Toll-Like Receptor 9-Dependent Macrophage Activation byEntamoeba histolyticaDNA

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Abstract
Activation of the innate immune system by bacterial DNA and DNA of other invertebrates represents a pathogen recognition mechanism. In this study we investigated macrophage responses to DNA from the intestinal protozoan parasiteEntamoeba histolytica.E. histolyticagenomic DNA was purified from log-phase trophozoites and tested with the mouse macrophage cell line RAW 264.7. RAW cells treated withE. histolyticaDNA demonstrated an increase in levels of tumor necrosis factor alpha (TNF-α) mRNA and protein production. TNF-α production was blocked by pretreatment with chloroquine or monensin. In fact, an NF-κB luciferase reporter assay in HEK cells transfected with human TLR9 demonstrated thatE. histolyticaDNA signaled through Toll-like receptor 9 (TLR9) in a manner similar to that seen with CpG-ODN. Immunofluorescence assays confirmed NF-κB activation in RAW cells, as seen by nuclear translocation of the p65 subunit. Western blot analysis demonstrated mitogen-activated protein kinase activation byE. histolyticaDNA.E. histolyticaDNA effects were abolished in MYD88−/−mouse-derived macrophages. In the context of disease, immunization withE. histolyticaDNA protected gerbils from anE. histolyticachallenge infection. Taken together, these results demonstrate thatE. histolyticaDNA is recognized by TLR9 to activate macrophages and may provide an innate defense mechanism characterized by the induction of the inflammatory mediator TNF-α.

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