Inhibition of endogenous sialidase activity suppressed LPS-induced NFκB activation in HEK cells.

<p>HEK293T cells were infected with recombinant adenoviruses expressing human Neu1 (Ad-Neu1) or Neu3 (Ad-Neu3), or with virus containing empty vector (Ad-GFP) and sialidase activity from cell lysates was assayed using 4-MUNANA as substrate in the absence or presence of the sialidase inhibitor 2-DN (250 µg/ml) (A). TLR4/CD14/MD2-transfected HEK293T cells were further infected with Ad-Neu1, Ad-Neu3 or Ad-GFP, for 2 days, stimulated with LPS (1 ng/ml) for 16 h, and evaluated for luciferase activity (B). TLR4/CD14/MD2-transfected HEK293T cells were incubated with 2-DN (250 µg/ml) or KDO (250 µg/ml) for 2 days, and stimulated with different concentrations of LPS for 16 h prior to analysis of cell lysates for luciferase activity (C). Results shown are representative of data from at least 3 independent experiments, each with similar results. ND: not done.</p

Effect of P714H, N35A/N173A, and N35A/N173A/N205A mutations on the ability of TLR4 to mediate LPS-induced activation.

<p>HEK293T cells were co-transfected with expression vectors encoding wild type TLR4 (WT) or P714H, N35A/N173A, and N35A/N173A/N205A mutants along with pEFBOS-MD2, pCDNA3-CD14, pELAM-luc, and pTK-<i>Renilla</i>-luc. Transfected cells were stimulated with LPS for 6 h, and firefly vs. renilla luciferase activities were measured in cell lysates. Data were processed using Student t-test. *p<0.005; **p<0.05 (vs. WT).</p

Optimal LPS-induced signaling when both TLR4/CD14 and MD2 treated with neuraminidase.

<p>HEK293T cells that were transfected as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032359#pone-0032359-g002" target="_blank">Figure 2</a> and TLR4/CD14-transfected cells were supplemented with NA-treated SNT from MD2 transfected cells (MD2*), heated-inactivated NA-treated SNT (MD2**), or SNT from empty vector transfected cells (pcDNA) before LPS stimulation (5 ng/ml). Cell lysates were harvested after 16 h for reporter assay (A). TLR4/CD14-transfected HEK293T cells were treated with NA, heat-inactivated NA (ΔNA), or were untreated (-) for one hour at 37°C, stimulated with LPS (5 ng/ml) for 16 h in the presence of SNT from MD2 transfected cells (sMD2), which had been pre-treated with NA agarose (NA), heated-inactivated NA agarose (ΔNA), or were untreated, and luciferase activities were determined in cell lysates (B). TLR4/MD2-transfected HEK293T cells were supplemented with NA treated recombinant human CD14 (rhCD14[NA]), untreated CD14 (rhCD14), or medium (none) prior to LPS stimulation (1–5 ng/ml). Cell lysates were harvested after 16 h for reporter assay (C). Results shown are representative of data from at least 3 independent experiments, each with similar results.</p

Sialylation of TLR4 and MD2.

<p>Recombinant human TLR4-His/MD2-His proteins expressed in the mammalian NS0 cell line were separated by SDS-PAGE and analyzed on immunoblot for binding to lectin SNA, MAAII and to anti-His antibody (A). Recombinant human TLR4-His/MD2-His expressed in NS0 cells (lane 1) or recombinant human MD2-His expressed in <i>E. coli</i> (lane 2) were separated by SDS-PAGE and probed by lectin blot with SNA (left) and on immunoblot with anti-His antibody (right) (B). Recombinant human CD14 (lane 1) were separated by SDS-PAGE and probed by lectin blot with SNA (left) and MAAII (right). The highly sialylated glycoprotein, fetuin (lane 2) and asialofetuin (lane 3) were included as positive and negative controls respectively (C). HEK293T cells were transfected with control pcDNA, or plasmids encoding TLR4-YFP alone or with MD2 and CD14 expression plasmids, and proteins from cell lysates were immunoprecipitated with ant-GFP antibody and probed on immunoblot with either anti-GFP antibody (top) or SNA (bottom) (D). Proteins from the medium of MD2-transfected cells were immunoprecipitated with anti-FLAG antibody and probed on immunoblot with anti-FLAG antibody (left) or SNA (right) (E). The expected molecular weights of MD2 and TLR4 are indicated by arrows. Results shown are representative of data from at least 2 independent experiments, each with similar results.</p

Neuraminidase treatment enhances cell response to LPS stimulation.

<p>HEK293T cells were transfected with a mixture of expression vectors encoding TLR4-YFP, MD2-FLAG, CD14, and firefly and Renilla luciferase genes. After 48 h, the transfected cells were stimulated with LPS (1 to 10 ng/ml) for 16 h and firefly vs. renilla luciferase activities were measured in cell lysates (A). Transfected cells were treated with <i>C. perfringens</i> neuraminidase (NA) or mock-treated (PBS) for one hour at 37°C, stimulated with LPS (1 ng/ml) or no-stimulation (medium) for 16 h, and the lysates were evaluated for luciferase activity (B), and production of IL-8 (C). Transfected cells were mock-treated (PBS) or treated with NA alone, NA with specific inhibitor (NA+2-DN), or heat-inactivated NA (ΔNA), stimulated with LPS (1 ng/ml) for 16 h, and the lysates were evaluated for luciferase activity (D). Results shown are representative of data from at least 3 independent experiments, each with similar results. RLU (relative luciferase units) represent each sample's firefly vs. renilla luciferase activity.</p

Requirement of MD2 in TLR4 signaling.

<p>HEK293T cells were transfected with a mixture of all plasmids as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032359#pone-0032359-g001" target="_blank">Figure 1</a> (TLR4/CD14/MD2), plasmids encoding only TLR4 and CD14 (TLR4/CD14), or control plasmid (pcDNA), stimulated with LPS (1 ng/ml) for 16 h and the lysates were evaluated for luciferase activity (A). TLR4/CD14-transfected HEK293T cells were stimulated with LPS in culture in the presence of increasing amount of recombinant human MD2, and the lysates were evaluated for luciferase activity (B). Culture supernatants (SNT) from MD2- or empty vector- (pcDNA) transfected cells were added to TLR4/CD14-transfected cells prior to LPS stimulation and luciferase activity was determined in cell lysates (C). A specific amount of affinity-purified sMD2 was added in culture media of TLR4/CD14-transfected cells before LPS stimulation (dark bars). TLR4/CD14/MD2-transfected cells with LPS stimulation were included as positive control (open bars) (D). Results shown are representative of data from at least 3 independent experiments, each with similar results.</p