Figure 5. PHADs induce TLR4-dependent MyD88- and TRIF-pathway signaling in bone marrow derived macrophages, and 3D 6-Acyl PHAD mediates partial immune function in TLR4 knockout mice.

(A) Bone marrow derived macrophages from wild type and TLR4 knockout mice were incubated with vehicle or PHADs for 45 minutes. IKK phosphorylation and total IKK, and IRF3 phosphorylation and total IRF3 were determined by Western blotting. IKK and IRF3 phosphorylation was quantified via densitometry (ImageJ 1.52a). (B) Wild type or knockout mice received vehicle or 20 μg of 3D 6-Acyl PHAD or vehicle via IP injection 48 and 24 hours prior to IP injection with 1 × 10⁸ CFU of P. aeruginosa. Six hours after infection, blood was collected for plasma extraction and peritoneal lavage was performed. Hourly core body temperature was measured after infection and continued until euthanasia. P. aeruginosa numbers were quantified in peritoneal lavage fluid as a measure of bacterial burden. (B) Peritoneal cells were stained for F4/80, Ly6G, and Ly6C. Neutrophils were identified as F4/80⁻Ly6G⁺. Monocytes were identified as F4/80⁺/Ly6C⁺. Total neutrophils and monocytes were measured from peritoneal lavage fluid*p<0.05 compared to control, ‡p<0.05 compared to 3D PHAD, and +p<0.05 compared to wild type. Results represent 3 separate experiments for Western blots, n=6, and 2 separate experiments for infection studies, n=10.