Microbiota y la respuesta immune - Dra Romina Goldszmid

1. MMiiccrroobbiioottaa yy llaa rreessppuueessttaa iimmmmuunnee
RRoommiinnaa GGoollddsszzmmiidd,, PPhhDD

2. 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
OOeessoopphhaagguuss
SSttoommaacchh
VVaaggiinn
aa
MMoouutthh
SSkkiinn
CCoolloonn
Firmicutes
Bacteroidetes
Actinobacteria
Proteobacteria
Other phyla

3. Microorganisms inhabit all barrier surfaces of the organism
Nat Immunol. 2013 Jul;14(7
Compartmentalized and systemic control of tissue immunity by commensals.
Belkaid Y, Naik S.

4. Healthy skin bacterial survey
Grice et al., Science 2009

5. Development of the microbiota from the first inoculum as an infant
through continuous change, modified by diet, genetics and the
environment, through life
• Changes in the last
trimester of pregnancy
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.

6. Changes iinn oouurr mmiiccrroobbiioottaa……
AAnnttiibbiioottiiccss
LLiiffeessttyyllee NNuuttrriittiioonn HHyyggiieennee
IInnffeeccttiioonnss
DDyyssbbiioossiiss
Geographical
location
Host
Genetics
PPrroobbiioottiiccss
AAggee
Mode of
delivery

7. 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.

8. Modulation of adaptive immune responses in the gut by the
Nadine Cerf-Bensussan and Valerie Gaboriau-Routhiau
The immune system and the gut: friends or foes?
Nature Reviews Immunology 10:735 (2010)
commensal microbiota

9. The intestinal microbiota enhances colonization resistance to intestinal
pathogens by both direct and indirect (immune-mediated) mechanisms of
action.
Charlie G. Buffie & Eric G. Pamer1
Microbiota-mediated colonization resistance against intestinal pathogens
Nature Reviews Immunology 13:790 (2013)

10. HHooww CCoommmmeennssaall BBaacctteerriiaa aaffffeecctt
LLooccaall aanndd SSyysstteemmiicc IInnffllaammmmaattiioonn//IImmmmuunniittyy??
CCoommppaarrttmmeennttaalliizzeedd ccoonnttrrooll ooff bbaarrrriieerr ssiittee
iimmmmuunniittyy bbyy rreessiiddeenntt ccoommmmeennssaallss
SSkkiinn IImmmmuunniittyy
IInntteessttiinnaall mmiiccrroobbiioottaa
MMuuccoossaall IImmmmuunniittyy
SSkkiinn mmiiccrroobbiioottaa
IInntteessttiinnee
SSkkiinn
MMoouutthh
PPhhaarryynnxx
RReessppiirraattoorryy
ssyysstteemm
UUrrooggeenniittaall
ttrraacctt

11. IL-17A production in skin tissue is not impacted by distinct
gut commensal populations
8 Taconic
7
6
5
4
3
2
1
0 Gut Skin
Jackson
% IL-17A+ CD45+
NS
Science 337, 1115 (2012)
Taconic
Farms
SFB
Jackson Labs
8
7
CD45+
6
5
17A+ 4
3
IL-2
% 1
0 Gut
Taconic
Jackson

12. Altered T cell inflammatory and regulatory
profile in skin tissue of germ free mice.
Skin
12 **
Germ
free
SPF GF
8
4
32
24
16
8
0
25 **
SPF GF
20
15
10
5
70
60
50
40
30
20
10
0
% IL-17A+ gd T Cells
% IFNg+ ab T Cells
% IL-17A+ab T Cells
% Foxp3+ Tregs
SPF GF
0
SPF GF
0
* **
SPF- Specific Pathogen Free GF- Germ Free
4 weeks
Oral Antibiotic
(ATB)
Ampicillin
Metronidazole
Neomycin
Vancomycin
GUT ***
% IL-17A+CD45+ % IL-17A+CD45+
25
20
15
10
5
0
30
20
10
0
% IFNg+ ab T Cells % IFNg+ ab T Cells
**
8
6
4
2
0
5
4
3
2
1
0
SKI
N
Vehicle
ATB

13. CCoolloonn rreeccttaall ccaarrcciinnoommaa
SSttoommaacchh ccaanncceerr
MMaalltt llyymmpphhoommaa
HHeeppaattoocceelllluullaarr ccaarrcciinnoommaa
MMaammmmaarryy ccaarrcciinnoommaa
TThhyymmiicc llyymmpphhoommaa
Tumors

14. HHuummaannss aarree
mmeettaaoorrggaanniissmmss
((ssyymmbbiioonnttss))
ccoommppoosseedd ooff hhoosstt
aanndd mmiiccrroobbiiaall cceellllss
wwiitthh tthheeiirr oowwnn
ggeenneess
((mmeettaaggeennoommee)) aanndd
sshhaarreedd mmeettaabboolliicc
pprroocceesssseess aanndd
pprroodduuccttss
((mmeettaabboolloommee))..

15. MMeettaabboolliissmm
CCaarrddiioovvaassccuullaarr,,
EExxccrreettoorryy,,
MMuussccuulloosskkeelleettaall,,
aanndd AAddiippoossee ttiissssuuee
ffuunnccttiioonnss
NNeeuurroollooggiiccaall..
bbeehhaavviioorraall
aanndd ccooggnniittiivvee
ffuunnccttiioonnss
AAggiinngg
HHeemmaattooppooiieessiiss
CCiirrccaaddiiaann rrhhyytthhmm
IInnffllaammmmaattiioonn aanndd
IImmmmuunniittyy
CCaanncceerr iinniittiiaattiioonn,,
pprrooggrreessssiioonn aanndd
rreessppoonnssee ttoo
tthheerraappyy
TThhee hhuummaann
mmeettaaoorrggaanniissmm
BBootthh mmiiccrroobbiiaall aanndd
hhuummaann cceellllss aacctt aass
sseennssoorrss ffoorr
eennvviirroonnmmeennttaall cchhaannggeess
ccoommmmuunniiccaattiinngg
rreecciipprrooccaallllyy vviiaa ssiiggnnaalliinngg
ppaatthhwwaayyss tthhaatt,, iinn ppaarrtt,,
uuttiilliizzee iinnnnaattee iimmmmuunniittyy
mmeecchhaanniissmmss..
EEnnvviirroonnmmeennttaall
ffaaccttoorrss
FFoooodd
CChheemmiiccaallss
TTeemmppeerraattuurree
RRaaddiiaattiioonn
PPhhyyssiiccaall aanndd
ppssyyccoollooggiiccaall
ssttrreessss
PPaatthhooggeennss
………………

16. TTHHEE CCHHAALLLLEENNGGEE::
To uunnddeerrssttaanndd tthhee pprreecciissee mmeecchhaanniissmmss tthhaatt aaffffeecctt
hheeaalltthh aanndd ddiisseeaassee aanndd ttoo ttaarrggeett tthheemm ffoorr ddiisseeaassee
pprreevveennttiioonn aanndd ccuurree

17. GGuutt mmiiccrroobbiioottaa ccoonnttrrooll ooff
ssyysstteemmiicc iimmmmuunniittyy//iinnffllaammmmaattiioonn

18. MMiiccrroobbiioottaa MMiiccrroobbiioottaa
PPaatthhoobbiioonnttss
PPaatthhooggeennss
Mets
GGeennoommiicc
mmuuttaattiioonnss
TTuummoorr
pprroommoottiioonn
CChhrroonniicc
iinnffllaammmmaattiioonn
((iinnffeeccttiioonnss,,
aasseeppttiicc))
IInnttrriinnssiicc //
oonnccooggeennee
iinndduucceedd
iinnffllaammmmaattiioonn
PPrreeddiissppoossiinngg ccoonnddiittiioonnss
((oobbeessiittyy,, mmeettaabboolliicc ssyynnddrroommee))
CCaanncceerr
aassssoocciiaatteedd
iinnffllaammmmaattiioonn
TTuummoorr ggrroowwtthh
AAnnggiiooggeenneessiiss
TTiissssuuee rreemmooddeelliinngg
IInnffiillttrraattiioonn aanndd
MMeettaassttaassiiss
IImmmmuunnoo
eevvaassiioonn
CCoo--mmoorrbbiiddiittiieess
RReessppoonnssee ttoo
tthheerraappyy
AAnnttii--ccaanncceerr
iimmmmuunnee
rreessppoonnssee
PPrriimmaarryy
ttuummoorr
MMiiccrroobbiioottaa
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

19. Is the response to cancer therapy regulated by the
commensal bacteria?
SSyysstteemmiicc aannttii--IILL--1100RR ++ IInnttrraattuummoorr CCppGG--OOGGNN iimmmmuunnootthheerraappyy
PPllaattiinnuumm ccoommppoouunndd ((ooxxaalliippllaattiinn,, cciissppllaattiinn)) cchheemmootthheerraappyy
IInntteessttiinnaall mmiiccrroobbiioottaa
SStteerriillee ssuubbccuuttaanneeoouuss
ttrraannssppllaanntteedd
ttuummoorr
AANNTTIIBBIIOOTTIICCSS
NNeeoommyycciinn
VVaannccoommyycciinn
IImmiippeenneemm
oorr GGeerrmm--ffrreeee mmiiccee
Noriho Iida, Amiran Dzutsev, C. Andrew Stewart, ……… Giorgio
Trinchieri, Romina S. Goldszmid
Commensal bacteria control cancer response to therapy by
modulating the tumor microenvironment
Science, 2013; 342:967-70

20. CTX, oxaliplatin aanndd CCppGG ((++//-- aannttii--IILL--
1100RR)) bbuutt nnoott cciissppllaattiinn iinndduuccee
IImmmmuunnooggeenniicc CCeellll DDeeaatthh
Direct toxicity
(Innate) (0-48h) Adaptive (>7 days)
IInnttrraattuummoorraall CCppGG ++ ssyysstteemmiicc aannttii--IILL--1100RR
SSyysstteemmiicc ooxxaalliippllaattiinn oorr cciissppllaattiinn
CCyycclloopphhoosspphhaammiiddee ((CCTTXX))

21. Response to Cancer Immunotherapy and Chemotherapy
Requires the Commensal Microbiota
CpG-CyclophosphamidCpG-OGN Oxaliplatin Cisplatin Cyclophosphamidee
Days after treatment
Tx
Tx + Antibiotics
Untreated
Noriho Iida, Amiran Dzutsev, C. Andrew Stewart, ………
Giorgio Trinchieri, Romina S. Goldszmid
Commensal bacteria control cancer response to therapy by
modulating the tumor microenvironment
Science, 2013; 342:967-70
Sophie Viaud, ……….and Laurence Zitvogel
The intestinal microbiota modulates the anticancer
immune effects of cyclophosphamide
Science, 2013, 342:9671-74
Sterile subcutaneous mouse
transplantable tumors

22. Antibiotics (ABX) suppress TNF-mediated early necrosis of the tumor and
decrease inflammatory cytokine production following anti-IL-10R/CpG
WT (BL6Ncr) TNFKO
H2O
untreated
H2O
aIL-10R
CpG
ABX
aIL-10R
CpG
1 cm
1 ABX decrease TNF and IL-122 pprroodduuccttiioonn bbyy ttuummoorr--
iinnffiillttrraattiinngg mmyyeellooiidd cceellllss ffoolllloowwiinngg aaIILL--1100RR//CCppGG
H2O aIL-10R/CpG
ABX aIL-10R/CpG
ABX untreated
H2O untreated
Most inflammatory but not anti-inflammatory (e.g. IL10)
genes are lower in ABX treated mice
MC38 tumor, 72 h after CpG treatment

23. Oral LPS partially restores the TNF production impaired
by ABX and TLR4 signaling is required for the effective
Cytokine production by tumor
infiltrating myeloid cells
** P=0.05
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
Tlr4-/- mice fail to respond to aIL-10R/CpG

24. Composition of fecal microbiota can be used to segregate mice
with high and low intratumoral TNF in response to CpG
High TNF
Low TNF
Unweighted Unifrac
H2O- drinking mice
PCA1
PCA2
PCA3
PCA1
16S rDNA analysis using 454 pyrosequencing

25. Identification of bacterial genera positively correlating with intratumoral
TNF levels after CpG in microbiota perturbation experiments
AA.. sshhaahhiiii
SSiinnggllee
uunnccllaassssiiffiieedd
OOTTUU
Alistipes (Gram-)
Ruminococcus (Gram+)

26. Identification of bacterial genera negatively correlating with intratumoral
TNF levels after CpG in microbiota perturbation experiments
L. murinum
L. intestinalis
LL.. ffeerrmmeennttuumm

27. ABX treatment blocks oxaliplatin-induced gene espression changes
Oxaliplatin:
ROS production Nox1
Cybb
Sod1
Sod2
microarray analysis ooff ttoottaall EELL44 ttuummoorrss

28. Antibiotics (ABX) impair oxaliplatin chemotherapy bbyy pprreevveennttiinngg RROOSS
pprroodduuccttiioonn ffrroomm NNOOXX22 ((CCyybbbb)) eexxpprreessssiinngg mmyyeellooiidd cceellllss
Oxaliplatin induces ROS
production in tumors of control
but not ABX-treated mice
EL4 tumors-bearing B6 mice were treated with 10mg/kg
oxaliplatin
ROS-induced bioluminescence using the L-012 probe was
analyzed 24 hours after oxaliplatin injection
MFI ROS x 10-3
80
60
40
20
0
Oxaliplatin induces NOX2 (Cybb)-
mediated ROS production in
tumor-associated myeloid cells
WT Cybb-/-
Ctrl Oxp Ctrl Oxp
H2O
ABX
Ctrl Oxp
H2O
ROS+ Gr1hi
(neutrophils)
60
40
20
0
ROS+ Ly6C+F4/80+
(monocyte-derived)
WT Cybb-/-
Ctrl Oxp Ctrl Oxp
H2O
ABX
Ctrl Oxp
ROS- production analyzed by flow cytofluorimetry in EL-4 tumor-infiltrating
H2O
myeloid cells ex-vivo 24 hours after oxaliplatin injection

29. Cyclophosphamide (CTX) induces mucosal bacterial translocation
that is required for anti-tumor Th17 response
Sophie Viaud, ……….and Laurence Zitvogel
The intestinal microbiota modulates the
anticancer immune effects of cyclophosphamide
Science, 2013, in press

30. 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.
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

31. 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
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

32. CpG-ODN +
anti-IL-10R TNF
MyD88, Cybb
TBI and
adoptive T cell
transfer
Myeloid cell Tumor
CTL
CTX
Th17 CTL
Gut
microbiota
TBI or CTX induced transmucosal
bacterial translocation
ROS
Oxaliplatin
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
Modified from:
Goldszmid R.S., Dzutsev A., Trinchieri G.
Host immune response to infection and
cancer: unexpected commonalities
Cell Host & Microbe, 15, 295-305, 2014
TLR4
TLR4
MyD88

33. IImmmmuunnootthheerraappyy
CChheemmootthheerraappyy
RReessoolluuttiioonn ooff
IInnffeeccttiioonn,,
IInnffllaammmmaattiioonn
aanndd IImmmmuunniittyy
IInnffeeccttiioonn
((aaccuuttee
iinnffllaammmmaattiioonn))
TTuummoorr
((cchhrroonniicc
iinnffllaammmmaattiioonn))
Modified from:
Goldszmid R.S., Dzutsev A., Trinchieri G.
Host immune response to infection and cancer:
unexpected commonalities
Cell Host & Microbe, 15, 295-305, 2014

34. 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.
IImmmmuunniittyy
JJuullyy 22001122
Resistance to
respiratory viral
infection
Gut microbiota

35. Intestinal microbiota regulates granulocytosis, neutrophil
homeostasis and resistance to sepsis in neonates
Ajitha Thanabalasuriar & Paul Kubes
Neonates, antibiotics and the microbiome
Nature Medicine 20, 469–470 (2014)
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 Worthen
The microbiota regulates neutrophil homeostasis and host
resistance to Escherichia coli K1 sepsis in neonatal mice

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

37. Role of the microbiome in GVHD
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
TBI and/or chemotherapy

39. The effects of intestinal tract bacterial diversity on mortality following
allogeneic hematopoietic stem cell transplantation
Taur 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.

40. GGiioorrggiioo TTrriinncchhiieerrii
AAmmiirraann DDzzuuttsseevv
NNoorriihhoo IIiiddaa
AAnnddyy SStteewwaarrtt
SSaarraahh CCrraammeerr
LLoorreettttaa SSmmiitthh
RRoossii SSaallcceeddoo
MMiinn--RReenn DDaaii
JJeessssiiee KKiiuu
CCaanncceerr aanndd IInnffllaammmmaattiioonn
PPrrooggrraamm,,
CCCCRR,, NNCCII,, FFrreeddeerriicckk,, MMDD
YYaassmmiinnee BBeellkkaaiidd
SShhrruuttii NNaaiikk
NNiiccoollaass BBoouullaaddoouuxx
FFrraannccoo MMaarriinnccoollaa
Ennaa WWaanngg
KKaarreenn FFrraannkk
RReebbeeccccaa Weingarten
NNIIHH,, BBeetthheessddaa,, MMDD
TTHHAANNKKSS ttoo::
DDaann SSooppppeett
TTeerrii PPlloonnaa,,
KKrriisstteenn PPiikkee
AAnniill PPaattrrii
AATTRRFF,, SSAAIICC,, FFrreeddeerriicckk
Daniel A Molina
TRI, Inc.