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. 2015 Apr;144(2):284-97.
doi: 10.1093/toxsci/kfu315. Epub 2015 Jan 5.

Role of miRNA in the Regulation of Inflammatory Genes in Staphylococcal Enterotoxin B-induced Acute Inflammatory Lung Injury and Mortality

Affiliations
Free PMC article

Role of miRNA in the Regulation of Inflammatory Genes in Staphylococcal Enterotoxin B-induced Acute Inflammatory Lung Injury and Mortality

Roshni Rao et al. Toxicol Sci. .
Free PMC article

Abstract

Exposure to Staphylococcal enterotoxin B (SEB) causes food poisoning, acute inflammatory lung injury, toxic shock syndrome, and often death. In this study, we investigated whether microRNA (miRNA) play a role in regulating SEB-driven inflammation in the lungs. Exposure to SEB caused immune cell infiltration, robust cytokine and chemokine production, compromised lung function, and 100% mortality in mice. We assessed miRNA and mRNA expression in lung infiltrating mononuclear cells following exposure to SEB and found 89 miRNA that were dysregulated (>2-fold) compared with vehicle controls. In silico analysis revealed that the miRNA exhibited biological functions pertaining to cell death and survival, cellular proliferation, and cell cycle progression. Through the use of q-RT PCR, we validated 9 specific miRNA (miR-155, miR-132, miR-31, miR-222, miR-20b, miR-34a, miR-192, miR-193*, and let-7e) and observed that they were predicted to bind the 3'-UTR of a number of genes that were either involved in the stringent regulation of inflammation (Smad3, Tgfb, Runx1, and Foxo3) or those that contributed to its exacerbation (Stat3, Ptgs2, Ccnd1, Ccne1, NfκB, and Tbx21). Further, by increasing or decreasing the levels of miR-132 (a miRNA highly induced by SEB), we noted the corresponding decrease or increase in the levels of its predicted target FOXO3. As a result of FOXO3 suppression by miR-132, we saw increase in Ifn-γ, Ccnd, and Ccne1. Taken together, our data support the role for miRNA in actively participating and orchestrating SEB-mediated inflammation in the lungs and provide several therapeutic targets for the treatment of SEB-driven toxicity via the modulation of miRNA.

Keywords: ALI; SEB; inflammation; microRNA; mortality.

Figures

FIG. 1.
FIG. 1.
SEB exposure results in pulmonary inflammation and mortality of mice. C3H/HeJ mice were exposed to a “Dual Hit” of SEB and euthanized 72 h post-exposure. A, Survival curve of mice exposed to either vehicle or SEB. B, Measurement of vascular leak 24 h after exposure to the second dose of SEB. Mice were administered Evans Blue dye and following perfusion, lungs were placed in formamide. Absorbance was recorded at 620 nm and the percentage of vascular leak was calculated and graphically represented. C, Representative H&E (×40) staining of sections of the lung demonstrating immune cell infiltration. D, Total number of mononuclear cells infiltrating the lungs in vehicle or SEB exposed mice as determined by trypan blue exclusion method. E, Phenotypic characterization of mononuclear cells infiltrating the lung determined by staining cells with fluorescein-conjugated antibodies against CD4, CD8, and Vβ8 and conducting flow cytometric analysis. Absolute cell counts are represented graphically. Data are represented as mean ± SEM (n = 5) from 3 independent experiments. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (when compared with vehicle).
FIG. 2.
FIG. 2.
Exaggerated expression of chemokines and cytokines after SEB exposure. Mice were exposed to either vehicle or SEB for 72 h and euthanized. The trachea was bound with a suture. Following the excision of the lung along with the trachea, 1 ml of ice-cold PBS was flushed through the trachea and collected as the broncheoalveolar lavage fluid (BALF). Cytokine and chemokine expression was analyzed using a Bioplex and the concentration quantified in pg/ml. Data are represented as mean ± SEM (n = 5) from 2 independent experiments. Statistical significance is indicated as follows *P < 0.05, **P < 0.01, ***P < 0.001 (when compared with vehicle).
FIG. 3.
FIG. 3.
Effect of SEB activation in vitro. Splenocytes seeded at a density of 1 × 106 cells were activated with 1 μg/ml of SEB. A, Twenty-four hours after activation, cells were harvested and triple stained in the following combinations: CD3+Vβ8+CD69+, CD3+Vβ8+CD25+, and CD3+Vβ8+CD28+. The representative dotplots shown are gated on CD3+ cells. B, Cell proliferation assay as measured by the incorporation of thymidine in splenocytes that were activated with SEB for 48 h. C, IFN-γ expression determined by ELISA. Samples were obtained from collecting the supernatants following SEB activation of splenocytes. Data are represented as mean ± SEM (n = 3) from 2 independent experiments. Statistical significance is indicated as follows: **P < 0.01, ***P < 0.001, ****P < 0.0001 (when compared with vehicle).
FIG. 4.
FIG. 4.
SEB exposure leads to dysregulation of microRNA. Seventy-two hours after SEB exposure, lungs were perfused with heparinized PBS and were homogenized. After washing with sterile PBS, the mononuclear cell layer was isolated by density gradient centrifugation and total RNA was isolated. A, A heatmap depicting the differential expression of miRNA in the lungs of SEB exposed mice compared with vehicle. The accompanying color scape depicts mean expression values that are overexpressed (red) or underexpressed (green) above or below the mean, respectively. B, Fold change distribution plot of 1111 mouse-specific miRNA indicating several unchanged, upregulated, or downregulated miRNA. C, A proportional Venn diagram showing the number of miRNA that are overexpressed, underexpressed (<2-fold), or unchanged after SEB exposure.
FIG. 5.
FIG. 5.
Experimental validation of Top overexpressed and underexpressed miRNA. Seventy-two hours after SEB exposure, lungs were perfused with heparinized PBS and were homogenized. After washing with sterile PBS, the mononuclear cell layer was isolated by density gradient centrifugation and total RNA was isolated. A, q-RT PCR validation of the IPA generated Top overexpressed miRNA. B, q-RT PCR validation of the IPA generated Top underexpressed miRNA. Snord96a was used as the small endogenous control and expression of miRNA was normalized to vehicle. Data are represented as mean ± SEM from replicate samples. Lung infiltrating mononuclear cells were pooled from 5 mice in each group. Statistical significance is indicated as follows: *P < 0.05, ***P < 0.001, ****P < 0.0001 (when compared with vehicle).
FIG. 6.
FIG. 6.
In silico analysis of SEB-deregulated miRNA. Ingenuity pathway analysis (IPA) was used to analyze a combined set of the highly predicted and experimentally observed miRNA target genes for miRNA that were over or underexpressed (<2-fold). A, Bar graph highlighting the enrichment of target genes into functional annotation categories with the enrichment P-values plotted on the Y-axis. B, Horizontal slice plot depicting the percentage of the miRNA-associated molecules that can be attributed to very specific biological functions.
FIG. 7.
FIG. 7.
In silico analysis of the predicted mRNA targets. IPA generated network highlighting the interaction between validated miRNA (yellow) and their respective target genes (blue).
FIG. 8.
FIG. 8.
Experimental validation of miRNA target genes. A, Schematic illustration of SEB-induced overexpressed miRNA targeting the 3′-UTR of potential target genes. B, q-RT PCR validation of SEB-induced miRNA mRNA targets. C, Schematic illustration of SEB-induced underexpressed miRNA that target the 3′-UTR of potential target genes. D, q-RT PCR validation of SEB-induced miRNA mRNA targets. Data are represented as mean ± SEM from replicate samples. Lung infiltrating mononuclear cells were pooled from 5 mice in each group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (when compared with vehicle).
FIG. 9.
FIG. 9.
miR-132 targets Foxo3 and leads to increased IFN-γ, CCND1, and CCNE1 levels. A, Splenocytes obtained from mice were transfected either with miR-132 mimic (Mimic) or Mock transfection control (Mock) for 24 h. miR-132 and Foxo3 levels were determined by RT PCR and Western blot. B, Splenocytes were activated with (1 μg/ml) and for 24 h. Cells were then transfected with 100nM miR-132 inhibitor (Inhibitor) or Mock transfection control (Mock) for another 24 h. Foxo3 levels were determined via RT PCR and Western blot. For miRNA normalization, Snord96_a was used as internal control. For mRNA, β-actin was used as the internal control. C, Levels of Ifn-γ after transfection with miR-132 mimic and miR-132 inhibitor were assessed by RT PCR. D, Expression of cell cycle progression genes Ccnd1 and Ccne1 by RT PCR after transfection with miR-132 mimic and inhibitor. Data are represented as mean ± SEM from replicate samples. Statistical significance is indicated as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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