Working Group #01 Consolidated Pre-Conference Call Report.doc

NCDD Overview of the Digestive System Working Group (WG 1)Consolidated Pre-Conference Call Report

Chair: Richard S. Blumberg, MD, Brigham and Women’s Hospital, BostonVice Chair: Eugene B. Chang, MD, University of Chicago

This document is a collection of the reports submitted by the above-named NCDD Working Group (WG) members in preparation for their conference call scheduled for:

Monday, February 26, 20072:00 to 4:00 pm EST

The reports contain the members’ completed templates for research advances, recommended goals, and challenges and steps to achieve those goals in the group’s topic area, or, for some WGs, by a subgroup area. If the WG has subgroups, the reports are organized by subgroup; otherwise, they are alphabetical by author.

The reports have simply been collected into one document and are included here just as they were received, with only minor formatting for consistency. These documents are submitted so you can review the WG 1 members’ selections in preparation for their conference call discussions. The goal of that conference call will be to discuss the recommended science-based goals, relevant advances, and challenges/steps for achieving the goals and to reach consensus on what should be included in the group’s chapter of the NCDD long-range plan for digestive diseases research at NIH.

Dr. Davidison’s report that was in preparation and revised reports from Dr. Carey, Dr. Salyers, and Dr. Strober have been included in this revised consolidated report dated February 27, 2007.

List of Current ReportsPage

Development of the GI Tract, Ramesh Shivdasani, MD, PhD......................................................................2Growth and Integrative Physiology, Hannah Carey, PhD.............................................................................7Digestion, Nicholas Davidson, MD.............................................................................................................14Nutrient Fluid Absorption/Secretion, Marshall Montrose, PhD..................................................................19Neurophysiology and Endocrinology, Chung Owyang, MD.......................................................................23Intestinal Microbiota and Digestive Health, Abigail, Salyers, PhD............................................................32Mucosal Immunology, Warren Strober, MD...............................................................................................36

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NAME: Ramesh Shivdasani, MD, PhD, Harvard Medical School; Dana Farber Cancer InstituteWORKING GROUP: Overview of the Digestive System (WG 1)SUBGROUP: Development of the GI Tract

OVERVIEW: Many GI disorders, ranging from congenital malformations to inflammatory bowel disease, and malabsorption to metaplasia and cancer, are thought to have some basis in development of the embryonic gut. Accordingly, GI development has long been a priority for the NIH, which supported visionary research leading to improved understanding of endoderm specification, patterning, stem/progenitor cell kinetics, and crypt-villus organization. These advances permit the field to take the next vital steps and dissect the molecular basis of GI development, which holds enormous potential for pathophysiology and therapy. In the face of unprecedented scientific opportunities, the NIH is positioned to draft a bold agenda for molecular investigation of development of the gut and its affiliated organs. Although the research advances cited below are primarily in the realm of basic research, they touch on common problems in human health, especially birth defects, cancer and inflammation, and are highly relevant to emerging ideas and technologies in regenerative medicine.

1. RESEARCH ADVANCES

Research Advance #1

Elucidation of seminal pathways in development and maintenance of the mammalian gut and improved understanding of interactions between these developmental pathways in genesis of the GI tract. These advances help delineate how widely expressed signaling pathways act in concert to generate tissue- and organ-specific structures and functions and establish the GI tract as an exceptional model system to study developmental mechanisms.

Selected citations:

1. Ramalho-Santos, M., Melton, D. A. and McMahon, A. P. (2000). Hedgehog signals regulate multiple aspects of gastrointestinal development. Development 127, 2763-2772.

2. van den Brink, G. R., Hardwick, J. C., Tytgat, G. N., Brink, M. A., Ten Kate, F. J., Van Deventer, S. J. and Peppelenbosch, M. P. (2001). Sonic hedgehog regulates gastric gland morphogenesis in man and mouse. Gastroenterology 121, 317-328.

3. Batlle, E., Henderson, J. T., Beghtel, H., van den Born, M. M., Sancho, E., Huls, G., Meeldijk, J., Robertson, J., van de Wetering, M., Pawson, T. et al. (2002). Beta-catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB. Cell 111, 251-263.

4. Sansom, O. J., Reed, K. R., Hayes, A. J., Ireland, H., Brinkmann, H., Newton, I. P., Batlle, E., Simon-Assmann, P., Clevers, H., Nathke, I. S. et al. (2004). Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration. Genes Dev 18, 1385-1390.

5. Fre, S., Huyghe, M., Mourikis, P., Robine, S., Louvard, D. and Artavanis-Tsakonas, S. (2005). Notch signals control the fate of immature progenitor cells in the intestine. Nature 435, 964-968.

Research Advance #2

Deeper understanding of the molecular underpinnings of the intestinal crypt-villus axis, its relation to intestinal carcinomas, and of information transmitted by the underlying stroma. These advances relate in powerful ways the genetic basis of colorectal cancer, the second leading cause of U.S. cancer deaths, to epithelial stem cell homeostatic mechanisms.

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Selected citations:

1. van de Wetering, M., Sancho, E., Verweij, C., de Lau, W., Oving, I., Hurlstone, A., van der Horn, K., Batlle, E., Coudreuse, D., Haramis, A. P. et al. (2002). The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 111, 241-250.

2. Haramis, A. P., Begthel, H., van den Born, M., van Es, J., Jonkheer, S., Offerhaus, G. J. and Clevers, H. (2004). De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine. Science 303, 1684-1686.

3. He, X. C., Zhang, J., Tong, W. G., Tawfik, O., Ross, J., Scoville, D. H., Tian, Q., Zeng, X., He, X., Wiedemann, L. M. et al. (2004). BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-beta-catenin signaling. Nat Genet 36, 1117-1121.

4. Perreault, N., Sackett, S. D., Katz, J. P., Furth, E. E. and Kaestner, K. H. (2005). Foxl1 is a mesenchymal Modifier of Min in carcinogenesis of stomach and colon. Genes Dev 19, 311-315.

5. Stappenbeck, T. S., Mills, J. C. and Gordon, J. I. (2003). Molecular features of adult mouse small intestinal epithelial progenitors. Proc Natl Acad Sci USA 100, 1004-1009.

Research Advance #3

The GI tract and its evaginated derivatives (liver, pancreas, and biliary system) are a paradigm for inductive tissue interactions in development and, in particular, epithelial-mesenchymal interactions in organogenesis. Recent studies help define how undifferentiated endoderm is specified during embryogenesis in response to extraneous signals and the activities of tissue-restricted transcription factors. These studies indicate how signaling and transcription factors combine to confer tissue and organ identities. In parallel, cancer biologists are gaining a better understanding of epithelial-stromal (mesenchymal) interactions in neoplasia, and the principles of developmental interactions are likely to extend into the realm of tumor biology.

Selected citations:

1. Rossi, J. M., Dunn, N. R., Hogan, B. L. and Zaret, K. S. (2001). Distinct mesodermal signals, including BMPs from the septum transversum mesenchyme, are required in combination for hepatogenesis from the endoderm. Genes Dev 15, 1998-2009.

2. Bhowmick, N. A., Chytil, A., Plieth, D., Gorska, A. E., Dumont, N., Shappell, S., Washington, M. K., Neilson, E. G. and Moses, H. L. (2004). TGF-beta signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia. Science 303, 848-851.

3. Kim, B. M., Buchner, G., Miletich, I., Sharpe, P. T. and Shivdasani, R. A. (2005). The stomach mesenchymal transcription factor Barx1 specifies gastric epithelial identity through inhibition of transient Wnt signaling. Dev Cell 8, 611-622.

4. Fujitani, Y., Fujitani, S., Boyer, D. F., Gannon, M., Kawaguchi, Y., Ray, M., Shiota, M., Stein, R. W., Magnuson, M. A. and Wright, C. V. (2006). Targeted deletion of a cis-regulatory region reveals differential gene dosage requirements for Pdx1 in foregut organ differentiation and pancreas formation. Genes Dev 20, 253-266.

5. Calmont, A., Wandzioch, E., Tremblay, K. D., Minowada, G., Kaestner, K. H., Martin, G. R. and Zaret, K. S. (2006). An FGF response pathway that mediates hepatic gene induction in embryonic endoderm cells. Dev Cell 11, 339-348.

Research Advance #4

Successful application of assorted model systems to investigate aspects of gut development that are best approached through biochemical, genetic, and developmental studies in diverse species, including Drosophila, chicken, and zebrafish. Whereas the advances emphasized in #1 through #3 are

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based largely on studies in the laboratory mouse, other model systems provide unique advantages and can lead to insights not readily obtained in mice.

Selected citations:

1. Farber, S. A., Pack, M., Ho, S. Y., Johnson, I. D., Wagner, D. S., Dosch, R., Mullins, M. C., Hendrickson, H. S., Hendrickson, E. K. and Halpern, M. E. (2001). Genetic analysis of digestive physiology using fluorescent phospholipid reporters. Science 292, 1385-1388.

2. Kikuchi, Y., Agathon, A., Alexander, J., Thisse, C., Waldron, S., Yelon, D., Thisse, B. and Stainier, D. Y. (2001). casanova encodes a novel Sox-related protein necessary and sufficient for early endoderm formation in zebrafish. Genes Dev 15, 1493-1505.

3. Gaudet, J., Muttumu, S., Horner, M. and Mango, S. E. (2004). Whole-genome analysis of temporal gene expression during foregut development. PLoS Biol 2, e352.

4. Matsuda, Y., Wakamatsu, Y., Kohyama, J., Okano, H., Fukuda, K. and Yasugi, S. (2005). Notch signaling functions as a binary switch for the determination of glandular and luminal fates of endodermal epithelium during chicken stomach development. Development 132, 2783-2793.

5. Theodosiou, N. A. and Tabin, C. J. (2005). Sox9 and Nkx2.5 determine the pyloric sphincter epithelium under the control of BMP signaling. Dev Biol 279, 481-490.

6. Micchelli, C. A. and Perrimon, N. (2006). Evidence that stem cells reside in the adult Drosophila midgut epithelium. Nature 439, 475-479.

2. GOALS FOR RESEARCH

Short-Term Goals (0-3 years): CAPITALIZE ON RECENT ADVANCES AND DEVELOP TOOLS TO INTEGRATE THE RAPIDLY EMERGING UNDERSTANDING OF GUT DEVELOPMENT.

1. Encourage research aimed to understand how particular cell/tissue niches are generated and maintained: regions of the endoderm that are fated to develop into pancreas, liver, biliary tree, and digestive tract; prospective fetal gut segments (esophagus, stomach and its sub-regions, duodenum, ileum, etc.); and the distinctive domains of small bowel crypts and villi.

2. Develop tools that permit accurately targeted genetic studies in the mouse stomach and specific intestinal segments. It would be particularly useful to have a stable repertoire of transgenic mice that faithfully express the Cre recombinase, reporter genes such as GFP and -galactosidase, or toxigenes such as Diphtheria toxin, ideally in inducible forms.

3. Exploit the advanced understanding of the role of the Wnt-APC--catenin pathway in human colorectal cancer to develop new and effective treatment strategies.

4. Recruit (and retain) multi-disciplinary talent that is dedicated to answer important outstanding questions in gut development using diverse models and approaches.

Intermediate-Term Goals (4-6 years): DEVELOP A REASONABLY COMPLETE UNDERSTANDING OF THE PATHWAYS AND INTERACTIONS THAT MEDIATE CRITICAL PATTERNING EVENTS IN GUT ENDODERM AND GENERATE AND MAINTAIN THE SELF-RENEWING EPITHELIA OF THE STOMACH AND INTESTINE.

Develop a sophisticated appreciation of the major molecular pathways in development of the stomach, intestine, liver, and pancreas and attempt to relate these pathways to specific human diseases, including cancer. Scientific developments in colorectal cancer illustrate the value of such integration and suggest effective experimental strategies.

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Improve understanding of how the major signaling pathways implicated in gut development interact with each other (the proverbial “cross-talk”) and determine how the particular confluence of ubiquitous signals helps generate specific structures and tissues.

Delineate the relative contributions of specific signaling pathways and transcription factors in gut development and study how the intersection between extraneous and cell-intrinsic signals mediates particular developmental processes.

Enable wide distribution of genetically engineered animal models that can be intercrossed or studied using different methods in different laboratories.

Integrate molecular databases (gene expression, chromatin-IP, cis-element analyses) with functional studies (siRNA, genetically engineered mice) to capture new pathways and to better appreciate the underlying circuitry.

Long-Term Goals (7-10 years): APPLY THE UNDERSTANDING FROM BASIC RESEARCH IN GI DEVELOPMENT TO DISSECT DISEASE MECHANISMS AND IDENTIFY TARGETS FOR THERAPY.

1. To identify the key factors required to specify each digestive organ and begin to appreciate how the knowledge may be applied to facilitate tissue regeneration in vivo or ex vivo.

2. To distinguish between factors whose functions are restricted to the developmental period and those that continue to influence critical activities in the adult organs.

3. Recognize the specific molecular defects associated with particular congenital malformations and with tissue metaplasias and cancer, especially Barrett’s esophagus, gastric intestinal metaplasia, intestine-type gastric cancer, pancreatic in situ neoplasia and adenocarcinoma, and a range of non-infectious hepatic disorders.

3. MAJOR CHALLENGES AND STEPS TO ACHIEVE GOALS

Goal: Research aimed to understand how particular cell/tissue niches are generated and maintained.Challenge/Steps: (1) Develop powerful but faithful ex vivo approaches to replicate tissue niches in a manner that is amenable to experimental manipulation; (2) Detailed gene expression profiles of selected, highly enriched cell populations to identify molecular markers and candidate regulators.

Goal: To develop tools that permit accurately targeted genetic studies in the mouse stomach and specific intestinal segments.Challenge/Steps: (1) Validation of specific promoters/minigenes that drive tissue- or segment-specific gene expression; (2) Communication within the research community to identify, characterize, and distribute suitable mouse lines.

Goal: Capitalize on advanced understanding of the role of the Wnt-APC--catenin pathway in human colorectal cancer to develop new and effective treatment strategies.Challenges/Steps: (1) Small-molecule screens to disrupt protein-protein interactions; (2) Identification of additional pathway components such as kinases or other enzymes that may be more suitable candidates for targeting by small molecules.

Goals: (A) To develop sophisticated appreciation of the major molecular pathways in development of the stomach, intestine, liver, and pancreas; (B) To improve understanding of how the major signaling pathways implicated in gut development interact with each other; (C) To delineate the relative contributions of specific signaling pathways and transcription factors in gut development.

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Challenges/Steps: (1) Continued investment in sound investigator-initiated projects to delineate and study pathways in depth; (2) Detailed enumeration of the expression domains of specific signaling components at the level of RNA in situ hybridization on embryonic and adult tissue sections.

Goal: Functional evaluation of newly identified molecules (from gene expression, chromatin-IP analyses, etc.) in development of the GI tract.Challenges/Steps: (1) Develop innovative experimental approaches that permit functional evaluation in vivo and ex vivo.

Goals: To evaluate the extent to which molecules that mediate development of the GI tract are also active in adult gut and may be targets for therapy of specific disorders.Challenges/Steps: (1) Detailed enumeration of the expression domains of specific signaling components at the level of RNA in situ hybridization on embryonic and adult tissue sections; (2) Analysis of genetically engineered mouse strains at embryonic and adult stages, possibly in combination with selected Cre-expressing strains to enable tissue-specific recombination.

Goals: To recognize the specific molecular defects associated with particular congenital malformations and with tissue metaplasias and cancer.Challenges/Steps: (1) Working with tissue and tumor banks to study well-preserved and well-annotated clinical specimens for evidence of activation or disruption of specific pathways; (2) Generation of animal models of specified GI disorders to study molecular pathophysiology.

4. PATIENT PROFILE TOPIC

To be considered after the conference call.

5. GRAPHICS AND IMAGES

To be considered after the conference call.

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NAME: Hannah Carey, PhD, University of Wisconsin School of Veterinary Medicine, MadisonWORKING GROUP: Overview of the Digestive System (WG 1)SUBGROUP: Growth and Integrative Physiology

[Individuals contributing include: Denise Ney, Helen Raybould, Jeff Gordon lab (particularly Justin Sonnenburg and Peter Crawford)]

Research Advance #1Regulation of Intestinal Growth: Roles of Nutrients, Trophic Factors, and Neurohumoral Signaling

Glucagon-like peptide-2 (GLP-2) is a nutrient-dependent proglucagon-derived hormone that stimulates intestinal growth by poorly understood cellular mechanisms. Evidence in animal models and human subjects with short bowel syndrome suggests that GLP-2 is the key hormonal mediator of intestinal adaptive growth. The intestinotrophic actions of GLP-2 are indirect and likely involve a downstream mediator(s) released from one or more cell types separate from the enterocyte. Recent reports support a role for neural regulation of GLP-2 action induced by enteral nutrients, with insulin-like growth factor-I acting as a downstream mediator. Better understanding of the cellular mechanisms responsible for GLP-2 action may lead to improved treatments for individuals with intestinal failure who require parenteral nutrition.

Citations:

Estall, JL and Drucker, DJ. (2006). Glucagon-like peptide-2. Annu Rev Nutr 26:391-411.

Dube, PE, Forse, CL, Bahrami, J, and Brubaker PL. (2006). The essential role of insulin-like growth factor-I in the intestinal trophic effects of glucagon-like peptide-2 in mice. Gastroenterology 131:589-605.

Nelson, DW, Liu, X, Holst, JJ, Raybould, HE, and Ney, DM. (2006). Vagal afferents are essential for maximal resection-induced intestinal adaptive growth in orally fed rats. Am J Physiol Regul Integr Comp Physiol 291:R1256-R1264.

Short-Term Goals (1-3 yrs)

Obtain accurate measures of circulating and tissue concentrations of GLP-2 in normal health and after bowel resection.

Localize expression of the GLP-2 receptor in animal models and human subjects with short bowel syndrome before and after intestinal transplantation.

Intermediate-Term Goals (4-6 years)

Identify the intestinal stem cell populations that are upregulated by GLP-2.

Identify the nutrients that act synergistically with GLP-2 to facilitate intestinal growth.

Characterize the neural pathways and downstream mediators that regulate GLP-2 action.

Evaluate the efficacy of GLP-2 given in conjunction with other GI drugs.

Long-Term Goals (7-10 years)

Identify the major signaling pathways involved in GLP-2 action.

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Demonstrate the efficacy of GLP-2 and downstream mediators of GLP-2 in clinical trials, including short bowel syndrome, inflammatory bowel disease, intestinal damage induced by cancer chemotherapy, and ischemic injury.

Steps To Achieve Goals

Develop an antibody that recognizes the bioactive GLP-2 peptide.

Research Advance #2Molecular Events Underlying Intestinal Growth and Adaptation

There has been significant progress in our understanding of the changes in gene expression and cell signaling pathways that are induced by the loss of intestinal mass, such as occurs during bowel resection, and during the adaptive response that follows. Maintenance of intestinal homeostasis during development and adult life is a complex process that requires a proper balance among cell proliferation, apoptosis, and differentiation, and involves interactions between epithelial and other cell types in the intestinal wall including mesenchyme and fibroblasts. Understanding the molecular pathways that mediate normal intestinal growth and the response to injury, and how extrinsic stimuli such as nutrients affect their activity is crucial for development of interventions to maintain intestinal mass and functional capacity. In particular, studies that have begun to elucidate the stem cell niche response following gut resection or injury (e.g., from ischemia, radiation, or trauma) may provide novel therapeutic targets to enhance gut mass and function.

Citations:

Sheng G, Bernabe KQ, Guo J, Warner BW. 2006. Epidermal growth factor receptor-mediated proliferation of enterocytes requires p21waf1/cip1 expression. Gastroenterology 131: 153-164.

Erwin CR, Jarboe MD, Sartor MA, Medvedovic M, Stringer KF, Warner BW, Bates MD. 2006. Developmental characteristics of adapting mouse small intestine crypt cells. Gastroenterology 130: 1324-1332.

Tang Y, Swietlicki EA, Jiang S, Buhman KK, Davidson NO, Burkly LC, Levin MS, Rubin DC. 2006. Increased apoptosis and accelerated epithelial migration following inhibition of hedgehog signaling in adaptive small bowel postresection. Am J Physiol Gastrointest Liver Physiol 290: G1280-G1288.

Wang Y, Wang L, Iordanov H, Swietlicki EA, Zheng Q, Jiang S, Tang Y, Levin MS, Rubin DC. 2006. Epimorphin(-/-) mice have increased intestinal growth, decreased susceptibility to dextran sodium sulfate colitis, and impaired spermatogenesis. J Clin Invest 116: 1535-1546.

Brown SL, Riehl TE, Walker MR, Geske MJ, Doherty JM, Stenson WF, Stappenbeck TS. Myd88-dependent positioning of Ptgs2-expressing stromal cells maintains colonic epithelial proliferation during injury. J Clin Invest. 2007 Jan;117(1):258-69.

Helmrath MA, Fong JJ, Dekaney CM, Henning SJ Rapid expansion of intestinal secretory lineages following a massive small bowel resection in mice. Am J Physiol Gastrointest Liver Physiol. 2007 Jan;292(1):G215-22. Epub 2006 Aug 17. 

Short-Term Goals (1-3 yrs)

Determine downstream mediators of growth factor signaling (e.g., EGFR) that affect intestinal epithelial proliferation, and intrinsic as well as extrinsic death pathways (i.e., Bcl-2 family proteins).

Develop transgenic mouse model that over expresses hedgehog in the gut.

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Develop cell-specific promoters to permit overexpression or deletion of critical gut stromal (myofibroblast) molecules.

Intermediate-Term Goals (4-6 years)

Identify local and circulating factors that activate EGFR and other receptors associated with epithelial growth

Determine the mechanism by which epimorphin deficiency and alterations in other stromal molecules affect DSS-induced colitis and other small bowel and colonic injury models

Characterize the molecular basis of stromal-epithelial interactions in gut injury and repair to identify potential therapeutic targets, (using microarray and proteomics approaches for global gene and protein expression analyses)

Characterization of the cellular composition of the stem cell niche and alterations niche-stem cell interactions in response to resection and regeneration

Determine role of hedgehog signaling in normal intestinal growth and the response to resection

Long-Term Goals (7-10 years)

Target epimorphin and other mesenchymal-epithelial signaling pathways to enhance mucosal regeneration in inflammatory or ischemic conditions

Develop therapeutic approaches that use the EGFR and other growth factor signaling pathways to enhance gut growth after resection

Develop novel methods of tissue engineering utilizing knowledge of the stem cell and its niche, to create functional neomucosa.

Steps To Achieve Goals

Develop transgenic rodent lines with altered expression of molecules suspected to be involved in normal intestinal growth and adaptive response to resection and other disease states. Combine with appropriate models of abnormalities in intestinal morphogenesis/growth/differentiation to assess relative roles of each.

Develop and maintain database of molecular mechanisms identified in intestinal growth and cell differentiation pathways. Include phenotypes of transgenic mouse models and results from other model organisms, including zebrafish, Xenopus, and Drosophila, that target specific genetic pathways and disease conditions.

Research Advance #3Integration of Gut Microbiota and Host Physiology, Nutrition, and Metabolism

Our knowledge of the symbiotic relationships between the gut microbiome and human hosts is rapidly expanding, but there is much still to be elucidated. Microbes ferment dietary constituents and produce short chain fatty acids and other metabolites that supply energy and nutrients to the host. Gut microbes also influence host epithelial and immune function. The presence of gut microbes influences the architecture of the gut mucosa and induces expression of host genes that regulate nutrient processing and absorption. New research indicates that obesity in humans and animal models is associated with changes in the gut microbiome, suggesting that manipulation of the

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microbial population may be used to regulate body weight and obesity-related symptoms in the future. Understanding the functions of the gut microbiome and how they are integrated into host physiology is essential for digestive and whole-body health, and for treatment of disease.

Citations:

Backhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI. 2005. Host-bacterial mutualism in the human intestine. Science 307: 1915-1920.

Hooper LV, Xu J, Falk PG, Midtvedt T, Gordon JI. 1999. A molecular sensor that allows a gut commensal to control its nutrient foundation in a competitive ecosystem. Proc Natl Acad Sci USA 96: 9833-9838.

Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. 2006. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444: 1027-1031.

Short-Term Goals (1-3 yrs)

Build a knowledge base using experimental modelsCreate a human tissue bank

Identify candidate biomarkers of host and microbiota that are associated with specific microbial communities, metabolic processes, disease progression, etc. (employing systems/“-omics” approaches) using experimental models; investigation should focus on how changes in microbiota structure/function affect host biomarkers and associated metabolic and canonical signaling pathways related to nutrient processing, energetics, and immune function.

Expand identification of host physiological processes, phenotypes, and diseases influenced by microbiota structure/function by promoting the development of high-throughput/systems approaches:

Employ established animal models amenable to high-throughput screens (HTS); (e.g. genetic screens in Drosophila, or reporter screens in zebrafish to establish response to various components of the microbiota);

Develop cell-based assays for human cell-line-based HTS.

Characterize microbial responses to differences/perturbations in habitat (e.g. community composition, nutrient environment, host genotype) in vitro, in animal models, and in humans. Characterization may include enumerations of species, metagenomics, transcriptional profiling (employing custom-made microarrays in gnotobiotic models), and metabolomics (using mass spectrometry, NMR, or traditional enzymatic assays).

Create a human tissue bank centered on digestive diseases in a multi-center effort:

Procure tissue sets (e.g. serum, urine, fecal and colonic mucosal microbiota) from healthy and diseased individuals (focused on digestive diseases);

Employ a standardized chip or sequencing-based method to define fecal and/or colonic mucosal microbiota composition associated with each set;

Create a centralized tissue bank (or centralized database of tissues) that can be accessed by investigators through a competitive grant process that will be initiated in “intermediate” term.

Intermediate-Term Goals (4-6 years)

Extend knowledge base using human samplesCreate and mine integrated database

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Employ experimental models in small molecule HTS

Initiate studies that utilize human tissue bank/microbiota profiles outlined in “short” term section; focus of studies will be informed by findings from experimental models. Couple tissue-based studies to human trials of pre- and probiotics to establish plasticity of microbiota structure/function and effects of treatments on host (assessed by biomarkers, disease progression, etc.).

Develop integrated database of measured parameters of microbiota-host associations (“-omics” data) and promote application of computational approaches to further integrate and identify molecular networks and pathways of host and microbiota.

Employ small-molecule HTS focused on human cell lines or microbial species to generate chemical probes for (i) pursuing chemical biology approach to basic questions of host-microbiota interaction in experimental models and (ii) identifying drug targets and candidate compounds. This goal will interface with the Molecular Libraries Initiative (http://mli.nih.gov/).

Long-Term Goals (7-10 years)

Clinical Applicability

Establish correlations between biomarkers of microbiota and host from human data using computational approaches to (i) aid predictive and preventative medicine and (ii) drive animal studies aimed at establishing mechanism of findings/correlations. This iterative cycle should fuel a pipeline in which findings in experimental models are continually translated to improve human health.

Establish clinical diagnostic tools using biomarkers derived from human tissue studies.

Develop treatments using pre- and probiotics using knowledge of microbial plasticity (community structure and microbial physiology) from experimental models and human studies.

Begin trials using small molecules (or derivatives) identified in “intermediate” term, for the rational manipulation of microbiota, microbial physiology, and/or host biology.

Major Challenges and Steps To Achieve Goals

1. Unwieldy “–omics” datasets will need to be compressed, clustered, and analyzed to generate a database that is manageable, accessible and dynamic.

2. Massive datasets will require significant data storage infrastructure.

3. The repertoire of culturable and genetically-manipulatable organisms from the microbiota needs to be expanded.

4. Technology for high throughput metabolite identification (e.g., mass-spectrometry-based metabolomics) needs to be improved and widely available.

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Research Advance #4Integration of Brain-Gut Signaling, Metabolism, and Mucosal Biology in the

Regulation of Body Mass

Considerable progress has been made in our understanding of the way in which the presence of nutrient stimuli in the gut lumen is sensed by endocrine cells and nerves. This information is crucial in the normal digestive processes that occur in the gut and may be altered in disease. The presence of luminal nutrients is also important in the short-term regulation of food intake. There is good evidence from both rodent models and from human studies that two GI hormones, CCK and PYY, are involved in the regulation of food intake via activation of neural substrates in the gut-brain axis. Moreover, long-term changes in the macronutrient content of the diet can alter the sensitivity of the gut-brain axis and thus may lead to long-term changes in body mass.

Citations:

Murphy, KG, Dillo WS, Bloom SR. 2006. Gut peptides in the regulation of food intake and energy homeostasis. Endocrinology Rev 27:719-727.

Stader, AD and SC Woods. 2005. Gastrointestinal hormones and food intake. Gastroenterology 128:175-191

Short-Term Goals (1-3 yrs)

None.

Intermediate-Term Goals (4-6 years)

Understand the interactions between adipokines and the gut-brain axis.

Determine the role of inflammatory mediators in the gut wall on the sensitivity of neural signaling in gut-brain axis.

Understand the mechanisms by which bariatric surgery leads to changes in body mass.

Long-Term Goals (7-10 years)

Develop an effective, peripherally active substance for control of food intake and body weight.

Develop therapeutic interventions to mimic the effects of bariatric surgery on body mass.

Steps To Achieve Goals

Use systems biology approach, integrating physiology with proteomics/metabolomics and other technologies to identify adipokines that influence gut function, and how signals originating from the gut affect adipose tissue biology and metabolism.

Research Advance #5Role of the Vagus Nerve in Anti-Inflammatory Pathways

Recent evidence has demonstrated the importance of the vagal efferent pathway in the activation of an anti-inflammatory pathway, in addition to its established and important role in the regulation of gastrointestinal motility and secretion. Activation of this pathway decreases release of pro-inflammatory mediators including TNFα and other cytokines via activation of nicotinic receptors on peripheral macrophages. Electrical stimulation of the vagus nerve and/or administration of α7nAchR

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agonists have been successfully applied in models of inflammatory diseases, including endotoxaemia, sepsis, ischaemia reperfusion, postoperative ileus, haemorrhagic shock, subcutaneous and gastrointestinal inflammation, and pancreatitis. Importantly, it has recently been shown that this anti-inflammatory pathway can be activated by vagal afferent stimulation in response to intestinal perfusion with lipid.

Citations:

Pavlov, VA and Tracey, KJ. 2006. Controlling inflammation: the cholinergic anti-inflammatory pathway. Biochemical Society Transactions 34:1037-1040.

Luyer MD, Jacobs JA, Vreugdenhil AC, Hadfoune M, Dejong CH, Buurman WA, Greve JW. 2004. Enteral administration of high-fat nutrition before and directly after hemorrhagic shock reduces endotoxemia and bacterial translocation. Ann. Surg. 239:257-64.

Tracey KJ. 2007. Physiology and immunology of the cholinergic antiinflammatory pathway. J Clin Invest 117:289-296.

Short-Term Goals (1-3 yrs)

Determine effects of manipulation of cholinergic pathways on the release of cytokines and/or their downstream effects and effects on outcomes of experimentally induced GI inflammatory conditions.

Intermediate-Term Goals (4-6 years)

Understand the role of nutrition, including lipid-based diets, in the cholinergic anti-inflammatory pathway as it relates to GI inflammation and other gut pathologies.

Begin development of therapeutics based on cholinergic anti-inflammatory pathway targeted towards GI disease (e.g., IBD) and pathologies that have GI effects (e.g., shock).

Long-Term Goals (7-10 years)

Clinical trials with cholinergic agents and/or vagal stimulation to activate the anti-inflammatory pathway in IBD and other patients with GI disorders.

Steps To Achieve Goals Use animal models of GI inflammatory conditions for manipulation of cholinergic pathways through

pharmacological, electrical or nutritional interventions and identify mechanisms and effects on morbidity/mortality.

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NAME: Nicholas Davidson, MD, Washington University School of Medicine, St. LouisWORKING GROUP: Overview of the Digestive System (WG 1)SUBGROUP: Digestion

OVERVIEW: Understanding the fundamental mechanisms and pathways by which nutrients, vitamins, and minerals are processed and assimilated by the digestive tract is key to developing approaches to disorders of the small intestine, biliary tract, and pancreas in which malnutrition is among the most prominent features and remains one of the least tractable. The last few years have witnessed increased understanding of many of the pathways involved by expanding a hierarchy of membrane transporters with distinct substrate specificity, as well as refined subcellular itineraries and destinations. In addition, there has been finer definition of intracellular acceptor molecules and nuclear hormone receptors that participate in metabolic channeling as well as nutrient sensing and that signal through both import and export pumps. Finally emerging new data strongly suggests that these conserved pathways for nutrient processing and delivery may serve heretofore unanticipated roles in innate immunity.

1. RESEARCH ADVANCES

Research Advance #1Diversity in genetic pathways for absorption of cholesterol and other sterols

Identification of enterocyte membrane transporter (NPC1L1) specific for intestinal cholesterol uptake. Sensing and discrimination of subtle structural differences between cholesterol and plant sterols (sitosterol) and identification of sterol efflux pumps (ABCG5/G8) that minimize entry of non-authentic cholesterol through selective intestinal and biliary epithelial export into the lumen rather than into the systemic circulation. Identification of the basolateral cholesterol efflux pump ABCA1 and demonstration of its importance in the production of plasma HDL. Further advances have highlighted integrated regulation throughout the enterohepatic circulation in maintaining absorptive function. Collectively, these advances have greatly expanded our understanding of the complexities of whole body cholesterol homeostasis in health and disease.

Selected citations:

1. Altmann SW, Davis HRJr., Zhu L, Yao X, Hoos LM, et al. Niemann-Pick C1 like 1 protein is critical for intestinal cholesterol absorption. Science 2004; 303: 1201 - 1204.

2. Davis HRJr, Zhu L, Hoos LM, Tetzloff G, Maguire M, et al. Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis. J Biol Chem 2004; 279: 33586 - 33592.

3. Yu L, Li-Hawkins J, Hammer RE, Berge KE, Horton JD, Cohen JC, Hobbs HH. Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol. J Clin Invest. 110:671-80, 2002.

4. Brunham LR, Kruit JK, Iqbal J, Fievet C, Timmins JM, Pape TD, Coburn BA, Bissada N, Staels B, Groen AK, Hussain MM, Parks JS, Kuipers F, Hayden MR. Intestinal ABCA1 directly contributes to HDL biogenesis in vivo. J Clin Invest.;116:1052-62, 2006

Research Advance #2Expanded understanding of developmentally regulated cell-cell communication pathways and

absorptive function

Understanding of the complexities of cell-cell signaling within the small intestine as a model for providing dialog in nutrient sensing, absorption and delivery into the systemic circulation. This includes newly recognized functions for integral structural proteins in establishing and preserving absorptive function. In addition, several lines of evidence point to unanticipated roles for Hedgehog signaling in intestinal nutrient (particularly lipid) absorption function. The mechanisms and pathways for these absorptive phenotypes are incompletely understood. A more complete understanding of how

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diverse developmental pathways modulate elements of absorptive function will have important implications for our understanding of integrated intestinal digestive physiology.

Selected citations:

1. Jones RG, Li X, Gray PD, Kuang J, Clayton F, Samowitz WS, Madison BB, Gumucio DL, Kuwada SK. Conditional deletion of beta1 integrins in the intestinal epithelium causes a loss of Hedgehog expression, intestinal hyperplasia, and early postnatal lethality. J Cell Biol. 175:505-14, 2006

2. Wang LC, Nassir F, Liu ZY, Ling L, Kuo F, Crowell T, Olson D, Davidson NO, Burkly LC. Disruption of hedgehog signaling reveals a novel role in intestinal morphogenesis and intestinal-specific lipid metabolism in mice. Gastroenterology. 122:469-82, 2002

3. Wang CC, Biben C, Robb L, Nassir F, Barnett L, Davidson NO, Koentgen F, Tarlinton D, Harvey RP. Homeodomain factor Nkx2-3 controls regional expression of leukocyte homing coreceptor MAdCAM-1 in specialized endothelial cells of the viscera. Dev Biol. 15:152-67, 2000

4 Madison BB, Braunstein K, Kuizon E, Portman K, Qiao XT, Gumucio DL. Epithelial hedgehog signals pattern the intestinal crypt-villus axis. Development. 132:279-89, 2005

Research Advance #3Emerging hierarchy of ligands and receptors for intracellular signaling, metabolic

compartmentalization of nutrients

There has been new appreciation for the role of nuclear hormone receptors (FXR, LXR, PPARs) and other transporter/acceptor proteins (FABPs/FATPs) in energy sensing and in the maintenance of weight. In addition, new information concerning the metabolic compartmentalization of fatty acids (DGAT1/DGAT2) and monoglycerides (MGAT1/MGAT2) has provided new targets for obesity. Finally, advances in developing systems for understanding the dialog between host and luminal bacteria have expanded understanding of the relevance of the luminal bacterial environment in digestion and absorptive functions.

Selected citations:

1. Watanabe M, Houten SM, Mataki C, Christoffolete MA, Kim BW, Sato H, Messaddeq N, Harney JW, Ezaki O, Kodama T, Schoonjans K, Bianco AC, Auwerx J. Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation. Nature. 439:484-9, 2006

2. Houten SM, Watanabe M, Auwerx J. Endocrine functions of bile acids. EMBO J. 25:1419-25, 20063. Buhman KK, Smith SJ, Stone SJ, Repa JJ, Wong JS, Knapp FF Jr, Burri BJ, Hamilton RL, Abumrad

NA, Farese RV Jr. DGAT1 is not essential for intestinal triacylglycerol absorption or chylomicron synthesis. J Biol Chem. 277:25474-9. 2002

4. Yen CL, Farese RV Jr. MGAT2, a monoacylglycerol acyltransferase expressed in the small intestine. J Biol Chem.;278:18532-7, 2003.

5. Stahl A, Gimeno RE, Tartaglia LA, Lodish HF. Fatty acid transport proteins: a current view of a growing family. Trends Endocrinol Metab.12:266-73, 2001

6. Backhed F, Ding H, Wang T, Hooper LV, Koh GY, Nagy A, Semenkovich CF, Gordon JI. The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci USA. 101:15718-23, 2004

Research Advance #4Novel functions for genes involved in intestinal lipid absorption

A surprising series of discoveries has implicated conserved pathways in intestinal and hepatic lipid absorption in lipid antigen presentation and a role in innate immunity. In particular, emerging new information strongly suggests that the microsomal triglyceride transfer protein is responsible not only for lipidation of the export protein apoB, but also for lipid antigen presentation by CD1d. In addition,

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unanticipated findings have implicated apolipoproteins involved in lipid export (apoE) in lipid antigen presentation by CD1 molecules.

Selected citations:

1. van den Elzen P, Garg S, Leon L, Brigl M, Leadbetter EA, Gumperz JE, Dascher CC, Cheng TY, Sacks FM, Illarionov PA, Besra GS, Kent SC, Moody DB, Brenner MB. Apolipoprotein-mediated pathways of lipid antigen presentation. Nature.;437:906-10, 2005

2. Dougan SK, Salas A, Rava P, Agyemang A, Kaser A, Morrison J, Khurana A, Kronenberg M, Johnson C, Exley M, Hussain MM, Blumberg RS. Microsomal triglyceride transfer protein lipidation and control of CD1d on antigen-presenting cells. J Exp Med. 15;202(4):529-39, 2005

3. Brozovic S, Nagaishi T, Yoshida M, Betz S, Salas A, Chen D, Kaser A, Glickman J, Kuo T, Little A, Morrison J, Corazza N, Kim JY, Colgan SP, Young SG, Exley M, Blumberg RS. CD1d function is regulated by microsomal triglyceride transfer protein. Nat Med. 10:535-9, 2004.

2. GOALS FOR RESEARCH

Short-Term Goals (0-3 years): MAINTAIN AND EXPAND RESEARCH TOOLS AVAILABILITY AND INFRASTRUCTURE WITH ACCURATE AND HIGH-QUALITY PHENOTYPING

1. Encourage research programs aimed at generating and characterizing new and tractable models of digestive and absorptive functions in particular focusing on testing gain and loss of function phenotypes in defined genetic backgrounds.

2. Develop tools that will permit temporal and regional specific gain and loss of function testing of candidate genes in vivo, using inducible Cre-lox technology.

3. Identify cell membrane topology and biochemical mechanisms of known transporter function including substrate recognition, trafficking and regulation.

4. Identify mechanisms that control metabolic compartmentalization of substrates within the enterocyte and that regulate trafficking to distinct secretory pathways.

5. Encourage work that will identify and characterize novel genes that participate in the absorption of luminal substrates and xenobiotics.

Intermediate-Term Goals (4-6 years): DEVELOP A COMPREHENSIVE PROFILE OF INTESTINAL GENES THAT REGULATE MAMMALIAN ABSORPTIVE FUNCTIONS

1. Extend studies of candidate genes to examining selected absorptive and metabolic pathways (eg cholesterol, bile acid, micronutrients) from human populations using humanized knock-ins of informative polymorphisms.

2. Develop targeted approaches to obesity, hyperlipidemia, diabetes, through testing candidate small molecule inhibitors of gene function using mouse and other models.

3. Integrate advances in developmental biology to understanding regional differentiation of intestinal absorptive function (eg ileal bile acid transporter, duodenal iron absorption) and possible plasticity.

4. Encourage development of selective siRNA and other tractable knockdown methodologies for widespread use in digestive/absorptive pathway interrogation.

5. Obtain more complete understanding of the dialog between host and luminal bacteria and the signaling pathways involved.

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Long-Term Goals (7-10 years): TRANSITION FROM BASIC MOUSE AND OTHER ORGANISMAL MODELS INTO TESTABLE PATHWAYS WITH RELEVANCE FOR HUMAN DISEASE

1. Identify targeted therapeutics based on informative pathways that predict development of obesity, hyperlipidemia, diabetes.

2. Identify serum and tissue biomarkers that predict alterations in pathways identified above.

3. Recognize the specific molecular defects associated with nutrient malabsorption (including obesity) and defective or inappropriately increased intestinal nutrient delivery.

4. Initiate trials of selected pre and probiotics genetically selected to optimize luminal microbiome composition.

3. MAJOR CHALLENGES AND STEPS TO ACHIEVE GOALS

Challenge 1. Current animal models inadequate. Existing knockout models for intestine-specific deletion or transgene expression allow global intestinal lineage expression or deletion (villin) or mosaic patterns of expression (Fabp) while some promoter models allow duodenal expression at high levels (Adenosine deaminase). Needs include development of enteroendocrine specific promoters, ileal specific promoters. In addition, ability to direct siRNA knockdown to cells of the luminal GI tract would be major advance. Finally, mouse phenotyping is costly and resource intensive, precluding rapid development of useful functional databases.

Steps to achieve goal 1: Encourage development of needed animal and technical resources. Sharing of resources across programs, through center mechanisms as well as other centralized resources needs to be actively promoted and encouraged. Expand mouse phenotyping cores to digestive sciences. Encourage development of regional and national centers of excellence for methodologic development, for example with limited RFAs for pilot funding.

Challenge 2. Bioinformatics databases inadequate. Despite widespread use of arraying technologies, bioinformatics has lagged. This reflects complexities in developing algorithms for developing pathway based array outcomes and also the lack of trained human resource personnel. Development of new and useful mouse and other animal models could be linked to array-based phenotyping and proteomic surveying that would dramatically accelerate the pace of discovery.

Steps to achieve goal 2: Invest in developing computational biologists and bioinformatics infrastructure. Encourage career development pathways for PhD scientists, not currently engaged in targeted areas of biomedical sciences, in order to generate useful approaches that can be rapidly disseminated into the scientific community.

Challenge 3. Population-based screening approaches and biomarker development is extremely high risk for individual investigators. Obesity, diabetes and other disorders of nutrition and malabsorption are heterogeneous in origin and there is a paucity of well characterized populations. Undertaking to develop long range strategies for characterization of biomarkers currently beyond scope of academic investigators.

Steps to achieve goal 3. Encourage and support academia-industry dialog to support development of expanded understanding of human populations. Long range efforts will require coordination through regional and national database development and appropriate serum and tissue banking. Could be opportunity for translational investigator development, if coupled with formal training, partnership with basic scientific program, loan forgiveness etc.

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4. PATIENT PROFILE TOPIC

To be considered after the conference call.

5. GRAPHICS AND IMAGES

To be considered after the conference call.

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NAME: Marshall Montrose, PhD, University of CincinnatiWORKING GROUP: Overview of the Digestive System (WG 1)SUBGROUP: Nutrient Fluid Absorption/Secretion

State of the Field

Using both functional and genetic approaches, many of the membrane transport proteins mediating intestinal salt, solute, and water transport were defined over the past 10 years. We are in the middle of defining the proteins that contribute to barrier function, in addition to revealing some surprising new pathways of intestinal fat and metal ion absorption. While there will be a continuing need to identify the proteins mediating and regulating absorption and secretion, the real challenge of the next decade is leveraging this information about the building blocks into an integrated view of epithelial function in health and disease. If the goal is maximal impact on human health, this cannot be done efficiently by separate investigations of tissue culture cells, animal modesl, and clinical outcomes. Experimental approaches must be integrated much the same as the experimental outcomes must be integrated.

1. RESEARCH ADVANCES

Research Advance #1Blurring of Classical Boundaries of Absorptive and Secretory Cell Types

The intestinal epithelium is composed of a variety of cell types that are intermingled in the single layer of cells that separate the gut lumen from the body. Recent studies of intestinal epithelial development have revealed new pathways that regulate the balance among these different cell types (most notably Wnt signaling regulating epithelial proliferation, and Math-1 and Gfi in regulating the census of secretory cell types). With the constellation of absorptive and secretory proteins being defined, there is also increased ability to distinguish gradations of function. Recent studies have revealed surprising fluid and sodium absorptive functions in the structures of the colonic crypt, challenging our understanding of how the intestinal epithelium works in health and disease. Studies of epithelial repair and tissue remodeling in disease all emphasize the need to understand the complex interplay among epithelial cell types and their neighbors that results in the homeostasis of epithelial cell types (and thus epithelial function). It must be noted that the molecular pathway for intestinal epithelial differentiation to absorptive cell types has yet to be established. Our classic identification of intestinal epithelial cell types and functions is crude and limits our ability to understand deviations during disease and tissue remodeling.

Research Advance #2Regulation of Epithelial Barrier Function in Health and Disease

An accelerating body of work is aimed at understanding the molecular basis for barrier function thru elucidation of the function of claudins, occluding, and other tight junctional proteins. The regulation of tight junctions is now recognized as a major mechanism by which TNF causes diarrhea, providing a significant step forward in understanding a major symptom in IBD that brings patients to the clinic. The coordinate action of TNF on barrier function and absorptive transporters without effect on chloride secretion is a new concept and important for our understanding of inflammatory bowel disease. There is also expanding information about the specialized mechanisms by which intestinal epithelial cells actively contribute to body defense thru secretion of antibacterial peptides and repair of a disrupted epithelial layer. Complementary to understanding of barrier is the understanding of the molecular basis of cellular water transport. Cloning of water channel molecules in 1991 opened the door for advances in understanding cell and tissue fluid transport thru an expanding array of experiments that defined the impact of human aquaporin mutations on water channel function, crystal structure of aquaporins, and regulation of aquaporins. This led to the awarding of the 2003 Nobel prize in Chemistry to Peter Agre for discovery of the aquaporins. The advanced understanding of

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water channels will contribute greatly to understanding the pathways regulating barrier function versus fluid transport function.

Research Advance #3Gut Factors and Epi-Genetic Regulation of Food Intake and Energy Metabolism

Our understanding of cell and tissue regulation of intestinal transport has broadened in several notable areas. The discovery of non-mutational regulation of gene expression that can be passed through generations has ushered in an era of new understanding about disease predisposition. For example, studies have shown that the offspring of European women undergoing starvation during World War II are predisposed to a higher incidence of diabetes. These observations validate a need for more studies to evaluate this mode of regulation as a risk factor for obesity and other diseases that may involve changes in ion and nutrient transporters.

The study of nutrient absorption is now integrating information at the level of studying the regulation of food intake (satiety). Ghrelin is a recently discovered peptide hormone produced by the stomach that displays strong growth hormone-releasing activity and has a stimulatory effect on food intake and digestive function while reducing energy expenditure. Research in ghrelin has led to new insights into how this hormone produced by the stomach connects the endocrine control of nutritional homeostasis through the gut-brain interactions. Finally, epithelial cells have been shown to express Toll-like receptors (TLRs) for bacterial-epithelial recognition and signaling. This provides a hook to allow further studies how nutrients modulate epithelial/bacterial interactions in the gut.

Finally, recent research has revealed surprising regulation of intestinal iron absorption by the liver. It is widely recognized that iron homeostasis in the body is almost exclusively regulated at the level of intestinal iron absorption, because while the liver can store iron, the ability to excrete excess iron is poor. Prior to 2002, the main identified route to regulate iron uptake was iron regulatory elements on DNA and RNA that were sensitive to iron status, and affected the long-term response by modulating expression of proteins in the iron absorption pathway. Iron overload and iron deficiency syndromes have revealed unanticipated proteins originating from the liver that play a key role in the regulation of iron absorption. Hepcidin and haephestin were ascribed a role in iron absorption in 2002 and 1999, respectively, and ~500 publications have now explored these topics. Exploiting these discoveries will require a more integrated approach to understanding intestinal absorptive function than we can currently pursue, as well as a stronger understanding of the cell types mediating absorption and their response to specific stimuli.

Research Advance #4Intestinal Transporters Involved in the Absorption of Cholesterol and Fatty Acids

A surprising opportunity to explore an unexpected route for fat absorption came with the discovery of the drug Ezetimibe (generic name Zetia), which effectively inhibits cholesterol absorption. Since 2002, there have been ~400 publications on the subject. It is prescribed to patients to lower the amount of total cholesterol, LDL (bad) cholesterol, and apolipoprotein B (a protein needed to make cholesterol) in the blood. Zetia appears to bind to the Niemann-Pick C1-Like 1 protein (NPC1L1) on the gastrointestinal epithelium, a membrane transporter mediating cholesterol absorption. The decreased cholesterol absorption leads to an increase in LDL-cholesterol uptake into cells, thus decreasing levels of cholesterol in the blood plasma. If transporters are involved in the absorption of cholesterol and fatty acids (a new paradigm), then they are obvious targets for the development of drugs to combat the obesity epidemic. Both the cell types mediating cholesterol and fatty acid absorption by these new routes, and their response to specific stimuli, remain speculative.

2. GOALS FOR RESEARCH

Short-Term Goals (0-3 years)The short-term goals all use existing research tools.

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1. Define pathways mediating regulation of barrier function versus transport function.2. Identification of membrane transport proteins and intracellular chaperones of micronutrient and metal

ion absorption (iron, calcium, magnesium, etc.).3. The role of nutrients in modulating epithelial/bacterial interactions in the gut and bacterial ecosystems

within the gut lumen.4. Pilot test expanding use of non-mammalian models to studies of GI absorptive/secretory function

(e.g., zebrafish excellent for developmental studies, C. elegans good for candidate gene function analysis, drosophila mutant collection extensive).

Intermediate-Term Goals (4-6 years)The intermediate- term goals often require development of research tools and thus some enabling technologies must be initiated immediately to achieve these goals.

1. Appraise value and potential impact of outcomes from non-mammalian models prior to moving forward with more extensive programs.

2. Development of advanced mutant mouse models (tissue specific, knock-in, inducible mutations, humanized models) to minimize lethality of mutations, epi-effects driven by changes outside tissue of interest, adaptive responses, and to get animal models for studying human proteins.

3. Translate what the diversity of epithelial cell absorptive and secretory functions means at a proteomics level.

Long-Term Goals (7-10 years) The long-term goals all use the tools, concepts and information developed under intermediate goals.

1. Integrate information on role of cellular and protein diversity in creating efficient absorptive and secretory function in healthy tissue (human and mouse mandatory, non-mammalian systems as warranted).

2. Develop understanding of epithelial development and remodeling in response to injury, especially related to signals and pathways creating a balanced population of absorptive and secretory cells.

3. Understand the molecular and functional adaptation of individual epithelial cells of the intestine to challenge (surgery, inflammatory, diabetes, obesity, or experimental manipulation).

3. MAJOR CHALLENGES AND STEPS TO ACHIEVE GOALS

1. Lack of appropriate animal models . There are two issues here: (1) Most knockouts are constitutive and throughout the body; (2) Developing new mammalian models is slow. a) Need superior gene delivery methods for cells of intestinal tract and methods to study individual

transfected cells in situ.b) Develop mammalian models providing systems biology resolution. Such models would allow

integration of information from molecular regulation directly into studies defining organismal impact without needing to create new model (e.g., find role of protein without needing to create tissue-specific conditional knockout or transgene).i) Foster interdisciplinary research between gastrointestinal physiologists (including

immunologists and bacteriologists) and computational biologists.

2. Human workforce inadequate . Increasing emphasis on translational research needs cadre of trained, committed MD researchers and PhD researchers who can develop clinical research projects, as well as the committed involvement of bioinformatics researchers.a) Foster interdisciplinary research between gastrointestinal researchers and chemists and

biomedical engineers.b) Identify a mechanism to specifically encourage research that asks questions for both human and

advanced mammalian animal models.i) Foster PhD graduate study in translational GI research.ii) Foster MD careers and training in basic research.

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iii) Assist investigators needing to reduce reliance on tissue culture models (via development of novel animal models and/or re-training to use such models).

3. Increased success of translational research requires experimental approaches in animal models that can be more directly compared with human outcomes. Many experimental approaches to analyze cellular function not directly comparable between mouse and man. a) Develop equipment and chemical probes permitting parallel live tissue analyses in human

endoscopy and mouse intestinal tract.i) (REPEAT) Foster interdisciplinary research between gastrointestinal researchers and chemists

and biomedical engineersb) (REPEAT) Need superior gene delivery methods for cells of intestinal tract and methods to study

individual transfected cells in situ.

4. Genomic/proteomic approaches in GI research arenas are poorly developed. a) Establish centers for cell-type specific protein profiling, disease state profiling with standardized

procedures and outcomes. b) Develop a proteome fingerprint of cell types important to GI absorptive and secretory functions.

Must be performed in mouse and man, with some pilot testing in non-mammalian models.i) (REPEAT) Foster interdisciplinary research between gastrointestinal physiologists (including

immunologists and bacteriologists) and computational biologists.c) (REPEAT) Develop mammalian models providing systems biology resolution. Such models

would allow integration of information from molecular regulation directly into studies defining organismal impact without needing to create new model (e.g., find role of protein without needing to create tissue-specific conditional knockout or transgene).

4. PATIENT PROFILE TOPICS

Cystic fibrosis improvement in quality of life and life span informed by better understanding of CFTR function as a chloride channel and regulator of other membrane functions.

Obesity hardships improved by Zetia.

Oral rehydration solution improvements informed by basic science understanding of sodium-sugar cotransport function and stoichiometry.

5. GRAPHICS AND IMAGES

Crystal structure of aquaporin water channel (request from Peter Agre, winner of 2003 Nobel Prize, along with a picture of Peter).

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NAME: Chung Owyang, MD, University of Michigan Medical Center, Ann ArborWORKING GROUP: Overview of the Digestive System (WG 1)SUBGROUP: Neurophysiology and Endocrinology

Overview

In the last 5 years we have gained sophistication in our understanding of neuro-hormone control of gut functions and energy homeostasis. The unraveling of the complexity of signaling between diverse cells in the ENS provides the cellular and molecular basis for understanding some of the disorders affecting the enteric nervous system. The neurobiology of brain-gut interactions has become further characterized. This provides the necessary conceptual framework for scientist and clinician in their understanding and quest for new treatment of diseases and motility disorders. There is renewed and expanded interest in the role of the GI tract in the regulation of satiety and energy homeostasis. Increased understanding of the mechanisms governing nutrient sensing and peptide secretion by enteroendocrine cells allow investigators to exploit these pathways in their development of new agents to combat obesity and diabetes. A better understanding of the molecular mechanisms leading to disease and age-related apoptotic cell death provides hope for preventive and/or regenerative therapy. Finally a clear elucidation that neural crest stem cells persist in the adult gut and undergo changes in self renewal suggests that neuron replacement therapy can become a reality. Because of the diversity of the topic, it is difficult to cover all the major research advances in this area. The following are examples of the impact made as a result of basic and translational science research in the field.

1. RESEARCH ADVANCES

Research Advance #1

The multiple constituents of the enteric nervous system (ENS) can have profound effects on its functioning. In addition to the nerves and smooth muscle cells, the normal functioning of the system requires participation of the interstitial cell of Cajal, glial cells, and enteroendocrine cells. For example, research on the interstitial cell of Cajal has dramatically altered the way we look at regulation of smooth muscle. Interstitial cells of Cajal pace gastrointestinal muscle by initiating slow waves in both muscle layers and appear to be the preferred sites for reception of neurotransmitters. These specialized cells in intramuscular layer also provide regenerative responses to and amplification of pacing messages from cells of Cajal in the myenteric plexus.

Recent studies indicate that interstitial cells of Cajal mediate mechanosensitive response in the stomach. Furthermore, lack of ICC in the pylorus explains distinct peristaltic motor patterns in the stomach and small intestine. These animal studies clearly indicate that it would not be possible to generate the motor program stored in the ENS without patterned electrical activity and synaptic connectivity provided by ICC. These findings have profound implications in human physiology and pathophysiology as abnormal networks have been reported in GI muscles from patients with motility disorders. These include achalasia, diabetic gastropathy, infantile pyloric stenosis, idiopathic gastric perforation, pseudo-obstruction and slow-transit constipation

Another example is the glial cells. In the CNS, glial cells have an important role in synaptic transmission plasticity and immunoprotection. It is likely that glia may play similar roles in the ENS. Recent studies show that ablation of the enterial glia in mice leads to a fulminent hemorrhagic jejunoileitis, suggesting a central role of the enteric glia in the maintenance of gut mucosal integrity. This possibility is further supported by the observation that development of enterocolitis occurs in mice after autoimmune targeting of glial cells. In human Crohn’s disease and experimental colitis in rats, the glial-derived neurotrophic facter (GDNF) is upregulated. This neurotrophic factor has strong anti-apoptotic effects on colonic epithelial cells which may be responsible for its protective action on the epithelial lining during mucosal inflammation.

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Citations:1. Won KJ, Sanders KM, Ward SM. Interstitial cells of Cajal mediate mechanosensitive responses in the stomach. Proceedings of the National Academy of Science (USA) 102:14913-14918, 2005.2. Wang XY, Lammers WJEP, Bercik P, Huizinga JD. Lack of pyloric interstitial cells of Cajal explains distinct peristaltic motor patterns in stomach and small intestine. Am J Physiol 289:G539-G549, 2005.3. Steinkamp M, Geerling I, Seufferlein T, von Boyen G, Egger B, Grossmann J, Ludwig L, Adler G, Reinshagen M. Glial-derived neutrophic factor regulates apoptosis in colon epithelial cells. Gastroenterology 124:1748-1757, 2003.4. Clerc N, Gola M, Vogalis F, Furness JB. Controlling the excitability of IPANs: a possible route to therapeutics. Curr Opin Pharm 2:657-664, 2002.5. Gianino S, Grider JR, Cresswell J, Enomoto H, Heuckeroth RD. GDNF availability determines enteric neuron number by controlling precursor proliferation. Development 130:2187-2198.6. Rossi J, Herzig KH, Voikar V, Hiltunen PH, Segerstrale M, Airaksinen MS. Alimentary tract innervation deficits and dysfunction in mice lacking GDNF family receptor alpha2. J Clin Invest 112:707-716, 2003.

Research Advance #2

During the last decade accumulating laboratory and clinical evidence indicate that IBS is a real clinical entity and not a psychosomatic disorder. Much progress can be attributed to (i) a better characterization of the neurobiology of brain gut interactions, (ii) advances in neuroimaging to identify the brain regions responsible for perception and modulation of visceral afferent signals from the upper and lower GI tract ,and (iii) elucidation of the roles of peripheral serotonin/5HT receptor signaling system in the ENS. The corticotropin-releasing factor (CRF) signaling pathway perhaps is the best described brain gut circuit closely related to the pathogenesis of IBS.

This pathway coordinates endocrine, behavioral, and immune responses to stress. Activation of CRF receptor 1 and 2 produces differential effect on gastric and colonic motility. Clinical studies show that stimulation of CRF1 pathways produces the key symptoms of IBS diarrhea-predominant patients. These symptoms are alleviated by CRF1 receptor antagonists supporting the involvement of the CRF1 system at the central and peripheral sites in the pathogenesis of IBS triggered by stress. The CRF1 receptor antagonist that is directed at normalizing a sensitized CRF system holds great promise for a variety of stress-related GI disorders including IBS and cyclical vomiting syndrome.

Another major advance is the ability to image the living human brain with various neuroimaging modulaties. This has greatly enhanced our ability to study brain gut interactions in health and in disease conditions. The brain regions involved in conscious perception of sensory information coming from the peripheral (insular cortex) have been identified and should be contrasted with the dorsal anterior cingulated cortex (dACC) which mediates the effective response and motivational drive. The magnitude and gain of signals going to these regions is highly influenced by central arousal and cortico-limbic systems. Symptom components of IBS can be dissected and attributed to specific areas of the brain that mediate cognitive, emotional and motivational component of the discomfort. This new approach to the study of functional GI disorders provides more insightful information on the pathophysiology of this group of disorders. The ability to study a neurobiological substrate with imaging modulaties rather than relying on highly variable subjective symptoms (e.g., Rome criteria) makes it possible to investigate the role of genetic factors and receptor physiology on the pathophysiology of symptoms. We should have a precise endpoint to evaluate therapeutic interventions on distinct brain networks involved in afferent processing and modulation. Meaningful results from such studies can be obtained from much smaller samples of subjects compared with epidemiological or traditional pharmacological studies.

Lastly the characterization of the peripheral serotonin/5HT receptor signaling system in the ENS represents another significant advance in our endeavor to treat functional bowel disorders. Multiple 5HT receptor subtypes (5HT1, 5HT2, 5HT3 and 5HT4) are present in the ENS. Five differential

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distributions of 5HT receptor subtypes make it possible to use 5HT3 antagonists and 5HT4 agonist to treat motility disorders and IBS. When all the signaling by 5HT is over, its action is terminated by uptake into enterocytes or neurons that is mediated by the serotonin reuptake transporter (SERT). A number of investigators have found that the colonic mucosa of IBS patients had reduced expression of SERT (mRNA and immunohistochemistry), whereas the number of enteroendocrine cells and the release of serotonin under baseline conditions or in response to stimulation are normal. Thus the serotonergic network appears to play a key role in the neurohormonal brain-gut axis in health and disease, and should be an important target for new therapeutic approaches.

Citations:1. Tache Y and Bonaz B. Corticotropin-relasing factor receptors and stress-related alterations of gut motor function. J Clin Invest 117:33-40, 2007.2. Martinez V, Tache Y. CRF1 receptors as a therapeutic target for irritable bowel syndrome. Curr Pharm Des 12:4071-4088, 2006.3. Gravanis A and Margioris AN. The corticotrophin-releasing factor family of neuropeptide in inflammation: potential therapeutic applications. Curr Med Chem 12:1503-1512, 2005.4. Mayer EA, Naliboff BD, Craig ADB. Neuroimaging of the brain-gut axis: from basic understanding to treatment of functional GI disorders. Gastroenterology 131:1925-1942, 2006.5. Morgan V, Pickens D, Gautam S, Kessler R, Mertz H. Amitriptyline reduces rectal pain related activation of the anterior cingulated cortex in patients with irritable bowel syndrome. Gut 54:601-607, 2005.6. Hobson AR, Furlong PL, Worthen SF, Hillebrand A, Barnes GR, Sign KD, Aziz Q. Real time imaging of human cortical activity evoked by painful esophageal stimulation. Gastroenterology 128:610-619, 2005.7. Berman SM, Chang L, Suyenbu B, Derbyshire SW, Stains J, Fitzgerald L, Mandelkern M, Hamm L, Vogt B, Nalboff BD, Mayer EA. Condition-specific deactivation of brain regions by 5HT3 receptor antagonist alosetron. Gastroenterology 123:969-977, 2002.8. Hicks GA, Coldwell JR, Schindler M, et al. Excitation of rat colonic afferent fibers by 5HT3 receptors. J Physiol 544:861-869, 2002.9. Gershon MD. Plasticity in serotonin control mechanisms in the gut. Curr Opin Pharmacol 3:600-607, 2003.10. Coates MD, Mahoney CR, Linden DR, et al. Molecular defects in mucosal serotonin content and decreased serotonin reuptake transporter in ulcerative colitis and irritable bowel syndrome. Gastroenterology 126:1657-1664, 2004.

Research Advance #3

Until recently, it was generally believed that neural crest stem cells (NCSCs) undergo progressive restrictions in developmental potential and terminally differentiate soon after reaching post migratory sites. The postnatal peripheral nervous system was thought to lack stem cells. A number of recent studies show that NCSCs persist in the adult gut and they undergo changes in self renewal. Functionally the postnatal gut NCSCs make neurons that express a variety of neurotransmitters but lost the ability to make certain subtypes of neurons that are generated during fetal development. These include serotonergic and adrenergic neurons. This may be due to the loss of responsiveness of postnatal gut NCSCs to the neurogenic effects of BMPs. It should also be noted that cell-intrinsic differences between stem cells from different regions of the peripheral nervous system regulate the generation of neural diversity. For example upon transplantation of uncultured NCSCs into developing peripheral nerve in vivo, sciatic nerve NCSCs gave rise only to glia, while gut NCSCs gave rise primarily to neurons. Thus cell fate in the nerve is stem cell determined. Although we are still a long way from using stem cell technology to do replacement therapy, the demonstration that neural crest stem cells persist in the adult enteric nervous system and undergo self renewal opens up a new possibility for regeneration after injury or disease.

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Citations:1. Kruger GM, Mosher JT, Bixby S, Joseph N, Iwashita T and Morrison S. Neural crest stem cells persist in the adult gut but undergo changes in self-renewal, neuronal subtype potential and factor responsiveness. Neuron 35:657-669, 2002.2. Bixby S, Kruger GM, Mosher JT, Joseph NM, Morrison SJ. Cell intrinsic differences between stem cells from different regions of the peripheral nervous system regulate the generation of neural diversity. Neuron 35:643-656, 2002.3. Joseph NM, Mukouyama YS, Mosher JT, Jaegle M, Crone SA, Dormand EL, Lee KF, Meijer D, Anderson DJ and Morrison SJ. Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells. Development 131:5599-5612, 2004.

Research Advance #4

The recognition of the pivotal role of gut hormone in glucose homeostasis has opened up new therapeutic options in the treatment of type 2 diabetes mellitus. Among the various gut hormones, both GLP1 and GIP promote insulin biosynthesis and islet cell survival. Additionally, GLP1 also inhibits glucagons secretion and gastric emptying and induces satiety. GIP engages receptors on adipocytes coupled to energy storage. On the other hand, CCK and gastrin do not seem to acutely regulate levels of plasma glucose but might be important for stimulating the formation of new cells by stimulating islet neogenesis. These pleiotropic actions of gut hormones may be exploited to develop normal therapeutics in the treatment of disorders of energy homeostasis.

Recognition of the GI tract’s crucial role in satiety signaling and control of energy homeostasis is important in the formation of new approaches to combat obesity. Most of the satiation and orexigenic peptides are found in the GI tract. Rationale manipulation of the neuroendocrine pathways regulating appetite may be used to treat obesity. Furthermore gut microbiota can be an important contributing factor to the pathophysiology of obesity. Metagenomic and biochemical analyses show that mouse gut microbiota have different capacities to harvest energy from the diet. This trait is transmissible as colonization of germ-free mice with an “obsese microbiota” results in a significantly greater increase in total body fat than colonization with a “lean microbiota.” This observation if confirmed in humans will strongly suggest that gut microbiome may be a biomarker, a mediator, and a new therapeutic target for people suffering from obesity.

Citations:1.Drucker DJ. The role of gut hormones in glucose homeostasis. J Clin Invest 117:24-32, 2007.2. Ahren B et al. Inhibition of dipeptidyl peptidase-4 reduces glycemia sustains insulin levels, and reduces glucagons levels in type 2 diabetes. J Clin Endocrinol Metab 89:2078-2084, 2004.3. Zander M, Madsbad S, Maden JL and Holst JJ. Effect of 6 week course of glucagon-like peptide 1 on glycemic control, insulin sensitivity, and beta-cell function in type 2 diabetes: a parallel group study. Lancet 359:824-830, 2002. 4. Meier JJ et al. Stimulation of insulin secretion by intravenous bolus injection and continuous infusion of gastric inhibitory polypeptide in patients with type 2 diabetes and healthy control subjects. Diabetes 53 (Suppl 3): S220-S224, 2004.5. Rooman I, Bouwens L. Combined gastrin and epidermal growth factor treatment induces islet regeneration and restore normoglucemia in C57 B16/J mice treated with alloxan. Diabetologia.6. Farilla L et al. Glucagon-like peptide 1 inhibits cell apoptosis and improves glucose responsiveness of freshly isolated human islets. Endocrinology 144:5149-5158, 2003.7. Morton GJ, Cummings DE, Baskin DG, Barsh GS, Schwartz MW. Central nervous system control of food intake and body weight. Nature 443:289-295, 2006.8. Batterham RL. Inhibition of food intake in obese subjects by peptide 443-36. N Engl J Med 349:941-948, 2003.9. Turnbaugh PJ, Ley RE, Mahowald MA, Margrini V, Mardis ER, Gordon JI. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444:1027-1031, 2006.

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2. GOALS FOR RESEARCH

Short-Term Goals (1-3 years)

The structural and functional organization of ENS continues to evolve. We need to more fully characterize the enteric neural networks; understand the cross-talk between the enteric neurons, glial cells, interstitial cell of Cajal, smooth muscle cells, and enteroendocrine cells; and define the signal processing within the ENS in response to nutrient, mechanical stimulation, inflammation, metabolic stress, and changes in bacterial flora. Technically, we must develop the tools to visualize state of activity of relevant cells (ICC, enteric neurons, muscle cells, and neuroendocrine cells) in a live context of tissue (in vitro or in vivo), organ and system (in vivo) and develop quantitative analysis of spatio-temporal patterns.

We must investigate the molecular and electrophysiological characteristics of the various cellular components of the ENS. This includes identification of receptors, channels and signal transduction systems unique to different cell types in the ENS as they may be targets for new drug developments to treat motility disorders.

Among the neurons in the ENS, there is much to learn about the interneurons. Available evidence suggests they mediate motility reflexes and are the real site of integration. We need to identify the molecular and electrophysiological characteristics of these neurons and learn how they process information and coordinate sensory and motor reflexes. To visualize the state of activity of these neurons in live tissues and organs may help us to understand their roles in initiating specific motility patterns.

In the CNS, glial-derived neurotrophins and neurotrophic factors such as GDNF have strong effects on neurite outgrowth and differentiation and are able to protect neurons from apoptosis under various inflammatory conditions. It is likely that glial cells may play similar roles in the ENS. This needs to be thoroughly investigated. The mechanisms responsible for upregulation of the GDNF in inflammatory bowel conditions should be examined and its cellular actions to promote epithelial restitution should be investigated. Alterations of this novel gut neuroepithelial crosstalk may contribute to more severe course of disease in IBD.

Because the intrinsic primary afferent neurons (IPANs) are the initiating neurons for enteric reflexes, they are strategically placed to influence the intensity of the reflexes. We need to fully characterize the plasticity of the channels, receptors, and intracellular control systems in response to inflammation, metabolic stress, and changes in luminal bacterial flora. Altered properties of IPANs might be involved in the development of intestinal hypersensitivity and changed motility in IBS. This should be evaluated using an appropriate animal model and subsequently in human tissues.

Currently little is known about the site and mechanisms by which alterations in blood glucose levels is sensed. The glucose sensing neurons in the CNS are likely to play a major role in mediating postprandial digestive and metabolic function in response to physiological changes in circulating glucose as the glucose level in the CSF only ranges between 10-30% of blood glucose level. Comprehensive studies must be performed to investigate the peripheral mechanisms of glucose sensing. The site is likely to be at the vagal afferent fibers that may mediate gastric motility, satiety, and hepatic glucose uptake and outputs. Abnormality of this pathway may occur in patients with diabetes and/or obesity.

Characterize alterations in the gut-based 5HT signaling system in IBS and motility disorders. This may include SERT, modulators of SERT expression, and number of enterochromaffin cells and contents. This mechanism responsible for diminished serotonergic signaling in inflammation should be investigated. Transcripts encoding tryptophan hydroxylase-1 and SERT can be decreased. Successive potentiation of 5HT and/or desensitization of its receptor could account for the symptoms seen in

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diarrhea-predominant and constipation-predominant IBS, respectively. This possibility should be investigated.

Progress in the research of functional bowel disease is partly hampered by lack of suitable animal models to mimic the disease conditions. Although animal models of visceral hypersensitivity are available, they are not ideal as the pathophysiology may be quite different. We need to establish scientific criteria to determine the validity of animal models for IBS and other motility disorders.

Intermediate-Term Goals (4-6 years)

Define the roles of various cellular components of the ENS in the mediation of physiological events such as motility, sensory transmission, secretion, and blood flow. There is a need to integrate cellular events with whole system physiology.

Our understanding of ICC pathology is still in its infancy, but a picture is beginning to emerge that many disparate motility disorders result in loss of ICC. One approach is to make large-scale screenings of genetic changes that occur in tissues undergoing loss of ICC and investigate whether there are common genetic fingerprints that either produce or result from loss of ICC. Pathophysiological models coupled with genomics analyses may offer the opportunity to discover why ICC is vulnerable in apparently disparate motor disorders and how to recover ICC networks in disease organs.

Identify the distinct neural circuits involved in mediating different motility patterns. Define the principle receptors, neurotransmitters, and synaptic connections for each circuit. Identify the “switch” responsible to change one motility pattern to another. Examine how mechanical stimulation, metabolic stress, and inflammation can alter these circuits. This type of information may be important to define the pathophysiology of IBS.

Over the last decades, numerous molecules, pathways, and mechanisms linking the brain and the gut are now emerging but there is as yet comparatively little understanding of how these may be involved in organ dysfunction and disease. From neurochemical substrata and pathways that have been dissected, we have yet to find out which are utilized in health and whether these differ in disease and, if so, whether such differences are causes or merely consequences of the disorder. Expanded research effort should aim at unraveling the pathophysiology of brain-gut interactions.

We need to have a better understanding of the molecular mechanisms underlying age-related apoptosis of ENS neurons. Contemporary techniques for probing genetic and proteomic changes that occur with age, such as the use of oligonucleotide microarray and protein chip technologies, will help the field to evolve from phenomenology to hypothesis-driven research. It is important to establish the mechanisms that maintain the integrity of the adult ENS and its capacity to respond to altered function or “placticity” in adulthood and old age. Studies of neurotrophic and growth factors and the associated signaling pathways may provide some clues for this type of investigation.

We should continue our efforts to identify distinct brain circuits involved in autonomic regulation, appetite control, pain perception, and emotional and cognitive modulation. Characterize the networks in healthy control populations and various patient populations.

Investigate the channels and signal transduction pathways that mediate cell mass, resistance to apoptosis, and stimulation of biosynthesis and secretion of insulin. Agents with these biological properties may become therapeutics to treat diabetes.

Over the last decade, there were many major advances in the understanding of the molecules that regulate the development of the ENS. However, much remains to be learned about how the various signals interact to yield a “normal enteric nervous system.” We should continue to search for clues as we begin to identify developmental defects resulting in neuronal loss such as in Hirschsrpung’s disease

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and hypertrophic pyloric stenosis. We need to identify molecules and pathways that promote proliferation and differentiation of enteric neurons and/or guide the growth of enteric axons to their targets.

Long-Term Goals (7-10 years)

Genomic analysis of animal model of ICC loss may demonstrate common endpoints for the fate of ICC in a variety of motility disorders. From these studies, it may be possible to determine specific gene profiles that occur in tissues that have suffered ICC loss or in the tissues in the process of losing ICC. This information may allow scientists to design specialized tests that reliably evaluate the status of ICC networks from biopsy material. Development of a molecular test that could pick up pathological changes more reliably and at earlier time points may be highly beneficial as we may be able to halt the degeneration and minimize the damage and loss of function.

The fate of ICC in variety of motility disorders is unknown. Cells may undergo apoptosis or redifferentiation into muscle cells. Understanding the molecular mechanisms responsible for differentiation and redifferentiation of ICC may allow scientists to perform tissue engineering and to restore functional populations of ICC in patients with ICC loss.

Following identification of distinct brain circuits responsible for various GI functions and pain perception, we should characterize the signaling systems and receptors within these neural circuits using PET ligand imaging in rodents and in humans. Ultimately, we should be able to correlate the individual circuits identified with symptom production in patients (“intermediate phenotypes”) and establish correlation with distinct genotypes that include genome-wide search for polymorphism and haplotypes.

With the goals established above, ultimately we need to be able to fully characterize the pathomechanisms underlying symptom generation in common functional and motility disorders, including identification of genetic and early environmental influences and natural course of the disease. This should lead to more effective therapeutic intervention.

Degeneration in the enteric nervous system resulting in motility disorders is one of the hallmarks of aging and can be a major determinant of quality of life for the aged. Neuron replacement therapy should be our targeted goal. Once we have obtained the blueprint of the key neural circuits in the ENS, we should utilize our knowledge gained in developmental biology and organ genesis to manipulate the neural crest stem cells in the bowel wall to proliferate and differentiate and provide the signaling molecules to guide the growth of enteric axons to their targets.

Obesity research remains the top priority for investigators in the United States. The capacity to adjust food intake in response to changing energy requirements is essential for survival and good health. We need to continue to characterize the molecular, cellular, and behavioral mechanisms that link changes of body fat store to adaptive adjustments of feeding behaviors. We need to define the diverse blood-borne and affective neural signals that transmit information regarding nutrient status and energy stores to the brain where it is integrated with cognitive, visual, olfactory, and taste cues. Understanding the complexity of this energy homeostasis system will then allow us to start to search for mutation of key molecules mediating these pathways in patients with severe obesity.

3. MAJOR CHALLENGES AND STEPS TO ACHIEVE GOALS

General Comments

Advances in the molecular biology sciences and proteomics represent an unprecedented opportunity for us to understand key biologic pathways pertaining to the neuroendocrine system of the GI tract. However, much remains to be done. The limitations of NIH funding, hesitation of young trainees to embark on a

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scientific career, the lack of a cohesive national research agenda, and the inability to translate observations made at the bench to bedside practice all contribute to the challenges before us.

The search for novel technologies and development of national core facilities should be encouraged. We should put major emphasis on the development of new technology to visualize states of activity or relevant cells in live context of tissues, organ, and system and develop quantitative analysis of spatio-temporal patents. Application of bioinformatics to motility disorders and further refinements of proteomics and metabolic technologies to the gut and motility will be important. The development of multi-institutional consortium agreements to share resources should be fostered. This will help us take advantage of national core resources laboratories for expansive technologies such as neuro-imaging, genotyping of microflora, and advanced cell and in-vivo imaging techniques. Emphasis on translational research approaches cannot be overstated. The funding agencies should insist that the studies mechanism have a clear relevance for clinical medicine.

To best utilize our limited talent and financial resources, in the most effective manner, we need to develop a national agenda of priorities and fund research programs that have different clinical themes. A consortium of research teams consisting of scientists with different backgrounds and expertise from different disciplines working together to solve a common problem should be developed. These researchers may be recruited from institutions and laboratories with different skills to contribute to solving a clinical disorder. The research should be complementary, interdisciplinary, and coordinated with a common purpose and targets. This approach may be far superior to the current use of funding silos of purely specialty-based research. This will foster thinking outside the box and promote creative solutions for many perplexing issues in neurogastroenterology.

Goal: To integrate cellular events with whole system physiology and pathological conditions. Challenge/Steps: The “bench to bedside” approach is difficult to achieve by any single laboratory. This requires interdisciplinary research effort. Research on the pathophysiology of GI motility disorders continues to be hampered by lack of tissue from patients who are carefully genotyped and phenotyped. Generation of large national and international data and tissue banks may help to address this issue.

Goal: To understand how pathways linking brain and gut may explain organ dysfunction and disease. Challenge/Steps: Neuroplasticity and redundancy of pathways may obscure the physiological and/or pathophysiological significance of the brain-gut axis. Need animal models and watch for compensatory responses over time.

Goal: Redundancy of both GI and CNS pathways governing energy homeostasis poses major challenges for scientists designing anti-obesity drugs.Challenge/Steps: Treatment of obesity may require drug combinations that target discrete components of energy homeostasis, satiety, or food reward system.

Goal: To understand the physiology of functional bowel disease and search for new therapeutic optionsChallenge/Steps: IBS is a heterogeneous group of disorders with different pathophysiology for different subgroups. Currently there are no biomarkers and therapeutic target. Patients need to be better phenotyped based on clinical symptoms and pathophysiology consideration. Need to understand cellular mechanisms of disease to identify drug targets. The same is true for most motility disorders.

4. PATIENT PROFILE TOPICPlease give brief description of an idea for a patient profile relevant to this research topic.

None

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5. GRAPHICS AND IMAGES If you have access to the graphic/image, please send as email attachment; if not, please provide information on the source of the graphic/image.

None

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NAME: Abigail Salyers, PhD, University of Illinois at Urbana-ChampaignWORKING GROUP: Overview of the Digestive System (WG 1)SUBGROUP: Intestinal Microbiota and Digestive Health

1. RESEARH ADVANCES

Research Advance #1A workable and reproducible system for assessing the microbial population of the human colon

The human colonic microbiota clearly has a number of effects on human physiology and health. These include such proven effects as the role of the human microbiota in human nutrition through the colonic fermentation and the role of intestinal microbes as opportunistic pathogens. Less clear, but more exciting, are suspected but still unproven links to such intestinal diseases as inflammatory bowel disease, colon cancer, and such nonintestinal effects as obesity and autism. In the past, testing hypotheses about the effects of the colonic microflora has been hampered by the need for cultivation-based methods for enumerating the colonic microbiota, which are both time-consuming and unreliable. Molecular methods are beginning to be applied to assessing the composition of the colonic microbiota, based on a ribosomal RNA gene census approach but such methods need to be made more user-friendly and widely available.

Short-Term Goals (1-3 years)

1. Collect the accumulating mass of rDNA data from the human colon, the murine colon and the colons of any animals such as pigs that might be used as models for humans.

2. Develop a microarray that contains rDNA sequences from all of the known human intestinal species. This may have to be divided into separate microarrays for major and minor species. Develop reliable methods for detecting the population structure of the microbiota by isolating rRNA from fecal material and hybridizing it to the microarray.

3. Assess the relevance of animals such as mice and humans as models for the human microbiota. This would include testing of “humanized mice”, i.e. germfree mice that have been colonized with human fecal material. In the case of humans the microarray could be used but in the case of nonhuman animals it might first be prudent to do a classical PCR/cloning and sequencing of rDNA to assess the broad structure of the microbial population.

4. Make microarrays based on existing DNA sequences of colonic bacteria affordable and available to as many research groups as possible.

Intermediate-Term Goals (4-6 years)

1. Begin to take the “census” data provided by the rDNA sequencing studies to the level of physiology by obtaining the genomic sequences of the “unknown majority” of colonic bacteria, the gram positive anaerobic bacteria. Although many of these species, which comprise at least 60% of all colonic bacteria, have been cultivated, cultivation is difficult. Obtaining the genome sequence of the leading representatives of this group of bacteria, whose numbers are undisputedly large but whose roles in the colon are unknown, would help to develop hypotheses about what they might be doing.

Long-Term Goals (7-10 years)

1. Use the technology developed in the previous years to:

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(1) Do a systematic study of individual to individual and time to time variation in the microbiota in order to answer the question: What is “normal”? This should include studies of populations that are not in developing countries.

(2) Assess associations between diseases suspected to be caused or exacerbated by changes in the intestinal microbiota. This has already been started, but is not occurring in a very organized manner.

2. Begin to test interventions, whether from antibiotic treatment or from probiotics, for their short-term and long-term effects on the colonic microbiota. Realistically, this will have to be done first in animals but some studies such as the effect of probiotic administration or diet could be assessed in humans.

Research Advance #2Moving from “who’s there?” to “what are they doing?”

Taking a “census” of the bacteria that are present in the colon at any particular time is important, but this approach has an important limitation. The species identity of a microbe does not usually reveal its metabolic potential because of the incredible metabolic diversity of bacteria. The metabolic potential of a microbial population can be assessed in two ways. First, a metagenomic analysis, in which the whole genomic DNA of bacteria in a population is determined by random sequencing, can suggest metabolic potential by determining what genes are present in the population. This approach has two important limitations. There are many genes of unknown function (usually at least 20% of the genes in any sequenced bacterial genome) and the not all of these genes are being expressed at any one time. The former limitation may be overcome as more information becomes available about bacterial physiology and as the sequence databases become larger. The second limitation can be addressed by a second approach to analysis of the metabolic potential of a bacterial population: microarray analysis of gene expression. In some cases, this approach can resolve the “gene of unknown function” problem by identifying the effect of different environmental parameters on expression of the gene.

Short-Term Goals (1-3 years)

1. Begin a metagenomic analysis of the NORMAL human intestinal (and perhaps murine intestinal) population. In doing this, the latest advances in DNA sequencing technology and bioinformatics technology need to be brought to bear. Thus, much of the work done in this initial period would focus on assessing the best way to design and analyze metagenomic analyses. In this effort, it would be prudent to consult with soil and water microbiologists who are far in advance of microbiologists who work on the human microbial populations.

Intermediate-Term Goals (4-6 years)

1. Continue to expand the metagenomic analysis, now extending it to various diets and disease states.

2. Develop bioinformatic analytical procedures to assess the significance of the differences between metagenomes obtained in connection with different dietary, disease and age conditions.

3. Begin to develop microarrays based on the emerging metagenomic data and the genome sequences arising from Research Advance #1, and – most importantly – make these arrays available to a wide variety of scientists including nutritionists as well as clinicians.

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Long-Term Goals (7-10 years)

1. Figure out how to assess the mass of data flowing in from the previous years. Based on this experience, develop new algorithms and hypotheses that can help to refine and streamline future studies.

Research Advance #3Evaluating the archaea and eukaryotes as members of the colonic microbiota

The human colon contains microbes other than bacteria. Two known non-bacterial groups are the methanogenic archaea and the protozoal and metazoal eukaryotes. The methanogenic archaea, although much lower in numbers than the bacteria, produce enough methane in about 20% of the population for methane to be detected in breath samples. The human colonic methanogenic archaea have been characterized to some extent by culture based studies but it is not clear whether all of the colonic archaea, whether methanogenic or not, are known. Similarly, we know that protozoa and helminthes are found widely in the colons of people in developing countries but thought to be absent in people in developed countries. In fact, some scientists have suggested that diseases like asthma and inflammatory bowel disease are more prevalent in developed countries precisely because the LOSS of these eukaryotic organisms has altered what used to be the normal stimulation of the enteric immune system in a way that predisposes to allergic diseases. The view that protists and helminthes are absent from the intestines of people in developed countries is based on very inefficient microscopic analyses. Molecular methods could be applied to assess how prevalent these organisms actually are in the intestines of people from developed countries and might even reveal new, unsuspected eukaryotic denizens of the intestinal tract.

Short-Term Goals (1-3 years)

1. Assess the existing primers for a rDNA census of archaea and eukaryotic microbes. (Helminths are considered here to be honorary microbes even though many of them are large enough to be seen without a microscope.) Fungi should also be included in these exploratory studies.

2. Conduct a “census” of the normal human colonic microbiota using these primers. Parallel studies could also be done with a population from a developing country to see how many differences actually exist. It may be that some of the protists and helminthes and yeasts that are supposed to be absent in people from developing countries are actually present at very low levels. Or there could be substantial differences in the eukaryotic microbes found in the intestine depending on nutritional and sanitation factors.

Intermediate-Term Goals (4-6 years)

Extend these studies to people with diseases such as asthma, inflammatory bowel disease, and other inflammatory or allergic conditions. Autism might be included in this list since this is another disease that is more prevalent in developed countries.

Long-Term Goals (7-10 years)

1. These goals are dependent on what is found in the earlier years. Explore possible therapies based on earlier insights or put to rest forever the “hygiene hypothesis.”

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Research Advance #4Is there a normal viral microbiota of the human intestine?

This is a really outrageous question, but one that needs to asked. We know that influenza virus is shed from the intestinal tracts of otherwise healthy birds. In addition, viruses that infect bacteria often carry genes that encode toxins that are harmful to humans.

Short-Term Goals (1-3 years)

1. Use metagenomic technology to assess the viral population of the human intestine. This is already being done by the marine microbiologists for the ocean, so the techniques are available.

Intermediate (4-6 years) to Long-Range Goals (7-10 years)

Depends on outcome of short term goals.

Research Advance #5Genetic interactions between members of the microbiota and between members of the microbiota

and human intestinal cells

We now know know that bacteria are interacting genetically with each other and with lower eukaryotes by a process called conjugation. Are they also interacting with human cells in the same way?

Short-Term Goals (1-3 years)

This is another case in which the short-term goals will influence how far work proceeds. It would be straightforward to test in tissue culture the ability of several bacterial conjugative elements to transfer DNA to mammalian cells. If the answer is yes, the question of significance arises. Most of the intestinal cells are terminally differentiated. Would bacterial DNA affect them in any significant way? Colonic stem cells are a different matter.

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NAME: Warren Strober, MD, National Institute of Allergy and Infectious Diseases, NIHWORKING GROUP: Overview of the Digestive System (WG 1)SUBGROUP: Mucosal Immunology

The understanding of mucosal immune mechanisms will inevitably have a major impact on the understanding of immune diseases affecting the gastrointestinal tract and on both mucosal and system infections.

Research Advance #1The role of the intestinal microflora in the maintenence of mucosal immune homeostasis

The mucosal immune system is unique in that it lies in juxtaposition to an enormous consortium of commensal organisms that play multiple roles in maintaining gut homeostasis, including the prevention of colonization by pathogens and in the promotion of epithelial cell repair following damage. These organisms are separated from mucosal lymphoid elements by a single layer of epithelium and an over-lying mucus that prevents wholesale entry of the bacteria. Nevertheless, it has shown that commensal organisms do enter the mucosa via Peyer’s patches and are picked up by dendritic cells in these lymphoid structures. An additional mode of entry of commensal organisms is via CD11c+ dendritic cells in the lamina propria that extent processes between epithelial cells and take up organisms; this process is enhanced in epithelium exposed to TLR ligands. The function of such limited commensal uptake is several fold. First it leads to the production of IgA that functions to limit further uptake of organisms by coating organisms and preventing colonization. Second, it leads to the induction of regulatory T cells that control T cell responses in the mucosal and thus prevent the flora from inducing inflammation.

Citations:

Rakoff-Nahoum, S., Paglino, J., Eslami-Varzaneh, F., Edberg, S., and Medzhitov R (2004) Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 118: 229-241.

Macpherson, A.J. and Uhr, T. (2004) Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria. Science 303:1662-1665.

Chieppa, M., Rescigno,M. Huang, A.Y. and Germain, R.N. (2006) Dynamic Imaging of dendritic cell extension into the small bowel lumen in response to epithelial cell TLR engagement. J.Exp. Med., 203:254-258.

Tringe, S.G., von Mering, C., Kobayashi, A., Salamov, A.A., Chen, K., Chang, M., Podar, J.M., Short, E.J., Mathur, Detter, J.C., Bork, P., Hugenholtz, P and Rubin, E.M. (2005) Comparative metagenomics of microbial communities. Science 308:554-557.

Short Term Goals (1-3 years):

Further advances in the understanding of the relationship between the commensal organisms will depend in part on a better definition of the nature of the organisms in the mucosal flora in health and disease. Recently, there has been a revolution in the technical ability to characterize the gut flora with high output molecular techniques. Use this methodology to define possible difference in the mucosal flora amoung healthy individuals and individuals with inflammation or neoplasms of the GI tract.

Intermediate Term Goals (4-7 years)

Simultaneously track the growth of multiple organisms following introduction into an axenic environment and thus to define the effects of individual members of the microflora on mucosal immune responses.

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Extend the above study of commensal microflora to murine models of gastrointestinal inflammation to better characterize which organisms contribute to the prevention/induction of inflammation.

Gain a better understanding of the differences between innate responses to commensal organisms and pathogens so as to elucidate why commensal organisms generally lead to protective and anti-inflammatory responses in normal individual and pathogens lead to inflammation.

Long Term Goals (7-10 years)

Identify the antigens and TLR ligands that are specific for particular organisms and how they affect gut mucosal immune function at the level of innate and adaptive immune responses.

Develop approaches to the manipulation of commensal microflora populations so the population protects the host from the development of infection and inflammation and or reverses on-going inflammation.

Research Advance #2The role of epithelial cells in mucosal host defense and inflammation

It is now apparent that epithelial cells are not passive participants in the mucosal immune response, but on the contrary, play active and perhaps key roles in the shaping/initiation of that response. This manifests itself in the ability of epithelial cells to produce chemokines and cytokines that initiate innate immune cell responses and thus set up a first line of defense against the intrusion of organisms into the mucosa. Many of these responses are induced by Toll-like receptors and nucleotide oligomerization domain-LRR receptors interacting with microbial components. There is also increasing evidence that epithelial cells produce substances such as thymic stromal lymphopoietin (TSLP) that influence dendritic cell function and thus determine the nature of T cell differentiation that occurs in relation to mucosal antigenic stimulation. On another level, epithelial cells transport IgA via the polyimmunoglobulin receptor and IgG via the neonatal Fc receptor and in doing so carry anti-bacterial agents to the luminal surface and/or move immunoglobulin/antigen complexes in a bi-directional manner across the epithelium.finally, epithelial cells produce a variety of anti-bacterial substances including defensins and lectins (cryptins) that regulate the bacterial population in intestinal crypts and thus contribute to the development of inflammatory bowel disease. Regarding the latter, there is initial genetic evidence that susceptibility genes in IBD act through effects on the barrier function of the epithelium so that changes in the way the epithelium controls the local commensal flora and/or allow the penetration of bacteria, may influence the development of inflammation in the underlying mucosa.

Citations:

Benn, M.C. Darfeuille-Michaud, A., Egan, L.J., Miyamoto, Y. and Kagnoff, M.F. (2002) Role of EHEC O157:H7 virulence factors in the activation of intestinal epithelial cell NF-kappa B and MAP kinase pathways and the upregulated expression of interleukin 8. Cell Microbiol. 4:635-648.

Rimoldi, M., Chieppa, M., Salucci, V., Avogadri, F., Sonzogni, A., Sampietro, G.M., Nespoli, A., Viale., G., Allavena, P., and Rescigno, M (2005) Intestinal immune homeostasis is regulated by the crosstalk between epithelial cells and dendritic cells. Nature Immunol. 6:507-514.

Yoshida, M., Kobayashi, K., Kuo, T.T., Bry, L., Glickman, J.N., Claypool, S.M., Kaser, A., Nagaishi, T., Higgins, D.E., Mizoguchi, E., Watatsuki, Y., Roopenian, D.C., Mizoguchi, A., Lencer, W.I. and Blumberg, R.S. (2006) Neonatal Fc receptor for IgG regulates mucosal immune responses to luminal bacteria. J. Clin. Invest. 116:2142-2151.

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Cash, H.L., Whitham., C.V., Behrendt C.L., and Hooper, L.V. (2006) Symbiotic bacteria direct expression of an intestinal bactericidal lectin. Science 313: 1126-1130.

Cario E., Gerken, G., and Podolsky, DK (2004) Toll-like Receptor 2 enhances ZO-1-associated intestinal epithelial barrier integrity via protein kinase C. Gastroenterology 127:224-238.

Short-Term Goals (1-3 years)

Elucidation of the intra-cellular signaling pathways involved in the production of chemokines, cytokines and cryptins produced by epithelial cells.

Definition of the factors that regulate the expression of TLR and NLR on/in epithelial cells and the effect of stimulation of these receptors on epithelial barrier function, chemokine/cyokine production and cryptin production.

Exploration of the role of the neonatal Fc receptor in mucosal immune responses and induction of mucosal unresponsiveness (tolerance).

Elucidation of the factors produced by epithelial cells including TSLP, IL-10 and TGF-beta that effect dendritic cell function and/or T cell differentiation.

Intermediate-Term Goals (3-7 years)

Production mice expressing epithelial cell-specific knock-outs of key genes involved in epithelial mucosal immune function and barrier function followed by extensive evaluation of the effect of the loss of these genes on gut homeostasis, particularly with respect to understanding IBD and/or infection of the GI tract.

Definition of the function of genes affecting epithelial cell immune function or barrier function identified as susceptibility genes in IBD.

Long-Term Goals (7-10 years)

Characterization of embryonic and adult stem cell differentiation into epithelial cells focusing particularly on the acquisition of properties that relate to epithelial immune function. This includes the factors that control the differentiation of M cells and Paneth cells.

Development of methods for the long term cultivation of primary epithelial cells and the development mice with humanized epithelial cells.

Development of model systems for the elucidation of epithelial cell-lymphoid cell interactions.

Research Advance #3The role of antigen-presenting cells in the mucosal immune system

The dendritic cell is a key cellular player in the mucosal immune response as it is in immune responses in general; as such it plays a role in mucosal host defense and in the pathogenesis of inflammatory bowel disease. Studies of the function of the mucosal dendritic cell revealed that these cells are, as a population, somewhat unique. For example, the CD11chi population is made up of several sub-populations including a CD8lo sub-population that have an increased propensity to produce IL-10 and stimulate Th2 responses as compared to phenotypically-similar cells in the spleen; in addition, a CD11clo (plasmacytoid dendritic cell population is present but in this enviroment does not produce IFN-; finally, there are the aforementioned dendritic cells that sample antigens in the mucosal lumen via the extension of dendrites. Dendritic cells in the Peyer’s patches process antigen released from infected and apoptotic epithelial cells and then present

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these antigens to CD4+ T cells; dendritic cells in the lamina propria, perhaps those that engage in luminal antigen sampling, migrate to draining lymph nodes for presentation of antigens to T cells which then demonstrate a unique propensity to migrate back to the mucosa; these dendritic cells (as well as those from the Peyer’s patches can also migrate back to the lamina propria where they take part in the differentitation of B cells into IgA producing cells through the elaboration of B cell differentiation factors such as Baff and APRIL. Finally, evidence has recently emerged that mucosal dendritic cells may be uniquely involved in the induction of regulatory T cells in the mucosa via the production of TGF- as well as the induction of Th17 producing cells via production of IL-6 and TGF-. They thus control the balance of effector cells and regulatory cells at mucosal sites.

Citations:

Iwasaki, A., Kelsall, B.L. (2000) Localization of distinct Peyer’s patch dendritic cell subsets and their recruitment by chemokines macrophage inflammatory protein (MIP)-3alpha, MIP-3beta, and secondary lymphoid organ chemokine. J.Exp.Med; 191:1381-1394.

Rimoldi, M., Chieppa, M., Salucci, V., Avogadri, F., Sonzogni, A., Sampietro, G.M., Nespoli, A., Viale., G., Allavena, P., and Rescigno, M (2005) Intestinal immune homeostasis is regulated by the crosstalk between epithelial cells and dendritic cells. Nature Immunol. 6:507-514.

Johansson-Lindbom, B., Svensson, M., Pabst, O., Palmqvist, C., Marquez, G., Forster, R., and Agace, W.W. (2005) Functional specialization of gut CD103+ dendritic cells in the regulation of tissue-selective T cell homing. J.Exp. Med., 202:1063-1073.

Huang, F.P., Platt, N., Wykes, M., Major, J.R., Powell, T.J., Jenkins, C.D. and MacPherson, G.G. (2000) A discrete population of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes. J.Exp. Med. 191:432-444.

Watanabe, T., Kitani, A., Murray, P.J. and Strober,W. (2004) NOD2 is a negative regulator of Toll-like receptor 2-mediated T helper I responses. Nat. Immunol. 5:800-808.

Short-Term Goals (1-3 years)

Definition of the factors that influence dendritic cell maturation/function in the mucosal environment including factors derived from epithelial cells and the commensal microflora.

Elucidation of the chemokines that control the movement of dendritic cells into mucosal sites and around mucosal sites.

Definition of the factors that influence production of factors that differentially influence effector cell and regulatory cell development including the cell-cell interactions involved.

Intermediate-Term Goals (3-7 years)

Construction of mice that lack dendritic cell-specific surface molecule and intra-cellular molecules that control recognition of microbial components (TLR ligands/antigens) and are involved in essentially signaling functions that regulate dendritic cell maturation and activation.

Examination of the function of dendritic cells in murine models of inflammation with relation to changes in the sub-populations of dendritic cells present in the inflamed mucosa and the functions of these dendritic cells.

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Elucidation of the function of TLR and NLR microbial recognition molecules as these relate to positive and negative dendritic cell responses. This includes the propensity of these molecules to induce inhibitory factors that down-regulate responses.

Long -Term Goals (8-10 years)

In-depth elucidation of the intra-cellular signaling pathways that define TLR, NLR, CD40, IL-1b, TNF and other relevant signals that act on dendritic cells and determine the unique behavior of mucosal dendritic cells.

Definition of the effects of co-ordinate signaling on the overall response of mucosal dendritic cells.

Creation of mouse models characterized by dendritic cell dysfunction that lead to mucosal inflammation. Comparison of these mice with the function of dendritic cells derived from normal humans and those with IBD.

Research Advance #4The traffic of mucosal cells to various parts of the mucosal immune system

The last decade has seen major advances in the understanding of how and why the mucosal immune system is unified by a cell circulation system that ensures that cells generated with the inductive areas of the system, the Peyer’s patches and other lymphoid follicles, “home” back to the effector areas of the system, the GI lamina propria and other “diffuse” mucosal areas in other organs. Older studies focused on the role of integrin/integrin receptors in this process, particularly the role of the 47/MAdCAM-1 combination in gut home. However, in newer work it has been established that regional expression of epithelial chemokines in the small and large intestine play an indispensable part in the homing process: IgA plasma cell migration to the small intestine requires the CCL25/CCR9 (ligand/receptor) pair and the CCL28/CCR10 pair for migration to the colon and other mucosal tissues. In addition, the CCL25/CCR9 pair governs the corresponding migration of gut homing T cells to the small intestine; however, in this case, the chemokine pair guiding migration of these cells to the large intestine is still not defined. A major new finding is that retinoic acid (vitamin A) acting through the retinoic acid receptor (RAR) induces IgA plasmablasts and T cells to express homing receptors for the gut. This finding complements the older finding that TGF- induces the expression of the 47 integrin by completing our knowledge of the inducer of B cell gut homing receptors. Finally, the role of chemokine ligand-receptor interactions governs other aspects of mucosal immune function, including the exit of T cells and dendritic cells from the mucosal tissues into draining lymph nodes, the entry of CD8+ thymic emigrants into the mucosal to become intra-epithelial lymphocytes and the movement of dendritic cells to sub-epithelial locations. It is now apparent that the traffic of cells in, around and out of the mucosal immune system is a highly choreographed series of events that depends in great measure on chemokine ligands and their interaction with chemokine receptors.

Citations:

Svensson, M., Marsal, J., Ericsson, A., Carramolino, Broden, T., Marquez, G., and Agace, W.W. (2002) CCL25 mediates the localization of recently activated CD8alphabeta(+) lymphocytes to the small-intestinal mucosa. J. Clin. Invest. 110:1113.

Kunkel, E.J., Kim, C.H., Lazarus, N.H., Vierra, M.A., Soler, D., Bowman, E.P., and Butcher, E.C. (2003) CCR10 expression is a common feature of circulating and mucosal epithelial tissue IgA Ab-secreting cells. J. Clin. Invest. 111:1001.

Hieshima, K., Kawasaki, Y., Hanamoto, H., Nakayama, T., Nagakubo, D., Kanmaru, A., and Yoshie, O. (2004) CC chemokine ligands 25 and 28 play essential roles in intestinal extravasation of IgA antibody-secreting cells. J. Immunol. 173: 3668.

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Iwata, M., Hirakiyama, A., Eshima, Y., Kagechika, H., Kato, C., and Song, S.Y. (2004) Retinoic acid imprints gut-homing specificity on T cells. Immunity 21: 527.

Debes, G.F., Arnold, C.N., Young, A.J., Krautwald, S., Lipp, M., Hay, J.B., and Butcher, E.C. (2005) Chemokine receptor CCR7 required for T lymphocyte exit from peripheral tissues. Nat. Immunol. 6:889.

Short-Term Goals (1-3 years)

Definition of chemokine-chemokine receptor interactions that are critical for the entry of T cells into the large bowel and the entry of CD8+ thymic emigrants into the mucosa to become intra-epithelial lymphocytes. Likewise, definition of the interactions necessary for the entry of cells into other mucosal organs such as stomach, liver and oral cavity.

Definition of the chemokine-chemokine receptor interactions or other cell-cell interactions that contribute to the retention of lymphocytes and other cells in mucosal tissue.

Elucidation of the migration of various classes of regulatory T cells into and out of mucosal tissues.

Exploration of the molecular signaling and gene activation pathways that are involved in retinoic acid-ROR signaling giving rise to mucosal homing receptors.

Intermediate-Term Goals (4-7 years)

Continued exploration the value of blocking gut homing receptors in the prevention/amelioration of IBD and GvHD. These studies are a continuation of extensive studies already performed or underway of antibodies or other agents that impede the activity of the integrin homing receptor, 47 .

Construction of tissue-specific knock-out mice that lack key components of the gut homing apparatus and thus manifest various kinds of mucosal immune abnormalities.

Long-Term Goals (8-10 years)

Development of systems approach to the study of lymphocyte and dendritic cell homing that integrates the many factors that affect this process. This approach would integrate the combined effects on integrin, chemokine effects with the effects of other influences such as the state of cell differentiation, the presence of various cytokine and the influence of Toll-like receptors.

Research Advance #5Mucosal unresponsiveness (oral tolerance) and mucosal regulatory T cell development

It has long been known that oral administration of a protein antigen leads to a subsequent state of unresponsiveness to that antigen as a result of a phenomenon known as oral tolerance induction. Oral tolerance is also manifest in the lack of response to the mucosal microflora, despite the proximity and persistence of this source of potential antigens. The last decade has witnessed a great advance in the understanding of this phenomenon and the possible harnessing of its underlying mechanisms to the therapy of GI inflammation. A significant step forward came with the demonstration that while oral tolerance could be due to exposure of mucosal cells to high doses of antigen (in the absence of adequate T cell co-stimulation), it is more characteristically due to exposure of mucosal cells to low doses of antigen and the induction of regulatory T cells. Further work has established that the most important type of regulatory cell mediating oral tolerance is the so-called “natural” regulatory T cell that are defined by their expression of surface markers such CD25, CTLA-4 and CD103 and GITR and, more importantly, by their expression of a particular transcription factor known at foxp3. While still controversial, it is likely that natural regulatory T cells cause inhibition of responses because they express surface TGF-b and

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secrete TGF-b and brimg this suppressive cytokine to bear on antigen presenting cells. For the most part, these cells develop in the thymus (under the influence of IL-2) and bear antigen receptors recognizing self-antigens; however, it is likely that the also bear receptors recognizing antigens associated with the gut microflora because these antigens have access to the circulation and can enter the thymus. Thus, one scenario for the high prevalence of regulatory cells in the mucosal is that such cells develop in the thymus and then migrate to the mucosa where they re-encounter microflora antigens and undergo clonal expansion. A second possible reason for their high prevalence in the gut is that natural suppressor T cells can be induced to expand in peripheral tissues by TGF-b, a cytokine that is produced by epithelial cells and thus is present at a high steady-state level in the gut tissue. Another type of regulatory cell that can develop in the mucosa and that may also mediate oral tolerance is the so called Tr1 regulatory cell. In contrast to the natural regulatory cell, the Tr1 cell lacks high level foxp3 expression and develops in relation to exogenous rather than self antigens; thus, it may be induced by protein antigen feeding or by infection of the GI tract. This regulatory T cell is induced by IL-10 and other cytokines and produces high amounts of IL-10. The factors that determine whether a given mucosal antigenic stimulus will result in (positive) immune effector response important for host defense or a (negative) regulatory T cell response important for maintainence of an unresponsive state and prevention of mucosal inflammation are still poorly understood. One working hypothesis is that the regulatory response is a default response that occurs in the absence of a strong innate immune response driven by TLR ligands. The latter tends to drive high IL-12/IL-23 responses that over-ride and even inhibit regulatory responses. The importance of the regulatory response has become apparent in the study of murine models of inflammation in which it was shown in a number of models that lack of regulatory cell generation leads to colonic inflammation. In one model, the cell transfer model it has been shown that transfer of cell populations lacking regulatory T cells to RAG-2 KO mice leads to colonic inflammation, whereas cell populations containing regulatory T cells remain free of colonic inflammation. In this system, the regulatory cells seem to require TGF-b, since they do not prevent inflammation in recipients that lack TGF-b receptors. In another model, the hapten induced model of TNBS-colitis, oral feeding of TNP-substituted protein prevent colitis and this prevention is also TGF-b mediated. This model ties the regulation of experimental colitis to oral tolerance. Finally, using the cell transfer model, it has been shown that provision of regulatory T cells to mice with established colitis, abrogates the colitis. This finding raises the question of whether human IBD is caused by lack of sufficient regulatory T cell activity and whether IBD can be treated by enhancing such activity.

Citations:

Weiner, H.L. (2000) Oral tolerance, an active immunologic process mediated by multiple mechanisms. J. Clin. Invest.106:935-957.

Nakamura, K., Kitani, A., Fuss, I., Pedersen, A., Harada, N. and Strober, W. (2004) TGF-beta 1 plays an important role in the mechanism of CD4+CD23+ regulatory T cell activity in both humans and mice. J. Immunol. 172:834-842.

Fuss, I.J., Boirivant, M., Lacy, B., and Strober, W. (2002) The interrelated roles of TGF-beta and IL-10 in the regulation of experimental colitis. J. Immunol. 168:900-908.

Fahlen L., Read, S., Gorelik, L., Hurst, S.D., Coffman, R.L., Flavell, R.A., and Powrie, F. (2005) T cells that cannot respond to TGF-beta escape control by CD4(+)CD25(+) regulatory T cells. J. Exp. Med. 201:737-746.

Mottet C., Uhlig, H.H., and Powrie, F. (2003) Cutting Edge: Cure of colitis by CD4+CD25+ regulatory T cells. J. Immunol. 170:3939-3943.

Zheng, S.G., Grey, J.D., Ohtsuka, K., Yamagiwa, S., and Horwitz, D.A. (2002) Generation ex vivo of TGF-beta-producing regulatory T cells from CD4+CD25+ precursors. J. Immunol. 169:4183-4189.

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Short-Term Goals (1-3 years)

Elucidation of the innate immune responses that determine whether a mucosal immune response will result in an effector cell response characterized by inflammation or a regulatory cell response characterized by tolerance.

Discovery of the dendritic cell interactions that result in the generation of regulatory T cells and the relation of these interactions to microflora antigens and TLR ligands.

Further exploration of the nature of the immunological milieu of the mucosa enhancing or retarding the development of regulatory T cells. Acquisition of definitive data on the production of cytokines by epithelial cells that either enhance or retard regulatory T cell development.

Discovery of markers for the identification of regulatory T cells in the mucosal tissues and the measurement of the number and function of regulatory T cells in IBD tissues.

Intermediate-Term Goals (4-7 years)

Elucidation of methods of generating regulatory cells ex vivo for administration and treatment of patients with IBD.

Exploration of the signaling pathways involved in the generation of regulatory T cells via TGF-b or the inhibition of such generation by IL-6.

Achievement of a better understanding of the biology of regulatory T cells with relation to the function of foxp3 and other intra-cellular factors that control regulatory cell function.

Long-Term Goals (8-10 years)

Elucidation of the genetic basis of regulatory T cell function. Identification of genes that affect how regulatory T cells are generated in the periphery and how regulatory T cell function is maintained and inhibited.

Development of gene therapy approaches to the enhancement of regulatory T cell function in the treatment of chronic inflammatory states.

Research Advance #6Effector T cell responses in the GI tract and the pathogenesis of IBD

There is general agreement that chronic inflammation of the GI tract (both ulcerative colitis and Crohn’s disease) represents an abnormal immunologic response to antigens (?TLR ligands) in the mucosal microflora. This abnormal response can arise from an excessive effector T cell response to such antigens or as a normal effector T cell response to such antigens that is not sufficiently restrained by regulatory T cells. Compelling evidence for this formulation comes from the extensive study of murine models of mucosal inflammation over the last decade that shows that experimental inflammation arising from diverse causes does not occur if the mice are maintained in a germ-free environment. Recently additional evidence favoring this view has come from studies of the basis of inflammation in 15% of Crohn’s disease patients who bear mutation in the CARD 15 gene that encodes NOD2, an intra-cellular member of the NLR family of proteins that recognizes and responds to a peptide, muramyl dipeptide (MDP) derived from the peptidoglycan component of the bacterial cell wall. In these studies it was shown that while NOD2 stimulated by MDP has a modest ability to activate NF-B and induce cytokine synthesis, its major function is that of down-regulation of TLR responses, particularly those induced by its parent molecule, peptidoglycan, acting through the TLR2 receptor. Thus, these studies support the view that Crohn’s disease susceptibility arising from CARD 15 mutations is due to faulty down-regulation of TLR

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responses leading to excessive responses to antigens associated with the microflora. One additional and important point is that CARD 15 mutations may be a necessary but not a sufficient factor in the pathogenesis of Crohn’s disease. This view arises from studies showing that mice with NOD2 deficiency do not develop gut inflammation unless they are exposed to microbial antigen and have resident T cells that can respond to that antigen. This suggests a “two hit” theory of Crohn’s disease pathogenesis: a first hit involving a dysregulated innate immune response represented in this case by NOD2 deficiency and a second hit involving the presence of effector T cells that can respond to one or another antigen in the microbial microflora. This theory is currently supported by work showing that in both human and murine systems, adaptive responses to flagellin antigen seems to be a characteristic of some patients with Crohn’s disease. Another factor leading to excessive responses to microflora antigens that is likely to be involved in the pathogenesis of disease involves the barrier function of the gut epithelium. There is now some evidence that while the bacterial microflora in IBD may not in itself be qualitatively abnormal, the microflora has an abnormal relation to the mucosal immune system such that the latter becomes over-stimulated and inflammation results. Evidence for this comes from the fact the recent discovery that defective production of -defensins is associated with Crohn’s disease and that this abnormality is aggravated in patients with one type of CARD 15 mutation, possibly due to the fact that NOD2 is also expressed in Paneth cells and may regulate the production of -defensins in such cells. In addition, mice with defective epithelial barrier function are susceptible to experimental colitis and certain of identified genes associated with Crohn’s disease, notably the OCT/N gene, is thought to affect epithelial cell function. Whether these epithelial cells defects are sufficient to cause disease or require a concomitant abnormal of the mucosal immune system remains to be seen.

The fundamental defects resulting in IBD, result from re-enforcing genetically determined defects in immune function (or epithelial cell function) some of which are described above or others that are as yet poorly understood. Regardless of this verisimilitude, the immunologic effector cell response mediating the inflammation is channeled through a common final pathway, one specific for Crohn’s disease and the other for ulcerative colitis. The effector cell response in Crohn’s disease was presaged by studies of mouse models of colitis resembling Crohn’s disease that were universally shown to respond to treatment with antibody to IL-12 (anti-IL-12p40). This, plus the fact that the cells in the lesions produced high amounts of IFN-g but little or no IL-4 seemed to indicate that the inflammation was a Th1-mediated effector cell process. Ultimately, this conclusion was reinforced by a clinical study of the efficacy of anti-IL-12p40 in patients with Crohn’s disease that showed that most patients were remarkably responsive to this therapy. However, recently evidence has accumulated that in the cell transfer model of colitis mentioned above, most of the inflammation can be attributed to IL-23 rather than IL-12 and that the IL-23 was acting through its capacity to maintain and support the differentiation of cells producing IL-17 (Th17 cells). In the light of this finding, it was felt that the efficacy of anti-IL-12p40 was due to the fact that the p40 chain is shared by IL-12 and IL-23 and thus the antibody was mainly blocking IL-23 rather than IL-12 is previous mouse and human studies of this antibody. This seemed to be supported by the observation that anti-IL23p19, an antibody specific for IL-23 was as effective as anti-IL-12p40 in treating cell transfer colitis. One caveat to these new data is that in another model of colitis, the hapten-induced TNBS-colitis, absence of IL-23 actually resulted in more severe disease; in this model, there is evidence that IL-12p40 is indeed the major driving cytokine and that role of IL-23, if any, is to down-regulate the IL-12p40 response. Studies conducted in humans show that both IL-12 and IL-23 (as well as IFN- and IL-17) are elevated in lesional tissue, suggesting that both the Th1 and Th17 systems are operative in the human disease. The inflammation in the second major form of IBD, ulcerative colitis, is clearly not a Th1 or Th17-mediated process since in this disease neither IL-12 nor IL-23 is elevated. Recently, it has been shown that this disease resembles a second hapten-induced experimental colitis, namely oxazolone colitis, in that increased numbers of NKT cells are found in lesions and these cells produce increase amounts of IL-13. Furthermore, it has been shown that IL-13 can mediate tissue injury by acting as an autocrine factor that induces NKT cells to manifest increased cytotoxic activity toward epithelial cells and by acting directly on epithelial cells to increase permeability and apoptosis. On the basis of these data, the working hypothesis on the pathogenesis of ulcerative colitis is that it is a Th2-like process mediated by NKT cells producting IL-13.

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Citations:

Bouma, G., Strober, W. (2003) The immunological and genetic basis of inflammatory bowel disease. Nat Rev Immunol 3: 521-533.

Ogura, Y., Bonen, D.K., Inohara, N., Nicolae, D.L., Chen, F.F., Ramos, R., Britton, H., Moran, T., Karaliuskas, R., Duerr, R.H., Achkar, J.P., Brant, S.R., Bayless, T.M., Kirschner, B.S., Hanauer, S.B., Nunez, G., and Cho, J.H. (2001) A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature 411: 603-606.

Watanabe, T., Kitani, A., Murray, P.J., Wakatsuki, Y., Fuss, I.J., and Strober, W. (2006) Nucleotide binding oligomerization domain 2 deficiency leads to dysregulated TLR2 signaling and induction of antigen-specific colitis. Immunity 25: 473-485.

Wehkamp, J., Salzman, N.YH., Porter, E., Nuding, S., Weichenthal, M., Petras, R.E., Shen, B., Schaeffeler, E., Schwab, M., Linzmeier, R., Feathers, R.W., Chu, H., Lima, H., Jr., Fellermann, K., Ganz, T., Stange, E.F., and Bevins, C.L. (2005) Reduced Paneth cell alpha-defensins in ileal Crohn’s disease. Proc Natl Acad Sci USA 102: 18129-18134.

Mannon, P.J., Fuss, I.J., Mayer, L., Elson, C.O., Sandborn, W.J., Present, D., Dolin, B., Goodman, N., Groden, C., Hornung, R.L., Quezado, M., Yang, Z., Neurath, M.F., Salfeld, J., Veldman, G.M., Schwertschlag, U., and Strober, W. (2004) Anti-interleukin-12 antibody for active Crohn’s disease. N Engl J Med 351: 2069-2079.

Hue, S., Ahern, P., Buonocore, S., Kullberg, M.C., Cua, D.J., McKenzie, B.S., Powrie, F., and Maloy, K.J. (2006) Interleukin-23 drives innate and T cell-mediated intestinal inflammation. J Exp Med 203: 2473-2483.

Fuss, I.J., Heller, F., Boirivant, M., Leon, F., Yoshida, M., Fichtner-Feigl, S., Yang, Z., Exley, M., Kitani, A., Blumberg, R.S., Mannon, P., and Strober, W. (2004) Nonclassical CD1d-restricted NK T cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis. 113: 1490-1497.

Short-Term Goals (1-3 years)

Elucidate the function of NOD2 in murine systems with the use of NOD2 KO and KI mice as well as NOD2 transgenic mice with cell-specific over-expression. In particular, define the mechanism of NOD2 inhibition of TLR responses.

Create murine knock-out models of other genes so-far identified as susceptibility genes in IBD to determine the role of these genes in mucosal immune responses and/or epithelial barrier function.

Explore the function of novel genes that affect mucosal responses to the gut microflora, particularly those genes that have a direct or indirect impact on epithelial barrier function.

Create new models of mucosal inflammation that more closely mimic human Crohn’s disease and ulcerative colitis to determine the role of IL-12 and IL-23 in Crohn’s disease and IL-13 in ulcerative colitis.

Intermediate-Term Goals (4-7 years)

Identify genetically-determined hyper-responsiveness to candidate antigens in the mucosal microflora. Create murine models to verify that such hyper-responsiveness can lead to mucosal inflammation.

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Evaluate the efficacy of novel agents that affect the final common pathways of mucosal inflammation in inflammatory bowel disease including agents that affect the traffic of cell in the mucosal immune system, key effector cytokines such as IL-23p19, IL-17, IL-22 and IL-13. Complete on-going studies of IL-12p40 to determine if this can be a viable alternative for the treatment of Crohn’s disease.

Long-Term Goals (8-10 years)

Create mice with multiple gene defects simulating the congeries of defects found in patients to determine if such mice develop inflammatory bowel disease under appropriate environmental conditions.

Evaluate the treatment of patients with Crohn’s disease (or ulcerative colitis) with identified gene defects using hematopoietic stems repleted with normal genes.

Research Advance #7The IgA response and mucosal vaccination

Undoubtably the most unique aspect of the mucosal immune system is that the B cell response characteristic of this system is an IgA B cell response. The reason for this mucosal isotype skewing remains unclear; however, since class-switch recombination (CSR) resulting in IgA B cells has an absolute requirement for TGF- and since TGF- is particularly associated with cells present in the mucosal environment, a working hypothesis is that mucosal IgA dominance is a direct result of the availability of this cytokine in mucosal tissues. In an older view, IgA B cells were thought to arise exclusively in mucosal follicles present mainly in the Peyer’s patches or in isolated intestinal lymphoid nodules scattered through the intestine and indeed it could be shown that these follicles were the only ones containing substantial numbers of developing IgA B cells. There was substantial evidence that IgA B cell stimulation at these sites was initiated by B cell receptor (BCR)-mediated stimulation by protein antigens entering the follicle and required cognate interactions between T cells and conventional (B2) B cells, including interactions involving CD40L and CD40. Following such stimulation, the B cells were imprinted to migrate through the draining nodes and the lymph system back to the “diffuse” mucosal lymphoid areas in the lamina propria of mucosa. It is possible, but still unproven, that such cognate T cell-B cell interactions resulting in IgA B cells requires a T cell bearing surface TGF- or secreting TGF- as do regulatory T cells. On this basis, the regulatory T cell may be uniquely involved in IgA B cell development. In recent years, this follicle and T cell-centered view of IgA B cell differentiation has had to make room for a second pathway of IgA B cell development since it is clear that IgA B cells develop in relation to exposure to components of the commensal microflora in the absence of T cells or CD40L or, indeed of mucosal follicles. This pathway of IgA B cell development could be viewed as a more “innate” pathway given evidence that it occurs in response to innate receptors such as TLR receptors and may be triggered by T cell- independent non-protein antigens. In addition, this pathway may utilize, at least in part, an unconventional B cell, the B1 B cell, that follows an independent line of development from conventional B cells. It remains unclear where this type of IgA B cell actually undergoes IgA-specific CSR since initial reports that this occurs in the lamina propria have recently been refuted. On the other hand, there is a very recent report that the CSR occurs in close juxtaposition to epithelial cells (in epithelial pockets) and that the switching in these areas occurs when the epithelial cells are stimulated by TLR ligands and secrete TSLP (mentioned above in relation to epithelial effects on dendritic cells) as well as BAFF, a factor that may induce CSR as well as terminal B cell differention. The TGF-b necessary for IgA-specific CSR could be coming from epithelial cells or from near-by dendritic cells.

The fact that a single cytokine, TGF-, is at once involved in the induction of regulatory cell responses and in the induction of IgA responses is probably not fortuitous, since the mucosal immune system, as already alluded to above, is oriented toward regulation of the commensal organisms in the gut lumen. Indeed, the IgA produced by the T cell-independent process discussed above may have a particular role in controlling the entry of antigens derived from commensal organisms into the area beneath the epithelium since such antigens would be susceptible to interception by IgA and removal from the system via

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polyimmunoglobulin receptor transport across the epithelium. Thus, if we turn our attention to the induction of IgA antibody responses to protein components of potential pathogens via the administration of oral or intra-nasal (or intra-rectal) vaccines we may be dealing mainly with the IgA responses occurring in the follicles via cognate T cell interactions.

In the latter context it has been known for some time that such responses require the use of mucosal adjuvants, i.e., substance that when administered via the mucosal immune system (i.e., orally or intra-nasally) have properties that allow them to stimulate the production of factors that promote IgA-specific CSR and the expansion of IgA B cells resulting from the switch, rather than the default response discussed above that give rise to oral tolerance. Extensive study of one particularly potent mucosal adjuvant, cholera toxin, has provided important insights into the way mucosal adjuvants function. Cholera toxin (the holotoxin) consists of two chains, an A chain that has potent ADP ribosyltransferase activity and thus stimulates cells via a G protein-mediated activation process. The second or B chain is the anchor chain because of this ability to bind to cell surface GM1-ganglioside. The holotoxin cannot be used as an adjuvant because it’s binding to epithelial cells leads to GI fluid loss and diarrhea. Recently, this problem has been overcome by fusing the A chain to staphylococcus protein A to form a protein (CTA-1DD) that binds avidly to B cells and other antigen presenting cells but does not cause GI symptoms. Studies of the mechanism of action of CTA-1DD show antigen bound to CTA-1DD binds to marginal zone dendritic cells that then migrate to the T cell zone and express the co-stimulatory molecules, CD86. This induces the movement of antigen-specific CD4+ T cells into B cell follicles and subsequent germinal center formation. CTA-1DD lacking ribosylation capability does not have this effect and is a poor adjuvant and while CTB binds to dendritic cells, it does not induce their migration or maturation; this may explain why CTB given orally is actually an inducer of oral tolerance rather than immunization. Thus, the picture that emerges is that mucosal adjuvants induce mucosal immunization rather than tolerance because they activate dendritic cells to express surface molecules and cytokines that activate effector T cells rather than regulatory cells. These fruitful studies of cholera toxin-based mucosal adjuvants provide a template for the study of other mucosal adjuvants, including those that address all-important mucosal T cell responses.

A very practical aspect of pursuing increased knowledge concerning in the induction of IgA and other mucosal responses arises from the fact that the mucosal system is somewhat separate from the “systemic” immune system by virtue of the homing receptors that mandate the traffic of cells originating in the inductive sites of the system to effector sites. This carries the important implication that only mucosal immunization can effectively deal with pathogenic invasion of the mucosa. This point is particularly relevant to the prevention of HIV disease given recent evidence that the gastrointestinal tract in a major site of initial HIV development and a major reservoir of established HIV infection.

Citations:

Strober, W., Fagarasan, S., and Lycke, N., (2005) IgA B cell development. In: Mucosal Immunology, 3rd Edition. J. Mestecky, M.E. Lamm, W. Strober, J. Bienenstock, J.R. McGhee and L. Mayer, eds. Academic Press, Boston, pp. 583-616.

MacPherson, A.J., Gatto, E., Sainsbury, E., Harriman, G.R., Hengartner, H., and Zinkernagel, R.M. (2000) A primitive T cell-independent mechanism of intestinal IgA responses to commensal bacteria. Science 288:2222-2226.

Fagarasan, S., Muramatsu, M., Suzuki, K., Nagaoka, H., Hiai, H., and Honjo, T. (2002) Critical roles of activation-induced cytidine deaminase in the homeostasis of gut flora. Science 298:1424-1427

Bergqvist, P., Gardby, E., Stensson, A., Bemark, M., and Lycke, L. (2006) Gut IgA class switch recombination in the absence of CD40 does not occur in the lamina propria and is independent of germinal centers. J. Immunol. 177:7772-7783.

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Grdic, D., Ekman, L., Schon, K., Lindgren, K., Mattsson, J., Magnusson, K.E., Ricciardi-Castignoli, P., and Lycke, N. (2005) Splenic marginal zone dendritic cells mediate cholera toxin adjuvant effect: dependence on ADP ribosylation activity of the holotoxin. J. Immunol. 175:5192-5202.

Xu, W.,Bing, H., Chiu, A.,Chadburn, A., Shan, M., Buldys, M., Ding, A., Knowles, D.M., Santini, P.A., and Cerutti, A. (2007) Epithelial cells trigger frontline immunoglobulin class switching through a pathway regulated by the inhibitor SLPI. Nat. Immunol. 8:294-303.

Holmgren, J., and Czerkinsky, C. (2005) Mucosal Immunity and Vaccines. Nature Medicine, supplement.

Short Term Goals (1-3 years)

Elucidation of the full range of epithelial or stromal cell factors/cytokines that are involved in the elaboration of IgA B cells. Determination of whether these factors/cytokines operate at the level of class switch or terminal differentiation.

Delineation of the extent of T cell-independent IgA produced in the mucosa and its role in maintaining mucosal barrier function and/or antigen sampling. Delineation of the regulation of commensal organisms in the absence of igA production.

Clarification of the types of T cells involved in cognate interactions leading to IgA differentiation and regulatory T cells.

Development of anti-immunization agents administered orally that induce tolerizaiton rather than immunization such as cholera toxin B chain.

Intermediate-Term Goals (4-7 years)

Accumulation of more specific knowledge of the mechanism of action of various mucosal adjuvants at the level of dendritic cells and T cells.

Development of new adjuvants that target particular aspects of the mucosal immune response: TLR ligands alone or bound to cholera toxin adjuvants; ISCOMs for the targeting of antigen to dendritic cells; and, various cytokines, such as IL-12, IL-23, IL-15, etc.

Development of optimized vaccine schedules for particular infectious agents including intra-nasal and intra-rectal vaccine administration.

Long-Term Goals (8-10 years)

Development of effective vaccines that induce IgA and/or T cell responses via mucosal immunization; this includes the development of mucosal vaccines for many of the major epidemic viral infections and for HIV disease.

Development of mucosal vaccines that induce high level “innate” IgA responses addressing the entry of the intestinal microflora into the mucosa for use in inflammatory bowel disease due to defects of barrier function.

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