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Investigation of the induction mechanisms and function of secretory IgA against commensal bacteria using reversible germ-free colonisation

English title Investigation of the induction mechanisms and function of secretory IgA against commensal bacteria using reversible germ-free colonisation
Applicant Macpherson Andrew
Number 124732
Funding scheme Project funding (Div. I-III)
Research institution Institut für klinische Immunologie
Institution of higher education University of Berne - BE
Main discipline Immunology, Immunopathology
Start/End 01.04.2009 - 31.03.2012
Approved amount 675'000.00
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Keywords (5)

IgA; commensal bacteria; intestine; B lymphocytes;

Lay Summary (English)

Lead
Lay summary
The intestine is a continuous tube from mouth to anus, and the bacteria live inside this tube, but because the lining of the intestine is extremely thin - a layer just one cell thick - small numbers of commensal bacteria and their molecules are continuously penetrating the body tissues. The cells and antibodies that make up the body's defences against foreign microbes are called the immune system, and a major part of this system is in the lining of the intestine. We know that the mucosal immune system is important for protection against commensal intestinal bacteria, because we can follow its development as germ-free animals without any intestinal microbes are colonised with commensal bacteria. Unfortunately, antibiotics reduce but do not eliminate intestinal microbes, so there has been no way back to a germ-free condition once bacteria have populated the intestine. We have engineered new bacterial strains that cannot survive long in the intestine, so colonised mice become germ-free again. We are using this system to measure the threshold for inducing an IgA antibody response against commensals, and whether the immune system in the gut 'remembers' previous encounters with commensal bacteria and generates a stronger response on the second occasion (as in medical vaccinations). We are also working out how effective this immune response is, on its own, to protect the tissues of a germ-free animal from bacterial penetration, without competition from resident intestinal bacteria.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Homeland security: IgA immunity at the frontiers of the body.
Macpherson Andrew J, Geuking Markus B, McCoy Kathy D (2012), Homeland security: IgA immunity at the frontiers of the body., in Trends in immunology, 33(4), 160-7.
The function of secretory IgA in the context of the intestinal continuum of adaptive immune responses in host-microbial mutualism.
Geuking M B, McCoy K D, Macpherson A J (2012), The function of secretory IgA in the context of the intestinal continuum of adaptive immune responses in host-microbial mutualism., in Seminars in immunology, 24(1), 36-42.
Age, microbiota, and T cells shape diverse individual IgA repertoires in the intestine.
Lindner Cornelia, Wahl Benjamin, Föhse Lisa, Suerbaum Sebastian, Macpherson Andrew J, Prinz Immo, Pabst Oliver (2012), Age, microbiota, and T cells shape diverse individual IgA repertoires in the intestine., in The Journal of experimental medicine, 209(2), 365-77.
The habitat, double life, citizenship, and forgetfulness of IgA.
Macpherson Andrew J, Geuking Markus B, Slack Emma, Hapfelmeier Siegfried, McCoy Kathy D (2012), The habitat, double life, citizenship, and forgetfulness of IgA., in Immunological reviews, 245(1), 132-46.
Acquisition of a multifunctional IgA(+) plasma cell phenotype in the gut
Fritz JH, Rojas OL, Simard N, McCarthy DD, Hapfelmeier S, Rubino S, Robertson SJ, Larijani M, Gosselin J, Ivanov II, Martin A, Casellas R, Philpott DJ, Girardin SE, McCoy KD, Macpherson AJ, Paige CJ, Gommerman JL (2012), Acquisition of a multifunctional IgA(+) plasma cell phenotype in the gut, in NATURE, 481(7380), 199-199.
Crosstalk between B lymphocytes, microbiota and the intestinal epithelium governs immunity versus metabolism in the gut.
Shulzhenko Natalia, Morgun Andrey, Hsiao William, Battle Michele, Yao Michael, Gavrilova Oksana, Orandle Marlene, Mayer Lloyd, Macpherson Andrew J, McCoy Kathy D, Fraser-Liggett Claire, Matzinger Polly (2011), Crosstalk between B lymphocytes, microbiota and the intestinal epithelium governs immunity versus metabolism in the gut., in Nature medicine, 17(12), 1585-93.
Depletion of murine intestinal microbiota: effects on gut mucosa and epithelial gene expression.
Reikvam Dag Henrik, Erofeev Alexander, Sandvik Anders, Grcic Vedrana, Jahnsen Frode Lars, Gaustad Peter, McCoy Kathy D, Macpherson Andrew J, Meza-Zepeda Leonardo A, Johansen Finn-Eirik (2011), Depletion of murine intestinal microbiota: effects on gut mucosa and epithelial gene expression., in PloS one, 6(3), 17996-17996.
Immunoglobulin A: a bridge between innate and adaptive immunity.
Macpherson Andrew J, Geuking Markus B, McCoy Kathy D (2011), Immunoglobulin A: a bridge between innate and adaptive immunity., in Current opinion in gastroenterology, 27(6), 529-33.
Mice overexpressing BAFF develop a commensal flora-dependent, IgA-associated nephropathy.
McCarthy Douglas D, Kujawa Julie, Wilson Cheryl, Papandile Adrian, Poreci Urjana, Porfilio Elisa A, Ward Lesley, Lawson Melissa A E, Macpherson Andrew J, McCoy Kathy D, Pei York, Novak Lea, Lee Jeannette Y, Julian Bruce A, Novak Jan, Ranger Ann, Gommerman Jennifer L, Browning Jeffrey L (2011), Mice overexpressing BAFF develop a commensal flora-dependent, IgA-associated nephropathy., in The Journal of clinical investigation, 121(10), 3991-4002.
Intestinal bacterial colonization induces mutualistic regulatory T cell responses.
Geuking Markus B, Cahenzli Julia, Lawson Melissa A E, Ng Derek C K, Slack Emma, Hapfelmeier Siegfried, McCoy Kathy D, Macpherson Andrew J (2011), Intestinal bacterial colonization induces mutualistic regulatory T cell responses., in Immunity, 34(5), 794-806.
The microbiota mediates pathogen clearance from the gut lumen after non-typhoidal Salmonella diarrhea.
Endt Kathrin, Stecher Bärbel, Chaffron Samuel, Slack Emma, Tchitchek Nicolas, Benecke Arndt, Van Maele Laurye, Sirard Jean-Claude, Mueller Andreas J, Heikenwalder Mathias, Macpherson Andrew J, Strugnell Richard, von Mering Christian, Hardt Wolf-Dietrich (2010), The microbiota mediates pathogen clearance from the gut lumen after non-typhoidal Salmonella diarrhea., in PLoS pathogens, 6(9), 1001097-1001097.
The microbiota mediates pathogen clearance from the gut lumen after non-typhoidal Salmonella diarrhea.
Endt Kathrin, Stecher Bärbel, Chaffron Samuel, Slack Emma, Tchitchek Nicolas, Benecke Arndt, Van Maele Laurye, Sirard Jean-Claude, Mueller Andreas J, Heikenwalder Mathias, Macpherson Andrew J, Strugnell Richard, von Mering Christian, Hardt Wolf-Dietrich (2010), The microbiota mediates pathogen clearance from the gut lumen after non-typhoidal Salmonella diarrhea., in PLoS pathogens, 6(9), 1001097-1001097.
In remembrance of commensal intestinal microbes.
Hapfelmeier Siegfried, Macpherson Andrew J (2010), In remembrance of commensal intestinal microbes., in Communicative & integrative biology, 3(6), 569-71.
Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses.
Hapfelmeier Siegfried, Lawson Melissa A E, Slack Emma, Kirundi Jorum K, Stoel Maaike, Heikenwalder Mathias, Cahenzli Julia, Velykoredko Yuliya, Balmer Maria L, Endt Kathrin, Geuking Markus B, Curtiss Roy, McCoy Kathy D, Macpherson Andrew J (2010), Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses., in Science (New York, N.Y.), 328(5986), 1705-9.
Immune adaptations that maintain homeostasis with the intestinal microbiota.
Hooper Lora V, Macpherson Andrew J (2010), Immune adaptations that maintain homeostasis with the intestinal microbiota., in Nature reviews. Immunology, 10(3), 159-69.
Like will to like: abundances of closely related species can predict susceptibility to intestinal colonization by pathogenic and commensal bacteria.
Stecher Bärbel, Chaffron Samuel, Käppeli Rina, Hapfelmeier Siegfried, Freedrich Susanne, Weber Thomas C, Kirundi Jorum, Suar Mrutyunjay, McCoy Kathy D, von Mering Christian, Macpherson Andrew J, Hardt Wolf-Dietrich (2010), Like will to like: abundances of closely related species can predict susceptibility to intestinal colonization by pathogenic and commensal bacteria., in PLoS pathogens, 6(1), 1000711-1000711.
Innate and adaptive immunity cooperate flexibly to maintain host-microbiota mutualism.
Slack Emma, Hapfelmeier Siegfried, Stecher Bärbel, Velykoredko Yuliya, Stoel Maaike, Lawson Melissa A E, Geuking Markus B, Beutler Bruce, Tedder Thomas F, Hardt Wolf-Dietrich, Bercik Premysl, Verdu Elena F, McCoy Kathy D, Macpherson Andrew J (2009), Innate and adaptive immunity cooperate flexibly to maintain host-microbiota mutualism., in Science (New York, N.Y.), 325(5940), 617-20.

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
EMBO/EMPL Sympsoium Heidelberg 19.05.2012 Heidelberg Deutschland
World Immune Regulation Meeting -VI 18.03.2012 Davos
Keystone Symposia, Innate Immunity, Sensing the Microbes and Damage Signals/The Microbiome 04.03.2012 Colorado, USA
EASL Monothematic Conference IMLI 19.01.2012 Stratford-upon-Avon, UK
Hengsberger Symposium, University of Heidelberg 09.01.2012 Heidelberg Deutschland
Seminar Immunology & Medicine, Cambridge Institut for Medical Research 02.12.2011 Cambridge UK
Cancer Research UK: 24.10.2011 London
ESF Conference, B Cells and Protection: Back to Basics 12.06.2011 St. Feliu de Guixols, Spain
Microbiota Symposium PIWAS- College de France 23.05.2011 Paris
Microbiota and Mucosal Immunology, Nature Conference 14.04.2011 San Francisco
Swiss Society for Allergology and Immunology 17.03.2011 Lugano


Communication with the public

Communication Title Media Place Year
Talks/events/exhibitions Exploration of the Mucosal IgA: Response against Commensal Bacteria with Reversible Colonization International
Talks/events/exhibitions Immune Compartmentalisation in Mutualism with the Intestinal Commensal Microbiota International
Talks/events/exhibitions Immune Regulation of Microbiota Composition Italian-speaking Switzerland
Talks/events/exhibitions Innate and Adaptive Immunity in Host-Microbiota Mutualism. Rhaeto-Romanic Switzerland
Talks/events/exhibitions Insights from Reversible Intestinal Colonization International
Talks/events/exhibitions New Perspectives on Immunity to Infection International
Talks/events/exhibitions Novel Insights into Liver disease provided by the Microbiome International
Talks/events/exhibitions The Continuum between Innate and Adaptive Immunity in the Response to Commensal Intestinal Bacteria International
Talks/events/exhibitions The Intestinal Microbiome Instructs T Regulatory Responses in the Intestine International
Talks/events/exhibitions The TLR-dependent Continuum of Innate and Adaptive Immunity Ensuring Mutualism ... International
Talks/events/exhibitions Uncoupling Immune Responses against Commensal Bacterial from Intestinal Colonisation International

Associated projects

Number Title Start Funding scheme
139231 Advancement of functional genomics research at the University of Bern by extension of LC-MS platform 01.07.2012 R'EQUIP
133813 Next generation sequencing platform, University of Bern 01.12.2010 R'EQUIP
140700 Compartmentalisation of commensal intestinal microbes and host IgA immunity in maintaining host-microbial mutualism 01.04.2012 Project funding (Div. I-III)
100421 Mechanisms of intestinal IgA induction and protection of adults and neonates against the commensal intestinal flora 01.04.2003 Project funding (Div. I-III)

Abstract

BackgroundIgA is the dominant immunoglobulin isotype produced in mammals, mostly secreted across mucous membranes, especially in the intestinal mucosa. It is clear that IgA induction is driven by the enormous load of commensal bacteria resident in the intestine, because germ free animals have very low levels of IgA expression, yet IgA is produced normally within a few weeks if the intestines are colonised with a commensal microbiota. We have previously shown that IgA can be produced by both T cell-dependent and T-independent pathways, and that IgA induction follows sampling of commensal bacteria by intestinal dendritic cells. Despite the enormous amount of IgA produced in the intestine (about 3g/day in most humans) there are important deficiencies in our understanding of the system. We do not know how important immune memory is for the IgA response against commensal bacteria, how long-lived a single response can be, or how important plasticity of the responses are in protecting the host against the luxuriant load of intestinal luminal commensal microbes and their abundant proinflammatory molecules.Current methodological limitationsAlthough colonisation of germ free animals is an excellent way to study the IgA response because there is consistent strong induction, it has serious limitations, because once bacteria have been introduced to the germ free intestine there has been no way back to the germ free status. This means that individual separated doses of live commensal bacteria could not be given to test different induction mechanisms and immune memory, and that any experiments to test IgA function are compromised because the very bacteria that induce a mucosal IgA response compete with the test bacteria used to assess IgA function. Although important functional data have been obtained using IgA hybridomas implanted into subcutaneous tissues of mice, this is an experimental compromise, because IgA is not being produced locally in the intestinal mucosa of these animals.Experimental approachWe have solved the problem of the irreversibility of germ free colonisation by engineering bacteria with multiple auxotrophic mutations that result in them being non-viable in vivo. This means that live bacteria can be introduced into the germ free intestine, yet after approximately 72 hours, the mice become germ free again. Repeated single doses of the engineered bacteria can be given, and the system is completely ‘tight’ as a germ free status is always recovered. In preliminary experiments we have also shown that 14 days after the mice have become germ free again there is a specific IgA response to the organism used for reversible germ-free colonisation. These IgA levels are quantitatively similar to (irreversible) colonisation of germ free mice using the defined microbiota of 8 organisms in the ‘modified Schaedler flora’, although the specificities are completely different. We also know that induction of IgA using reversible colonisation with live bacteria is far more effective than with equivalent doses of killed organisms.Specific aims and experimental design1. To define the thresholds, kinetics and resultant affinities for induction of a specific secretory IgA response by commensal intestinal bacteria. This will be carried out by titrating the intestinal gavage dose of live auxotrophic bacteria (which cannot divide in vivo, and therefore do not persist). The kinetics of induction and disappearance of a specific IgA response will be determined by ex vivo analysis of intestinal induction sites by immunohistochemistry; total IgA by FACS, ELISA and ELISPOT and specific IgA by bacterial flow cytometry. Because the animals become germ free again, the disappearance/longevity of the response can also be measured. The key questions are i) whether the system works by DC sampling of a tiny proportion of bacteria present in the intestine (which can be measured when competition from a resident microbiota is excluded) and ii) whether a very long lived specific IgA response is induced. If these hypotheses are correct, there is an averaging of IgA secreted in response to intestinal bacteria appropriate to the high diversity that is normally present. Secondary aims in this section are described in the main proposal to determine the affinities of the antibody responses generated, using hybridoma methodology, and to distinguish kinetic parameters for T cell-dependent and T-independent pathways of IgA induction by carrying out experiments in T cell deficient strains.2. To determine whether immune memory is important in IgA induction against commensal bacteria. This can be addressed by timed intestinal immunisation with non-persistent bacteria using the ex vivo readouts described above. Our hypothesis is that, in contrast to responses against pathogens or toxins, there is limited memory for commensals, so that the IgA response does not become excessively biased by past immune experience. The practical importance of this is in the use of commensals or probiotics in an attempt to manipulate mucosal responses, which may only work well in the face of persistent exposure to the organism.3. To determine the functional consequences of specific IgA induction in establishing host bacterial mutualism in the intestine. Since we can uncouple specific mucosal induction of antibacterial secretory IgA from intestinal bacterial colonisation we have a new approach to addressing the functional aspects of IgA secretion. We will look at the consequences of challenge doses of bacteria in the germ free mouse, with or without prior induction of SIgA specific for that bacterial strain. The readouts will be i) measurement of translocation of live bacteria by culture and fluorescence microscopy on tissue sections, mucosal or mesenteric lymph node biopsies or purified dendritic cell or leukocyte fractions. We will also assess epithelial cell responses by measurement of bacterial-induced alterations in gene expression profiles. In a second set of experiments we will look at whether there is an additional luminal function of IgA to retain consortia of commensal bacteria within the intestine.Expected value of the projectThis project will employ novel methodology to carry out fundamental studies of the way in which the mucosal immune system responds to the luxuriant load of commensal bacteria with specific IgA responses and how this is important in preserving host bacterial mutualism. Our commensal bacteria outnumber our own eukaryotic cells by an order of magnitude, so mutualism with these organisms is crucial to our existence. The potential practical impact is in human patients with immune deficiency, aplasia or inflammatory bowel disease who can have serious sepsis or immunopathology from their commensals. The potential practical impact is in understanding the mechanisms of humoral protection against commensal bacteria which may fail in immune deficiency, aplasia or as a complication of malnutrition or inflammatory bowel disease. Understanding commensal induced IgA mechanisms is important in a rational attempt to use probiotic microbes as therapeutics.
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