Sialyl Residues Modulate LPS-Mediated Signaling through the Toll-Like Receptor 4 Complex

We previously reported that neuraminidase (NA) pretreatment of human PBMCs markedly increased their cytokine response to lipopolysaccharide (LPS). To study the mechanisms by which this occurs, we transfected HEK293T cells with plasmids encoding TLR4, CD14, and MD2 (three components of the LPS receptor complex), as well as a NFκB luciferase reporting system. Both TLR4 and MD2 encoded by the plasmids are α-2,6 sialylated. HEK293T cells transfected with TLR4/MD2/CD14 responded robustly to the addition of LPS; however, omission of the MD2 plasmid abrogated this response. Addition of culture supernatants from MD2 (sMD2)-transfected HEK293T cells, but not recombinant, non-glycosylated MD2 reconstituted this response. NA treatment of sMD2 enhanced the LPS response as did NA treatment of the TLR4/CD14-transfected cell supplemented with untreated sMD2, but optimal LPS-initiated responses were observed with NA-treated TLR4/CD14-transfected cells supplemented with NA-treated sMD2. We hypothesized that removal of negatively charged sialyl residues from glycans on the TLR4 complex would hasten the dimerization of TLR4 monomers required for signaling. Co-transfection of HEK293T cells with separate plasmids encoding either YFP- or FLAG-tagged TLR4, followed by treatment with NA and stimulation with LPS, led to an earlier and more robust time-dependent dimerization of TLR4 monomers on co-immunoprecipitation, compared to untreated cells. These findings were confirmed by fluorescence resonance energy transfer (FRET) analysis. Overexpression of human Neu1 increased LPS-initiated TLR4-mediated NFκB activation and a NA inhibitor suppressed its activation. We conclude that (1) sialyl residues on TLR4 modulate LPS responsiveness, perhaps by facilitating clustering of the homodimers, and that (2) sialic acid, and perhaps other glycosyl species, regulate MD2 activity required for LPS-mediated signaling. We speculate that endogenous sialidase activity mobilized during cell activation may play a role in this regulation

Polysialylation controls immune function of myeloid cells in murine model of pneumococcal pneumonia

Summary: Polysialic acid (polySia) is a post-translational modification of a select group of cell-surface proteins that guides cellular interactions. As the overall impact of changes in expression of this glycan on leukocytes during infection is not known, we evaluate the immune response of polySia-deficient ST8SiaIV−/− mice infected with Streptococcus pneumoniae (Spn). Compared with wild-type (WT) mice, ST8SiaIV−/− mice are less susceptible to infection and clear Spn from airways faster, with alveolar macrophages demonstrating greater viability and phagocytic activity. Leukocyte pulmonary recruitment, paradoxically, is diminished in infected ST8SiaIV−/− mice, corroborated by adoptive cell transfer, microfluidic migration experiments, and intravital microscopy, and possibly explained by dysregulated ERK1/2 signaling. PolySia is progressively lost from neutrophils and monocytes migrating from bone marrow to alveoli in Spn-infected WT mice, consistent with changing cellular functions. These data highlight multidimensional effects of polySia on leukocytes during an immune response and suggest therapeutic interventions for optimizing immunity

Expression of sialyltransferase activity on intact human neutrophils

Endogenous polymorphonuclear leukocyte (PMN)-associated sialidase activity enhances PMN adhesion to and migration across the endothelium through the removal of sialylated cell-surface residues. We tested the hypothesis that PMNs also express sialyltransferase (ST) activity that restores sialyl residues to the PMN surface. We developed a highly sensitive fluorometric assay to demonstrate that intact human PMNs can mediate and accept sialyl residue transfer. This ST activity is inhibited by a ST inhibitor, CMP, which also inhibits the transendothelial migration of PMNs in response to IL-8 in vitro and in vivo. We conclude that intact PMNs express sialidase and ST activities that permit rapid modulation of their surface sialylation and their ability to adhere to and migrate across the endothelium

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