Microbial hitchhikers And the leaky gut - Gut Microbes · Microbial hitchhikers And the leaky gut...
Transcript of Microbial hitchhikers And the leaky gut - Gut Microbes · Microbial hitchhikers And the leaky gut...
Microbial hitchhikers
And the leaky gut
Suzanne Devkota Ph.D. Cedars-Sinai Medical Center
Gut Microbiome Conference September 25. 2016
Defining Microbiome Research
Evolutionary Biology
(The principle)
Microbiology (Microbe)
Physiology (Host)
Ecology
Mucosal immunology
Microbiome Research
Gastroenterology
Genetics
Infectious diseases
Global Prevalence of
Diabetes- 2014
52M
7.9%
22M
5.1%
138M
8.5%
39M
11.4%
75M
8.7%
25M
8.1%
Source data: IDF Atlas 6th Edition Update, 2014 and CDC.gov
2011
2005
2000
1995
1990
1985
8% 6% 4% 0% 2%
2.7%
2.7%
3.1%
4.4%
5.6%
6.9%
2.5% 1980
Diabetes incidence in the U.S.
1980-2011
0
200,000
400,000
600,000
800,000
Denmark Canada U.K. China United States
Crohn's Disease Ulcerative Colitis
Trends in Incidence Rate of CD and UC in the US 1970-2011
6
9
12
15
Rate
of
new
ly d
iagnosed
cases p
er1
00,0
00 p
eople
1970-1979 1980-1989 1990-1999 2000-2009
Crohn’s
Disease
Ulcerative
Colitis
Prevalence and
Incidence of IBD-
Crohn’s and
Ulcerative Colitis
Nations with highest prevalence of IBD
Tota
l dia
gnosed c
ases
Source data: CCFA.org
Highest rates are in industrialized nations
• A move away from agrarian diets
• Higher energy density of foods
• More food additives
• Hyper-cleanliness
• Overuse of antibiotics
• Lack of physical activity
A shifting microbial landscape
Dietary Inputs
Fatty acids, Fibers, Amino
acids, Sugars, Food
additives, Choline, Carnitine,
Gluten, Fasting, Starvation
Gut Microbial Reactions
B. wadsworthia, B. fragilis,
SFB, A. muciniphila,
M.smithii, Clostridium cluster
XIV, Desulfovibrio
(Fungi and Viruses too!)
Mucosal Immune Responses
T- cell differentiation,
Foxp3+Treg pool and IL10
production, antimicrobial
peptides, serum amyloid A
Disease State
Byproducts/Metabolites
H2S, methane, SCFAs,
indoles, biotin, deoxycholic
acid, polysaccharide A
The mucosal barrier: our only protection from the outside world
Food particles
Bacteria, viruses, fungi Toxins
Microbial byproducts
Amerindia
United States
Malawi
Yatsunenko et al, Nature (2012)
Amerindia
United States
Malawi
Malawian
Yatsunenko et al, Nature (2012)
U.S.
Malawian
U.S.
Amerindian
Amerindian
Malawian
Sonnenburg, et al. Nature, 2016
Characteristics of the American diet
(NHANES, 2011)
• 2,618 kcal/d
• 49% carbohydrate
• 15.5% protein
• 32.8% fat
• 10.9% saturated fat (of
total kcal)
Study Diets Low-fat
(AIN-93M)
Saturated-
Milk Fat
Saturated-
Lard Fat
PUFA
(Safflower oil)
Fat (%kcal)
-Saturated
(%total)
-PUFA
Protein
Carbohydrate
Micronutrients/
Fiber
5
0.6
3.9
19
66
*ND
38
65
3.5
16
47
*ND
38
39
11
16
47
*ND
38
9
78
16
47
*ND
Fatty acid composition of common dietary fats
Milkfat Lard Safflower
Oil
4:0 Butyric 3.8
6:0 Caproic 2.3
8:0 Caprylic 1.1
10:0 Capric 2
12:0 Lauric 3.1
14:0 Myristic 11.7 1.5
16:0 Palmitic 26.2 24.8 6.5
16:1 Palmitoleic 1.9 3.1
17:0 Margaric 0.7
18:0 Stearic 12.5 12.3 2.4
18:1 Oleic 28.2 45.1 13.1
18:2 Linoleic 2.9 10 78
20:0 Arachidic 0.2
20:4 Arachadonic 0.1 0.4
20:5 EPA
22:5 DPA
22:6 DHA
Devkota et al., Nature 2012
Sartor, Nat Rev Gastro Hep. 2012
High Fat Feeding
Change Gut Flora
Increased Permeability
Increased systemic LPS/Endotoxemia
Inflammation
Diet-induced gut permeability
Metabolic Disorders
Samples: Macaques, Mice, & Humans
Unpublished (Macaque: Lean vs. Obese)
Subcut aneous
fa t
Inguina l
LN
M esent eric
LNM esent eric
Fat
Liver
Serum
St ool
Int est ine
Om ent a l
Fat
Skin
Unpublished (Mouse: Normal chow vs. HFD)
Epididym al
fa t
Subcut aneous
fa t
M esent eric
Fat
St ool
Int est ine
Unpublished (Human: Healthy vs. T2DM)
Subcut aneous
fa t
Om ent a l
Fat
Adipose tissue bacterial culture
16 weeks
High Fat Diet
Animals: (n = 22)
Adipose depot 1,000 ul PBS
+
sterile beads
Homogenize
800 ul PBS
Plate
Aerobic Anaerobic
5 days
(37oC)
Readout 1:
- CFU
Readout 2:
- Pool colony to ID taxa via 16S sequencing
High Fat Diet
vs.
Normal Chow
Sample Collection
Gut contents:
- Stool
Adipose:
- Mesenteric
- Inguinal
- Subcutaneous
Live, culturable bacteria in adipose! Mesenteric > subcutaneous > inguinal
Marta Wlodarska (Broad/Novartis) & Koji Yasuda (Harvard School of Public Health)
Unpublished
108 – 1010 / gram of stool Gut
102 – 104 / gram of fat Adipose
MES SQ ING1
10
100
1000
10000
100000
CF
U/g
ram
fa
t
Aerobic
anaerobic
Could Bifidobacteria be conferring beneficial effects in adipose?
Greater enrichment of anaerobic species in HFD adipose
anaerobic aerobic
Bifidobacterium
Bacteroides
Parabacteroides
Bacteroidets
Staphylococcus
Enterococcus
Lactobacillus
Lactococcus
Streptococcae
Clostridiacae
Peptostreptococcae
Allobaculus
Sutterella
Enterobacteraceae
Pasturellaceae
Hemophilus
Adipose tissue 16s sequencing
16 weeks
High Fat Diet
Animals: (n = 16)
High Fat Diet
vs.
Normal Chow
Sample Collection
Gut contents:
- Stool
- Colon
- Cecum
- Ileum
Adipose:
- Mesenteric
- Inguinal
- Subcutaneous
Adipose depot
DNA extraction
PCR amplification
(V4 region of 16S)
Miseq sequencing
AGCTAGCT
…GTC
TTCGATTC…
AAA
TCGATCGG
…GCG
CCGATCGC
…
Figure S4, related to Experimental Procedures. Study design and survey of primate gut microbial biogeography and microbial functional
potentials with and without Helicobacter A) Paired intestinal mucosal and lumenal contents were collected from the ileum, ascending, transverse,
and descending colon of 15 clinically-healthy rhesus macaques, in addition to stool and a sample of jejunal mucosa. The microbiome of the samples
was profiled by sequencing the V4 region of the 16S rRNA gene. B) After sequencing, community structure, function, and covariation with
biogeography were characterized by ordination (Caporaso et al., 2010), univariate (Segata et al., 2011a) and multivariate (Morgan et al., 2012)
association testing, metagenomic inference (Langille et al., 2013), and logistic regression. C) Comparison of the gastrointestinal tracts of humans and
rhesus macaques. In contrast to macaques, humans lack a prominent cecum. The total length of the GI tract is 6-7 m for an adult human and 1.5-2m
for an adult rhesus macaque. Comparison of intestinal microbial mass (Solnick et al., 2006; Walter and Ley, 2011), pH (Mercier et al., 2007; Walter
and Ley, 2011) and transit time (Dubois et al., 1977; Mercier et al., 2007). Percent of intestinal length is normalized to an intestinal length of 6.5 m
for humans and 1.75 m for a macaque, for comparison purposes. D) PICRUSt (Langille et al., 2013) was used to infer community function, and
LEFSe (Segata et al., 2011b) was used to determine which functions were most differential between the mucosa and lumen and LI and SI. Due to the
high abundance of Helicobacter, this analysis was repeated with Helicobacter removed. The ten largest LDA effects are shown here. The top and
bottom two panels are derived from 16S data including Helicobacter, and excluding Helicobacter OTUs, respectively.!
Microbial communities are determined primarily by diet and location
HFD
Control Gut
Adipose
High-fat vs. Low-fat Gut vs. Adipose
Pronounced HFD-induced changes in genus across body sites R
ela
tive a
bundance (
%)
Ileum Cecum Colon Stool Mesenteric Fat Inguinal Fat SQ Fat
In humans, diabetic adipose microbiota appear indistinguishable from healthy
T2DM
Normal
Omental fat (PC1 vs. PC2)
Subcutaneous fat (PC1 vs. PC2)
T2DM
Normal
Frozen samples, no diet records, no gut comparison
Extr
acti
on
P
CR
P
osit
ive
No
rmal o
men
tal
T2D
M o
men
tal
No
rmal sq
T2D
M s
q Human fat
Pre
-t2D
M(5
), u
nko
wn
(3),
T1 (
1),
om
Pre
-t2D
M(5
), u
nko
wn
(3),
T1 (
1),
sq
Macaque fat Macaque gut
Liv
er
ILN
ML
N
Seru
m
Skin
Macaque Mouse fat gut
Genus level relative abundance without contaminant
without Ralstonia
24
Prevalence of oligotypes among different tissue types and
hosts
HUMAN: Bacteria_unclassified_phylum MACAQUE: Streptococcus
Bacterial taxa absent in controls but are unique to host species MACAQUE:
Christensenellaceae
25
Prevalence of oligotypes among different tissues and hosts
Turicibacter oligotype 1 Turicibacter oligotype 2
Two similar taxa behaving differently across sample types
Blood
Adipose and stool collection from gastric bypass patients
Study Workflow
Human Adipose Tissue Microbiome – 2016P- 000259 Color designation = study staff responsible: =AV =GC =AV+GC =KY =Misc.
Visit 1:
Pre-Op: Input Description: Output Follow-Up
Source document - questionnaire
2 copies, consent form Pre-op sample collection
kit: Saliva & Stool kit Instructions Biohazard bag Freezer packs
Blood work CBC, A1C, CMP, lipids
Identify potential patients Fill out questionnaire Consent patients Provide collection kit Order pre-op blood work
if not typical
Scan consent Scan questionnaire Input data into
REDCap
Visit 2:
Morning of:
Input Description Output Follow-Up
Verify pre-qualifying exclusionary criteria questions
Blood work CBC, A1C, CMP, lipids
Post-op sample collection kit: Saliva kit & Stool kit Instructions Biohazard bag Freezer packs
Order pre-op blood work if not typical
Collect saliva & stool samples from patient
Pre-op samples Saliva sample Stool sample Blood sample
Input sample collection data into REDCap
Joslin Diabetes Ctr: Whole blood + serum: -
80°C freezer
Harvard School of PH: RNA+DNA (bact)
Intra-Op:
Input Description Output Follow-Up
Intra-op sample collection Aerobic culture tube Anaerobic culture tube Tissue collection cup
PBS, RNA later, liquid nitrogen, cryotubes
Mesenteric adipose [1-3] Near and/or far
Omental adipose [1-3] Near and/or far
Subcutaneous adipose [1-3]
Adipose tissue samples (#3-12)
PBS, RNA later, Liquid Nitrogen, cryotubes
Input sample collection data into REDCap
BWH Microbiology: Culture
Joslin Diabetes Ctr: Tissue: -80°C freezer
Harvard School of PH:
RNA+DNA (bact)
Visit 3:
Post-Op:
Input Description Output Follow-Up
Blood work CBC, A1C, CMP, lipids
Order pre-op bloodwork if not typical
Collect saliva & stool samples from patient
Post-op samples Saliva sample Stool sample Blood sample
Input sample collection data into REDCap
Joslin Diabetes Ctr: Whole blood + serum: -
80°C freezer
Harvard School of PH:
RNA+DNA (bact)
Cedars cultures
Subject Metadata
Record ID
0259-001 AGES
0259-002 BOAT
0259-003 LACE
0259-004 TOWN
0259-005 MEMO
0259-006 OATS
Gender Diabetic HbA1c BMI Waist (") Surgery DOS Probiotics Date, probx EtOH
F No 5.2 54.24331361 Sleeve 5/9/2016 No No
F No 6.1 42.86369363 Sleeve 5/25/2016 No Yes
F No 5.4 53.99506836 35 RYGB 6/8/2016 No No
F No 5.2 58.36455042 48 Sleeve 5/16/2016 No No
M Yes 7.3 76.42325685 71 Sleeve 6/6/2016 Yes 5/17/2016 No
F No 5.1 42.92883379 49 Sleeve 6/13/2016 No Yes
1 M : 5 F
1 DM : 5
No
0 Lean : 6
Obese
[2 not
obtaine
d pre-
op]
1 RYGB :
5
sleeve
1 probiotics : 5
NONE
2
EtOH
: 4
No
Smoking Stop Date PPD Age, wt gain MAX wt Pre-op Body type PRE Saliva HH:MM elapsed PRE Stool HH:MM elapsed
No 8 340 Apple Yes 0:04 Yes 4:00
Yes Yes 2009 0.25 30 260 Apple Yes 0:05 Yes 14:00
No 7 315 Apple Yes 2:00 Yes 11:00
No 9 311 Pear No No
Yes Yes 2009 1.5 1 521 Apple Yes 0:01 No
No 12 274 Apple Yes 13:00 Yes 13:00
2 Past
smoker :
4 Never
1 No PRE :
5 Yes PRE
SALIVA All w/in 24 HRS
2 No PRE
: 4 Yes
PRE
STOOL All w/in 24 HRS
Amt/Freq
4/mo
1/mo
Record ID
0259-001 AGES
0259-002 BOAT
0259-003 LACE
0259-004 TOWN
0259-005 MEMO
0259-006 OATS
Growth so far…
DOS Subject Subject Acronym BWH core ID Location BMI Sx Type Diabetic GROWTH
5/9/2016 001 AGES 1 SQ 54.33 SLEEVE
5/9/2016 001 AGES 2 Gastro-colic 54.33 SLEEVE
5/16/2016 004 TOWN 3 SQ 58.34 SLEEVE
5/16/2016 004 TOWN 4 OM 58.34 SLEEVE
5/16/2016 004 TOWN 5 Gastro-colic 58.34 SLEEVE
5/25/2016 002 BOAT 6 SQ 42.95 SLEEVE
5/25/2016 002 BOAT 7 Gastro-colic 42.95 SLEEVE
5/25/2016 002 BOAT 8 OM 42.95 SLEEVE
6/6/2016 005 MEMO 9 SQ 76.44 SLEEVE YES
6/6/2016 005 MEMO 10 GC 76.44 SLEEVE YES YES
W 6/6/2016 007 ANTS 11 SQ 25.87 CHOLECYSTECOMY
W 6/6/2016 007 ANTS 12 GC 25.87 CHOLECYSTECOMY
6/8/2016 003 LACE 13 OM 55.74 R-YGB
6/8/2016 003 LACE 14 GC 55.74 R-YGB
6/13/2016 006 OATS 15 GC 43.00 SLEEVE
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
YES
Acknowledgements
Cedars-Sinai
Connie Ha
Anthony Martin
Joslin Diabetes Center
Allison Goldfine
Brigham and Women’s Hospital
Lynn Bry
Ashley Vernon
Gemma Carter
Harvard School of Public Health
Curtis Huttenhower
Koji Yasuda
Broad Institute/Novartis
Marta Wlodarska
Funding: