Human neutrophil Siglec-9 is a lectin that recognizes sialic acids (Sias) via an amino-terminal V-set Ig domain and possesses tyrosine-based inhibitory motifs in its cytoplasmic tail. We hypothesized that Siglec-9 recognizes host Sias as "self," including in cis interactions with Sias on the neutrophil's own surface, thereby dampening unwanted neutrophil reactivity. Here we show that neutrophils presented with immobilized multimerized Sia␣2-3Gal␤1-4GlcNAc units engage them in trans via Siglec-9. The sialylated capsular polysaccharide of group B Streptococcus (GBS) also presents terminal Sia␣2-3Gal␤1-4GlcNAc units, and similarly engages neutrophil Siglec-9, dampening neutrophil responses in a Sia-and Siglec-9-dependent manner. Reduction in the neutrophil oxidative burst, diminished formation of neutrophil extracellular DNA traps, and increased bacterial survival are also facilitated by GBS sialylated capsular polysaccharide interactions with Siglec-9. Thus, GBS can impair neutrophil defense functions by coopting a host inhibitory receptor via sialoglycan molecular mimicry, a novel mechanism of bacterial immune evasion. (Blood. 2009; 113:3333-3336) IntroductionAlthough antimicrobial properties of vertebrate innate immune cells are extensively studied, less is understood about mechanisms dampening inflammatory responses. Such negative regulatory systems can be subverted by microbes. Of relevant interest is the "molecular mimicry" of mammalian sialic acid (Sia)-terminated sialoglycans by microbes that are obligate commensals or potential pathogens of humans. 1 Surface Sia expression can blunt alternative pathway complement activation and reduce immunogenicity. 1 However, this may not fully explain convergent bacterial evolution of near-perfect mimicry of vertebrate sialoglycans. For example, the human-specific commensal/pathogen group B Streptococcus (GBS) has a capsular polysaccharide (CPS) that displays the structure Sia␣2-3Gal␤1-4GlcNAc, 2 a sequence identical to one common at termini of human glycoproteins.Sia-recognizing immunoglobulin superfamily lectins (Siglecs) are type I transmembrane proteins expressed on immune cells. 3,4 The rapidly evolving subgroup of CD33-related Siglecs (CD33rSiglecs) are postulated (but not proven) to negatively regulate inflammatory responses by recognizing host sialoglycans. 3 Many CD33rSiglecs have conserved cytoplasmic tyrosine-based motifs, comprising a membrane-proximal immunoreceptor tyrosinebased inhibitory motif (ITIM) and a membrane-distal ITIM-like motif. 3,4 The wide expression of host Sias and the prominence of cognate ITIM-bearing CD33rSiglecs on immune cells suggest that they may function in "self"-recognition, dampening innate immune responses to prevent autoreactivity. 3 The functional outcome of CD33rSiglec binding to sialylated ligands remains poorly understood. Cross-linking antibodies and/or Siglec transfection into Siglec-deficient cell lines has demonstrated the importance of the ITIM and ITIM-like motifs for inhibiting cellular activation and proliferation, ...

SUMMARYShort interfering RNA (siRNA) induced RNA interference (RNAi) responses allow for discovery research and performing large scale screening1-5; however, due to their size and anionic charge, siRNAs have no bioavailability to enter cells4,5. Current approaches fail to deliver siRNAs into a high percentage of primary cells in a non-cytotoxic fashion. Here we report an efficient siRNA delivery approach that utilizes a Peptide Transduction Domain-dsRNA Binding Domain (PTD-DRBD) fusion protein. DRBDs bind to siRNAs with high avidity, masking the siRNA negative charge and allow for PTD-mediated cellular uptake. PTD-DRBD delivered siRNAs induced rapid RNAi responses in a non-cytotoxic manner in the entire cell population of primary and transformed cells, including T cells, HUVECs and hESCs. Whole genome microarray analysis showed minimal transcriptional changes by PTD-DRBD and we did not detect any innate immune responses in PBMCs. Thus, PTD-DRBD mediated siRNA delivery allows efficient RNAi manipulation of difficult primary cell types.

Group B Streptococcus (GBS) is a leading cause of invasive bacterial infections in human newborns. A key GBS virulence factor is its capsular polysaccharide (CPS), displaying terminal sialic acid (Sia) residues which block deposition and activation of complement on the bacterial surface. We recently demonstrated that GBS Sia can bind human CD33-related Sia-recognizing immunoglobulin (Ig) superfamily lectins (hCD33rSiglecs), a family of inhibitory receptors expressed on the surface of leukocytes. We report the unexpected discovery that certain GBS strains may bind one such receptor, hSiglec-5, in a Sia-independent manner, via the cell wall–anchored β protein, resulting in recruitment of SHP protein tyrosine phosphatases. Using a panel of WT and mutant GBS strains together with Siglec-expressing cells and soluble Siglec-Fc chimeras, we show that GBS β protein binding to Siglec-5 functions to impair human leukocyte phagocytosis, oxidative burst, and extracellular trap production, promoting bacterial survival. We conclude that protein-mediated functional engagement of an inhibitory host lectin receptor promotes bacterial innate immune evasion.

Decoy receptor 3 (DcR3) is a member of the TNF receptor superfamily and is up-regulated in tumors originating from a diversity of lineages. DcR3 is capable of promoting angiogenesis, inducing dendritic cell apoptosis, and modulating macrophage differentiation. Since tumor-associated macrophages (TAMs) are the major infiltrating leukocytes in most malignant tumors, we used microarray technology to investigate whether DcR3 contributes to the development of TAMs. Among the DcR3-modulated genes expressed by TAMs, those that encode proteins involved in MHC class II (MHC-II)-dependent antigen presentation were down-regulated substantially, together with the master regulator of MHC-II expression (the class II transactivator, CIITA). The ERK- and JNK-induced deacetylation of histones associated with the CIITA promoters was responsible for DcR3-mediated down-regulation of MHC-II expression. Furthermore, the expression level of DcR3 in cancer cells correlated inversely with HLA-DR levels on TAMs and with the overall survival time of pancreatic cancer patients. The role of DcR3 in the development of TAMs was further confirmed using transgenic mice overexpressing DcR3. This elucidates the molecular mechanism of impaired MHC-II-mediated antigen presentation by TAMs, and raises the possibility that subversion of TAM-induced immunosuppression via inhibition of DcR3 expression might represent a target for the design of new therapeutics.