wound healing to longevity: harnessing microbe-induced ...€¦ · lactic acid bacteria *...

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