Gut microbiota and lipopolysaccharide content of the diet influence development of regulatory T cells: studies in germ-free mice

BMC Immunology BioMed Central Research article Open Access Gut microbiota and lipopolysaccharide content of the diet influence development of regulatory T cells: studies in germ-free mice Tomas Hrncir1, Renata Stepankova1, Hana Kozakova1, Tomas Hudcovic1 and Helena Tlaskalova-Hogenova*1,2 Address: 1Department of Immunology and Gnotobiology, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague and Novy Hradek, Czech Republic and 2Institute of Immunology and Microbiology, 1st Faculty of Medicine, Charles University in Prague, Czech Republic Email: Tomas Hrncir - ; Renata Stepankova - ; Hana Kozakova - ; Tomas Hudcovic - ; Helena Tlaskalova-Hogenova* - * Corresponding author Published: 6 November 2008 BMC Immunology 2008, 9:65 doi:10.1186/1471-2172-9-65 Received: 20 May 2008 Accepted: 6 November 2008 This article is available from: http://www.biomedcentral.com/1471-2172/9/65 © 2008 Hrncir et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Mammals are essentially born germ-free but the epithelial surfaces are promptly colonized by astounding numbers of bacteria soon after birth. The most extensive microbial community is harbored by the distal intestine. The gut microbiota outnumber ~10 times the total number of our somatic and germ cells. The host-microbiota relationship has evolved to become mutually beneficial. Studies in germ-free mice have shown that gut microbiota play a crucial role in the development of the immune system. The principal aim of the present study was to elucidate whether the presence of gut microbiota and the quality of a sterile diet containing various amounts of bacterial contaminants, measured by lipopolysaccharide (LPS) content, can influence maturation of the immune system in gnotobiotic mice. Results: We have found that the presence of gut microbiota and to a lesser extent also the LPSrich sterile diet drive the expansion of B and T cells in Peyer's patches and mesenteric lymph nodes. The most prominent was the expansion of CD4+ T cells including Foxp3-expressing T cells in mesenteric lymph nodes. Further, we have observed that both the presence of gut microbiota and the LPS-rich sterile diet influence in vitro cytokine profile of spleen cells. Both gut microbiota and LPS-rich diet increase the production of interleukin-12 and decrease the production of interleukin4. In addition, the presence of gut microbiota increases the production of interleukin-10 and interferon-γ. Conclusion: Our data clearly show that not only live gut microbiota but also microbial components (LPS) contained in sterile diet stimulate the development, expansion and function of the immune system. Finally, we would like to emphasize that the composition of diet should be regularly tested especially in all gnotobiotic models as the LPS content and other microbial components present in the diet may significantly alter the outcome of experiments. Page 1 of 11 (page number not for citation purposes) BMC Immunology 2008, 9:65 Background The mammalian gut harbors a vast and complex microbial community. The human intestinal microflora is estimated to contain 500 to 1000 species and the size of the population is ~10 times greater than the total number of our somatic and germ cells. The role of microbiota in many physiological processes has been demonstrated by using animal models reared under gnotobiological conditions [1-5]. Studies in germ-free (GF) animals have shown that gut microbiota play a crucial role in the development and maturation of the immune system [6-23]. It was demonstrated that the gut-associated lymphoid tissue (GALT), which is the largest immune organ, is immature in GF mice. The content of the lamina propria CD4+ T cells, IgA producing B cells and intraepithelial T cells is reduced in GF animals [8-10,12,13,16,22]. Comparative experiments have also shown that the gene expression profiles of the intestinal epithelial cells are shaped by the presence of gut microbiota and that upregulated genes contribute to secretion of antibacterial molecules at the intestinal surface and the regulation of intestinal angiogenesis [5,15]. The effects of gut microbiota are not only limited to the GALT but systemic immunity is also affected. GF mice have decreased serum immunoglobulin levels and their mesenteric lymph nodes and spleens are smaller and less cellular [11,20,24]. A role of gut microbiota in establishing equilibrium between TH1 and TH2 immunological responses, which is critical to overall human and animal health, has been postulated [20,25-28]. It is not yet clear whether gut microbiota and microbial components play a role in the development and function of Tregs [29-33] which were recently suggested to be a crucial factor in establishing immunological homeostasis. It has been demonstrated that cells with regulatory function are Foxp3-expressing CD4+ T cells [34-36]. Regulatory T cells (Tregs) suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens and harmless exogenous antigens [34-42]. Depletion or functional abrogation of these cells can cause inflammatory diseases [37-39]. Enormous amount of gut microbiota and their products are in an intimate contact with epithelial surface of the intestinal mucosa. Microbe-associated molecular patterns (MAMPs) present in the intestinal content are sampled mainly by DCs and recognized by their receptors – pattern recognition receptors (PRRs), which include the transmembrane Toll-like receptors (TLRs) [43] and C-type lec- http://www.biomedcentral.com/1471-2172/9/65 tin receptors (CLRs) [44], and the cytoplasmic Nod-like receptors (NLRs) [45]. The activated DCs traffic from the intestinal epithelium and Peyer's patches into the mesenteric lymph nodes, where they activate cells of the adaptive immune system. Gnotobiotic (germ-free) models represent an important tool for unraveling the function of gut microbiota, especially their effects on the mucosal and systemic immunity. Germ-free animals are free of live bacteria but their sterile food contains microbial components and other immunogenic components. To exclude the effects of these components a chemically defined ultrafiltered antigen-free diet was introduced but is rarely used due to technical and financial obstacles [6,24,46-48]. The aim of the present study was to investigate the effects of live gut microbiota and LPS content of the sterile diet as one of the markers of bacterial contamination on the development of the immune system. Our preliminary data have shown that the effect of a low LPS diet (AIN93G) is negligible in conventional (CV) mice. To address the specific aims of the study we have establ (...truncated)