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HBeAg induces the expression of macrophage miR-155 to accelerate liver injury via promoting production of inflammatory cytokines

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Abstract

Activation of Kupffer cells (KCs) induced that inflammatory cytokine production plays a central role in the pathogenesis of HBV infection. The previous studies from our and other laboratory demonstrated miRNAs can regulate TLR-inducing inflammatory responses to macrophage. However, the involvement of miRNAs in HBV-associated antigen-induced macrophage activation is still not thoroughly understood. Here, we evaluated the effects and mechanisms of miR-155 in HBV-associated antigen-induced macrophage activation. First, co-culture assay of HepG2 or HepG2.2.15 cells and RAW264.7 macrophages showed that HepG2.2.15 cells could significantly promote macrophages to produce inflammatory cytokines. Furthermore, we, respectively, stimulated RAW264.7 macrophages, mouse primary peritoneal macrophages, or healthy human peripheral blood monocytes with HBV-associated antigens, including HBcAg, HBeAg, and HBsAg, and found that only HBeAg could steadily enhance the production of inflammatory cytokines in these cells. Subsequently, miRNAs sequencing presented the up- or down-regulated expression of multiple miRNAs in HBeAg-stimulated RAW264.7 cells. In addition, we verified the expression of miR-155 and its precursors BIC gene with q-PCR in the system of co-culture or HBeAg-stimulated macrophages. Meanwhile, the increased miR-155 expression was positively correlation with serum ALT, AST, and HBeAg levels in AHB patients. Although MAPK, PI3K, and NF-κB signal pathways were all activated during HBeAg treatment, only PI3K and NF-κB pathways were involved in miR-155 expression induced by HBeAg stimulation. Consistently, miR-155 over-expression inhibited production of inflammatory cytokines, which could be reversed by knocking down miR-155. Moreover, we demonstrated that miR-155 regulated HBeAg-induced cytokine production by targeting BCL-6, SHIP-1, and SOCS-1. In conclusion, our data revealed that HBeAg augments the expression of miR-155 in macrophages via PI3K and NF-κB signal pathway and the increased miR-155 promotes HBeAg-induced inflammatory cytokine production by inhibiting the expression of BCL-6, SHIP-1, and SOCS-1.

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Abbreviations

HBV:

Hepatitis B virus

HAV:

Hepatitis A virus

HCV:

Hepatitis C virus

HDV:

Hepatitis D virus

HEV:

Hepatitis E virus

HIV:

Human immunodeficiency virus

HBcAg:

Hepatitis B core antigen

HBeAg:

Hepatitis B e antigen

HBsAg:

Hepatitis B surface antigen

IL-6:

Interleukin-6

TNF-α:

Tumor necrosis factor-α

miR-155:

MicroRNA-155

q-PCR:

Quantitative real-time polymerase chain reaction

ALT:

Alanine aminotransferase

AST:

Aspartate aminotransferase

AHB:

Acute hepatitis B

CHB:

Chronic hepatitis B

MAPK:

Mitogen-activated protein kinase

BCL-6:

B-cell lymphoma 6

SHIP-1:

Src homology-2 domain-containing inositol 5-phosphatase 1

SOCS-1:

Suppressor of cytokine signaling-1

TLR:

Toll-like receptors

NK:

Natural killer

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (81600469, 81472685, and 81772626), the Science and Technology Development Projects of Shandong Province (2017GSF218053 and 2016GSF201126), the Clinical Medical Science and Technology Innovation Program (201704114), the Major Special Plan of Science and Technology of Shandong Province (2015ZDXX0802A01), and the Shandong Province medical and health science and technology development project (2017WS194).

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  1. Wenwen Wang and Hongjun Bian have contributed equally to this work.

Authors and Affiliations

  1. Shandong Provincial Hospital Affiliated to Shandong University, Jinan, 250021, Shandong, China

    Wenwen Wang, Hongjun Bian, Feifei Li, Xiao Li, Di Zhang, Shuohuan Sun, Shouyang Song, Qiang Zhu, Wanhua Ren, Chengyong Qin & Jianni Qi

  2. Shandong Provincial Engineering and Technological Research Center for Liver Diseases Prevention and Control, Jinan, 250021, Shandong, China

    Wenwen Wang, Hongjun Bian, Feifei Li, Xiao Li, Di Zhang, Shuohuan Sun, Shouyang Song, Qiang Zhu, Wanhua Ren, Chengyong Qin & Jianni Qi

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  1. Wenwen Wang
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18_2018_2753_MOESM1_ESM.tif

Supplementary material 1 (TIFF 17571 kb) Supplementary Fig. 1 RAW264.7 macrophages were stimulated with HBeAg (2000 µg/ml) for 24 h and collected with Trizol. U6, snoRNA202 (another commonly internal control) and miR-155 were reverse transcribed with TapMan-related kit and these microRNA expressions were measured using TaqMan miRNA assays (A–D). Data are representative of three independent experiments (mean ± S.D. of triplicates in A-D)

18_2018_2753_MOESM2_ESM.tif

Supplementary material 2 (TIFF 25690 kb) Supplementary Fig. 2 RAW264.7 macrophages were stimulated with different concentrations HBeAg (including 0, 25, 50, 100, 300, 500, 1000, and 2000 ng/ml) for 24 h and the expression of IL-6, TNF-α, and miR-155 were detected with q-PCR (A, C, E) and the secretion of IL-6 and TNF-α was measured with ELISA (B, D). HBeAg was diluted to different concentrations (0, 25, 50, 100, 300, 500, 1000, and 2000 ng/ml) and detected with chemiluminescence microparticle immunoassay (CMIA) (F). Data are representative of three independent experiments (mean ± S.D. of triplicates in A–E). Data are representative of one experiment (mean ± S.D. of triplicates in F). *p < 0.05, **p < 0.01, ***p < 0.001

18_2018_2753_MOESM3_ESM.tif

Supplementary material 3 (TIFF 54744 kb) Supplementary Fig. 3 RAW264.7 macrophages were stimulated with HBeAg for 24 h and some more magnification photos were recorded with light microscope (original magnification, ×100 or ×200. White box: the specific zoom position)

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Wang, W., Bian, H., Li, F. et al. HBeAg induces the expression of macrophage miR-155 to accelerate liver injury via promoting production of inflammatory cytokines. Cell. Mol. Life Sci. 75, 2627–2641 (2018). https://doi.org/10.1007/s00018-018-2753-8

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