imiquimod- induced psoriasis in vivo using MAB-mR3.

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The data that support the findings of this study are available upon request to the corresponding author.

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Acknowledgements

We thank J. Domenico and M. Wade for their technical assistance, and S.H. Kim (Laboratory of Cytokine Immunology, Konkuk Univ., Republic of Korea) and M. Fujita for providing cell lines. We thank the OLAR Vivarium, the ClinImmune Flow Core Facility and the Histology Shared Resource Center funded by the University of Colorado Cancer Center NIH grant (no. P30CA046934) at the University of Colorado Anschutz Medical Campus. We also thank the University of Colorado School of Medicine Biological Mass Spectrometry Facility for analyzing IP samples. J.F.H. was supported by the Oticon Foundation, Lundbeck Foundation, Knud Højgaard Foundation and the Interleukin Foundation. M.L.V.K and B.J.S. was supported by the Interleukin Foundation. A.S.M. was funded by NIH grants no. HL126736, ES025534 and HL135872-01. C.A.D. was funded by NIH grant no. AI-15614.

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Authors and Affiliations

  1. Department of Infectious Diseases, Aarhus University Hospital, Aarhus, Denmark

    Jesper Falkesgaard Højen & Martin Tolstrup

  2. Department of Clinical Medicine, Aarhus University, Aarhus, Denmark

    Jesper Falkesgaard Højen & Martin Tolstrup

  3. Department of Medicine, University of Colorado Denver, Aurora, CO, USA

    Jesper Falkesgaard Højen, Marie Louise Vindvad Kristensen, Amy S. McKee, Megan Taylor Wade, Tania Azam, Dennis M. de Graaf, Benjamin J. Swartzwelter & Charles A. Dinarello

  4. Department of Microbiology and Immunology, University of Colorado Denver, Aurora, CO, USA

    Amy S. McKee & Mayumi Fujita

  5. Division of Asthma Exacerbation & Regulation, Priority Area Asthma and Allergy, Leibniz Lung Center Borstel, Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Borstel, Germany

    Lars P. Lunding & Michael Wegmann

  6. Department of Internal Medicine, Radboud University Medical Center, Nijmegen, the Netherlands

    Dennis M. de Graaf & Charles A. Dinarello

  7. MAB Discovery GmbH, Neuried, Germany

    Karsten Beckman & Stephan Fischer

  8. Department of Dermatology, University of Colorado Denver, Aurora, CO, USA

    Mayumi Fujita

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  1. Jesper Falkesgaard Højen
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Contributions

J.F.H. designed and performed experiments, analyzed data and wrote the manuscript. M.L.V.K. designed and performed experiments and analyzed data and assisted with manuscript preparation. A.S.M. and M.T.W. designed and performed experiments and analyzed data. T.A., L.P.L, D.M.D.G., B.J.S. and M.W. performed experiments and analyzed data. M.T. assisted in the experimental design and manuscript preparation. K.B. designed experiments and analyzed data. M.F. designed and supervised experiments and analyzed data. S.F. produced the human and mouse anti-IL-1R3 antibodies, designed experiments and analyzed data. C.A.D. designed experiments, analyzed data and edited the manuscript. All authors read and accepted the final manuscript.

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Correspondence to Charles A. Dinarello.

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Competing interests

K.B. is employed by MAB Discovery GmbH, Neuried, Germany. S.F. is the CEO of MAB Discovery GmbH. All other authors declare no competing interests.

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Peer review information: 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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Integrated supplementary information

Supplementary Figure 1 Properties of MAB-hR3.

(a) Gating strategy for FACS analysis of MAB-hR3 and isotype binding to SK-MEL-30 and NIH-3T3 cell lines (Fig. 1b) (b) MAB-hR3 immunoprecipitate from A549 cell lysate. Gel electrophoresis of immunoprecipitate stained with collodial coomassie. The arrow at 80kDa indicate the primary location of IL-1R3 peptides as analyzed by MS. IL-1R3 peptides, presumably proteolytic breakdown products, were similar found at 60kDa (5 peptides) and 20kDa (3 peptides). (c) Viability of PBMCs using MAB-hR3 (20μg/mL or 1μg/mL) compared to media alone (percentage) (left y-axis) and viability of media alone as compared to 24hrs (right y-axis), using a WST-1 cell proliferation reagent at 24 hours, 3 days and 5 days, respectively. (d) IL-6 production in unstimulated PBMCs using MAB-hR3 (20μg/mL or 1μg/mL) alone. Assayed at 24 hours, 3 days and 5 days, respectively. Mean +SEM, data from 3 donors, all in triplicates.

Supplementary Figure 2 Properties of anti-mouse IL-1R3 antibody (MAB-mR3) and comparison to MAB-hR3.

(a+b) MAB-mR3 (a) and MAB-hR3 (b) in a murine NIH-3T3 luciferase reporter assay (NF-κB activation) stimulated with 50pg/mL of mIL-1β, relative luciferase units (RLU) depicted. (a) IC50: 281.7 ng/mL. (c+d) MAB-mR3 (c) and MAB-hR3 (d) inhibition of IL-6 production in murine NIH-3T3 cells stimulated with hIL-1β (50 pg/mL). (c) IC50: 1276 ng/mL. (e+f) MAB-mR3 in a murine NIH-3T3 luciferase reporter assay (NF-κB activation) stimulated with (e) 1ng/mL of mIL-33 (IC50: 559.5ng/mL) or (f) 170ng/mL of mIL-36γ (IC50: 65.3ng/mL), RLU depicted. (g) MAB-mR3 kinetics analyzed using SPR (1 measurement depicted). Single-cycle kinetics were done using increasing concentrations of murine IL-1R3 (0.185nM - 15nM). Data are from one representative experiment, repeated once to confirm results. (a-d) Data made in triplicates. OD; optical density. Mean + SEM depicted.

Supplementary Figure 3 Gating Strategy for analysis of bronchoalveolar cells.

Singlet cells were gated for cells to eliminate debris and analyzed for live vs dead cells. Live cells were analyzed for the presence of alveolar macrophages (Siglec F+, CD11chi) and eosinophils (Siglec F-, CD11clo). The Siglec F negative cells were analyzed for the presence of neutrophils (Ly6Ghi CD11bhi) and monocytes (Ly6Glo CD11bhi). The scatter of each population of cells was confirmed by backgating as shown.

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Højen, J.F., Kristensen, M.L.V., McKee, A.S. et al. IL-1R3 blockade broadly attenuates the functions of six members of the IL-1 family, revealing their contribution to models of disease. Nat Immunol 20, 1138–1149 (2019). https://doi.org/10.1038/s41590-019-0467-1

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