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. 2014 Jul;82(7):2971-9.
doi: 10.1128/IAI.01666-14. Epub 2014 Apr 28.

Staphylococcal Enterotoxin B-induced microRNA-155 Targets SOCS1 to Promote Acute Inflammatory Lung Injury

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Free PMC article

Staphylococcal Enterotoxin B-induced microRNA-155 Targets SOCS1 to Promote Acute Inflammatory Lung Injury

Roshni Rao et al. Infect Immun. .
Free PMC article

Erratum in

  • Infect Immun. 2014 Sep;82(9):3986. Rieder, Sadiye Amcaoglu [added]

Abstract

Staphylococcal enterotoxin B (SEB) causes food poisoning in humans. It is considered a biological weapon, and inhalation can trigger lung injury and sometimes respiratory failure. Being a superantigen, SEB initiates an exaggerated inflammatory response. While the role of microRNAs (miRNAs) in immune cell activation is getting increasing recognition, their role in the regulation of inflammatory disease induced by SEB has not been studied. In this investigation, we demonstrate that exposure to SEB by inhalation results in acute inflammatory lung injury accompanied by an altered miRNA expression profile in lung-infiltrating cells. Among the miRNAs that were significantly elevated, miR-155 was the most overexpressed. Interestingly, miR-155(-/-) mice were protected from SEB-mediated inflammation and lung injury. Further studies revealed a functional link between SEB-induced miR-155 and proinflammatory cytokine gamma interferon (IFN-γ). Through the use of bioinformatics tools, suppressor of cytokine signaling 1 (SOCS1), a negative regulator of IFN-γ, was identified as a potential target of miR-155. While miR-155(-/-) mice displayed increased expression of Socs1, the overexpression of miR-155 led to its suppression, thereby enhancing IFN-γ levels. Additionally, the inhibition of miR-155 resulted in restored Socs1expression. Together, our data demonstrate an important role for miR-155 in promoting SEB-mediated inflammation in the lungs through Socs1 suppression and suggest that miR-155 may be an important target in preventing SEB-mediated inflammation and tissue injury.

Figures

FIG 1
FIG 1
SEB induces lung inflammation. (A) Representative H&E images (×20) of cross sections of the lung from mice exposed to either vehicle or SEB. (B) Lung-infiltrating mononuclear cells obtained by density gradient centrifugation and total number of viable cells were counted using a hemocytometer. Cells were further stained with monoclonal antibody (MAb) to identify CD4+ and CD8+ cells and were analyzed on a flow cytometer. The percentage of the immune cell subsets was multiplied by the total number of cells found in the lung and divided by 100 to yield the absolute cell numbers shown. (C) The concentration of IFN-γ protein present in the BALF was determined using a standard ELISA kit. Data are means ± SEM (n = 5) and are representative of three independent experiments. Statistical significance compared to SEB-vehicle is indicated as follows: **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
FIG 2
FIG 2
SEB exposure leads to dysregulation of miRNA. Forty-eight hours after vehicle or SEB administration, miRNA was isolated from lung-infiltrating mononuclear cells. (A) Heatmap depicting differential expression of miRNA in the lungs of mice exposed to SEB-vehicle compared to vehicle; (B) fold change distribution of the 609 miRNAs, indicating several upregulated and downregulated miRNAs; (C) ingenuity pathway analysis-generated “top network” with network function denoted as “inflammatory response, cellular development, cellular growth, and proliferation”; (D) Cytoscape-generated gene ontology (GO) network based on immunological processes for the molecules associated in the top network using the ClueGo 2.0.7 application. Analysis criteria consisted of a two-sided hypergeometric test with Benjamini Hochberg correction. Only results with a kappa score of 0.3 are displayed. (E) qRT-PCR validation of the IPA-generated top upregulated miRNA. Total RNA was isolated from lung-infiltrating mononuclear cells. Snord96a was used as the small RNA endogenous control, and the expression level of SEB-induced miRNA shown here was normalized to vehicle. Data are represented as means ± SEM from replicate samples (***, P < 0.05; ****, P < 0.01 compared to vehicle).
FIG 3
FIG 3
miR-155 plays a critical role in SEB-induced ALI. WT (C57BL/6) and miR-155−/− (B6.Cg-Mir155 tm1.1 Rsky/J) mice were exposed to SEB and euthanized 48 h later. (A) Representative H&E images (×20) of sections of lung indicating immune cell infiltration; (B) phenotypic characterization of cells infiltrating the lung was determined by staining of mononuclear cells with fluorescent-conjugated MAb against CD4 and CD8; (C) levels of IFN-γ cytokine in the BALF was determined by ELISA. Data are means ± SEM (n = 5) and are representative of two independent experiments. Statistical significance compared to WT is indicated as follows: **, P < 0.01.
FIG 4
FIG 4
IFN-γ forms a critical link between SEB and subsequent miR-155 induction. (A) Lymph node (LN) T cells obtained from naive wild-type (WT) mice were transfected either with miR-155 mimic (Mimic) or mimic control (Control) for 24 h. IFN-γ levels were determined by RT-PCR. (B) LN T cells obtained from miR-155−/− mice were also transfected with 40 nM miR-155 mimic or mimic control as indicated for 24 h, and IFN-γ levels were assessed. (C) LN cells were activated with SEB (1 μg/ml) for 24 h. Cells were then transfected with 100 nM miR-155 inhibitor (Inh) or inhibitor control (Inh Control) for another 24 h. IFN-γ levels were determined via RT-PCR. Data are represented as means ± SEM from replicate samples. Statistical significance is indicated as follows: **, P < 0.01; ****, P < 0.0001.
FIG 5
FIG 5
Identification of SEB-induced miR-155 targets. (A) miR-155 targets were filtered based on their role in cytokine signaling, cell growth and proliferation, and cellular immune response using IPA. A proportional Venn diagram indicating the miR-155 targets common to all three filtering criteria was generated. The list of targets is indicated within brackets, and Socs1, a highly predicted target, is in red. (B) IPA network was generated highlighting the highly predicted (yellow) and experimentally observed (brown) miR-155 targets, in addition to Socs1 (red), the target of interest. (C) Schematic illustration of the predicted target site for miR-155 within the 3′UTR of Socs1 mRNA. (D) Total mRNA was isolated from lung-infiltrating mononuclear cells of WT and miR-155−/− mice exposed to SEB. Relative expression of Socs1 mRNA was determined by qRT-PCR using β-actin as the endogenous control. Data are represented as means ± SEM from replicate samples. Statistical significance is indicated as follows: ****, P < 0.0001.
FIG 6
FIG 6
miR-155 targets Socs1. (A) Chinese hamster ovary (CHO) cells were cotransfected with miR-155 mimic or mimic control along with plasmid containing either the 3′UTR of Socs1 or control plasmid for 24 h. Relative luciferase activity (firefly normalized to renilla) was determined following transfection. (B) Lymph nodes (LN) cells obtained from naive wild-type (WT) mice were transfected either with 40 nM miR-155 mimic (Mimic) or mimic control (Control) for 24 h. Socs1 levels were determined by RT-PCR. (C) LN cells obtained from miR-155−/− were also transfected with 40 nM miR-155 mimic or mimic control as indicated for 24 h, and Socs1 levels were assessed. (D) LN cells were activated with SEB (1 μg/ml) for 24 h. Cells were then transfected with 100 nM miR-155 inhibitor (Inh) or inhibitor control (Inh Control) for another 24 h. Socs1 levels were determined via qRT-PCR. Data are represented as means ± SEM from replicate samples statistical significance is indicated as follows: *, P < 0.05; **, P < 0.01; ****, P < 0.0001.
FIG 7
FIG 7
Schematic of SEB-mediated downregulation of Socs1 via miR-155. SEB exposure leads to the release of IFN-γ and subsequent expression of miR-155. miR-155-mediated suppression of Socs1 prevents appropriate control of IFN-γ, leading to cell proliferation and sustained cytokine signaling and damage to the lung.

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