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