Fig 1: HB0043 retained the affinities and blockade activity to both targets. (A) Schematics of the anti-IL-36R×IL-17A BsAb (HB0043) structure. HB0043 was constructed by linking a scFv targeting IL-36R to the C-terminal domain of the anti-IL-17A IgG backbone. (B) The kinetics profile of HB0043 and HB0017 (the parental anti-IL-17A mAb) binding to human IL-17A, and human IL-36R binding to HB0043 and HB0034 (the parental anti-IL-36R mAb) measured by SPR assay. (C) The ability of HB0043 to neutralize IL-17A signaling was measured and compared to that of HB0017, using an IL-17A blockade cell-based assay. The secretion of IL-6 was monitored, which was induced by human IL-17A (0.3 nM) and TNF-α (0.6 nM) in HT-1080 cell. (D) The inhibition of IL-6 releases from NCI/ADR-RES cells was used as an evaluation of IL-36R signaling inhibition. NCI/ADR-RES cells were stimulated with human IL36α (13.5 nM) in the presence of HB0043 (32 pM to 245 nM) and HB0034 (4 pM to 336 nM).
Fig 2: Deficiency in CAD accelerates spontaneous immortalization of mouse embryonic fibroblasts.(A) Cell numbers of cultures of wild-type (wt) and CAD-deficient MEFs. Cells were counted and replated at the same cell number every 3 days, and cumulative cell numbers were calculated. Significant growth differences were detected after about 30 days of culture. Numbers are means/SD of three MEF preparations from individual mice for each genotype. Pictures show cells stained for β-galactosidase after 33 days of culture (scale bar: 50 µm). (B) Expression of senescence-associated genes analyzed at the start of the experiment (day 1) and after 33 days in culture. mRNA was extracted and gene expression was analyzed by RT-PCR. Gene expression was normalized to day 1 wt MEFs. (C) Supernatants from cells cultured from 33 days were analyzed by bead array for IL-6, CCL2, CCL5, CXCL1, and CXCL10. Data are from three pairs of MEFs derived from individual littermate embryos that were tested in parallel. Data are means/SD of three MEF preparations from individual mice for each genotype. Data represent means/SD. Unpaired parametric t test (with Welch’s correction) was used to calculate statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure.
Fig 3: Sub-lethal activation of CAD can trigger senescence.(A) WI-38 fibroblasts (carrying a non-targeting gRNA (Ctrl) or CAD-deficient) were treated with 5 µM ABT-737. Media was replaced with fresh ABT every 48 h over a period of 21 days. Bright-field images show cells after 21 days of ABT treatment. White stars highlight cells with enlarged and flat morphology. Scale bar: 50 µm. Cells were stained with β-galactosidase staining solution, and Edu and percentages of SA-β-Gal+ and Edu+ cells were quantified by microscopy. (B) Cells were treated as in (A). Lamin B1 and γH2A.X protein expression was analyzed by western blot. GAPDH was used as a loading control. (C) Expression of senescence-associated genes was measured by RT-PCR. (D) Supernatants from cells cultured for 21 days were analyzed by ELISA for IL-6. (E) MEF (from wild-type or CAD-deficient embryos) were treated as in (A), except the duration of the experiment was 7 days. Bright-field images show cells after 7 days of ABT treatment. White stars highlight cells with enlarge and flat morphology. Scale bar: 50 µm. Cells were stained with a β-galactosidase staining solution, and Edu, and percentages of SA-β-Gal+ and Edu+ cells were quantified by microscopy. (F) Lamin B1 and γH2A.X protein expression were analyzed by western blot. GAPDH was used as loading control. (G) Expression of senescence-associated genes was measured by RT-PCR. (H) Supernatants from cells cultured for 7 days were analyzed by ELISA for IL-6. Each symbol shows the result from one independent experiment. Data represent the mean/SD. An unpaired parametric t test (with Welch’s correction) was used to calculate statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are available online for this figure.
Fig 4: IL-36 synergized with IL-17A to promote inflammatory cytokines expression in NHDF. (A, B) RNA sequencing was performed for NHDF cultured with human IL-17A (100 ng/ml), IL-36 ligand (IL-36α 30 ng/ml + IL-36β 30 ng/ml + IL-36γ 30 ng/ml), and IL-36 ligand (IL-36α 30 ng/ml + IL-36β 30 ng/ml + IL-36γ 30 ng/ml) plus IL-17A (100 ng/ml) proteins for 24 hours. Differential gene expression profiling (FCH ≥ 2, FDR < 0.05). FCH, fold change; FDR, false discovery rate. (A) Venn diagram displaying the number and overlap of differentially expressed genes compared with those of the control. (B) Hierarchically clustered heatmap of inflammatory related differential gene expression of these groups. (C) The synergy effect of IL-17A and IL-36 ligand in the induction of IL-6 secretion. NHDF cells were cultured with 0.5 ng/ml IL-17A and 15 ng/ml IL-36α, 1 ng/ml IL-36β or 2 ng/ml IL-36γ overnight. (D) Various gene expression was analyzed by RT-PCR in NHDF cell following stimulation by IL-17A, IL-36 or IL-17A&IL-36 combined. Results demonstrated the synergistic effect of IL-17A and IL-36 in inducing inflammatory related gene expression. *P<0.05, **P<0.01, ***P<0.01, ****P<0.0001, ns, no significance.
Fig 5: HB0043 effectively antagonized the production of IL-6 induced by IL-17A and IL-36 in NHDF cells. (A–C) The ability of HB0043 to block IL-36R signaling and IL-17A signaling was measured and compared to that of HB0034 and HB0017, using IL-17A and IL-36R blockade cell-based assay. The secretion of IL-6 was monitored, which was induced by 0.5 ng/ml human IL-17A and 15 ng/ml IL-36α (A), 1 ng/ml IL-36β (B) or 2 ng/ml IL-36γ (C) in NHDF cells overnight. Data are expressed as means ± SEM, n=3. *P<0.05, **P<0.01, ***P<0.01, ****P < 0.0001, as determined by one-way ANOVA.
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