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

Jingjing Tong, Hongmin Wang, Xiang Xu, Zhihong Wan, Hongbin Fang, Jing Chen, Xiuying Mu, Zifeng Liu, Jing Chen, Haibin Su, Xiaoyan Liu, Chen Li, Xiaowen Huang, Jinhua Hu, Jingjing Tong, Hongmin Wang, Xiang Xu, Zhihong Wan, Hongbin Fang, Jing Chen, Xiuying Mu, Zifeng Liu, Jing Chen, Haibin Su, Xiaoyan Liu, Chen Li, Xiaowen Huang, Jinhua Hu

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.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Tong, Wang, Xu, Wan, Fang, Chen, Mu, Liu, Chen, Su, Liu, Li, Huang and Hu.

Figures

Figure 1
Figure 1
Flowchart of screening and recruitment of patients with HBV-ACLF.
Figure 2
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
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
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
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
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
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.

References

    1. Sarin SK, Choudhury A, Sharma MK, Maiwall R, Al Mahtab M, Rahman S, et al. . Acute–on–Chronic Liver Failure: Consensus Recommendations of the Asian Pacific Association for the Study of the Liver (APASL): An Update. Hepatol Int (2019) 13(4):353–90. doi: 10.1007/s12072-019-09946-3
    1. Sarin SK, Choudhury A. Acute–on–Chronic Liver Failure: Terminology, Mechanisms and Management. Nat Rev Gastroenterol hepatol (2016) 13(3):131–49. doi: 10.1038/nrgastro.2015.219
    1. Moreau R, Jalan R, Gines P, Pavesi M, Angeli P, Cordoba J, et al. . Acute–on–Chronic Liver Failure is a Distinct Syndrome That Develops in Patients With Acute Decompensation of Cirrhosis. Gastroenterol (2013) 144(7):1426–37.e1421–1429. doi: :10.1053/j.gastro.2013.02.042
    1. Bernal W, Jalan R, Quaglia A, Simpson K, Wendon J, Burroughs A. Acute–on–Chronic Liver Failure. Lancet (Lond Engl) (2015) 386(10003):1576–87. doi: 10.1016/S0140-6736(15)00309-8
    1. Duan XZ, Liu FF, Tong JJ, Yang HZ, Chen J, Liu XY, et al. . Granulocyte–Colony Stimulating Factor Therapy Improves Survival in Patients With Hepatitis B Virus–Associated Acute–on–Chronic Liver Failure. World J Gastroenterol (2013) 19(7):1104–10. doi: 10.3748/wjg.v19.i7.1104
    1. Garg V, Garg H, Khan A, Trehanpati N, Kumar A, Sharma BC, et al. . Granulocyte Colony–Stimulating Factor Mobilizes CD34(+) Cells and Improves Survival of Patients With Acute–on–Chronic Liver Failure. Gastroenterol (2012) 142(3):505–12.e501. doi: 10.1053/j.gastro.2011.11.027
    1. Singh V, Sharma AK, Narasimhan RL, Bhalla A, Sharma N, Sharma R. Granulocyte Colony–Stimulating Factor in Severe Alcoholic Hepatitis: A Randomized Pilot Study. Am J Gastroenterol (2014) 109(9):1417–23. doi: 10.1038/ajg.2014.154
    1. Di Campli C, Zocco MA, Saulnier N, Grieco A, Rapaccini G, Addolorato G, et al. . Safety and Efficacy Profile of G–CSF Therapy in Patients With Acute on Chronic Liver Failure. Digest Liver Dis (2007) 39(12):1071–6. doi: 10.1016/j.dld.2007.08.006
    1. Saha BK, Mahtab MA, Akbar SMF, Noor ESM, Mamun AA, Hossain SMS, et al. . Therapeutic Implications of Granulocyte Colony Stimulating Factor in Patients With Acute–on–Chronic Liver Failure: Increased Survival and Containment of Liver Damage. Hepatol Int (2017) 11(6):540–6. doi: 10.1007/s12072-017-9814-1
    1. Engelmann C, Herber A, Franke A, Bruns T, Reuken P, Schiefke I, et al. . Granulocyte–Colony Stimulating Factor (G–CSF) to Treat Acute–on–Chronic Liver Failure: A Multicenter Randomized Trial (GRAFT Study). J Hepatol (2021) 75(6):1346–54. doi: 10.1016/j.jhep.2021.07.033
    1. Theocharis SE, Papadimitriou LJ, Retsou ZP, Margeli AP, Ninos SS, Papadimitriou JD. Granulocyte–Colony Stimulating Factor Administration Ameliorates Liver Regeneration in Animal Model of Fulminant Hepatic Failure and Encephalopathy. Digest Dis Sci (2003) 48(9):1797–803. doi: 10.1023/a:1025463532521
    1. Spahr L, Lambert JF, Rubbia-Brandt L, Chalandon Y, Frossard JL, Giostra E, et al. . Granulocyte–Colony Stimulating Factor Induces Proliferation of Hepatic Progenitors in Alcoholic Steatohepatitis: A Randomized Trial. Hepatol (Baltimore Md) (2008) 48(1):221–9. doi: 10.1002/hep.22317
    1. Khanam A, Trehanpati N, Garg V, Kumar C, Garg H, Sharma BC, et al. . Altered Frequencies of Dendritic Cells and IFN–Gamma–Secreting T Cells With Granulocyte Colony–Stimulating Factor (G–CSF) Therapy in Acute–on– Chronic Liver Failure. Liver Int (2014) 34(4):505–13. doi: 10.1111/liv.12415
    1. Moreau R, Rautou PE. G–CSF Therapy for Severe Alcoholic Hepatitis: Targeting Liver Regeneration or Neutrophil Function? Am J Gastroenterol (2014) 109(9):1424–6. doi: 10.1038/ajg.2014.250
    1. Krenkel O, Tacke F. Liver Macrophages in Tissue Homeostasis and Disease. Nat Rev Immunol (2017) 17(5):306–21. doi: 10.1038/nri.2017.11
    1. Heymann F, Tacke F. Immunology in the Liver–From Homeostasis to Disease. Nat Rev Gastroenterol Hepatol (2016) 13(2):88–110. doi: 10.1038/nrgastro.2015.200
    1. Triantafyllou E, Woollard KJ, McPhail MJW, Antoniades CG, Possamai LA. The Role of Monocytes and Macrophages in Acute and Acute–On–Chronic Liver Failure. Front Immunol (2018) 9:2948. doi: 10.3389/fimmu.2018.02948
    1. Youn JI, Kumar V, Collazo M, Nefedova Y, Condamine T, Cheng P, et al. . Epigenetic Silencing of Retinoblastoma Gene Regulates Pathologic Differentiation of Myeloid Cells in Cancer. Nat Immunol (2013) 14(3):211–20. doi: 10.1038/ni.2526
    1. Jakubzick CV, Randolph GJ, Henson PM. Monocyte Differentiation and Antigen–Presenting Functions. Nat Rev Immunol (2017) 17(6):349–62. doi: 10.1038/nri.2017.28
    1. Bernsmeier C, van der Merwe S, Périanin A. Innate Immune Cells in Cirrhosis. J Hepatol (2020) 73(1):186–201. doi: 10.1016/j.jhep.2020.03.027
    1. You S, Rong Y, Zhu B, Zhang A, Zang H, Liu H, et al. . Changing Etiology of Liver Failure in 3,916 Patients From Northern China: A 10–Year Survey. Hepatol Int (2013) 7(2):714–20. doi: 10.1007/s12072-013-9424-5
    1. Matsushita M, Freigang S, Schneider C, Conrad M, Bornkamm GW, Kopf M. T Cell Lipid Peroxidation Induces Ferroptosis and Prevents Immunity to Infection. J Exp Med (2015) 212(4):555–68. doi: 10.1084/jem.20140857
    1. Yu Z, Li J, Ren Z, Sun R, Zhou Y, Zhang Q, et al. . Switching From Fatty Acid Oxidation to Glycolysis Improves the Outcome of Acute–On–Chronic Liver Failure. Adv Sci (Weinheim Baden–Wurttemberg Germany) (2020) 7(7):1902996. doi: 10.1002/advs.201902996
    1. Sarin SK, Kumar A, Almeida JA, Chawla YK, Fan ST, Garg H, et al. . Acute–On–Chronic Liver Failure: Consensus Recommendations of the Asian Pacific Association for the Study of the Liver (APASL). Hepatol Int (2009) 3(1):269–82. doi: 10.1007/s12072-008-9106-x
    1. Angeli P, Ginès P, Wong F, Bernardi M, Boyer TD, Gerbes A, et al. . Diagnosis and Management of Acute Kidney Injury in Patients With Cirrhosis: Revised Consensus Recommendations of the International Club of Ascites. J Hepatol (2015) 62(4):968–74. doi: 10.1016/j.jhep.2014.12.029
    1. Patidar KR, Bajaj JS. Covert and Overt Hepatic Encephalopathy: Diagnosis and Management. Clin Gastroenterol Hepatol (2015) 13(12):2048–61. doi: 10.1016/j.cgh.2015.06.039
    1. Kamath PS, Kim WR. The Model for End–Stage Liver Disease (MELD). Hepatol (Baltimore Md) (2007) 45(3):797–805. doi: 10.1002/hep.21563
    1. Kim WR, Biggins SW, Kremers WK, Wiesner RH, Kamath PS, Benson JT, et al. . Hyponatremia and Mortality Among Patients on the Liver–Transplant Waiting List. N Engl J Med (2008) 359(10):1018–26. doi: 10.1056/NEJMoa0801209
    1. Piano S, Singh V, Caraceni P, Maiwall R, Alessandria C, Fernandez J, et al. . Epidemiology and Effects of Bacterial Infections in Patients With Cirrhosis Worldwide. Gastroenterol (2019) 156(5):1368–80.e1310. doi: 10.1053/j.gastro.2018.12.005
    1. Zhang Z, Yang Z, Cheng Q, Hu X, Liu M, Liu Y, et al. . Establishment and Validation of a Prognostic Model for Hepatitis B Virus−Related Acute–on–Chronic Liver Failure Patients With Bacterial Infection. Hepatol Int (2022) 16(1):38–47. doi: 10.1007/s12072-021-10268-6
    1. Bajaj JS. Defining Acute–on–Chronic Liver Failure: Will East and West Ever Meet? Gastroenterol (2013) 144(7):1337–9. doi: 10.1053/j.gastro.2013.04.024
    1. Jindal A, Sarin SK. G–CSF in Acute–on–Chronic Liver Failure – Art of 'Patient Selection' is Paramount! J Hepatol (2022) 76(2):472–3. doi: 10.1016/j.jhep.2021.08.022
    1. Weise G, Pösel C, Möller K, Kranz A, Didwischus N, Boltze J, et al. . High–Dosage Granulocyte Colony Stimulating Factor Treatment Alters Monocyte Trafficking to the Brain After Experimental Stroke. Brain Behav Immun (2017) 60:15–26. doi: 10.1016/j.bbi.2016.08.008
    1. Fadini GP, De Kreutzenberg SV, Boscaro E, Albiero M, Cappellari R, Kränkel N, et al. . An Unbalanced Monocyte Polarisation in Peripheral Blood and Bone Marrow of Patients With Type 2 Diabetes has an Impact on Microangiopathy. Diabetologia (2013) 56(8):1856–66. doi: 10.1007/s00125-013-2918-9
    1. Boneberg EM, Hareng L, Gantner F, Wendel A, Hartung T. Human Monocytes Express Functional Receptors for Granulocyte Colony–Stimulating Factor That Mediate Suppression of Monokines and Interferon–Gamma. Blood (2000) 95(1):270–6. doi: 10.1182/blood.V95.1.270.001k39_270_276
    1. Nishiki S, Hato F, Kamata N, Sakamoto E, Hasegawa T, Kimura-Eto A, et al. . Selective Activation of STAT3 in Human Monocytes Stimulated by G–CSF: Implication in Inhibition of LPS–Induced TNF–Alpha Production. Am J Physiol Cell Physiol (2004) 286(6):C1302–1311. doi: 10.1152/ajpcell.00387.2003
    1. Chen P, Wang YY, Chen C, Guan J, Zhu HH, Chen Z. The Immunological Roles in Acute–on–Chronic Liver Failure: An Update. Hepatobil Pancreat Dis Int: HBPD Int (2019) 18(5):403–11. doi: 10.1016/j.hbpd.2019.07.003

Source: PubMed

Sponsorer og samarbejdspartnere

Medicinske tilstande

Narkotikainterventioner

3
Abonner