Alimentary Pharmacology and Therapeutics
The severity of circulating neutrophil dysfunction in patients with cirrhosis is associated with 90-day and 1-year mortality N. J. Taylor*,†, G. K. Manakkat Vijay†, R. D. Abeles*,†, G. Auzinger*, W. Bernal*,†, Y. Ma†, J. A. Wendon*,† & D. L. Shawcross*,†
*Liver Intensive Care Unit, King’s College London School of Medicine at King’s College Hospital, London, UK. † Institute of Liver Studies and Transplantation, King’s College London School of Medicine at King’s College Hospital, London, UK.
Correspondence to: Dr D. L. Shawcross, Institute of Liver Studies and Transplantation, King’s College London School of Medicine at King’s College Hospital Denmark Hill, London SE5 9RS, UK. E-mail: [email protected]
Publication data Submitted 4 May 2014 First decision 23 May 2014 Resubmitted 30 June 2014 Accepted 2 July 2014 EV Pub Online 25 July 2014 This article was accepted for publication after full peer-review.
SUMMARY Background Patients with cirrhosis are susceptible to sepsis, pre-disposing to the development of encephalopathy, bleeding and organ dysfunction with associated high mortality. Aim To characterise circulating neutrophil function in a cirrhotic cohort as a determinant of 90-day and 1-year mortality. Methods Sixty-two patients with cirrhosis [49 stable (Child–Pugh A/B/C = 24%/39%/ 37%); 13 acute-on-chronic liver failure] were prospectively studied and compared with 11 healthy controls. Neutrophil function was evaluated at baseline and repeated at critical points during the course of the patient’s illness until death/transplantation. Neutrophil phenotype was determined using fluorochrome-labelled antibodies to CD16/CD11b and assessed by flow cytometry. Neutrophil phagocytic activity (NPA) and capacity (NPC) were determined using FITC-labelled opsonised Escherichia coli. Oxidative burst (OB) was quantified by the percentage of neutrophils producing reactive oxygen species (ROS) and mean fluorescence intensity at rest, and after stimulation with E. coli. Physiological variables, biochemistry, microbiology and outcomes were collected. Plasma pro- and anti-inflammatory cytokine profiles were performed by ELISA. Results NPA/NPC was impaired in cirrhosis with the most significant dysfunction being observed in those with advanced disease and in those treated with propranolol. NPC predicted survival in stable cirrhosis [AUROC 0.83 (95% CI 0.68–0.97); P = 0.021] and differentiated survivors from nonsurvivors (90-day P = 0.01; 1 year P < 0.001). Resting OB ≥12% predicted 90-day mortality with 80% sensitivity and 71% specificity [AUROC 0.81 (95% CI 0.64–0.97); P = 0.026 and differentiated survivors from nonsurvivors; P = 0.015]. Conclusion Circulating neutrophils in patients with cirrhosis are dysfunctional and predict the development of infection, organ dysfunction and survival at 90 days and 1 year. Aliment Pharmacol Ther 2014; 40: 705–715
ª 2014 John Wiley & Sons Ltd doi:10.1111/apt.12886
705
N. J. Taylor et al. INTRODUCTION Cirrhosis is associated with an increased incidence of microbial infection resulting in hospitalisation and complicating hospital admissions in up to 40% of cases.1 Infection can lead to worsening liver function and precipitate complications including variceal bleeding, hepatic encephalopathy (HE), acute kidney injury (AKI) and multi-organ dysfunction syndrome (MODS), contributing to high mortality. The exponential increase in the incidence of alcohol-related and non-alcoholic fatty liver cirrhosis in the UK2 and US poses a major challenge in managing these patients particularly with respect to utilisation of intensive care unit (ICU) beds and requirement for liver transplantation (LT). With poor outcomes following sepsis and its associated complications, and increasing waiting list mortality for LT, there is an urgent need for novel approaches to reducing the rate of infection in cirrhosis. Cirrhosis represents a state of functional immune paresis with defects in opsonisation, innate and adaptive immune responses and up-regulation of pro-inflammatory cytokine production akin to that observed in patients with sepsis.3 Neutrophils are key effector cells of the innate immune response and show defects in phagocytic capacity and oxidative burst (OB) in cirrhosis.4, 5 In patients with cirrhosis bacterial translocation, porto-systemic shunting and reduced reticulo-endothelial function culminate in increased levels of circulating endotoxin and other pathogen-associated molecular pattern (PAMPs) molecules. The interaction of PAMPs with pattern-recognition receptors such as Toll-like receptors (TLR) expressed on the neutrophil surface, results in the priming of circulating neutrophils.6 Neutrophil priming also occurs in response to circulating chemokines and pro-inflammatory cytokines.7 In the primed state, circulating neutrophils show altered surface receptor expression, increased basal superoxide production and reduced thresholds for undergoing phagocytosis and OB.8, 9 Primed circulating neutrophils have been postulated to play a central role in the development of AKI and lung injury in sepsis,10, 11 as well as accumulating in other organs in patients who died of MODS.12 The generation and release of reactive oxygen species (ROS) and serine proteases (e.g. elastase and matrix-metalloproteinases) by neutrophils at these sites result in tissue damage and the development of MODS.13 Antibiotic prophylaxis is inadequate at reducing the incidence of infection in patients with cirrhosis and may be associated with the development of multi-drug resistant organisms.1 A greater understanding of the pathogenic 706
mechanisms underpinning neutrophil dysfunction in cirrhosis might facilitate better identification of those at risk of developing infection and promote the development of neutrophil-targeted therapies. The primary aim of this longitudinal prospective study was to investigate whether defective neutrophil functioning and bacteriocidal capacity in patients with cirrhosis predicts the development of infection and extra-hepatic organ dysfunction. To test the hypothesis, we characterised neutrophil activity and functional bacteriocidal capacity in a cohort of cirrhotic patients in relation to clinical characteristics, plasma biochemical and cytokine profile. The development of infection and organ dysfunction at 90 days and 1 year was a secondary outcome.
PATIENTS AND METHODS Study population and design A prospective cross-sectional longitudinal cohort study was performed on 62 patients with cirrhosis [49-stable cirrhosis and 13-acute-on-chronic liver failure (ACLF)] recruited from the liver out-patient clinic and wards (including liver ICU) at King’s College Hospital between October 2008 and August 2010. Neutrophil phenotype, neutrophil phagocytic activity (NPA), neutrophil phagocytic capacity (NPC) and OB (resting and E. coli stimulated) were determined and compared to healthy controls [HC] (n = 11). The dynamics of neutrophil function during the course of the illness were compared between patient groups and in relation to those who survived and those who did not survive. Neutrophil sampling was performed and repeated at critical points during the time course of the patient’s illness such as following the development of infection and MODS until recovery, death or LT. Patients were followed up until October 2012 and episodes of infection were documented along with other relevant patient outcomes including the development of organ failure and survival at 90 days, and 1 year. Inclusion criteria Patients were included if they were aged >18 and
The severity of circulating neutrophil dysfunction in patients with cirrhosis is associated with 90-day and 1-year mortality N. J. Taylor*,†, G. K. Manakkat Vijay†, R. D. Abeles*,†, G. Auzinger*, W. Bernal*,†, Y. Ma†, J. A. Wendon*,† & D. L. Shawcross*,†
*Liver Intensive Care Unit, King’s College London School of Medicine at King’s College Hospital, London, UK. † Institute of Liver Studies and Transplantation, King’s College London School of Medicine at King’s College Hospital, London, UK.
Correspondence to: Dr D. L. Shawcross, Institute of Liver Studies and Transplantation, King’s College London School of Medicine at King’s College Hospital Denmark Hill, London SE5 9RS, UK. E-mail: [email protected]
Publication data Submitted 4 May 2014 First decision 23 May 2014 Resubmitted 30 June 2014 Accepted 2 July 2014 EV Pub Online 25 July 2014 This article was accepted for publication after full peer-review.
SUMMARY Background Patients with cirrhosis are susceptible to sepsis, pre-disposing to the development of encephalopathy, bleeding and organ dysfunction with associated high mortality. Aim To characterise circulating neutrophil function in a cirrhotic cohort as a determinant of 90-day and 1-year mortality. Methods Sixty-two patients with cirrhosis [49 stable (Child–Pugh A/B/C = 24%/39%/ 37%); 13 acute-on-chronic liver failure] were prospectively studied and compared with 11 healthy controls. Neutrophil function was evaluated at baseline and repeated at critical points during the course of the patient’s illness until death/transplantation. Neutrophil phenotype was determined using fluorochrome-labelled antibodies to CD16/CD11b and assessed by flow cytometry. Neutrophil phagocytic activity (NPA) and capacity (NPC) were determined using FITC-labelled opsonised Escherichia coli. Oxidative burst (OB) was quantified by the percentage of neutrophils producing reactive oxygen species (ROS) and mean fluorescence intensity at rest, and after stimulation with E. coli. Physiological variables, biochemistry, microbiology and outcomes were collected. Plasma pro- and anti-inflammatory cytokine profiles were performed by ELISA. Results NPA/NPC was impaired in cirrhosis with the most significant dysfunction being observed in those with advanced disease and in those treated with propranolol. NPC predicted survival in stable cirrhosis [AUROC 0.83 (95% CI 0.68–0.97); P = 0.021] and differentiated survivors from nonsurvivors (90-day P = 0.01; 1 year P < 0.001). Resting OB ≥12% predicted 90-day mortality with 80% sensitivity and 71% specificity [AUROC 0.81 (95% CI 0.64–0.97); P = 0.026 and differentiated survivors from nonsurvivors; P = 0.015]. Conclusion Circulating neutrophils in patients with cirrhosis are dysfunctional and predict the development of infection, organ dysfunction and survival at 90 days and 1 year. Aliment Pharmacol Ther 2014; 40: 705–715
ª 2014 John Wiley & Sons Ltd doi:10.1111/apt.12886
705
N. J. Taylor et al. INTRODUCTION Cirrhosis is associated with an increased incidence of microbial infection resulting in hospitalisation and complicating hospital admissions in up to 40% of cases.1 Infection can lead to worsening liver function and precipitate complications including variceal bleeding, hepatic encephalopathy (HE), acute kidney injury (AKI) and multi-organ dysfunction syndrome (MODS), contributing to high mortality. The exponential increase in the incidence of alcohol-related and non-alcoholic fatty liver cirrhosis in the UK2 and US poses a major challenge in managing these patients particularly with respect to utilisation of intensive care unit (ICU) beds and requirement for liver transplantation (LT). With poor outcomes following sepsis and its associated complications, and increasing waiting list mortality for LT, there is an urgent need for novel approaches to reducing the rate of infection in cirrhosis. Cirrhosis represents a state of functional immune paresis with defects in opsonisation, innate and adaptive immune responses and up-regulation of pro-inflammatory cytokine production akin to that observed in patients with sepsis.3 Neutrophils are key effector cells of the innate immune response and show defects in phagocytic capacity and oxidative burst (OB) in cirrhosis.4, 5 In patients with cirrhosis bacterial translocation, porto-systemic shunting and reduced reticulo-endothelial function culminate in increased levels of circulating endotoxin and other pathogen-associated molecular pattern (PAMPs) molecules. The interaction of PAMPs with pattern-recognition receptors such as Toll-like receptors (TLR) expressed on the neutrophil surface, results in the priming of circulating neutrophils.6 Neutrophil priming also occurs in response to circulating chemokines and pro-inflammatory cytokines.7 In the primed state, circulating neutrophils show altered surface receptor expression, increased basal superoxide production and reduced thresholds for undergoing phagocytosis and OB.8, 9 Primed circulating neutrophils have been postulated to play a central role in the development of AKI and lung injury in sepsis,10, 11 as well as accumulating in other organs in patients who died of MODS.12 The generation and release of reactive oxygen species (ROS) and serine proteases (e.g. elastase and matrix-metalloproteinases) by neutrophils at these sites result in tissue damage and the development of MODS.13 Antibiotic prophylaxis is inadequate at reducing the incidence of infection in patients with cirrhosis and may be associated with the development of multi-drug resistant organisms.1 A greater understanding of the pathogenic 706
mechanisms underpinning neutrophil dysfunction in cirrhosis might facilitate better identification of those at risk of developing infection and promote the development of neutrophil-targeted therapies. The primary aim of this longitudinal prospective study was to investigate whether defective neutrophil functioning and bacteriocidal capacity in patients with cirrhosis predicts the development of infection and extra-hepatic organ dysfunction. To test the hypothesis, we characterised neutrophil activity and functional bacteriocidal capacity in a cohort of cirrhotic patients in relation to clinical characteristics, plasma biochemical and cytokine profile. The development of infection and organ dysfunction at 90 days and 1 year was a secondary outcome.
PATIENTS AND METHODS Study population and design A prospective cross-sectional longitudinal cohort study was performed on 62 patients with cirrhosis [49-stable cirrhosis and 13-acute-on-chronic liver failure (ACLF)] recruited from the liver out-patient clinic and wards (including liver ICU) at King’s College Hospital between October 2008 and August 2010. Neutrophil phenotype, neutrophil phagocytic activity (NPA), neutrophil phagocytic capacity (NPC) and OB (resting and E. coli stimulated) were determined and compared to healthy controls [HC] (n = 11). The dynamics of neutrophil function during the course of the illness were compared between patient groups and in relation to those who survived and those who did not survive. Neutrophil sampling was performed and repeated at critical points during the time course of the patient’s illness such as following the development of infection and MODS until recovery, death or LT. Patients were followed up until October 2012 and episodes of infection were documented along with other relevant patient outcomes including the development of organ failure and survival at 90 days, and 1 year. Inclusion criteria Patients were included if they were aged >18 and
