Figure 3. Immunomodulatory regulation of p16Ink4a and SAβG in macrophages. Peritoneal lavage cells elicited by alginate-encapsulated SCs from p16Ink4a/Luc mice were treated ex vivo with immunomodulatory agents for 72 hours. (A) p16Ink4a promoter-driven luciferase activity (black bars) and β-galactosidase activity (via 4-MUG hydrolysis) (gray bars) were measured following treatment with M1- and M2-polarizing stimuli: LPS at 1 ng/mL, IFN-γ at 10 ng/mL, LPS/IFN-γ co-treatment, Poly(I:C) at 10 μg/mL, IFN-α at 100 U/mL, IL-4 at 20 ng/mL, IL-13 at 10 ng/ml, and IL-4/IL-13 co-treatment. Results are shown as the mean ± standard deviation for at least 3 independent experiments with statistical significance between treated and non-treated samples depicted. (B) Microphotograph of SAβG-stained adherence-selected macrophages with or without stimulation with LPS (1 ng/mL) for 72 hours (10x objective). (C) mRNA expression of p16Ink4a and β-galactosidase (Glb1) (relative to B2m expression) in macrophages from wild type mice with or without LPS stimulation for 72 hours analyzed via qPCR, as normalized to non-treated controls. Results depicted as mean ± standard deviation (n=3). (D&E) Kinetics of p16Ink4a promoter-driven luciferase activity per cell with or without LPS stimulation (D) or IL-4 stimulation (E), normalized to activity from non-treated cells at time zero. Results are shown as the mean ± standard deviation (n=3). Statistical significance with respect to non-treated control at time zero is indicated. (F) Luciferase activity and β-galactosidase activity (via 4-MUG hydrolysis) from proteose peptone-elicited lavage cells following stimulation with IL-4 (20 ng/mL) for 72 hours, normalized to non-treated controls. Results depicted as mean ± standard deviation (n=3). (G-J) Dose-dependent response of JAK1/2 inhibitor Ruxolitinib (G&H) and STAT6 inhibitor AS1517499 (I&J) on luciferase activity (G&I) and viability via CyQuant Direct assay (H&J) following 72 hours treatment of AB-elicited macrophages in the presence (gray bars) or absence (black bars) of IL-4 (10 ng/mL) stimulation. Results of luciferase activity and viability are representative of two independent experiments, depicted as mean ± standard deviation of data normalized to respective controls lacking inhibitors (with or without IL-4). Luciferase activity and viability are depicted as the percent signal relative to non-treated (NT) controls. Statistical significance between IL-4 stimulated and non-stimulated cells at each concentration of inhibitor is shown. Results are representative of three independent experiments, depicted as mean ± standard deviation. (K) Relative luciferase activity per cell following repolarization of AB-elicited macrophages (via adherence-enriched peritoneal lavage) with M1- and M2-inducing agents. Macrophages were left non-treated (NT) or treated with either LPS (1 ng/ml) or IL-4 (20 ng/ml) for 72 hours (days 1-3), as indicated. For each treatment set, samples were collected at 72 hours (no further treatment; days 4-6 = x). Alternatively, cells were washed and placed in fresh medium (-), medium containing LPS (1 ng/mL) and IFN-γ (10 ng/mL), or medium containing IL-4 (20 ng/mL) and IL-13 (10 ng/mL) and incubated for an additional 72 hours prior to sample collection (as indicated for days 4-6). Luciferase activity is expressed as the percent activity per cell relative to non-treated (NT) controls after the first 72 hours. Results are representative of two independent experiments. *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001.