The immunomodulatory effects of THP are exerted by a TLR4-dependent mechanism. Bone marrow–derived murine macrophages (BM-Mo) (A) or murine splenocytes (B) from C57BL/6 WT, TLR2, TLR4, TLR9, or Myd88 KO mice were stimulated with THP, LPS, or Pam3Cys. Cell-free supernatants were collected 18 hours after addition of LPS, THP, or Pam3Cys and analyzed for TNF-α by ELISA. nd, not determined. *Significantly different from the values for activated controls in WT mice; P < 0.001. (C) Analysis of in vivo cytokine production in serum of C57BL/6 mice or TLR4 KO mice after i.v. administration of LPS or THP. One-hour postinjection serum levels of TNF-α were analyzed by ELISA. bd, below detection limit. Similar results were obtained in another independent experiment. (D) THP was injected i.v. on days 0, 1, 2, and 7 into C57BL/6 mice or TLR4 KO mice, and the subsequent THP-specific Ab response (IgG) was determined by ELISA. Ab response is shown at a serum dilution of 1:500.