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. 2024 May 8;16(5):741.
doi: 10.3390/v16050741.

Cytokine Response of Natural Killer Cells to Hepatitis B Virus Infection Depends on Monocyte Co-Stimulation

Paul Kupke  1 , Johanna Brucker  1 , Jochen M Wettengel  2   3 , Ulrike Protzer  2   3 , Jürgen J Wenzel  4 , Hans J Schlitt  1 , Edward K Geissler  1 , Jens M Werner  1
Affiliations

Cytokine Response of Natural Killer Cells to Hepatitis B Virus Infection Depends on Monocyte Co-Stimulation

Paul Kupke et al. Viruses. .

Abstract

Hepatitis B virus (HBV) is a major driver of chronic hepatic inflammation, which regularly leads to liver cirrhosis or hepatocellular carcinoma. Immediate innate immune cell response is crucial for the rapid clearance of the infection. Here, natural killer (NK) cells play a pivotal role in direct cytotoxicity and the secretion of antiviral cytokines as well as regulatory function. The aim of this study was to further elucidate NK cell responses triggered by an HBV infection. Therefore, we optimized HBV in vitro models that reliably stimulate NK cells using hepatocyte-like HepG2 cells expressing the Na+-taurocholate co-transporting polypeptide (NTCP) and HepaRG cells. Immune cells were acquired from healthy platelet donors. Initially, HepG2-NTCP cells demonstrated higher viral replication compared to HepaRG cells. Co-cultures with immune cells revealed increased production of interferon-γ and tumor necrosis factor-α by NK cells, which was no longer evident in isolated NK cells. Likewise, the depletion of monocytes and spatial separation from target cells led to the absence of the antiviral cytokine production of NK cells. Eventually, the combined co-culture of isolated NK cells and monocytes led to a sufficient cytokine response of NK cells, which was also apparent when communication between the two immune cell subpopulations was restricted to soluble factors. In summary, our study demonstrates antiviral cytokine production by NK cells in response to HBV+ HepG2-NTCP cells, which is dependent on monocyte bystander activation.

Keywords: HBV; HepG2; HepaRG; NK cells; NTCP; bystander activation; hepatitis B virus; monocytes; natural killer cells.

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Conflict of interest statement

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Verification of HBV replication: (A) the progression of HBV DNA and HBeAg in the cell culture supernatants of HepG2-NTCP and HepaRG cells as a median of two independent experiments; (B) the effect of PBMCs and isolated NK cells on HBeAg after 24 h of co-culture (n = 18). Appearance: median with interquartile range (whiskers min to max); statistical analysis: Friedman test with Dunn’s multiple-comparison test.
Figure 2
Figure 2
Antiviral cytokine response by NK cells after contact with HBV+/− HepG2-NTCP cells for 24 h: (A) the frequency of IFN-γ+ NK cells and (B) the respective MFI values (n = 15); (C) the change in frequency (HBV+ – HBV−) of IFN-γ+ NK cells comparing CD56bright and CD56dim NK cells (n = 15); (D) the frequency of TNF-α+ NK cells and (E) the respective MFI values (n = 12); (F) the change in frequency (HBV+ – HBV−) of TNF-α+ NK cells comparing CD56bright and CD56dim NK cells (n = 12). Appearance: median with interquartile range (C,F); statistical analysis: Wilcoxon matched-pair signed-rank test.
Figure 3
Figure 3
Loss of antiviral cytokine response after isolation of CD56+ NK cells; co-culture with HBV+/− HepG2-NTCP cells for 24 h: (A) the change in frequency (HBV+ – HBV−) of isolated IFN-γ+ NK cells compared to NK cells within PBMCs, also separated in (B) CD56bright NK cells and (C) CD56dim NK cells (n = 15 and 10); (D) the change in frequency (HBV+ – HBV−) of isolated TNF-α+ NK cells compared to NK cells within PBMCs, also separated in (E) CD56bright NK cells and (F) CD56dim NK cells (n = 12 and 10). Appearance: median with interquartile range (whiskers min to max); statistical analysis: Wilcoxon matched-pair signed-rank test.
Figure 4
Figure 4
Loss of antiviral cytokine response after depletion of CD14+ monocytes and spatial separation by transwells; co-culture with HBV+/- HepG2-NTCP cells for 24 h: (A) the frequency of IFN-γ+ NK cells following the depletion of CD14+ monocytes and (B) the respective frequency of TNF-α+ NK cells; (C) the frequency of IFN-γ+ NK cells following spatial separation by transwells and (D) the respective frequency of TNF-α+ NK cells (n = 7). Appearance: median with interquartile range (whiskers min to max); statistical analysis: Friedman test with Dunn’s multiple-comparison test.
Figure 5
Figure 5
Restored cytokine production of CD56+ NK cells after combination with CD14+ monocytes depending on the communication possible; co-culture with HBV+/− HepG2-NTCP cells for 24 h: (A) the change in frequency (HBV+ − HBV−) of IFN-γ+ NK cells depending on the usage of transwells (TW) and (B) the respective frequency of TNF-α+ NK cells (n = 7). Appearance: median with interquartile range; statistical analysis: one-sample Wilcoxon signed-rank test. * indicates p < 0.05.
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