Microglial activation by microbial neuraminidase through TLR2 and TLR4 receptors

María Del Mar Fernández-Arjona,Jesús M Grondona,Pedro Fernández-Llebrez,María Dolores López-Ávalos

doi:10.1186/s12974-019-1643-9

María Del Mar Fernández-Arjona, Jesús M Grondona + Show 2 more

Open Access

https://doi.org/10.1186/s12974-019-1643-9

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Abstract

BackgroundNeuraminidase (NA) is a sialidase present, among various locations, in the envelope/membrane of some bacteria/viruses (e.g., influenza virus), and is involved in infectiveness and/or dispersion. The administration of NA within the brain lateral ventricle represents a model of acute sterile inflammation. The relevance of the Toll-like receptors TLR2 and TLR4 (particularly those in microglial cells) in such process was investigated.MethodsMouse strains deficient in either TLR2 (TLR2-/-) or TLR4 (TLR4-/-) were used. NA was injected in the lateral ventricle, and the inflammatory reaction was studied by immunohistochemistry (IBA1 and IL-1β) and qPCR (cytokine response). Also, microglia was isolated from those strains and in vitro stimulated with NA, or with TLR2/TLR4 agonists as positive controls (P3C and LPS respectively). The relevance of the sialidase activity of NA was investigated by stimulating microglia with heat-inactivated NA, or with native NA in the presence of sialidase inhibitors (oseltamivir phosphate and N-acetyl-2,3-dehydro-2-deoxyneuraminic acid).ResultsIn septofimbria and hypothalamus, IBA1-positive and IL-1β-positive cell counts increased after NA injection in wild type (WT) mice. In TLR4-/- mice, such increases were largely abolished, while were only slightly diminished in TLR2-/- mice. Similarly, the NA-induced expression of IL-1β, TNFα, and IL-6 was completely blocked in TLR4-/- mice, and only partially reduced in TLR2-/- mice. In isolated cultured microglia, NA induced a cytokine response (IL-1β, TNFα, and IL-6) in WT microglia, but was unable to do so in TLR4-/- microglia; TLR2 deficiency partially affected the NA-induced microglial response. When WT microglia was exposed in vitro to heat-inactivated NA or to native NA along with sialidase inhibitors, the NA-induced microglia activation was almost completely abrogated.ConclusionsNA is able to directly activate microglial cells, and it does so mostly acting through the TLR4 receptor, while TLR2 has a secondary role. Accordingly, the inflammatory reaction induced by NA in vivo is partially dependent on TLR2, while TLR4 plays a crucial role. Also, the sialidase activity of NA is critical for microglial activation. These results highlight the relevance of microbial NA in the neuroinflammation provoked by NA-bearing pathogens and the possibility of targeting its sialidase activity to ameliorate its impact.

Highlights

Toll-like receptors (TLRs) are a large family of membrane proteins which recognizes conserved structural motifs found in microbes, the so-called pathogen-associated molecular patterns (PAMPs), as well as patterns belonging to the individual itself, the damage-associated molecular patterns (DAMPs) [1, 2]
Toll-like receptor 4 (TLR4)-dependent manner Wild-type, Toll-like receptor 2 (TLR2)-/- and TLR4-/- mice were ICV injected with NA, and sacrificed 24 h later
Brain sections were subjected to immunolabeling with selected markers: i) Ionized calcium-binding adaptor molecule 1 (IBA1), which labels microglia and peripheral, perivascular, and meningeal macrophages; ii) Interleukin 1 beta (IL-1β), a pro-inflammatory cytokine expressed in microglia which indicates its proinflammatory activation

Summary

Introduction

Toll-like receptors (TLRs) are a large family of membrane proteins which recognizes conserved structural motifs found in microbes, the so-called pathogen-associated molecular patterns (PAMPs), as well as patterns belonging to the individual itself, the damage-associated molecular patterns (DAMPs) [1, 2] They serve as sensitive sensors and contribute to a first line of defense in the immune response. Peptidoglycans of bacterial cell wall, and lipopolysaccharide (LPS) of the outer membrane of Gram-negative bacteria, are well known specific ligands of TLR2 and TLR4 respectively Both trigger intracellular signaling pathways, a MyD88-dependent pathway that ends in the activation of the nuclear factor kappa B (NF-κB), with the subsequent increase in the expression of proinflammatory cytokines such as interleukin 1 beta ( IL-1β) and tumor necrosis factor alpha (TNFα) [3, 5, 7]. The relevance of the sialidase activity of NA was investigated by stimulating microglia with heat-inactivated NA, or with native NA in the presence of sialidase inhibitors (oseltamivir phosphate and N-acetyl-2,3-dehydro-2-deoxyneuraminic acid)

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