Timely graduated TLR4–TLR2 activation induces maximal cell activation and correlates with TLR4- and MyD88-dependent release of IFN-γ by NK and NKT cells to enhance TLR2-specific sensitivity and surface TLR2 expression upon Gram-negative bacterial infection. (A) Mice were challenged by i.p. injection of 50 μg LPS or 50 μg dipalmitoylated hexapeptide (P₂C), or they were left untreated (none; n = 6 per experimental group). After a second challenge (or none) at 180 min, serum was drawn at 270 min and analyzed by ELISA. *, P < 0.004. ND, not detected. (B) Mice were infected with 5 × 10⁸ CFU E. coli or 10⁸ CFU S. enterica (ent.) for 3 h, after which serum was sampled and analyzed by ELISA. *, P < 0.004 (inf., infected; WT, n = 4; n = 3 for each TLR4^−/− group). (C and D) Murine splenocytes were prepared 2 h after systemic infection with 10⁸ CFU S. enterica. Viable CD3⁻ and CD3⁺ fractions of splenocytes were analyzed for NK 1.1 (NK and NKT, respectively) and intracellular IFN-γ expression. (E) Splenocytes from wild-type (♦, white bar), TRIF^−/− (▪, black bar), MyD88^−/− (•, light gray bar), and TRIF^−/−/MyD88^−/− (▴, dark gray bar) mice were challenged for 24 h ex vivo upon which supernatants were analyzed by ELISA (3.5 μg/ml αCD3 as control, represents two independent experiments). (F) Primary macrophages were IFN-γ primed for 3 h or left untreated (−). Cells were washed twice and challenged with 1 ng/ml lipopeptide (Pam₃CSK₄, white bars), 10 ng/ml (gray bars), or 100 ng/ml (black bars) for an additional 6 h and analyzed by ELISA. *, P < 0.006 (represents three independent experiments). (G) Splenocytes were isolated 3 h after S. enterica infection of mice (10⁸ CFU) with indicated genotypes and the CD11b⁺ fraction was analyzed for surface TLR2 expression (representative of at least three independent experiments).