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, 30 (1), 35-44

Hepatitis B Virus E Antigen Regulates Monocyte Function and Promotes B Lymphocyte Activation

Affiliations

Hepatitis B Virus E Antigen Regulates Monocyte Function and Promotes B Lymphocyte Activation

Bingru Lu et al. Viral Immunol.

Abstract

Hepatitis B virus (HBV) e (HBe) antigen is a nonstructural virus component with great immune regulation roles. It regulates adaptive immunity response and participates in persistent infection development. However, its roles on monocytes and B lymphocytes were rarely studied. Herein, we studied HBe roles on U937 and Hmy2.CIR by creating HBe stably transfected cells using lentivirus. We detected the motility of HBe-U937 through transwell migration assay. Cytokines that primarily produced by monocytes, including BAFF, B-cell activating factor (BAFF), interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, and A proliferation inducing ligand (APRIL), were measured in culture supernatants of transfected U937, and serum BAFF, IL-6, and IL-10 were detected in HBe-positive and HBe-negative HBV-infected patients. Among these, BAFF mRNA and membrane-bound BAFF were further detected. Activation and inhibition markers of B lymphocytes on HBe-Hmy2.CIR and proliferation of transfected Hmy2.CIR after coculture with transfected U937 were also detected. We found that U937 migration was inhibited by HBe. BAFF expression was increased in HBe-U937, however, membrane-bound BAFF on HBe-U937 was decreased. In addition, Serum BAFF in HBe-positive patients was higher than in HBe-negative patients. IL-6 and IL-10 were increased in HBe-U937 after being stimulated by lipopolysaccharide (LPS), however, serum IL-6 and IL-10 were not associated with HBe status in patients. Besides, TNF-α and APRIL expression were basically the same in GV166-U937 and HBe-U937. B lymphocyte activation markers CD86 and Tspan33 were raised in HBe-Hmy2.CIR. However, inhibition markers Lyn and CD32b had no differences between HBe-Hmy2.CIR and control. Proliferation of transfected Hmy2.CIR was not affected by coculture with transfected U937, however, HBe transfection itself enhanced Hmy2.CIR proliferation. Altogether, these revealed that HBe can inhibit U937 migration and promote cytokines, including BAFF, IL-6, and IL-10, production in U937. Besides, HBe enhances BAFF release from U937 and increases BAFF concentration in vivo. In addition, HBe antigen facilitates Hmy2.CIR activation and proliferation through direct induction.

Keywords: B cell activating factor; B lymphocyte; HBV e antigen; cytokine; monocyte.

Conflict of interest statement

Author Disclosure Statement No competing financial interests exist.

Figures

<b>FIG. 1.</b>
FIG. 1.
HBe was successfully transfected into U937 and Hmy2.CIR cells and expressed in these cells. (A) DNA extracted from Hmy2.CIR and transfected Hmy2.CIR cells were used as templates to amplify HBe DNA fragments. The result of DNA agarose gel electrophoresis showed that HBe DNA fragment was successfully integrated in Hmy2.CIR cells. Lane 1: empty lentivirus carrier—Hmy2.CIR; Lane 2: HBe-Hmy2.CIR; Lane 3: Hmy2.CIR; Lane 4 and Lane 5: negative control. (B) DNA extracted from U937 and transfected U937 cells were used as templates to amplify HBe DNA fragments. DNA agarose gel electrophoresis showed that HBe DNA fragment was successfully integrated in U937 cells also. Lane 1: empty lentivirus carrier—U937; Lane 2: HBe-U937; Lane 3: U937; Lane 4 and Lane 5: negative control. HBe DNA fragment is 552 bp in length. (C) HBe mRNA expression in Hmy2.CIR and U937 cells detected by reverse transcription PCR. Lane 1: empty lentivirus carrier—Hmy2.CIR; Lane 2: HBe-Hmy2.CIR; Lane 3: Hmy2.CIR; Lane 4 and Lane 5: negative control; Lane 6: DNA Marker; Lane 7: empty lentivirus carrier—U937; Lane 8: HBe-U937, Lane 9: U937; Lane 10 and Lane 11: negative control; Lane 12: DNA marker. The product of the reverse transcription PCR is 156 bp in length. This result proved that HBe DNA was successfully transcribed in transfected Hmy2.CIR and U937 cells. (D) Expression of HBe protein in transfected Hmy2.CIR cells detected by immunofluorescence. There was weak fluorescence in cytoplasm of HBe-Hmy2.CIR cells after incubation with HBe antibody and FITC-labeled second antibody (the first lane). It verified that HBe protein was weakly expressed in cytoplasm of HBe-Hmy2.CIR cells. Hmy2.CIR cells incubated with PBS was done as negative control. PCR, polymerase chain reaction. Color images available online at www.liebertpub.com/vim
<b>FIG. 2.</b>
FIG. 2.
HBe inhibited U937 motility and increased BAFF, IL-6, and IL-10 expression in monocytes. (A) Impaired migration ability of HBe-transfected U937 cells. The percentage of migratory cells in HBe-U937 group was sufficiently decreased when compared with GV166-HBe group (GV166-U937 vs. HBe-U937: 21.9% vs. 14.4%; p = 0.0010). This revealed a significant inhibitory role of HBe in the migration ability of U937 cells. (B) BAFF mRNA expression in HBe-transfected U937 cells. Semiquantitative RT-PCR showed that the BAFF mRNA expression level in HBe-U937 cells was significantly increased, which indicated that HBe promoted BAFF expression in U937 cells. GAPDH was used for reference gene to normalize BAFF mRNA level. (C) Soluble BAFF concentration in culture supernatants of U937 and transfected U937 cells. It showed that soluble BAFF secretion was increased in HBe-transfected U937 cells. (D) Serum BAFF levels in HBe-positive and HBe-negative patients with HBV infection. Serum samples were collected from patients and BAFF levels were measured by ELISA. This result confirmed that BAFF secretion increased in HBe-positive patients. (E) Other cytokines in culture supernatant of U937 and transfected U937 cells. IL-6 and IL-10 expression in lentivirus-transfected U937 were inhibited compared with U937. However, when compared with GV166-U937, it showed that both IL-6 and IL-10 were increased in HBe-U937 cells. TNF-α and APRIL expression had no significant difference between groups. (F) Serum IL-6 and IL-10 in HBe-positive and HBe-negative CHB patients. It showed that IL-6 and IL-10 expression were not associated with HBe antigen status in HBV-infected patients. All experiments were repeated thrice independently. Differences between groups were analyzed using the independent sample Student's t-test and considered to be significant if p < 0.05. APRIL, A proliferation inducing ligand; BAFF, B cell activating factor; CHB, chronic hepatitis B; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; RT, real time; TNF, tumor necrosis factor.
<b>FIG. 3.</b>
FIG. 3.
Membrane-bound BAFF on U937 cells and BAFF-R on Hmy2.CIR cells detected by flow cytometry. (A) Presentation of one single experiment about mBAFF expression on U937 and transfected U937 cells detected by flow cytometry. (B) Presentation of one single experiment about BAFF-R expression on Hmy2.CIR and transfected Hmy2.CIR cells detected by flow cytometry. (C) mBAFF expression on U937 and transfected U937 cells. Statistical results of triple experiments about mBAFF expression on U937 and transfected U937 cells were presented, which revealed that there was a significant decrease in mBAFF expression on HBe-U937 cells (GV166-U937 vs. HBe-U937: 87.83% vs. 73.77%, p = 0.0056). (D) Statistical results of triple experiments about BAFF-R expression on Hmy2.CIR and transfected Hmy2.CIR cells. There was no significant difference in BAFF-R expression on HBe-Hmy2.CIR cells compared with Hmy2.CIR cells. All experiments were repeated thrice independently. Differences between groups were analyzed using the independent sample Student's t-test and considered to be significant if p < 0.05. BAFF-R, B-cell activating factor receptor; mBAFF, membrane-bound BAFF. Color images available online at www.liebertpub.com/vim
<b>FIG. 4.</b>
FIG. 4.
B lymphocyte activation markers and inhibitory regulation molecules determined by semiquantitative RT-PCR. (A, B) mRNA expression of B cell activation markers CD69, CD86, and TSPAN33 in Hmy2.CIR and transfected Hmy2.CIR cells. (A) Without stimulation. This result showed that in the resting state, the expression of two B lymphocyte activation markers CD86 and TSPAN33 was slightly increased in HBe-Hmy2.CIR cells. (B) After stimulation with CD40L (1.5 μg/mL) and IL-4 (3 ng/mL) for 12 h. This result presented a significant increase of CD86 and TSPAN33 expression in HBe-Hmy2.CIR cells after these cells were activated. (C, D) mRNA expression of B cell inhibitory regulation molecules CD32b and Lyn in Hmy2.CIR and transfected Hmy2.CIR cells. (C) Without stimulation, (D) stimulated with IgM μ chain (2 μg/mL) for 8 h. No significant differences of these two inhibition markers of B lymphocytes were detected between HBe-Hmy2.CIR and GV166-Hmy2.CIR cells. All experiments were repeated thrice independently. Differences between groups were analyzed using the independent samples Student's t-test and considered to be significant if p < 0.05. *p < 0.05, **p < 0.01.
<b>FIG. 5.</b>
FIG. 5.
HBe-positive and HBe-negative Hmy2.CIR proliferation in coculture systems with HBe-positive or HBe-negative U937. GV166-Hmy2.CIR and HBe-Hmy2.CIR were cocultured with GV166-U937 or HBe-U937 for indicated times. After that, proliferation of GV166-Hmy2.CIR and HBe-Hmy2.CIR was detected using Cell Counting Kit-8. Absorbance at 450 nm of each group was used to reflect cell proliferation. It showed that coculture with GV166-U937 or HBe-U937 did not influence GV166-Hmy2.CIR and HBe-Hmy2.CIR proliferation; however, HBe-Hmy2.CIR proliferation was significantly increased compared with GV166-Hmy2.CIR, whether cocultured with GV166-U937 or HBe-U937.

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