Microbial Flora: Mechanistic and therapeutic implications€¦ · Microbial Flora: Mechanistic and therapeutic implications R. Balfour Sartor, M.D. Midgette Distinguished Professor

Microbial Flora: Mechanistic and therapeutic implications

R. Balfour Sartor, M.D.Midgette Distinguished Professor of Medicine,

Microbiology & ImmunologyDirector, Multidisciplinary Center for IBD

Research and TreatmentUniversity of North Carolina

Chapel Hill, NC USA

Gastrointestinal Bacteria in Normal Humans

Stomach 0Stomach 0--101022LactobacillusLactobacillusCandidaCandidaStreptococcusStreptococcusHelicobacter pyloriHelicobacter pyloriPeptostreptococcusPeptostreptococcus

Colon 10Colon 101111BacteroidesBacteroidesBifidobacteriumBifidobacteriumClostridium coccoidesClostridium coccoidesClostridium Clostridium leptumleptum/ / FusobacteriumFusobacteriumColiformsColiforms (10(1088))

Distal Ileum 10Distal Ileum 1077--101088ClostridiumClostridiumBacteroides Bacteroides sp sp ColiformsColiforms

Duodenum 10Duodenum 1022StreptococcusStreptococcusLactobacillusLactobacillus

Jejunum 10Jejunum 1022StreptococcusStreptococcusLactobacillusLactobacillus

Proximal Ileum 10Proximal Ileum 1033StreptococcusStreptococcusLactobacillusLactobacillus

Bacteria

The mucosal surface is the interface between the host and environment

Bacteria1011/gm

300-500Species

2-4 milliongenes

IgA: 3-4 gm/day

T cells: 1010/m

Bloodflow

Bloodflow

Normal Mucosal Defenses

BacteriaBacteria

AntigensAntigens

sIgA, IgM

sIgA, IgM

Intestinal trefoil factor

Intestinal trefoil factor

IgAIgA Mucuslayer

Mucuslayer

Tight junctions

Tight junctions

RestitutionRestitution

TGF-βFGF 20

?GM-CSF

TGF-βFGF 20

?GM-CSF

KGFKGFPGE2PGE2

MucusMucus

Defensins (α and β)Defensins (α and β)

Luminal factorsGastric acidPancreatic enzymesBile acidsPeristalsisBiofilm

SCFA

Mucosal Response to Infection and Tolerance to Commensal Bacteria

Bacterial antigens, microbial pathogens

IFN-γ

IL-17TNF

IL-12, 18 IL-23, IL-6

(+)

(-)

IL-10 +TGF-β

Th1Tr1

APC

Th1

Th1Tr1

Microbial antigens are sampled by standard and specialized epithelial cells and interdigitating dendritic

cells, then presented to T and B cells

Science 2005 307:1920

Etiologic Hypotheses of IBD:Microbial- Host Interactions

Etiologic Hypotheses of IBD:Etiologic Hypotheses of IBD:MicrobialMicrobial-- Host InteractionsHost Interactions

Crohn’s UC

CrohnCrohn’’s s UCUC

Defective mucosal barrier• Altered mucus• Increased permeability• Cellular starvation• Impaired restitution• Defective bacterial killing and export of bacterial products• Deficient zinc

Dysregulated immune response

• Loss of tolerance to bacteria• Aggressive cellular activation• Defective apoptosis• Defective bacterial killing

Persistent infection• Mycobacteria• Helicobacter sp.• Measles-mumps• Listeria• Adherent/invasive E. coli

Dysbiosis• ↓ protective bacteria• ↑ aggressive commensals

Mycobacterium paratuberculosis: ProClinical similarities Johne’s disease and Crohn’s diseaseMycobacteria are in the diet! (milk, water, ? meat)Detectible by culture: Crohn’s > controls (resected

intestinal tissues, mucosal biopsies, blood)Detectible by PCR (tissues): Crohn’s > controlsDetectible by in situ hybridization in granulomas, tissuesSerologic responsesTransmitted by human breast milk??Antimycobacterial antibiotics can have beneficial effects32 kDa HupB cross reacts with ANCA

Mycobacterium paratuberculosis: ConOrganisms absent or very rare in tissues by histology

(paucibacillary infection)No evidence of T cell responseHighly variable PCR, serology, antibioticsNo worsening with immunosuppression (steroids, anti-TNF

Ab)No evidence of transmission (farm workers, farm families,

meat processors, veterinarians, offspring of mothers vs. fathers with Crohn’s)

No cure with antibiotics, no documentation of preferential response to pts with ⊕ DNA or serology

Anti-mycobacterial antibiotics transiently suppress activity of Crohn’s disease

Week Antibiotics Placebo16 66% 50%52 41%* 25%104 33%* 18%156 19% 12%

Selby et al, 2005 (abstract)

Response Rate (%)

Methods: Oral treatment with clarithromycin, rifabutin and ethambutol in addition to standard therapy in a blinded , prospective placebo trial for 2 years. Follow up for 1 year after treatment.

ConclusionsM. paratuberculosis is common in environment,

may selectively colonize/ lodge in or even penetrate ulcerated mucosa of Crohn’s disease patientsi,.e. may be present but not cause disease

? pathogen in subset of genetically susceptible hosts (NOD2/CARD 15) or could potentiate disease driven by commensal bacterial antigens

Etiologic Hypotheses of IBDEtiologic Hypotheses of IBDEtiologic Hypotheses of IBD

Crohn’s UC




• Loss of tolerance• Aggressive cellular activation• Defective apoptosis• Defective bacterial killing



Intestinal homeostasis vs. inflammation depends on the relative balance of beneficial vs. detrimental enteric bacteria

Injurious Pro-inflammatory

Bacteroides vulgatus, B. thetaEnterococcus faecalis

E. coli - enteroadherent / invasive

Klebsiella pneumoniaeFusobacterium varium

Intestinal Helicobacter species

Lactobacillus sp.Bifidobacterium sp.

Non-pathogenic E. coliSaccharomyces boulardii

Bacteroides thetaiotaomicron

ProtectiveProbiotic

Etiologic Hypotheses of IBDEtiologic Hypotheses of IBDEtiologic Hypotheses of IBD

Crohn’s UC




• Loss of tolerance• Aggressive cellular activation• Defective apoptosis• Defective bacterial killing



Etiologic Hypothesis of Crohn’s Disease

• Chronic intestinal and extraintestinal inflammation is due to overly aggressive cellular immune responses to a subset of luminal bacteria

• Susceptibility is determined by genes encoding immune responses, barrier function or bacterial clearance

• Onset/reactivation is triggered by environmental stimuli that transiently break the mucosal barrier and initiate inflammation

Microbial antigens and adjuvants

Environmental triggers

Effector immune response

Genetic susceptibility

Pathogenesis of IBD

Epithelial cell gene mutations associated with human IBD

• OCTN 1/2– organic cation (carnitine) transporter

• DLG5– Scaffolding protein, epithelium

• MDR1α (UC)– Drug, ? Xenobiotic compound

transporter, epithelium• Gαi2

– G protein alpha i2• NOD2/CARD15

- intracellular bacterial receptor-defective α defensin production, clearance of intracellular infections,

regulation of response to peptidoglycan

1 28 124 220 273 577577 744 1044LRRCARD CARD

Arg702Trp

Gly908Arg

Leu1007insCPro268Ser

NBD

• NOD2 is on chromosome 16q12 and is mutated in 25-30% of western Europeans with Crohn’s disease (ileal phenotype). RR is 2-3 for single polymorphism, 20-30 for 2 polymorphisms

• Leucine rich repeat region (LRR) interacts with the bacterial peptidoglycan moiety muramyl dipeptide to activate NF-κB

• Expressed in Paneth cells, monocytes, dendritic cells and activated intestinal epithelial cells

Polymorphisms in the bacterial binding region of NOD2 are associated with Crohn’s disease

NOD2/Card 15 binds intracellular MDP and activates NFκB: Crohn’s disease mutant NOD2 polymorphisms lose this function

NF-κB

NO

DC

AR

DC

AR

DLR

R

NO

DC

AR

DC

AR

D

LRRMDP

Chamaillard, PNAS 2003;3455

NO

DC

AR

DC

AR

D

NO

DC

AR

DC

AR

DLR

R LRRMDP

Consequences of loss of NOD2 function:• Defective clearance of intracellular bacteria• Defective production of anti-microbial defensins• Defective inhibition of TLR2- induced NFκB activation

MDP= muramyl dipeptide

Nod2 is constitutively expressed in Paneth cells: Crohn’s variants are associated with decreased antimicrobial defensin production

? Explanation for ileal phenotype ?

Decreased human defensin 5 (HD5) in Crohn’s ileitis, especially patients with NOD2 polymorphisms

WehkampWehkamp J et al. PNAS 2005; 102:18129J et al. PNAS 2005; 102:18129

MucosallyMucosally-- associated bacteria are increased in associated bacteria are increased in patients with active IBD patients with active IBD

SwidsinskiSwidsinski, , et.al. Gastroenterology. et.al. Gastroenterology. 122: 44122: 44--54, 200254, 2002

(FISH analysis: BL=Basal lamina; G=Goblet cell; N= Nucleus)(FISH analysis: BL=Basal lamina; G=Goblet cell; N= Nucleus)

Low mucosal bacterial concentrationLow mucosal bacterial concentrationin normal biopsyin normal biopsy

Increased bacterial concentrationsIncreased bacterial concentrationsin mucosa of Crohnin mucosa of Crohn’’s disease patients disease patient

Phenotypes in murine Nod2 engineered models

Commensal bacteriaCommensal bacteriaNod2-/-

Pathogen ( oral Listeria)Pathogen ( oral Listeria)Nod2-/-

Kobayashi, K. S., M. Chamaillard, et al. (2005). Science 307(5710): 731-4.

DSSDSS--treatmenttreatmentNod22939iC

Maeda, S., L. C. Hsu, et al. (2005). Science 307(5710): 734-8.

Pauleau, A. L. and P. J. Murray (2003). Molecular & Cellular Biology. 23(21): 7531-9

normal

death

colitis

TLRTLR--22

MyD88

IRAKTRAF6 ECSIT

IKK

NIK

NF-κBIκB

NF-κB

RICK

Peptidoglycan

NOD2CARD CARD LRR

••Chronic mucosal and Chronic mucosal and intracellular bacterial intracellular bacterial

stimulationstimulation••Lack of downregulation Lack of downregulation

of adaptive immunityof adaptive immunity

NOD2 model

Environmental Triggers of IBD

IBDIBDOnset and Onset and

ReactivationReactivation

AntibioticsAntibiotics

DietDiet

NSAIDsNSAIDs

Acute infections

Acute infections

StressStress

SmokingSmoking

Altered microflora

Altered mucosal barrier function

and/or immunoregulation

Microbial antigens and adjuvants

Environmental triggers

Immune response

Genetic susceptibility

Pathogenesis of IBD

Clinical evidence implicating the commensal Clinical evidence implicating the commensal enteric flora in the pathogenesis of IBDenteric flora in the pathogenesis of IBD

IBD occurs in intestinal segments with the highest bacterial IBD occurs in intestinal segments with the highest bacterial concentrations concentrations

Increased numbers of bacteria in the mucosa of IBDIncreased numbers of bacteria in the mucosa of IBDpatientspatients-- secondary invasion, translocation, mucosal secondary invasion, translocation, mucosal adherenceadherence

Fecal stream diversion prevents CD relapse, disease Fecal stream diversion prevents CD relapse, disease recurs upon restoration of fecal flowrecurs upon restoration of fecal flow

Maintenance of remission of UC and Maintenance of remission of UC and pouchitispouchitis by antibiotics by antibiotics and probioticsand probiotics

Immunological reactivity against bacterial antigens inImmunological reactivity against bacterial antigens inIBD patients IBD patients

Crohn’s disease depends on the presence of luminal contents

Ileocecal resection,

10 anastamosis, recurrence

85%

Takedown of proximal

ostomy recurrence <1

month

Resection, 10 anastamosis, proximal diversionNo recurrence

Infusion of luminal contents inflammation within 1 week

D’Haens et. al, Gastroenterology, 1998; 114:262

Evidence implicating the commensal enteric flora in Evidence implicating the commensal enteric flora in the pathogenesis of IBDthe pathogenesis of IBD

IBD occurs in gut segments with the highest bacterial IBD occurs in gut segments with the highest bacterial concentrations concentrations

Increased numbers of bacteria in the mucosa of IBDIncreased numbers of bacteria in the mucosa of IBDpatientspatients-- secondary invasion, translocation, mucosal secondary invasion, translocation, mucosal adherenceadherence

Fecal stream diversion prevents CD relapse, disease Fecal stream diversion prevents CD relapse, disease recurs upon restoration of fecal flowrecurs upon restoration of fecal flow

Maintenance of remission of UC and Maintenance of remission of UC and pouchitispouchitis by antibiotics by antibiotics and probioticsand probiotics

Immunological reactivity against bacterial antigens inImmunological reactivity against bacterial antigens inIBD patientsIBD patients-- serologic and T cell responses serologic and T cell responses

All Negative

20.1%

anti-OmpC

5.6%

anti-I210.6%

ASCA10.2%

11.2%

4.6%12.9%

24.8%

79.9% of Patients Mow et al. Gastroenterology 2004

Serologic responses to microbial antigens are positive in 80% of Crohn’s disease patients

No immune activation

Macrophage and TH1immune

activation

No colitisNo colitis ColitisColitis

Resident bacteriaResident bacteriaNo bacteriaNo bacteria

Resident bacteria are essential for chronic intestinal Resident bacteria are essential for chronic intestinal inflammation in animal modelsinflammation in animal models

MiceIL-2KO

IL-10KO

TCRα KO

CD3Ε26TGSAMP1/Yit (?)

CD45RBhi SCIDRats

HLA-B27 TGIndomethacinGuinea pigsCarrageenan

Non-human primateCotton top tamarin

Rapid onset of colitis following colonizationof germ-free IL-10-/- mice

(Sellon et al, Infection & Immunity, 66:5224-31, 1998)

Duration of Cecal histologiccolonization (weeks) score (0-4)

0 0.2 ± 0.11 2.3 ± 0.1*2 2.8 ± 0.3*5 3.8 ± 0.2*

*p < 0.001

Transmural inflammation in IL-10-/- mice colonized with SPF bacteria

Sellon et al, Infect Immunity 1998

Differential ability of various bacterial speciesto induce or prevent experimental colitisAll bacteria are not equal!

Cecal bacteriaHLA B27

transgenic ratHLA B27

transgenic rat

Bacteroides vulgatus

E. coli

Cecal bacteria + Lactobacillus GG

Aggressive colitisAggressive colitis

ProtectionProtection

No colitisNo colitis

Moderate colitisModerate colitis

Rath et al., J Clin Invest 1996;98:945Rath et al., Infect Immunity 1999; 67:2969 Dieleman et.al., Gut 2003; 52:370

Germ free, no colitis

Host-specific responses to selected entericcommensal bacterial species (Sandy Kim DDW 2005)

Host Monoassociated bacteria Outcome

HLA B27 TG rat Bacteroides vulgatus Colitis (cecal predominant)E. coli No inflammationEnterococcus faecalis No inflammation

IL-10 deficient mouse B. vulgatus No inflammationE. coli Colitis (cecal predominant)E. faecalis Colitis (distal),

duodenal obstruction

BM → CD3ε TG mouse B. vulgatus No inflammationE. coli No inflammationE. faecalis No inflammation

Different bacterial species induce different phenotypes of colitis in IL-10 KO 129SvEv mice

GermGerm--FreeFree

Commensal BacteriaCommensal Bacteria

E. faecalisE. faecalis

E. coliE. coli

Kim et al. Gastroenterology 2005 Kim et al. Gastroenterology 2004 abstract

IL-10-/-

IL-1 IL0-/-

IL-10-/-

10-/-

IL-10-/-

0KO10KO

No colitis

Pancolitis(rt. sided)Onset 1 wk

Left sided10-12 wks

Rt. sided3 weeks

E. coli + E. faecalisE. coli + E. faecalis Pancolitis1 week

Flagellin from a commensal Clostridium sp is a dominant antigen in murine colitis (C3H/Hej Bir mice) and human Crohn’s disease

(Lodes et al, JCI 113:1296-1306, 2004)

CBACBA--specificspecific

Conclusions: Commensal bacteria in pathogenesis Conclusions: Commensal bacteria in pathogenesis of chronic intestinal inflammationof chronic intestinal inflammation

1.1. Various commensal bacterial species selectively induce Various commensal bacterial species selectively induce disease in hosts with different genetic backgrounds and disease in hosts with different genetic backgrounds and cause different phenotypes of disease in a single genetically cause different phenotypes of disease in a single genetically susceptible host. susceptible host.

2. Some bacterial species are aggressive, some protective and some neutral. Species specificity within a genus

3. No single species induces as aggressive colitis as the complex microflora of the distal intestine- additive effects

4. Virulence factors affect ability of commensals to induce disease (Adherent/invasive E. coli, B. fragilis cytotoxin, E. faecalis superoxide)

5. Normal hosts effectively downregulate innate immune responses to commensal bacteria- defective host immunoregulation leads to persistent immune activation and chronic colitis driven by normal bacteria

How do bacterial products induce immune responses?

•Innate

•Acquired

•Protective vs. proinflammatory

Bacterial components activate innate and adaptive immune responses

CD-80CD-86CD-80CD-86

CD-28CD-28

CTLA-4CTLA-4MHCMHC

TCRTCR

MHCMHC

TCRTCRAg

AgCD40CD40

CD40LCD40L

IL-10IL-10IL-6,12,18, 23

IL-6,12,18, 23

IL-10TGFβIL-10TGFβ

IFNγIL-17IFNγIL-17 TH1

TH17

TH1TH17

TR1, TH3Treg

TR1, TH3Treg

Antigen presenting cells (APC)process and present bacterial

antigens, are activated by bacterial adjuvants


CD25CD25

MAPK NFκB

TLR9TNFR

TLR5

TLR3 TLR4/MD2

TLR2

IL-1R

TNF

IL-1

Lipopeptides

Peptidoglycan ds RNA LPSHSP60

Flagellin

CpG

DNA

CARD4/NOD1

CARD15/NOD2

Transcription of inflammatory and protective genes

P50 P65ERK1/2

JNKP38 DAP

MDP

IBD - Susceptible Host

Luminalantigens and

adjuvants(Bacterial LPS, PG,

flagellin, DNA, chemotactic peptides,

antigens)

TNFIL-6IL-8

TH1, TH17,TH1, TH17,

TNFIL-17

IFNγIL-12IL-23

IgGIgGLeaky

mucosal barrier

Leaky mucosal barrier

IL-1β

Dysregulated immune

response

Loss of tolerance to

bacteria

Impaired mucosal defense

(permeability, healing,bacterial

clearance)

TH2TH2

Normal host: Basal TLR/ NFkB/ NOD2 activation by commensal bacteria protects against acute epithelial

injury by inducing products that promote epithelial healing and bacterial killing

Epithelial barrierEpithelial barrier

AcuteAcute injuryinjury(DSS, radiation, infection,(DSS, radiation, infection,

ischemia/ reperfusion)ischemia/ reperfusion)

Com

men

sals

Com

men

sals

Healing ↑Barrier protection ↑

Tolerance ↑Defensins ↑

TLR2,4,9

PGE2IFNα/βDefensins

LPSPG,MDPCpG

Chen et al, Nat Med 2003Nakoff-Nahoum Cell 2004Egan et al, PNAS 2004Katakurat al, JCI 2005Kobiashi, Science 2005

NOD2NFκB

Com

men

sals

Com

men

sals

Lamina propria

Lamina propria

Invasion

NFκB

TH1, TH17TH1, TH17immune immune responsesresponsesCytokinesCytokines

In the In the lamina propria,lamina propria, induction ofinduction of TLR /MyD88 /TLR /MyD88 /NFkBNFkBsignalingsignaling by commensal bacteria causes intestinal by commensal bacteria causes intestinal

inflammationinflammation

Inflammation ↑Barrier destruction ↑

Loss of tolerance ↑

Disrupted epithelial barrierDisrupted epithelial barrier

IL-6, 12, 23TNFIFNγ, IL-17

Chen et al, Nat Med 2003

LPSPG

Flagellin

TLR/ NFkB signaling is protective in the normal gut/ epithelium but inflammatory in the

lamina propria/ genetically susceptible host

TLRTLRNFNFκκBB

EpitheliumEpithelium

Barrier functionBarrier functionEpithelial repairEpithelial repair

Immune toleranceImmune tolerance

Lamina propriaLamina propria

InflammationInflammationHost Host protectionprotectionImmune activationTissue destructionLoss of tolerance

Clinical Implications for NFkB/ TLR blockade

• Use NFkB inhibitors during active disease to induce a remission

• Avoid blocking NFkB during quiescent disease states

• Target NFkB in lamina propria rather than epithelial cells

• Consider stimulating epithelial TLR/ NFkBsignaling to maintain remission (probiotics, CpG)

Bacterial components activate adaptive immune responses


CD-28CD-28

CTLA-4CTLA-4MHCMHC

TCRTCR

MHCMHC

TCRTCRAg

AgCD40CD40

CD40LCD40L

IL-10IL-10IL-6, 12, 18, 23

IL-6, 12, 18, 23

IL-10TGFβIL-10TGFβ

IFNγIL-17IL-13

IFNγIL-17IL-13

TH1TH17, TH2

TH1TH17, TH2

TR1, TH3Treg

TR1, TH3Treg

Antigen presenting cells (APC)process and present bacterial

antigens, are activated by bacterial adjuvants


CD25CD25

Immunoregulation by bacterial responsive T cells

Bacterial antigen specific T-cell

clones

Bacterial antigen specific T-cell

clones

Transfer to immunodeficient

mice

Transfer to immunodeficient

mice

ColitisColitis

ProtectionProtection

No colitisNo colitis

TH1TH1

TR1TR1

IFNγIFNγ

IL-10IL-10

Cong et. al. J Immunology 2002

CD4 T Cell Lineage Development

Tp

Th1 Th2

Effector T Cells

+ AntigenAPCIL-12 IL-4

IFNγTNF

Crohn’sdisease

IL-4IL-5IL-10IL-13UC

Homeostasis(Health)

Regulatory T Cells

Tr1

IL-10

IL-10 mTGF−β

CD4+CD25+Treg

Contact

Th3

CD4+CD25+Thymocyte?CD4+CD25-

Tp

ThIL-17

IL-17TNF

Crohn’s disease

TGF−β

IL-23

Types of regulatory T cells in the intestine

Type Marker Location of Type of Mechanism ofstimulation stimuli suppression

T reg CD2, 5 Thymus Self antigens Cell/cell contactFox P3 (? TGFβ)

TH3 ? Intestine Luminal antigen TGFβ, IL-10(dietary)

Tr1 ? Intestine Luminal antigen IL-10(bacteria)

Endogenous Flora - Immune InteractionsResident intestinal bacteria profoundly influence the mucosal

and systemic immune response. Homeostasis vs. chronic intestinal inflammation is determined by the host’s genetically determined immunologic response to luminal antigens and adjuvants

Regulated immune response: tolerance mediated by regulatory T cells, dendritic cells, B cells and epithelial cells(TGF-β, IL-10, INFα/β, PGE2 , PGJ2, PPARγ, Α20)

Dysregulated immune response: chronic relapsing intestinal inflammation mediated by macrophages, TH1 and possibly NKT cells (IL-1β, TNF, IL-6, IL-12, IL-13, IL-17, IL-23, IFNγ)

Response to NonResponse to Non--specific Injuryspecific Injury

IBD- Pathogenesis

Tolerance,controlled

inflammation

Acute injuryAcute injury

Environmental trigger

(infection, NSAID)

Tolerance

Loss of tolerance,

failure of repair or

bacterial clearance

Normal gut

Normal gut

Chronic inflammation

Normal host

Normal host

Genetically susceptible

host

Genetically susceptible

host

Luminal bacteria

Complete healing

Acute inflammation

Etiologic Hypothesis of Crohn’s Disease

• Chronic intestinal and extraintestinal inflammation is due to overly aggressive T cell responses to discrete antigens from a subset of luminal bacteria (most likely commensal)

• Susceptibility is determined by genes encoding immune responses, barrier function or bacterial clearance

• Onset/reactivation is triggered by environmental stimuli

Etiologic Hypothesis of Ulcerative Colitis

• Chronic intestinal and extraintestinal inflammation is due to overly aggressive T cell immune responses to unknown antigens

• Susceptibility is determined by genes encoding immune responses and mucosal barrier function (Mdr 1, PPARγ, HLA)

• Onset/reactivation are triggered by environmental stimuli

Therapeutic Manipulation of Intestinal Bacteria:Selectively Alter Beneficial vs. Detrimental Species by Antibiotics, Probiotics and/ or Prebiotics









ProtectiveProbiotic

Rationale of antibiotic therapy in IBD

↓ luminal bacterial concentrations

Selectively eliminate bacterial subsets that induce or perpetuate disease

↓ tissue invasion, microabcesses

↓ bacterial translocation, dissemination

Mechanisms of action of antibiotics in IBD

• Presumably by direct effects on luminal and tissue bacteria (bacteriocidal, static properties)

• Can also have immunomodulatory activities (decrease proinflammatory cytokine production, affect enteric neurons/ neuropeptides)

Therapy of active Crohn’s disease with metronidazole:regional specific responses (colon > small intestine)

Change in CDAI

Change in CDAI

-100-100

-50-50

00

5050

100100

150150

IleitisIleitis Ileo-colitisIleo-colitis ColitisColitis

PlaceboPlaceboMetronidazoleMetronidazole

No difference in clinical remission ratesNo difference in clinical remission ratesSutherland L et al, Gut 1992; 32:1071

Protocol: 130 patients with active Crohn’s ileitis ± right colitisreceived budesonide (9 mg/d) plus ciprofloxacin and metronidazole (500 mg b.i.d.) vs placebo × 8 weeks

Remission rate (%)

D/C Rx 2°All patients Ileocolitis side effects

Budesonide + placebo (n = 66) 38% (n = 17) 25% 0%Budesonide 33% (n = 16) 53% 20%

+ ciprofloxacin/metronidazole (P = 0.10) (n = 64)

Combined Budesonide and Ciprofloxacin/ Metronidazole for Crohn’s Disease-

No benefit in the small intestine

Steinhart H et al. Gastroenterology. 2002;123:33-40.

Selectively Alter Beneficial vs. Detrimental Species by Antibiotics, Probiotics and/ or Prebiotics










ProtectiveProbiotic

ProtectiveProbiotic

• 15/20 (75%) remained in remission at 12 months following treatment with VSL#3 6g (1800 billion bacteria)/day for 12 months

• No side effects0

2

4

6

8

10

12

020

days

60 da

ys90

days

12 m

onths

15 day

s afte

r tre

at.

Log

10 C

FU/g

dry

wei

ght

Bifidobacteria

Lactobacilli

Streptococcus

VSL#3 is effective for maintenance of UCpatients intolerant of 5-ASA products, but

doesn’t have lasting effects

Venturi et al., Aliment Pharmacol Ther 1999Venturi et al., Aliment Pharmacol Ther 1999

Lymphocyte Transfer ModelCD45Rbhigh T cell SCID mice

75

80

85

90

95

100

105

110

0 1 4 6 8 10 13 15 17 20 22 24 27 29Days

% W

eigh

t Los

s

PlaceboBifidobacterium 1Bifidobacterium 2Bifidobacterium 3Lactobacillus

****** *

Effects of various probiotic species are not uniform in experimental colitis: each species is different

Shanahan et al

Probiotics: Issues for discussion

•Are all probiotic species equally effective?- No!•Are combinations of antibiotics, probiotics and prebiotics more effective than single agents alone? Yes- antibiotics followed by probiotics, synbiotics (pre and probiotics)•Do they need to be alive?•How do they work?

MAPK NFκB

TLR9TNFR

TLR5

TLR3 TLR4/MD2

TLR2

IL-1R

TNF

IL-1

Lipopeptides

Peptidoglycan ds RNA LPSHSP60

Flagellin

CpG

DNA

CARD4/NOD1

CARD15/NOD2

Transcription

P50 P65ERK1/2

JNKP38 DAP

MDP

Mechanisms of mucosal protection by bacterial DNA (CpG)

Rachmilewitz et al, Gastro 2004, Katakura et al, JCI 2005

• Mediated by TLR-9

• Activates NFκB, increased production of COX 2 and PGE2

(but indomethacin doesn’t block protection)

• Decreases epithelial apoptosis following DSS

• *Induces interferon α/β by plasmacytoid dendritic cells, which

mediate protection

Probiotics: Mechanisms of action

Inhibit pathogenic bacteria Improve epithelial function ↑ Immunoregulation

↓ luminal pH ↑ SCFA (butyrate) ↑ IL-10, TGF-β

bacteriocidal proteins ↑ healing ↓ TNF, IL-12

colonization resistance ↑ mucus ↓ Τ cell prolif.

↓ epithelial binding ↓ apoptosis ↑ apoptosis TH1

↓ epithelial invasion ↑ barrier integrity ↑ sIgA

↑ β defensins ↑ HSP 25, 72 ↓ ΝFκΒ

Induction of β defensin 2 by E coli Nissle in Caco 2 cells (NFκB- dependant)

Wehkamp et al, Infect Immun. 2004 72(10):5750-8.

E. coli Nissle selectively inducesHuman β defensin 2 (HBD-2)in Caco-2 cells

Probiotics: Mechanisms of action

Inhibit pathogenic bacteria Improve epithelial function ↑ Immunoregulation

↓ luminal pH ↑ SCFA (butyrate) ↑ IL-10, TGF-β

bacteriocidal proteins ↑ healing ↓ TNF, IL-12

colonization resistance ↑ mucus ↓ Τ cell prolif.

↓ epithelial binding ↓ apoptosis ↑ apoptosis TH1

↓ epithelial invasion ↑ barrier integrity ↑ sIgA

↑ β defensins ↑ HSP 25, 72 ↓ ΝFκΒ

0.0

1.0

2.0

3.0

4.0

Distal colon Proximal colon Cecum Doudnem

His

tolo

gica

l sco

re

** ** **p<0.0001p<0.0001

129 WT

IL-10 KO

B. B. animalisanimalis monoassociation causes inflammation in the monoassociation causes inflammation in the colon and duodenum of ILcolon and duodenum of IL--10 KO mice10 KO mice

B. B. animalisanimalis monoassociation causes colitis in ILmonoassociation causes colitis in IL--10 KO mice (23 weeks)10 KO mice (23 weeks)

WT distal colon IL-10 KO distal colon

B. B. animalisanimalis monoassociation causes severe monoassociation causes severe duodenalduodenal inflammation in ILinflammation in IL--10 KO mice10 KO mice

WT 11 wks IL-10 KO 11 wks IL-10 KO 23 wks

Inflammation vs. Homeostasis Depends on the Balance of Beneficial vs. Detrimental Commensal Bacterial Species



Bifidobacterium animalisE. coli (enteroadherent /

invasive)Klebsiella pneumoniaeFusobacterium varium





ProtectiveProbiotic

Turbocharged Probiotics: Genetic engineeringof bacteria to secrete IL-10 and ITF

Genetically engineered Lactococcus lactis secreting Interleukin-10 decreases experimental colitis

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Histologic Score, Week 4

UntreatedVectorIL-10

Steidler, Science 2000;289:1352-55

Selectively Alter Beneficial vs. Detrimental Species by Antibiotics, Probiotics and/ or Prebiotics










ProtectiveProbiotic

ProtectiveProbiotic

PrebioticsDefinition: dietary substances, usually

nondigested carbohydrates, which foster the growth and metabolic activity of beneficial enteric bacteria

Mechanisms of action:↑ concentrations Bifidobacteria, ↑ SCFA (butyrate), ↓ pH

↑ water-holding capacity stool

Therapeutic lessons from rodent models: antibiotics, probiotics and prebiotics

• Broad spectrum antibiotics can both prevent and reverse established disease, narrow spectrum agents can only attenuate the onset of colitis

• Antibiotics can have regional specificity (Rt. vs. left colon, ileum vs. colon)

• All Bifidobacterium and Lactobacillus species aren’t equally effective as probiotics

• Host specificity can be important- probiotics may need to be individualized

• Antibiotics and probiotics (and possibly prebiotics) can work synergistically (antibiotics induce remission, probiotics maintain remission)

• Prevention of onset or relapse of disease is easier to accomplish than treatment

Therapeutic implications

Antibiotics , probiotics, prebiotics and synbiotics with selective spectra of activities can be used to treat IBD patients and to prevent relapse, but each subset of patients may respond selectively to various agents

(need to individualize treatments for each patient)

Synergistic Therapy of IBD: Induce and maintain long term remission

IL-1βTNFIL-1βTNF

IL-12IL-12IFNγTNFIFNγTNF

Eliminate antigenic driveAntibiotics, probiotics, prebiotics, bacterial binding site blockade,enhance bacterial killing

(stimulate defensins,give GM-CSF)

Restore mucosal barrier function Growth factors, trefoil factor,

Promote T cell regulatory activity (TR1, TH3 , Treg, DC)

Promote T cell regulatory activity (TR1, TH3 , Treg, DC)

Inhibit TH1, macrophage responses

Inhibit TH1, macrophage responses

Innate and AdaptiveImmune Responses Polarized Cytokine

Responses (Th1,Th2, Treg)

LeukocyteHoming/integrins(α4β7)

Tissue Destruction/Repair

Individualizing treatment of heterogeneous IBDsubsets based on genetic, clinical, immunologic

and microbial patternsGenetic Clinical Serologic & Fecal Optimal

defect phenotype T cell response profile treatmentA X 1 15 ZB Y 2 21 Q+M*C O 3 35 R+T

* Q for induction of remission, M for maintenance

Documents

Microbial Flora: Mechanistic and therapeutic implications€¦ · Microbial Flora: Mechanistic and therapeutic implications R. Balfour Sartor, M.D. Midgette Distinguished Professor