key: cord-0892458-zr9ntz2g authors: Riva, Antonio; Gray, Elizabeth H.; Azarian, Sarah; Zamalloa, Ane; McPhail, Mark JW.; Vincent, Royce P.; Williams, Roger; Chokshi, Shilpa; Patel, Vishal C.; Edwards, Lindsey A. title: Faecal cytokine profiling as a marker of intestinal inflammation in acutely decompensated cirrhosis date: 2020-07-30 journal: JHEP Rep DOI: 10.1016/j.jhepr.2020.100151 sha: 183ccca2cd397a84c6d5bcbae48587071331417b doc_id: 892458 cord_uid: zr9ntz2g BACKGROUND & AIMS: Gut dysbiosis and inflammation perpetuates loss of gut-barrier integrity (GBI) and pathological bacterial translocation (BT) in cirrhosis, contributing to infection risk. Little is known about gut inflammation in cirrhosis and how this differs in acute decompensation (AD). We developed a novel approach to characterise intestinal immunopathology by quantifying faecal cytokines (FC) and GBI markers. METHODS: Faeces and plasma were obtained from patients with stable cirrhosis (SC; n=16), AD (n=47), and healthy controls (HC; n=31). A panel of 15 cytokines and GBI markers including intestinal fatty acid-binding protein-2 (FABP2), D-lactate and faecal calprotectin (FCAL) were quantified by electrochemiluminescence/ELISA. Correlations between analytes and clinical metadata with univariate and multivariate analyses were performed. RESULTS: Faecal (F) IL-1β, IFNγ, TNFα, IL-21, IL-17A/F and IL-22 were significantly elevated in AD vs SC (q<0.01). F-IL-23 was significantly elevated in AD vs HC (p=0.0007). FABP2/D-lactate were significantly increased in faeces in AD vs SC and AD vs HC (p<0.0001) and in plasma (p=0.0004; p=0.011). F-FABP2 correlated most strongly with disease severity (Spearman’s rho: Child-Pugh 0.466, p<0.0001; MELD 0.488, p<0.0001). FCAL correlated with plasma IL-21, IL-1β and IL-17F only and none of the faecal analytes. F-cytokines and F-GBI markers were more accurate than plasma in discriminating AD from SC. CONCLUSIONS: FC-profiling represents an innovative approach to investigating the localised intestinal cytokine microenvironment in cirrhosis. These data reveal that AD is associated with a highly inflamed and permeable gut-barrier. FC profiles are very different from the classical innate-like features of systemic inflammation. There is non-specific upregulation of T(H)1/T(H)17 effector cytokines and those known to mediate intestinal barrier damage. This prevents mucosal healing in AD and further propagates BT and systemic inflammation. Background & Aims: Gut dysbiosis and inflammation perpetuates loss of gut-barrier integrity (GBI) and pathological bacterial translocation (BT) in cirrhosis, contributing to infection risk. Little is known about gut inflammation in cirrhosis and how this differs in acute decompensation (AD). We developed a novel approach to characterise intestinal immunopathology by quantifying faecal cytokines (FC) and GBI markers. Please refer to separate Powerpoint file for graphical abstract. • The intestinal cytokine microenvironment in cirrhosis primes mucosal immune responses determining inflammatory outcomes. • Exacerbated gut mucosal immune responses in acutely decompensated (AD) cirrhosis perpetuate intestinal barrier damage and prevent mucosal healing. • Intestinal cytokine profiles are different in pattern and absolute quantity from the classical features of systemic inflammation in AD vs stable cirrhosis. There is an upregulation of T-cell meditated type-1 and type-17 effector faecal cytokines in AD but no increase in either faecal IL-8 or faecal IL-10 which are both innate-like cytokines that are increased in the circulation. • Faecal cytokines and markers of gut barrier inflammation (FABP2) and integrity (D-lactate) accurately differentiate between stable cirrhosis and AD. The gut barrier is crucial in liver cirrhosis in preventing infection-causing bacteria that normally live in the gut from accessing the liver and other organs via the bloodstream. Inflammation affecting the lining of the gut is thought to play an important role in this process and leads to complications seen in cirrhosis due to disturbances of this 'gut-liver axis'. Gut inflammation is difficult to study in cirrhosis patients without the use of invasive procedures. Most of the current knowledge is therefore based on changes measured in the bloodstream although these are not representative of the local problems happening in the gut itself. This work characterised these processes by measuring different markers in stool samples from patients at different stages of cirrhosis and compared this to healthy people. These markers, when compared to equivalent markers usually measured in blood, were found to be very different in pattern and absolute levels, suggesting that there is significant gut inflammation in cirrhosis related to different immune system pathways to that seen outside of the gut. This provides new insights into gut-specific immune disturbances that predispose to complications of cirrhosis and emphasises that a better understanding of the gut-liver axis is necessary to develop better targeted therapies. Please refer to separate MS Word file for highlights. Short Summary Box Gut dysbiosis and microbial overgrowth contribute to increased intestinal inflammation and loss of barrier integrity in cirrhosis. Gut barrier dysfunction enables pathological bacterial translocation, which contributes to cirrhosis associated immune dysfunction, heightened susceptibility to infection and predisposes to hepatic decompensation. Little is known about gut-specific inflammation in cirrhosis, and how this differs in patients with and without acutely decompensated (AD) cirrhosis. Intestinal cytokine profiles are different in pattern and absolute quantity from the classical features of systemic inflammation in AD, with no upregulation of either faecal IL-8 or faecal IL-10, involved in pro-or anti-inflammatory pathways, respectively, in comparison to stable cirrhosis (SC). Multiple faecal cytokines are elevated in AD in keeping with a generalised and non-specific upregulation of type-1 and type-17 effector cytokines, and those known to be drivers of innate immune-mediated inflammation in the gut. Faecal D-lactate and FABP2 -markers of gut barrier damage and intestinal inflammationaccurately differentiate between AD and SC patients, with greater sensitivity and specificity than equivalent plasma measurements. These data highlight the importance of the role of intestinal inflammation and barrier dysfunction which affect the gut-liver axis in cirrhosis. Gut-specific targets may better enable non-antibiotic based therapies to be developed that prevent progression to AD, particularly in an era of ever increasing antimicrobial multidrug resistance. This technique can be applied to other disease paradigms, where gut inflammation and epithelial barrier integrity are thought to play an important role. In health, the gut barrier is crucial in the defence against the extensive and continuous exposure of the liver to the intestinal microbiota, their immunogenic products (pathogen-associated molecular patterns, PAMPs) and microbial metabolites. However, in cirrhosis, there is increasing evidence that this barrier is dysfunctional, more permeable and highly inflamed [1] . The gut barrier consists of several layers which determine the extent to which microbes and their PAMPs can access the host circulation. The first line of defence is the mucus layer [2] which physically separates the microbiota from the next layer consisting of intestinal epithelial cells (IECs) that are bound by tight junctions [3] . Below this is the lamina propria which in addition to consisting of non-cellular connective tissue elements is an immune-dense layer where several types of innate and adaptive immune cells are concentrated and where aggregations of lymphoid nodules give rise to the specialised areas known as Peyer's patches [4] . The gut vascular barrier (GVB) represents the final layer controlling the entry of microbes and PAMPs into the portal circulation and therefore the liver [5] . IEC and GVB disruption has been shown to be crucial in the development of non-alcoholic fatty steatohepatitis [6] . This dysfunctional 'gut-liver axis' is driven by intestinal microbial dysbiosis, translocation of pathogenic gut microbes and their PAMPS, a process termed bacterial translocation (BT), which initiates both intestinal mucosal dysfunction and systemic immuneparesis [7] [8] [9] . The relative contribution of each component is, however, not well understood [10, 11] . Increased BT has been shown to be a key process that contributes to acute hepatic decompensation (AD) and acute-on-chronic liver failure (ACLF), the latter being associated with extra-hepatic organ failure(s) and very high short-term mortality [12, 13] . How gut barrier dysfunction and inflammation affects the clinico-pathological transition from stable cirrhosis to AD and/or ACLF is unknown. Pathogenic gut bacterial and fungal species -such as those observed in cirrhosis -are able to intimately adhere to the intestinal mucosa, induce barrier disruption and alter host mucosal immune responses [14] [15] [16] [17] . In particular, IFNγ, IL-22, IL-17A and IL-17F play a critical role in establishing host anti-microbial immunity [18] . How intestinal inflammation promotes systemic inflammation in relation to gut-derived BT in cirrhosis remains unclear and requires elucidation. Limited data exists on faecal calprotectin (FCAL) levels in cirrhosis as a surrogate marker of gut inflammation. Calprotectin is a calcium-and zinc-binding protein of granulocytes, accounting for over 60% the soluble cytosolic proteins found in human neutrophil granulocytes [19] . FCAL correlates positively with the degree of intestinal neutrophil migration [20] and as such has become established as a diagnostic and prognostic marker for the assessment of localised intestinal inflammation [21] in inflammatory bowel disease (IBD) [22, 23] as it is resistant to degradation during intestinal transit [24] . Very limited data are available in cirrhosis where FCAL is reported to be elevated in cirrhotic patients (some with hepatic encephalopathy) when comparing to healthy controls [25] . The intestinal immune system is regionally specialised due to conditioning by the gut microenvironment and inhabiting microbiome. Adaptations are reflected in its complex gut-associated lymphoid tissues and isolated immune cells, including an almost exclusive population of antigenexperienced T-cells scattered throughout the intestinal epithelial compartment. A further layer of specialisation is local priming of these effector lymphocytes due to the complex cytokine milieu generated as a result of pattern recognition receptor (PRR) activation. IECs or antigen-presenting cells of the gut lamina propria help prime T-cell differentiation into protective T-cell subsets, which together with the innate immune system, form the first line of defence against invading pathogens [26] [27] [28] and play a crucial role in maintaining gut barrier integrity [29] . The healthy gut microbiome contributes to the constitutive development of T H 17 cells in the intestinal lamina propria [30, 31] . T H 17 cells induce the recruitment of neutrophils and activation of IECs, enhancing the clearance of extracellular pathogens in concert with other immune cells such as IgA-secreting plasma cells and group-3 innate lymphoid cells (ILC3s) [15] . Peripheral circulating cytokine profiling has been shown to be related to prognosis at different stages of cirrhosis [32] , differentiates patients with and without AD and ACLF, and clinical outcomes including short-term mortality [33] . However, relatively little is known about localised gut inflammation and the key immunological events that mediate barrier disruption in cirrhosis using similar cytokine markers, and how this relates to BT and clinically relevant outcomes. This lack of knowledge stems primarily from difficulty in obtaining intestinal tissue in patients with cirrhosis and a paucity of non-invasive techniques. The aim of the current investigation was to develop a method of characterising and differentiating gut mucosal inflammation and injury in stable cirrhosis and AD patients by utilising faeces as a biological matrix. A panel of cytokines and markers of gut-barrier-integrity and inflammation were measured and compared in both faeces and plasma, which in combination are herein referred to as analytes. Patients were consecutively recruited at King's College Hospital after admission to the ward or when reviewed in the hepatology out-patient clinic. The study was granted ethics approval by the national Faecal lysates (FL) were produced from frozen faecal samples by combined chemical and mechanical homogenisation using an optimised extraction method. Sample collection and preparation, including FL generation, are described in Supplementary Methods. The following cytokines were quantified in paired faecal and plasma samples, to enable a comparison between the gut and the systemic compartments: ii. Innate/adaptive cytokines belonging to the type-1/type-17 anti-microbial axis: IL-12p70, IL-23, IFNγ, IL-17A, IL-17F [39, 40, 42, 43, 45] . iii. Cytokines conventionally associated with systemic inflammatory responses to infection: IL-1β, IL-6, IL-8, TNFα [39, 40, 46, 47] . Cytokines were measured in plasma or neat FL using an electro-chemiluminescence platform or by ELISA as per manufacturer instructions, as is described in detail in Supplementary Methods. Intestinal fatty acid-binding protein (FABP2) [48] and the microbial metabolite D-lactate [49] [50] [51] were quantified to serve as gut-specific markers of intestinal barrier integrity and BT, to assess whether these differentiated AD from stable cirrhosis, and in the healthy control cohort to define whether 'physiological' or basal levels were detectable. FABP2 was quantified using the human FABP2/I-FABP Quantikine ELISA Kit (R&D Systems). Plasma D-lactate was measured using a colourimetric assay Analyte values were obtained using 4-/5-parameter logistic regression standard curves as appropriate. AD, stable cirrhosis and healthy control groups were compared using (i) the When comparing across all three groups (AD vs. stable cirrhosis vs. healthy controls) by post-hoc comparisons, faecal FABP2 ( Figure 1A ) was significantly different across groups (q=0.000025), with higher levels in AD compared to stable cirrhosis (Dunn's p=0.000092) and healthy controls (Dunn's p=0.000088). Faecal D-lactate ( Figure 1A) Table 1 ). FABP2 was consistently higher while D-lactate was consistently lower in plasma in all three groups when compared to faecal levels ( Figure 1A ). Mucosal-associated IL-17E and IL-21 were higher in faeces in all three groups, whilst IL-22 was comparable between the two compartments ( Figure 2A ). Cytokines belonging to the type-1/type-17 antimicrobial axis (IL-12p70, IL-23) had elevated plasma levels in healthy controls and AD ( Figure 1B) . Conversely, levels of effector cytokines IL-17A, IL-17F (and IFNγ to a lesser extent) were elevated in faeces compared to plasma in all three groups ( Figure 2B ). Amongst typical pro-and anti-inflammatory cytokines, IL-1β, TNFα and IL-10 were overall higher in faeces compared to plasma in all groups, but IL-6 and IL-8 levels were comparable between the two compartments ( Figure 1C) ; only AD had moderately more plasma than faecal IL-6 in comparison to healthy controls. Having identified significant correlations within faecal or plasma analytes, we sought to investigate latent discrimination potential by multivariate methods. Supplementary Figures 1 and 3 illustrate results for faecal and plasma analytes, respectively. When using faecal analytes, clinical distinction between stable cirrhosis and AD patients mapped with PCA clusters (PC 1+2 =72.1% variance explained) (supplementary Figure 1A) , and OPLS-DA analysis identified faecal FABP2 and D-lactate as the main contributors to this discrimination (supplementary Figure 1B) . The OPLS-DA model was significant by OPLS-DA diagnostics (supplementary Figure 1B) . When examining plasma analytes, the separation between AD and stable cirrhosis was still present (PCA variance explained, PC 1+2 =69.3%) (supplementary Figure 3A ), but in contrast to faecal analytes, the plasma-based OPLS-DA identified plasma cytokines as the main discriminating factors, while plasma FABP2 and D-lactate were found to have no discriminatory ability (supplementary Figure 3B ). This plasma-based model was also significant by OPLS-DA diagnostics (supplementary Figure 3B ). This highlights a critical difference between the two anatomical compartments and provides further support for the independent faecal and plasma co-regulation clusters previously discussed. The separation between stable cirrhosis and AD patients was next investigated by ROC curve analysis (supplementary Figure 2) . Faecal FABP2 and D-lactate were identified as first-and second-best discriminators with areas under the curve (AUROC) of 0.886±0.059 and 0.875±0.059 respectively (q=0.004 for both) and cut-offs with sensitivities ≥84% and specificities ≥78% (supplementary Table 2A ) Table 2B, individual DS equations in supplementary Table 3 ). The DS model based on faecal FABP2 plus faecal Dlactate improved the AUROC to 0.940±0.035 (q=0.00084) with higher sensitivity (89%) and specificity (100%) compared to FABP2 alone. Sequential addition of other faecal cytokines (IL-21, IL-1β, IL-6) diluted this effect, leading to less powerful models. Thus, faecal FABP2 and faecal D-lactate as markers of gut barrier integrity and intestinal inflammation appear to be highly effective discriminators of stable cirrhosis and AD patients. We also performed the same analysis with plasma analytes (supplementary Figure 4) , but in contrast to the faecal findings, plasma-based AUROC analysis identified plasma FABP2 and plasma D-lactate as the only two analytes lacking stable cirrhosis/AD discrimination. Plasma IL-21 was the strongest discriminator (AUROC 0.860±0.057, q=0.0038, sensitivity/specificity=77/82%), followed by all the other 12 plasma cytokines (TNFα, IL-23, IL-17F, IL-1β, IL-8, IL-12p70, IL-22, IFNγ, IL-17A, IL-17E, IL-10 and IL-6) (supplementary Table 4A ). Discriminant scores built sequentially combining all 13 significant plasma cytokines showed that all the top 5 plasma parameters (IL-21, TNFα, IL-23, IL-17F, IL-1β) were necessary to achieve the best AUROC (0.922±0.037, q=0.000016, sensitivity/specificity=82/92%) compared to all other models (supplementary Figure 4 , supplementary Table 4B , individual DS equations in supplementary Table 5 ). Notably, the DS model using only faecal FABP2 and D-lactate achieved better discrimination than the DS model using the top-5 plasma parameters. In this study, we describe for the first-time profiling of faecal cytokines and faecal markers of gut barrier integrity in cirrhosis. We demonstrate that intestinal inflammation involving the gut-liver axis is strongly associated with acute decompensation and more so than with equivalent plasma markers. In fact, we found that faecal cytokines and gut-barrier integrity markers discriminated between stable cirrhosis and AD patients with superior sensitivity (95%) and extremely high specificity (100%) when compared to the same circulating plasma analytes. to Noroviruses [52] and TNFα in Crohn's disease [53] but not in the context of cirrhosis. Other conventional markers of gut inflammation such as FCAL [20] have also been shown to be nonspecifically elevated in decompensated cirrhosis [25, 54, 55] . A relative limitation of FCAL, however, is that it is representative of mainly neutrophil activity and not of the other critical innate (e.g. innate lymphoid cells) and adaptive (e.g. T-regulatory and T-helper) immune cell subsets that are increasingly recognised as involved in gut mucosal homeostasis and dysregulation. Increased levels of mucosal cytokines in the IL-23-T H 17 axis were detected in the faeces of AD patients. Mucosal IL-12p70-T H 1 axis was also upregulated in AD, but to a lesser extent, with IL-12p70 and IL-23 representing two intimately related master regulators of T-cell mediated type-1/type-17 effector balance [45] . Our results suggest a generalised and non-specific upregulation of type-1 and type-17 effector cytokines, which may be more detrimental in propagating nonspecific gut mucosal inflammation [42, 45] . In this context, faecal IL-1β, IL-6 and TNFα were also elevated in AD compared to stable cirrhosis and healthy controls; these cytokines are considered promiscuous innate drivers of inflammation throughout the T H 1-T H 17 spectrum, with increased tissue concentrations observed in chronic inflammatory pathologies affecting the gut [42, 45] . the epithelial layer is severely disrupted and bacterial invasion had occurred [15] , providing context to our findings for evidence of similar dual T H 1/T H 17 pathway induction in cirrhotic patients and detrimental effect on the gut barrier. Even under physiological conditions, the gut has a basal level of inflammation as evidenced by our healthy control data, requiring finely tuned interactions between the different cytokines and their receptors to support intestinal mucosal homeostasis. In addition, many of the cytokines measured have dichotomous pro-and anti-inflammatory roles in mucosal immunity [56] . For example, IL-1β, IL-6, TNFα and IL-17A are cytokines with well-known pro-inflammatory roles but are also involved in promoting epithelial proliferation, crucial for both wound closure and replacing cells lost through homeostatic and likely pathological shedding [57] [58] [59] . IL-1β levels correlate with the severity of intestinal inflammation in Crohn's disease due to increases in IEC tight junction permeability [60] . IL-22 is involved in repair and protection of barrier surfaces, especially in conjunction with IL-17A/F, IL-36γ and IL-23, which during intestinal injury, collectively drive antimicrobial peptide secretion, recruitment and activation of immune cells and barrier protection [42, 43, 61] . However, IL-22 can also increase IECs tight junction permeability and enhance the pro-inflammatory capacity of TNFα depending on the microenvironment, whilst IL-17A can be destructive by promoting neutrophilic inflammation [62, 63] . IL-21 has also been found to beneficially control inflammatory pathways in the intestine, yet is upregulated in IBD, stimulating the secretion of extracellular matrix-degrading enzymes by fibroblasts and enhancing T-cell recruitment by IECs [43, 44] . Prolonged combined IFNγ and TNFα expression have been shown to contribute to an impairment of barrier function of IECs [43, 64] , with the latter inducing IEC damage by excessive neutrophil adhesion and degranulation [43] . Similarly, IL-1β and TNFα activate immune responses suppressing intestinal pathogens, but excessive levels exacerbate inflammation [56, 65] . Collectively, in AD we observe a complex cytokine microenvironment that can drive intestinal inflammation and perpetuate intestinal barrier disruption that is evident in these patients given the panel of gut-barrier integrity markers measured in tandem. This combination if left unchecked can lead to pathological BT, hepatic inflammation and fibrotic transformation, and may even predispose to the development of hepatocellular carcinoma [66, 67] . The findings in this study strongly support the role of these faecal cytokines in driving disease progression in cirrhosis which, given their pattern, appear to be derived mainly from T-cell adaptive immune-mediated processes. The plasma cytokine profiles in AD, conversely, appear to be driven more by innate immune responses which are well characterised [68] . These data highlight the need for more focused studies to elucidate the precise origins, effects and roles of these cytokines at the gut interface. There may be a need to therapeutically rebalance these dichotomously acting cytokines in the gut mucosa and, equally, targeting these cytokines in the systemic circulation may not be the way forward [69] . For example, patients with alcoholic hepatitis and underlying cirrhosis who were treated with systemic TNFα inhibitors experienced a higher rate of adverse events including serious infections and overall mortality [70] . It is noteworthy that faecal IL-8 and IL-10 were not found to be elevated in AD patients compared to stable cirrhosis or healthy controls in contrast to what was observed in matched plasma samples. This may be indicative of a defective AD-specific response in keeping with cirrhosisassociated immune dysfunction and which may be associated with deleterious downstream consequences such as (i) hampering differentiation of anti-inflammatory T-regulatory cells [45] , (ii) limiting IL-8-mediated recruitment of neutrophils to the gut epithelium [71] , and (iii) an inability to recover from gut barrier injury [72] , in combination perpetuating intestinal injury and inflammation and propagating BT. D-lactate and FABP2 were quantified to reflect gut barrier damage and intestinal inflammation when conventionally measured in plasma [49] . Measurement of faecal FABP2 is novel and may represent IEC shedding from the epithelial monolayer into the lumen, causing transient gaps or microerosions in the gut barrier, resulting in increased intestinal permeability [73] and contributing to pathological BT. A six-fold increase in faecal FABP2 levels was found in AD when compared to stable cirrhosis and healthy controls, suggesting that gut barrier injury is involved in the progression from stable to decompensated cirrhosis. FABP2, when elevated in plasma conventionally indicates enterocyte damage [74] [75] [76] , was overall higher than that measured in faeces, but failed to significantly distinguish between AD and stable cirrhosis despite a trend towards higher levels in AD. Faecal and not plasma measurement of FABP2 provided a more sensitive assessment of gut mucosal injury in cirrhosis, and the ability to differentiate between AD and stable cirrhosis. D-lactate is found in high levels in pathological states due to increased gut microbial production [51, 77] and elevated levels in plasma are considered to be an indicator of bacterial translocation [50] due to a breach in the gut epithelial barrier. This metabolite has been found elevated in the plasma of patients with gut ischaemia [78] and alcohol-related liver disease [49] . Faecal D-lactate measurement in this study is new and levels were significantly different across all three groups but, unlike FABP2, lower levels were detected in AD when compared to stable cirrhosis and healthy controls. In contrast, plasma D-lactate levels were comparable across all groups with a trend towards higher levels in AD. Lower faecal D-lactate levels in AD is previously unreported and may reflect several mechanisms: (i) increased translocation from the intestinal lumen into the systemic circulation via an impaired gut barrier; (ii) enrichment of D-lactate-metabolising gut microbial species [79] due to gut dysbiosis in AD patients, such that faecal levels of this bacterial metabolic substrate fall; (iii) loss of D-lactate-producing gut bacterial species (such as Lactobacillus spp. [80] ), which has been reported to occur in alcohol-induced liver injury [81] . FCAL was measured as a conventional marker of gut epithelial inflammation [20] and in this context was employed as a clinical measure with which to compare the faecal and plasma analytes. FCAL has been shown to be elevated in decompensated cirrhosis in this study and previously [25, 54] . FCAL broadly and non-specifically quantifies intestinal inflammation and has been shown in other conditions such as Crohn's disease where gut inflammation is the pathologically defining hallmark to positively correlate with plasma cytokines such as IFNγ, IL-6, TNFβ and IL-17A [82] . Recently, patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing COVID-19 disease were reported to have elevated FCAL levels depending on the presence of diarrhoeal symptoms and where FCAL positively correlated with plasma IL-6 levels [83] . Given that FCAL is derived mainly from neutrophils and relates to the presence of neutrophils in the epithelium and intestinal erosions or ulcers [84] , it cannot provide insights into the other increasingly recognised and important inflammatory pathways linked to the various aforementioned innate and in particular adaptive immune cell subtypes, especially of the T H 1/T H 17 axis. That FCAL was significantly higher in AD vs stable cirrhosis and correlated positively with conventional markers of liver disease severity (Child-Pugh and MELD) and with some plasma cytokines but did not with any of the faecal analytes suggests that the pathways driving intestinal inflammation in AD are T-cell mediated and likely more biologically relevant to those related to neutrophil activity. What is striking is that no significant differences were found in any of the analytes by way of cytokines, D-lactate, FABP2 and FCAL when comparing between stable cirrhosis and healthy control groups, regardless of the faecal or blood matrix in which they were measured. This is suggestive of there being relative biological equipoise in compensated cirrhosis in gut barrier integrity and systemic pro-and compensatory anti-inflammatory processes, which once disturbed are associated with progression to hepatic decompensation and cirrhosis-associated immune dysfunction [85] . These findings differ from existing reports, where concentrations of IL-6, IL-10 and IL-17A were higher in the plasma of stable cirrhosis patients than in healthy controls [32] ; notably in the same study, only these three cytokines were measured and no faecal analysis was undertaken. As to the origin of these faecal cytokines, there are several mechanisms that are likely to be contributory and require further investigation: (i) leakage across the epithelium due to gut barrier disruption [53] , (ii) vectorial (apical) secretion, previously reported for IL-1β, IL-6 and IL-8, which mediate autocrine epithelial restitution [86] , (iii) immune cells, such as neutrophils and macrophages, passing into the gut lumen [87] and/or (iv) IEC present in the faeces, which are shed due to mucosal injury in AD as evidenced by elevated faecal FABP2 levels. Equilibrium between the rate of epithelial shedding at the villus tip and generation of new cells in the crypt is key to maintaining intestinal tissue homeostasis. However, in intestinal inflammatory states, pathological IEC shedding causes microerosions in the epithelial barrier, resulting in increased intestinal permeability [73] . Enhanced mucosal expression of IL-23, IL-1β, IL-21, IFNγ, TNFα, IL-17E and IL-17F in conjunction with reduced IL-8 and IL-10 expression, as detected in the faeces of AD patients, would provide a microenvironment propagating gut barrier disruption [39, 42, 43, 72] . The levels and combinations of cytokines detected in the faeces of AD patients may, therefore, contribute to increased pathological IEC shedding and enhanced BT, as evidenced by elevated faecal FABP2 and plasma D-lactate levels, respectively. Confounding factors that may have impacted on the findings reported are the higher proportion of patients in the AD group that were more likely to be actively-drinking with alcohol as the primary cause of cirrhosis and also more likely to be treated with antibiotics. Existing data shows deleterious changes in the gut microbiome, bile acid and other metabolic profiling in actively drinking cirrhotics [88] and that alcohol directly increases gut permeability and mediates barrier disruption [49, 89, 90] , as well as the more widespread deleterious impact of antibiotics on the gut microbiome [91] . Both alcohol ingestion and antibiotic treatment play important roles in both gut epithelial barrier function and systemic inflammation. Whilst the impact of these factors require further investigation, it is important to note for the latter that analyses of the impact of antibiotic therapy on the various analytes measured in the AD patient group did not reveal any significant effect on any of the faecal or plasma markers. In conclusion, profiling of cytokines and gut-barrier integrity markers in faeces as a biological matrix represents an innovative approach to the localised assessment of the intestinal cytokine microenvironment in cirrhosis with simultaneous evaluation of gut mucosal inflammation and barrier dysfunction. Our data demonstrate that AD is associated with a highly inflamed and damaged gut barrier, and that faecal cytokine and gut-barrier integrity marker profiles which appear to be T-cell driven are very different from the classical and innate-like features of systemic inflammation in cirrhosis, as determined by plasma-based assays. This study begins to delineate the complex mechanisms governing intestinal inflammation in cirrhosis, which are increasingly recognised as a major driver in disease progression and hepatic decompensation and which have been elusive and challenging to study to date. This is an important area that warrants immediate attention and further study focusing on the underlying mechanisms at a cellular level. A more complete understanding of how cytokine biology promotes intestinal mucosal homeostasis and damage at different stages of cirrhosis also presents an opportunity for developing treatments. Similarly, pharmacological modulation of faecal cytokine production by gut-targeting therapies [56] needs further exploration. Pearson correlation and 'complete' method as clustering parameters. 3B. Significance plot representing the p-values associated with the correlation levels from panel 3A, using the following thresholds: white: non-significant; pink: p≤0.05 (trend); red: p≤0.00012 (significant). The significance threshold was determined by Bonferroni FWER correction. 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School of Immunology and Microbial Sciences, Faculty of Life Sciences and Medicine King's College Hospital NHS Foundation Trust, Denmark Hill, London, SE5 9RS. UK 4. Department of Clinical Biochemistry, King's College Hospital NHS Foundation Trust Joint first authors § Joint senior authors # Corresponding author: Vishal C Patel Corresponding author address Corresponding author telephone Corresponding author email: vish.patel@kcl.ac.uk Corresponding author ORCiD • The gut microenvironment in cirrhosis primes mucosal and systemic immune responses Gut inflammation is challenging to study in cirrhosis with a paucity of targeted assays Exacerbated gut immune responses and barrier damage occur in acutely decompensated (AD) cirrhosis Gut cytokine profiles in AD have very different patterns to innate-like systemic inflammation Upregulation of faecal T-cell meditated type-1 and type-17 effector cytokines occurs in AD • Faecal cytokines and gut barrier markers accurately differentiate between stable cirrhosis and AD