1. Normal Intestine vs. IBD Intestine
Environmental
Triggers (infection,
bacterial products)
Failure to down
regulate
Chronic uncontrolled
inflammation = IBD
Moderately
inflamed
Down regulate
Normal gut
Controlled inflammation
Normal gut
Controlled inflammation

2. The Mucosal Immune System
Anatomy M-cells
Unique Cell Types
IgA
Oral Tolerance

3. Characteristic features of MALT

4. Mechanisms for preferential migration of mucosal-derived lymphocytes to mucosal sites.
- Preferential migration is believed to result from expression of unique complementary adhesion molecules by mucosal lymphocytes and endothelial cells that target mucosal endothelium for traffic.
- Lymphocyte: a4b7 integrin
- Mucosal endothelium: mucosal addressin cell adhesion molecule (MAdCAM-1).

5. Unique Features of IgA Immunity
- In the human, IgA is found in both monomeric and dimeric forms.
- Monomeric IgA is produced mostly in bone marrow and found mainly in blood.
- Dimeric IgA is produced mostly in lamina propria of mucosal tissues and found mainly in external secretions.
- Dimeric IgA is actively transported into external secretions via the polymeric immunoglobulin receptor (Pig-R).

6. Oral Tolerance
- Oral tolerance is the generation of systemic immune unresponsiveness by feeding of antigen. The antigen is usually soluble and without adjuvant or proinflammatory activity.
- Oral tolerance is likely a mechanism for prevention of harmful immune responses to harmless antigens such as foods.
- A number of mechanisms may underlie oral tolerance, including clonal deletion, clonal anergy, or active suppression by T cells (cytotoxic, TH2, or TGF-b producing)

7. Oral tolerance as a treatment for experimental allergic encephalomyelits. Induction of oral tolerance is being studied for use clinically.

8. The Spectrum of IBD

9. Multifactorial Basis of IBD
Immune factors
Nonspecific inflammation
Tissue injury
Genetic Environmental factors factors
Restitution and repair
Elson CO et al. In: Proceedings of V International
Symposium on Inflammatory Bowel Diseases, 1998.

10. Etiologic Factors in IBD
Genetics
Antigenic triggers
Microbial pathogens
Dietary factors
Autoantigens
Other environmental factors
Smoking
Use of oral contraceptives, NSAIDs?
Stenson W. In: Yamada T (ed). Textbook of Gastroenterology. 1999:1775.
Sandler et al. In: Kirsner JB (ed). Inflammatory Bowel Disease. 2000: 98.

11. IBD: Evidence of Genetic Influence
Racial differences in incidence: White > Black > Asian
Ethnic influences: Jewish > non-Jewish; Ashkanazi > Sephardic
Twin studies: Monozygotic > dizygotic
Association with specific HLA antigens
Susceptibility loci on chromosomes 1, 3, 5, 6, 16
Sartor RB. Inflammatory Bowel Dis. 1995;24:475.

12. NOD2 First identified Crohn’s disease susceptibility gene.
Chromosome 16q12.
One frameshift mutation (Europe and USA) and 2 point mutations (Europe) identified.
Allele frequency 8.2% in CD, 4.0% in controls, 3.2% in UC.
Ougura et al; Nature 2001; 411: 603
Hugot et al; Nature 2001; 411: 599

13. LRR
TIR
TLR
NF-kB
TNF
Rip2
IL-1
IL-12
LRR
CARD
NOD2

14. “Loss of Function” Mutations in Innate Immune Receptors
How could this lead to IBD?
Bacterial products from commensal flora deliver anti-inflammatory signals
Intracellular defect in LPS signaling (NOD2) may lead to increased extracellular signaling (TLRs)
Decreased apoptosis of bacterially activated cells
Defective killing of bacteria leading to persistent immune response

15. Toll-Like Receptors
Pattern recognition receptors of the innate immune system.
Conserved from plants through humans.
TLR4: Functional LPS receptor.
Activation of inflammatory gene expression (NF-kB) and apoptosis.

16. TLR Ligands TLR2 TLR3 TLR4 TLR5 TLR9 Gram+ PG LP ds RNA LPS Flagellin
Bacterial DNA
TLR2
TLR3
TLR4
TLR5
TLR9

17. TLR4 NF-kB MyD88 IRAK1, 2, M TRAF6 Ecsit MAP Kinases CD14 MD2 TIRAP
Tollip
TRIKA1/TAB/TAK
PKR
Ecsit
MAP Kinases
CD14
MD2

18. Enteric Pathogens Cytokines IL-1 C-X-C Chemokines TNF C-C Chemokines
GM-CSF

19. Tp Th1 Tr ? ? IL-7 IL-18 IL-10 b-defensin IL-15 TLRs Non-Classical MHC
NOD1
TLRs
Neurohormones
IL-7
IL-18
IL-10
b-defensin
Non-Classical MHC
IL-15 ? ? Tp
Th1 Tr

20. Local Environmental Factors in Animals
Germ-free-derived colitis-prone animals do not develop IBD unless commensal enteric bacteria are re-introduced
Restriction of bacterial colonization reduces the incidence of IBD
Antibiotic administration reduces the incidence of IBD
Enteric bacterial antigens activate T cells and induce colitis
A selected subset of bacterial proteins triggers immune reactions
No specific bacterial or viral infection has been identified

21. Potential Infectious Triggers of IBD
Chlamydia
All forms of Pseudomonas maltophilia
RNA retrovirus
Mycobacterium kansasii
Mycobacterium paratuberculosis
Others
Sartor RB. In: Inflammatory Bowel Diseases. 1995:96.

23. Koch's Postulates
The specific organism should be shown to be present in all cases of animals suffering from a specific disease but should not be found in healthy animals.
The specific microorganism should be isolated from the diseased animal and grown in pure culture on artificial laboratory media.
This freshly isolated microorganism, when inoculated into a healthy laboratory animal, should cause the same disease seen in the original animal.
The microorganism should be re-isolated in pure culture from the experimental infection.

24. Limitations of Koch's Postulates
Bacteria that are part of the normal flora or is common in the environment may become pathogenic in certain situations:
Bacteria from the normal flora may acquire extra virulence factors.
Harmless bacteria can cause disease if they gain access to deep tissues.
In immunocompromised patients, components of the normal flora can cause disease.
Not all of those infected or colonized by pathogenic bacteria will develop disease - Subclinical infection is usually more common.
Some bacteria are not culturable in vitro or there may be no suitable animal model of infection.

25. Mycobacterium avium subsp. paratuberculosis and Crohn’s disease
Environmental exposure
Water, meat, dairy
Host genetic determinants of infection outcome?
MAP detection in tissue
Conflicting results
MAP-specific immune responses
p35/p36 antigens, HupB
Anti-mycobacterial therapy
Need large placebo controlled trials with active agents

26. Pathogenesis of IBD
IBD is caused by genetically determined immune dysregulation to luminal antigens, which produces
Overly active upregulation of immune response?
Inadequate downregulation of immune response?
Stenson WB. In: Yamata T et al (eds): Textbook of Gastroenterology (3rd ed). 1999:1779.

27. Chronic Inflammation: Imbalance Between Mediators
IFN-g
IL-10
IL-12
TGF-b
IL-1ra
IL-8
IL-1b
IL-4/IL-13
TNF-a
Anti-inflammatory
Pro-inflammatory

28. T Cell Subsets
Th1
Th2
IL-4
IFN-g
IL-5
IL-2
IL-10

29. T Cell Subsets in IBD
Th1
Th2
Inhibitory
Tissue Damage

30. Th3
Tr1 T
TGFb

31. Th1 Regulatory Cytokines
IL-12
IL-18

32. IL-12
T cell independent
T cell dependent
Bacteria T
TLR2
CD40

33. Leishmania
Anti-IL-12
Th1
Th2
IL-12
Immunity

34. IL-12 Family Members
IL-12
p40
p35
IL-23
p19
IL-27
EBI3
p28

35. Th1 Treg T Cell Subsets in IBD Inhibitory IL-10 TGFb Tissue Damage
Tbet
GATA-3
IL-10
TGFb
Tissue Damage

36. Th1 Cytokines in Mouse Models of IBD
Virtually all IBD mouse models demonstrate Th1-mediated mucosal inflammation.
Critical role for intestinal bacterial flora in Th1-mediated mucosal inflammation.

37. Mouse Models: “Chemical”
Th1
TNBS
Th2
Oxazolone

38. TNBS Experimental Colitis
Chronic transmural Th1-mediated colitis.
Anti-IL-12 abrogated colitis early and late after induction. (Neurath et al, J Exp Med 1995)
Mononuclear cells are reactive to autologous enteric bacteria; tolerance is restored by anti-IL-12. (Duchmann et al, Eur J Immunol 1996)
Anti-CD40 ligand attenuates colitis by inhibiting IL-12. (Stuber et al, J Exp Med 1996)

39. Mouse Models: “Immunologic”
CD45RB hi
TH1
scid
Treg
CD45RB lo

40. TH1 Cytokines in IBD TH1 scid Treg Evidence from Animal Models
Anti-IL-12
Anti-IFN-g
Anti-TNF
scid
Treg

41. Mouse Models: “Genetic”
Th1
IL-10 -/-
IL-10R -/-
TGFb -/-
IL-2 -/-
Gi2a -/-
TNF ARE TG
Th2
TCRa -/-
TCRb -/-

42. Animal Models: “Spontaneous”
Cotton top tamarin
Th2 colitis
Stress induced
C3H/HeJ Bir
Th1
Transferred by T cells
SAMP1/Yit
Focal ileitis and fistulas

43. C3H/HeJBir Mice Develop spontaneous colitis.
Th1 response to enteric microbial flora.
CD4+ T cells activated with enteric bacteria/APC transfer colitis. (Cong et al, J Exp Med 1998)
Transfer of colitis mediated by CD40-dependent IL-12 production by APC.

44. Th1 Cytokines in IBD CD: Increased mucosal IFN-g, IL-2, TNF-a.
Distinct cytokine patterns in early and chronic ileal CD lesions (Desreumaux et al, Gastro. 1997)
UC: Non-Th1 bias mucosally.
Heterogeneous responses amongst patients.

45. IL-12 in IBD
Increased mucosal IL-12 p40 and p35 in CD, but not UC (Monteleone et al, Gastro. 1997; Parronchi et al, Am J Path 1997)

46. IL-18 in IBD IGIF (IFN-g Inducing Factor)
Precursor IL-18 processed by ICE to bioactive IL-18.
Increased mRNA and protein in mucosal macrophages and IEC from CD, possibly UC. (Pizarro et al, J Immunol 1999; Monteleone et al, J Immunol 1999)

47. Transcription Factor and Signal Transduction Targets
NF-kB
T-bet
GATA-3
JAK/STAT
TLR Pathway
NOD Pathway
MAP Kinase Pathways (Hommes. Gastroenterology. 2001)

48. MAP Kinases and IL-12
The p38 pathway is important for IL-12 and IL-10 gene expression.
JNK activation is involved in IL-10, but not IL-12 gene expression.
The ERK pathway activates IL-10, but inhibits IL-12 gene expression.

49. Adhesion and Recruitment
Activated
PMN
Activated
Macrophage
Chemokines
Selectins
Integrins
ICAM-1
IL-8
MAdCAM-1
a4b7
Lymphocyte

50. IL-10 IL-12 IL-23 STAT3 STAT4 STAT4 IL-12R b1 b2 IL-12Rb1 IL-10R

51. Bone Marrow Transplanted CD3e Transgenic Mice
Bone marrow transplantation results in Th1-mediated IBD. (Wang et al, Immunol Rev 1997)
STAT4-/- transplanted CD3e TG mice don’t develop IBD.
Germ-free mice don’t develop IBD.
Anti-IL-12 attenuates IBD.

52. STAT4 Transgenic Mice
Th1-mediated transmural colitis in mice immunized with DNP-KLH. (Wirtz et al, J Immunol 1999)
CD4+ T cells respond to enteric bacterial antigens/APC and transfer colitis.

53. Mice With IBD and Normal (or absent) T Cells
Dominant negative N-cadherin (Hermiston et al, Science 1995)
Macrophage-specific STAT3 knockout (Takeda et al, Immunity 1999)
RAG -/- mice with Helicobacter hepaticus (von Freeden-Jeffry et al, J Immunol 1998)

54. IL-23 and Th17 cells in Colitis
J. Clin. Invest. 116: (2006). doi: /JCI21404

55. IL-17 and IL-6 in Colitis

56. CD4+ T-cells x x IL-4 IL-4 IL-5 TH2 IL-10 IL-2 IFN-g TNF TH1 IFN-g
IL-17A
IL-6
TNF
IL-22
TGF-beta, IL-6, IL-23

57. Structures of Infliximab and Etanercept
Infliximab Structure
Etanercept Structure
L chain
Human p75 TNF receptor
H chain
Fab SS
Site of fusion SS S S S S S S S S
Complement binding region Fc Fc
Affinity
Specificity
Avidity
High
High (TNF-a only)
High
Low (TNF-a, lymphotoxin)
Low
Knight DM et al. Mol Immunol. 1993;30:1443.
Mohler KM et al. J Immunol. 1993;151:1548.

58. Adhesion and Recruitment
Activated
PMN
Activated
Macrophage
Selectins
Integrins
ICAM-1
IL-8
MAdCAM-1
Lymphocyte

59. Anti-Adhesion Strategies
Anti-leukocyte adhesion therapies
Anti-a4 integrin (natalizumab)
Anti- a4 b7 (LDP-02)
Antisense to ICAM-1 (Isis 2302)

60. Daclizumab: Mechanism of Action
IL-2
IL-2
CD25
IL-2
IL-2
CD25
Daclizumab binds to CD25 (IL-2 receptor)
The immune response to antigen follows from the interaction of resting T cells with antigen-presenting cells
Subsequent activation and proliferation of T cells is regulated by the interaction of IL-2 with the high-affinity IL-2 receptor ( subunit)
On activation, IL-2 synthesis by T cells is upregulated and IL-2 subunit (CD25 or Tac) synthesis increases in parallel, resulting in high-affinity IL-2 binding
Inhibition of T-cell proliferation is thought to be central in the treatment of T-cellmediated disease
However, long-term use of agents known to inhibit the proliferation of T cells (eg, cyclosporine) is limited by toxicity
An agent that blocks T-cell proliferation in a selective manner would hold much potential for a wide range of T-cellmediated disorders
Daclizumab specifically binds to the  subunit of the high-affinity IL-2 receptor and functions as an IL-2 receptor antagonist
T cell
IL-2
Daclizumab
IL-2

61. Probiotics Lactobacillus Bifidobacterium Streptococcus E. Coli
Saccharomyces
Enterococcus

62. Probiotics and IBD
Lactobacillus prevents colitis in mouse models of IBD.
E. Coli as effective as mesalazine in remission maintenance in UC.
VSL#3 demonstrated 75% remission rate at 1 year in UC with increased fecal concentrations of probiotic bacteria.

64. Worms in IBD Enteric helminths generate a strong Th2 mucosal response.
Enteric helminths are effective anti-inflammatory agents in mouse models of IBD.
Epidemiologic data suggest that the incidence of IBD increases with improved sanitation.
Clinical trials in human IBD.

66. Th2-Mediated Chronic Mucosal Inflammation
TCR deficient mice.
Oxazolone-induced murine colitis.
EBI3 transgenic mice?
Ulcerative Colitis?
Allergic asthma.