Human macrophages utilize a wide range of pathogen recognition receptors to respond to Legionella pneumophila

Grigoryeva, Lubov Sergeevna

doi:https://doi.org/10.21985/n2-qgsy-az41

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Human macrophages utilize a wide range of pathogen recognition receptors to respond to Legionella pneumophila

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Cytokines made by macrophages play a critical role in determining the course of Legionella pneumophila infection. Prior, murine-based modeling indicated that the cytokine response initiated upon recognition of L. pneumophila involves a subset of Toll-like receptors, namely TLR2, TLR5, and TLR9. Using shRNA/siRNA knockdowns and subsequently CRISPR/Cas9 knockouts (KO), I 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, I 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 lipopolysaccharide (LPS) was dependent upon TLR4, a further contradiction to murine-based studies, which had concluded that TLR2 is the receptor for Legionella LPS. I also confirmed that unlike human cells, TRIF/TLR3/TLR4 pathways were not required for murine macrophages to sense L. pneumophila. Given the role of TLR3 in sensing nucleic acid (i.e., dsRNA), I utilized CRISPR/Cas9 KO U937 cells to document that DNA-sensing by cGAS-STING and DNA-PK and RNA-sensing by PKR and DEAD-box helicase DDX3 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. Furthermore, I identified both novel recognition pathways and pathways of inflammation that have heretofore gone unrecognized in past murine-based studies as being important during human L. pneumophila infection. Ultimately, future studies can therapeutically target these novel pathways as a means decreasing severity of L. pneumophila infection.

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