wound healing to longevity: harnessing microbe-induced ...€¦ · lactic acid bacteria *...
TRANSCRIPT
Wound healing to longevity: Microbe-induced immune
proficiency in human health
Susan E. Erdman Division of Comparative Medicine
Massachusetts Institute of Technology
Human Microbiome Science Vision for the Future
July 24-26, 2013
Can microbe-based restructuring of immune networks
remedy our public health inflammatory disease crisis?
TREG TEFF
sick
healthy
TEFF
TREG
TREG
Healthful longevity Th-2 Th-1 Th-17
Gut microbiota community restructuring in human subjects
Christina Clarke-Dur CPIC &
Stanford University
Eric J Alm Massachusetts Institute of Technology
& Broad Institute
David A Hafler Yale University & Broad Institute
NIH U01 CA164337. GI Tract Dysbiosis and Breast Cancer. [PIs: Erdman, Alm & Wang] 2012 – 2017
California Teacher Study 60,000+ women
TREG TEFF
Does maternal and infant microbial ecology offer opportunity to impart good health to future generations?
Why use animal models?
HUMAN GENERATION TIME: 20 YRS
MOUSE GENERATION TIME: 10 WEEKS
3 6 9 12 15 18 21 24 0 YEARS
NIEHS P30 ES002109. [P30 PIs: Leona Samson John Essigmann] Perinatal microbe exposures. Pilot project co-PIs: SE Erdman & EJ Alm
2011 – 2013
Bronchoalveolar adenocarcinoma in 3/3 ‘grandchild’ outbred Swiss mice (age = 5 months)
Preliminary cancer outcomes in ‘grandchildren’ mice: • 1/3 liver cancer (hepatocellular carcinoma) • 2/3 lymphoma (high grade) • 3/3 lung cancer (bronchoalveolar adenocarcinoma)
Grandma’s microbial ecology may put grandchild at risk for cancer?
Gut microbe-triggered systemic events
(modified from Coombs, et al 2005; Fiona Powrie lab)
Neutrophil recruitment
Macrophages
Inflammatory cytokines
Migration and maturation
Dendritic Cells
TR
IL10
IL10
IL10
TE
TE
CD40-CD40L TR
TGFb IL10
TGFb IL10
Gut microbe-triggered systemic events
(modified from Coombs, et al 2005; Fiona Powrie lab)
Neutrophil recruitment
Macrophages
Inflammatory cytokines
Migration and maturation
Dendritic Cells
CD40-CD40L
Rag2-KO mouse recipient
Transplantable anti-inflammatory CD4+CD45RBloCD25+ lymphocytes
adoptive cell transfer
Cells extracted from immune-competent
donor mouse
Cells injected into recipient without it’s own lymphocytes
Wild type mouse donor
Adoptive Cell Transfer Paradigm
pro-inflammatory CD4+CD45RBhiCD25-
Cells extracted from immune-competent
donor mouse
Cells injected into Rag2-KO recipient without it’s own lymphocytes
Co-transfer anti-inflammatory CD4+CD45RBloCD25+
Sham
Wild type mouse donor
Cells extracted from immune-competent
donor mouse
Microbe infection into Rag2-KO recipient without it’s own lymphocytes
Wild type mouse donor
Transfer anti-inflammatory CD4+CD45RBloCD25+
Sham
Helicobacter hepaticus
We thank Bruce H Horwitz & James G Fox
H. hepaticus-infected
cecu
m
colo
n
Uninfected H. hepaticus-infected
3 6 9 12 15 18 21 24 Start
3-4 wks
Cells of adaptive immunity suppress IBD and carcinoma
Rag2-KO
(Erdman et al 2003)
Gut microbe-triggered systemic events
(modified from Coombs, et al 2005; Fiona Powrie lab)
Neutrophil recruitment
Macrophages
Inflammatory cytokines
Migration and maturation
Dendritic Cells
CD40-CD40L
(modified from Coombs, et al 2005; Fiona Powrie lab)
Neutrophil recruitment
Macrophages
Inflammatory cytokines
Migration and maturation
Dendritic Cells
TR
IL10
IL10
IL10
CD40-CD40L TR
TGFb IL10
TGFb IL10
Gut microbe-triggered systemic events
Inva
sive
col
onic
car
cino
ma
in
H. h
epat
icus
-infe
cted
Rag
2-/-
mic
e
Erdman, et al, Am J Path, 2003
3 6 9 12 15 18 21 24 Start
6-12 wks
Rag2-KO
Infect with H. hepaticus
Mucinous carcinoma
Poorly-differentiated carcinoma
uninfected Rag2-/-
H hepaticus-infected Rag2-/-
Col
onic
car
cino
ma
in
H. h
epat
icus
-infe
cted
Rag
2-/-
mic
e
Erdman, et al, 2003
3 6 9 12 15 18 21 24 Start
24 wks
Rag2-KO
3 wks 6 9 12 15 18 21 24 wks Start
24 wks
Rag2-KO
0.0
4.0
8.0
12.0
16.0
-4.0 **
20.0
* 24.0
IL-6
**
0.0
2.0
4.0
6.0
8.0
-2.0 ***
TNF-α
***
P=0.44 10.0
Rag2-/- control Rag2-/- +Hh
Rag2-/- +Hh + IL10-/-F TReg
Rag2-/- +Hh + IL10-/- TReg + IL10 *** = p<0.001 ** = p<0.01 * = p<0.05
fold
cha
nge
in e
xpre
ssio
n
0
10
20
30
120
-10 **
150 *
180
IL-17 ***
fold
cha
nge
in e
xpre
ssio
n
fold
cha
nge
in e
xpre
ssio
n
H. hepaticus infection up-regulates pro-inflammatory cytokines
Reciprocal relationships exist between Interleukin (IL)-10 and IL-17
Erdman et al 2010 We thank Theofilos Poutahidis
Blocking inflammation leads to total remission of established invasive colonic carcinoma
Pro-inflammatory cells & cytokines Tumor growth
Interleukin-10
TREG
Inter-related roles for IL-10 and TGF-β in colon cancer
PI = SE Erdman
NIH R01 CA108854
[2005 – 2015]
ingestion
H. hepaticus-fed donor
adoptive cell transfer
Prior microbe exposures convey health benefits transplantable via purified immune cells
H. hepaticus dosed orally by gastric gavage
Rag2-KO mouse recipient
Transplantable anti-inflammatory CD4+CD45RBloCD25+ lymphocytes
Cells injected into recipient without it’s own lymphocytes
19 JANUARY 1990 SCIENCE, VOL. 247
we thank Werner Olipitz
Min mouse microbiomes deviate from co-housed Wild type littermate mice
Significant differences exist between microbiota in Min mice and their co-housed wild type littermates
We thank Mark Burnham-Smith (EJ Alm lab) for microbiome analyses
Apc Min/+ mouse recipient
Transplantable anti-inflammatory CD4+CD45RBloCD25+ lymphocytes
adoptive cell transfer
Cells extracted from immune-competent
donor mouse
Cells injected into recipient Wild type litter mate
mouse donor
Adoptive Cell Transfer Paradigm
TREG cells require IL-10 to prevent intestinal adenomas in ApcMin/+ mice
Erdman, et al, 2005 We thank David B Schauer
3 wks 6 9 12 15 18 21 24 wks Start
2-5 days
ApcMin
Sham-treated ApcMin ApcMin + 3ooK wt Treg cells
TREG cells induce regression of intestinal adenomas in ApcMin/+ mice
untreated Apc Min/+ Apc Min/+ plus TREG cells
Age at treatment 4.5 - 6 months (µ age = 5.6 mos)
N = 14; total intestinal adenomas µ = 58 (37-91) N = 14; total intestinal adenomas µ =12(6-43)
µ = 12 adenomas µ = 58 adenomas
(p<0.01)
TREG cells induce regression of intestinal adenomas in Apc Min/+ mice
Erdman, et al, 2005
TREG cells induce apoptosis within intestinal adenomas
Erdman, et al, 2005
P=0.011
Cells extracted from immune-competent
wt donor mouse Wild type mouse donor
Transfer anti-inflammatory CD4+CD45RBloCD25+
Sham
Helicobacter hepaticus
Microbe infection in Min or Min/Rag2-KO recipients
Cells extracted from immune-competent
wt donor mouse Wild type mouse donor
Transfer anti-inflammatory CD4+CD45RBloCD25+
Sham
Helicobacter hepaticus
Microbe infection in Min or Min/Rag2-KO recipients
Cells of adaptive immunity suppress IBD and carcinoma
H. hepaticus-infected ApcMin/+ mice rapidly develop mammary tumors
head
abdominal cavity
forelimb 2 cm
1 wks 2 3 4 5 6 7 8 wks Start
DOD Award W81XWH-05-1-0460. Anti-inflammatory regulatory cells and breast cancer.
[PI: Susan E Erdman]. 2005 – 2006.
Systemic elevation in cytokines or activated cells?
Translocation of intestinal bacteria or antigens?
IL1β MDSC cancer
IL10 TREG TEFF
IL6
TCW
SEE SEE
DAH
TMEN U01 U01 CA164337 SE Erdman (MIT) and EJ Alm (MIT) and TC Wang (Columbia U)
CCD
TC Wang (Columbia)
SE Erdman and EJ Alm (MIT)
C Clarke-Dur (CPIC)
DA Hafler (Yale)
EJA
2.67±0.49
4.91±0.30
0.88±0.21 0.96±0.12
3.44±0.56 2.73±0.38
Spleen Mammary gland LN
Mesenteric LN
H. hepaticus triggers accumulation of MDSCs
Gr-
1
CD11b
Mammary gland LN Mesenteric LN Spleen
ApcMin/+
ApcMin/+ H. hepaticus
P=0.0172
P=0.0127 P=0.0211 *
* *
(we thank TC Wang)
ApcMin/+
ApcMin/+ sham-dosed mammary gland
H. hepaticus infection promotes mammary tumorigenesis in C57BL/6 ApcMin/+ mice
ApcMin/+ sham-dosed mammary LN
ApcMin/+ + Hh
ApcMin/+ sham mammary gland ApcMin/+ + Hh mammary gland (tumor)
ApcMin/+ + Hh mammary gland
ApcMin/+ + Hh mammary LN (we thank TC Wang)
Bacteria
ingestion
RagMin or Min recipient
Transplantable anti-inflammatory CD4+CD45RBloCD25+ lymphocytes
Microbial benefits
adoptive cell transfer
Cells extracted from immune-competent
donor mouse
Cells injected into recipient Microbe-fed wt donor
Adoptive Cell Transfer Paradigm
Pro-inflammatory cells & cytokines Tumor growth
Interleukin-10
TREG
Do intestinal bacteria modulate breast cancer?
IL-10
Systemic inflammatory response
In h
ealth
TREG
INTESTINE
In p
atho
geni
c gu
t inf
ectio
n
normal
BREAST carcinoma
D
C
B
A
Translocation of bacteria or antigens?
Bow
el e
pith
elia
Bre
ast e
pith
elia
l inj
ury
& d
ysre
gula
tion
Systemic elevation in cytokines or activated cells?
Earlier exposures to gut microbes
impact potency of CD4+ lymphocytes to modulate extra-intestinal cancers.
We thank Mark Burnham-Smith (EJ Alm lab) for microbiome analyses
p<0.05
Increased polyposis in Min mice after adoptive transfer of
‘hygienic’ T cells
Significant changes occur in Min mice after adoptive transfer of ‘hygienic’ T cells
(Erdman et al 2010)
Microbe-educated lymphocytes impart longevity
hygienic’ cells
‘primed’ cells
IL1β MDSC cancer
IL10 TREG TEFF
IL6
TCW
SEE SEE
DAH
TMEN U01 U01 CA164337 SE Erdman (MIT) and EJ Alm (MIT) and TC Wang (Columbia U)
CCD
TC Wang (Columbia)
SE Erdman and EJ Alm (MIT)
C Clarke-Dur (CPIC)
DA Hafler (Yale)
EJA
inflammation
TREG
breast cancer ovarian cancer endometrial cancer thyroid cancer melanoma
Hygienic rearing & GI tract dysbiosis (Christina Clarke-Dur)
Gut microbiota community restructuring in human subjects after novel exposures
Lactic Acid Bacteria *
ingestion
Probiotic-fed donor Rag2-KO recipient
Transplantable CD4+gfp-Foxp3+ lymphocytes
Probiotic benefits adoptive cell transfer
Microbe benefits are transplantable via immune cells
* We thank James Versalovic for the gift of Lactobacillus reuteri ATCC 6475
Yassin Ibrahim Bernard Varian Tatiana Levkovich
Jessica Lakritz Kelsey Cappelle
Growing luxuriant hair
Hair follicles (HF) in subcutis
Hair follicles (HF) in subcutis
Glow of health
3 wks 6 9 12 15 18 21 24 Birth
20 wks
1 2 3 4 5 6 7 8 wks Cell transfer
Rag2-KO
Probiotic benefits transplantable
via lymphocytes Cells injected into recipient
without it’s own lymphocytes
Gfp-Foxp3+ cells
Pro-inflammatory cells & cytokines Obesity
Interleukin-10
TREG
frequent = at least once a week up to daily
Humans Eating at Fast Food Restaurants * P<0.05
The one that eats probiotics daily, left, is slimmer and shinier than his brother
These mice are wild type litter mate brothers …..
v
v
v
v
v
vv
v
vv
v
Serum thyroid hormone (free T4) levels remain in high-normal range even with increasing age
age of mice
thyr
oid
horm
one
(free
T4)
ng/
dl
2 months 8 months
v control
L. reuteri
L. reuteri-treated mouse paired-testes weights
Survival to wean (control) = 67% (351/563) (L. reuteri) = 98% (622/630)
Reproductive Fitness
Reproductive Fitness
“my bacteria made me do it”
Importance of wound healing capability
Aonghus O’Loughlin and Timothy O’Brien (2011). Topical Stem and Progenitor Cell Therapy for Diabetic Foot Ulcers, Stem Cells in Clinic and Research, Dr. Ali Gholamrezanezhad (Ed.), ISBN: 978-953-307-797-0, InTech, DOI: 10.5772/19070.
1 day 2 3 4 5 6 7 8 Biopsy
Skin wounds heal twice-as-fast when mice are eating L. reuteri
1 day 2 3 4 5 6 7 8 Biopsy
Skin wounds heal twice-as-fast when mice are eating L. reuteri
ingestion
Rag2-KO recipient
Transplantation of CD4+Foxp3+gfp lymphocytes
Probiotic benefits
adoptive cell transfer
Cells extracted from gfp donor mouse
Cells injected into Rag-KO recipient without it’s own lymphocytes Probiotic-fed donor
Lactic Acid Bacteria
Lactobacillus reuteri-induced phenotypes are transplantable
1 day 2 3 4 5 6 7 8 days Biopsy
Rag2-KO
Probiotic benefits transplantable
via lymphocytes Cells injected into recipient
without it’s own lymphocytes
Gfp-Foxp3+ cells
Pro-inflammatory cells & cytokines Tumor growth
Interleukin-10
TREG
“my bacteria made me do it”
1 day 2 3 4 5 6 7 8 days Biopsy
L. reuteri-induced improvement in wound repair requires oxytocin
NIEHS P30 ES002109. [P30 PIs: Leona Samson John Essigmann] Perinatal microbe exposures. Pilot project co-PIs: SE Erdman & EJ Alm
2011 – 2013
Bronchoalveolar adenocarcinoma in 3/3 ‘grandchild’ outbred Swiss mice (age = 5 months)
Preliminary cancer outcomes in ‘grandchildren’ mice: • 1/3 liver cancer (hepatocellular carcinoma) • 2/3 lymphoma (high grade) • 3/3 lung cancer (bronchoalveolar adenocarcinoma)
Grandma’s microbial ecology may put grandchild at risk for cancer?
TREG TEFF
Does human maternal and infant microbial ecology offer opportunity to impart good health to future generations?
Can microbe-based restructuring
of immune networks improve our public health?
Harnessing microbes for public health
Do lactic acid bacteria offer an immediate palatable public health remedy ?
Harnessing microbes for public health
Prospective longitudinal studies to identify hygienic signatures for risk assessment
and microbial rescue strategies ?
Harnessing microbes for public health
Probe epigenetic/genetic impact of microbes upon host progeny and their offspring
Thank you! • Eric Alm • Karen Sue Anderson • Christina Clarke-Dur • Bevin Engelward • John Essigmann • James G Fox • David A Hafler • Bruce H Horwitz • Theofilos Poutahidis • Leona D Samson • David B Schauer • James Versalovic • Jerrold Ward • Timothy C Wang