ACE2 and TMPRSS2 Expression in Hepatocytes of Chronic HBV Infection Patients

Abstract

Background: Pre-existing liver disease is a risk factor for the worse prognosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We aimed to evaluate whether chronic hepatitis B (CHB) and hepatocellular carcinoma (HCC) affect the expression of viral receptor angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) in the liver.

Methods: Twelve pairs of matched liver tissues of HCC and para-carcinoma were collected from the First Affiliated Hospital of Zhejiang University School of Medicine. And 20 liver biopsies from CHB patients were collected from Peking University People's Hospital. The expression of ACE2 and TMRPSS2 were detected using immunofluorescence staining, western blot, and RT-qPCR. The effects of hepatitis B virus (HBV) replication or interferon on ACE2 and TMPRSS2 expression were tested in hepatic cell lines.

Results: The mRNA expression of TMPRSS2 in HCC tissues was six-fold higher than that of para-carcinoma tissues (P = 0.002), whereas that of ACE2 was not statistically different between HCC and para-carcinoma tissues. Hepatocellular ACE2 expression was detected in 35% (7/20) of CHB patients and mostly distributed in the inflammatory areas. However, there was no difference in TMPRSS2 expression between areas with or without inflammation. IFN-α2b slightly induced ACE2 expression (2.4-fold, P = 0.033) in HepG2 cells but not in Huh-7, QSG-7701, and L-02 cells. IFN-α2b did not affect TMPRSS2 expression in these cell lines. In addition, HBV replication did not alter ACE2 expression in HepAD38 cells.

Conclusions: Although HBV replication does not directly affect the expression of ACE2 and TMPRSS2, intrahepatic inflammation and carcinogenesis may increase their expression in some patients, which, in turn, may facilitate SARS-CoV-2 infection in hepatocytes.

Keywords: ACE2; Liver diseases; SARS-CoV-2; TMPRSS2.

Figure 1

Intrahepatic expression of ACE2 and TMPRSS2 in patients with hepatocellular carcinoma. The levels of (A) ACE2 mRNA and (B) TMPRSS2 mRNA in 12 pairs of matched hepatocellular carcinoma and para-carcinoma tissues were analyzed using RT-qPCR and were compared using paired Student t-test. The value of ΔCt was the Ct value of actin minus that of target genes of ACE2 or TMPRSS2. The differences in (C) ACE2 and (D) TMPRSS2 mRNA expression between hepatocellular carcinoma and para-carcinoma tissues were compared using Mann-Whitney U test. P < 0.05 was considered statistically significant. ACE2: Angiotensin-converting enzyme 2; RT-qPCR: Quantitative reverse-transcription polymerase chain reaction; TMPRSS2: Transmembrane serine protease 2.

Figure 2

ACE2 and TMPRSS2 expression in the liver biopsies of CHB patients. The proteins were detected using immunofluorescence staining. CK18 was used as a marker to indicate hepatocyte distribution. The inflammatory areas were indicated with concentrated cell nuclei stained with Hoechst33342 (white arrows). These representative figures were collected from at least 10 visual fields with or without immunocytes infiltration in each slide. (A) In the controls, anti-HCV NS3 was used as the primary antibody (Rabbit-IgG). (B) The comparison of ALT levels in the CHB patients with or without ACE2 expression of hepatocyte. Student t-test was used to evaluate differences. P < 0.05 was considered statistically significant. ACE2: Angiotensin-converting enzyme 2; ALT: Alanine aminotransferase; CHB: Chronic hepatitis B; TMPRSS2: Transmembrane serine protease 2.

Figure 3

(A) to (D) Changes in ACE2 and TMPRSS2 mRNA of the hepatic cell lines treated with interferon. HepG2, Huh-7, QSG-7701, and L-02 cells were treated with 100 IU/mL of IFN-α2b for 2 days. The housekeeping gene β-actin was used as a control to normalize variations. (E) ACE2 protein levels were calculated based on the ratio of gray values of ACE2 to actin (n = 3). Mann-Whitney U test was used to evaluate differences. P < 0.05 was considered statistically significant. ACE2: Angiotensin-converting enzyme 2; TMPRSS2: Transmembrane serine protease 2.

Figure 4

HBV replication does not alter ACE2 expression of HepAD38 cells. To support a stable production of HBV, tetracycline was removed from the culture medium 2 weeks prior. HepAD38 cells cultured with or without tetracycline were labeled as (+) and (−), respectively. The housekeeping gene β-actin was used as a control to normalize variations. Mann-Whitney U test was used to evaluate differences (n = 3). P < 0.05 was considered statistically significant. ACE2: Angiotensin-converting enzyme 2; HBV: Hepatitis B virus.