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Rio Negro
Nature 2009;426:266
Science 2010;327:928
Avanços científicos nas doenças
inflamatórias intestinais:
o que vai ajudar o paciente?
Claudio Fiocchi
Department of Pathobiology, Lerner Research Institute
Department of Gastroenterology & Hepatology
The Cleveland Clinic Foundation
Cleveland, Ohio, USA
CCF
The clinical spectrum of
inflammatory bowel diseases (IBD)
Ulcerative
colitis
(UC)
Crohn’s
disease
(CD)
Other colitides and IBDIBD-like conditions
Ulcerative colitis
Inflamed
Normal
Crohn’s cobblestone mucosa
Distribution of IBD: pre-World War II
Distribution of IBD: 1950-1970
Distribution of IBD: 1970-1990
Distribution of IBD: 1990-present
Childhood mortality due to infectious diarrhea
Petri WA et al. J Clin Invest 2008:118:1277-1290
The hygiene hypothesis:
the incidence of infectious diseases has decreased and
of immune disorders has increased over the last fifty years
%
100
%
300
Hepatitis A
Tuberculosis
50
200
Rheumatic
fever
Asthma
IDDM
Crohn's
disease
Measles
Multiple
sclerosis
100
0
1955 65
75
85
95
Infectious diseases
1955 65
75
85
95
Immune disorders
Bach J-F. N Engl J Med 2002; 347:911
Geography and social status
Stress
Microbes and
enteric flora
Drugs
Permeability
Diet
Environmental factors
Smoking
Immune
system
Genes
Appendectomy
“Physiological”
inflammation
Pathological
inflammation
Normal
IBD
Psoriasis
Depression
Schizophrenia
Strong
genetic
influence
IQ
Neurosis
Diabetes
Asthma
Cardiac
disease
Cancer
Multiple
sclerosis
Adapted from Chakravarti and Little, Nature 2003
Weak
genetic
influence
Major genetic variations detected by GWAs in IBD
Chr
Gene
Product function
CD UC
1p31
IL
IL--23 receptor
Immune inflammatory response
+
+
5q33
IL
IL--12b (p40)
Immune inflammatory response
+
+
9p24
JAK2
Signaling molecules
+
+
17q21
STAT3
Transcription factor
+
+
18p11
PTPN2
T cell tyrosine phosphatase
+
-
9q32
TNFS15
Immune inflammatory response
+
-
6q27
CCR6
Chemokine receptor
+
-
3p21
MST1
Macrophage chemotaxis
+
-
2q37
ATG16L1
+
-
5q33
IRGM
Autophagosome pathway
+
-
16q12
NOD2/CARD15
Bacterial recognition
+
-
20q13
TNFRSF6B
Inflammatory response, apoptosis
+
+
21q22
PSMG1
Proteasome-related protein
+
+
12q12
MUC19
Epithelial integrity
-
+
1q32
IL
IL--10
Immunoregulation
-
+
Autophagosome pathway
All >50 genetic
associations
reported so far
account for <30%
of all IBD cases
Disruption of the Paneth cell granule exocytosis pathway
in mice and humans carrying Atg16L1 mutations
WT
Atg16L1HM*
Murine ileum
*HM: hypomorphic
Normal
Atg16L1 mutant
Human ileum
Cadwell K et al. Curr Topics Microbiol & Immunol. 2009;335:141
Morphological and functional abnormalities in macrophages
and Paneth cells carrying autophagy gene mutations
Cadwell K et al. Curr Topics Microbiol & Immunol. 2009;335:141
Modulation of autophagy by small molecules
Rubinzstein D et al. Nat Rev Drug Discovery. 2007;6:304
Use of sirolimus (rapamycin) to treat refractory Crohn’s disease
Massey D et al. Gut 2008;57:1294
Clinical predictive value of complex genetic signatures
Gene expression signatures in breast cancer
Gene expression signatures in IBD
Children carrying 34 or more of the
common CD risk alleles have a
~13-fold increased risk of
developing CD, while children
carrying 20 or more of the common
UC risk alleles have a ~7-fold
increased risk of developing UC
Imielinski M et al. Nat Genetics 2009;41:1335
Sotiriou C, Pusztai L. NEJM 2009;360:790
Gut 2009;58:1612
Inflam Bowel Dis 2010 [Epub ahead of print]
IBD: what will benefit the patient?
Genetics
•
•
Genetic screening
•
Creation of IBD, CD, and UC genetic
signatures
•
Prediction of clinical course, response to
therapy, and disease outcome
•
Potential for targeted gene therapy
Identification of patient subsetsubset-specific
genetic variations
The enteric commensal flora
1014 resident
microbes
109 probiotic
Courtesy of F. Shanahan
The Human Microbiome Project
“Characterize the microbiome and
the factors that influence the
distribution and evolution of
constituent microorganisms
to understand the range
of human genetic,
physiological and
pathological
diversity”
IBD:
“Think
in terms of
interacting microbial
communities rather than
individual pathogenic organisms”
Turnbaugh PT et al. Nature 2007; 449:804
Eckburg P & Relman D. Clin Infect Dis Nature 2007; 44:256
Microbial communities shape intestinal immune responses in health and disease
Round JL & Mazmanian SR. Nat Rev Immunol 2009;9:313
Result of hosthost-bacterial interactions
in the gastrointestinal tract
Beneficial interaction
Detrimental interaction
Antibiotics
Probiotics
Prebiotics
“Physiological”
inflammation
Pathological
inflammation (IBD)
Antimicrobial, immune and nonnon-immune functions of defensins
T-cell, monocyte,
mast cell chemotaxis
Mast cell
Epithelial cell
degranulation
proliferation
Dendritic cell
recruitment
and maturation
Neovascularization,
wound closure
CpG (TLR9)
Bacteria
MDP (NOD2)
LPS (TLR4)
Selsted ME & Oulette AJ, Nat Immunol 2005;5:551
LTA
α-defensins,
sPLA2,
lysozyme, TNF-α,
α,
matrilysin, CRIP
Reduced Paneth cell α-defensins and antimicrobial activity
in Nod2 mutated ileal Crohn’s disease
HD5
Controls CD ileitis
Controls CD ileitis
Controls CD ileitis
Is there a role for defensin supplementation in IBD therapy?
Wehkamp J et al. PNAS 2005; 102:18129
B. fragilis PSA*-mediated prevention of experimental colitis
*PSA: polysaccharide A
Mechanism of action:
induction of ILIL-10
10-producing Treg cells
Mazmanian S et al. Nature 2008;453:620
Different frequency (%) of various
serologic markers in CD and UC
Antibody
Controls
CD
UC
ASCA
5
5555-65
5
pANCA
<5
1010-25
5050-65
Anti-OmpC
<5
3838-50
2
Anti-I2
<5
54
2
Anti-pancreas
<5
3030-40
4
Beaven SW and Abreu MT. Curr Opinion Gastroenterol 2004;20:318
Clinical predictive value of combined
serologic and genetic parameters
Inflam Bowel Dis 2010 [Epub ahead of print]
Inflam Bowel Dis 2010 [Epub ahead of print]
IBD: what will benefit the patient?
Gut flora
•
Identification of IBDIBD-promoting microbial
communities (microbial signatures)
•
Identification of bacteriabacteria-derived homeostatic
or protective molecules
•
Identification of microbial signatures predictive
of disease course and clinical outcome
•
Complementation of antimicrobial therapy with
natural antimicrobials (defensins, etc.)
T cell differentiation pathways:
one--way, twoone
two-way, or going
around traffic?
Traditional
one-way
rigid
view
Novel
two-way
flexible
view
O’ Shea H & Paul W. Science 2010;327:1098
Distinct properties of CD and UC mucosal T cells
CD: Th1/17
UC: Th2/17
Proliferation
Higher
Lower
ILIL-2Rα expression
Higher
Lower
Cytotoxicity
Higher
Lower
ILIL-2, IFNIFN-γ production
Higher
Lower
ILIL-12 and ILIL-18 production
Higher
Lower
ILIL-4-mediated suppression
No
Yes
Fas ligand expression
Lower
Higher
Bax expression
Decreased
Normal
Telomerase activity
Increased
Decreased
Apoptosis
Resistant
Susceptible
Survivin expression
Increased
Normal
Induction of in vivo T cell apoptosis by azathioprine
therapy in IBD patients
Tiede I et al. J Clin Invest 2003;111:1133
Antibodies against T cell activation
molecules and receptors
Antibodies against molecules in the
IL-12/IL-23 pathway
Antibodies against cell adhesion molecules
Antibodies against chemokine receptors
Rutgeerts P et al. Gastroenterology, 2009;136:1182
Impact of environmental factors on T cell subset balance
Environmental
toxins
Cytokines
Th17
T reg
AHR
Hormones
Th17
T reg
Inflammation
autoimmunity
AHR: aryl hydrocarbon receptor
Stevens EA & Bradfield CA. Nature 2008:453:46
Defective innate immunity in Crohn’s disease:
impaired erythema, swelling and blood flow
upon E. coli injection
Control
Control
Crohn’s disease
Marks DJB et al., Lancet 2006;367:668
Altered conditioning of mucosal dendritic in infection & inflammation
Tolerogenic DC
Immunogenic DC
Coombes JL & Powrie F. Nat Rev Immunol 2008;8:435
Regulation of intestinal inflammation by mucosal DCs
Food
Flora
Tolerogenic
Immunogenic
(Regulatory)
(inflammatory)
DC
DC
Host response
Physiologic / controlled
inflammation
IL
IL--10
Vitamin D3
Probiotics
Corticosteroids
Mycophenolate
Estrogen
Tracolimus
Rapamycin
Chloroquine
Pathologic / uncontrolled
inflammation (IBD)
Potential therapeutic value of stem cell transplantation
Therapeutic targeting of innate immunity with TLR agonists and antagonists
X
X
X
X
X
X
X
X
X
X
X
X
X
Liew FY et al. Nat Rev Immunol, 2005;5:446
Kinases as therapeutic targets for IBD
Canonical
Non--canonical
Non
* Dominguez C et al., Curr Opin Drug Disc Devel 2005;8:421
Gaestel M et al. Nat Rev Drugs 2009;8:480
Damage--associated molecular patterns (DAMPs)
Damage
PAMPs
DAMPs
Rubartelli A & Lotze M. Trends Immunol 2007;28:429
EPIGENETICS
Definition and relevance to IBD
•
The study of changes in gene function that are stable
and heritable (or potentially heritable) and do not entail changes
in DNA sequence
•
The adaptation of chromosomal regions allowing to perpetuate a
local activation state, whether of short or long duration, and
whether inherited or not
•
In IBD an epigenetic stimulus could be environmental, infectious,
inflammatory, or immune
Maternal factors
Genetics
In utero nutrition
Courtesy of A. Beaudet
Toxins
Diet
Epigenotype
(birth)
Toxins,drugs
Aging
Health
Epigenotype
(adult)
Illness
IBD
Wilson AG. J Periodontol 2008;79:1514
Inflammatory stimuli-induced transcription
and chromatin remodelling
IL-1β
β
LPS
TNF-α
α
Inactive promoter:
condensed chromatin
Ac
Ac
Histone-coded enzymatically
modified (Acetylated) promoter
Ac
Ac
Ac
Remodeled & accessible promoter:
decondensed chromatin
Active promoter
H3
H4
Ac
Pol II
Ac
gene transcription
Courtesy of E. Stylianou
Histone deacetylase inhibitors
(HDACi) expand and increase the
activity of Tregs
Reddy P & Zou W. Nature 2007;13:1281 / Tao et al. Nature 2007;13:1307
Inhibition of HDAC9 increases
Treg function and prevents
murine colitis
De Zoeten E et al. Gastroenterology 2010;138:583
IBD: what will benefit the patient?
Immune response
•
Identification of IBDIBD-specific immune signatures
(Th1, Th2, Th17, ThX, etc.)
•
Blockade of IBD subtypesubtype-specific inflammatory
pathways
•
•
Therapeutic modulation of epigenetic changes
•
Selective deactivation or reconstitution of innate
and adaptive immune pathways
Cell based therapies (tolerogenic DCs, stem
cells, etc.)
Angiogenesis blockade as biological therapy for IBD
ATN-161 decreases vascular density and improves colitis in experimental IBD
ATN-163
ATN-161
15
60
*
40
20
0
10
*
5
0
ATN-163
Danese S et al. Gut 2007;56:855
Histological score
Vascular density
80
ATN-161
ATN-163
ATN-161
Impaired lymphatic flow in IBD
Inflamed mucosa
Increased
blood
flow
Decreased
lymphatic
flow
Lymphatic flow
Blood flow
OUT
IN
Leukocytes
Courtesy of Dr. S. Danese
Fibrostenotic Crohn’s disease
Antifibrotic activity of the protein kinase inhibitor imatinib mesylate
TGF--β
TGF
Potential antifibrotic agents
Soluble TGFβRII fragments
TGFβ antibodies
TGFβRII kinase inhibitors
TGFβ mRNA inhibitors
PKCδ inhibitors
Angiotensin inhibitors
Rosenbloom J et al. Ann Inter Med 2010; 152:159
IBD: what will benefit the patient?
Tissue response
• Appreciation of the contribution of the
chronic tissue reaction
• Prevention of stimulation of nonnon-immune
cells by bacterial products
• Angiogenesis blockade
• Stimulation of lymphatic flow
• Blockade of selective propro-fibrogenic
pathways
Lifetime evolution of IBD
?
Unknown
ultimate
outcome
Remission
recurrences
medications
hospitalizations
operations
cancer, etc.
Life before IBD
Clinical
disease
Microscopic
disease
Abnormal
permeability
Response / no response
to therapy
Therapy
Diagnosis
Immunoregulatory
imbalance
Birth: Exposure
to environment
Conception:
Genetic
susceptibility
Triggering event
Life after IBD
Priming event
Early disease
Life with IBD
Late disease
2.5
5
2.0
IL
IL--12 p40 (ng/ml)
6
4
3
2
1
20
15
1.5
10
1.0
5
0.5
0
0
0
Pre Early Late
Pre Early Late
300
IL
IL--13 (pg/ml)
IL
IL--4 (pg/ml)
Disease activity index
Time--dependent immune response in experimental IBD
Time
200
100
0
Pre
Early
Late
Pre Early Late
300
200
100
0
Pre
Early
Late
Spencer D et al. Gastroenterology 2002;122:94
Top-down (early
Top(early combined immunosuppression)
immunosuppression) vs
step--up ((conventional
step
conventional management):
management): induction of remission
*
*
*
D’Haens G et al. Lancet 371:660; 2008
IBD: what will benefit the patients?
Timing of disease
• Appreciate the evolution of IBD over
time
• Understand and differentiate
mechanisms of early and late IBD
• Customize therapy according to the
stage of disease evolution
Functional networks among components of IBD pathogenesis
Environment
Genes
Microbes
Immunity
Environment
Genes
Microbes
Immunity
Environment
Genes
Microbes
Immunity
Environment
Genes
Microbes
Immunity
Volume 10, Number 4, April 2009
Systems biology:
complex solutions for
complex diseases
Gardy JL, et al. Trends Immunol 2009;30:249
Evolution of IBD therapy:
from empirical to pathophysiologypathophysiology-based
Year
Agent
1940’s
1950’s
1960’s
1970’s
1980’s
Sulfasalazine
ACTH, cortisone
Azathioprine
5-ASA
6-MP
Metronidazole
Elemental diets
Cyclosporine
Budenoside
Methotrexate
Antibiotics
Biologicals
Probiotics, prebiotics
Leukapheresis
Combination therapies
1990’s (early)
1990’s (late)
2000’s
Specificity of action
No
No
No
No
No
No
No
Yes
No
No
No
Yes
No
No
Yes, No
Combination of innate and adaptive immunity modulation
in experimental IBD
LL-mIL10 treatment
Healthy control
IL-10-/- colitis
DSS colitis
No treatment
Steidler L, et al. Science 2000;298:1352
IBD: what will benefit the patient?
Complexity of disease
• Recognize and accept the complexity
of mechanisms of IBD
• Integrate data from multiple disease
pathways
• Start using system biology approaches
for diagnosis and therapy
• Routine use of bioinformatics
Scientific advances in IBD:
what will benefit the patient?
•
All disciplines investigating “IBD science” at
the bench will eventually help clinical
practice
•
How much each discipline may contribute
will vary according to the underlying
pathogenesis of each type of IBD
•
A possible “cure” for IBD can only be
expected from highly integrated,
complementary, and pathophysiologypathophysiologybased therapies
CCF
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