Accepted Manuscript. Acute-On-Chronic Liver Failure, Human Serum Albumin and Immune-Modulation: The Beginning of an Exciting Adventure

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1 Accepted Manuscript Acute-On-Chronic Liver Failure, Human Serum Albumin and Immune-Modulation: The Beginning of an Exciting Adventure Vicente Arroyo, Joan Clària PII: S (17) DOI: /j.cgh Reference: YJCGH To appear in: Clinical Gastroenterology and Hepatology Accepted Date: 3 December 2017 Please cite this article as: Arroyo V, Clària J, Acute-On-Chronic Liver Failure, Human Serum Albumin and Immune-Modulation: The Beginning of an Exciting Adventure, Clinical Gastroenterology and Hepatology (2018), doi: /j.cgh This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

2 ACUTE-ON-CHRONIC LIVER FAILURE, HUMAN SERUM ALBUMIN AND IMMUNE-MODULATION: THE BEGINNING OF AN EXCITING ADVENTURE. Vicente Arroyo 1,3 and Joan Clària 1,2,3 European Association for the Study of the Liver-Chronic Liver Failure (EASL-CLIF) Consortium and Grifols 1 Chair, European Foundation for the Study of Chronic Liver Failure (EF-CLIF) 1, Hospital Clinic-IDIBAPS 2 and University of Barcelona 3. Short title: ALBUMIN, IMMUNE MODULATION AND CIRRHOSIS Corresponding author: Vicente Arroyo, European Foundation for the Study of Chronic Liver Failure (EF Clif), Travessera del Gràcia 11 7º, Barcelona, Spain. vicente.arroyo@efclif.com; telephone Disclosures. EF-CLIF is supported by unrestricted donations from Cellex Foundation (Barcelona) and Grifols and is partner in several projects financed by the HORIZON 2020 program of the European Union. Work at the authors laboratory is also supported by Spanish MEC (SAF R and PIE14/00045) under European Regional Development Funds. Abbreviations. ACLF: Acute-on-chronic liver failure; HR: Hepatorenal syndrome; IL: Interleukin; RCT: Randomized controlled trial; LM: Lipid mediator; LPS: Lipopolysaccharide; MOF: multiorgan failure; PG: prostaglandin; SMT: Standard Medical Therapy; PAVH: Peripheral arterial vasodilation hypothesis; SI: Systemic inflammation; TNF-α: Tumor necrosis factor-α.

3 The clinical course of cirrhosis follows three clear-cut phases. The pre-cirrhotic-phase includes a longterm process (10-25 years) of continuous tissue injury, fibrosis and disruption of the organ architecture. In the second phase (compensated cirrhosis, 5-15 years) there is asymptomatic progression of the histological lesions. Finally, the third phase (decompensated cirrhosis) starts when ascites, variceal 2 bleeding and/or encephalopathy develop. In addition to liver failure, this phase is characterized by progressive impairment in the function of most extrahepatic organs and systems (heart, lungs, circulation, respiration, coagulation, kidneys, brain, immune system, endocrine organs, intestine and muscles) and short survival (< 3-5 years). The mechanism by which the diseased liver induces extrahepatic organ failures is a topic of major interest. The story started 25 years ago with the peripheral arterial vasodilation hypothesis (PAVH) of ascites and hepatorenal syndrome (HRS)(1). According to this hypothesis, the initial event is a failure of the intestines to prevent translocation of bacteria and/or bacterial products from intestinal lumen to the lamina propria and the circulation, leading to local release of vasoactive inflammatory mediators (nitric oxide, kinins and prostaglandins (PGs)), splanchnic arterial vasodilation, effective arterial hypovolemia, sodium and water retention and, in extreme cases, renal hypo-perfusion. Impairment in intestinal permeability, qualitative and quantitative changes in microbiota, and impairment of the intestinal immune system are now well recognized mechanisms of bacterial translocation; cirrhotic cardiomyopathy is also recognized as a contributory factor of circulatory dysfunction (2). The PAVH inspired the re-introduction of therapeutic paracentesis for the management of ascites; it also underscored the importance of treating circulatory dysfunction and HRS, mostly using intravenous administration of albumin. Acute-of Chronic Liver Failure (ACLF), a recently defined (3) highly prevalent syndrome among patients hospitalized for acute decompensation (30%), is the second piece of this story. Its main differential characteristics are the presence of single or multiple organ failures (MFO; liver, kidney, brain, coagulation, circulation and/or respiration) and the association with high short-term (28-day) mortality rate (>30% versus 1.9% in decompensated patients without ACLF) (2). ACLF is frequently but not always

4 preceded by precipitating events (mainly bacterial infections or alcoholic liver injury). The third piece of the story is systemic inflammation (SI), a recognized but relatively neglected feature in decompensated cirrhosis. European investigators have recently proposed that SI could be the mechanism linking cirrhosis with extrahepatic organ dysfunction (4). There is strong evidence for this contention. First, SI is 3 absent or moderate in compensated cirrhosis, chronic and intense in decompensated cirrhosis, and acute and very intense (similar to sepsis) in ACLF (5). Second, severity of ACLF (as estimated by the number of organ failures) correlates closely with severity of SI (4). Finally, the course of ACLF (resolution, improvement, no change or worsening) and mortality correlates closely with parallel changes in SI (5). There are several mechanisms by which SI may induce MOF: 1. Arterial vasodilation, left ventricular dysfunction and organ hypo-perfusion; 2. Endothelial dysfunction and impairment of organ microcirculation (increased shunting of blood through precapillary vessels and reduction in the density of perfused capillaries); and 3. The extension of SI into the organs with direct deleterious effects of the inflammatory mediators on cell mitochondrial function, bio-energetics, redox equilibrium and death (Figure 1A). SI in decompensated cirrhosis might be due to chronic bacterial translocation or translocation of bacterial products, namely pathogen-associated molecular patterns (PAMPs). SI in ACLF is probably due to acute bursts of SI induced by the precipitating events or, in cases without triggers, acute episodes of severe bacterial translocations. SI in ACLF might also be triggered by damageassociated molecular patterns (DAMPs) passively released from dying cells in failing organs. Albumin may have potential immune-modulatory effects in decompensated cirrhosis. This function relies on the ability of albumin to bind PAMPs (i.e. lipopolysaccharide (LPS), lipoteichoic acid, peptidoglycans, DNA fragments), DAMPs (hyaluronic acid, mtdna) and inflammatory mediators (lipid mediators (LM), kynurenines and reactive oxygen and nitrogen species) in the extracellular space, thus preventing their interaction with specific membrane-bound and cytoplasmic pattern recognition receptors in endothelial and immune cells (6). It is likely that albumin also blocks inflammatory signaling pathways by acting intracellularly through the internalization into endocytic vesicles. There are at least

5 seven putative membrane-associated albumin-binding proteins and receptors in endothelial and immune cells that regulate albumin binding and endocytotic process that results in transcytosis (7). These receptors transport albumin to the interstitial cell membrane, a mechanism that is used by 50% of circulating albumin to traverse the endothelial barrier. In addition to these, there are also three 4 different endosomal pathways for albumin which transport irreversible conformational-modified albumin molecules (i.e. oxidized or glycated albumin) into the lysosomes where they are degraded (7). Finally, there is an alternative pathway that protects non-modified native albumin from lysosomal destruction, recycling them into the intravascular compartment. Since the endosomal membrane contains a number of pattern recognition receptors involved in inflammation (toll-like receptors 3, 7 and 9), albumin may also modulate SI at this intracellular site. Two randomized controlled trials (RCTs) have presented solid data indicating that albumin treatment is highly effective in preventing extrahepatic organ failure in cirrhosis. The first, performed in 126 patients with spontaneous bacterial peritonitis, showed that treatment with antibiotics plus albumin significantly reduced HRS and hospital mortality (from 30% in the control group to 10%) (8). The second RCT (ANSWER study, still unpublished), compares standard medical therapy (SMT) versus SMT plus weekly administration of albumin during 18 months in 386 patients with decompensated cirrhosis. The incidence of HRS and grade III-IV hepatic encephalopathy was significantly reduced (0.72 and 0.62) and the probability of survival significantly increased (22% vs 34%) in patients receiving albumin. There are two additional ongoing RCTs, the ATTIRE study (866 patients) and the PRECIOSA study (410 patients), aimed to ascertain if increasing serum albumin concentration to over 30 g/l (14-days) or 35 g/l (1-year), reduces the rate of nosocomial bacterial infections (ATTIRE) or the 1-year prevalence of ACLF/mortality (PRECIOSA). The article by China et al in the current issue of Clinical Gastroenterology and Hepatology (9), describes the first mechanistic human study so far published assessing the effects of albumin treatment on the innate immune system function in cirrhosis. The study was performed in samples collected from 52 patients with decompensated cirrhosis with and without ACLF included in the ATTIRE feasibility trial. The

6 rationale of the study relies in three features: 1. SI in sepsis follows a two phase immune-response, a short initial period of intense immune stimulation and a second period of immune suppression during which patients may develop aggravation of the primary infection or new nosocomial infections; 2. The prevalence of nosocomial infections in patients with acute decompensation or ACLF is high (20% and 5 60%, respectively) (10); 3. A prior investigation by O Brien et al. (11) showing that plasma levels of PGE 2, a pleiotropic PG released in response to inflammatory stimuli that inhibits immune (lymphocyte and phagocyte) function and circulates mainly bound to plasma albumin, are markedly elevated in patients and experimental animals with decompensated cirrhosis in association with features of immunesuppression (inhibition of TNFα release by LPS-primed macrophages incubated with patients plasma and of bacterial killing capacity in cirrhotic mice with sepsis). These in-vitro and in-vivo actions were prevented by the prior administration of albumin and secondary reduction of free plasma PGE 2 levels. The authors interpretation of the data was that albumin treatment by reducing the circulating free PGE 2 levels, attenuates immune suppression and reduce the risk of nosocomial bacterial infection. Concepts derived from experimental investigations may not be confirmed when assessed in the clinical setting and this occurred in the study of China et al. In contrast to what should be expected according to the study of O Brien, in this study no significant changes in total (free+bound) plasma PGE 2 levels (although it reduced free PGE 2 levels) and pro- and anti-inflammatory cytokines (TNFα, IL-6, IL-8, IL-10, and IL-1β) were observed after albumin treatment suggesting that the beneficial effect of albumin in decompensated cirrhosis does not occur via direct modulation or scavenging of these circulating inflammatory mediators. In contrast, they observed specific changes in the circulating levels of a novel class of pro-resolving LM generated from long-chain omega-3 polyunsaturated fatty acids (12) in 10 patients with decompensated cirrhosis, suggesting that modulation of pro-resolving LM mediators by albumin could also be responsible for the immune-modulatory effects of this protein. Interestingly enough, the observed changes in LM cannot be merely explained by the scavenger properties of albumin since the plasma levels of some of these LM were increased in some patients while decreased in others.

7 Patients with cirrhosis and acute decompensation or ACLF constitute an extremely heterogeneous population and require of highly powered investigations to get reliable results. This may explain some discrepant results of the study of China et al in with other studies. For example, the reduced plasma levels of TNFα in decompensated cirrhosis found by China et al is not in agreement with a recent study 6 showing markedly elevated levels of this cytokine in 522 patients with acute decompensation (232 with ACLF) (5). On the other hand, the percent reduction induced by albumin treatment of the plasma levels of IL-8, the most sensitive pro-inflammatory cytokine in cirrhosis was relatively high (34%). These limitations, however, do not decrease the interest of the O Brien et al and China el al contributions, which have opened new spaces in our understanding on the mechanism of albumin function in decompensated cirrhosis and ACLF. A second study from China et al in this issue (13), describing the characteristics of the 79 patients included in the Attire feasibility trial, is clear example of how complex it is to perform pathophysiological investigations in heterogeneous groups of patients with cirrhosis and acute decompensation or ACLF. Seventeen patients had extrahepatic organ failure and 21 had bacterial infections at the time of recruitment. Eleven patients did not respond to albumin treatment (dosage adjusted according to baseline serum albumin concentration to achieve serum levels > 30 g/l). Finally, 21 patients developed a new bacterial infection; 7, 19, 15 and 1 patients, respectively, met renal, respiratory, circulatory and renal dysfunction criteria within the first 2 weeks of hospital stay. In summary, the studies of China et al, are in agreement with recent data indicating that systemic inflammation is a major issue in patients with decompensated cirrhosis and that albumin is emerging as a potent modulator of the innate immune system that could be useful in the management of ACLF. The studies also show that serum albumin concentration is probably the most appropriated target for albumin dosage. These features should be taken into account for future pathophysiological and therapeutic studies on albumin in cirrhosis.

8 7 References 1. Schrier RW, Arroyo V, Bernardi M, et al. Peripheral arterial vasodilation hypothesis: a proposal for the initiation of renal sodium and water retention in cirrhosis. Hepatology 1988; 8: Arroyo V, Moreau R, Kamath P, et al. Acute-on-chronic liver failure in cirrhosis. Nat Rev Dis Primers Jun 9;2: doi: /nrdp Moreau, R. Jalan R, Ginès P, et al. Acute-on-chronic liver failure is a distinct syndrome that develops in patients with acute decompensation of cirrhosis. Gastroenterology 2013; 144, Bernardi M, Moreau R, Angeli P, et al. Mechanisms of organ failure Mechanisms of decompensation and organ failure in cirrhosis: From peripheral arterial vasodilation to systemic inflammation hypothesis. J Hepatol 2015, 63: Clària J, Stauber RE, Coenraad MJ, et al. Systemic inflammation in decompensated cirrhosis: Characterization and role in acute-on-chronic liver failure. Hepatology 2016; 64: Arroyo V, García-Martinez R, Salvatella X, Human serum albumin, systemic inflammation and cirrhosis. J Hepatol 2014; 61: Merlot AM, Kalinowski DS, Richardson DR. Unraveling the mysteries of serum albumin-more than just a serum protein. Front Physiol. 2014;5: Sort P, Navasa M, Arroyo V, et al. Effect of intravenous albumin on renal impairment and mortality in patients with cirrhosis and spontaneous bacterial peritonitis. N Engl J Med.; 1999; 341: China L, Maini A, Skene SS, et al. Albumin counteracts immunosuppressive effects of lipid mediators in patients with advanced liver diseases. Clin Gastroenterol Hepatol 2018; (in press). 10. Fernández J, Acevedo J, Wiest R, et al. Bacterial and fungal infections in acute-on-chronic liver failure: prevalence, characteristics and impact on prognosis. Gut Aug 28. pii: gutjnl doi: /gutjnl O Brien AJ, Fullerton JN, Massey KA, et al. Immunosuppression in acutely decompensated cirrhosis is mediated by prostaglandin E2. Nat Med 2014; 20:

9 12. Spite M, Clària J, Serhan CN. Resolvins, specialized proresolving lipid mediators, and their potential roles in metabolic diseases. Cell Metab. 2014;19: China L, Skene SS, Shabir Z, et al. Albumin prevents infections in patients with chronic liver failure in a single-arm feasibility trial. Clin Gastroenterol Hepatol 2018; (in press) Schrier RW, Arroyo V, Bernardi M, et al. Peripheral arterial vasodilation hypothesis: a proposal for the initiation of renal sodium and water retention in cirrhosis. Hepatology 1988; 8: Arroyo V, Moreau R, Kamath P, et al. Acute-on-chronic liver failure in cirrhosis. Nat Rev Dis Primers Jun 9;2: doi: /nrdp Moreau, R. Jalan R, Ginès P, et al. Acute-on-chronic liver failure is a distinct syndrome that develops in patients with acute decompensation of cirrhosis. Gastroenterology 2013; 144, Bernardi M, Moreau R, Angeli P, et al. Mechanisms of organ failure Mechanisms of decompensation and organ failure in cirrhosis: From peripheral arterial vasodilation to systemic inflammation hypothesis. J Hepatol 2015, 63: Clària J, Stauber RE, Coenraad MJ, et al. Systemic inflammation in decompensated cirrhosis: Characterization and role in acute-on-chronic liver failure. Hepatology 2016; 64: Arroyo V, García-Martinez R, Salvatella X, Human serum albumin, systemic inflammation and cirrhosis. J Hepatol 2014; 61: Merlot AM, Kalinowski DS, Richardson DR. Unraveling the mysteries of serum albumin-more than just a serum protein. Front Physiol. 2014;5: Sort P, Navasa M, Arroyo V, et al. Effect of intravenous albumin on renal impairment and mortality in patients with cirrhosis and spontaneous bacterial peritonitis. N Engl J Med.; 1999; 341: China L, Maini A, Skene SS, et al. Albumin counteracts immunosuppressive effects of lipid mediators in patients with advanced liver diseases. Clin Gastroenterol Hepatol 2018; (in press). 23. Fernández J, Acevedo J, Wiest R, et al. Bacterial and fungal infections in acute-on-chronic liver failure: prevalence, characteristics and impact on prognosis. Gut Aug 28. pii: gutjnl doi: /gutjnl O Brien AJ, Fullerton JN, Massey KA, et al. Immunosuppression in acutely decompensated cirrhosis is mediated by prostaglandin E2. Nat Med 2014; 20:

10 25. Spite M, Clària J, Serhan CN. Resolvins, specialized proresolving lipid mediators, and their potential roles in metabolic diseases. Cell Metab. 2014;19: Legend of figures Figure 1. Panel A. Decompensation of cirrhosis is characterized by the development of specific complication and the impairment in the function of extra-hepatic organs/systems which may be moderate (dysfunction) or severe (failure). Several pathophysiological events (steps) contribute to cirrhosis decompensation. Step 1 consists in an increased intestinal translocation of viable bacteria and pathogen-associated molecular patterns (PAMPs). At the early phase of cirrhosis, inflammation is predominantly located at the lamina propia where it induces arterial vasodilation. However, as the disease progresses, inflammation extends systemically affecting also the extra-splanchnic organs. Systemic inflammation may also develop by the release of damage-associated molecular patterns (DAMPs) from the diseased liver (for example in the case of acute liver injury superimposed to cirrhosis). Organ hypo-perfusion related to circulatory dysfunction and direct deleterious effects of inflammatory mediators on tissue homeostasis are the two predominant mechanisms or multi-organ dysfunction/failure in cirrhosis. Panel B. Time-course of systemic inflammation, acute decompensation and ACLF development in cirrhosis. Systemic inflammation which is absent or moderate in compensated cirrhosis, rises rapidly during the transition to decompensated cirrhosis and then follows a relatively steady course. ACLF develops in the setting of a burst of systemic inflammation (represented as yellow figures) promoted by a precipitating event (PE). Three inflammatory bursts are represented in compensated and decompensated cirrhosis. There are thresholds for systemic inflammation for ACLF development. Please note that the magnitude of the systemic inflammatory reaction (inverted red triangles) required for ACLF development is greater in patients with compensated cirrhosis than in those with decompensated cirrhosis. If the inflammatory reaction promoted by a PE is insufficient to reach the threshold level, no ACLF develops.

11 Figure 1 A PROGRESSIVE LIVER DYSFUNCTION & PORTAL HYPERTENSION INTESTINAL DYSBIOSIS, LOST OF INTEGRITY OF GUT BARRIER, IMPAIRED INTESTINAL IMMUNE FUNCTION SYSTEMIC INFLAMMATION AFFECTING NON SPLANCHNIC ORGANS MICROCIRCULATORY DYSFUNCTION, MITOCHONDRIAL AND CELL DYSFUNCTION, CELL DEATH TRANSLOCATION BACTERIA AND PAMPs TO THE LAMINA PROPIA AND THE CIRCULATION SPLANCHNIC ARTERIAL VASODILATION, SYSTEMIC CIRCULATORY DYSFUNCTION, ORGAN HYPOPERFUSION ACUTE DECOMPENSATION, MULTI ORGAN DYSFUNCTION (DECOMPENSATED CIRRHOSIS) AND MULTI ORGAN FAILURE (ACLF)

12 Figure 1 B PE ACLF ACLF ACLF Threshold of systemic inflammation for ACLF developmet PE Threshold of systemic inflammation for AD developmet Time course curve of systemic inflammation COMPENSATED CIRRHOSIS DECOMPENSATED CIRRHOSIS PE H