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HBeAg induces neutrophils activation impairing NK cells function in patients with chronic hepatitis B

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The role of neutrophils in hepatitis B virus (HBV) infection has been a subject of debate due to their involvement in antiviral responses and immune regulation. This study aimed to elucidate the neutrophil characteristics in patients with chronic hepatitis B (CHB).


Through flow cytometry and ribonucleic acid-sequencing analysis, the phenotypes and counts of neutrophils were analyzed in patients with CHB. Moreover, the effects of HBeAg on neutrophils and the corresponding pattern recognition receptors were identified. Simultaneously, the cross-talk between neutrophils and natural killer (NK) cells was investigated.


Neutrophils were activated in patients with CHB, characterized by higher expression levels of programmed death-ligand 1 (PD-L1), cluster of differentiation 86, and interleukin-8, and lower levels of CXC motif chemokine receptor (CXCR) 1 and CXCR2. Hepatitis B e antigen (HBeAg) partially induces neutrophil activation through the Toll-like receptor 2 (TLR2). A consistent upregulation of the TLR2 and HBeAg expression was observed in patients with CHB. Notably, the genes encoding molecules pivotal for NK-cell function upon NK receptor engagement enriched in neutrophils after HBeAg activation. The HBeAg-activated neutrophils demonstrated the ability to decrease the production of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) in NK cells, while the PD-1 and PD-L1 pathways partially mediated the immunosuppression.


The immunosuppression of neutrophils induced by HBeAg suggests a novel pathogenic mechanism contributing to immune tolerance in patients with CHB.

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Data availability

The data that support the findings of the current study are available from the corresponding author upon reasonable request.



Hepatitis B virus






Reactive oxygen species


Neutrophil extracellular traps




Hepatitis B e antigen.


Hepatitis B surface antigen


Natural killer


Tumor necrosis factor


Programmed cell death protein 1


Programmed Cell Death-Ligand 1


Healthy controls


Chronic hepatitis B

eAg + CInf:

HBeAg + chronic infection

eAg + CHep:

HBeAg + chronic hepatitis

eAg- CInf:

HBeAg- chronic infection


Peripheral blood mononuclear cells


RNA integrity number


Gene ontology


Kyoto encyclopedia of genes and genomes


Differentially expressed genes




Pattern recognition receptors


NOD-like receptors


  1. Hutin Y, Nasrullah M, Easterbrook P, Nguimfack BD, Burrone E, Averhoff F, et al. Access to treatment for hepatitis B virus infection—worldwide, 2016. MMWR Morb Mortal Wkly Rep. 2018;67(28):773–777.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Spyrou E, Smith CI, Ghany MG. Hepatitis b: current status of therapy and future therapies. Gastroenterol Clin North Am. 2020;49(2):215–238.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Khanam A, Chua JV, Kottilil S. Immunopathology of chronic hepatitis b infection: role of innate and adaptive immune response in disease progression. Int J Mol Sci. 2021.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Yang G, Wan P, Zhang Y, Tan Q, Qudus MS, Yue Z, et al. Innate immunity, inflammation, and intervention in HBV infection. Viruses. 2022.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Maini MK, Gehring AJ. The role of innate immunity in the immunopathology and treatment of HBV infection. J Hepatol. 2016;64(1 Suppl):S60–S70.

    Article  CAS  PubMed  Google Scholar 

  6. Liu K, Wang FS, Xu R. Neutrophils in liver diseases: Pathogenesis and therapeutic targets. Cell Mol Immunol. 2021;18(1):38–44.

    Article  CAS  PubMed  Google Scholar 

  7. Galani IE, Andreakos E. Neutrophils in viral infections: current concepts and caveats. J Leukoc Biol. 2015;98(4):557–564.

    Article  CAS  PubMed  Google Scholar 

  8. Hayashi F, Means TK, Luster AD. Toll-like receptors stimulate human neutrophil function. Blood. 2003;102(7):2660–2669.

    Article  CAS  PubMed  Google Scholar 

  9. Zhou R, Liu L, Wang Y. Viral proteins recognized by different TLRs. J Med Virol. 2021;93(11):6116–6123.

    Article  CAS  PubMed  Google Scholar 

  10. Hoar DI, Bowen T, Matheson D, Poon MC. Hepatitis B virus DNA is enriched in polymorphonuclear leukocytes. Blood. 1985;66(6):1251–1253

    Article  CAS  PubMed  Google Scholar 

  11. Leu CM, Lu YC, Peng WL, Chu HT, Hu CP. The hepatitis B virus e antigen suppresses the respiratory burst and mobility of human monocytes and neutrophils. Immunobiology. 2014;219(11):880–887.

    Article  CAS  PubMed  Google Scholar 

  12. Hu S, Liu X, Gao Y, Zhou R, Wei M, Dong J, et al. Hepatitis b virus inhibits neutrophil extracellular trap release by modulating reactive oxygen species production and autophagy. J Immunol. 2019;202(3):805–815.

    Article  CAS  PubMed  Google Scholar 

  13. Mitra B, Wang J, Kim ES, Mao R, Dong M, Liu Y, et al. Hepatitis b virus precore protein p22 inhibits alpha interferon signaling by blocking STAT nuclear translocation. J Virol. 2019.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Block TM, Guo H, Guo JT. Molecular virology of hepatitis B virus for clinicians. Clin Liver Dis. 2007;11(4):685–706.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Park JJ, Wong DK, Wahed AS, Lee WM, Feld JJ, Terrault N, et al. Hepatitis b virus-specific and global T-Cell dysfunction in chronic hepatitis b. Gastroenterology. 2016;150(3):684-695.e5.

    Article  CAS  PubMed  Google Scholar 

  16. Yang F, Yu X, Zhou C, Mao R, Zhu M, Zhu H, et al. Hepatitis B e antigen induces the expansion of monocytic myeloid-derived suppressor cells to dampen T-cell function in chronic hepatitis B virus infection. Plos Pathog. 2019;15(4):e1007690.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. de Groen RA, Hou J, van Oord GW, Groothuismink Z, van der Heide M, de Knegt RJ, et al. NK cell phenotypic and functional shifts coincide with specific clinical phases in the natural history of chronic HBV infection. Antiviral Res. 2017;140:18–24.

    Article  CAS  PubMed  Google Scholar 

  18. Kayesh M, Kohara M, Tsukiyama-Kohara K. Toll-Like receptor response to hepatitis b virus infection and potential of TLR agonists as immunomodulators for treating chronic hepatitis b: an overview. Int J Mol Sci. 2021.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Lang T, Lo C, Skinner N, Locarnini S, Visvanathan K, Mansell A. The hepatitis B e antigen (HBeAg) targets and suppresses activation of the toll-like receptor signaling pathway. J Hepatol. 2011;55(4):762–769.

    Article  CAS  PubMed  Google Scholar 

  20. Xie X, Lv H, Liu C, Su X, Yu Z, Song S, et al. HBeAg mediates inflammatory functions of macrophages by TLR2 contributing to hepatic fibrosis. Bmc Med. 2021;19(1):247.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Sarin SK, Kumar M, Lau GK, Abbas Z, Chan HL, Chen CJ, et al. Asian-Pacific clinical practice guidelines on the management of hepatitis B: A 2015 update. Hepatol Int. 2016;10(1):1–98.

    Article  CAS  PubMed  Google Scholar 

  22. Liu K, Huang HH, Yang T, Jiao YM, Zhang C, Song JW, et al. Increased neutrophil aging contributes to t cell immune suppression by PD-L1 and arginase-1 in HIV-1 treatment naïve patients. Front Immunol. 2021;12:670616.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Zhang LX, Jiao YM, Zhang C, Song JW, Fan X, Xu RN, et al. HIV reservoir decay and CD4 recovery associated with high CD8 counts in immune restored patients on long-term ART. Front Immunol. 2020;11:1541.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Zhao R, Wang TZ, Kong D, Zhang L, Meng HX, Jiang Y, et al. Hepatoma cell line HepG2.2.15 demonstrates distinct biological features compared with parental HepG2. World J Gastroenterol. 2011;17(9):1152–9.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Zhang J, Zheng H, Diao Y. Natural killer cells and current applications of chimeric antigen Receptor-Modified NK-92 cells in tumor immunotherapy. Int J Mol Sci. 2019.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Bryceson YT, March ME, Ljunggren HG, Long EO. Activation, coactivation, and costimulation of resting human natural killer cells. Immunol Rev. 2006;214:73–91.

    Article  CAS  PubMed  Google Scholar 

  27. Alfarra H, Weir J, Grieve S, Reiman T. Targeting NK cell inhibitory receptors for precision multiple myeloma immunotherapy. Front Immunol. 2020;11:575609.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Gehring AJ, Protzer U. Targeting innate and adaptive immune responses to cure chronic HBV infection. Gastroenterology. 2019;156(2):325–337.

    Article  CAS  PubMed  Google Scholar 

  29. George ST, Lai J, Ma J, Stacey HD, Miller MS, Mullarkey CE. Neutrophils and influenza: a thin line between helpful and harmful. Vaccines (Basel). 2021.

    Article  PubMed  Google Scholar 

  30. Naumenko V, Turk M, Jenne CN, Kim SJ. Neutrophils in viral infection. Cell Tissue Res. 2018;371(3):505–516.

    Article  CAS  PubMed  Google Scholar 

  31. Xu Y, Zhang Q, Zhao Y. The functional diversity of neutrophils and clustered polarization of immunity. Cell Mol Immunol. 2020;17(11):1212–1214.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Goh JG, Ravikumar S, Win MS, Cao Q, Tan AL, Lim J, et al. Neutrophils differentially attenuate immune response to Aspergillus infection through complement receptor 3 and induction of myeloperoxidase. Cell Microbiol. 2018.

    Article  PubMed  Google Scholar 

  33. Pillay J, Kamp VM, van Hoffen E, Visser T, Tak T, Lammers JW, et al. A subset of neutrophils in human systemic inflammation inhibits T cell responses through Mac-1. J Clin Invest. 2012;122(1):327–336.

    Article  CAS  PubMed  Google Scholar 

  34. Xu R, Lin F, Bao C, Huang H, Ji C, Wang S, et al. Complement 5a receptor-mediated neutrophil dysfunction is associated with a poor outcome in sepsis. Cell Mol Immunol. 2016;13(1):103–109.

    Article  CAS  PubMed  Google Scholar 

  35. Milich D, Liang TJ. Exploring the biological basis of hepatitis B e antigen in hepatitis B virus infection. Hepatology. 2003;38(5):1075–1086.

    Article  CAS  PubMed  Google Scholar 

  36. Tsai KN, Ou JJ. Hepatitis B virus e antigen and viral persistence. Curr Opin Virol. 2021;51:158–163.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Milich DR, Jones JE, Hughes JL, Price J, Raney AK, Mclachlan A. Is a function of the secreted hepatitis B e antigen to induce immunologic tolerance in utero? Proc Natl Acad Sci U S A. 1990;87(17):6599–6603.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Tsai SL, Chen PJ, Lai MY, Yang PM, Sung JL, Huang JH, et al. Acute exacerbations of chronic type B hepatitis are accompanied by increased T cell responses to hepatitis B core and e antigens Implications for hepatitis B e antigen seroconversion. J Clin Invest. 1992;89(1):87–96.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Zhu SS, Dong Y, Xu ZQ, Wang LM, Chen DW, Gan Y, et al. a retrospective study on HBsAg clearance rate after antiviral therapy in children with HBeAg-positive chronic hepatitis B aged 1–7 years. Zhonghua Gan Zang Bing Za Zhi. 2016;24(10):738–743.

    Article  CAS  PubMed  Google Scholar 

  40. Yang Y, Han Q, Zhang C, Xiao M, Zhang J. Hepatitis B virus antigens impair NK cell function. Int Immunopharmacol. 2016;38:291–297.

    Article  CAS  PubMed  Google Scholar 

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This study is supported by National Natural Science Foundation of China (No. 82171732), (No. 82272311), (81721002), National Key Research and Development Program of China (2023YFC2306804) and Natural Science Foundation of Shandong Province (ZR2022QH329). Figure 6 was created by BioRender software.

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Authors and Affiliations



Fu-Sheng Wang and Ruonan Xu conceived the study, wrote the manuscript, and constructed the figures with Zhiqian Feng and Junliang Fu. Junliang Fu, Chunmei Bao and Honghong Liu recruited participants and provided samples. jin-Hong Yuan and Chun-Bao Zhou performed flow cytometry. Lili Tang, Kai Liu and Chao Zhang contributed to scientific planning. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Ruonan Xu or Fu-Sheng Wang.

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Conflict of interest

The authors Zhiqian Feng, Junliang Fu, Lili Tang, Chunmei Bao, Honghong Liu, Kai Liu, Tao Yang, Jin-Hong Yuan, Chun-Bao Zhou, Chao Zhang, Ruonan Xu, Fu-Sheng Wang declare no competing interests.

Ethical approval

The study was approved by the Ethics Committee of the Fifth Medical Center of Chinese PLA General Hospital (ethics number: 2020053D). All participants signed informed consent for the study.

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Feng, Z., Fu, J., Tang, L. et al. HBeAg induces neutrophils activation impairing NK cells function in patients with chronic hepatitis B. Hepatol Int (2024).

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