Faecal cytokine profiling as a marker of intestinal inflammation in acutely decompensated cirrhosis

Abstract

Background & aims: Gut dysbiosis and inflammation perpetuate 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 (FCs) and GBI markers.

Methods: Faeces and plasma were obtained from patients with stable cirrhosis (SC; n = 16), AD (n = 47), and healthy controls (HCs; 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, interferon gamma, tumour necrosis factor alpha, 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; model for end-stage liver disease 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 micro-environment 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_H1/T_H17 effector cytokines and those known to mediate intestinal barrier damage. This prevents mucosal healing in AD and further propagates BT and systemic inflammation.

Lay summary: The gut barrier is crucial in cirrhosis in preventing infection-causing bacteria that normally live in the gut from accessing the liver and other organs via the bloodstream. Herein, we characterised gut inflammation by measuring different markers in stool samples from patients at different stages of cirrhosis and comparing this to healthy people. These markers, when compared with 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.

Keywords: ACLF, acute-on-chronic liver failure; AD, acute decompensation; AUROC, area under the receiver operating characteristic; BT, bacterial translocation; Bacterial translocation; CLIF-C AD, Chronic Liver Failure Consortium-acute decompensation; Chronic liver disease; Cytokines; DS, discriminant score; FABP2, fatty-acid-binding protein-2; FCAL, faecal calprotectin; FDR, false discovery rate; FL, faecal lysate; FWER, family-wise error rate; GVB, gut vascular barrier; Gut inflammation; HC, healthy control; IBD, inflammatory bowel disease; IEC, intestinal epithelial cell; Intestinal barrier function; MELD, model for end-stage liver disease; OPLS-DA, orthogonal projection to latent structures discriminant analysis; PAMP, pathogen-associated molecular pattern; PCA, principal component analysis; ROC, receiver operating characteristic; SC, stable cirrhosis; UKELD, United Kingdom model for end-stage liver disease.

Graphical abstract

Fig. 1

Faecal and plasma cytokine, FABP2, and d-lactate concentrations comparing acutely decompensated and stable cirrhosis to healthy controls. KWp and BHq values: Kruskal-Wallis p values and BH adjusted q values for overall intergroup comparisons. Bracketed high values: Dunn's corrected p values for multiple comparisons, when KWp is significant. Base purple values: BH adjusted q values for paired faeces vs. plasma (Wilcoxon) comparisons for each group. (A) Surrogate markers of intestinal barrier damage, gut inflammation, and bacterial translocation. (B) Type 1/type 17 balance, effector cytokines. (C) Conventional markers of inflammation. AD, acute decompensation; BH, Benjamini-Hochberg; FABP2, fatty-acid-binding protein-2; FC, faecal; FCAL, faecal calprotectin; HC, healthy control; PL, plasma; SC, stable cirrhosis.

Fig. 2

Faecal and plasma cytokine, FABP2, and d-lactate concentrations comparing acutely decompensated and stable cirrhosis to healthy controls. KWp and BHq values: Kruskal-Wallis p values and BH adjusted q values for overall intergroup comparisons. Bracketed high values: Dunn's corrected p values for multiple comparisons, when KWp is significant. Base purple values: BH adjusted q values for paired faeces vs. plasma (Wilcoxon) comparisons for each group. (A) Cytokines relevant to mucosal immune modulation. (B) IL-12/23 balance, master regulation of type 1 vs. type 17 responses. AD, acute decompensation; BH, Benjamini-Hochberg; FABP2, fatty-acid-binding protein-2; FC, faecal; HC, healthy control; IFNγ, interferon gamma; PL, plasma; SC, stable cirrhosis.

Fig. 3

Intercorrelations between all the analytes, faecal and plasma cytokines, FABP2, d-lactate, and faecal calprotectin. (A) Intercorrelation matrix obtained by hierarchical clustering, using Pearson's correlation and ‘complete’ method as clustering parameters. (B) Significance plot representing the p values associated with the correlation levels from (A), 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. FABP2, fatty-acid-binding protein-2; FWER, family-wise error rate; IFNγ, interferon gamma; TNFα, tumour necrosis factor alpha.

Fig. 4

Correlation between faecal/plasma analytes and clinical parameters. (A) Correlation level plot of faecal and plasma cytokines, FABP2, and d-lactate with individual clinical parameters, including faecal calprotectin and disease composite severity and prognostication scores. Pearson's correlation or Spearman's correlation coefficients were calculated as appropriate, and only correlations with p ≤0.05 are represented. (B) Significance plot representing the p values associated with the correlation levels using the following thresholds: white, non-significant; pink, p ≤0.05 (trend); red, p ≤0.00013 (significant). The significance threshold was determined by Bonferroni FWER correction. CLIF-C AD, Chronic Liver Failure Consortium-acute decompensation; FABP2, fatty-acid-binding protein-2; FWER, family-wise error rate; IFNγ, interferon gamma; MELD, model for end-stage liver disease; TNFα, tumour necrosis factor alpha; UKELD, United Kingdom model for end-stage liver disease.