MiR-155 Induction by F. novicida but Not the Virulent F. tularensis Results in SHIP Down-Regulation and Enhanced Pro-Inflammatory Cytokine Response

The intracellular Gram-negative bacterium Francisella tularensis causes the disease tularemia and is known for its ability to subvert host immune responses. Previous work from our laboratory identified the PI3K/Akt pathway and SHIP as critical modulators of host resistance to Francisella. Here, we show that SHIP expression is strongly down-regulated in monocytes and macrophages following infection with F. tularensis novicida (F.n.). To account for this negative regulation we explored the possibility that microRNAs (miRs) that target SHIP may be induced during infection. There is one miR that is predicted to target SHIP, miR-155. We tested for induction and found that F.n. induced miR-155 both in primary monocytes/macrophages and in vivo. Using luciferase reporter assays we confirmed that miR-155 led to down-regulation of SHIP, showing that it specifically targets the SHIP 3′UTR. Further experiments showed that miR-155 and BIC, the gene that encodes miR-155, were induced as early as four hours post-infection in primary human monocytes. This expression was dependent on TLR2/MyD88 and did not require inflammasome activation. Importantly, miR-155 positively regulated pro-inflammatory cytokine release in human monocytes infected with Francisella. In sharp contrast, we found that the highly virulent type A SCHU S4 strain of Francisella tularensis (F.t.) led to a significantly lower miR-155 response than the less virulent F.n. Hence, F.n. induces miR-155 expression and leads to down-regulation of SHIP, resulting in enhanced pro-inflammatory responses. However, impaired miR-155 induction by SCHU S4 may help explain the lack of both SHIP down-regulation and pro-inflammatory response and may account for the virulence of Type A Francisella

Detection of microRNA Expression in Human Peripheral Blood Microvesicles

MicroRNAs (miRNA) are small non-coding RNAs that regulate translation of mRNA and protein. Loss or enhanced expression of miRNAs is associated with several diseases, including cancer. However, the identification of circulating miRNA in healthy donors is not well characterized. Microvesicles, also known as exosomes or microparticles, circulate in the peripheral blood and can stimulate cellular signaling. In this study, we hypothesized that under normal healthy conditions, microvesicles contain miRNAs, contributing to biological homeostasis.Microvesicles were isolated from the plasma of normal healthy individuals. RNA was isolated from both the microvesicles and matched mononuclear cells and profiled for 420 known mature miRNAs by real-time PCR. Hierarchical clustering of the data sets indicated significant differences in miRNA expression between peripheral blood mononuclear cells (PBMC) and plasma microvesicles. We observed 71 miRNAs co-expressed between microvesicles and PBMC. Notably, we found 33 and 4 significantly differentially expressed miRNAs in the plasma microvesicles and mononuclear cells, respectively. Prediction of the gene targets and associated biological pathways regulated by the detected miRNAs was performed. The majority of the miRNAs expressed in the microvesicles from the blood were predicted to regulate cellular differentiation of blood cells and metabolic pathways. Interestingly, a select few miRNAs were also predicted to be important modulators of immune function.This study is the first to identify and define miRNA expression in circulating plasma microvesicles of normal subjects. The data generated from this study provides a basis for future studies to determine the predictive role of peripheral blood miRNA signatures in human disease and will enable the definition of the biological processes regulated by these miRNA

Virulent <i>F.t.</i> elicits suboptimal miR-155 and pro-inflammatory cytokine responses.

<p>(A–B) PBM (n = 7) were infected with <i>F.n.</i> or <i>F.t.</i> at an MOI of 100 for 24 hours. Relative miR-155 (A) and <i>BIC</i> (B) expression were measured by qRT-PCR. (C) PBM were infected with <i>F.n.</i> or <i>F.t.</i> at an MOI of 50 for 24 hours, then cell lysates probed for SHIP by Western blotting (top panel). The lower panel is a reprobe of the same membrane with anti-actin antibody. (D–E) PBM were infected with <i>F.n.</i> or <i>F.t.</i> at an MOI of 50 for 24 hours. TNFα (D) and (E) IL-6 in supernatants were measured by ELISA. Graphs represent the mean and standard deviation of samples from three independent infections. Data were analyzed by a paired Student <i>t</i> test. An asterisk (*) indicates a <i>p</i>-value<0.05.</p

SHIP expression is down-regulated in response to <i>F.n.</i> infection.

<p>(A) PBM were infected with <i>F.n.</i> at an MOI of 50 for 24 hours. Cell lysates were analyzed by Western blotting use an anti-SHIP antibody in the top panel. ‘R’ designates resting/uninfected cells and ‘24 h’ designates infected with <i>F.n.</i> The lower panel is a reprobe of the same membrane with an anti-actin antibody. (B) THP-1 were infected with <i>F.n.</i> at an MOI of 100 for 24 hours. Cell lysates were resolved as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008508#pone-0008508-g001" target="_blank">Figure 1A</a>. (C) Western blots for SHIP in murine bone marrow-derived macrophages infected with <i>F.n.</i> at an MOI of 50 for 24 hours. Cell lysates were resolved as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008508#pone-0008508-g001" target="_blank">Figure 1A</a>. (D) Predicted interaction between miR-155 and the 3′UTR of SHIP (INPP5D) mRNA. (E) Normalized luciferase activity in cells transfected with the 3′UTR of SHIP (psiCHECK_INPP5D) or with vector alone (psiCHECK), and cotransfected with a control <i>Renilla</i> luciferase vector. Synthetic miR-155 or non-specific (scrambled) miRs were subsequently transfected at concentrations of 0, 10, 25 and 50 nM. Luminometer readings were taken 48 hours post-transfection. The graph represents <i>f-luc</i> expression normalized to <i>r-luc</i> expression, then normalized to percent maximal response.</p

Model of miR-155 induction and function during <i>Francisella</i> infection.

<p><i>Francisella</i> is recognized on the host cell surface by TLR2. The signal is transmitted through the adaptor protein MyD88. Subsequently MAPKs, PI3K and Akt are activated, which leads to enhanced NFκB activity, inflammatory cytokine production, and effective host response. SHIP negatively regulates the activation of Akt to prevent effective host response. During <i>Francisella</i> infection miR-155 is induced through the TLR signaling pathway, PI3K/Akt, ERK, JNK, and NFκB. MiR-155 induction in turn down-regulates SHIP to promote the activation of the PI3K/Akt pathway and inflammatory cytokine production. The highly virulent <i>F. tularensis</i> suppresses or subverts the induction of miR-155 in human monocytes, while the relatively avirulent <i>F. novicida</i> does not.</p