Project

IgA; commensal bacteria; intestine; B lymphocytes;

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.

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Last update: 21.02.2013

Active participation

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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.