Organogenesis of Ileal Peyer's Patches Is Initiated Prenatally and Accelerated Postnatally With Comprehensive Proliferation of B Cells in Pigs.

Morphogenesis and differentiation of organs is required for subsequent functional maturation. The morphological features of Peyer's patches vary among species. In pigs, they develop extensively in the ileum as ileal Peyer's patches (IPPs). However, the role of IPPs in the porcine immune system remains to be elucidated because of a lack of complete understanding of IPP organogenesis. Results of the present study revealed that development of porcine IPPs is initiated prenatally between embryonic days 76 and 91. The process of IPP organogenesis is concomitant with increased transcriptional patterns of CXCL13 and CCL19. IPPs undergo further development postnatally by forming central, marginal, and subepithelial zones. Importantly, a large number of proliferating B cells and apoptotic cells are found in porcine IPPs postnatally, but not prenatally. The expression level of IgM in proliferating B cells depends on the zone in which distinct B cells are separately localized after birth. Specifically, IgM+ cells are predominantly found in the central zone, whereas IgM-/low cells are abundant in the marginal zone. Importantly, the cellular feature of IPPs differs from that of mesenteric lymph nodes (MLNs) where such distinct zones are not formed both prenatally and postnatally. Our findings suggest that IPPs (not MLNs) in postnatal pigs are involved in complementing functions of the primary lymphoid tissue that promotes the differentiation and maturation of B cells.

Differentiation and function of group 3 innate lymphoid cells, from embryo to adult.

Group 3 innate lymphoid cells (ILC3) represent a heterogeneous population of cells that share the nuclear hormone receptor RORγt (retinoic acid receptor-related orphan receptor γt) as a master regulator for differentiation and function. ILC3 can be divided into two major subsets based on the cell surface expression of the natural cytotoxicity receptor (NCR), NKp46. A subset of NCR(-) ILC3 includes the previously known lymphoid-tissue inducer cells that are essential for the embryonic formation of peripheral lymph nodes and Peyer's patches. After birth, the NCR(-) and NCR(+) ILC3 contribute to the maintenance of health but also to inflammation in mucosal tissues. This review will describe the differentiation pathways of ILC3, their involvement in the development of the adaptive immune system and their role in the establishment and maintenance of gut immunity.

Divergence of Vascular Specification in Visceral Lymphoid Organs-Genetic Determinants and Differentiation Checkpoints.

Despite their functional similarities, peripheral lymphoid tissues are remarkably different according to their developmental properties and structural characteristics, including their specified vasculature. Access of leukocytes to these organs critically depends on their interactions with the local endothelium, where endothelial cells are patterned to display a restricted set of adhesion molecules and other regulatory compounds necessary for extravasation. Recent advances in high throughput analyses of highly purified endothelial subsets in various lymphoid tissues as well as the expansion of various transgenic animal models have shed new light on the transcriptional complexities of lymphoid tissue vascular endothelium. This review is aimed at providing a comprehensive analysis linking the functional competence of spleen and intestinal lymphoid tissues with the developmental programming and functional divergence of their vascular specification, with particular emphasis on the transcriptional control of endothelial cells exerted by Nkx2.3 homeodomain transcription factor.

Differential RET signaling pathways drive development of the enteric lymphoid and nervous systems.

During the early development of the gastrointestinal tract, signaling through the receptor tyrosine kinase RET is required for initiation of lymphoid organ (Peyer's patch) formation and for intestinal innervation by enteric neurons. RET signaling occurs through glial cell line-derived neurotrophic factor (GDNF) family receptor α co-receptors present in the same cell (signaling in cis). It is unclear whether RET signaling in trans, which occurs in vitro through co-receptors from other cells, has a biological role. We showed that the initial aggregation of hematopoietic cells to form lymphoid clusters occurred in a RET-dependent, chemokine-independent manner through adhesion-mediated arrest of lymphoid tissue initiator (LTin) cells. Lymphoid tissue inducer cells were not necessary for this initiation phase. LTin cells responded to all RET ligands in trans, requiring factors from other cells, whereas RET was activated in enteric neurons exclusively by GDNF in cis. Furthermore, genetic and molecular approaches revealed that the versatile RET responses in LTin cells were determined by distinct patterns of expression of the genes encoding RET and its co-receptors. Our study shows that a trans RET response in LTin cells determines the initial phase of enteric lymphoid organ morphogenesis, and suggests that differential co-expression of Ret and Gfra can control the specificity of RET signaling.

Peyer's patch M cells derived from Lgr5(+) stem cells require SpiB and are induced by RankL in cultured "miniguts".

Peyer's patches consist of domains of specialized intestinal epithelium overlying gut-associated lymphoid tissue (GALT). Luminal antigens reach the GALT by translocation through epithelial gatekeeper cells, the so-called M cells. We recently demonstrated that all epithelial cells required for the digestive functions of the intestine are generated from Lgr5-expressing stem cells. Here, we show that M cells also derive from these crypt-based Lgr5 stem cells. The Ets family transcription factor SpiB, known to control effector functions of bone marrow-derived immune cells, is specifically expressed in M cells. In SpiB(-/-) mice, M cells are entirely absent, which occurs in a cell-autonomous fashion. It has been shown that Tnfsf11 (RankL) can induce M cell development in vivo. We show that in intestinal organoid ("minigut") cultures, stimulation with RankL induces SpiB expression within 24 h and expression of other M cell markers subsequently. We conclude that RankL-induced expression of SpiB is essential for Lgr5 stem cell-derived epithelial precursors to develop into M cells.

Expression pattern changes and function of RANKL during mouse lymph node microarchitecture development.

Receptor activator of nuclear factor kappa-B ligand (RANKL) expression was examined during the development of mouse fetal peripheral lymphoid organs. A shift in the expression pattern was detected during the transition from lymphoid tissue inducer (LTi) cells to lymphoid tissue organizer (LTo) cells in the lymph node (LN) anlagen but not in the Peyer's patch anlagen. In order to understand the functional impact of these changes in the fetal expression of RANKL, the RANKL function was blocked by a blocking antibody. Excess anti-RANKL antibody was administered to pregnant mice between 13.5 and 16.5 dpc and was found to completely block LN anlagen development, suggesting that RANKL function during this period is critical for LN development. In addition, small amounts of anti-RANKL antibodies were injected directly into the amniotic space at 13.5 dpc, resulting in perturbed B-cell follicle formation and high endothelial venule differentiation after birth. These results suggest that RANKL expression on LTi cells during the early phase of LN development is critical for the development LN microarchitecture.

Kit ligand and Il7 differentially regulate Peyer's patch and lymph node development.

Hematopoietic lymphoid tissue inducer (LTi) cells initiate lymph node (LN) and Peyer's patch (PP) development during fetal life by inducing the differentiation of mesenchymal organizer cells. The growth factor signals underlying LTi cell development and LN and PP organogenesis remain poorly understood. LTi cells express the Il7r and the receptor tyrosine kinase Kit, whereas organizer cells express their cognate ligands. To determine the relative significance of Il7 and Kit signaling in LTi cell homeostasis and PP and LN development, we have analyzed mice deficient for Kit (Kit(W/Wv)), Il7 (Il7(-/-)), or both (Il7(-/-) Kit(W/Wv)). Unlike Kit(W/Wv) and Il7(-/-) single mutants, Il7(-/-) Kit(W/Wv) mice were almost devoid of LTi cells in their mesenteric LN anlage. This LTi deficiency was associated with a block in mesenchymal LN organizer cell generation and the absence of almost all LNs. In contrast, intestinal LTi cell numbers, PP organizer cell generation, and PP development were strongly affected by impaired Kit signaling, but were independent of Il7. Hence, Kit and Il7 act synergistically in LN organogenesis, whereas Kit signaling, but not Il7, critically regulates PP organogenesis and LTi cell numbers in the intestine. Consistent with these differential growth factor requirements for PP and LN development, PP organizer cells expressed higher Kitl and lower Il7 levels than did LN organizer cells. Collectively, these results demonstrate that Kit and Il7 differentially control PP and LN organogenesis through the local growth factor-driven regulation of LTi cell numbers.

Comparative studies on the secondary lymphoid tissue areas in the chicken bursa of Fabricius and calf ileal Peyer's patch.

The chicken bursa of Fabricius and calf ileal Peyer's patch are thought to be the primary lymphoid organs of B cell development. In the bursa, the existence of secondary lymphoid tissue, called the diffusely infiltrated area, has been recognized. Recently, we have found the presence of a region of secondary lymphoid tissue in the ileal Peyer's patch at the period of the most rapid growth of this organ. In this study, we compared the development of these secondary lymphoid tissue regions in the bursa and ileal Peyer's patch histologically. Before hatching, lymphatic follicle formation occurred in the bursa, but not in the diffusely infiltrated area, where only a small number of lymphoid cells were found. However, during fetal calf development, lymphatic follicle formation occurred not only in the primary lymphoid organ but also in the secondary lymphoid tissue regions. Therefore, the prenatal development of the secondary lymphoid tissue regions of the bursa and ileal Peyer's patch were distinct. After hatching, formation of the germinal center, which contained many CD4+ cells, was observed in the diffusely infiltrated area of the bursa. After birth, many CD4+ cells and IgG mRNA expression were observed in the lymphatic follicle of the secondary lymphoid tissue regions in the ileal Peyer's patch, but rarely in the ileal Peyer's patch lymphatic follicles. The change of character of these secondary lymphoid tissue regions at the postnatal stage might be dependent on external antigens.

The prenatal development and histochemistry of the ileal mucins in the bovine fetuses.

Few studies exist regarding the distribution of intestinal mucins in fetuses of mammalians such as cattle and sheep. In this study, we aimed to describe the changes in the mucin production by ileal epithelium of bovine fetuses during their prenatal development. The goblet cells showed heterogeneity in mucins and the apical cytoplasm of the enterocytes demonstrated Periodic acid Schiff-positive reaction which declined gradually towards the birth. Moreover, the number of the goblet cells containing acidic and mixed mucins augmented, whereas those containing neutral mucins decreased with advancing gestational age. After sixth month of gestation, with the initiation of the ileal Peyer patches and follicle-associated epithelium development, a gradual increase in the number of goblet cells containing sulfomucins was also noticed towards the birth. The presence of different mucins in the ileum of bovine fetuses throughout prenatal development might play a role in the protection of the intestinal mucosa against urinary waste products in swallowed amniotic fluid and bile. Furthermore, mucins can also contribute for the formation of meconium in intra-uterine life and building of strong intestinal barrier with predominating sulfomucins, protecting the intestine against potential pathogens and digestive enzymes after birth.

Development of human lymph nodes and Peyer's patches.

In contrast to our understanding of murine lymphoid organogenesis, detailed knowledge on the mechanisms of human lymph node development is virtually lacking. This is mainly due to the obvious difficulties that accompany research using human fetal organs. In this review we will highlight current knowledge on human lymph node and Peyer's patch development and will temporally align observations made in humans with data available from murine studies. In the final paragraphs we will put this knowledge in the context of human malignancies in which interactions between lymphocytes and stroma, resembling those seen in lymphoid organs, are recapitulated.

Homeostatic chemokines in development, plasticity, and functional organization of the intestinal immune system.

In the past decade accumulating evidence supported the view that the immune system should be regarded as trust consisting of several branches. In this review, we will first introduce the architectural features comprising the intestinal immune system emphasising its plasticity and subsequently discuss the concepts describing its development. We then focus on the chemokine/receptor system as a key integrator managing coordinated migration of and communication among the cells mediating intestinal immunity. Thus, chemokines control development and maintain functionality of the intestinal immune system that is required to perform the unique balancing act between tolerating food, curtailing commensals activities and eliminating pathogenic infections.

Development of secondary lymphoid organs.

Secondary lymphoid organs develop during embryogenesis or in the first few weeks after birth according to a highly coordinated series of interactions between newly emerging hematopoietic cells and immature mesenchymal or stromal cells. These interactions are orchestrated by homeostatic chemokines, cytokines, and growth factors that attract hematopoietic cells to sites of future lymphoid organ development and promote their survival and differentiation. In turn, lymphotoxin-expressing hematopoietic cells trigger the differentiation of stromal and endothelial cells that make up the scaffolding of secondary lymphoid organs. Lymphotoxin signaling also maintains the expression of adhesion molecules and chemokines that govern the ultimate structure and function of secondary lymphoid organs. Here we describe the current paradigm of secondary lymphoid organ development and discuss the subtle differences in the timing, molecular interactions, and cell types involved in the development of each secondary lymphoid organ.

[Development and formation of the mucosal immune system of the small intestine].

Morphologic and morphometric characteristics of the grouped lymphoid nodules (Peyer's patches) and of the small intestine lamina propria were studied in rats at the 19 prenatal and 1, 7, 14, 21, 90 postnatal days. The development of these structures was found to be heterochronic and fragmentary. The development of the individual components of the mucosal immune system was interrelated. The integration of the afferent and efferent limbs of the mucosal immune system with the processes of digestion and absorption, is regarded as its adaptation to the peculiarities of postnatal development of mammals and as the property of the functional system, maintaining the homeostasis of the internal milieu of the organism.

Distinct activities of stromal cells involved in the organogenesis of lymph nodes and Peyer's patches.

It is now well established that the interaction between "inducer" cells of hemopoietic origin and "organizer" cells of mesenchymal lineage is involved in the organogenesis of lymph node (LN) and Peyer's patch (PP). Organizer cells are defined by the expression of VCAM-1 and ICAM-1 and the production of homeostatic chemokines. However, several studies suggested the presence of a diversity among these cells from different lymphoid tissues. Thus, we attempted to define the difference of organizer cells of LN and PP in terms of gene expression profile. Microarray analyses of organizer cells revealed that these cells isolated from embryonic mesenteric LN expressed higher levels of genes that are related to inflammation, tissue remodeling, and development of mesenchymal lineage compared with those from PP. Several transcription factors related to epithelial-mesenchymal interactions were also up-regulated in organizer cells from LN. These results indicate that organizer cells in LN and PP are indeed distinct and suggest that the organizer cells in LN are at a more activated stage than those in PP.

Tyrosine kinase receptor RET is a key regulator of Peyer's patch organogenesis.

Normal organogenesis requires co-ordinate development and interaction of multiple cell types, and is seemingly governed by tissue specific factors. Lymphoid organogenesis during embryonic life is dependent on molecules the temporal expression of which is tightly regulated. During this process, haematopoietic 'inducer' cells interact with stromal 'organizer' cells, giving rise to the lymphoid organ primordia. Here we show that the haematopoietic cells in the gut exhibit a random pattern of motility before aggregation into the primordia of Peyer's patches, a major component of the gut-associated lymphoid tissue. We further show that a CD45+CD4-CD3-Il7Ralpha-c-Kit+CD11c+ haematopoietic population expressing lymphotoxin has an important role in the formation of Peyer's patches. A subset of these cells expresses the receptor tyrosine kinase RET, which is essential for mammalian enteric nervous system formation. We demonstrate that RET signalling is also crucial for Peyer's patch formation. Functional genetic analysis revealed that Gfra3-deficiency results in impairment of Peyer's patch development, suggesting that the signalling axis RET/GFRalpha3/ARTN is involved in this process. To support this hypothesis, we show that the RET ligand ARTN is a strong attractant of gut haematopoietic cells, inducing the formation of ectopic Peyer's patch-like structures. Our work strongly suggests that the RET signalling pathway, by regulating the development of both the nervous and lymphoid system in the gut, has a key role in the molecular mechanisms that orchestrate intestine organogenesis.

TNF receptor-associated factor 2-dependent canonical pathway is crucial for the development of Peyer's patches.

Activation of the noncanonical pathway through the interaction of lymphotoxin (LT)-alpha(1)beta(2) and LT-betaR is essential for the development of secondary lymphoid organs including lymph nodes (LN) and Peyer's patches (PP). Although TNFR-associated factor (TRAF) 2 and TRAF5 were identified as signal transducers for the LT-betaR, roles for TRAF2 and TRAF5 in the development of secondary lymphoid organs remain obscure. In this study, we show that PP but not mesenteric LN development is severely impaired in traf2(-/-) and traf2(-/-)traf5(-/-) mice. Development of VCAM-1(+) and ICAM-1(+) mesenchymal cells and expression of CXCL13, a crucial chemokine for the development of PP, are severely impaired in PP anlagen in the intestines of traf2(-/-) mice. Surprisingly, TNF-alpha stimulation potently up-regulates cxcl13 mRNA expression in wild-type murine embryonic fibroblasts, which is impaired in traf2(-/-) and relA(-/-) murine embryonic fibroblasts. Moreover, RelA is recruited to the promoter of cxcl13 gene upon TNF-alpha stimulation and PP development is impaired in TNFR type 1 (tnfr1)(-/-) mice. These results underscore a crucial role for the TNFR1-TRAF2-RelA-dependent canonical pathway in the development of PP through up-regulation of cxcl13 mRNA.

Cutting edge: Uniqueness of lymphoid chemokine requirement for the initiation and maturation of nasopharynx-associated lymphoid tissue organogenesis.

CD3(-)CD4(+)CD45(+) inducer cells are required for the initiation of mucosa-associated organogenesis of both nasopharynx-associated lymphoid tissues (NALT) and Peyer's patches (PP) in the aerodigestive tract. CXCL13(-/-) mice and mice carrying the paucity of lymph node T cell (plt) mutation and lacking expression of CCL19 and CCL21 accumulate CD3(-)CD4(+)CD45(+) cells at the site of NALT but not of PP genesis. Although NALT was observed to develop in adult CXCL13(-/-) and plt/plt mice, the formation of germinal centers in CXCL13(-/-) mice was affected, and their population of B cells was much lower than in the NALT of CXCL13(+/-) mice. Similarly, fewer T cells were observed in the NALT of plt/plt mice than in control mice. These findings indicate that the initiation of NALT organogenesis is independent of CXCL13, CCL19, and CCL21. However, the expression of these lymphoid chemokines is essential for the maturation of NALT microarchitecture.

Molecular networks orchestrating GALT development.

During evolution, the development of secondary lymphoid organs has evolved as a strategy to promote adaptive immune responses at sites of antigen sequestration. Mesenteric lymph nodes (LNs) and Peyer's patches (PPs) are localized in proximity to mucosal surfaces, and their development is coordinated by a series of temporally and spatially regulated molecular events involving the collaboration between hematopoietic, mesenchymal, and, for PPs, epithelial cells. Transcriptional control of cellular differentiation, production of cytokines as well as adhesion molecules are mandatory for organogenesis, recruitment of mature leukocytes, and lymphoid tissue organization. Similar to fetal and neonatal organogenesis, lymphoid tissue neoformation can occur in adult individuals at sites of chronic stimulation via cytokines and TNF-family member molecules. These molecules represent new therapeutic targets to manipulate the microenvironment during autoimmune diseases.

Mammalian Polycomb complexes are required for Peyer's patch development by regulating lymphoid cell proliferation.

The vertebrate Polycomb Group (PcG) genes encode proteins that form large multimeric and chromatin-associated complexes implicated in the stable repression of developmentally essential genes. Rnf110 and Phc2 are shown to be components of mammalian PcG multimeric complexes in HeLa cells. Here we report defects in Peyer's patch (PP) development in Rnf110 mutant mice, which is synergically exaggerated by Phc2 mutation. PP development involves a series of inductive interactions and subsequent differentiation and proliferation between lymphoid and mesenchymal cells in late gestational stage. Rnf110 and Phc2 mutations impair development of PP anlagen by affecting proliferation of lymphoid lineage cells populated in PP anlagen in gene-dosage dependent manner. We suggest that PcG complexes may act to mediate certain inductive signals maybe through IL-7Ralpha to allow sufficient proliferation of lymphoid inducer cells during PP organogenesis.

Inducible lymphoid tissues in the adult gut: recapitulation of a fetal developmental pathway?

The intestinal immune system faces an extraordinary challenge from the large numbers of commensal bacteria and potential pathogens that are restrained by only a single layer of epithelial cells. Here, I discuss evidence that the intestinal immune system develops an extensive network of inducible, reversible lymphoid tissues that contributes to the vital equilibrium between the gut and the bacterial flora. I propose that this network is induced by cryptopatches, which are small clusters of dendritic cells and lymphoid cells that are identical to fetal inducers of lymph-node and Peyer's-patch development.