Sorted B cell transcriptomes point towards actively regulated B cell responses during ongoing chronic hepatitis B infections

Highlights

• Four clinical phases describe the natural course of a chronic hepatitis B (CHB) infection.

• The immune cells that govern these clinical disease transitions remain unknown.

• We performed RNA sequencing on B cells of CHB patients and healthy controls.

• We found distinct gene expression profiles between healthy controls and CHB patients.

• Our study corroborates an important role for B cells during ongoing CHB infections.

Abstract

The natural course of chronic hepatitis B virus (HBV) infections follows distinct clinical disease phases, characterized by fluctuating levels of serum HBV DNA and ALT. The immune cells and their features that govern these clinical disease transitions remain unknown. In the current study, we performed RNA sequencing on purified B cells from blood (n = 42) and liver (n = 10) of healthy controls and chronic HBV patients. We found distinct gene expression profiles between healthy controls and chronic HBV patients, as evidenced by 190 differentially expressed genes (DEG), but also between the clinical phenotypes of a chronic HBV infection (17–110 DEG between each phase). Numerous immune pathways, including the B cell receptor pathway were upregulated in liver B cells when compared to peripheral B cells. Further investigation of the detected DEG suggested an activation of B cells during HBeAg seroconversion and an active regulation of B cell signalling in the liver.

Introduction

Increasing evidence suggests an important role for B cells in the pathogenesis of chronic hepatitis B virus (HBV) infections [1], [2], [3], [4], [5]. Treatment with B cell depleting agents such as the anti-CD20 monoclonal antibody rituximab is well known to reactivate HBV replication risking hepatic flares and fatal outcomes, even in patients with a resolved infection [6]. Peripheral blood B cells were shown to be phenotypically activated during a chronic HBV infection, with an increased expression of the surface markers CD69 and CD83 and antibody production, but a reduced proliferative capacity [7], [8], [9].

The natural course of a chronic HBV infection discerns four distinct clinical phases with alternating HBV load and Alanine Aminotransferase (ALT) levels: a HBeAg positive Immune Tolerant (IT) and Immune Active (IA) phase and a HBeAg negative Inactive Carrier (IC) and HBeAg negative hepatitis (ENEG) phase [10]. Significant viral replication is observed in the IT, IA and ENEG, but not in the IC phase [10]. ALT elevations, indicating ongoing liver damage, are mostly seen in the IA and ENEG phases. As such, the length of both phases predisposes to rapid fibrosis progression [11]. In contrast, patients in the IC phase show an almost negligible risk of fibrosis progression [11].

The nature of the viral or immune mechanisms discriminating these clinical phases and their involvement in the transition between consecutive phases is a matter of intense research. Our group identified an immune gene signature consisting of many B cell related genes in whole blood and liver tissue of chronic HBV patients that correlates with the distinct clinical phases, and was found to be upregulated in the IA and ENEG phase [12], [13]. In line with this, in a subsequent study, we found elevated levels of antibodies against the hepatitis B core antigen (HBcAg) in the IA and ENEG phase, whereas levels of antibodies against the hepatitis B surface antigen (HBsAg) did not significantly differ between clinical phases [4].

Using RNA sequencing, we previously uncovered important differences between sorted NK cells from viraemic patients and healthy controls [14]. The sorted NK cell transcriptome signatures differed substantially between viraemic HBV patients, HCV patients and HIV patients when compared to matched healthy controls, but barely any differences between HBV’s clinical phases could be detected [14]. To further unravel the role of B cells during the different phases of an ongoing chronic HBV infection, we now use the same approach by performing RNA sequencing on purified B cells obtained from blood and liver of chronic HBV patients. This technique may uncover genes that are activated in vivo during chronic HBV infections, but that are not identified through biased phenotypic approaches.