Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Apr 8;17(4):535.
doi: 10.3390/ijms17040535.

Inhibition of IFN-γ-Induced Nitric Oxide Dependent Antimycobacterial Activity by miR-155 and C/EBPβ

Yongwei Qin  1   2 , Qinglan Wang  3 , Youlang Zhou  4 , Yinong Duan  5 , Qian Gao  6
Affiliations

Inhibition of IFN-γ-Induced Nitric Oxide Dependent Antimycobacterial Activity by miR-155 and C/EBPβ

Yongwei Qin et al. Int J Mol Sci. .

Abstract

miR-155 (microRNA-155) is an important non-coding RNA in regulating host crucial biological regulators. However, its regulatory function in mycobacterium infection remains unclear. Our study demonstrates that miR-155 expression is significantly increased in macrophages after Mycobacterium marinum (M.m) infection. Transfection with anti-miR-155 enhances nitric oxide (NO) synthesis and decreases the mycobacterium burden, and vice versa, in interferon γ (IFN-γ) activated macrophages. More importantly, miR-155 can directly bind to the 3'UTR of CCAAT/enhancer binding protein β (C/EBPβ), a positive transcriptional regulator of nitric oxide synthase (NOS2), and regulate C/EBPβ expression negatively. Knockdown of C/EBPβ inhibit the production of nitric oxide synthase and promoted mycobacterium survival. Collectively, these data suggest that M.m-induced upregulation of miR-155 downregulated the expression of C/EBPβ, thus decreasing the production of NO and promoting mycobacterium survival, which may provide an insight into the function of miRNA in subverting the host innate immune response by using mycobacterium for its own profit. Understanding how miRNAs partly regulate microbicidal mechanisms may represent an attractive way to control tuberculosis infectious.

Keywords: C/EBPβ; IFN-γ; antibacteria; microRNA-155; mycobacterium.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Distinguishing expression of miRNAs in M.m infected macrophages. (a–e) Expression of miR-125b, miR-146a, miR-129, Let-7a and miR-155 in RAW 264.7 cells after M.m infection for the indicated times; (f) Expression of miR-155 in RAW 264.7 cells at different multiplicities of infection (MOI) for 24 h; (g) miR-155 expression levels were detected in THP-1 cells after M.m infection or lipopolysaccharide (LPS) (100 ng/mL) treatment; (h) miR-155 expression level was examined in mouse peritoneal macrophages (MPMs); (i) miR-155 expression was detected in RAW 264.7 cells after transfected with miR-155 Negative Control (NC), miR-155, anti-miR-155 NC and anti-miR-155 prior to infection with M.m or treatment with IFN-γ/M.m for 24 h. All miRNAs were detected by quantitative real-time polymerase chain reaction (PCRs) (qPCRs). U6 snRNA was used as endogenous control. Experiments were executed with at least three biologically independent replicates, data were shown as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. FC: fold change.
Figure 2
Figure 2
miR-155 negatively regulates the antimycobacterial activity of IFN-γ activated macrophages. RAW 264.7 cells (a,b) and MPM (c,d) were transfected with miR-155 NC, miR-155 or anti-miR-155 NC, anti-miR-155 prior to infection with IFN-γ/M.m for 48 and 96 h, and intracellular M.m survival was quantified by Colony Forming Unit (CFU) assays at the indicated time points. All data represent the mean ± SEM from three independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 3
Figure 3
miR-155 promote M.m survival in macrophages by inhibition of NO. (a) NOS2 expression in the cytoplasm of RAW 264.7 cells. The co-localization of GFP-labeled M.m (green) and TRITC-labeled NOS2 (red) in the cytoplasm was examined by confocal microscopy. Arrows indicate M.m. Nucleus of RAW 264.7 cells were stained with Hoechst (purple); (b) Immunoblot blot analysis of NOS2 in RAW 264.7 cells; (c) NO synthesis in RAW 264.7 cell. Cells were transfected with miR-155 NC, miR-155, anti-miR-155 NC and anti-miR-155 followed by IFN-γ treatment and infected with M.m for 24 h, supernatant nitrite was determined by the Griess test; (d) NOS2 expression in the cytoplasm of MPM cells. Cells were transfected with miR-155 NC, miR-155, anti-miR-155, and infected with GFP-labeled M.m/IFN-γ for 24 h. The co-localization of M.m (green) and TRITC-labeled NOS2 (red) in the cytoplasm were detected by confocal microscopy. Nucleuses of MPMs were stained with Hoechst (purple); (e,f) The effect of miR-155, anti-miR-155 and NOS2 inhibitor on M.m survival in RAW 264.7 cells. Cells were transfected with miR-155, anti-miR-155 and corresponding NC, pretreated with 1 mM SMT for 2 h, infected with IFN-γ/M.m for 48 h. Intracellular M.m survival was determined by CFU assay. Data are shown as the means ± SEM of three independent experiments, * p < 0.05, ** p < 0.01.
Figure 4
Figure 4
Translational inhibition of C/EBPβ by miR-155. (a) Schematic drawing of the C/EBPβ transcript. Sequence alignment and evolutionary conservation between miR-155 and its theoretical binding sites in the 3′UTR of C/EBPβ mRNA from several indicated species, with sequences recognized by miR-155 seed sequence shown in bold. The sequence of C/EBPβ 3′UTR mutant used for reporter assay is also shown (asterisk); (b) luciferase reporter constructs containing 3′UTR (psiCHECK2-C/EBPβ 3′UTR) or mutant 3′UTR (psiCHECK2-C/EBPβ 3′UTR-mut) of C/EBPβ gene was cotransfected with miR-155 or miR-155 NC in HEK 293T cell and the luciferase activities (Renilla/firefly) were assayed; (c) Western blot analysis of C/EBPβ in RAW 264.7 cells; (d) integrated band densities were obtained by scanning using a densitometer. Data are representative of three independent experiments. Results shown are the mean ± SEM. * p < 0.05.
Figure 5
Figure 5
miR-155 promote M.m survival in RAW 264.7 cells by inhibition of C/EBPβ and NO. (a) Western blot confirmation of shRNA-mediated knockdown of C/EBPβ in RAW 264.7 cell; (b) the effect of C/EBPβ on NOS2 expression in RAW 264.7 cells. Cells were infected with either LV-copGFP-scramble or LV-copGFP-shRNA-C/EBPβ. After 48 h, cells were infected with IFN-γ/M.m for 24 h and cell lysates were analyzed via immunoblot; (c) RAW 264.7 cells infected with either LV-copGFP-scramble or LV-copGFP-shRNA-C/EBPβ were treated with IFN-γ and infected with M.m for the indicated times, and intracellular M.m survival was quantified by CFU test at the indicated times; (d,e) RAW 264.7 cells infected with either LV-copGFP-scramble or LV-copGFP-shRNA-C/EBPβ were respectively transfected with miR-155 NC, miR-155 (d), anti-miR-155 NC or anti-miR-155 (e), followed by infection with IFN-γ/M.m for 96 h, and intracellular M.m survival was determined by CFU assay. All data are shown as the means ± SEM of three independent experiments, * p < 0.05, ** p < 0.01.
Figure 6
Figure 6
Schematic outline of miR-155-regulated bactericidal activity. miR-155 expression is induced by M.m in IFN-γ activated macrophages and mediates translational inhibition of C/EBPβ by targeting its 3′UTR. Successively, C/EBPβ is induced and mediates NO-dependent antibacterial mechanism. Thereby, miR-155 can negatively regulate the NO-dependent antibacterial mechanism by C/EBPβ. Black dash line arrow: direct effect; black solid line arrow: indirect effect; blue arrow: activated.
See this image and copyright information in PMC

References

    1. Russell D.G. Mycobacterium tuberculosis: Here today, and here tomorrow. Nat. Rev. Mol. Cell Biol. 2001;2:569–577. doi: 10.1038/35085034. - DOI - PubMed
    1. Koul A., Herget T., Klebl B., Ullrich A. Interplay between mycobacteria and host signalling pathways. Nat. Rev. Microbiol. 2004;2:189–202. doi: 10.1038/nrmicro840. - DOI - PubMed
    1. Krutzik S.R., Modlin R.L. The role of Toll-like receptors in combating mycobacteria. Semin. Immunol. 2004;16:35–41. doi: 10.1016/j.smim.2003.10.005. - DOI - PubMed
    1. Huntzinger E., Izaurralde E. Gene silencing by microRNAs: Contributions of translational repression and mRNA decay. Nat. Rev. Genet. 2011;12:99–110. doi: 10.1038/nrg2936. - DOI - PubMed
    1. Inui M., Martello G., Piccolo S. MicroRNA control of signal transduction. Nat. Rev. Mol. Cell Biol. 2010;11:252–263. doi: 10.1038/nrm2868. - DOI - PubMed

Publication types

MeSH terms

LinkOut - more resources