Human macrophages utilize a wide range of pathogen recognition receptors to recognize Legionella pneumophila, including Toll-Like Receptor 4 engaging Legionella lipopolysaccharide and the Toll-like Receptor 3 nucleic-acid sensor.

Lubov S Grigoryeva,Nicholas P Cianciotto,Sunny Shin

doi:10.1371/journal.ppat.1009781

Lubov S Grigoryeva, Nicholas P Cianciotto + Show 1 more

Open Access

https://doi.org/10.1371/journal.ppat.1009781

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Journal: PLoS pathogens	Publication Date: Jul 19, 2021
Citations: 9	License type: CC BY 4.0

Affiliation: Northwestern University

Abstract

Cytokines made by macrophages play a critical role in determining the course of Legionella pneumophila infection. Prior murine-based modeling indicated that this cytokine response is initiated upon recognition of L. pneumophila by a subset of Toll-like receptors, namely TLR2, TLR5, and TLR9. Through the use of shRNA/siRNA knockdowns and subsequently CRISPR/Cas9 knockouts (KO), we determined that TRIF, an adaptor downstream of endosomal TLR3 and TLR4, is required for full cytokine secretion by human primary and cell-line macrophages. By characterizing a further set of TLR KO’s in human U937 cells, we discerned that, contrary to the viewpoint garnered from murine-based studies, TLR3 and TLR4 (along with TLR2 and TLR5) are in fact vital to the macrophage response in the early stages of L. pneumophila infection. This conclusion was bolstered by showing that i) chemical inhibitors of TLR3 and TLR4 dampen the cytokine output of primary human macrophages and ii) transfection of TLR3 and TLR4 into HEK cells conferred an ability to sense L. pneumophila. TLR3- and TLR4-dependent cytokines promoted migration of human HL-60 neutrophils across an epithelial layer, pointing to the biological importance for the newfound signaling pathway. The response of U937 cells to L. pneumophila LPS was dependent upon TLR4, a further contradiction to murine-based studies, which had concluded that TLR2 is the receptor for Legionella LPS. Given the role of TLR3 in sensing nucleic acid (i.e., dsRNA), we utilized newly-made KO U937 cells to document that DNA-sensing by cGAS-STING and DNA-PK are also needed for the response of human macrophages to L. pneumophila. Given the lack of attention given them in the bacterial field, C-type lectin receptors were similarly examined; but, they were not required. Overall, this study arguably represents the most extensive, single-characterization of Legionella-recognition receptors within human macrophages.

Highlights

Legionella pneumophila is a Gram-negative bacterium that flourishes in natural and manmade water systems [1,2,3]
Using a range of genetic and biochemical approaches, we identified molecular receptors that serve critical roles in the ability of human macrophages to recognize and respond to L. pneumophila infection
Contrary to long-standing conclusions from past murine-based studies, we found that among these key host factors are the so-called Toll-like receptors TLR3 and TLR4, with the latter being responsible for sensing Legionella LPS/endotoxin

Summary

Introduction

Legionella pneumophila is a Gram-negative bacterium that flourishes in natural and manmade water systems [1,2,3]. L. pneumophila grows in amoebae [4]. If aerosolized by man-made devices, L. pneumophila can be inhaled and replicate within both resident alveolar macrophages and monocyte-derived macrophages that migrate into the infected lung [5]. L. pneumophila infection is most often manifest as a severe form of pneumonia known as Legionnaires’ Disease (LD) [4,6,7,8,9]. The mechanistic causes of human susceptibility to serious L. pneumophila infection is still largely unclear, they likely include the manner in which the macrophage responds to the pathogen [13]

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Conclusion