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. Author manuscript; available in PMC: 2020 Feb 19.
Published in final edited form as: Nat Immunol. 2019 Aug 19;20(9):1138–1149. doi: 10.1038/s41590-019-0467-1

Figure 2. MAB-hR3 inhibits IL-1R1, IL-1R4 and IL-1R6 signaling in vitro and is functional ex vivo.

Figure 2.

(a-c) Suppression of cytokine signaling by MAB-hR3 compared to polyclonal goat anti-IL-1R3. (-a) Inhibition of IL-1α signaling in an A549 luciferase reporter cell line (NF-κB activation) (IC50: MAB-hR3=146ng/mL, polyclonal=2609ng/mL; p<0.0001). (-b) IL-33 signaling inhibition in an HEK293 SEAP reporter cell line (NF-κB/ AP-1 activation) (IC50: MAB-hR3=749ng/mL, polyclonal=20986ng/mL, p<0.0001). (-c) IL-36γ signaling inhibition in an HEK293/17 luciferase reporter cell line (NF-κB activation) (IC50: MAB-hR3=17.1ng/mL, polyclonal=769ng/mL, p<0.0001). (d-f) Comparison of cytokine production by MAB-hR3 to IL-1Ra (-d) IL-1α induced IL-6 production in a human lung epithelial cell line (A549). (-e) IL-33 induced IL-8 production in a human mast cell line (HMC-1). (-f) IL-36α induced IL-8 production in a human keratinocytic cell line (HaCaT). (g) The levels of soluble receptors in whole-blood cultures after 24hrs of incubation in media (h+i) Heat-killed (hk.) C. albicans stimulation (0.5 ×106/mL, 24hrs) of IL-17 and IL-6 production in whole-blood cultures. (j) Pre-incubation (14hrs) of MAB-hR3 (1μg/mL) with recombinant human soluble IL-1R3 (sIL-1R3) in increasing concentrations, before evaluating IL-6 production in hk. C. albicans stimulated whole blood. (k) The anti-inflammatory effect of various concentrations of sIL-1R3 alone on hk. C. albicans stimulated whole blood. UT; untreated. IL-1Ra; 10μg/mL= 578nM. MAB-hR3; 10μg/mL=69nM. Inhibitors compared to stimulation alone. *p<0.05, **p<0.01 (Paired t-test, two-sided). Mean ±SEM depicted. Data are from one representative experiment (a-c) confirmed once with similar results, n=3 independent experiments (d-f), n=11 (g), n=10 (h,i) and n=9 (j,k) donors.