Interplay Between Sialic Acids, Siglec-E, and Neu1 Regulates MyD88- and TRIF-Dependent Pathways for TLR4-Activation During Leishmania donovani Infection.

Joyshree Karmakar,Chitra Mandal

doi:10.3389/fimmu.2021.626110

Joyshree Karmakar, Chitra Mandal

Open Access

https://doi.org/10.3389/fimmu.2021.626110

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Abstract

TLR4 activates two distinct signaling pathways involving adaptors MyD88 and TRIF to produce proinflammatory cytokines and type-I interferon respectively. How Leishmania donovani 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.

Highlights

Leishmaniasis is a protozoan parasitic disease that manifests in three different forms-cutaneous, mucocutaneous and lifethreatening visceral leishmaniasis (VL)
We examined whether such association between siglec-E and TLR4 exists during L.donovani infection
TLR4 is a well-known sialylated cell surface molecule on the macrophage, which induce the activation of both MyD88 and TRIF-dependent pathways with distinct cytokine repertoire [10,11,12]

Summary

Introduction

Leishmaniasis is a protozoan parasitic disease that manifests in three different forms-cutaneous, mucocutaneous and lifethreatening visceral leishmaniasis (VL). VL is a deadly neglected tropical disease ranking second to malaria in causing deaths by a protozoal pathogen [3]. Leishmania is intra-macrophage parasite whose initial interaction with the macrophages through the pattern recognition receptors (PRRs) play a vital role in pathogen recognition [4, 5]. The toll-like receptors (TLRs) is a wellknown PRR that identifies pathogen-associated molecular patterns (PAMPs) and serves as important regulators of the innate immune response [6, 7]. The initial ability of the parasite to gain entry and ensure its survival relies on the interaction between cell surface molecules and those on the parasite

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