Nagala Manjula, McKenzie Emma, Richards Hannah, Sharma Ritu, Thomson Sarah, Mastroeni Pietro, Crocker Paul R
Division of Cell Signalling and Immunology, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.
Front Immunol. 2018 Jan 15;8:1926. doi: 10.3389/fimmu.2017.01926. eCollection 2017.
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 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 and , 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 serovar Typhimurium in the liver following intravenous infection, but macrophages lacking siglec-E did not show altered uptake or killing of bacteria . 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 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.
唾液酸结合免疫球蛋白样凝集素E（Siglec-E）是一种与小鼠CD33相关的唾液酸结合免疫球蛋白样凝集素，作为一种抑制性受体发挥作用，主要表达于中性粒细胞和巨噬细胞群体。最近的研究表明，Siglec-E是脂多糖（LPS）-Toll样受体4（TLR4）信号传导的重要负调节因子，并且有一份报告称，巨噬细胞和树突状细胞（DC）摄取LPS后，TLR4内吞作用需要Siglec-E。我们尝试使用野生型（WT）和Siglec-E缺陷型小鼠的细胞来重现这些观察结果，但未成功。我们使用了多种检测方法来确定不同巨噬细胞群体表达的Siglec-E是否能够调节对LPS的TLR4信号传导，但在将三种不同品系的Siglec-E缺陷型小鼠与匹配的WT对照进行比较时，未发现细胞因子分泌方面的一致差异。没有证据表明Siglec-E缺陷会由其他与小鼠抑制性CD33相关的唾液酸结合免疫球蛋白样凝集素Siglec-F和-G的表达来补偿。定量蛋白质组学被用作一种无偏差的方法，并提供了额外的证据表明Siglec-E不会抑制炎症性TLR4信号传导。有趣的是，蛋白质组学揭示了巨噬细胞蛋白质组成中与Siglec-E相关的改变，这可能与宿主防御中的功能反应有关。支持这一点的是，Siglec-E缺陷型小鼠在静脉感染后肝脏中鼠伤寒沙门氏菌血清型的生长增强，但缺乏Siglec-E的巨噬细胞对细菌的摄取或杀伤没有改变。使用包括野生型和Siglec-E缺陷型小鼠的骨髓来源DC（BMDC）、脾DC和巨噬细胞在内的各种细胞类型，我们表明在摄取LPS或LPS刺激后，TLR4内吞作用不需要Siglec-E。即使在LPS诱导成熟后，我们也未在BMDC中看到Siglec-E的表达，但证实了先前的研究，即脾DC表达低水平的Siglec-E。综上所述，我们的发现不支持Siglec-E在调节巨噬细胞或DC中的TLR4信号传导功能或TLR4内吞作用中起主要作用。相反，它们揭示了LPS诱导的Siglec-E可调节巨噬细胞的表型，其功能意义目前尚不清楚。
