TLR8 agonists induce robust phosphorylation of p38 MAP kinase in both neonatal and adult blood monocytes. Blood was stimulated with TLR agonists for 10 minutes prior to red cell lysis, fixation, and permeabilization. Representative examples of imiquimod (TLR7)–induced (A) and 3M-002 (TLR8)–induced (B) phosphorylation of p38 in newborn monocytes are provided with isotype (open), buffer control (gray), and TLR-stimulated (black) conditions indicated. Phosphorylation of p38 MAPK in CD14⁺ monocytes was detected by flow cytometry using a PE-conjugated anti–phospho-p38 mAb and depicted as an expression index (EI) calculated as a product of percent positive and mean fluorescent intensity (MFI). In these examples, TLR7 activation (A) induced a pp38 expression index (EI) of 0.3, whereas TLR8 activation (B) induced an EI of 584. (C) Composite analysis of such data reveals that TLR8 and TLR7/8 agonists induce substantially and significantly greater p38 phosphorylation than do TLR7 agonists. (D) Additional blood samples were allowed to incubate for 5 hours to measure TNF-α production in parallel. Data represent means ± SEM. Statistical comparisons were made within groups. n = 3-4, *P < .05 in comparison to both of the TLR7 agonists tested. Spearman analysis indicates that TLR-induced p38 phosphorylation and TNF-α production were significantly correlated (adults: r = 0.7973, r 2 = 0.6357, P < .001; newborns: r = 0.8322, r 2 = 0.6926; P <.001).