Granulocyte Colony-Stimulating Factor Accelerates the Recovery of Hepatitis B Virus-Related Acute-on-Chronic Liver Failure by Promoting M2-Like Transition of Monocytes

Abstract

Background and aim: Acute-on-chronic liver failure (ACLF) has a high mortality rate. The role of granulocyte colony-stimulating factor (G-CSF) in ACLF remains controversial. Monocytes/macrophages are core immune cells, which are involved in the initiation and progression of liver failure; however, the effect of G-CSF on monocytes/macrophages is unclear. The study aimed to verify the clinical efficacy of G-CSF and explore the effect of it on monocytes in hepatitis B virus (HBV)-related ACLF (HBV-ACLF) paitents.

Methods: We performed a large randomized controlled clinical trial for the treatment of HBV-ACLF using G-CSF. A total of 111 patients with HBV-ACLF were prospectively randomized into the G-CSF group (5 μg/kg G-CSF every day for 6 days, then every other day until day 18) or the control group (standard therapy). All participants were followed up for at least 180 days. The relationship between monocyte count and mortality risk was analyzed. The effect of G-CSF on the phenotype and function of monocytes from patients with HBV-ACLF was evaluated using flow cytometry in vivo and in vitro experiments.

Results: The survival probability of the G-CSF group at 180 days was higher than that of the control group (72.2% vs. 53.8%, P = 0.0142). In the G-CSF-treated group, the monocyte counts on days 0 and 7 were independently associated with an evaluated mortality risk in the fully adjusted model (Model 3) [at day 0: hazard ratio (HR) 95% confidence interval (CI): 15.48 (3.60, 66.66), P = 0.0002; at day 7: HR (95% CI): 1.10 (0.50, 2.43), P=0.8080]. Further analysis showed that after treatment with G-CSF in HBV-ACLF patients, the expression of M1-like markers (HLA-DR and CD86) in monocytes decreased (HLA-DR: P = 0.0148; CD86: P = 0.0764). The expression of MerTK (M2-like marker) increased (P = 0.0002). The secretion of TNF-α, IL-6, and IL-10 from monocytes decreased without lipopolysaccharide (LPS) stimulation (TNF-α: P < 0.0001; IL-6: P= 0.0025; IL-10: P = 0.0004) or with LPS stimulation (TNF-α: P = 0.0439; P = 0.0611; IL-10: P = 0.0099). Similar effects were observed in vitro experiments.

Conclusion: G-CSF therapy confers a survival benefit to patients with HBV-ACLF. G-CSF can promote the anti-inflammatory/pro-restorative phenotype (M2-like) transition of monocytes, which may contribute to the recovery of ACLF.Clinical Trial Registration Number: ClinicalTrials.gov, identifier (NCT02331745).

Keywords: acute-on-chronic liver failure; cytokine; granulocyte colony stimulating factor; hepatitis B virus; inflammation; monocytes; prognosis.

Figure 1

Flowchart of screening and recruitment of patients with HBV-ACLF.

Figure 2

Kaplan–Meier curve showing the 180-day survival in G-CSF group, compared with the control group. G-CSF, Granulocyte-colony stimulating factor.

Figure 3

Gating of monocytes and effect of G-CSF on monocyte subtypes in patients with HBV-ACLF. (A) Flow cytometry analysis and gating strategy used to determine monocytes and their subsets. (B) Effect of G-CSF on monocyte subtypes in patients with HBV-ACLF (n=12). Non-parametric (Wilcoxon’s matched-pair test) statistical analysis was used. Data presented as median with interquartile range. ns represents P >0.05; G-CSF, Granulocyte-colony stimulating factor; HBV-ACLF, hepatitis B virus-related acute-on -chronic liver failure.

Figure 4

Phenotype of circulating monocytes in HBV-ACLF patients (n=12) before (day 0) and after G-CSF treatment. (A) Expression of CD86、HLA-DR、MerTK, and CD163 on monocytes in HBV-ACLF before and after G-CSF treatment. (B) Phenotypic alterations on monocytes after treated with G-CSF. (C) Expression of tissue-homing receptors on monocytes after treated with G-CSF. Non-parametric (Wilcoxon’s matched-pair test) statistical analysis was used. Data presented as median with interquartile range. Compared with day 0, *P < 0.05, **P < 0.01, ***P < 0.001. ns represents P >0.05. G-CSF, Granulocyte-colony stimulating factor; HBV-ACLF, hepatitis B virus-related acute-on -chronic liver failure; FSC, forward scatter.

Figure 5

G-CSF therapy attenuated cytokine secretion in monocytes with or without LPS stimulation in HBV-ACLF patients (n=12). (A) Cytokine secretion in monocytes without LPS stimulation. (B) Cytokine secretion in monocytes with LPS stimulation. Non-parametric (Wilcoxon’s matched-pair test) statistical analysis was used. Data presented as median with interquartile range. Compared with day 0, *P < 0.05, **P < 0.01, ***P < 0.001. G-CSF, Granulocyte-colony stimulating factor; HBV-ACLF, hepatitis B virus-related acute-on-chronic liver failure; LPS, lipopolysaccharide.

Figure 6

G-CSF induces M2-like phenotype and functional transition of monocytes from HBV-ACLF patients in vitro. (A) G-CSF decreased the expression of pro-inflammatory markers on monocytes (n=9). (B) G-CSF elevated the expression of anti-inflammatory/pro-restorative markers on monocytes (n=9). (C) Effect of G-CSF on the expression of homing receptors on monocytes (n=9). (D) G-CSF attenuated pro-inflammatory cytokine secretion in monocytes (n=5). Non-parametric (Wilcoxon’s matched-pair test) statistical analysis was used. Data presented as median with interquartile range. ** represents compared with day 0, P<0.01; ns represents P >0.05; G-CSF, Granulocyte-colony stimulating factor; HBV-ACLF, hepatitis B virus-related acute-on -chronic liver failure.

Figure 7

Influence of G-CSF on phagocytosis and oxidative burst function of monocytes in HBV-ACLF. (A) Effect of G-CSF on phagocytosis function of monocytes (n=5). (B) Effect of G-CSF on oxidative burst function of monocytes (n=3). ns represents P >0.05; G-CSF, Granulocyte-colony stimulating factor; HBV-ACLF, hepatitis B virus-related acute-on -chronic liver failure.