Alternative activation potentiates N meningitidis–induced proinflammatory cytokine secretion via MyD88, independent of SRA and MARCO. (A) WT ThioMΦs were cultivated 48 hours in the presence or absence of IL-4 and incubated for 24 hours with or without N meningitidis (Nm; 100 bacteria/cell). The culture supernatant was analyzed for production of TNF-α and IL-6 by ELISA and by FlowCytomix for IL-12 p70 production. Data represent the mean ± SEM of replicates from 1 experiment, representative of 3 experiments. (B) Increased proinflammatory secretion depended on the IL-4 receptor pathway but was independent of pathogen recognition receptor expression. (Left) WT (■) and IL-4Rα^−/− (□) ThioMΦs were cultivated 48 hours in the presence or absence of IL-4 and incubated for 24 hours with or without N meningitidis (100 bacteria/cell). The culture supernatant was analyzed for production of TNF-α and IL-6 by ELISA. Data represent the mean ± SEM of replicates from 1 experiment, representative of 3 experiments. (Right) WT, SRA^−/−, MARCO^−/−, and SRA^−/−/MARCO^−/− ThioMΦs were cultivated for 48 hours in the presence or absence of IL-4 and challenged with or without N meningitidis (100 bacteria/cells) for 24 hours. The culture supernatant was harvested and analyzed for IL-6 secretion by ELISA. (C) MyD88^−/− ThioMΦs were cultivated for 48 hours in the presence or absence of IL-4 and challenged with or without N meningitidis for 24 hours. Cell supernatants were assayed for TNF-α by ELISA. (D) Flow cytometry of ingestion of RdGnX-N meningitidis by WT and MyD88^−/− ThioMΦs. ThioMΦs were treated for 48 hours with different concentrations of IL-4 and incubated for 2 hours at 37°C with RdGnX-N meningitidis (100 bacteria/MΦ). The average mean fluorescence intensity of 3 independent experiments is shown as a bar diagram. Error bars indicate SDs.