MicroRNA-Regulated Rickettsial Invasion into Host Endothelium via Fibroblast Growth Factor 2 and Its Receptor FGFR1

Abstract

Microvascular endothelial cells (ECs) represent the primary target cells during human rickettsioses and respond to infection via the activation of immediate⁻early signaling cascades and the resultant induction of gene expression. As small noncoding RNAs dispersed throughout the genome, microRNAs (miRNAs) regulate gene expression post-transcriptionally to govern a wide range of biological processes. Based on our recent findings demonstrating the involvement of fibroblast growth factor receptor 1 (FGFR1) in facilitating rickettsial invasion into host cells and published reports suggesting miR-424 and miR-503 as regulators of FGF2/FGFR1, we measured the expression of miR-424 and miR-503 during R. conorii infection of human dermal microvascular endothelial cells (HMECs). Our results revealed a significant decrease in miR-424 and miR-503 expression in apparent correlation with increased expression of FGF2 and FGFR1. Considering the established phenomenon of endothelial heterogeneity and pulmonary and cerebral edema as the prominent pathogenic features of rickettsial infections, and significant pathogen burden in the lungs and brain in established mouse models of disease, we next quantified miR-424 and miR-503 expression in pulmonary and cerebral microvascular ECs. Again, R. conorii infection dramatically downregulated both miRNAs in these tissue-specific ECs as early as 30 min post-infection in correlation with higher FGF2/FGFR1 expression. Changes in the expression of both miRNAs and FGF2/FGFR1 were next confirmed in a mouse model of R. conorii infection. Furthermore, miR-424 overexpression via transfection of a mimic into host ECs reduced the expression of FGF2/FGFR1 and gave a corresponding decrease in R. conorii invasion, while an inhibitor of miR-424 had the expected opposite effect. Together, these findings implicate the rickettsial manipulation of host gene expression via regulatory miRNAs to ensure efficient cellular entry as the critical requirement to establish intracellular infection.

Keywords: FGF2; FGFR1; Rickettsia; endothelial cells; miRNA.

Figure 1

Expression levels of miR-424 and miR-503 in different types of endothelial cells infected with R. conorii. HMECs (A), HLMECs (B), and HCECs (C) were infected with R. conorii (Rc) for various time periods up to 6 h. RNA was extracted and qRT-PCR assays were performed to measure the expression of miR-424 and miR-503. The data was normalized to 18S RNA, and relative expression was calculated by the ^ΔΔCt method. The results are presented as the mean ± standard error (SE) of three independent experiments. The asterisk indicates statistically significant change (p ≤ 0.01). HMECs: human dermal microvascular endothelial cells; HLMECs: human lung microvascular endothelial cells; HCECs: human cerebral microvascular endothelial cells; Con: control.

Figure 2

Expression levels of FGF2 and FGFR1 mRNA in R. conorii-infected host endothelial cells. HMECs (A), HLMECs (B), and HCECs (C) were infected with R. conorii for various time periods up to 6 h. RNA was extracted, and qRT-PCR assays were performed to measure the FGF2 and FGFR1 expression. The data was normalized to 18S rRNA and relative expression was calculated by the ^ΔΔCt method. The results are presented as the mean ± SE of three independent experiments. The asterisk indicates statistically significant change (p ≤ 0.01).

Figure 3

Expression of miR-424(322), miR-503, and FGF2/FGFR1 in R. conorii-infected C3H/HeN mice in vivo. Mice were infected with R. conorii (2.25 × 10⁵ pfu) intravenously (IV). Control animals received IV injection of saline. On day 3 post-infection, mice were anesthetized, blood and lungs were collected, RNA was isolated, and expression of both miRNAs in the lungs (A) and blood (C) and FGF2/FGFR1 in the lungs (B) was measured by qRT-PCR. The asterisks indicate statistically significant change (p ≤ 0.01).

Figure 4

Effect of miRNA mimics on the expression levels of FGF2/FGFR1. ECs were transfected with miR-424 or miR-503 mimic (1 nM) for 24 h using Lipofectamine RNAiMAX according to the manufacturer’s instructions prior to infection with R. conorii for 6 h. Cells were lysed in Tri Reagent for isolation of total RNA. The expression of miR-424 and miR-503 (A) and FGF2/FGFR1 (B) was measured by qRT-PCR. The data are presented as the mean ± SE of three independent experiments, and the asterisk (* p ≤ 0.01) indicates statistically significant change.

Figure 5

Effect of miRNA inhibitors on expression levels of FGF2/FGFR1. ECs were transfected with miR-424 or miR-503 inhibitor (200 nM) for 72 h using Lipofectamine RNAiMAX according to the manufacturer’s instructions and then infected with R. conorii for 6 h. Cells were lysed in Tri Reagent and total RNA was isolated for measuring the expression of miR-424 and miR-503 (A) and FGF2/FGFR1 (B) by qRT-PCR. The data are presented as the mean ± SE of three separate experiments. The asterisk (* p ≤ 0.01) indicates statistically significant change.

Figure 6

miRNA-mediated rickettsial internalization into host endothelial cells: ECs were transfected with a miR-424 mimic (1 nM) or inhibitor (200 nM) prior to infection with R. conorii for 6 h. Cells were lysed and DNA was isolated using a Qiagen kit for determination of the copy number of rickettsiae by qPCR. The data are presented as the mean ± SE of three separate experiments. The asterisk (* p ≤ 0.01) indicates statistically significant change.