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

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

Characteristic features of MALT

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).

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).

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)

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

The Spectrum of IBD

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.

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.

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.

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

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

“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

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.

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

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

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

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

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

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.

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.

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.

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

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.

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

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

T Cell Subsets in IBD Th1 Th2 Inhibitory Tissue Damage

Th3 Tr1 T TGFb

Th1 Regulatory Cytokines IL-12 IL-18

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

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

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

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

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.

Mouse Models: “Chemical” Th1 TNBS Th2 Oxazolone

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)

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

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

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

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

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.

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.

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)

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)

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

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.

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

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

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.

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.

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)

IL-23 and Th17 cells in Colitis J. Clin. Invest. 116:1310-1316 (2006). doi:10.1172/JCI21404

IL-17 and IL-6 in Colitis

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

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.

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

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

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

Probiotics Lactobacillus Bifidobacterium Streptococcus E. Coli Saccharomyces Enterococcus

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.

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.

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