HS promotes DC maturation and production of proinflammatory cytokines via the TLR4-MyD88 pathway. (A) WT, TLR4^−/−, or MyD88^−/− BALB/c DCs (2 × 10⁵/well) were stimulated with LPS (100 ng/mL), HS (100 μg/mL), or Pam3CSK4 (2 μg/mL), or left unstimulated (media) for 24 hours and then measured for surface expression of costimulatory molecules CD40 and CD80 by FACS analysis. (B-C) WT, MyD88^−/−, and TLR4^−/− BALB/c cultured DCs were cocultured with media alone, LPS, HS, or Pam3CSK4 as described in panel A, and culture supernatants were tested for IL-6 (B) and IL-12 (C) by ELISA. Data are representative of 3 independent experiments (*P < .05 compared with media alone). (D-E) Assay of DC production of IL-6 and IL-12 ± the addition of the LPS inhibitor PMB (10 μg/mL) after stimulation with media, HS, or LPS (*P < .05).