Abstract
Siglec-E is a murine CD33-related siglec that functions as an inhibitory receptor and is expressed mainly on neutrophils and macrophage populations. Recent studies have suggested that siglec-E is an important negative regulator of lipopolysaccharide (LPS)-toll-like receptor 4 (TLR4) signaling and one report (1) claimed that siglec-E is required for TLR4 endocytosis following uptake of Escherichia coli by macrophages and dendritic cells (DCs). Our attempts to reproduce these observations using cells from wild-type (WT) and siglec-E-deficient mice were unsuccessful. We used a variety of assays to determine if siglec-E expressed by different macrophage populations can regulate TLR4 signaling in response to LPS, but found no consistent differences in cytokine secretion in vitro and in vivo, comparing three different strains of siglec-E-deficient mice with matched WT controls. No evidence was found that the siglec-E deficiency was compensated by expression of siglecs-F and -G, the other murine inhibitory CD33-related siglecs. Quantitative proteomics was used as an unbiased approach and provided additional evidence that siglec-E does not suppress inflammatory TLR4 signaling. Interestingly, proteomics revealed a siglec-E-dependent alteration in macrophage protein composition that could be relevant to functional responses in host defense. In support of this, siglec-E-deficient mice exhibited enhanced growth of Salmonella enterica serovar Typhimurium in the liver following intravenous infection, but macrophages lacking siglec-E did not show altered uptake or killing of bacteria in vitro. Using various cell types including bone marrow-derived DCs (BMDCs), splenic DCs, and macrophages from WT and siglec-E-deficient mice, we showed that siglec-E is not required for TLR4 endocytosis following E. coli uptake or LPS challenge. We failed to see expression of siglec-E by BMDC even after LPS-induced maturation, but confirmed previous studies that splenic DCs express low levels of siglec-E. Taken together, our findings do not support a major role of siglec-E in regulation of TLR4 signaling functions or TLR4 endocytosis in macrophages or DCs. Instead, they reveal that induction of siglec-E by LPS can modulate the phenotype of macrophages, the functional significance of which is currently unclear.
Highlights
Innate immune cells express toll-like receptors (TLRs) which play critical roles in recognition of various pathogen-associated molecular patterns (PAMPs)
Quantitative proteomics revealed differences in the levels of many proteins shared by WT and siglec-E-deficient macrophages following LPS activation and we cannot exclude the possibility that some of these differences could mask siglec-E dependent effects on toll-like receptor 4 (TLR4) signaling
Their importance has been clearly documented in the case of CD22 and siglec-G on B cells using genetic and biochemical approaches where CD22 has been shown to associate with other CD22 molecules and siglec-G has been shown to associate with the B cell receptor complex [reviewed in Ref. [11]]
Summary
Innate immune cells express toll-like receptors (TLRs) which play critical roles in recognition of various pathogen-associated molecular patterns (PAMPs). Exposure of macrophages and dendritic cells (DCs) to PAMPS, such as Gram-negative bacterial lipopolysaccharide (LPS), which triggers through TLR4, can orchestrate a diverse gene expression program required for shaping the innate and adaptive arms of the immune response [2,3,4]. These changes include the induction or repression of a wide range of genes that regulate pro-inflammatory cytokines, chemokines, inflammatory mediators, polarization, migration, and cell survival. Siglecs can interact with sialic acid ligands in trans, for example, on encountering another cell or a pathogen expressing highaffinity/avidity ligands and this can trigger siglec-dependent signaling functions and endocytosis
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