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. 2014 Apr 10:5:3619.
doi: 10.1038/ncomms4619.

MicroRNA-302b augments host defense to bacteria by regulating inflammatory responses via feedback to TLR/IRAK4 circuits

Affiliations

MicroRNA-302b augments host defense to bacteria by regulating inflammatory responses via feedback to TLR/IRAK4 circuits

Xikun Zhou et al. Nat Commun. .

Erratum in

Abstract

MicroRNAs (miRNAs) have been implicated in a spectrum of physiological and pathological conditions, including immune responses. miR-302b has been implicated in stem cell differentiation but its role in immunity remains unknown. Here we show that miR-302b is induced by Toll-like receptor 2 (TLR2) and TLR4 through ERK-p38-NF-κB signalling upon Gram-negative bacterium Pseudomonas aeruginosa infection. Suppression of inflammatory responses to bacterial infection is mediated by targeting IRAK4, a protein required for the activation and nuclear translocation of NF-κB. Through negative feedback, enforced expression of miR-302b or IRAK4 siRNA silencing inhibits downstream NF-κB signalling and airway leukocyte infiltration, thereby alleviating lung injury and increasing survival in P. aeruginosa-infected mice. In contrast, miR-302b inhibitors exacerbate inflammatory responses and decrease survival in P. aeruginosa-infected mice and lung cells. These findings reveal that miR-302b is a novel inflammatory regulator of NF-κB activation in respiratory bacterial infections by providing negative feedback to TLRs-mediated immunity. VSports手机版.

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Conflict of interest statement

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. miR-302b expression was up-regulated after bacterial infection
(a) Microarray analysis of miRNA expression in PAO1-infected MH-S cells vs. medium only controls (sham). Genes with >2-fold change among subsets and p<0.05 are highlighted (n=3 biological replicates). (b) Real-time qPCR analysis of miR-302b expression in lung tissues from the mice, MLE-12 cells, and MH-S cells infected with PAO1, PAK and Kp. (c) Time-dependent manner of miR-302b expression in mice lung tissues, MH-S, and MLE-12 cells. Mice were treated with 1 × 107 CFU of PAO1 and the lung tissues samples (n=5) were collected at a different time points from 3 to 72 h. MH-S and MLE-12 cells were infected with PAO1 at MOI 10:1 for 1 h and polymyxin B (100 μg/ml) was added for another 1 h to kill bacteria outside of the cells. The cell samples were also collected at a different time points from 3 to 72 h. The miR-302b expression in lung tissues samples, MH-S cells and MLE-12 cells were detected by real-time qPCR. Cell data are representative of three experiments and are shown as means ± SDEV (* p<0.05 by One-Way ANOVA with Tukey’s post-hoc). Average values and SDEVs for animal data (n=5) were calculated from triplicate samples.
Figure 2
Figure 2. miR-302b was induced by TLR2 and TLR4 via the ERK, p38 and NF-κB signaling pathway
(a) MH-S and MLE-12 cells were transfected with control siRNA, TLR4 siRNA or TLR2 siRNA at 10 nM, respectively. Protein lysates prepared at 48 h post-transfection were analyzed for TLR4 or TLR2 expression by western blotting. (b) 24 h later, the transfected cells were infected with PAO1 at MOI 10:1 for 1 h and polymyxin B (100 μg/ml) was added for another 1 h to kill bacteria outside of the cells. Real-time qPCR analysis of miR-302b expression in MH-S and MLE-12 cells. (c) WT and TLR4−/− mice were infected with 1 × 107 CFU/mouse of PAO1 and sacrificed at 12 h post-infection (n=3). Lung tissues were lysed to assess the levels of miR-302b. MH-S (d) and MLE-12 cells (e) were cultured for 2 h with medium only (sham) or PAO1 in the presence or absence of SN50, SP600125, SB203580, GSK690693, and FR180204, respectively. The expression of miR-302b was analyzed by real-time qPCR analysis. Cell data are representative of three experiments and are shown as means ± SDEV (* p<0.05 by One-Way ANOVA with Tukey’s post-hoc). Average values and SDEVs for animal data (n=3) were calculated from triplicate samples.
Figure 3
Figure 3. miR-302b repressed bacterium-induced proinflammatory cytokine gene expression in vitro
(a) Real-time qPCR analysis of IL-1β, IL-6, and TNF-α mRNA levels in MLE-12 cells transfected with miRNA negative control (NS-m) or miR-302b mimics (302b-m), miRNA inhibitor negative control (NS-i), or miR-302b inhibitor (302b-i), respectively and infected with PAO1, PAK and Kp. (b) ELISA analysis of IL-1β, IL-6, and TNF-α protein levels in cell culture medium 16 h after PAO1, PAK and Kp treatment. MLE-12 cells were transfected as indicated in (a). (c) Western blot analysis of IL-1β, IL-6, and TNF-α in MLE-12 cells transfected with NS-m or 302b-m, NS-i, or 302b-i, after treatment with PAO1, PAK and Kp treatment for 8 h. Densitometry was performed and fold changes of protein expression are shown below the corresponding band. (d) Transwell assay for MH-S cells plated on the upper cell culture inserts, with culture medium from untreated-MLE-12 cells, MLE-12 cells transfected with NS-m or 302b-m as indicated in (b) in the lower chambers. Scale bars, 100 μm. These data are representative of three experiments and are shown as means ± SDEV (* p<0.05 by One-Way ANOVA with Tukey’s post-hoc).
Figure 4
Figure 4. miR-302b inhibited bacterium--induced inflammatory responses in vivo
Mice were i.v. injected with NS-m or 302b-m (50 μg/mouse). 24 h later, mice were treated with or without 1 × 107 CFU of PAO1 for 12 h (a) Real-time qPCR analysis of the IL-1β mRNA level in indicated tissues was performed. (b) Lungs were harvested for Western blot analysis of IL-1β, IL-6, and TNF-α protein levels. Densitometry was performed and fold changes of protein expression were quantified. (c) BAL fluid was collected and different cytokines were measured by a standard ELISA (Biosources Sci, Carlsbad, CA). (d) Lung inflammation as assessed by morphologic analysis. The lungs were embedded in formalin and sections were analyzed by H&E staining. Scale bars, 200 μm. (e) Leukocyte infiltration score in lungs. These data are representative of five animals in each group. Error bars indicate ± SDEV (* p<0.05 by One-Way ANOVA with Tukey’s post-hoc).
Figure 5
Figure 5. miR-302b altered the inflammatory responses of AM cells to PAO1
(a) MH-S cells transfected with NS-m or 302b-m, NS-i, or 302b-i. 24 h later, cells were infected with PAO1-GFP at MOI 10:1 for 1 h. Fluorescence intensity was calculated from triplicate samples. (b) The proliferative ability of AMs from the mice of (a) was measured by the MTT assay. (c) MH-S cells were transfected with NS-m, 302b-m, NS-I or 302b-i for 24 h. Then the cells were infected with P. aeruginosa at an MOI 10:1 for 1 h and polymyxin B (100 μg/ml) was added for another 7 h. The medium supernatant was collected and different cytokines were measured by a standard ELISA. (d) Mice were i.v. injected with NS-m and 302b-m (50 μg/mouse) for twice. 24 h after the last dose, AM cells were collected from BAL fluid. The cells were infected with PAO1-GFP at MOI 10:1 for 1 h. Fluorescence intensity was calculated from triplicate samples. (e) The proliferative ability of AMs from the mice of (a) was measured by the MTT assay. (f) AM cells were infected with P. aeruginosa at MOI 10:1 for 1 h and polymyxin B (100 μg/ml) was added for another 8 h to kill bacteria outside of the cells. The medium supernatant was collected and different cytokines were measured by a standard ELISA. These data are representative of three experiments and are shown as means ± SDEV (* p<0.05 by One-Way ANOVA with Tukey’s post-hoc).
Figure 6
Figure 6. miR-302b suppressed the activation of TLR4 signaling pathway
MLE-12 cells were transfected with NS-m or 302b-m for 24 h. Then the cells were infected with PAO1 at MOI 10:1 for 1 h and polymyxin B (100 μg/ml) was added for another indicated time to kill bacteria outside of the cells. (a) Western blot analysis of TLR4, NF-κB p65 and phosphorylated NF-κB p65 in MLE-12 cells 1 h after PAO1 treatment. Densitometric quantification of the western blotting gel data was used by Quantity one software. (b) Luciferase activity of reporters containing the NF-κB promoter in MLE-12 cells after 12 h infected with PAO1. The cell lysates were subjected to luciferase activity analysis using the Dual-Luciferase Reporter Assay System. (c) Confocal results showed the translocation of p-NF-κB (p-p65) in MLE-12 cells from (a) using immune staining. DAPI was used to stain the nucleus (arrows showing the nuclear translocation). Scale bars, 20 μm. These data are representative of three experiments and are shown as means ± SDEV (* p<0.05 by One-Way ANOVA with Tukey’s post-hoc).
Figure 7
Figure 7. IRAK4 is the functional target of miR-302b
(a) IRAK4 3′UTRs contain one predicted miR-302b binding. The figure shows predicted duplex formations between IRAK4 3′UTR (bottom) and miR-302b (middle). The sites of target mutagenesis (top) are also indicated. (b) MLE-12 cells were transfected with NS-m or 302b-m. 24 h later, cells were infected with PAO1 at MOI 10:1 for 1 h and polymyxin B (100 μg/ml) was added for another 1 h to kill bacteria outside of the cells. Real time qPCR analysis of IRAK4 mRNA level in MLE-12 cells transfected with NS-m or 302b-m. (c) Western blot analysis of total and phosphorylated IRAK4 protein levels in MLE-12 cells treated as in (b). (d) Normalized luciferase activity of a reporter containing the wild-type or point-mutated 3′ UTR reporter constructs (wt UTR or mutant UTR) of IRAK4 in MH-S cells co-transfected with NS-m or 302b-m. Average values and SDEVs were calculated from triplicate samples. (e and f) Mice were i.v. injected with vehicle, NS-m or 302b-m (50 μg/mouse) for twice times. 24 h after the last dose, mice were treated with or without 1 × 107 CFU/mouse of PAO1 for 12 h. Lungs were harvested for (e) real-time qPCR analysis of the expression of miR-302b and IRAK4 and (f) western blot analysis of total and phosphorylated IRAK4 protein levels. (g) Real-time qPCR and western blot analysis of IRAK4 mRNA level in MLE-12 cells transfected with siRNA negative control (NS) or IRAK4 siRNA. (h) Western blot analysis of total and phosphorylated NF-κB p65 in MLE-12 cells treated as in (g). (i) ELISA analysis of elaborated IL-1β, IL-6, and TNF-α protein levels in cell culture medium 16 h after PAO1 infection. MLE-12 cells were transfected as indicated in (g). These data are representative of three experiments and are shown as means ± SDEV (* p<0.05 by One-Way ANOVA with Tukey’s post-hoc).
Figure 8
Figure 8. miR-302b decreased susceptibility and mortality rate following P. aeruginosa infection
(a) Mice were i.v. injected with vehicle, NS-i, or 302b-i (50 μg/mouse) 24 and 48 h before bacteria challenge. Then, the mice were sacrificed 12 h after 1 × 107 CFU of PAO1 infection. Lungs were harvested for real-time qPCR analysis of the expression of miR-302b. (b) BAL fluid of the mice from (a) was collected and different cytokines were measured by a standard ELISA. These data are representative of three experiments and are shown as means ± SDEV (* p<0.05 by One-Way ANOVA with Tukey’s post-hoc). (c) Kaplan-Meier survival curves of PAO1 infected C57BL/6 mice (1 × 107 CFU/mouse). Mice were i.v. injected with vehicle, NS-i, 302b-i, NS-m, 302b-m, NS-si (control siRNA), IRAK4-si (IRAK4 siRNA) (50 μg/mouse) 24 and 48 h before bacterial challenge. Survival was determined up to 80 h. (p=0.013, log-rank test, n=6). (d) Schematic model of the critical role of miR-302b in respiratory bacterial infection pathogenesis. miR-302b is induced by TLR2 and TLR4 through the ERK-p38-NF-κB signaling pathway upon Gram-negative bacterial infection. It functions as a negative feedback regulator in TLR signaling by targeting IRAK4.

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