Development Of Gut-associated Lymphoid Tissue Research Articles

Impact of early posthatch feeding on the immune system and selected hematological, biochemical, and hormonal parameters in broiler chickens

Under commercial conditions, chicks hatch within a 24 to 48 h window, a period known as the hatching window. Subsequently, they undergo various treatments before finally being transported to the broiler farm. These procedures may delay the chicks' access to food and water, sometimes receiving them as late as 72 h after hatching. Previous studies have indicated that fasting during this initial period is detrimental, leading to impaired body growth, compromised immune system response, and hindered muscle development. The objective of this study was to assess the impact of early posthatch feeding on immune system organs and selected hematological, biochemical, and hormonal parameters. The experiment utilized Ross 308 broiler eggs incubated under typical commercial hatchery conditions. The experimental group's eggs were hatched in HatchCare hatchers (HC) with immediate access to feed and water, while the control group's eggs were hatched under standard conditions (ST). Thirty chickens from each group were assessed on the 1st (D1), 7th (D7), 21st (D21), and 35th (D35) day after hatching. On D1, the HC group exhibited lower hemoglobin, hematocrit, and total serum protein values, suggesting that early access to water prevents initial dehydration in newborn chicks. Conversely, the ST group showed a stress reaction on D1 due to feed deprivation, leading to an almost 2-fold higher serum corticosterone concentration compared to the HC group. However, this increase did not result in a significant change in the heterophil/lymphocyte ratio. Furthermore, the HC group displayed an increase in triglyceride concentration and a decrease in HDL concentration on D1. On D7, the HC group exhibited an increased relative weight of the bursa and a higher CD4+ cell number in the cecal tonsil (CT), indicating a more rapid development of these organs resulting from early stimulation of the gastrointestinal tract. However, early feeding did not influence the numbers of Bu-1+, CD4+, and CD8+ cells or the germinal center (GC) areas in the spleen. In conclusion, early feeding contributes to the welfare of newborn chicks by reducing dehydration and stress levels and stimulating the development of gut-associated lymphoid tissue.

The Lipid-Soluble Forms of Choline Enhance Ex Vivo Responses from the Gut-Associated Immune System in Young Female Rat Offspring

In humans, the development of gut-associated lymphoid tissue (GALT) occurs in the first years of life and can be influenced by diet. The objective of this study was to determine the effect of dietary choline on the development of gut-associated lymphoid tissue (GALT). Three feeding trials were conducted in female Sprague-Dawley rats. Beginning 3 d before parturition (studies 1 and 3) or at day 10 of gestation (study 2), control dams consumed a 100% free choline (FC) diet until the end of the lactation period. In studies 1 and 3, test dams consumed a high-glycerophosphocholine (HGPC) diet [75% glycerophosphocholine (GPC), 12.5% phosphatidylcholine (PC), 12.5% FC] and a 100% PC diet, respectively (both 1 g of choline/kg diet). In study 2, test dams consumed a high-sphingomyelin (SM) and PC (SMPC) diet (34% SM, 37% PC, 17% GPC, 7% FC, 5% phosphocholine) or a 50% PC diet (50% PC, 25% FC, 25% GPC), both 1.7 g of choline/kg diet. Immune cell phenotypes and ex vivo cytokine production by mitogen-stimulated immune cells were measured. Feeding of the HGPC diet lowered T-cell IL-2 (44%), IFN-γ (34%), and TNF-α (55%) production in mesenteric lymph nodes (MLNs) compared with control. Feeding both SMPC and 50% PC diets during the lactation and weaning periods increased IL-2 (54%) and TNF-α (46%) production after T-cell stimulation compared with control. There was a lower production of IL-2 (46%), IL-6 (66%), and TNF-α (45%), and a higher production of IL-10 (44%) in both SMPC and 50% PC groups following ovalbumin stimulation compared with control in MLNs. Feeding a diet containing 100% PC increased the production of IFN-γ by 52% after T-cell stimulation compared with control. Feeding a diet containing a mixture of choline forms with a high content of lipid-soluble forms during both the lactation and weaning periods enhances ex vivo immune responses from the GALT in female Sprague-Dawley offspring.

The impact of the gut microbiota on T cell ontogeny in the thymus.

The intestinal microbiota is critical for the development of gut-associated lymphoid tissues, including Peyer's patches and mesenteric lymph nodes, and is instrumental in educating the local as well as systemic immune system. In addition, it also impacts the development and function of peripheral organs, such as liver, lung, and the brain, in health and disease. However, whether and how the intestinal microbiota has an impact on T cell ontogeny in the hymus remains largely unclear. Recently, the impact of molecules and metabolites derived from the intestinal microbiota on T cell ontogeny in the thymus has been investigated in more detail. In this review, we will discuss the recent findings in the emerging field of the gut-thymus axis and we will highlight the current questions and challenges in the field.

The porcine large intestine contains developmentally distinct submucosal lymphoid clusters and mucosal isolated lymphoid follicles

Gut-associated lymphoid tissues (GALT) serve as key priming sites for intestinal adaptive immune responses. Most of our understanding of GALT function and development arises from studies in mice. However, the diversity, structure and cellular composition of GALT differs markedly between mammalian species and the developmental window in which distinct GALT structures develop in large mammals remains poorly understood. Given the importance of pigs as models of human disease, as well as their role in livestock production, we adapted a recently developed protocol for the isolation of human GALT to assess the diversity, development and immune composition of large intestinal GALT in neonatal and adult pigs. We demonstrate that the large intestine of adult pigs contains two major GALT types; multifollicular submucosal GALT that we term submucosal lymphoid clusters (SLC) which develop prenatally, and as yet undescribed mucosal isolated lymphoid follicles (M-ILF), which arise after birth. Using confocal laser microscopy and flow cytometry, we additionally assess the microanatomy and lymphocyte composition of SLC and M-ILF, compare them to jejunal Peyer's patches (PP), and describe the maturation of these structures.Collectively, our results provide a deeper understanding of the diversity and development of GALT within the porcine large intestine.

Immune translational control by CPEB4 regulates intestinal inflammation resolution and colorectal cancer development

SummaryUpon tissue injury, cytokine expression reprogramming transiently remodels the inflammatory immune microenvironment to activate repair processes and subsequently return to homeostasis. However, chronic inflammation induces permanent changes in cytokine production which exacerbate tissue damage and may even favor tumor development. Here, we address the contribution of post-transcriptional regulation, by the RNA-binding protein CPEB4, to intestinal immune homeostasis and its role in inflammatory bowel diseases (IBD) and colorectal cancer (CRC) development. We found that intestinal damage induces CPEB4 expression in adaptive and innate immune cells, which is required for the translation of cytokine mRNA(s) such as the one encoding interleukin-22. Accordingly, CPEB4 is required for the development of gut-associated lymphoid tissues and to maintain intestinal immune homeostasis, mediating repair and remodeling after acute inflammatory tissue damage and promoting the resolution of intestinal inflammation. CPEB4 is chronically overexpressed in inflammatory cells in patients with IBD and in CRC, favoring tumor development.

Fetal environment and fetal intestine are sterile during the third trimester of pregnancy

Recent next generation sequencing studies on host-associated microbiomes generated debatable conclusions regarding the central dogma of fetal gut sterility. These observations challenge the concepts that microbial colonization of the gut begins during and after birth as well as the concept of antigen-independent prenatal maturation of mucosal-associated lymphoid tissue in ruminants and humans. The placental barrier varies markedly among mammalian species with mice and humans having haemochorial placentas (fetal tissue in direct contact with maternal blood) versus epitheliochorial placentation (maternal and fetal blood separated by six tissue layers) in ruminants. Therefore, this study re-examined the question of fetal gut sterility using the fetal lamb as a model ruminant species with the most complete placental barrier. Use of PCR and quantitative real-time PCR with three different pairs of universal bacterial primers (27 F and 1492R, HDA1 and HDA2, U2F and U2R) to amplify 16S rRNA gene did not generate detectable PCR products from samples collected from the fetal environment (placenta, amniotic fluid) and fetal intestine during the third trimester of pregnancy. Procedures to further enrich microbial DNA from total extracted DNA also resulted in no detectable genomic DNA. Moreover, use of 16S amplicon sequencing confirmed the absence of bacteria in the fetal environment during the third trimester of pregnancy. A ‘No Template’ control containing only PCR reagents generated sequences that could be clustered into OTUs at 97% similarity and assigned to bacterial genera, including Staphylococcus, Lactobacillus and Escherichia-Shigella. Use of multiple molecular-based approaches to profile fetal environment-associated microbiota supports the conclusion that the fetal environment and fetal intestine remain sterile during the third trimester of pregnancy. The use of appropriate controls, both positive and no template, revealed inherent contamination in reagents and that variations in the data analysis pipeline can produce artificial microbial profiles from host tissues containing low microbial biomass. Finally, these findings confirm that extensive development of gut-associated lymphoid tissue in the ruminant fetal intestine, characterized by active B cell proliferation and immunoglobulin V gene somatic mutation, is not associated with exposure to bacterial DNA and antigens.

Transmission of integrin β7 transmembrane domain topology enables gut lymphoid tissue development.

Integrin activation regulates adhesion, extracellular matrix assembly, and cell migration, thereby playing an indispensable role in development and in many pathological processes. A proline mutation in the central integrin β3 transmembrane domain (TMD) creates a flexible kink that uncouples the topology of the inner half of the TMD from the outer half. In this study, using leukocyte integrin α4β7, which enables development of gut-associated lymphoid tissue (GALT), we examined the biological effect of such a proline mutation and report that it impairs agonist-induced talin-mediated activation of integrin α4β7, thereby inhibiting rolling lymphocyte arrest, a key step in transmigration. Furthermore, the α4β7(L721P) mutation blocks lymphocyte homing to and development of the GALT. These studies show that impairing the ability of an integrin β TMD to transmit talin-induced TMD topology inhibits agonist-induced physiological integrin activation and biological function in development.

Intestinal dysbiosis and probiotic applications in autoimmune diseases.

In humans, a complex interaction between the host immune system and commensal microbiota is required to maintain gut homeostasis. In this symbiotic relationship, the microbiota provides carbohydrate fermentation and digestion, vitamin synthesis and gut-associated lymphoid tissue development, as well as preventing colonization by pathobionts, whereas the host offers a niche and nutrients for the survival of the microbiota. However, when this mutualistic relationship is compromised and an altered interaction between immune cells and microorganisms occurs, the gut microbiota may cause or contribute to the establishment of infectious diseases and trigger autoimmune diseases. Researchers have made efforts to clarify the role of the microbiota in autoimmune disease development and find new therapeutic approaches to treat immune-mediated diseases. However, the exact mechanisms involved in the dysbiosis and breakdown of the gut epithelial barrier are currently unknown. Here, we provide a general overview of studies describing gut microbiota perturbations in animal models of autoimmune diseases, such as type 1 diabetes, multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus. Moreover, we include the main studies concerning dysbiosis in humans and a critical discussion of the existing data on the use of probiotics in these autoimmune diseases.

ATP-Gated Ionotropic P2X7 Receptor Controls Follicular T Helper Cell Numbers in Peyer’s Patches to Promote Host-Microbiota Mutualism

Microbial colonization of the gut induces the development of gut-associated lymphoid tissue (GALT). The molecular mechanisms that regulate GALT function and result in gut-commensal homeostasis are poorly defined. T follicular helper (Tfh) cells in Peyer's patches (PPs) promote high-affinity IgA responses. Here we found that the ATP-gated ionotropic P2X7 receptor controls Tfh cell numbers in PPs. Lack of P2X7 in Tfh cells enhanced germinal center reactions and high-affinity IgA secretion and binding to commensals. The ensuing depletion of mucosal bacteria resulted in reduced systemic translocation of microbial components, lowering B1 cell stimulation and serum IgM concentrations. Mice lacking P2X7 had increased susceptibility to polymicrobial sepsis, which was rescued by Tfh cell depletion or administration of purified IgM. Thus, regulation of Tfh cells by P2X7 activity is important for mucosal colonization, which in turn results in IgM serum concentrations necessary to protect the host from bacteremia.

Nonredundant Function of Soluble LTα 3 Produced by Innate Lymphoid Cells in Intestinal Homeostasis

Immunoglobulin A (IgA) production at mucosal surfaces contributes to protection against pathogens and controls intestinal microbiota composition. However, mechanisms regulating IgA induction are not completely defined. We show that soluble lymphotoxin α (sLTα3) produced by RORγt(+) innate lymphoid cells (ILCs) controls T cell-dependent IgA induction in the lamina propria via regulation of T cell homing to the gut. By contrast, membrane-bound lymphotoxin β (LTα1β2) produced by RORγt(+) ILCs is critical for T cell-independent IgA induction in the lamina propria via control of dendritic cell functions. Ablation of LTα in RORγt(+) cells abrogated IgA production in the gut and altered microbiota composition. Thus, soluble and membrane-bound lymphotoxins produced by ILCs distinctly organize adaptive immune responses in the gut and control commensal microbiota composition.

IgA production in the large intestine is modulated by a different mechanism than in the small intestine: Bacteroides acidifaciens promotes IgA production in the large intestine by inducing germinal center formation and increasing the number of IgA+ B cells

It has been demonstrated that intestinal commensal bacteria induce immunoglobulin (Ig) A production by promoting the development of gut-associated lymphoid tissues in the small intestine. However, the precise mechanism whereby these bacteria modulate IgA production in the large intestine, which harbors the majority of intestinal commensals, is poorly understood. In addition, it is not known which commensal bacteria induce IgA production in the small intestine and which induce production in the large intestine. To address these issues, we generated gnotobiotic mice mono-associated with different murine commensal bacteria by inoculating germ-free (GF) mice with Lactobacillus johnsonii or Bacteroides acidifaciens. In GF mice, IgA production was barely detectable in the small intestine and was not detected in the large intestine. Interestingly, total IgA secretion in the large intestinal mucosa of B. acidifaciens mono-associated (BA) mice was significantly greater than that of GF and L. johnsonii mono-associated (LJ) mice. However, there was no difference in total IgA production in the small intestine of GF, LJ and BA mice. In addition, in the large intestine of BA mice, the expression of IgA+ cells and germinal center formation were more remarkable than in GF and LJ mice. Furthermore, B. acidifaciens-specific IgA was detected in the large intestine of BA mice. These results suggest that the production of IgA in the large intestine may be modulated by a different mechanism than that in the small intestine, and that B. acidifaciens is one of the predominant bacteria responsible for promoting IgA production in the large intestine.

Dietary effect of short-chain organic acids on growth performance, mortality and development of intestinal lymphoid tissues in young non-medicated rabbits

This work aimed to test the effect of a dietary inclusion of formic and citric acids on growth performance, mortality, jejunal histology and development of intestinal lymphoid tissues in growing non-medicated rabbits. For that purpose, a diet including the acids (diet A) was compared to a control diet (diet C). Sixty rabbits weaned at 28 days were submitted to each diet. At 56 and 77 days, ten rabbits were slaughtered to assess caecal traits, jejunal histology and follicular development in the caudal ileal Peyer’s patch and in the appendix. In the 56-77 d period, average daily gain of rabbits fed diet A was greater than that of control rabbits (48.0 vs. 43.9 g, P=0.019). Mortality rate was not affected by the diet (6.12% on average). Caecal pH was lower at 77 than at 56 days (6.02 vs. 6.19, P=0.016). The concentration of ammonia in the caecal contents increased from 9.62 to 14.2 mmol/l (P=0.003) when rabbits reached 77 days of age. The appendix was heavier (9.75 vs. 4.30 g, P<0.001), longer (13.3 vs. 10.4 cm, P<0.001) and wider (1.74 vs. 1.45 cm, P=0.006) at 77 than at 56 days. Rabbits of 56 days of age fed diet C had shorter villi than the mean value of the other three treatments (662 vs. 807 µm, P<0.001). In the Peyer’s patch, the average follicle area was greater at 77 than at 56 days of age (118 vs. 88.4 x 103 µm2, P<0.001) and was also greater in rabbits fed diet C than in those fed diet A (109 vs. 97.5 x 103 µm2, P=0.049). In the appendix, no differences on the average follicle area were found at 56 days of age (115 x 103 µm2) whereas, at 77 days, the area increase was higher for rabbits fed diet C than for those fed diet A (95.5 vs. 50.8%, P<0.001). In conclusion, including formic and citric acids in growing rabbit diets improves weight gain, has a trophic effect on the jejunal mucosa and controls the development of gut-associated lymphoid tissues.

Beneficial health effects of milk and fermented dairy products — Review

Milk is a complex physiological liquid that simultaneously provides nutrients and bioactive components that facilitate the successful postnatal adaptation of the newborn infant by stimulating cellular growth and digestive maturation, the establishment of symbiotic microflora, and the development of gut-associated lymphoid tissues. The number, the potency, and the importance of bioactive compounds in milk and especially in fermented milk products are probably greater than previously thought. They include certain vitamins, specific proteins, bioactive peptides, oligosaccharides, organic (including fatty) acids. Some of them are normal milk components, others emerge during digestive or fermentation processes. Fermented dairy products and probiotic bacteria decrease the absorption of cholesterol. Whey proteins, medium-chain fatty acids and in particular calcium and other minerals may contribute to the beneficial effect of dairy food on body fat and body mass. There has been growing evidence of the role that dairy proteins play in the regulation of satiety, food intake and obesity-related metabolic disorders. Milk proteins, peptides, probiotic lactic acid bacteria, calcium and other minerals can significantly reduce blood pressure. Milk fat contains a number of components having functional properties. Sphingolipids and their active metabolites may exert antimicrobial effects either directly or upon digestion.

Ontogeny and induction of adhesion molecule expression in human fetal intestine.

In this study we examined the distribution of the adhesion molecules ICAM-1, VCAM-1 and E-selectin in human fetal intestine, to determine whether they may have a role in the development of gut-associated lymphoid tissue. Secondly, we studied the tempo of induction of these molecules after T cell activation in explants of human fetal intestine cultured in vitro. In the fetus from 11 to 20 weeks gestation, endothelial expression of ICAM-1 and diffuse staining of VCAM-1 was observed in the lamina propria. In contrast, there was intense expression of ICAM-1 and VCAM-1 in the developing Peyer's patches, suggesting that these molecules may be involved in the accumulation or organization of lymphoid tissue in the gut. After T cell activation in fetal intestinal explants, the expression of ICAM-1 and VCAM-1 was increased on most endothelial cells, leucocytes, and stromal cells in the lamina propria. Expression was maintained for at least 4 days. In contrast, the induction of E-selectin was rapid, and the expression was transient, despite the continuing presence of activated T cells and macrophages. This suggests that other factors are required to prevent the down-regulation of E-selectin to maintain the sustained expression sometimes observed in vivo.

Is There Evidence That the Gut Contributes to Mucosal Immunity in Humans?

Our understanding of the common mucosal immune system derives from animal studies. Antigen-sensitized lymphocytes in the gut-associated lymphoid tissue (GALT) migrate via the blood to mucosal tissues to generate the mucosal-associated lymphoid tissue (MALT). In these sites, B cells differentiate into plasma cells and produce antigen-specific secretory IgA, the principal specific immune antiviral and antibacterial defense of moist mucosal surfaces. Responses to oral intake seem necessary to actively maintain this system in health. Experimentally, lack of enteral stimulation with parenteral feeding alters GALT and MALT size and function. These alterations disturb intestinal and extraintestinal mucosal immunity. This review is an overview of current and classical studies demonstrating the human mucosal immune system and interactions with nutrition. Human evidence of the mucosal immune system exists, although most data are indirect. Gut stimulation after oral intake induces a generalized immune response in the human MALT through a mucosal-immune network. Examples include neonatal development of GALT influenced by enteral feeding, the presence of antigen-specific IgA and antigen-specific IgA-secreting plasma cells in distant mucosal effector sites such as the breast after gut luminal antigen exposure, and isolation of IgA-producing cells from circulating blood. It is unlikely that clinical studies will ever completely define the effect of route of feeding in all patient populations. This may be possible, however, if investigators understand, define and characterize nutrition-dependent immunologic mechanisms, allowing clinicians to examine clinical responses to nutrition in specific patient populations. This might allow generation of new approaches to protect mucosal immunity.

Regulation of immune response at intestinal and peripheral sites by probiotics

The gut associated lymphoid tissue (GALT) should protect intestinal mucosa against pathogens, but also avoid hypersensitivity reactions to food proteins, normal bacterial flora and other environmental macromolecules. The interaction between epithelial cells and microflora is fundamental to establish gut mucosal barrier and GALT development. The normal colonization of intestine by commensal bacteria is thus crucial for a correct development of mucosal immune system. Probiotic bacteria are normal inhabitants of microflora and may confer health benefits to the host. The modification of the intestinal microflora towards a healthier probiotics enriched microflora may generate beneficial mucosal immunomodulatory effects and may represent a new strategy to cure intestinal and allergic diseases. The health benefits may be specific for different probiotic strains. Ongoing research is providing new insights into the probiotic beneficial effects and related mechanisms. This review represents an update of immunomodulatory activity of different probiotics and of the more accredited mechanisms underlying such activities.

Role of human milk components in gastrointestinal development: Current knowledge and future NEEDS

Human milk (HM) is a complex physiological fluid with multifunctional roles within the gastrointestinal tract that facilitate the successful postnatal adaptation of the newborn by stimulating cellular growth and digestive maturation, the establishment of symbiotic microflora, and the development of gut-associated lymphoid tissues. An intricate mixture of bioactive proteins, lipids, and carbohydrates that is unique to milk supports these processes and provides important signals to the developing intestine. The complexity of HM composition and the potential interactions among milk components has hampered our understanding of how HM components affect gastrointestinal development. Furthermore, limited information exists about the fundamental mechanisms of postnatal intestinal growth of healthy infants, further hindering our ability to delineate the role of HM in regulating this process. In this paper, evidence for the role of HM components in early intestinal development is summarized, with a discussion of the limitations of existing data, and suggestions are made for future research that is needed to delineate the biology of HM, the physiology of postnatal intestinal development, and the complexity of potential interactions that occur between HM components and the developing neonate. Human milk (HM) is a complex physiological fluid with multifunctional roles within the gastrointestinal tract that facilitate the successful postnatal adaptation of the newborn by stimulating cellular growth and digestive maturation, the establishment of symbiotic microflora, and the development of gut-associated lymphoid tissues. An intricate mixture of bioactive proteins, lipids, and carbohydrates that is unique to milk supports these processes and provides important signals to the developing intestine. The complexity of HM composition and the potential interactions among milk components has hampered our understanding of how HM components affect gastrointestinal development. Furthermore, limited information exists about the fundamental mechanisms of postnatal intestinal growth of healthy infants, further hindering our ability to delineate the role of HM in regulating this process. In this paper, evidence for the role of HM components in early intestinal development is summarized, with a discussion of the limitations of existing data, and suggestions are made for future research that is needed to delineate the biology of HM, the physiology of postnatal intestinal development, and the complexity of potential interactions that occur between HM components and the developing neonate.

Impaired immune responses in broiler hatchling hindgut following delayed access to feed

One of the key stimulators of intestinal development in the chick is physical exposure to feed, while feed withholding delays the onset of gut development. A delay of 24-72 h in onset of feeding is quite common in the poultry industry due to variation in hatching time and hatchery treatments. As intestinal development occurs in concert with the development of the gut associated lymphoid tissue (GALT), we investigated the effects of short term feed withholding on development of GALT in broiler hatchlings. GALT activity was determined by antibody production (systemic and locally in the gut), distribution of B and T lymphocytes in the gut, expression of lymphocyte specific genes, and distribution of B and T lymphocytes in the cloacal bursa. Our findings show that while development of GALT in the foregut (duodenum, jejunum, ileum) was only slightly and temporarily impeded by feed withholding, GALT activity in the hindgut and the gut-related cloacal bursa was significantly delayed during the first 2 weeks of life: Systemic and intestinal antibody responses following rectal immunization to antigen were lower, colonization of the hindgut (cecum and colon) by T and B lymphocytes was delayed, as well as the expression of chIL-2 mRNA in hindgut T lymphocytes. We also found that the increase of B and T population size in the cloacal bursa was delayed with time. Full recovery occurred from 2 weeks of age. The 2-week vulnerable period should be seriously considered in circumstances where hatchlings are in transit for extended periods from hatcheries to farms.

Role of commensal bacteria in development of gut-associated lymphoid tissues and preimmune antibody repertoire.

Intestinal bacteria are required for development of gut-associated lymphoid tissues (GALT), which mediate a variety of host immune functions, such as mucosal immunity and oral tolerance. In rabbits, the intestinal microflora are also required for developing the preimmune Ab repertoire by promoting somatic diversification of Ig genes in B cells that have migrated to GALT. We studied the mechanism of bacteria-induced GALT development. Bacteria were introduced into rabbits in which the appendix had been rendered germfree by microsurgery (we refer to these rabbits as germfree-appendix rabbits). We then identified specific members of the intestinal flora that promote GALT development. The combination of Bacteroides fragilis and Bacillus subtilis consistently promoted GALT development and led to development of the preimmune Ab repertoire, as shown by an increase in somatic diversification of VDJ-C micro genes in appendix B cells. Neither species alone consistently induced GALT development, nor did Clostridium subterminale, Escherichia coli, or Staphylococcus epidermidis. B. fragilis, which by itself is immunogenic, did not promote GALT development; hence, GALT development in rabbits does not appear to be the result of an Ag-specific immune response. To identify bacterial pathways required for GALT development, we introduced B. fragilis along with stress-response mutants of B. subtilis into germfree-appendix rabbits. We identified two Spo0A-controlled stress responses, sporulation and secretion of the protein YqxM, which are required for GALT development. We conclude that specific members of the commensal, intestinal flora drive GALT development through a specific subset of stress responses.

Normal development of the gut-associated lymphoid tissue except Peyer's patch in MyD88-deficient mice.

MyD88 is a key adaptor molecule for signalling via Toll-like receptors (TLRs) and the response to gut commensal microbes. To investigate the role of TLRs/MyD88 pathway in the development of the gut-associated lymphoid tissue (GALT), we examined the development of Peyer's patches (PPs) and cryptopatch (CP), and also one of effector compartment, intraepithelial lymphocyte (IEL) in MyD88-/-, TLR2-/- and TLR4-/- mice. In MyD88-/- mice, the organogenesis of PPs was not disturbed. However, PPs in 2-week-old MyD88-/- mice were significantly smaller than those in MyD88+/- mice. Also, in 2-week-old TLR4-/-, but not TLR2-/- mice, PPs did not develop rapidly. The development of PPs in MyD88-/- and TLR4-/- mice was completely recovered in 10 weeks. PP cells from MyD88-/- mice showed significant decrease in proliferation when stimulated with lipopolysaccharide. The development of CP and IEL was also normal in 10-week-old MyD88-/- mice. These results suggest that the TLRs/MyD88 pathway might be involved in the development of PPs only at early postnatal stage, and TLRs/MyD88-independent signalling is critically involved in the development of GALT in adult mice.