sTLR2-mediated augmentation of pro-inflammatory cytokines during bacterial lipoprotein exposure.
(A) Immunodepletion of ∼38 kDa sTLR2 in breast milk (BM) described in materials and methods. Quantitative analysis using TLR2 ELISA indicated a significant (P<0.05) decrease in sTLR2-depleted (sTLR2-D) compared to mock-D breast milk (BM). Western blot analysis revealed that ∼38 kDa and ∼83 kDa sTLR2 were markedly reduced compared to mock-D BM. (B-D) 500 ng/mL Pam3CSK4 was incubated with media or BM that was either mock-D or sTLR2-D for 1 hr at 37°C before being placed on cells. Supernatants were collected for IL-8 ELISA after 18 hours. Results represent (B) U937, (C) HEK293-TLR2 and (D) HEK293. (E) Commercial rsTLR2 was used at varying concentration with or without sCD14 and showed no inhibition of IL-8 or (F) IL-6 production in HEK293-TLR2 cells. Significant increases in pro-inflammatory cytokine, IL-8, was observed in sTLR2-D compared to mock-D BM. *P<0.05, **P<0.01, ***P<0.001. Errors bars, SEM. A representative data set from triplicate experiments is shown.
