Microbiota y la respuesta immuneMicrobiota y la respuesta immune

Romina Goldszmid, PhDRomina Goldszmid, PhD

Humans have co-evolved with microbial partners

• We are a composite of species: bacteria, fungi, viruses, bacteriophages

• Microorganisms inhabit all barrier surfaces of the organism and outnumber the human cells by about 10 fold

• The total microbial DNA in our body (the microbiome) contains 100 times more genes than our ‘own’ human genome

• The microbiome is an integral part of our genetic landscape and plays a central role in the maintenance and control of host homeostasis

• The development of the immune system is dependent on interactions with the commensal microbiota

VaginVaginaa

StomachStomach

OesophagusOesophagus

MouthMouth

SkinSkin

ColonColon

FirmicutesBacteroidetesActinobacteriaProteobacteriaOther phyla

Nat Immunol. 2013 Jul;14(7Compartmentalized and systemic control of tissue immunity by commensals.Belkaid Y, Naik S.

Microorganisms inhabit all barrier surfaces of the organism

Healthy skin bacterial survey

Grice et al., Science 2009

• Changes in the last trimester of pregnancy

Development of the microbiota from the first inoculum as an infant through continuous change, modified by diet, genetics and the

environment, through life

Dominguez-Bello MG, Blaser MJ, Ley RE, Knight R. Development of the human gastrointestinal microbiota and insights from high-throughput sequencing. Gastroenterology. 2011;140:1713-9.

Changes in our microbiota…Changes in our microbiota…

AntibioticsAntibiotics

LifestyleLifestyle NutritionNutrition HygieneHygiene

InfectionsInfections

DysbiosisDysbiosisHost

GeneticsHost

Genetics

ProbioticsProbiotics

AgeAge

Mode of delivery

Mode of delivery

Geographical location

Geographical location

Microbiota-induced maturation of the mouse gastrointestinal tract

Sommer F, Bäckhed F. The gut microbiota--masters of host development and physiology. Nat Rev Microbiol. 2013;11:227-38.

Nadine Cerf-Bensussan and Valerie Gaboriau-RouthiauThe immune system and the gut: friends or foes?Nature Reviews Immunology 10:735 (2010)

Modulation of adaptive immune responses in the gut by the commensal microbiota

The intestinal microbiota enhances colonization resistance to intestinal pathogens by both direct and indirect (immune-mediated) mechanisms of

action.

Charlie G. Buffie & Eric G. Pamer1Microbiota-mediated colonization resistance against intestinal pathogensNature Reviews Immunology 13:790 (2013)

How Commensal Bacteria affect How Commensal Bacteria affect Local and Systemic Inflammation/Immunity?Local and Systemic Inflammation/Immunity?

Intestinal microbiotaIntestinal microbiota

Mucosal ImmunityMucosal Immunity

Skin ImmunitySkin ImmunitySkin microbiotaSkin microbiota

Intestinal microbiotaIntestinal microbiota

Compartmentalized control of barrier site Compartmentalized control of barrier site immunity by resident commensalsimmunity by resident commensals

IntestineIntestine

SkinSkin

Mouth Mouth Pharynx Pharynx

Respiratory Respiratory systemsystem

Urogenital Urogenital tracttract

IL-17A production in skin tissue is not impacted by distinct gut commensal populations

Gut Skin 01

23

4

567

8 TaconicJackson

% I

L-1

7A+ C

D45

+

NS

Science 337, 1115 (2012)

Taconic Farms

Jackson Labs

SFB

Gut 01

23

4

567

8

TaconicJackson

% I

L-1

7A+ C

D4

5+

Altered T cell inflammatory and regulatory profile in skin tissue of germ free mice.

Skin

SPF GF0

8

16

24

32

SPF GF 0

10

20

30

40

50

60

70

% IF

Nγ+

αβ T

Cel

ls

% F

oxp3

+ T

regs

% IL

-17A

+ γδ

T C

ells

% IL

-17A

+ αβ

T C

ells

SPF GF0

4

8

12 **

SPF GF0

5

10

15

20

25 **

* **

SPF- Specific Pathogen Free GF- Germ Free

Germfree

4 weeks

Oral Antibiotic (ATB)

AmpicillinMetronidazoleNeomycinVancomycin

% IL

-17A

+ C

D45

+%

IL-1

7A+ C

D45

+

0

5

10

15

20

25

0

10

20

30

% IF

Nγ+

αβ T

Cel

ls%

IFN

γ+ αβ

T C

ells

**

0

2

4

6

8

0

1

2

3

4

5

***GUT

SKIN

VehicleATB

Tumors

Colon rectal carcinomaColon rectal carcinomaStomach cancerStomach cancer

Malt lymphomaMalt lymphomaHepatocellular carcinomaHepatocellular carcinoma

Mammary carcinomaMammary carcinomaThymic lymphomaThymic lymphoma

Humans are Humans are

metaorganismsmetaorganisms(symbionts)(symbionts)

composed of host composed of host and microbial cells and microbial cells

with their own with their own genes genes

(metagenome) and (metagenome) and shared metabolic shared metabolic

processes and processes and products products

(metabolome). (metabolome).

MetabolismMetabolism

Cardiovascular, Cardiovascular, Excretory,Excretory,

Musculoskeletal,Musculoskeletal,and Adipose tissue and Adipose tissue

functionsfunctions

Neurological. Neurological. behavioral behavioral

and cognitive and cognitive functionsfunctions

AgingAging

HematopoiesisHematopoiesis

Circadian rhythmCircadian rhythm

Inflammation and Inflammation and ImmunityImmunity

Cancer initiation, Cancer initiation, progression and progression and

response to response to therapytherapy

The human The human metaorganismmetaorganism

Both microbial and Both microbial and human cells act as human cells act as

sensors for sensors for environmental changes environmental changes

communicating communicating reciprocally via signaling reciprocally via signaling pathways that, in part, pathways that, in part, utilize innate immunity utilize innate immunity

mechanisms.mechanisms.

Environmental Environmental factorsfactors

FoodFoodChemicalsChemicals

TemperatureTemperatureRadiationRadiation

Physical and Physical and psycological psycological

stressstressPathogensPathogens

………………

To understand the precise mechanisms that affect To understand the precise mechanisms that affect health and disease and to target them for disease health and disease and to target them for disease

prevention and cure prevention and cure

THE CHALLENGE:THE CHALLENGE:

Gut microbiota control of Gut microbiota control of systemic immunity/inflammation systemic immunity/inflammation

Mets

Genomic Genomic mutationsmutations

Tumor Tumor promotionpromotion

Chronic Chronic inflammation inflammation

(infections, (infections, aseptic)aseptic)

Intrinsic / Intrinsic / oncogene oncogene induced induced

inflammationinflammation

Predisposing conditions Predisposing conditions (obesity, metabolic syndrome)(obesity, metabolic syndrome)

Cancer Cancer associated associated

inflammationinflammation

Tumor growthTumor growth

AngiogenesisAngiogenesis

Tissue remodeling Tissue remodeling Infiltration and Infiltration and

MetastasisMetastasis

Immuno Immuno evasionevasion

Co-morbiditiesCo-morbidities

Response to Response to therapytherapy

Anti-cancer Anti-cancer immune immune responseresponse

Primary Primary tumortumor

MicrobiotaMicrobiota

MicrobiotaMicrobiotaMicrobiotaMicrobiotaPathobiontsPathobiontsPathogensPathogens

A Dzutsev, RS Goldszmid, S Viaud, L Zitvogel, G Trinchieri. The role of the microbiota in inflammation, carcinogenesis and cancer therapy. Eur J Immunol 2014 Oct 18 DOI: 10.1002/eji.201444972

Is the response to cancer therapy regulated by the commensal bacteria?

Intestinal microbiotaIntestinal microbiota

Sterile subcutaneousSterile subcutaneoustransplantedtransplanted

tumortumor

Systemic anti-IL-10R + Intratumor CpG-OGN immunotherapySystemic anti-IL-10R + Intratumor CpG-OGN immunotherapyPlatinum compound (oxaliplatin, cisplatin) chemotherapyPlatinum compound (oxaliplatin, cisplatin) chemotherapy

ANTIBIOTICSANTIBIOTICSNeomycinNeomycinVancomycinVancomycinImipenemImipenem

or Germ-free miceor Germ-free mice

Noriho Iida, Amiran Dzutsev, C. Andrew Stewart, ……… Giorgio Trinchieri, Romina S. Goldszmid Commensal bacteria control cancer response to therapy by modulating the tumor microenvironmentScience, 2013; 342:967-70

CTX, oxaliplatin and CpG (+/- anti-IL-CTX, oxaliplatin and CpG (+/- anti-IL-10R) but not cisplatin induce 10R) but not cisplatin induce

Immunogenic Cell DeathImmunogenic Cell Death

Direct toxicity(Innate) (0-48h) Adaptive (>7 days)

Intratumoral CpG + systemic anti-IL-10RIntratumoral CpG + systemic anti-IL-10R

Systemic oxaliplatin or cisplatinSystemic oxaliplatin or cisplatin

Cyclophosphamide (CTX) Cyclophosphamide (CTX)

Days after treatment

CpG-OGN Oxaliplatin Cisplatin CyclophosphamideCpG-OGN Oxaliplatin Cisplatin Cyclophosphamide

Response to Cancer Immunotherapy and Chemotherapy Requires the Commensal Microbiota

TxTx + AntibioticsUntreated

Sophie Viaud, ……….and Laurence ZitvogelThe intestinal microbiota modulates the anticancer immune effects of cyclophosphamideScience, 2013, 342:9671-74

Noriho Iida, Amiran Dzutsev, C. Andrew Stewart, ……… Giorgio Trinchieri, Romina S. Goldszmid Commensal bacteria control cancer response to therapy by modulating the tumor microenvironmentScience, 2013; 342:967-70

Sterile subcutaneous mouse transplantable tumors

WT (BL6Ncr) TNFKO

H2Ountreated

H2OaIL-10RCpG

ABXaIL-10RCpG

1 cm

Antibiotics (ABX) suppress TNF-mediated early necrosis of the tumor and decrease inflammatory cytokine production following anti-IL-10R/CpG

Most inflammatory but not anti-inflammatory (e.g. IL10) genes are lower in ABX treated mice

ABX decrease TNF and IL-12 production by tumor-ABX decrease TNF and IL-12 production by tumor-infiltrating myeloid cells following aIL-10R/CpGinfiltrating myeloid cells following aIL-10R/CpG

H2O aIL-10R/CpGABX aIL-10R/CpGABX untreatedH2O untreated

MC38 tumor, 72 h after CpG treatment

** P=0.05

Oral LPS partially restores the TNF production impaired by ABX and TLR4 signaling is required for the effective

anti-tumor response

aIL-10R/CpG

25mg/kg BW of LPS was orally administered 3 times/week, 2 weeks prior and 1week after tumor injection

Cytokine production by tumor infiltrating myeloid cells

Tlr4-/- mice fail to respond to aIL-10R/CpG

High TNF

Low TNF

Composition of fecal microbiota can be used to segregate mice with high and low intratumoral TNF in response to CpG

Unweighted UnifracH2O- drinking mice

PCA1

PCA

2

PCA

3

PCA1

16S rDNA analysis using 454 pyrosequencing

Identification of bacterial genera positively correlating with intratumoral TNF levels after CpG in microbiota perturbation experiments

Single Single unclassified unclassified

OTUOTU

A. shahiiA. shahii Alistipes (Gram-)

Ruminococcus (Gram+)

L. murinumL. intestinalis L. fermentumL. fermentum

Identification of bacterial genera negatively correlating with intratumoral TNF levels after CpG in microbiota perturbation experiments

Oxaliplatin:

ABX treatment blocks oxaliplatin-induced gene espression changes

ROS production Nox1CybbSod1Sod2

microarray analysis of total EL4 tumors microarray analysis of total EL4 tumors

Antibiotics (ABX) impair oxaliplatin chemotherapy by preventing ROS Antibiotics (ABX) impair oxaliplatin chemotherapy by preventing ROS production from NOX2 (production from NOX2 (CybbCybb) expressing myeloid cells) expressing myeloid cells

EL4 tumors-bearing B6 mice were treated with 10mg/kg oxaliplatinROS-induced bioluminescence using the L-012 probe was analyzed 24 hours after oxaliplatin injection

Oxaliplatin induces ROS production in tumors of control

but not ABX-treated mice

MFI

RO

S x

10-3

20

40

60

0

80

WT Cybb-/-

H2O

Ctrl Oxp Ctrl Oxp

ABX

Ctrl Oxp

H2O

ROS+ Gr1hi

(neutrophils)

ROS- production analyzed by flow cytofluorimetry in EL-4 tumor-infiltrating myeloid cells ex-vivo 24 hours after oxaliplatin injection

20

40

60

0

WT Cybb-/-

H2O

Ctrl Oxp Ctrl Oxp

ABX

Ctrl Oxp

H2O

ROS+ Ly6C+F4/80+

(monocyte-derived)

Oxaliplatin induces NOX2 (Cybb)-mediated ROS production in

tumor-associated myeloid cells

Sophie Viaud, ……….and Laurence ZitvogelThe intestinal microbiota modulates the anticancer immune effects of cyclophosphamideScience, 2013, in press

Cyclophosphamide (CTX) induces mucosal bacterial translocation that is required for anti-tumor Th17 response

Microbial translocation following total body irradiation augments the function of adoptively transferred tumor-specific CD8+ T cells

Paulos CM …..Restifo NP.

Microbial translocation augments the function of adoptively

transferred self/tumor-specific CD8+ T cells via TLR4 signaling.

J Clin Invest. 2007;117:2197-204

Adoptive transfer of pmel-1 tumor reactive CD8+ T cells into animal bearing established B16F10 melanoma tumors

TBI leads to: Destruction of established tumors

Enhanced autoimmune vitiligo

Increased pmel-1 T cells cytokine production 5 days after transfer

Paulos CM …..Restifo NP.

Microbial translocation augments the function of adoptively

transferred self/tumor-specific CD8+ T cells via TLR4 signaling.

J Clin Invest. 2007;117:2197-204

TBI causes mucosal barrier injury resulting in bacteria translocation, increased serum LPS and activation of innate immune response.

ABX leads to: Reduced systemic LPS level

Impairedactivation of antigen presenting cells

Reduced effectiveness of adoptive cell transfer therapy

Microbial translocation following total body irradiation augments the function of adoptively transferred tumor-specific CD8+ T cells

Myeloid cell Tumor

CTL

TBI and

adoptive T cell

transfer

CTX

Th17 CTL

Gut microbiota

TBI or CTX induced transmucosal bacterial translocation

ROS

Oxaliplatin

CpG-ODN + anti-IL-10R TNF

Modified from:Goldszmid R.S., Dzutsev A., Trinchieri G.Host immune response to infection and cancer: unexpected commonalitiesCell Host & Microbe, 15, 295-305, 2014

Iida N, Dzutsev A, Stewart CA ……, Trinchieri G,

Goldszmid RS.

Commensal bacteria control cancer response to

therapy by modulating the tumor microenvironment.

Science. 2013;342:967-70

Paulos CM …..Restifo NP.

Microbial translocation augments the function

of adoptively transferred self/tumor-specific

CD8+ T cells via TLR4 signaling.

J Clin Invest. 2007;117:2197-204

Viaud S …….. Zitvogel L.

The intestinal microbiota modulates the anticancer

immune effects of cyclophosphamide.

Science. 2013;342:971-6

TLR4

TLR4

MyD88, Cybb

MyD88

ImmunotherapyImmunotherapyChemotherapyChemotherapy

Resolution of Resolution of Infection, Infection, Inflammation Inflammation and Immunityand Immunity

Infection Infection (acute (acute

inflammation)inflammation)

Tumor Tumor (chronic (chronic inflammation)inflammation)

Modified from:Goldszmid R.S., Dzutsev A., Trinchieri G.Host immune response to infection and cancer: unexpected commonalitiesCell Host & Microbe, 15, 295-305, 2014

Commensal bacteria calibrate the activation threshold of innate antiviral immunity.Abt MC, Osborne LC, Monticelli LA, Doering TA, Alenghat T, Sonnenberg GF, Paley MA, Antenus M, Williams KL, Erikson J, Wherry EJ, Artis D.

Priming of natural killer cells by nonmucosal mononuclear phagocytes requires instructive signals from commensal microbiota.Ganal SC, Sanos SL, Kallfass C, Oberle K, Johner C, Kirschning C, Lienenklaus S, Weiss S, Staeheli P, Aichele P, Diefenbach A.

ImmunityImmunityJuly 2012July 2012

Gut microbiota

Resistance to respiratory viral infection

Nature Medicine 20, 469–470 (2014)

Intestinal microbiota regulates granulocytosis, neutrophil homeostasis and resistance to sepsis in neonates

Ajitha Thanabalasuriar & Paul KubesNeonates, antibiotics and the microbiome

Hitesh S Deshmukh, Yuhong Liu, Ogechukwu R Menkiti, Junjie Mei, Ning Dai, Claire E O'Leary,Paula M Oliver, Jay K Kolls, Jeffrey N Weiser & G Scott WorthenThe microbiota regulates neutrophil homeostasis and host resistance to Escherichia coli K1 sepsis in neonatal mice

Gut Microbiota Promote Hematopoiesis to Control Bacterial Infection.Arya Khosravi , Alberto Yáñez , Jeremy G. Price , Andrew Chow , Miriam Merad , Helen S. Goodridge , Sarkis K. Mazmanian.Cell Host & Microbe, Volume 15, Issue 3, 2014, 374 - 381

Gut microbes impact myelopoiesis and promote host resistance to systemic bacterial infection

Role of the microbiome in GVHD

TBI and/or chemotherapy

The primary target organs of acute GVHD (i.e. G.I. tract, skin, lung and liver) are in constant interaction with commensal and pathogenic bacteria

The effects of intestinal tract bacterial diversity on mortality following allogeneic hematopoietic stem cell transplantationTaur Y, Jenq RR, Perales MA, Littmann ER, Morjaria S, Ling L, No D, Gobourne A, Viale A, Dahi PB, Ponce DM, Barker JN, Giralt S, van den Brink M, Pamer EG.Ying Blood, 2014.

Key Points

Intestinal diversity is predictive of mortality in allo-HSCT.

Abstract

Highly diverse bacterial populations inhabit the gastrointestinal tract and modulate host inflammation and promote immune tolerance. In allogeneic hematopoietic stem cell transplantation (allo-HSCT), the gastrointestinal mucosa is damaged, and colonizing bacteria are impacted, leading to an impaired intestinal microbiota with reduced diversity. We examined the impact of intestinal diversity on subsequent mortality outcomes following transplantation. Fecal specimens were collected from 80 recipients of allo-HSCT at the time of stem cell engraftment. Bacterial 16S rRNA gene sequences were characterized, and microbial diversity was estimated using the inverse Simpson index. Subjects were classified into high, intermediate, and low diversity groups and assessed for differences in outcomes. Mortality outcomes were significantly worse in patients with lower intestinal diversity; overall survival at 3 years was 36%, 60%, and 67% for low, intermediate, and high diversity groups, respectively (P = .019, log-rank test). Low diversity showed a strong effect on mortality after multivariate adjustment for other clinical predictors (transplant related mortality: adjusted hazard ratio, 5.25; P = .014). In conclusion, the diversity of the intestinal microbiota at engraftment is an independent predictor of mortality in allo-HSCT recipients. These results indicate that the intestinal microbiota may be an important factor in the success or failure in allo-HSCT.

Giorgio TrinchieriGiorgio TrinchieriAmiran DzutsevAmiran Dzutsev

Noriho IidaNoriho IidaAndy StewartAndy StewartSarah CramerSarah CramerLoretta SmithLoretta SmithRosi SalcedoRosi SalcedoMin-Ren DaiMin-Ren Dai

Jessie KiuJessie Kiu

Cancer and Inflammation Cancer and Inflammation Program, Program,

CCR, NCI, Frederick, MDCCR, NCI, Frederick, MD

Yasmine BelkaidYasmine BelkaidShruti NaikShruti Naik

Nicolas BouladouxNicolas Bouladoux

Franco MarincolaFranco Marincola

Ena WangEna Wang

Karen FrankKaren FrankRebecca Rebecca Weingarten

NIH, Bethesda, MDNIH, Bethesda, MD

THANKS to:THANKS to:

Dan SoppetDan SoppetTeri Plona, Teri Plona,

Kristen Pike Kristen Pike

Anil PatriAnil Patri

ATRF, SAIC, FrederickATRF, SAIC, Frederick

Daniel A Molina

TRI, Inc.