IL-1b-Bearing NETs: Bridging Inflammation to Early Cirrhosis in Hepatitis B

Abstract

Hepatitis B virus (HBV) infection is one of the most dangerous viral diseases, with innate immunity representing the first line of defense against the virus. In this branch of the immune system, neutrophils are considered key cellular mediators. To better understand the implication of neutrophils in the distinct stages of the disease, HBV-infected patients were enrolled in this study and categorized into three groups: patients with acute infection, chronic infection under treatment, and at early cirrhotic stage. To elucidate the role of inflammatory mediators and cellular mechanisms of neutrophilic origin in the course of the infection, both ex vivo and in vitro studies were performed. Increased levels of C-C motif chemokine ligand 2 (CCL2), interleukin (IL)-18, IL-33, and citrullinated histone H3 (CitH3)-an accurate marker of neutrophil extracellular traps (NETs)-were detected in the circulation of patients with acute infection or early cirrhosis. In parallel, sera from the aforementioned patient groups induced the formation of IL-1b-bearing NETs in neutrophils from healthy individuals. These inflammatory NETs affected primary fibroblasts towards acquiring a pro-fibrotic phenotype. These results suggest that NETs could be regarded as mediators in hepatitis B manifestations, while their therapeutic targeting could enhance the management of early-stage cirrhotic patients.

Keywords: cirrhosis; hepatitis B virus (HBV); inflammation; interleukin (IL)-1b; liver fibrosis; neutrophil extracellular traps; neutrophils.

Figure 1

Inflammatory proteins and neutrophil extracellular traps (NETs) are present in the circulation of a-HBV and cir-HBV patients. Levels of (A) C-C motif chemokine ligand 2 (CCL2), (B) interleukin(IL)-18, and (C) interleukin(IL)-33 in the serum of HBV patients (n = 11 subjects per group). (D) Concentration of citrullinated histone H3 (CitH3), representing NET release, in the serum of HBV patients (n = 11 subjects per group). For (A–D), data are shown as mean ± SD, Kruskal–Wallis test, followed by Dunn’s multiple comparison test. All conditions were compared to HI. Statistically significant: p < 0.05. ns: non-statistically significant. HI: healthy individuals; a-HBV: acute HBV; chr-HBV: chronic HBV infection under treatment; cir-HBV: early cirrhotic stage.

Figure 2

The inflammatory a-HBV and cir-HBV microenvironment induces NET formation in vitro. (A) Fluorescence confocal microscopy images showing citrullinated histone H3 (CitH3) staining, representing NETs (blue: DAPI; green: CitH3; original magnification: 400×). A representative example of 6 independent experiments is shown. (B) Levels of CitH3, representing NET release, after in vitro stimulation of control neutrophils with serum from HBV patients (n = 11). For (B), data are shown as mean ± SD, Friedman test, followed by Dunn’s multiple comparison test. For (B), all conditions were compared to control NETs. Statistically significant: p < 0.05. ns: non-statistically significant. a-HBV: acute HBV; chr-HBV: chronic HBV infection under treatment; cir-HBV: early cirrhotic stage.

Figure 3

IL-1b-bearing NETs are formed in the presence of a-HBV or cir-HBV microenvironment in vitro. (A) Fluorescence confocal microscopy images showing neutrophil elastase (NE) and interleukin(IL)-1b staining (blue: DAPI; red: NE; green: IL-1b; original magnification: 400×). A representative example of 6 independent experiments is shown. (B) Concentration of IL-1b on in vitro isolated NETs, after stimulation of control neutrophils with serum from HBV patients (n = 11 independent experiments). (C) IL-1b expression in control neutrophils treated with HBV serum, as assessed by RT-qPCR (n = 6 independent experiments). For (B,C), data are shown as mean ± SD, Friedman test, followed by Dunn’s multiple comparison test. For (B,C), all conditions were compared to control NETs and untreated, respectively. Statistically significant: p < 0.05. a-HBV: acute HBV; cir-HBV: early cirrhotic stage.

Figure 4

The migratory/wound healing dynamic and collagen production of fibroblasts are enhanced by cir-HBV NETs in vitro. (A) Smooth muscle actin alpha 2 (ACTA2) expression (n = 6 independent experiments), (B) collagen production (n = 6 independent experiments), and (C) migratory/wound healing capacity of control fibroblasts (Fbs) upon stimulation with in vitro isolated cir-HBV NETs (original magnification: 40×). For (C), a representative example of 6 independent experiments is shown. In all experiments, NET structures were dismantled with DNase I and interleukin(IL)-1b signaling was blocked in Fbs using a recombinant human IL-1 receptor antagonist (Anakinra). For (A,B), data are shown as mean ± SD, Friedman test, followed by Dunn’s multiple comparison test. For (A,B), all conditions were compared to untreated. Statistically significant: p < 0.05. cir-HBV: early cirrhotic stage.