Abstract
Francisella tularensis is a Gram-negative, facultative intracellular pathogen that replicates in the cytosol of macrophages and is the causative agent of the potentially fatal disease tularemia. A characteristic feature of F. tularensis is its limited proinflammatory capacity, but the mechanisms that underlie the diminished host response to this organism are only partially defined. Recently, microRNAs have emerged as important regulators of immunity and inflammation. In the present study we investigated the microRNA response of primary human monocyte-derived macrophages (MDMs) to F. tularensis and identified 10 microRNAs that were significantly differentially expressed after infection with the live vaccine strain (LVS), as judged by Taqman Low Density Array profiling. Among the microRNAs identified, miR-155 is of particular interest as its established direct targets include components of the Toll-like receptor (TLR) pathway, which is essential for innate defense and proinflammatory cytokine production. Additional studies demonstrated that miR-155 acted by translational repression to downregulate the TLR adapter protein MyD88 and the inositol 5′-phosphatase SHIP-1 in MDMs infected with F. tularensis LVS or the fully virulent strain Schu S4. Kinetic analyses indicated that miR-155 increased progressively 3-18 hours after infection with LVS or Schu S4, and target proteins disappeared after 12–18 hours. Dynamic modulation of MyD88 and SHIP-1 was confirmed using specific pre-miRs and anti-miRs to increase and decrease miR-155 levels, respectively. Of note, miR-155 did not contribute to the attenuated cytokine response triggered by F. tularensis phagocytosis. Instead, this microRNA was required for the ability of LVS-infected cells to inhibit endotoxin-stimulated TNFα secretion 18–24 hours after infection. Thus, our data are consistent with the ability of miR-155 to act as a global negative regulator of the inflammatory response in F. tularensis-infected human macrophages.
Highlights
Francisella tularensis is a facultative intracellular Gramnegative bacterium that causes the zoonotic disease tularemia [1]
To determine the extent to which microRNAs were differentially expressed in human macrophages during F. tularensis infection, we isolated RNA from control cells and monocyte-derived macrophages (MDMs) that were infected with live vaccine strain (LVS) at an multiplicity of infection (MOI) of 100:1 for 18 h, with parallel samples analyzed by microscopy to assess infection efficiency and bacterial burden
In this study we undertook what is, to our knowledge, the first overall analysis of the human macrophage microRNA response to F. tularensis, and demonstrate that a small subset of microRNAs were significantly differentially regulated in MDMs within 18 hours of infection
Summary
Francisella tularensis is a facultative intracellular Gramnegative bacterium that causes the zoonotic disease tularemia [1]. This organism infects over 200 types of animals in nature and can be transmitted to humans through insect bites, ingestion of contaminated food and water, or direct contact with infected animals or aerosolized bacteria. Due to its high infectivity, ease of dissemination, and potential lethality, F. tularensis is considered a candidate bioweapon. Both type A and type B strains of this pathogen have been classified as Tier 1 select agents, and their possession and study are tightly regulated [1,3]. LVS retains many key features of virulent type A and type B F. tularensis during in vitro infection of eukaryotic cells but does not require biosafety level-3 (BSL-3) containment, and for this reason is an attractive model for studies of tularemia pathogenesis [1,4,5]
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