Faecal microbiota transplant restores intestinal barrier function and augments ammonia metabolism in patients with cirrhosis: a randomised single-blind placebo-controlled trial

doi:10.21203/rs.3.rs-3088449/v1

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Faecal microbiota transplant restores intestinal barrier function and augments ammonia metabolism in patients with cirrhosis: a randomised single-blind placebo-controlled trial

https://doi.org/10.21203/rs.3.rs-3088449/v1

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Patients with cirrhosis have reduced gut-bacterial diversity and microbiota enriched with pathobionts. This enrichment, coupled with increased gut permeability and bacterial translocation, increases susceptibility to infection and death. Faecal microbiota transplant [FMT] previously restored gut diversity and improved hepatic encephalopathy (HE) in small phase-I-trials, but its impact upon the disease process in cirrhosis is unexplored. We performed a randomised, placebo-controlled feasibility trial of jejunal FMT transplant in 32 advanced cirrhosis patients. The primary endpoint assessed safety, feasibility, and tolerability of FMT; secondary endpoints explored efficacy and mechanism. FMT was safe and well-tolerated with no serious adverse events. Deep-faecal-metagenomic sequencing confirmed FMT increased recipient species richness with significant donor engraftment. FMT reduced intestinal barrier damage and systemic inflammation. FMT decreased microbial-associated ammonia production and augmented ammonia excretion viaanaerobic metabolism of L-aspartate to hippurateproviding proof of concept that FMT enhances ammonia metabolism, central in the pathogenesis of HEin cirrhosis.

Health sciences/Medical research/Translational research

Biological sciences/Microbiology/Microbial communities/Microbiome

Biological sciences/Immunology/Antimicrobial responses

Ammonia

Cirrhosis

Faecal Microbiota Transplant

Gut dysbiosis

Gut microbiome

The burden of chronic liver disease is increasing worldwide, accounting for 2 million deaths annually; 3.5% of global mortality. (1, 2) Advanced liver cirrhosis brings a plethora of complications including hepatic encephalopathy (HE), variceal bleeding, ascites, and a propensity to develop infections. All of which can lead to multiorgan failure and death. (3) The gut microbiota becomes perturbed in patients with cirrhosis (4) with oralisation (5) resulting from translocation of mucin-degrading sialidase-rich species from the mouth to the gut e.g. Streptococcus spp., Veillonella atypica, and V. parvula. (6) Alongside increases in pathogens known to induce gut barrier damage, this contributes to increased gut permeability with bacterial translocation (BT) into the systemic circulation. (7) BT is a significant driver of cirrhosis-associated immune dysfunction (CAID) which increases susceptibility to infection. (8, 9)

Gut microbiota manipulation with the non-absorbable antibiotic rifaximin-α in cirrhotic patients led to a reduction in hospitalisation and 30-day readmission. (10, 11) Furthermore, we have recently shown that rifaximin-treated cirrhotic patients were less likely to develop infection. Rifaximin reduces oralisation of the gut with mucin-degrading species, attenuating systemic inflammation and promoting gut barrier repair. (6) These data constitute “proof of principle” that modifying the gut microbiota in patients with cirrhosis improves clinical outcomes. However, there is considerable concern that long-term antibiotic use is driving antimicrobial resistance (AMR). (12, 13) Faecal microbiota transplant (FMT) is a well-established treatment, acting as a “whole ecosystem” approach to restore the homeostatic balance of gut microbiota. It is safe (14) and cost-effective (15) in the treatment of recurrent Clostridioides difficile infection (where it has become standard of care), and shows promise for a wide range of other disease states associated with gut microbiota perturbation. (16) An open-label phase I pilot study of 10 cirrhotic patients treated with rectally-instilled FMT restored gut-microbiota diversity and improved HE. However, there are limitations in attributing results to FMT alone as patients were treated concurrently with broad-spectrum antibiotics, whereas those allocated to standard of care were not. (17) A further 10-patient phase I trial using FMT capsules was associated with improved duodenal mucosal microbial diversity, gut microbial diversity, antimicrobial peptide expression and reduced lipopolysaccharide-binding protein. (18) However, no exploration of the impact of FMT upon underlying pathogenic pathways in cirrhosis was reported. FMT, therefore, offers a promising non-antimicrobial therapeutic strategy to improve an array of clinical outcomes in cirrhotic patients, ranging from the development of encephalopathy and infection to reducing AMR rates.

Here we report the first randomised placebo-controlled trial of FMT in patients with advanced cirrhosis in Europe, without antibiotic pre-treatment or concurrent rifaximin therapy. (19) We assessed the safety and feasibility of liquid frozen FMT derived from > 50g raw stool versus placebo [allocated in a 3:1 ratio in a single-blinded fashion]. The treatment was delivered to the small bowel endoscopically via a nasojejunal tube following bowel lavage in 21 patients with advanced cirrhosis. The primary endpoints of the study were to assess twofold: 1) the feasibility of administering FMT, as determined by recruitment rates (including the acceptability of the intervention) and the tolerability of FMT, and 2) safety in patients with advanced cirrhosis. Patients were reviewed at 7, 30 and 90 days following intervention and blood, urine and stool were collected. Faecal deep sequencing metagenomics, faecal proteomics, plasma, urine and faecal metabolomics, whole blood 16S rDNA targeted metagenomic sequencing, plasma and faecal cytokine measurement, gut barrier biomarker assays, and plasma biochemical analyses were performed to assess secondary mechanistic endpoints; including whether FMT could restore gut bacterial diversity, gut barrier function, alter ammonia metabolism, reduce systemic inflammation and progression of liver disease.

Overview of trial enrolment

Thirty-two patients were recruited from outpatient clinics at King’s College Hospital or from suitable inpatients on the wards. From the 2nd March 2018 to 31st July 2019, 318 patients aged 18 to 75 years were pre-screened for eligibility; 88 fulfilled the inclusion criteria. 32 consented to be enrolled and of these, 23 patients were randomised, with 21 receiving treatment (15 FMT; 6 placebo) [Figure 1]. Randomisation was undertaken using a validated online randomisation programme in a 3:1 ratio of FMT: placebo. Treatment allocation was by block randomisation with randomly varying block sizes. All 15 patients receiving FMT completed the trial and attended all 4 follow-up visits. Five of the six patients receiving a placebo completed the trial, with the sixth patient attending all but the day 90 follow-up visit.

Characteristics of the study population

The randomised participants had a mean age of 57.1 +/- 11 years, 26% (6/23) were female and 78% (18/23) were Caucasian. 52% had alcohol-related cirrhosis. Overall, treatment arms were balanced with half of the participants in each arm having ascites and one-third having a history of overt HE with similar baseline levels of blood ammonia. [Table 1]

Table 1

Baseline characteristics of the treated study cohort
	Total		FMT		Placebo		p-value
	n	Mean (SD) or %	n	Mean (SD) or %	n	Mean (SD) or %	p-value
Age	21	57.38 (10.73)	15	57.80 (10.77)	6	56.33 (11.57)	0.70
Sex: female	5	23.8%	3	20.0%	2	33.3%	0.60
Ethnicity							0.032
White	16	76.2%	13	86.7%	3	50.0%	-
Black	1	4.8%	0	0.0%	1	16.7%	-
Asian	2	9.5%	0	0.0%	2	33.3%	-
Other	2	9.5%	2	13.3%	0	0.0%	-
Weight (kg)	21	83.32 (20.23)	15	87.16 (19.31)	6	73.72 (20.95)	0.15
Body Mass Index	21	29.15 (7.38)	15	30.10 (7.69)	6	26.79 (6.55)	0.23
Smoker	5	23.8%	4	26.7%	1	16.7%	1.00
Aetiology							0.51
Alcohol	12	57.1%	8	53.3%	4	66.7%	-
Metabolic/NAFLD	4	19.0%	3	20.0%	1	16.7%	-
Alcohol/Metabolic	1	4.8%	1	6.7%	0	0.0%	-
Hepatitis C (RNA negative)	3	14.3%	3	20.0%	0	0.0%	-
Primary biliary cholangitis	1	4.8%	0	0.0%	1	16.7%	-
Diabetes	4	19.0%	3	20.0%	1	16.7%	1.00
Prior Ascites	10	47.6%	7	46.7%	5	50.0%	1.00
Prior Spontaneous Bacterial Peritonitis	1	4.8%	1	6.7%	0	0.0%	1.00
Prior Hepatic Encephalopathy	13	61.9%	9	60.0%	4	66.7%	1.00
Prior Variceal Bleeding	8	38.1%	5	33.3%	3	50.0%	0.75
TIPS	1	4.8%	1	6.7%	0	0.0%	1.00
Encephalopathy grade at randomisation							0.62
0	15	71.4%	10	66.7%	5	83.3%	-
1	6	28.6%	5	33.3%	1	16.7%	-
2	0	0.0%	0	0.0%	0	0.0%	-
Ascites at randomisation	2	9.5%	2	13.3%	0	0.0%	1.00
White Blood Cell Count (x10⁹/L)	21	4.35 (1.66)	15	4.22 (1.33)	6	4.66 (2.43)	0.82
Neutrophils (x10⁹/L)	21	2.71 (1.08)	15	2.68 (0.88)	6	2.79 (1.58)	1.00
C-reactive protein (mg/L)	21	5.07 (6.12)	15	4.53 (3.67)	6	6.42 (10.43)	0.23
Haemoglobin (g/L)	21	123.3 (20.0)	15	123.5 (20.0)	6	123.0 (21.9)	0.91
Platelet count (x10⁹/L)	20	109.0 (61.1)	15	105.5 (50.6)	5	119.6 (92.5)	1.00
INR	20	1.46 (0.34)	14	1.48 (0.38)	6	1.40 (0.22)	0.97
Sodium (mmol/L)	21	137.0 (4.5)	15	137.1 (5.1)	6	136.5 (3.1)	0.41
Urea (mmol/L)	20	5.47 (2.38)	15	5.61 (2.70)	5	5.04 (1.03)	0.90
Creatinine (µmol/L)	21	73.57 (30.31)	15	75.33 (34.25)	6	69.17 (18.93)	0.67
ALT (IU/L)	21	33.00 (19.52)	15	29.27 (15.28)	6	42.33 (26.90)	0.15
AST (IU/L)	20	50.65 (27.16)	14	45.86 (14.75)	6	61.83 (44.97)	0.71
Total bilirubin (µmol/L)	20	32.30 (22.38)	14	28.64 (14.37)	6	40.83 (35.23)	0.71
Albumin (g/L)	20	36.85 (4.65)	14	37.93 (4.63)	6	34.33 (3.93)	0.11
Ammonia (µmol/L)	21	69.38 (27.55)	15	71.27 (28.22)	6	64.67 (27.73)	0.56
Lactate (mmol/L)	21	1.59 (0.53)	15	1.55 (0.52)	6	1.68 (0.58)	0.75
MELD Score	19	13.11 (2.16)	13	13.38 (2.10)	6	12.50 (2.35)	0.45

Primary Outcome:

(i) Feasibility

83% (95%CI 64–97%) of those that consented and screened were eligible. 87% (95%CI 66–97%) of all patients randomised were treated and followed up to completion at 90-days.

(ii) Tolerability

Overall, FMT was well tolerated with only minor transient gastrointestinal side effects reported within the following 7 days. One individual vomited within 2-hours of receiving the FMT (bile not faeces) and 3/15 (20%) passed a type 6/7 bowel motion within 2-hours of receiving the FMT. No patients receiving FMT reported a foul taste or smell, flatulence or reflux.

(iii) Safety

There were 9 serious adverse events (SAEs) occurring in 4/15 patients in the FMT arm and one SAE in 1/6 patients in the placebo arm. None of the SAEs were deemed related to the FMT by an independent data monitoring committee. Two patients treated with FMT developed suspected infections (no organisms cultured); one patient developed pedal cellulitis on day 15 post-FMT following a podiatry intervention and one patient developed a urinary tract infection on day 87 post-FMT. There were no infections in the placebo group [Supplementary Table 1].

Secondary clinical and mechanistic outcomes:

(i) FMT restored faecal microbial richness with temporal donor engraftment

Gut microbial metagenomic species (MGS) richness plays an essential role in regulating health and immunity and is reduced in cirrhosis. (4, 5) We confirmed patients with cirrhosis had reduced faecal MGS richness compared to healthy donors [p = 1.73e-5 Fig. 2a]. The faecal microbial composition was distinct from healthy donors [Figure 2d-e]. FMT treatment restored recipient MGS species richness [p = 0.038 Fig. 2b] with significant donor engraftment on day 7 [Figure 2f] recently linked to enhanced clinical success. (20) In line with previous studies, (21) we observed antibiotic treatment adversely impacted engraftment [subjects 1 and 7; Fig. 2f]. FMT modified the microbiome composition [Figure 2g] increasing the abundance of beneficial Alistipes putredinis (decreased A. putredinis correlates with liver decompensation), (22) Firmicutes bacterium and Faecalibacterium prausnitzii, a strict anaerobe that produces butyrate (short-chain fatty acid, SCFA) associated with gut health [Figure 2h]. (23, 24) Following separation into Cluster-1 enriched for beneficial obligate anaerobes and Cluster-2 containing pathobionts, FMT increased cluster-1 and reduced cluster-2 at day-7 [Figure 2j-k]. The changes were temporal as by day-30 post-FMT a resurgence (outgrowth) of pathobiont cluster 2 was observed which was further enhanced at day-90 post-FMT [Figure 2f, j and k]. However, there are some residual effects of FMT at D90 compared to baseline.

(ii) FMT ameliorates cirrhosis-associated immune dysfunction (CAID) and augments anti-bacterial cytokine responses

BT is a significant driver of CAID characterised by compromised immune responses to bacterial pathogens with lymphocyte anergy and dysregulated cytokine production including circulating interleukin (IL)-17A and increased susceptibility to developing infection. (8) FMT increased plasma IL-17A (day-7; p = 0.002) [Figure 3a] involved in antibacterial function, systemic immune modulation, and gut barrier repair. (25, 26) Conversely, plasma IL-1β concentrations were higher in placebo-treated patients than in FMT [Figure 3b]. IL-1β is integral to the host-response and resistance to pathogens, however, it also exacerbates acute tissue injury during chronic disease. (26) Indeed, FMT augmented systemic immune cell responses following bacterial stimulation (Heat-killed Escherichia coli 0111:B4 [HKEB] and Escherichia coli lipopolysaccharide [LPS]). Whole blood peripheral blood mononuclear cells incubated with HKEB or LPS 7-days post-FMT, increased tumour necrosis factor alpha (TNF-α) production (HKEB p = 0.0025; LPS p = 0.032) and IFN-γ-induced protein 10 (IP-10) (LPS p = 0.0002) production [Figure 3c-d]. IL-1β production was increased in response to HKEB (p = 0.0085) in the placebo arm compared to FMT on day-90 [Figure 3e]. Collectively FMT contributed to a reduction in immune paralysis and a drive towards the restoration of immune antibacterial function [Figure 3a-e].

(iii) FMT promotes microbial hyodeoxycholic acid and butyrate production promoting gut barrier repair

Gut microbial-host co-metabolites are understood to contribute to gut barrier function via impact upon host immune responses and direct impact upon epithelial cell homeostasis. Patients with high faecal butyrate levels post-FMT had lower plasma IFNγ and had higher levels of the faecal secondary bile acid hyodeoxycholic acid (HDCA) (p = 0.048; R² = 0.57) [Figure 3f]. Interferons are important in host defence against infection. (27) Gut microbiota-derived HDCA attenuates inflammatory responses to LPS via TGR5, AKT, and NF-κB suppression, decreasing TNF-α, IL-6, and IL-1. (28) HDCA also modifies gut barrier integrity by suppressing intestinal epithelial cell proliferation through FXR-PI3K/AKT, alongside increasing the abundance of the gut bacteria associated with bile acid metabolism. (29)

Fatty Acid Binding Protein 2 (FABP2) is expressed in small intestinal epithelial cells. It is a marker of epithelial shedding and when elevated in the blood is a marker of gut barrier damage. (24) Plasma FABP2 levels were higher in the placebo group compared to the FMT group at 7, 30 and 90 days (p = 0.014, p = 0.0062, p = 0.0315, respectively) [Figure 3g]. PCoA showed that FMT had a significant effect on components associated with intestinal barrier integrity. Albumin exerts several homeostatic functions as a potent scavenger, antioxidant, and immunomodulatory molecule, including the transport of fatty acids as well as modulating intestinal barrier function. (30) Plasma albumin rose across the 90-day follow-up period and faecal FABP2 levels fell (p = 0.014) following FMT but not placebo [Figure 3h]. Faecal FABP2 levels began to rise again in the FMT group compared to placebo at 30 days (p = 0.03) [Figure 3i] mirroring the resurgence of pathobiont cluster 2. Undigested carbohydrate is fermented to D-lactate by lactic acid–producing bacteria [LAB] present in the colon. In dysbiosis LAB species are either lost or, with excessive carbohydrate consumption, overgrown resulting in excessive D-lactate passing into the systemic circulation (a biomarker of BT). Plasma D-lactate was reduced at day-7 post-FMT compared to placebo (p = 0.0077) [Figure 3j]. Faecal D-lactate levels increased post-FMT treatment compared to placebo [p = 0.043 Extended data Fig. 1]. Indeed, enhanced proportions of the LAB-containing phylum Bacillota are observed in cluster 1 in these patients [Figure 2j-k]. Faecal proteomic analysis demonstrated FMT increased the abundance of human proteins [Supplementary Table 3] involved in antimicrobial mucosal barrier defence and suppression of microbiome and pathogen entry into the host including human zymogen granule proteins, secretory IgA and several neutrophil-derived molecules including calprotectin, neutrophil defensin 1 and cathepsin G [Figure 3k-n]. Over the duration of the study markers of barrier disruption (alpha-amylase 2B, lithstathine-1-α, intestinal-type alkaline phosphatase and neprilysin) were enhanced in the placebo stool but not following FMT [Supplementary Table 3, Fig. 3k-n].

FMT also initiated microbial and human metabolic reprogramming, particularly of glutamine. Intestinal epithelial cells and immune cells largely depend on glutamine availability to survive, proliferate, function, and ultimately defend against pathogen invasion [Supplementary Tables 3 & 8, Fig. 3k-n]. (31) Taken together, these data indicate that FMT is associated with a change in bacterial species resulting in microbial and human metabolic reprogramming that augments intestinal barrier integrity and immune defence.

(iv) FMT modulates the translocating microbiota towards health

In healthy individuals, the circulating human blood microbiota is mostly composed of members of the phyla, Pseudomonadota (synonym Proteobacteria) and to a lesser extent of Actinomycetota (synonym Actinobacteria) and Bacteroidota (synonym Bacteroidetes) phyla. (32, 33) Intestinal epithelial and immune cells limit the translocation of gut microbiota across the intestinal barrier. In patients with cirrhosis and portal hypertension, however, there is exaggerated BT of pathogenic organisms. Increased alpha-diversity of the blood microbiome (at genus and species level) was observed in the placebo group [Figure 4a, 4c and Extended Data Fig. 2a] over time but not in the FMT-treated cohort [Figure 4b and extended data Fig. 2b]. FMT modified the profile of the species translocating across the gut barrier [Figure 4e-f, Extended Data Fig. 2c-d and Supplementary Tables 5–6]. The relative abundance of Bacillota (synonym Firmicutes) such as Clostridia, including the Ruminococcaceae family, was increased in patients receiving FMT [Figure 4e-f; Supplementary Table 5]. Ruminococcaceae convert primary bile acids into secondary bile acids and are major butyrate producers such as butyrate and are depleted in cirrhosis. (34)

Blood 16S rDNA gene-targeted sequencing was further analysed with PICRUSt 2.0 to predict altered bacterial functions altered. Enhanced levels of microbial enzymes involved in butyrate metabolism, with an increase in butyrate kinase [EC.2.7.2.7] following FMT (day 30; p = 0.0013) and aldehyde dehydrogenase [EC.1.2.1.10] (day 90; p = 0.003) were observed [Figure 4e-g]. This enrichment was accompanied by a rise in faecal butyrate (day 90; p = 0.0258, Extended Data Fig. 2e) pointing to FMT promoting engraftment of butyrate-producing species. The local anti-inflammatory effects of butyrate render intestinal epithelial cells tolerant of commensal bacteria and additionally confer a protective role against unchecked inflammation. (35)

(v) FMT attenuated microbial-associated ammonia production and upregulated ammonia utilisation by augmenting the anaerobic metabolism of L-aspartate

Plasma ammonia reduced following FMT (day 30; p = 0.0006) [Figure 5a] and faecal ammonia rose (days 7, 30 and 90; p = 0.019, p = 0.011 and p = 0.025, respectively) [Figure 5b]. This was associated with amelioration in biomarkers of systemic inflammation [Figure 5c]. Patients with higher ammonia levels at baseline had higher levels of faecal calprotectin (R² = 0.79; p = 0.002). Calprotectin is a surrogate marker of intestinal inflammation and a consequence of neutrophil migration into an inflamed intestinal mucosa [Figure 5c]. The reduction in plasma ammonia was associated with an improvement in HE grade (R²= -0.601; p = 0.02) [Figure 5c] and the rise in faecal ammonia was associated with lower faecal LPS levels (R²= -0.71; p = 0.003) [Figure 5d]. In contrast, plasma TNF-α and IL-1β increased with placebo.

Metagenome prediction from blood 16s rDNA sequencing using PICRUSt 2.0 revealed increases in the abundance of microbial enzymes involved in nitrogen assimilation and a reduction in those required for denitrification and ammonification [Figure 5f]. Nitrite reductase [EC.1.7.2.1], which reduces nitrite to nitric oxide (NO), was increased from day-7 (p = 0.0043), day-30 (p = 0.0125) and day-90 (p = 0.0004) post-FMT [Figure 5g, Extended Data Fig. 3a.]. Nitrite reductase [EC.1.7.2.1] is usually expressed in obligate anaerobes (36) and coincided with an increase in Cluster-1 containing obligate anaerobes on day-7 post-FMT. The respiration and growth of E. coli are reversibly suppressed by NO. (37) NO produced by facultative or anaerobic bacteria diffuses into the mucosal microaerobic zones facilitating colonic bacterial production of SCFAs which provide respiratory fuels for intestinal epithelia and modulating mucosal immunity. (38) Nitrite reductase further reduces NO and hydroxylamine to ammonia, and sulphite to sulphide (39), was enriched in patients with higher plasma ammonia [Figure 5j] and increased post-FMT from day 30 to 90 coinciding with the resurgence of the pathogenic Cluster-2 [Figure 5h]. Nitrite reductase [EC.1.7.2.2] confers a selective advantage for pathogenic bacteria to colonise mucosa. (39) A decrease in the abundance of the ammonia-generating enzyme L-seryl-tRNASec selenium transferase [EC.2.9.1.1] which metabolises L-serine from dietary protein into glycine and ammonia was observed post-FMT (day-90; p = 0.0027) whilst an increase was observed with placebo (p = 0.025) [Figure 5k and m].

Aspartate ammonia-lyase [EC.4.3.1.1] was significantly enriched in the blood microbiome post-FMT (day-90; p = 0.0004) [Figure 5l; Extended Data Fig. 3c]. In anaerobic conditions, EC.4.3.1.1 combines ammonia with fumarate to produce L-aspartate which requires the transport of succinate but in aerobic growth conditions, L-aspartate is metabolised by aspA-encoded aspartase to produce ammonia. (40) The increase in Cluster-1 rich in obligate anaerobes post-FMT supports FMT-promoting growth conditions that drive the anaerobic catabolism of L-aspartate consuming ammonia.

Faecal proteomic analysis revealed that FMT increased the abundance of faecal microbial enzymes involved in denitrification and ammonification with a reduction in enzymes required for nitrogen assimilation and excretion via the urea cycle (Fig. 5n). Glutamine synthetase [EC.6.3.1.2] involved in the incorporation of ammonia into glutamate was increased in the FMT-treated group over the course of the trial, being 17.4-fold higher in the FMT group at day 90. Enzymes involved in ammonia excretion by the urea cycle were modified following FMT. Bacterial ornithine carbamoyltransferase [EC2.1.3.3, Q328S8, P75473, B2G653, Q93JF1, P0C0N1] which converts ammonia and L-aspartate via L-ornithine to urea were enhanced in the stool (p = 0.006, Q328S8). FMT increased urinary hippurate, a marker of gut diversity (41) and metabolic health (42) (day-30; p = 0.0299) [Figure 6a] and decreased urinary succinate (day-7; p = 0.0005, day-30; p = 0.0162) [Figure 6b]. Metagenome prediction from blood 16s rDNA sequencing using PICRUSt 2.0 revealed changes in the abundance of microbial enzymes involved in benzoate metabolism [Figure 6c-g] and glycine [Figure 6h] post-FMT culminating in increased hippurate production [Figure 6n] and consumption of ammonia indicative of urinary excretion.

In this first European randomised phase, I single-blinded placebo-controlled trial of jejunal FMT in patients with advanced cirrhosis, we show that FMT is a well-tolerated treatment. In contrast to previous published FMT studies, we did not pre-treat with antibiotics or use concurrent rifaximin therapy, which has been major confounders in evaluating the impact of FMT on the restoration of gut microbial diversity to date. This study also aimed to provide preliminary evidence of efficacy and mechanism of action.

Deep metagenomic sequencing confirmed FMT increased recipient species richness with significant donor engraftment up to 90-days associated with a reduction in systemic inflammation and surrogate markers of intestinal barrier damage such as faecal FABP2 and plasma D-lactate. FMT modified the microbiota profile decreasing the abundance of detrimental Blautia producta and Clostridium spp. whilst increasing the abundance of beneficial F. prausnitzii. (23) FMT led to an increase in obligate anaerobes (cluster 1) and a decrease in pathobionts (cluster 2). However, changes were time-limited, as by day 30 post-FMT, a resurgence (outgrowth) of pathobiont cluster 2 was observed and mirrored the loss of engrafted donor species at days 30 and 90. Future FMT trials should consider using repeat doses every 90 days.

The role of the intestinal innate immune system, in providing a first line of defence against enteric pathogens, is essential to maintaining health, and there is a wealth of data reporting dysregulation in these systems and loss of compartmentalization in patients with cirrhosis. The frequency and severity of bacterial infections in patients with cirrhosis are attributable to a complex interplay between the gut, liver and immune system, with systemic immunological disarray spanning the innate and humoral systems at multiple levels; cirrhosis-associated immune dysregulation (CAID). (43) CAID encapsulates a paradigm where patients with advanced cirrhosis exist in a state of persistent low-level systemic inflammation with high baseline levels of proinflammatory cytokines in addition to impaired pathogen surveillance and susceptibility to developing infection. Alongside increases in pathogens known to induce gut barrier damage, the gut becomes more permeable with an increased translocation of bacteria and bacterial products (metabolites, LPS, proteins etc) across the gut barrier, subsequently leading to endotoxemia, further inflammation, tissue damage, and organ failure. (8) In this study, we observed a restoration in systemic immune paralysis and enhanced antibacterial function of the gut mucosal compartment in response to FMT. We observed enhanced faecal butyrate in patients treated with FMT and increased levels of the faecal secondary bile acid, HDCA. HDCA is produced by the gut microbiota and attenuates inflammatory responses to LPS by decreasing the production of inflammatory mediators. (28)

FMT had a significant effect on components associated with intestinal barrier integrity. Biomarkers of intestinal barrier damage such as plasma D-lactate and FABP2 were reduced post-FMT. Plasma albumin rose, which exerts several homeostatic functions as a potent endotoxin scavenger, antioxidant and immunomodulatory molecule, including the transport of fatty acids as well as modulating intestinal barrier function. (44) This increase in plasma albumin may reflect reduced intestinal epithelial cell shedding from the epithelial monolayer into the lumen and consequently less protein loss reducing transient gaps or micro-erosions in the gut barrier, resulting in enhanced gut barrier integrity.

Whilst FMT did not reduce BT across the gut barrier per se, it did modulate the translocating microbiota towards health. FMT shifted the profile of the circulating microbiota away from species that damage the intestinal barrier, denitrify and produce ammonia, underpinning the development of HE (45) and other complications of cirrhosis. (4, 46)

FMT reduced plasma and increased faecal ammonia levels which was accompanied by a marked reduction in gut and systemic inflammation. Inflammation and ammonia exhibit synergism in the pathogenesis of HE (47, 48) and this may explain the reported observations of FMT reducing HE episodes in phase I studies. (17, 18) Blood microbiome 16s rDNA analysis showed that patients in the placebo group had significantly increased bacterial richness and diversity over time when FMT patients did not show a change in alpha diversity values. These results implicate FMT-related restoration of the intestinal barrier. In addition, the relative abundance of the Ruminococcaceae family was higher over time in the blood of FMT patients. This family is known to decrease in faeces of patients with cirrhosis and in the blood of patients with cirrhosis with a concomitant decrease of the primary to secondary bile acid conversion. (49, 50) The higher relative abundance of this family in the FMT group seems to confirm the correction of the dysbiosis and associated physiological impact due to the cirrhosis. Metagenome prediction from blood 16S rDNA sequencing using PICRUSt 2.0 suggests an increase following FMT in the abundance of bacterial enzymes involved in nitrogen assimilation and a reduction in those required for denitrification and ammonification. Furthermore, FMT led to a reduction in microbial-associated ammonia production and upregulated ammonia utilisation by augmenting the anaerobic metabolism of L-aspartate and excretion of hippurate, providing proof of concept that FMT augments ammonia metabolism. Hyperammonemia has recently been shown to increase hospitalisation and liver-related complications and mortality in stable patients with cirrhosis (44). These data point to FMT having utility in not only reducing blood ammonia but ameliorating gut-derived systemic inflammation in patients with cirrhosis and hepatic encephalopathy. This will now be explored in a large multicentre double-blind placebo-controlled trial [PROMISE Trial] exploring the impact of FMT on the development of infection, hospitalisation and liver-related complications such as HE and spontaneous bacterial peritonitis, known to be driven by reduced gut bacterial diversity over-represented by pathobionts and reduced gut barrier integrity.

Trial design overview

This phase I safety and feasibility ‘proof of concept’ trial was conducted in compliance with the principles of the Declaration of Helsinki (1996), principles of Good Clinical Practice, Research Governance Framework and the Medicines for Human Use (Clinical Trial) Regulations. Fully informed consent was obtained from all participants and safety monitoring was conducted by the King’s Health Partners Joint Clinical Trials Office. It was approved by the London South East Research Ethics Committee (17/LO/2081) and by the Medicines and Healthcare Products Regulatory Agency for Clinical Trial Authorisation [EudraCT number: 2017-003629-13; ClinicalTrials.gov NCT02862249].

A prospective, randomised placebo-controlled feasibility single-centre trial was performed recruiting 32 patients with stable cirrhosis from the liver outpatient clinic at King’s College Hospital NHS Foundation Trust. Patients were randomised in a single-blinded fashion in a 3:1 ratio with FMT or placebo. Patients were unaware of the intervention given, but investigators were not blinded to the treatment intervention. The trial protocol has previously been published. (19) The randomisation schedule was generated using a validated randomisation programme and verified for accuracy using strict quality control procedures. The treatment allocation was centrally coordinated by King’s Clinical Trials Unit. A Participant Identification Number (PIN) was generated by a MACRO database.

Study population

Patients aged 18 to 75 years with the capacity to consent and a diagnosis of cirrhosis and a Model for End-stage Liver Disease (MELD) score (45) of 10 to 16 were screened for eligibility. The diagnosis of cirrhosis was based on radiological, clinical or histological parameters.

Inclusion criteria

18–75 years
Confirmed cirrhosis of any aetiology with MELD score 10–16. (51)
Patients with alcohol-related liver cirrhosis must have been abstinent from alcohol for a minimum of 6 weeks.
Patients must be deemed to have the capacity to consent.

Exclusion Criteria

Severe or life-threatening food allergy.
Pregnancy or breastfeeding.
Treatment for active variceal bleeding, infection, bacterial peritonitis, overt hepatic encephalopathy or acute-on-chronic liver failure within the past 14 days.
Received antibiotics (including rifaximin) in the past 14 days.
Active alcohol consumption > 20g/day.
Previous liver transplant.
Hepatocellular carcinoma.
Inflammatory bowel disease.
Prior gastrointestinal resection e.g. gastric bypass.
Severe renal impairment (creatinine > 150µmol/L).
HIV positive.
Immunosuppressant therapy for > 2 weeks within 8 weeks of intervention.

Study endpoints

The primary objective of the study was to assess whether stabilising gut dysbiosis with FMT in patients with advanced cirrhosis is both feasible and safe.

The primary endpoints were twofold:

i) to assess the feasibility of FMT as determined by the recruitment rates (including the acceptability of the intervention) and tolerability of FMT.

ii) to assess the safety of FMT administration in advanced cirrhosis.

Success Criteria of Primary Endpoints:

> 25% consent rate (of all patients screened).
> 50% fulfil inclusion/exclusion criteria (of all patients consented).
> 80% of randomised patients were treated successfully and completing the study up to day 90.
Availability of sufficient stool donors for the study.
Reflux rates of transplanted material < 20% (e.g. foul taste, smell, nausea and vomiting and indigestion).
Significant gastrointestinal side effects (including diarrhoea, constipation, abdominal pain, flatulence and bloating) of < 20%.

Secondary objectives:

Provide preliminary evidence of efficacy for a larger randomised trial with the purpose of choosing the optimal primary outcome, and estimating the parameters for sample size calculation.

(a) Improvement in global liver synthetic function assessed by MELD score at 90 days. (51)

(b) Development of overt hepatic encephalopathy. (52)

(d) Development of infection.

(e) Stability of transplanted gut microbiota by comparing the percentage composition of the stool microbiota on days 7, 30 and 90 with the donor microbiome (engraftment).

(f) Comparison of the stool microbiome at baseline, day 7, day 30 and day 90 in response to treatment.

Mechanistic outcomes:

Blood, stool and urine samples were collected from the enrolled patients at baseline and days 7, 30 and 90 to assess the efficacy and mechanisms of action of FMT. Faecal deep sequencing metagenomics, plasma and faecal metabolomics, gut barrier biomarkers, whole blood bacterial DNA, plasma cytokines and plasma biochemistry were performed to assess whether FMT could restore gut bacterial diversity, alter ammonia metabolism, reduce systemic inflammation and progression of liver disease [Please see Supplementary Methods].

Interventions

FMT was prepared in accordance with the principles of good manufacturing practice and under Manufacturing Authorisation for an Investigational Medicinal Product from the Medicines and Healthcare Products Regulatory Agency. FMT donors were carefully screened healthy volunteers aged between 28 and 35 with a normal body mass index [Donor characteristics detailed in Supplementary Methods]. Donors underwent detailed questionnaire screening for risk factors and robust testing for a range of infectious agents [as previously published (19)] and in accordance with national guidelines published jointly by the British Society of Gastroenterology and the Healthcare Infection Society in the UK. (53) This includes testing for carriage of extended-spectrum beta-lactamase-producing bacteria. International expert consensus guidelines on FMT were adhered to at all times including the International Consensus Conference on stool banking for FMT in clinical practice, (54) and the United European Gastroenterology Consensus on standardisation of stool banking. (55) This ensured robust donor selection and suitability, screening and traceability.

FMT (200mLs less a small aliquot for archiving) was administered via a gastroscope into the jejunum following bowel preparation with two sachets of MoviPrep® administered the day before the procedure. Patients received xylocaine throat spray and sedation with a small dose of intravenous midazolam ranging between 1 and 4mg prior to the gastroscopy.

Placebo solution (200mLs 0.9% normal saline and 12.5% glycerol) was administered via a gastroscope into the jejunum following preparation of the bowel with two sachets of MoviPrep® administered the day before the procedure.

ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BT: Bacterial translocation; CAID: Cirrhosis-associated immune dysfunction; FMT: Faecal Microbiota Transplant; GGT: Gamma-glutamyl Transferase; Hepatic encephalopathy: HE; HKEB: Heat-killed Escherichia coli 0111:B4; IFN: interferon; IL: interleukin; INR: International normalised ratio; LAB: lactic acid–producing bacteria; LPS: lipopolysaccharide; MELD: Model For End-Stage Liver Disease; MGS: Metagenomic species; NAFLD: Non-alcoholic fatty liver disease; NLR: Neutrophil-Lymphocyte Ratio; Principal Component Analysis: PCA; PCoA: Principal Coordinate Analysis; RNA: Ribonucleic acid; Serious Adverse Event: SAE; Short-chain fatty acids: SCFA; TIPS: Transjugular portosystemic intrahepatic shunt; TNF-α: tumour necrosis factor alpha; WCC: white cell count.

Acknowledgements: This study/project is funded by the National Institute of Health and Care Research (NIHR) [Research for Patient Benefit Programme PB-PG-0215-36070]. Metabolomics studies were performed at the MRC-NIHR National Phenome Centre at Imperial College London; this centre receives financial support from the Medical Research Council (MRC) and National Institute of Health Research (NIHR) (grant number MC_PC_12025). BHM is the recipient of an NIHR Academic Clinical Lectureship (CL-962 2019-21-002). The Division of Digestive Diseases and MRC-NIHR National Phenome Centre at Imperial College London receive financial and infrastructure support from the NIHR Imperial Biomedical Research Centre (BRC) based at Imperial College Healthcare NHS Trust and Imperial College London. The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. We would also like to acknowledge the support of Professor Alastair O’Brien and Dr Louise China, University College London in the set-up of the trial. This study was also supported by the Science for Life Laboratory (SciLifeLab), Engineering and Physical Sciences Research Council (EPSRC), EP/S001301/1 and Biotechnology Biological Sciences Research Council (BBSRC) BB/S016899/1. Sequencing was undertaken by the SciLifeLab and the National Genomics Infrastructure (NGI), and high performance computing and bioinformatics by the Swedish National Infrastructure for Computing at SNIC through Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX) under Project SNIC 2020-5-222, SNIC 2019/3-226, SNIC 2020/6-153 and King’s College London computational infrastructure facility, Rosalind (https://rosalind.kcl.ac.uk).

Author contributions: LAE: assay development, data generation, data analysis and interpretation, figure generation and manuscript development; CAW: patient recruitment and consent, administration of placebo/FMT, sample acquisition, storage and analysis, trial management and data generation; SL: data analysis and figure generation; BHM: data generation and analysis; AL and CF: statistical analysis; AD: senior trial methodologist and statistician; TP, LM, VTK, XY, SL, CR, BL, SH, PT, JRM, SS, THT: data generation, analysis and interpretation; BM: FMT development and manufacture; AZ: patient recruitment and trial management; VCP: senior trial management; SDG: trial design and delivery and FMT development and manufacture; DLS: Chief Investigator, trial design and delivery, data analysis and interpretation, figure generation and manuscript writing. All authors approved the final version of the manuscript.

Competing interests statement: DLS has undertaken consultancy for Norgine Pharmaceuticals Ltd, EnteroBiotix, Mallinckrodt Pharmaceuticals and ONO Pharma UK Ltd and has delivered paid lectures for Norgine Pharmaceuticals Ltd, Falk Pharma and Aska Pharmaceutical Co. Ltd. JRM has undertaken consultancy for Cultech Ltd and Enterobiotix. BHM has undertaken consultancy for Finch Therapeutics Group. VCP has delivered paid lectures for Norgine Pharmaceuticals Ltd and Menarini Diagnostics Ltd. SDG has undertaken consultancy and delivered paid lectures for Enterobiotix, MSD, Pfizer, Novartis, Tillotts and Shionogi. BL is a shareholder of Vaiomer. SS is co-founder of Bash Biotech Inc and Gigabiome Ltd. All other authors have no interests to declare.

Data Availability: The shotgun metagenomics sequence raw data for this study can be found under the study accession PRJEB61882.

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Yes there is potential Competing Interest. DLS has undertaken consultancy for Norgine Pharmaceuticals Ltd, EnteroBiotix, Mallinckrodt Pharmaceuticals and ONO Pharma UK Ltd and has delivered paid lectures for Norgine Pharmaceuticals Ltd, Falk Pharma and Aska Pharmaceutical Co. Ltd. JRM has undertaken consultancy for Cultech Ltd and Enterobiotix. BHM has undertaken consultancy for Finch Therapeutics Group. VCP has delivered paid lectures for Norgine Pharmaceuticals Ltd and Menarini Diagnostics Ltd. SDG has undertaken consultancy and delivered paid lectures for Enterobiotix, MSD, Pfizer, Novartis, Tillotts and Shionogi. BL is a shareholder of Vaiomer. SS is co-founder of Bash Biotech Inc and Gigabiome Ltd. All other authors have no interests to declare.

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Faecal microbiota transplant restores intestinal barrier function and augments ammonia metabolism in patients with cirrhosis: a randomised single-blind placebo-controlled trial

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Abstract

Figures

Introduction

Results

Overview of trial enrolment

Characteristics of the study population

Primary Outcome:

(i) Feasibility

(ii) Tolerability

(iii) Safety

Secondary clinical and mechanistic outcomes:

(i) FMT restored faecal microbial richness with temporal donor engraftment

(ii) FMT ameliorates cirrhosis-associated immune dysfunction (CAID) and augments anti-bacterial cytokine responses

(iii) FMT promotes microbial hyodeoxycholic acid and butyrate production promoting gut barrier repair

(iv) FMT modulates the translocating microbiota towards health

Discussion

Methods

Trial design overview

Study population

Inclusion criteria

Exclusion Criteria

Study endpoints

Success Criteria of Primary Endpoints:

Secondary objectives:

Mechanistic outcomes:

Interventions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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