Alterations in the fecal microbiota in patients with advanced cystic fibrosis liver disease after 6 months of elexacaftor/tezacaftor/ivacaftor

Abstract

Background: Cystic fibrosis associated liver disease (CFLD) carries a significant disease burden with no effective preventive therapies. According to the gut-liver axis hypothesis for CFLD pathogenesis, dysbiosis and increased intestinal inflammation and permeability permit pathogenic bacterial translocation into the portal circulation, leading to hepatic inflammation and fibrosis. Evaluating the effect of CFTR (cystic fibrosis transmembrane conductance regulator) modulation with elexacaftor/tezacaftor/ivacaftor (ETI) may help determine the role of CFTR in CFLD and increase understanding of CFLD pathogenesis, which is critical for developing therapies. We aimed to characterize the fecal microbiota in participants with CF with and without advanced CFLD (aCFLD) before and after ETI.

Methods: This is an ancillary analysis of stool samples from participants ages ≥12 y/o enrolled in PROMISE (NCT04038047). Included participants had aCFLD (cirrhosis with or without portal hypertension, or non-cirrhotic portal hypertension) or CF without liver disease (CFnoLD). Fecal microbiota were defined by shotgun metagenomic sequencing at baseline and 1 and 6 months post-ETI.

Results: We analyzed 93 samples from 34 participants (11 aCFLD and 23 CFnoLD). Compared to CFnoLD, aCFLD had significantly higher baseline relative abundances of potential pathogens Streptococcus salivarius and Veillonella parvula. Four of 11 aCFLD participants had an initially abnormal fecal calprotectin that normalized 6 months post-ETI, correlating with a significant decrease in S. salivarius and a trend towards decreasing V. parvula.

Conclusions: These results support an association between dysbiosis and intestinal inflammation in CFLD with improvements in both post-ETI, lending further support to the gut-liver axis in aCFLD.

Keywords: Cystic fibrosis liver disease; Dysbiosis; Elexacaftor/tezacaftor/ivacaftor; Fecal microbiome; Gut-liver axis.

Figure 1.. Fecal microbial composition and diversity of participants with advanced cystic fibrosis associated liver disease (aCFLD) compared to participants with CF without liver disease (CFnoLD) at baseline

(a) Alpha Diversity. Boxplot of Shannon Index with individual data points. The red circle within the box indicates the mean, the line indicates the median, and the boxplot hinges indicate the first and third quartiles. The number of samples were aCFLD (n=11) and CFnoLD (n=23) (p= 0.07, two-sided Wilcoxon rank sum test). (b) Alpha diversity. Boxplot of Species Richness with individual data points. (p= 0.45, two-sided Wilcoxon rank sum test). (c) Beta Diversity. Principal coordinates analysis (PCoA) utilizing Bray-Curtis dissimilarity demonstrating fecal microbiota of participants with aCFLD and CFnoLD at the species level. Larger symbols represent centroids (average microbiota); smaller symbols represent individual sample microbiota. (p=0.66, PERMANOVA) (p=0.25, homogeneity of multivariate dispersions) (d) Stacked bar plot at the class level demonstrating fecal microbiota of participants with aCFLD compared to CFnoLD. Among the 11 participants with aCFLD, 2 had non-cirrhotic portal hypertension (PHTN), 8 had cirrhosis without PHTN, and 1 had both cirrhosis and PHTN.

Figure 2.. Baseline fecal microbial composition is altered in participants with advanced cystic fibrosis associated liver disease (aCFLD) as compared to participants with CF without liver disease (CFnoLD)

Boxplots indicating relative abundances of indicated taxa prior to elexacaftor/tezacaftor/ivacaftor (ETI) in participants with aCFLD (n=11) and CFnoLD (n=23). (a) Streptococcus (b) Veillonella (c) Rothia (d) Streptococcus salivarius (f) Veillonella parvula (f) Ruminococcus torques. The boxplot hinges indicate the first and third quartiles. The red circle within the box indicates the median. Statistical testing performed via two-sided Wilcoxon rank sum test. *p<0.05, **p<0.01

Figure 3.. Fecal microbiota changes in participants with advanced cystic fibrosis associated liver disease (aCFLD) after treatment with elexacaftor/tezacaftor/ivacaftor (ETI)

(a) Alpha Diversity. Boxplot of Shannon Index with individual data points. The red circle within the box indicates the mean, the line indicates the median, and the boxplot hinges indicate the first and third quartiles. The number of samples were baseline (n=11), 1 month post-ETI (n=10), and 6 months-post ETI (n=8) (p=0.20, Kruskal-Wallis). (b) Alpha diversity. Boxplot of Species Richness with individual data points. (p= 0.46, Kruskal-Wallis). (c) Beta Diversity. Principal coordinates analysis (PCoA) utilizing Bray-Curtis dissimilarity demonstrating fecal microbiota of participants with aCFLD before and after treatment with ETI at the species level. Larger symbols represent centroids (average microbiota); smaller symbols represent individual sample microbiota. (p=0.97, PERMANOVA) (p=0.95, homogeneity of multivariate dispersions). (d) Boxplot indicating relative abundances of Staphylococcus aureus, which demonstrated statistically significant changes in participants with aCFLD before and after treatment with ETI. Statistical testing performed via Kruskal-Wallis test with pairwise comparison using Dunn’s post hoc test and Bonferroni correction (p<0.001, Kruskal-Wallis). *p<0.05, **p<0.01

Figure 4.. Participants with advanced cystic fibrosis associated liver disease (aCFLD) and decrease in fecal calprotectin exhibit alternations in the fecal microbiota after 6 months of elexacaftor/tezacaftor/ivacaftor (ETI)

(a) Line plot of fecal calprotectin among participants with measurable decrease in fecal calprotectin (n=8). Dotted red lines denote levels of fecal calprotectin with values between 0 – 50 μg/g indicating normal, 50 – 120 μg/g borderline, and >120 μg/g abnormal. (b-c) Boxplots indicating relative abundances of Veillonella parvula and Staphylococcus aureus, which demonstrated statistically significant decreases in participants with aCFLD and calprotectin decrease before and after treatment with ETI. Statistical testing performed via Kruskal-Wallis test with pairwise comparison using Dunn’s post hoc test and Bonferroni correction. Veillonella parvula (p<0.05, Kruskal-Wallis). Staphylococcus aureus (p<0.05, Kruskal-Wallis). (d) Line plot of fecal calprotectin among participants with initially abnormal calprotectin that normalized after ETI (n=4). (e) Boxplot indicating relative abundances of Streptococcus salivarius, which demonstrated statistically significant decrease in participants with aCFLD and calprotectin normalization before and after treatment with ETI. (p<0.05, Kruskal-Wallis). (f) Boxplot indicating relative abundances of Veillonella parvula, which demonstrated decreasing trend in participants with aCFLD and calprotectin normalization before and after treatment with ETI. (p=0.31, Kruskal-Wallis). *p<0.05