Simultaneous blockade of TLR4 and TNFR1 attenuates TLR2 sensitivity in LPS-stimulated macrophages through TNFR2-mediated pathway

Background: Recent studies have found TLR2 to be a significant player in initiating immune responses in the host during bacterial infection. Macrophage polarization is one of the vital factors in the amelioration of sepsis. It is well established that recognition and binding of LPS with cell surface TLR4 could induce the production of a wide array of pro-inflammatory cytokines that initiate an organism’s inflammatory responses. Recent studies claimed that augmented expression of TLR2 shows better responsiveness to LPS, thus increasing its affinity to the ligand. Objectives: Our study attempts to demonstrate the underlying mechanisms of how TLR2 sensitivity is altered during the simultaneous blocking of TLR4 and TNFR1 and how TLR2 contributes towards the phenotypic switching of macrophages. We were also interested to see whether blocking TLR4, in any way, affects the LPS/TLR2 interactions and influences some major cytokine receptors. Materials and Methods: Murine peritoneal macrophages (5×106 cells/ml) were pre-treated with TLR4 and TNFR1 antibody (alone or in combination) and then stimulated with LPS for 60 min. FACS analyses were performed to determine M1 and M2 polarized cell populations. Assays from the cell-free supernatant determined ROS generation, and the activities of antioxidant enzymes were determined from the cell-free lysate. Western blot analysis was used to determine receptor expressions. Results: Our results indicated that blocking both receptors markedly reduced ROS levels due to its scavenging by the elevated antioxidant enzymes. Western blot data confirmed that combinatorial blockade of TLR4 and TNFR1 augmented TLR2 and TNFR2 expression in contrast to the attenuation of IL-1R. Conclusion: Therefore, the regulation of TLR2 expression was found to be TLR4-dependent, and it can show reduced NF-κB activation in response to LPS in TLR4 and TNFR1 blocked macrophages. Moreover, dual blocking can promote M2 polarization by up-regulating TNFR2. This approach could be taken as an alternative therapeutic strategy to treat LPS-sepsis.