PennCHOP MICROBIOME PROGRAM Physiologic implications of [PDF]

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David Shen, MD, PhD

Division of Gastroenterology Perelman School of Medicine University of Pennsylvania

MICROBIOME PROGRAM

PennCHOP

Physiologic implications of co-metabolism between the gut microbiome and its host

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Atherosclerosis
Asthma
Colon cancer
Inflammatory bowel diseases
Obesity and metabolic syndrome

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Gut Microbiome and Liver Diseases

Liver is first portal that emerges from intestinal mucosal surface: Receives approximately 75% of blood supply from splanchnic circulation Potential liver diseases/processes affected by gut microbiome

Cholestatic liver disease (PBC and PSC)
NASH and NAFLD
Cirrhosis
Hyperammonemia and hepatic encephalopathy
Hepatic drug metabolism

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Urea Bile Acids

The Human World

Biotransformation and alteration of receptor- ligand interactions via FXR and TGR5 Hydrolysis into ammonia and its use by both the host and the gut microbiota as a source of nitrogen

The Microbial World

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Agenda

FXR-dependent modulation of the human

small intestinal microbiome by the bile acid derivative obeticholic acid

Engineering the gut microbiota to treat

hyperammonemia

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Agenda

FXR-dependent modulation of the human

small intestinal microbiome by the bile acid derivative obeticholic acid

Engineering the gut microbiota to treat

hyperammonemia

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Enterohepatic circulation of bile acids

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Bile Salt Hydrolase (Deconjugation) 7α-Dehydroxylation

Bacteria Bile Acids

A Bidirectional Relationship Between Bacteria and Bile Acids

M. Begley et al. FEMS Microbiology Reviews 2005; 29: 625–651
Gram-positive bacteria are more

sensitive to the toxic effects of bile than Gram-negative bacteria (MacConkey agar contains bile)

Bile acid toxicity to bacteria is

multifactorial with membrane effects, DNA damage, oxidative stress, alterations in RNA structure, and protein denaturation

Bile salt hydrolases, found primarily

in bacteria that inhabit the intestinal tract of mammals, enhance colonization efficiency

Bacteria Bile Acids

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Schaap FG, et al. Nat Rev Gastroenterol Hepatol. 2014;11:55-67.

Bile acids are ligands for Farnesoid X Receptor (FXR)

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OCA (6α-ethyl chenodeoxycholic acid)

Erlinger S. 2017

Selective FXR agonist
Derived from CDCA, which is the strongest endogenous FXR ligand
Approximately 100 times more potent than CDCA in activating FXR

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Obeticholic Acid for the Treatment of Primary Biliary Cholangitis

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7α-Hydroxy-4-cholesten-3-one (C4): intermediate in the biochemical synthesis of bile acids from cholesterol

Plasma Levels of C4 as a Biomarker of OCA-Dependent Inhibition

f Bile Acid Synthesis

16 37 On OCA Off OCA

Controlled human subject study examining the effect of OCA treatment (n=8 per

group):

5 mg OCA
10 mg OCA
25 mg OCA

Friedman, Li, Shen, et al. Gastroenterology 2018

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Bacterial Taxonomic Associations with Bile Acid Synthesis-Specific Effects of Obeticholic Acid (10 mg/day): A general increase in Gram-positive bacteria and decrease in Gram-negative bacteria Plasma C4

S. thermophilus

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Bacterial Taxonomic Associations with Bile Acid Synthesis-Specific Effects of Obeticholic Acid (10 mg/day): A general increase in Gram-positive bacteria and decrease in Gram-negative bacteria

Table 1: GEE model identified 15 species significantly associated with C4 change over time Phylum Species P value of C4 FDR of C4 OCA Response Gram Firmicutes Streptococcus_thermophilus 1.87e-07 2.30e-05 Increase pos Actinobacteria Bifidobacterium_breve 4.46e-04 0.023 Increase pos Firmicutes Streptococcus_salivarius 0.001 0.023 Decrease pos Firmicutes Lactobacillus_casei_paracasei 0.001 0.03 Increase pos Firmicutes Lachnospiraceae_bacterium_5_1_63FAA 0.001 0.03 Increase pos Bacteroidetes Alistipes_putredinis 0.003 0.053 Decrease neg Firmicutes Lactococcus_lactis 0.01 0.172 Increase pos Bacteroidetes Bacteroidales_bacterium_ph8 0.022 0.316 Decrease neg Firmicutes Subdoligranulum_unclassified 0.024 0.316 Equivocal pos Firmicutes Dorea_longicatena 0.026 0.316 Increase pos Actinobacteria Bifidobacterium_longum 0.03 0.316 Increase pos Firmicutes Dialister_invisus 0.031 0.316 Decrease pos Bacteroidetes Bacteroides_plebeius 0.037 0.347 Decrease neg Firmicutes Ruminococcus_obeum 0.045 0.389 Decrease pos Bacteroidetes Paraprevotella_unclassified 0.049 0.389 Decrease neg

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Bacterial Gene Associations with OCA Administration

(time effect FDR <0.05)

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Uniref 90 Genomic Pathway Analysis

135 pathways with significant association with time

(Repeated Measure ANOVA, FDR <0.01)

* * *

Top Metabolic Pathways in common across taxa:

Nucleotide synthesis
Amino Acid Biosynthesis

Lactococcus lactis Streptococcus Thermophillus Lactobacillus casei/paracasei

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Hypothesis

FXR activation by OCA decreases endogenous bile acid synthesis, leading to the outgrowth of bile-sensitive gram positive organisms in the small intestine

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Blue=Physiologically relevant concentrations in the human small intestine

Minimal Inhibitory Concentrations of Two Conjugated Bile Acids

n the Growth of Gram-Positive Bacteria that Increase in

Abundance with OCA Treatment

GCDCA (uM) GCA (uM)

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Physiologically-relevant concentration of OCA* in the human small intestine (1-40 mM**)

Obeticholic Acid Has Minimal Effects on Bacterial Growth at Physiologically-Relevant Concentrations in Humans

*Unconjugated OCA equivalents (i.e., summation of unconjugated OCA, glyco-OCA, and tauro-OCA) **Concentrations based on estimates of: calculation of OCA dose distributed in small intestine; simulated steady-state total OCA concentrations by physiological compartment for 10 mg OCA daily administration1.

1From Intercept Pharmaceuticals.

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The Bacterial Taxonomic Signature in Response to OCA is due to Small Intestinal Bacteria

Dlugosz A. et al. Sci Rep. 2015;5:8508

Streptococcus spp. accounts for 19% of 454-pyrosequencing reads in the human small intestine

Pereira and Berry. Environ Microbiol 2017

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> 25, n M

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a l Bile Acids Endogenous Bile Acids Primary Bile Acids Secondary Bile Acids

Concentration (nM)

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600,000 500,000 400,000 300,000 20 , 100,000

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a l d i st a l sma l l i n t e st i n a l bile acid levels Concentration (nM)

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a l Bile Acids Endogenous Bile Acids Primary Bile Acids Secondary Bile Acids

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OCA treatment inhibits endogenous luminal bile acid levels and leads to an increase in Gram-positive bacteria specifically in the small intestine of mice

*p<0.05 **p<0.01 ***p<0.001

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a l f e c a l bile acid levels Concentration (nmol/g stool)

6,000 5,000 4,000 3,000 2, 1,000 7,000 T

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a l Bile Acids Endogenous Bile Acids Primary Bile Acids Secondary Bile Acids

d T T T T 66NP 67 NP 63NP 65NP 9 3NP

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C MC OC

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Proximal SI Distal SI Feces Control Methylcellulose OCA

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OCA treatment inhibits endogenous luminal bile acid levels and leads to an increase in Gram-positive bacteria specifically in the small intestine of mice

Control Methylcellulose OCA

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A robust taxonomic signature for FXR activation in the human gut microbiome: An opportunity for precision medicine

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Summary I

By inhibiting endogenous bile acid synthesis/secretion, OCA treatment

leads to a significant induction of bile acid sensitive Gram-positive bacterial taxa in the human small intestine whose signature can be detected in the stool

Implications

The fecal microbiota might be a useful biomarker of bile acid-dependent

effects of FXR agonists in humans and its association with diet—a glimpse into the dynamics of the human small intestinal microbiota

The composition and genomic representation, possibly along with elements
f the fecal metabolome, will likely have utility as a robust and biologically

dynamic biomarker of FXR (OCA) function for precision medicine

The ability of a FXR agonist to alter the environment of the human small

intestine might provide novel opportunities to “engineer” the gut microbial composition

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Agenda

FXR-dependent modulation of the

human small intestinal microbiome by the bile acid derivative obeticholic acid

Engineering the gut microbiota to treat

hyperammonemia

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Urea CO2 + NH3

Urease

Urea Cycle

Amino Acid Pool Dietary Protein Intake

Renal Urea Excretion

The Gut Microbiome and Host Nitrogen Balance

15-30% 74% 18% 4%

Urea Cycle

Fate of NH3 from colonic urea hydrolysis: 1) Excreted in feces 2) Used by gut bacteria to synthesize amino acids, proteins, and other small molecules 3) Absorbed by host and utilized in liver for protein or urea synthesis

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Hypothesis

Inoculation of a urease-deficient defined microbial consortium into a properly prepared mammalian host will reduce fecal ammonia production and ameliorate morbidity and mortality in a murine model

f liver disease.

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Method: Inoculation of a Defined Microbial Consortium into Previously Colonized Mice

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Method: Inoculation of a Defined Microbial Consortium into Previously Colonized Mice

Defined microbial consortium

assembled in 1970s and standardized by NCI in 1978

Comprises eight murine gut

commensal bacteria

Shown to be innocuous, with benefit of

inducing immune tolerance

Minimal urease gene content

Altered Schaedler’s Flora (ASF) Method of Host Preparation

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ASF colonizes and persists after inoculation into properly prepared hosts

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ASF develops into a new stable resilient microbial community over time

A

Day post transfer

Germfree + ASF Prepared + ASF

B

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Longitudinal Evolution of the Gut Microbiome after ASF Inoculation

Cho and Blaser. Nature Rev. Genet. 2012

ASF suppresses the return of Bacteroidetes

Day

ASF permits the selective return

f Firmicutes

Day

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The New Stable State Leads to a Durable Reduction in Fecal Urease and Fecal Ammonia Levels

Fecal Urease Activity After Inoculation Fecal Ammonia Levels After Inoculation

*p<0.05, **p<0.001

Host Urea + H2O Ammonia + CO2

Bacterial Urease

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ASF reduces morbidity and mortality in the thioacetamide (TAA) murine model of hepatic injury and fibrosis

Survival benefit of ASF in TAA model of hepatic injury

Control (no TAA) Prepared + Normal Microbiota (TAA) Prepared + ASF (TAA)

20 40 60 80 100

% Spontaneous alternation

ns

*

ASF improves performance in the Y-maze neurobehavior test

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 50 100

Time Post-FMT (wks) Percent survival Control (TAA) Prepared + ASF (TAA)

p < 0.05 Start of TAA Time Post-Transplantation (wks) *p<0.05, ANOVA p=0.04

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Summary II

Inoculation of a urease-deficient microbial consortium into a

properly prepared host reduces fecal urease activity and fecal ammonia production long-term and reduces morbidity and mortality in a murine model of liver disease

These results provide proof of concept that inoculation of a prepared

host with a defined gut microbiota can lead to durable metabolic changes with therapeutic utility

Human studies will be critically important in determining the efficacy

and impact on host physiology and metabolism

The development of drugs to target the gut microbiome and microbial

pathways carries therapeutic potential for diseases

Implications

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Gut Microbiome

Gut microbiome in disease diagnosis, drug metabolism, and targeted therapeutics

Biomarker Therapeutics

Disease diagnosis/staging (e.g. liver disease) Drug response (e.g. OCA) Diet, prebiotics, probiotics, synbiotics Antimicrobials Microbial transfer (FMT, ASF) Drug targets Therapeutic modifications Host physiologic response Drug ADME

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*Co-Principal Investigators

DNA sequencing, data analysis, and mathematical modeling Frederic D. Bushman, PhD (Penn) Hongzhe Li, PhD (Penn) Rob Knight, PhD (U of C, Boulder)

“Penn Intestinal Microbiome Project Group”

Patient/subject recruitment and phenotyping, dietary assessment, sample collection and processing *Gary D. Wu, MD (Penn) James D. Lewis, MD (Penn) Robert Baldassano, MD (CHOP)

Jun Chen, Sam Minot, Serena Dollive, Eric Chen, Meenakshi Bewtra, Christian Hoffmann, Ying-Yu Chen, Sue

A. Keilbaugh, Kyle Bittinger, Jennifer Hwang, Erin Gilroy, Kernika Gupta, Lisa Nessel, Lindsey Albenberg,

Judith Kelsen, Colleen Judge, Christel Chehoud, David Shen, Rohini Sinha, David Metz, Tatiana Esipova, Susan Parrott, Elliot Friedman, Josie Ni, Sarah Smith, Lillian Chau, Andrew Lin

Gary L. Lichtenstein, MD (Penn) Charlene Compher, PhD, RD (Penn) Anthony Otley, MD (Dalhousie) Anne Griffiths, MD (Toronto) Metabolomics Michael Bennett, PhD (CHOP) Marc Yudkoff, MD (CHOP) Biological Oxymetry Sergei Vinogradov, PhD (Penn) Stephen Thom, MD (Univ. of Maryland)

MICROBIOME PROGRAM

PennCHOP

NIH K08 DK106457 AGA Microbiome Junior Investigator Research Award Center for Molecular Studies in Digestive and Liver Diseases (P30 DK050306) The Joint Penn-CHOP Center for Digestive, Liver, and Pancreatic Medicine