Figure 2.

IKKβ maintains the alternative phenotype of tumor-polarized macrophages. WT, mock, IKKβ^DN or IKKβ^f/f, and IKKβ^Δ BMDMs were co-cultured with ID8 cells for 24 h. Supernatant was collected after 24 h and analyzed for IL-10, IL-12p70, TNF-α, and NO production by ELISA. (A) Co-cultured WT macrophages express an immunosuppressive IL-10^high IL-12p70^low TNF-α^high profile. However, supernatant from co-cultured IKKβ-deleted macrophages show a significant decrease in IL-10 and TNF-α (P < 0.01; t test with Welch's correction) but increase in IL-12p70 (P < 0.01; t test with Welch's correction). Data are represented as mean ± SD of n = 3. Representative data are shown from at least three independent experiments. (B) In parallel, total RNA was isolated for real-time PCR analysis of IL-12p40 and arginase-1 in co-cultured macrophages. Targeting IKKβ in macrophages switches their phenotype to an IL-12^high arginase-1^low NOS2^high profile, consistent with an M1 phenotype. In_addition, IKKβ-targeted macrophages secrete significantly higher levels of NO, measured as nitrite by Griess assay (P < 0.01; Mann-Whitney test). Data are represented as mean ± SD of n = 3. Representative data are shown from at least four independent experiments. (C) Macrophages were co-cultured with ID8 cells, and protein extracts were prepared at the indicated time points for biochemical analysis. Expression of NF-κB p65, serine 536 phosphorylation of p65, IKKβ, Stat1, tyrosine 701 phosphorylation of Stat1 (Tyr701), and NOS2 was measured by immunoblot analysis of cell lysates using β-actin as a loading control. Representative data are shown from at least three independent experiments. (D) [¹¹¹In]oxine release assay after co-culture of ID8 ovarian cancer cells with IKKβ^DN or IKKβ^Δ BMDMs. IKKβ-targeted macrophages promote increased cytotoxicity that was reversed by the addition of the selective NOS2 inhibitor 1400W. Data are represented as mean of n = 3. Representative data are shown from at least three independent experiments.