Desialylation of TLR4/MD2/CD14 enhances the response to LPS (135.16)

Abstract Objective: Since bacterial neuraminidase (NANase) treatment of intact human PBMCs led to a synergistic increase in cytokine expression, we hypothesized that components of the TLR4 receptor complex were sialylated, and that removal of sialyl residues enhanced LPS-initiated signaling. Methods: HEK293T cells were transfected with TLR4, MD2, CD14 and ELAM-Luc expression vectors. They were treated with NANase, stimulated with LPS, and assayed for NF-kB-driven luciferase expression. Sialylation was assayed by lectin blot after immunoprecipitation from lysed transfected cells. Results: LPS stimulated NF-kB activation and IL-8 expression only in cells transfected with TLR4/MD2/CD14 or in cells transfected with TLR4/CD14 supplemented with supernatants (SNT) of MD2-transfected cells. NANase treatment of TLR4/CD14/MD2 cells led to enhanced NF-kB activation. While NANase treatment of either TLR4/CD14 cells or MD2 SNTs each enhanced the LPS response in the reconstituted culture, optimal responses occurred when both were treated. Lectin blots revealed MD2 to be sialylated with a α2,6 linkage since SNA but not Maackia bound to MD2. Conclusions: We conclude that removal of sialyl residues from MD2 and perhaps from TLR4/CD14 enhances the response to LPS. Thus mobilization of endogenous sialidase in PBMCs could modulate the activity of the TLR4/CD14/MD2 complex and serve as a therapeutic target. Support: NIH grant no. HL086933-01A1