Cancer Biology & Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India.
Front Immunol. 2021 Mar 3;12:626110. doi: 10.3389/fimmu.2021.626110. eCollection 2021.
TLR4 activates two distinct signaling pathways involving adaptors MyD88 and TRIF to produce proinflammatory cytokines and type-I interferon respectively. How suppresses these pathways is not well studied. We earlier reported, TLR4 is hypersialylated due to reduced membrane-bound neuraminidase (Neu1) on infected-macrophages. We hypothesized that such enhanced sialoglycoconjugates on host cells may modulate the interactions with siglecs- which are the inhibitory receptors. Here, we examined the impact of such sialylation on overall TLR4 activation both in murine cell line J774A.1 and primary bone marrow derived macrophages (BMDM). Supporting this hypothesis, we demonstrated siglec-E engages hypersialylated TLR4 during infection. Such sialic acids-siglec-E interaction enhanced siglec-E phosphorylation that mediated its strong association with SHP1/SHP2 and also upregulated their phosphorylation in both types of macrophages. Pre-treatment of parasites and host cells with neuraminidase reduced SHP1/SHP2 phosphorylation and triggered TLR4 activation respectively through enhanced nuclear translocation of p-65. Moreover, a reciprocal interplay between Neu1 and siglec-E differentially regulates MyD88- and TRIF-pathways through sialic acids on TLR4 as their common substrate during infection. Correspondingly, Neu1 overexpression enhanced MyD88-signaling while still suppressing TRIF-activation. However, silencing siglec-E specifically activated TRIF-signaling. Pro-inflammatory cytokines corresponding to MyD88 and TRIF pathways were also upregulated respectively. Additionally, Neu1 overexpression or siglec-E silencing prevented TLR4 ubiquitination and subsequent degradation by Triad3A. Neu1-overexpression and siglec-E-silencing together followed by infection activated both MyD88 and TRIF-signaling through their enhanced TLR4-association. This elevated the MyD88-specific cytokines and TRIF-mediated IRF3 and IFN-β genes, thus upregulating the pro-inflammatory cytokines and nitric oxide levels and reduced anti-inflammatory cytokines. All these significantly inhibited parasite survival in macrophages thus demonstrating a previously unidentified dualistic regulation of TLR4signaling pathways activation through sialic acids by interplay of Neu1 and siglec-E during Leishmania infection.
TLR4 通过衔接蛋白 MyD88 和 TRIF 激活两条不同的信号通路，分别产生促炎细胞因子和 I 型干扰素。TLR4 如何抑制这些通路尚不清楚。我们之前的研究表明，感染巨噬细胞中膜结合神经氨酸酶（Neu1）减少导致 TLR4 过度唾液酸化。我们假设，宿主细胞上这种增强的唾液糖蛋白可能会调节与 Siglecs 的相互作用——Siglecs 是抑制受体。在这里，我们研究了这种唾液酸化对 J774A.1 细胞系和原代骨髓来源巨噬细胞（BMDM）中 TLR4 整体激活的影响。支持这一假设，我们证明了 Siglec-E 在感染过程中与过度唾液酸化的 TLR4 结合。这种唾液酸-Siglec-E 相互作用增强了 Siglec-E 的磷酸化，使其与 SHP1/SHP2 强烈结合，并上调了两种巨噬细胞中 SHP1/SHP2 的磷酸化。寄生虫和宿主细胞用神经氨酸酶预处理可分别降低 SHP1/SHP2 的磷酸化，并通过增强 p-65 的核易位触发 TLR4 激活。此外，Neu1 和 Siglec-E 之间的相互作用通过感染过程中 TLR4 上的唾液酸差异调节 MyD88 和 TRIF 通路，因为它们是共同的底物。相应地，Neu1 过表达增强了 MyD88 信号通路，同时仍然抑制了 TRIF 激活。然而，沉默 Siglec-E 特异性地激活了 TRIF 信号通路。与 MyD88 和 TRIF 通路相对应的促炎细胞因子也分别上调。此外，Neu1 过表达或 Siglec-E 沉默可防止 TLR4 被 Triad3A 泛素化和随后降解。Neu1 过表达和 Siglec-E 沉默共同作用，随后感染通过增强 TLR4 结合，激活了 MyD88 和 TRIF 信号通路。这增加了 MyD88 特异性细胞因子和 TRIF 介导的 IRF3 和 IFN-β 基因，从而上调了促炎细胞因子和一氧化氮水平，并减少了抗炎细胞因子。所有这些都显著抑制了巨噬细胞中的寄生虫存活，从而证明了在 Leishmania 感染过程中，Neu1 和 Siglec-E 的相互作用通过唾液酸对 TLR4 信号通路激活的一种先前未被识别的双重调节。
