Circulating neutrophil anti-pathogen dysfunction in cirrhosis

Summary Neutrophils are the largest population of leucocytes and are among the first cells of the innate immune system to fight against intruding pathogens. In patients with cirrhosis, neutrophils exhibit altered functionality, including changes in phagocytic ability, bacterial killing, chemotaxis, degranulation, reactive oxygen species production and NET (neutrophil extracellular trap) formation. This results in their inability to mount an adequate antibacterial response and protect the individual from infection. Prognosis and survival in patients with cirrhosis are greatly influenced by the development of infectious complications. Multidrug-resistant bacterial infections in patients with cirrhosis are currently a growing problem worldwide; therefore, alternative methods for the prevention and treatment of bacterial infections in cirrhosis are urgently needed. The prevention and treatment of neutrophil dysfunction could be a potential way to protect patients from bacterial infections. However, the reasons for changes in neutrophil function in cirrhosis are still not completely understood, which limits the development of efficient therapeutic strategies. Both cellular and serum factors have been proposed to contribute to the functional impairment of neutrophils. Herein, we review the current knowledge on features and proposed causes of neutrophil dysfunction in cirrhosis, with a focus on current knowledge gaps and limitations, as well as opportunities for future investigations in this field.


Introduction
Cirrhosis is a life-threatening chronic and progressive liver disease that develops primarily as a consequence of chronic hepatitis B or hepatitis C virus infections, or alcohol-related or non-alcoholic steatohepatitis, with other rarer causes including alpha-1-antitrypsin deficiency, haemochromatosis, primary sclerosis cholangitis, Wilson's disease, etc. 1,2 Being the 11th leading cause of death in the world, cirrhosis accounted for 2.2% of total deaths in 2016 3 and 2.4% of total deaths in 2017, 4 with an annually increasing mortality rate. 4,5Compensated patients with cirrhosis have an 5-fold increased mortality risk and decompensated patients a 10fold increased mortality risk compared to the general population. 6][9] Around one-third of hospitalized patients with cirrhosis suffer from bacterial infections, which leads to increased hospitalization time, 7 de-listing from the transplantation waiting list, 10 acute kidney injury 11 and, hence, a 4-fold increased mortality rate compared to non-infected patients with cirrhosis. 8Of those with cirrhosis who develop bacterial infections, 30% die within 1 month and more than 60% die within 1 year after the infection. 8fections in patients with cirrhosis are mostly caused by gram-negative bacteria (such as Escherichia coli and Klebsiella pneumoniae) of intestinal origin, but infections caused by gram-positive bacteria (such as Staphylococcus aureus and Enterococci) are on the rise, particularly in hospitalized patients. 12Multidrug-resistant bacterial infections in cirrhosis are currently a growing problem worldwide; therefore, alternative methods for the prevention and treatment of bacterial infections in cirrhosis are urgently needed. 13,14n important reason for the increased susceptibility to bacterial infections in patients with cirrhosis is the progressive development of immune dysfunction. 15nnate immune dysfunction, including impaired neutrophil functionality, is a predominant part of cirrhosis-associated immune dysfunction and, therefore, plays a major role in the development of bacterial infections in patients with cirrhosis. 16The number of circulating neutrophils is frequently altered in cirrhosis.Neutropenia is often described in cirrhosis, 17 whereas neutrophilia has been reported for acute-on-chronic liver failure (ACLF). 18,19The neutrophil-to-lymphocyte ratio is associated with liver-related mortality in patients with different stages of cirrhosis, 17,20 as well as in patients with ACLF. 19,21Furthermore, an increased neutrophil-to-lymphocyte ratio in patients with cirrhosis positively correlates with the number of

Features of neutrophil dysfunction in cirrhosis
Neutrophils are the largest population of leucocytes and are among the first cells of the innate immune system to fight against intruding pathogens.Neutrophils express a range of surface G protein-coupled receptors, which enable them to sense chemoattractants (inflammatory mediators or pathogen-related molecules) and, consequently, migrate to the site of the intruding pathogen (mostly bacteria and fungi).This process of directed migration is called "chemotaxis".As soon as neutrophils reach the site of infection, they use one of their defence mechanisms to protect the host organism from the infectious agent.They can internalise the pathogen via a process called "phagocytosis" and kill it inside the phagolysosome using the microbiocidal contents of their intracellular granules (e.g.various proteases) and reactive oxygen species (ROS).These substances can also be released into the extracellular space by neutrophils to enable extracellular bacterial killing, but this can also cause tissue damage.Neutrophils produce different anti-and pro-inflammatory cytokines, which help regulate inflammatory as well as other physiological and pathophysiological processes.Furthermore, neutrophils have recently been described as being able to extrude their DNA, which is coated with histones and cytoplasmic and granular proteins (e.g.neutrophil elastase, myeloperoxidase, etc.), i.e. the so-called "neutrophil extracellular traps" (NETs), in order to catch, immobilize and kill infectious agents.Neutrophils are cells with a relatively short lifespan, which is extended in the case of infection.Neutrophils undergo apoptosis and then are phagocytosed by macrophages and dendritic cells to promote the resolution of inflammation (reviewed in 27-29).
All the aforementioned functions allow neutrophils to fight bacterial infections.In patients with cirrhosis, neutrophils exhibit altered functionality, including changes in phagocytic ability, bacterial killing, chemotaxis, degranulation, reactive oxygen species production and NET formation (Fig. 1, Table S1).This results in their inability to mount an adequate antibacterial response and protect the patient from infection.

Chemotaxis
1][32][33][34][35] Neutrophils from patients with alcohol-associated cirrhosis show reduced migration towards healthy serum compared to healthy donor neutrophils.Serum from patients with cirrhosis had less chemoattractant activity compared to healthy serum in the same study.No correlation with bacterial infections has been found; 30 however, another study reported lower neutrophil migration ability in patients with cirrhosis with previous bacterial infections compared to those without previous infection. 34The decrease in serum chemoattractant activity might be explained by alterations in chemoattractant production found in patients with cirrhosis. 36nterestingly, another study shows that serum chemotactic inhibitory activity (which shows how effectively cirrhotic serum inhibits the chemoattractant activity of healthy serum mixed with zymosan A) is higher in alcohol-associated cirrhosis compared to non-alcoholic cirrhosis. 32Alcohol intake per se is a likely contributor to neutrophil dysfunction in alcohol-related cirrhosis as it has previously been shown to reduce neutrophil chemotaxis in rats, 37 transiently reduce neutrophil ROS production and phagocytosis in healthy volunteers, 38 and cause dysregulation of neutrophil function upon in vitro alcohol exposure. 39Other studies also report the presence of serum chemotactic inhibitory activity in patients with cirrhosis. 33,35Cirrhotic neutrophils show decreased transendothelial migration in response to N-formyl-met-leu-phe (fMLF), increased adhesion to endothelial cells (HMEC-1) and an altered expression of adhesion receptors, and higher CD11b and lower CD62L expression, which also indicates increased degranulation 40 and neutrophil ageing, 41 compared to healthy controls. 31ecreased neutrophil migration towards interleukin (IL)-8 has been shown in cirrhosis, probably due to the decreased expression of CXC motif chemokine receptor 2 (which senses IL-8). 42Also, decreased migration and adhesion of neutrophils isolated from patients with cirrhosis in response to leukotriene B4 has been Intrinsic and serum defects have been proposed to cause neutrophil dysfunction, although neither can fully describe the extent of neutrophil dysfunction and findings are contradictory.
Due to the lack of clinical studies with neutrophil function as a primary or secondary outcome, there is only a vague understanding of the best strategy to prevent and treat neutrophil dysfunction in cirrhosis.
Further research is needed to develop a neutrophil function screening panel and biomarkers for clinical practice, and to further describe neutrophil dysfunction, its causes and consequences, and approaches for its prevention and treatment.reported. 36Interestingly, the impairment in migration in the presence of fMLF is more pronounced in patients with acute decompensation and ACLF than in patients with compensated cirrhosis or healthy volunteers, which is less evident or absent (though with a tendency towards a decrease) when CXC motif ligand (CXCL)8 or CXCL1 is used as a chemoattractant.Furthermore, neutrophil migration patterns correlate with the incidence of adverse outcomes in this cohort of patients. 435][46] This might initially preserve chemotaxis due to ligand excess or may also cause the homologous desensitisation of their receptors on neutrophils due to the prolonged excess of ligands, their downregulation and the subsequent decrease in neutrophil migration.The higher grade of impairment of neutrophil migration towards fMLF might also be explained by the different mechanisms involved, e.g.another FPR1 ligand that is highly abundant in cirrhosis, such as chenodeoxycholic acid (CDCA), might compete with fMLF for binding to the receptor and, as a result, inhibit neutrophil chemotaxis. 47When casein is used as a chemoattractant, neutrophil chemotaxis is not affected in patients with cirrhosis.This indicates that defects in chemotaxis in cirrhosis are limited to certain pathways and depend on the chemoattractant used.The same study shows that the random migration of neutrophils is not affected in cirrhosis. 48

Swarming
Only one study so far implies that swarming (coordinated neutrophil communication and recruitment) of neutrophils in order to control Candida albicans hyphae growth is impaired in patients with cirrhosis compared to healthy controls. 49

Phagocytosis
][52][53][54][55][56][57][58] Defective phagocytosis of S. aureus has been detected in isolated neutrophils in alcohol-associated cirrhosis 50,56 and primary biliary cholangitis (PBC), 56 as has defective phagocytosis of E. coli but to a lesser extent. 50The decrease in neutrophil phagocytosis of E. coli is more pronounced in patients with cirrhosis who had previous bacterial infections. 34Neutrophil phagocytic capacity (number of internalised bacteria per cell) of E. coli measured in whole blood or isolated neutrophils of patients with cirrhosis is impaired 51,52,57,59,60 or unchanged. 61,62The percentage of phagocytic neutrophils is decreased in the blood of patients with cirrhosis 53,55,60,62,63 as well as in those with ACLF; 63 the degree of this dysfunction increases with increasing severity of cirrhosis and is higher in active drinkers compared to abstinent patients, but is not different between the aetiologies of cirrhosis (alcohol, HCV, autoimmune and other). 53No defect in the phagocytosis of C. albicans was observed in patients with cirrhosis. 48In ACLF, impaired phagocytosis of latex beads is reported and associated with 90-day survival. 58It remains to be clarified whether these subtle observed differences in phagocytosis are dependent on the method used or if they represent true differences in pathogen phagocytosis.

Killing capacity
Bacterial killing is reduced in neutrophils from patients with cirrhosis, which means that even those active neutrophils which manage to engulf bacteria cannot efficiently kill them. 50,64In particular, intracellular killing of S. aureus and E. coli, which are common causes of bacterial infections in cirrhosis, is impaired in

Killing capacity
Bacterial killing alcohol-associated cirrhosis. 50In contrast, another study could not find any differences in neutrophils' capacity for intracellular killing of S. aureus in alcohol-associated cirrhosis and PBC despite decreased total bacterial killing, which the authors put down to the decreased percentage of phagocytic neutrophils. 56In another study, neutrophils from patients with cirrhosis showed impaired killing capacity not only of bacteria but also of fungi, such as C. albicans; 49 however, another study found it unchanged. 48

ROS production
Impaired neutrophil ROS production [51][52][53][54][55]58,59,[65][66][67][68] and degranulation 50,64 have been described in cirrhosis and might contribute to impaired bacterial killing. 50 The alteation of ROS production in cirrhosis is characterised by an elevated percentage of neutrophils with basal ROS production (so-called "resting burst") 51,55,58,60,62,65 and elevated, 59,65 unchanged 61,62 or reduced 63,67 intracellular basal ROS production (intracellular ROS levels).63 Extracellular basal superoxide production is unchanged 50 or elevated in patients with cirrhosis.48 An increased percentage of neutrophils from patients with cirrhosis respond to a low physiological stimulus like fMLF, 51,52,55,62 which suggests that these neutrophils have previously been primed by persistent low-grade stimulation with priming agents like lipopolysaccharide (LPS) or tumour necrosis factor-a (TNF-a).However, other studies show it to be unchanged. 60The intracellular ROS pool of those neutrophils that respond to fMLF stimulation is slightly increased, 65 decreased 67 or unchanged 62 in patients with cirrhosis.Extracellular superoxide release in response to fMLF [66][67][68] and TNF-a 66 is decreased in patients with cirrhosis.The decrease in superoxide release in response to fMLF is significantly more pronounced in patients with ACLF than in patients with advanced cirrhosis.18 Notably, priming neutrophils from patients with cirrhosis with TNF-a does not increase their response to fMLF as is usually the case with healthy donor neutrophils.66 The number of neutrophils producing ROS in response to a potent stimulus (E. coli) is unchanged 51,53,55,60,61,63,65 or decreased 62 in patients with cirrhosis, whereas their intracellular ROS level is increased, 65 decreased 63 or unchanged.62 In ACLF, ROS production in response to E. coli has been shown to be unchanged compared to that in healthy controls. 63 Extracellular superoide production in response to zymosan (structural component of yeast cell wall) is reduced in patients with cirrhosis. 48,50,66,69Extracellular hydrogen peroxide levels produced by neutrophils in response to zymosan are not different or higher in patients with cirrhosis compared to healthy controls, which is reflected in the increased hydrogen peroxide/superoxide molar ratio.50 Interestingly, superoxide and hydrogen peroxide have different effects on cell apoptosis and necrosis: superoxide inhibits apoptosis, whereas hydrogen peroxide promotes cell apoptosis via intracellular acidification and even necrosis when present at very high concentrations.70,71 Nitric oxide production in response to opsonised zymosan is also increased in neutrophils from patients with cirrhosis.69 Changes in ROS production are different in patients with cirrhosis with active infection: basal ROS production and ROS production in response to fMLF have been shown to be unaltered, as have the percentage of neutrophils which produce ROS in response to E. coli stimulation; however, the intracellular ROS pool generated in response to E. coli is decreased, which might support a concept that cirrhotic neutrophils are exhausted via prior low-grade stimulation and cannot mount an augmented ROS response when they have to fight a real infection.65 The aforementioned changes have been shown in studies on cirrhosis of different aetiologies; however, most were performed with neutrophils from patients with alcohol-or HCV-associated cirrhosis.Several studies report no dependence of ROS production on the aetiology of cirrhosis. 53,66 Cotrasting findings have been reported on the correlation of changes in ROS production with cirrhosis severity: some studies report no correlation, 51,53 while some studies have found a correlation with disease severity.65,66

Degranulation
The intracellular enzyme contents (lysozyme, myeloperoxidase [MPO]) and their release from neutrophil granules upon stimulation with zymosan have been shown to be reduced in neutrophils from patients with cirrhosis; however, the authors of this study claim that the reduced release is not dependent on the reduced enzyme levels inside the granules. 50A decreased number of neutrophils producing MPO and reduced intracellular MPO levels have been observed in neutrophils isolated from patients with compensated cirrhosis, but not from those with ACLF. 63Another study reported that intracellular MPO content is not altered in neutrophils from patients with cirrhosis but that its extracellular release in response to fMLF is decreased. 64MPO activity has been shown to be either decreased 66 or unchanged 64 in neutrophils from patients with cirrhosis.In patients with alcohol-associated cirrhosis, increased mobilisation of MPO to the cell surface of the primary neutrophil granules has been observed. 72T formation NET formation is a recently discovered mechanism of neutrophil defence.73 To date, only scarce data are available regarding the role of NET formation in cirrhosis.75 Another group reported later that plasma of patients with decompensated cirrhosis can induce NET formation in neutrophils isolated from healthy controls.57 This finding is also supported by other studies that reported elevated NET markers, such as H3Cit-DNA 76 and MPO-DNA, 76,77 in the plasma of patients with cirrhosis and ACLF, correlating with cirrhosis severity in these patients.76 NET formation in response to E. coli, fMLF and PMA, as well as spontaneous NET formation, is elevated in patients with compensated cirrhosis and ACLF.63 Apoptosis and viability Current knowledge on neutrophil apoptosis in cirrhosis is not very broad.An increased rate of apoptosis and decreased viability of neutrophils 24 h after isolation from the whole blood of neutropenic patients with viral cirrhosis has been described.78 Increased apoptosis of neutrophils has also been reported in patients with decompensated cirrhosis.79

Summary
Deficiencies in chemotaxis and phagocytosis, as well as decreased neutrophil viability and increased apoptosis, have been reported fairly consistently in patients with cirrhosis across studies, irrespective of differences in cohort selection, or methods of neutrophil isolation and functional assessment.Although the majority of patients had alcohol-related cirrhosis, some studies show comparable results for other aetiologies of cirrhosis, such as in study cohorts of primarily viral aetiology 31,34,60,63 or patients with PBC. 56Although most studies have been performed in patients with decompensated cirrhosis, functional impairment has also been shown in patients with compensated cirrhosis. 62,63The findings regarding chemotaxis and phagocytosis are, however, specific to the stimulus used.For example, chemotaxis towards CXCL8, CXCL1 or casein, and phagocytosis of C. albicans are not impaired in cirrhosis, which might indicate that functional defects in cirrhosis are limited to certain pathways.Whether this indicates a true pathophysiological difference making patients with advanced chronic liver diseases more prone to bacterial than to fungal infections or whether this is a methodological bias needs to be further explored.In contrast, the changes in ROS production, degranulation, bacterial killing and NET formation are variable among studies.The most controversial is the nature of alterations in neutrophil ROS production in cirrhosis.Although most studies have reported an elevated number of neutrophils with basal ROS production 51,55,60,65 and ROS production in response to physiological stimuli with low potency like fMLF, 51,52,55 the results on ROS production are highly varied.Most studies have been performed in patients with an alcohol-related aetiology of cirrhosis and decompensated cirrhosis; however, these results are comparable to those obtained for other aetiologies and for compensated cirrhosis. 60,63Furthermore, some studies report no dependence of ROS production on the aetiology of cirrhosis. 53,66he variety of methods and experimental conditions used to study ROS production in patients with cirrhosis, often measuring different types of ROS, is the most likely explanation for these differences.The majority of studies measure neutrophil ROS production in cirrhosis with either flow cytometry (e.g. based on the conversion of dihydrorhodamine 123 to rhodamine 123, or similar), 58,59,63,65 cytochrome c reduction 18,[66][67][68] or luminolbased 67 assays.Flow cytometry-based assays measure only intracellular ROS production, detecting a mixture of different ROS but with almost no sensitivity for superoxide (e.g. in the case of dihydrorhodamine). 80The cytochrome c reduction assay is able mainly to detect extracellular ROS and, in contrast, detects only superoxide. 81Luminol-based assays can be used to measure either intracellular or total ROS production (mixture of intracellular and extracellular ROS), depending on the presence or absence of horseradish peroxidase, and to detect the mixture of different ROS. 81,82Furthermore, the neutrophil preparation technique, whether whole blood or isolated neutrophils are used, and the type of isolation procedure may contribute to the observed differences.When the literature is separated based on methodology used for ROS production assessment, the results become more consistent (Fig. 1).To date, there is no evidence to support the notion that one method is superior in cirrhosis; however, standardisation would be necessary to harmonise the findings and enable translation of neutrophil diagnostics to clinical practice.However, even after standardisation some discrepancies will remain.Despite the methodology used to study neutrophil function, other parameters, such as patient characteristics, cirrhosis aetiology and severity, presence of previous or active bacterial infections, active alcohol drinking, neutrophil isolation technique (given the ex vivo fragility of neutrophils), and the combination of all these factors can contribute to the discrepancies in results between studies (Table S1).
Interestingly, recent studies have revealed the importance of neutrophil heterogeneity in different diseases.Neutrophils are no longer perceived as one cell type, but rather as several "types" of cells with particular functions and roles in health and disease.For instance, distant molecular signatures have been described for eight neutrophil subpopulations 83 and different functional profiles have been described for five neutrophil subgroups. 84The role of neutrophil heterogeneity in cirrhosis and its interplay with neutrophil dysfunction are yet to be determined.
Taken together, circulating neutrophils in cirrhosis are altered numerically but, more importantly, exhibit multiple functional deficiencies, starting from the inability to migrate to the infection site and subsequent inability to internalise and kill pathogens.The loss of neutrophil antimicrobial function, particularly changes in ROS production and phagocytosis in cirrhosis, contributes to the development of bacterial infections, organ failure and mortality. 34,51,53his underscores the importance of identifying the mechanisms underlying neutrophil dysfunction in cirrhosis, which will allow for the development of targeted therapies for the prevention and treatment of bacterial infections in cirrhosis.

Causes of neutrophil dysfunction in cirrhosis
First, the course of cirrhosis per se contributes to altered neutrophil functionality.Portal hypertension is associated with upregulation of the neutrophil chemoattractant CXCL1 in primary liver sinusoidal endothelial cells from mice and with NET formation. 85Alterations in Toll-like receptor (TLR)4 expression and signalling not only contribute to inflammation and endothelial dysfunction but also neutrophil dysfunction in patients with cirrhosis. 86Thrombocytopenia is also commonly described in cirrhosis and its crosstalk with neutrophil function has been described, e.g.platelet transfusions in patients with thrombocytopenia result in further increases in CD11b expression on neutrophils. 87Despite this, one of the important questions is whether the neutrophil dysfunction is primarily a cellular problem or if it is caused by extracellular factors.There are multiple studies that attempt to support one or other of these concepts.

Proposed intrinsic defects
Some studies propose that cellular defects of neutrophils are responsible for their dysfunction in cirrhosis.In one study, sera from patients with decreased neutrophil locomotion does not cause a similar defect in chemotaxis in healthy control neutrophils. 30However, in the same study, sera from patients with cirrhosis exhibited reduced chemoattractant activity for healthy donor neutrophils compared to healthy donor serum, which could indicate that changes in serum content also potentially contribute to the decreased chemotaxis of patients' neutrophils. 30Reduced intracellular glutathione levelsa detoxifier of hydrogen peroxideare reported in neutrophils from patients with cirrhosis, which could explain the prevalence of hydrogen peroxide over superoxide.Lower levels of glutathione have been associated with higher levels of hydrogen peroxide production and lower hydrogen peroxide/superoxide ratio, degranulation and intracellular killing of E. coli.In this study, cirrhotic serum does not cause any changes in phagocytic function or intracellular bacterial killing of healthy donor neutrophils, suggesting underlying cellular defects, including intracellular glutathione deficiency. 50Dysfunctional neutrophils from patients with cirrhosis have decreased phospholipase C (which is involved in superoxide production) activity; however, the reason for this is unknown. 66Furthermore, changes in neutrophil glycolytic metabolism and other transcriptional profile alterations, including induction of granule genes and downregulation of cell migration and cell cycle genes have been reported in patients with decompensated cirrhosis with ACLF, 18 and are likely to be associated with the impaired anti-pathogenic function of neutrophils. 18,63,88oposed serum defects The majority of studies investigating neutrophil dysfunction have proposed that changes in serum content in patients with cirrhosis initiate the development of neutrophil dysfunction (Fig. 2, Table 1).Almost all of the functional deficiencies outlined have been shown to be transferrable with patient sera.Neutrophils from patients with cirrhosis have a defect in migration towards zymosan only in the presence of autologous plasma and not healthy control plasma, and these defects vary between the aetiologies of cirrhosis (defects are present in alcohol-associated and cryptogenic cirrhosis, but not PBC). 33Healthy donor neutrophils have been shown to have decreased capacity to kill C. albicans following incubation with cirrhotic serum. 49ecreased phagocytic capacity for E. coli, but unchanged (though a tendency toward an increase) basal ROS production (percentage of neutrophils), has been shown in healthy donor neutrophils after incubation with cirrhotic plasma, dependent on cirrhosis severity but independent of cirrhosis aetiology. 61In another study, incubation with cirrhotic plasma promoted an increase in the number of healthy donor neutrophils with basal ROS production and a decrease in phagocytosis.Interestingly, neutrophil dysfunction in cirrhosis seems to be reversible with a restoration of function observed following incubation with healthy donor plasma. 51Neutrophil phagocytosis and intracellular killing of S. aureus are not affected in cirrhotic neutrophils incubated with AB serum despite being dysfunctional in the presence of autologous serum. 56Patients' plasma also influences healthy donor neutrophil degranulation, decreasing MPO release in response to fMLF. 64Our research group recently showed that serum components more than 30 kD in size are responsible for the changes in neutrophil phagocytic function. 54ncreased gut permeability in cirrhosis results in various bacteria and bacteria-derived molecules, like endotoxins, getting from the gut lumen to the systemic circulation. 89These substances in the serum of patients with cirrhosis are thought to have an influence on neutrophil function via persistent lowgrade stimulation.

Endotoxins
Endotoxins cause an increase in the percentage of neutrophils with basal ROS production in in vitro experiments with healthy donor neutrophils and a decrease in phagocytic capacity in experiments with cirrhotic neutrophils.Plasma endotoxin removal strategies prevent the deleterious effects of patients' plasma on healthy donor neutrophils. 51However, despite in vitro effects of LPS on neutrophil function, the levels of bacterial endotoxin in the plasma of patients with cirrhosis do not correlate with the defects in phagocytic function 55,61 and basal ROS production. 61ndotoxin receptors TLR2 and TLR4 are upregulated in healthy donor neutrophils after incubation with cirrhotic plasma 61,90 and in neutrophils from patients with cirrhosis. 52TLR2 is upregulated only after incubation with plasma from patients with alcoholassociated cirrhosis, while TLR4 is upregulated in neutrophils treated with plasma from patients with both alcohol-associated and viral cirrhosis. 61Inhibition of TLR2 and TLR4 decreases the number of neutrophils with basal ROS production caused by incubation with cirrhotic serum, but further impairs phagocytic capacity. 90LPS-binding protein (LBP) levels are elevated in patients with cirrhosis compared to healthy controls, being associated with lower intracellular basal ROS production 65 and with the development of severe bacterial infections. 91LBP enhances the effects of LPS on immune cells. 92cterial DNA Bacterial DNA itself may also be responsible for changes in neutrophil function in cirrhosis.Bacterial DNA is present in the serum of some patients without active infection, [93][94][95] but is not associated with the Child-Pugh score nor clinical characteristics of these patients.93 The presence of bacterial DNA in the serum of patients with cirrhosis is associated with higher levels of cytokines such as TNF-a, IFN-c, IL-12 and nitric oxide.95 Interestingly, higher cytokine levels in the sera of patients with bacterial DNA are independent of their LPS or LBP serum levels.96 In one study, the bacterial DNA sensing receptor in neutrophils, TLR9, has been shown to be more highly expressed in neutrophils from patients with cirrhosis than healthy controls.52 However, in other studies, TLR9 has not been found to be upregulated in patients with cirrhosis 61,90 and circulating bacterial DNA measured in the plasma of patients with cirrhosis could neither be associated with the changes in neutrophil phagocytic and basal ROS production, nor with patient mortality.61 Albumin Albumin is synthesised in the liver and, therefore, its concentration is reduced in cirrhosis.97 Furthermore, the structure of albumin is also altered in cirrhosis, mainly via oxidation, plasma levels of oxidized albumin increase further in ACLF.98,99 Albumin binding capacity is decreased in patients with decompensated cirrhosis, which negatively correlates with the MELD (model for end-stage liver disease) score 100 and increased mortality.3 Given the ability of albumin to bind bacterial products, ROS, and nitric oxide, 104 the changes in its abundance and structure might contribute to neutrophil dysfunction.The addition of albumin to incubation media has been shown to decrease the number of neutrophils with high basal ROS production and reverse phagocytic capacity defects caused by plasma from patients with cirrhosis. However, one study shows that patients with cirrhosis with dysfunctional neutrophil phagocytosis and intracellular killing capacity have comparable serum albumin levels to patients with cirrhosis without neutrophil functional defects, indicating that it is not the quantity but the functionality of albumin that is important in Decreased phagocytosis 51 Beneficial effects of endotoxin removal from plasma 51 No correlation with defect in ROS production and phagocytosis 55,61 Bacterial DNA Correlation with higher levels of TNF-a, IFN-c, IL-12 and nitric oxide 95 No association with severity and clinical parameters 93 No association with ROS production and phagocytic function 61

Albumin
Decreased albumin binding capacity is associated with cirrhosis severity 100 and mortality 101 Decreased basal ROS production and improved phagocytosis in vitro 90 Decreased neutrophil superoxide production 105 No association with C. albicans killing capacity 49 No association with phagocytosis and intracellular killing capacity 50 Ammonia Impaired phagocytosis of E. coli, increased basal ROS production 107 Correlation with decreased phagocytic activity 53

Lipoproteins
Correlation with disease severity, increased levels of TNF-a, IL-8, IL-6 108 Higher levels of IgG autoantibodies against oxidised lowdensity lipoproteins correlated with higher integrated intracellular ROS production level in response to E. coli 65

Cytokines
Correlation with increased numbers of neutrophils with basal ROS production and ROS producing neutrophils in response to E. coli. 53nhibited neutrophil phagocytosis and bactericidal activity 112 Improved neutrophil chemotaxis towards IL-8 42,116 Calprotectin is predictive for survival and infections 120,121 Accelerate neutrophil clearance during inflammation and impair CXCL1 production 113 Increase neutrophil regress from the bone marrow and their recruitment to the tissues 114 Trigger degranulation and NETs formation 115,118 Delays neutrophil apoptosis in presence of PBMCs 117 Calprotectin levels similar between cirrhotic patients and healthy volunteers 120 IL-6 does not influence neutrophil apoptosis, priming or adhesion molecule expression 116
cirrhosis-associated neutrophil dysfunction. 50Furthermore, the C. albicans killing capacity of cirrhotic neutrophils does not correlate with serum albumin levels. 49monia Ammonia levels in the serum of patients with cirrhosis predict organ failure and mortality. 106Neutrophils from rats subjected to ammonia supplementation and healthy donor neutrophils incubated with ammonia exhibit impaired phagocytosis of E. coli and an increased number of neutrophils with basal ROS production, due to the ability of ammonia to cause cell swelling. 107ecreased neutrophil phagocytic activity correlates with increased plasma ammonia levels in patients with cirrhosis. 53poproteins Patients with cirrhosis have lower levels of high-density lipoprotein (HDL) cholesterol and apolipoprotein A1, which further decrease upon decompensation, correlating with increased levels of TNF-a, IL-8, IL-6 and severe bacterial infection, and predicting patient mortality. 108The important function of HDL, similar to that of albumin, is to neutralize LPS; 109 therefore, its low abundance in cirrhosis could be a reason for higher LPS serum concentrations and low-grade inflammation.Furthermore, HDL composition and function is altered in cirrhosis. 110igher levels of IgG autoantibodies against oxidised lowdensity lipoproteins are correlated with higher integrated intracellular ROS production in response to E. coli in neutrophils from patients with cirrhosis. 65tokines Elevated pro-and anti-inflammatory cytokines, such as TNF-a, IL-6, IL-1b, IL8 and IL10, have been reported in many but not all studies. 49,52,53,55,61Increasing numbers of neutrophils with basal ROS production correlate with increased TNF-a, IL-6, IL-8 and IL-10, whereas increasing numbers of neutrophils producing ROS in response to E. coli correlate with increased IL-1b, IL-8, IL-1 and IL-17 levels. 53Interestingly, patients with ACLF have even higher levels of TNF-a, IL-6, IL-8, IL-1b, IL-12, IL-1RA, IL-10, granulocyte colony stimulating factor (G-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF) in blood compared to patients with decompensated cirrhosis. 19,44,99TNF-a is a known priming agent. 111IL-10 inhibits neutrophil phagocytosis and bactericidal activity. 112IL-6 has been shown, on the one hand, to accelerate neutrophil clearance during inflammation and to impair CXCL1 production (which is a chemoattractant for neutrophils) via the IL-6/gp130/STAT3 pathway; 113 on the other hand, IL-6 has been shown to increase neutrophil regress from the bone marrow and recruitment to target tissues. 114IL-8 is a known neutrophil chemoattractant and has been shown to trigger degranulation and NET formation. 115IL-33 treatment of neutrophils from patients with cirrhosis improves their chemotaxis towards IL-8 42 and IL-6 has been reported to increase neutrophil migration towards IL-8; however, it does not influence neutrophil apoptosis, priming or adhesion molecule expression. 116IL-1b delays neutrophil apoptosis in the presence of peripheral blood mononuclear cells 117 and induces NET formation, which can be abrogated by IL-1RA. 118IL-17-activated pericytes produce chemokines, which stimulate neutrophil production of pro-inflammatory molecules, prolong neutrophil survival and increase neutrophil phagocytic capacity. 119Serum calprotectin is an important biomarker of neutrophil activation.
Serum levels of calprotectin in patients with compensated and decompensated alcohol-induced cirrhosis are similar to those from healthy volunteers and are predictive of survival and recurrent infections, independent of the severity of cirrhosis. 120nother study has shown increased calprotectin levels in patients with stable cirrhosis and acute decompensation of cirrhosis of different aetiologies, which correlates with the severity of the disease, ACLF and infection, and is associated with poor survival in acute decompensation but not in ACLF. 121on metabolism parameters Disturbances in iron metabolism are known in patients with cirrhosis. 122Iron metabolism parameters influence neutrophil function.Heme is a part of haemoglobin and excessive free heme is released to the circulation in the case of haemolysis, 123 which is common for patients with cirrhosis. 60Hemin, which is different from heme as it contains ferric and not ferrous ion, is also increased in haemolysis. 124Heme delays apoptosis of neutrophils 125 and induces migration of and ROS production by neutrophils. 126In contrast, another study shows impaired phagocytosis and migration of neutrophils in response to heme, which can be explained by completely different experimental design of chemotaxis experiments, with heme used not as a chemoattractant for human neutrophils in vitro, but as a treatment for mice in in vivo experiments. 127Hemin activates neutrophil chemotaxis, ROS production and IL-8 expression. 128he ferritin-containing fraction of serum significantly decreases neutrophil phagocytosis. 129High serum ferritin levels have been linked to impaired neutrophil phagocytosis and chemotaxis. 130ome authors have linked ferritin to reduced ROS formation. 131igher ferritin levels in serum are associated with higher risk of bacterial infections and lower ferritin levels are associated with disease progression in patients with cirrhosis. 132Intracellular ROS production in response to fMLF, opsonised zymosan, or PMA is increased and correlates positively with plasma transferrin saturation but not with ferritin level in patients with hereditary hemochromatosis (liver disease severity is unclear from the paper). 133Furthermore, this patient cohort also shows increased neutrophil phagocytic capacity and decreased Lselectin/CD62L surface expression compared to healthy controls. 133Mouse models of hereditary haemochromatosis show decreased NET formation and ROS production in response to PMA, but no defect in E. coli phagocytosis or mobilisation of azurophilic granules. 134Healthy donor neutrophils pre-treated with ferrous ions or holo-transferrin decrease NET formation, but holo-transferrin does not affect neutrophil ROS production, degranulation of azurophilic granules, phagocytosis nor bacterial killing. 134A high-iron diet in mice results in decreased NET formation and ROS production by neutrophils in response to PMA. 134

Serum opsonins
Opsonisation of bacteria helps neutrophils recognise pathogens and promotes phagocytosis and killing.The main opsonins are immunoglobulins and components of the complement system. 135Defects in serum opsonisation have been described in patients with cirrhosis due to deficiency of opsonisation factors. 136IgA, IgG and IgM have been shown to be increased in sera from patients with cirrhosis.The opsonic effects of patients' sera on E. coli were found to be decreased compared to controls in this study. 137However, IgG, IgM and IgA levels in serum from patients with and without defects in neutrophil chemotaxis, phagocytosis or intracellular killing capacity, have not been reported to differ in some studies. 30,50Some studies have reported no correlation of higher IgA with the level of serum chemotactic inhibitory activity 32 or defects in neutrophil locomotion, 33 while another study found a correlation of increased levels of IgA and IgG with chemotactic inhibitory activity in patients with cirrhosis, and demonstrated that IgA removal restores normal chemotactic activity. 35Patients with cirrhosis were shown to have normal levels of C3 and C4 in one study, 56 but decreased levels in another study. 137No correlation of defects in neutrophil locomotion with serum levels of C3 and C5 has been shown. 33tibiotics A large number of patients with cirrhosis are prescribed antibiotics; therefore, serum concentrations of antibiotics could also affect neutrophil function in these patients.Apart from their antibacterial effects, some antibiotics are also known to exhibit immunomodulatory properties.For example, ceftaroline induces CD11b and decreases CD62L expression, which tends to increase neutrophil survival in response to S. aureus-derived lipoteichoic acid.Vancomycin, as well as dalbavancin, teicoplanin, sulfametrole/trimethoprim and ceftazidime/avibactam have been shown to decrease CXCL8 release in neutrophils. 138,139Dalbavancin and teicoplanin inhibit neutrophil ROS production.Dalbavancin also inhibits neutrophil bactericidal activity.Ceftazidime/avibactam inhibit neutrophil burst in response to fMLF/cytochalasin B. 139 Azithromycin and chloramphenicol decrease NET formation in vitro. 140Further effects of different antibiotic types on neutrophil functions are reviewed in 141-143.
Other factors EMR2 (EGF-like molecule containing mucin-like hormone receptor 2) expression is increased in patients with cirrhosis, dependent on severity and the presence of bacterial infections, and is a predictor of mortality.However, ligation of EMR2 has failed to improve the phagocytic capacity of cirrhotic neutrophils despite increasing intracellular ROS production in response to E. coli. 59

Potential players in neutrophil function regulation in cirrhosis
The contribution of serum factors discussed above to cirrhosisassociated neutrophil dysfunction is rather controversial given the contrasting findings regarding their effects on neutrophils as well as their association with bacterial infections and mortality in cirrhosis.Further investigations of causes for neutrophil deficiency in cirrhosis are necessary.Particular attention should be given to serum components larger than 30 kDa. 54 Bile acids are among these components.In systemic circulation, they are bound mainly to albumin and lipoproteins, and are therefore found in the serum fraction larger than 30 kD. [144][145][146] Furthermore, serum bile acids are highly elevated in liver diseases, including cirrhosis, [147][148][149][150][151][152][153] which makes them a potential player in the regulation of neutrophil function in cirrhosis.
Bile acids are broadly known for their functions in the gastrointestinal tract, e.g.cholesterol elimination and lipid emulsification.However, the bile acid receptors farnesoid X receptor [154][155][156] and Takeda G protein-coupled receptor 5 157,158 have been shown to play a role in many metabolic processes and the immune response. 159,160Approximately 95% of bile acids are reabsorbed in the intestine by the apical sodium-dependent bile acid transporter or through passive diffusion, and around 0.5 mg of bile acids enter the systemic circulation each day. 161everal studies describe neutrophil function in rat or mouse cholestatic models, associating cholestasis with either increased 162 or decreased 163 ROS production, decreased bacterial killing, 163 neutrophil adhesion 164 and increased migration, 162 unchanged 163 or increased 162 phagocytosis, unchanged degranulation, 163 and increased Mac-1 expression and L-selectin shedding. 165Bile, unconjugated lithocholic acid, CDCA, deoxycholic acid and cholic acid (at very high concentrations) potentiate ROS release in primed rat neutrophils.Only unconjugated lithocholic acid also caused superoxide production in rat neutrophils that had not been pre-activated. 166The priming effect of lithocholic acid on rat neutrophils activated with PMA, fMLF or calcium ionophore has been shown, as has its inhibitory activity against beta-glucuronidase release from fMLF-activated neutrophils. 167][170] There are only a few studies describing the effects of bile acids on human neutrophils.CDCA and ursodeoxycholic acid (UDCA) serum levels have been associated with neutrophil phagocytosis and ROS production in patients with cirrhosis. 62Sera from patients with obstructive jaundice have been shown to induce ROS production in healthy donor neutrophils. 171Individual bile acids have been studied only in regard to chemotaxis, intracellular calcium mobilisation, phagocytosis and ROS production in human neutrophils.Unconjugated CDCA and UDCA have been shown to reversibly inhibit chemotaxis of human neutrophils in response to fMLF, but these effects have not been shown for lithocholic acid and cholic acid. 172In other studies, unconjugated CDCA, UDCA 47 and deoxycholic acid (DCA) 173 inhibit chemotaxis towards fMLF in human neutrophils, but DCA does not inhibit chemotaxis towards C5a or IL-8. 173Unconjugated and conjugated forms of CDCA, UDCA (at high concentrations) 47 and unconjugated DCA (reversibly) 173 have been shown to inhibit calcium flux in healthy donor neutrophils in response to fMLF, but not in response to C5a or IL-8.Bile acids differentially affect ROS production with total lithocholic acid triggering neutrophil ROS production in the absence of other stimuli.Total CDCA and lithocholic acid inhibit ROS production in response to fMLF while total CDCA and DCA inhibit ROS production and phagocytosis in response to E. coli 62 (Fig. 3).Hence, the effects of bile acids on human neutrophils are not yet well described.This underscores the necessity to further investigate the effects of bile acids on neutrophils as potential contributors to cirrhosis-associated immune dysfunction.
As mentioned, bile acids are transported in the systemic circulation with the help of proteins and lipoproteins, mainly albumin. 144,145Albumin plays a role in neutrophil dysfunction, including defects in phagocytosis and basal ROS production, 90 and is associated with bacterial infections in cirrhosis. 174Albumin has several identified bile acid-specific binding sites. 175,176he previously described albumin dysfunction in cirrhosis 177 might change its affinity for bile acids.Furthermore, in conditions of albumin deficiency, bile acids change their transporter preferences to lipoproteins, which in turn might facilitate their interaction with cells and tissues, including neutrophils, 146 and potentially cause their higher intracellular accumulation.Therefore, serum concentrations of bile acids, which are measured in cirrhosis, might underestimate the concentrations to which neutrophils are actually exposed, making bile acids that are present at relatively low serum concentrations, such as lithocholic acid and UDCA, more pathophysiologically relevant.As some lipoproteins are deficient in sera of patients with cirrhosis, 110 the pathophysiological importance of other potential bile acid protein transporters should be considered.Therefore, serum factors responsible for cirrhosis-associated immune dysfunction should be further studied together rather than separately, as they might be interdependent.
Apart from albumin, other serum proteins could contribute directly or indirectly to neutrophil dysfunction, e.g.via transporting bile acids.For instance, haptoglobin is one of the most downregulated proteins in sera of patients with cirrhosis compared to healthy controls and is associated with neutrophil function. 54Another study shows the whole range of serum proteins which are significantly associated with neutrophil function changes in patients with cirrhosis. 57urthermore, increased ANCA (anti-neutrophil cytoplasmic antibodies) levels have been observed in cirrhosis and were associated with disease severity and risk of infections. 178Moreover, ANCA induce NET release, 179,180 suggesting their possible contribution to the development of defects in neutrophil function in cirrhosis.

Proposed and potential therapeutic strategies
The search for ways to prevent and treat the development of cirrhosis-associated immune dysfunction, including neutrophil dysfunction, is ongoing.The reversibility of neutrophil dysfunc-tion reported in several studies 49,51 suggests that it could be possible to develop therapeutic strategies aimed at restoration of neutrophil function.

Therapeutic strategies targeting neutrophil dysfunction in cirrhosis supported by clinical studies
Previous studies have shown the beneficial effects of albumin administration on overall survival and bacterial infection rates in patients with cirrhosis; 181,174 however, its effects on neutrophil function have not been investigated within clinical studies.In vitro experiments have hinted at the possibility of restoring neutrophil function with albumin treatment. 90There is also indirect clinical evidence of possible albumin-related effects on neutrophil functionality, as some studies reported decreased plasma cytokine levels, 174,182,183 which have been shown to correlate with changes in neutrophil ROS production, 53 following albumin treatment.Albumin appears to be a promising immunomodulating strategy in cirrhosis, though more clinical evidence is required. 184x vivo studies assessing the removal of endotoxin from the sera of patients with cirrhosis support the hypothesis that this can be a potential therapeutic approach to combat neutrophil dysfunction in cirrhosis. 51We have identified only one study to date that explored the effects of endotoxin removal from the plasma of patients with cirrhosis; however, they have not yet reported whether they observed any effects on neutrophil function. 185urther strategies suggested in cirrhosis include treatment with G-CSF or GM-CSF.It has been shown in ex vivo studies, for instance, that treatment with either G-CSF or GM-CSF can restore neutrophil's ability to inhibit C. albicans growth 49 3. Current knowledge on how bile acids affect human neutrophil function.CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; fMLF, N-Formyl-metleu-phe; GCDCA, glycochenodeoxycholic acid; GLCA, glycolithocholic acid; GUDCA, glycoursodeoxycholic acid; LCA, lithocholica acid; RA, relative abundance in serum; ROS, reactive oxygen species; TCDCA, taurochenodeoxycholic acid; TLCA, taurolithocholica acid; total DCA, sum of deoxycholic, taurodeoxycholic and glycodeoxycholic acids; total LCA, sum of LCA, TLCA and GLCA; total CDCA, sum of CDCA, TCDCA and GCDCA; TUDCA, tauroursodeoxycholic acid; UDCA, ursodeoxycholic acid.8][189] Interestingly, beta-blockers have recently shown potential to improve neutrophil phagocytic capacity in cirrhosis. 190iven the existing interactions between gut microbial composition and neutrophil function, 191 probiotic interventions have been examined for their indirect modulating effects on neutrophil function.However, the results of clinical trials are conflicting, indicating either effects of probiotic supplementation only on the phagocytic capacity of neutrophils, 52 or on neutrophil ROS production, 55 or no effect. 192The origin of conflicting findings might be different sample sizes and different probiotics used for supplementation.A detailed overview of clinical studies is presented in Table 2.

Potential therapeutic strategies of neutrophil dysfunction in cirrhosis
Other potential therapeutic strategies include modulating bile acid composition via oral intake of bile acids, e.g.UDCA or cholic acid, or modulating gut microbial composition (as bile acids are metabolites of gut microbiota and the gut microbiome shapes serum bile acid composition 193 ) via probiotic supplementation.
A range of bile acids including UDCA and obeticholic acid have already been approved for use in clinical settings, representing a potential treatment option for neutrophil dysfunction in cirrhosis given the altered serum bile acid composition in patients with cirrhosis and in vitro evidence of bile acid effects on neutrophil function. 62However, to date, there have been no clinical studies on the effect of bile acid supplementation on neutrophil dysfunction either in cirrhosis or in any other diseases or healthy volunteers.
Furthermore, the normalisation of circulating bile acid composition can be achieved indirectly by modulating the composition of the gut microbiome.Different bacteria have been shown to be associated with bile acid metabolism.For example, Akkermansia abundance is affected by bile acids, 194 genus Prevotella is associated with plasma bile acid composition 195 and genus Streptococcus is involved in primary bile acid metabolism. 196,197UDCA is a result of 7a/b-isomerisation of CDCA by gut microbiota, 193 e.g. by Clostridium absonum, 198 Clostridium baratii 199 and strains from genera Eubacterium and Ruminococcus. 200Therefore, a desired decrease in toxic CDCA, which is highly elevated in sera of patients with cirrhosis, can potentially be reached via the modulation of microbial composition, e.g. with probiotic supplements, which aims at restoration of the balance in abundance of bacteria with 7a-and 7b-hydroxysteroid dehydrogenase activity and, thus, for epimerisation of CDCA to UDCA. 201Besides, this might be achieved by modulating the abundance of bacteria with bile salt hydrolase activity, which is responsible for bile acid deconjugation and was previously suggested to play a role in epimerisation. 199These bacteria include Bacteroides ovatus, 202 Clostridium perfringens, 203 Enterococcus faecalis 204 and different strains of Bifidobacterium 205 and Lactobacillus. 206,207Administering a combination of bacterial strains with bile salt hydrolase activity and 7a-/7b-hydroxysteroid dehydrogenase activity in order to increase UDCA production has already been proposed. 208nother potential therapeutic strategy to treat neutrophil dysfunction in cirrhosis may be the inhibition of PD1 (programmed cell death 1) and TIM3 (T-cell immunoglobulin and mucin domain-containing protein 3) receptors, which mediate immunosuppression.Ex vivo studies have shown that the presence of antibodies blocking PD1 and TIM3 increases neutrophil antimicrobial activities, such as phagocytic capacity and ROS production, in response to E. coli. 209Furthermore, strategies involving liver assist devices can potentially improve neutrophil function in cirrhosis by removing endotoxin, oxidised albumin 210 and cytokines 211 from the circulation.

Conclusion
Neutrophil dysfunction is a recognised feature of cirrhosis that predisposes patients with cirrhosis to bacterial infections and increases their mortality rate.Despite extensive studies in recent decades, significant knowledge gaps and problems remain to be solved, which are summarised in Table 3.There is a need for

Fig. 1 .
Fig. 1.Summary of neutrophil dysfunction features in cirrhosis.Findings consistent throughout the literature are shown.Created with Biorender.com.

Fig. 2 .
Fig. 2. Overview on proposed serum factors that might directly or indirectly affect neutrophil function in cirrhosis.Created with Biorender.com.

Table 1 .
Overview of serum factors thought to contribute to neutrophil dysfunction in cirrhosis.

Table 2 .
Clinical studies that evaluated different therapeutic strategies in cirrhosis with potential effects on neutrophil function.

Table 3 .
Current gaps and future research areas.standardisation of the approaches used to assess different neutrophil functions.There is also a need to develop neutrophil function screening panels that are useful for clinical studies and clinical practice as most of the currently available methodologies to study neutrophil function are extremely time-and labourconsuming and performer-sensitive due to the short lifespan and fragility of neutrophils ex vivo.In particular, more knowledge on recently described neutrophil behaviour, like NET formation or swarming, as well as on the molecular mechanisms involved in the development of neutrophil dysfunction, are needed.Furthermore, there is still a vague understanding of the best strategy to prevent and treat neutrophil dysfunction in cirrhosis, mostly because of the lack of clinical studies with neutrophil function as a primary or secondary outcome.So far, albumin supplementation and G-CSF have the most clinical evidence; however, although clinical benefits have been noted, a clear causal relationship between these therapies and improvement of neutrophil function needs to be established.Last but not least, it will be important to study neutrophil dysfunction in patients with cirrhosis as a multifactorial problem.Studies that investigate the potential causative factors of neutrophil dysfunction not individually but interdependently, e.g. the interdependent role of serum proteins, lipoproteins and bile acids, are needed.This will improve our understanding of the mechanisms behind defects in neutrophil function and guide efforts to improve the prevention and treatment of neutrophil dysfunction, decrease the development and consequences of bacterial infections and, ultimately, improve quality of life and survival in cirrhosis. Review