Elucidation of the Effects of a Current X-SCID Therapy on Intestinal Lymphoid Organogenesis Using an In Vivo Animal Model.

BACKGROUND & AIMS: Organ-level research using an animal model lacking Il2rg, the gene responsible for X-linked severe combined immunodeficiency (X-SCID), is clinically unavailable and would be a powerful tool to gain deeper insights into the symptoms of patients with X-SCID. METHODS: We used an X-SCID animal model, which was first established in our group by the deletion of Il2rg gene in pigs, to understand the clinical signs from multiple perspectives based on pathology, immunology, microbiology, and nutrition. We also treated the X-SCID pigs with bone marrow transplantation (BMT) for mimicking a current therapeutic treatment for patients with X-SCID and investigated the effect at the organ-level. Moreover, the results were confirmed using serum and fecal samples collected from patients with X-SCID. RESULTS: We demonstrated that X-SCID pigs completely lacked Peyer's patches (PPs) and IgA production in the small intestine, but possessed some dysfunctional intestinal T and B cells. Another novel discovery was that X-SCID pigs developed a heterogeneous intestinal microflora and possessed abnormal plasma metabolites, indicating that X-SCID could be an immune disorder that affects various in vivo functions. Importantly, the organogenesis of PPs in X-SCID pigs was not promoted by BMT. Although a few isolated lymphoid follicles developed in the small intestine of BMT-treated X-SCID pigs, there was no evidence that they contributed to IgA production and microflora formation. Consistently, most patients with X-SCID who received BMT possessed abnormal intestinal immune and microbial environments regardless of the presence of sufficient serum IgG. CONCLUSIONS: These results indicate that the current BMT therapies for patients with X-SCID may be insufficient to induce the organogenesis of intestinal lymphoid tissues that are associated with numerous functions in vivo.

4,4'-Diaponeurosporene-Producing Bacillus subtilis Promotes the Development of the Mucosal Immune System of the Piglet Gut.

Gut mucosal immune responses are known to act as the first line of defense against invasion of pathogenic microorganisms. Piglets have an incompletely developed gut mucosal immune system, making them sensitive to intestinal infections. Promoting the development of the mucosal immune system will increase the pathogen resistance of piglets. The aim of the present study was to investigate the effect of carotenoid (4,4'-diaponeurosporene)-producing Bacillus subtilis (B.s-Dia) on intestinal mucosal immunity in piglets. We showed that oral administration to piglets of B.s-Dia remarkably improved the development of Peyer's patches (PPs) (P < 0.01), and increased villus height (P < 0.01) and colon crypt depth (P < 0.01). In addition, B.s-Dia also increased the number of intraepithelial lymphocytes (P < 0.01), while Bacillus subtilis (B.s) had no significant influence on it (P > 0.05). Moreover, B.s-Dia also increased the number of SIgA+ cells (P < 0.01). Oral administration of either B.s or B.s-Dia increased the number of CD4+ and CD8+ cells in ileum lamina propria (P < 0.01). These results indicate that B.s-Dia contributes to a higher extent to porcine mucosal immune system development than B.s, and might serve as an immunopotentiator candidate. Anat Rec, 302:1800-1807, 2019. © 2019 American Association for Anatomy.

The porcine tonsils and Peyer's patches: A stereological morphometric analysis in conventionally and artificially reared piglets.

Selection for prolificacy in modern pig farming has resulted in increasing litter sizes. Since rearing large litters is challenging, artificial rearing of piglets with a milk replacer is an alternative strategy. It is hypothesized that the development of the piglets' mucosa-associated lymphoid tissues (MALT) is affected by these artificial conditions. Therefore, the stereologically estimated volumes of the tonsil of the soft palate, and the lingual, nasopharyngeal and paraepiglottic tonsils, as well as the jejunal and ileal Peyer's patches were statistically compared at day 21 postpartum between six conventionally reared piglets and six piglets that were artificially reared from day 7 onwards. In addition, six 7-day-old sow-fed piglets were examined to evaluate the effect of age. All tonsils and Peyer's patches significantly increased in volume with age. The rearing strategy had no significant effect on the volumes of the tonsil of the soft palate and the lingual tonsil. The former tonsil was by far the largest with a mean volume of 967.2 ± 122.4 mm3 and 822.3 ± 125.4 mm3 in the conventionally and artificially reared piglets, respectively. The lingual tonsil only measured 9.4 ± 6.4 mm3 and 6.3 ± 2.6 mm3 in conventionally and artificially reared groups, respectively. In contrast, the rearing strategy did affect the volumes of the nasopharyngeal and paraepiglottic tonsils, which had a mean volume of 137.1 ± 32.4 mm3 and 84.4 ± 26.9 mm3, and 30.7 ± 7.8 mm3 and 20.0 ± 3.9 mm3 in conventionally and artificially reared piglets, respectively. The rearing strategy did not affect the development of the Peyer's patches. At day 21, the jejunal Peyer's patches of the conventionally and artificially reared piglets presented a volume of 1.6 ± 0.4 cm3 and 1.3 ± 0.2 cm3, respectively. The volumes of the ileal Peyer's patch amounted to 15.1 ± 3.0 cm³ in conventionally reared piglets and 12.0 ± 2.6 cm³ in artificially reared piglets at day 21. The results showed that artificial rearing hampers the morphological development of the tonsils that are exposed to inhaled antigens, but the voluminous lymphoid tissues that sample oral antigens are not influenced. Since it is unlikely that the observed differences in both tonsils are due to the milk replacer, artificial rearing could be a valuable alternative for raising large litters. In addition, the presence of developing MALT in piglets allows for investigating the value of nasal and oral vaccination in this species for human or veterinary purposes.

Mechanism of M-cell differentiation accelerated by proliferation of indigenous bacteria in rat Peyer's patches.

The mechanism by which indigenous bacteria on the follicle-associated epithelium (FAE) of lymphatic follicles (LFs) accelerate the differentiation of microvillous columnar epithelial cells (MV) into M-cells was immunohistochemically investigated in rat Peyer's patches. The results showed that the number of Toll-like receptor (TLR) -4+ M-cells was greater in the FAE with expansion of bacterial colonies (LFs with bacterial colonies on the FAE: b-LF) than the FAE without expansion of bacterial colonies (nb-LF). TLR-4 was also expressed in the striated borders of MV upstream next to M-cells in the FAE of the b-LF. TLR-4+ vesicles were frequently detected in the cytoplasms of MV with TLR-4+ striated borders upstream next to TLR-4+ M-cells in the FAE of b-LF. These findings suggest that TLR-4+ MV take up TLR-4 ligands and differentiate into M-cells in the b-LF. Neither the distribution of RANK nor that of RANKL was coincident with that of M-cells in the b-LF. Moreover, RANK, but not RANKL, was expressed in intestinal villi, whereas cleaved caspase-3 was immunonegative in the MV and M-cells of the FAE, unlike in villous epithelial cells. Therefore, RANK/RANKL signaling in the LF might contribute to the down-regulation of epithelial apoptosis to facilitate the differentiation of MV into M-cells in rat Peyer's patches.

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.

Influence of an Enterococcus faecium probiotic on the development of Peyer's patches B cells in piglets.

To study the interactions between a probiotic bacterium and the host's immune system, we undertook a feeding trial with Enterococcus faecium SF68 (NCIMB 10415). Starting at an age of 1 day, piglets received a daily oral dose of the probiotic bacteria. Immune cells were isolated from the blood and the distal continuous Peyer's patch (PP) of the piglets. While the percentage of B cells in the distal continuous PP was not influenced by the probiotic treatment, an elevated expression of CD1 on Peyer's patch B cells was observed after probiotic treatment. Furthermore, the fraction of CD4-CD8+ cells was decreased in this organ. In blood lymphocyte fractions of the probiotic-treated piglets, the proportion of CD16-positive cells was also diminished, whereas the portion of γδ T cells and CD4-positive T cells increased. The data indicate that early administration of the probiotic Enterococcus faecium can modulate the composition of blood lymphocyte populations in piglets.

Unique microanatomy of ileal peyer's patches of the one humped camel (Camelus dromedarius) is not age-dependent.

The Peyer's patches (PP) have been intensely investigated in several species because this is an important entry site for antigens and infectious agents. There are many PP in the jejunum, and in some species such as ruminants, carnivores, and omnivores, a different continuous PP is found in the terminal ileum. This PP disappears with age in these species studied. So far the ileal PP (IPP) has only been examined in the camel by light microscopy. Therefore, the localization of ileal Peyer's patches in the dromedary camel at different ages, as well as the histology and ultrastructures were now investigated. The IPP were characteristically seen as dark rose-colored isolated structures in the shape of a cup, arranged in three irregular rows. The central row was antimesenteric. Each patch was formed by several mainly elongated dome regions flanked by intestinal villi. In cross-sections these domes appeared as short, wide villi. The domes were formed from lymphoid follicles covered with a typical dome-associated epithelium of enterocytes and M cells without any goblet cells. The M cells showed variable appearance depending on the functional status. The lymphoid follicles expressed clear germinal centers. High endothelial venules were localized in the interfollicular region. In contrast to other species the IPP were still present with a comparable macroscopic and histological structure in camels of 25 years of age.

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.

Effects of diets supplemented with organic acids and nucleotides on growth, immune responses and digestive tract development in weaned pigs.

Sixty-eight (Experiment 1, 46 days feeding) and sixteen (Experiment 2, 21 days feeding) 21-days-old weaned pigs were allotted to four dietary treatments including control, 0.6% organic acids (OA), 0.1% nucleotides (NA) and 0.6% OA plus 0.1% NA for determining the dietary effects. In Experiment 1, OA enhanced peripheral blood mononuclear cells proliferation on day 28 and 46. The plasma immunoglobulin (Ig) A level was elevated by OA (p < 0.06) and NA (p < 0.07), respectively. In Experiment 2, NA increased plasma IgM level, and had an interactive effect with OA on ileal Peyer's patches and mesenteric lymph node lymphocyte proliferation, bile and plasma IgA levels, and jejunal crypt depth. NA elevated gastric pepsin and jejunal alkaline phosphatase activities, however, decreased ileal aminopeptidase N, sucrase or maltase activity. These results suggest that OA and NA have synergistically enhanced the gut-associated lymphocyte responses and NA modulates the digestive tract development of weaned pigs.

Diet regulates the development of gut-associated lymphoid tissue in neonatal piglets.

BACKGROUND: Controversy exists concerning diet-induced changes to gut epithelia and immunocytes that occur during weaning. Furthermore, studies on dietary effects on the development of the neonatal immune system, especially gut-associated lymphoid tissue (GALT), are lacking. OBJECTIVE: The purpose of this study was to investigate growth and development, intestinal morphology, and GALT immune maturation in sow-reared littermates in comparison with early-weaned piglets fed a casein-based liquid diet. METHOD: Piglets were breast fed by the sow or were weaned at 48 h to a casein-based diet (formula) that provided the amount of nutrient requirements recommended by the National Research Council. RESULTS: Gross physical appearance and visual inspection of the gastrointestinal tract and other organs at necropsy revealed normal organogenesis in both cohorts. On postnatal day 21, body weight, liver and kidney weight relative to body weight, small intestine length, and weight-to-length ratio were greater in formula-fed piglets as compared with sow-reared piglets (p<0.05). The CD21+ B lymphocyte component of GALT and spleen was reduced in the formula-fed piglets. This was associated with lower circulating IgG and IgM levels in the formula-fed as compared with the breast-fed neonatal piglets (p<0.001). CONCLUSIONS: Feeding a casein-based formula to newborn piglets may compromise the development of GALT and systemic immune system. Further, the neonatal piglet model may be used to identify the effects of dietary factors on the development of the neonatal immune system.

Lymphotoxin-independent expression of TNF-related activation-induced cytokine by stromal cells in cryptopatches, isolated lymphoid follicles, and Peyer's patches.

Stromal cells play a crucial role in the organogenesis of lymphoid tissues. We previously identified VCAM-1(+) stromal cells in cryptopatches (CP) and isolated lymphoid follicles (ILF) in the small intestine of C57BL/6 mice. Nonhemopoietic stromal cell networks in CP and ILF of adult mice also expressed FDC-M1, CD157 (BP-3), and TNF-related activation-induced cytokine (TRANCE). Individual stromal cells were heterogeneous in their expression of these markers, with not all stromal cells expressing the entire set of stromal cell markers. Expression of VCAM-1, FDC-M1, and CD157 on CP stromal cells was absent in alymphoplasia mice deficient in NF-kappaB-inducing kinase (NIK) and NIK knockout mice. Administration of lymphotoxin beta receptor (LTbetaR)-Ig to wild-type mice on day 13 resulted in the absence of CP on day 20; delaying administration of LTbetaR-Ig until day 18 resulted in an 80% decrease in the number of CP on day 22 and diminished expression of VCAM-1, FDC-M1, and CD157 on the remaining CP. In sharp contrast, TRANCE expression by stromal cells was completely independent of NIK and LTbetaR. In addition, expression of TRANCE in ILF was concentrated just beneath the follicle-associated epithelium, a pattern of polarization that was also observed in Peyer's patches. These findings suggest that TRANCE on stromal cells contributes to the differentiation and maintenance of organized lymphoid aggregates in the small intestine.

Neuroregulator RET initiates Peyer's-patch tissue genesis.

The tyrosine kinase receptor RET regulates the intestinal nervous system. A recent paper by Veiga-Fernandes et al. (2007) demonstrates that RET is also involved in the intestinal immune system through the initiation of Peyer's-patch tissue genesis.

CC chemokine receptor 6 expression by B lymphocytes is essential for the development of isolated lymphoid follicles.

Isolated lymphoid follicles (ILFs) are organized lymphoid structures that facilitate the efficient interaction of antigen, antigen-presenting cells, and lymphocytes to generate controlled adaptive immune responses within the intestine. Because CC chemokine receptor 6 (CCR6) deficiency affects the generation of mucosal immune responses, we evaluated a potential role for CCR6 in the development of ILFs. We observed that CCR6 and its ligand CCL20 are highly expressed within ILFs and that B lymphocytes are the largest CCR6-expressing population within ILFs. ILF development was profoundly arrested in the absence of CCR6. Concordant with a block in ILF development at a stage corresponding to the influx of B lymphocytes, we observed that CCR6-deficient mice had a diminished population of intestinal B lymphocytes. Bone marrow reconstitution studies demonstrated that ILF development is dependent on CCR6-sufficient B lymphocytes, and adoptive transfers demonstrated that CCR6(-/-) B lymphocytes were inefficient at localizing to intestinal lymphoid structures. Paralleling these findings, we observed that CCR6-deficient mice had a reduced proportion of Peyer's patch B lymphocytes and an associated re-duction in the number and size of Peyer's patch follicular domes. These observations define an essential role for CCR6 expression by B lymphocytes in localizing to intestinal lymphoid structures and in ILF development.

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.

PanR1, a dominant negative missense allele of the gene encoding TNF-alpha (Tnf), does not impair lymphoid development.

A dominant hypomorphic allele of Tnf, PanR1, was identified in a population of G(1) mice born to N-ethyl-N-nitrosourea-mutagenized sires. Macrophages from homozygotes produced no detectable TNF bioactivity, although normal quantities of immunoreactive TNF were secreted. The phenotype was confined to a critical region on mouse chromosome 17, and then ascribed to a C-->A transversion at position 3480 of the Tnf gene, corresponding to the amino acid substitution P138T. As a result of subunit exchange, the protein exerts a dominant-negative effect on normal TNF trimers, interfering with the trimer/receptor interaction. Homozygotes are highly susceptible to infection by Listeria monocytogenes, confirming the essential role of TNF in innate immune defense. However, PanR1 mutant mice show normal architecture of the spleen and Peyer's patches, suggesting that TNF is not essential for the formation of these lymphoid structures.

The sheep and cattle Peyer's patch as a site of B-cell development.

In sheep and cattle, the ileal Peyer's patch (PP), which extends one-two meters along the terminal small intestine, is a primary lymphoid organ of B-cell development. B-cell diversity in the ileal PP is thought to develop by combinatorial mechanisms, gene conversion and/or point mutation. These species also have jejunal PP that function more like secondary lymphoid tissues concerned with mucosal immune reactions. These two types of PP differ significantly in their histology, ontogeny and the extent of lymphocyte traffic. The prenatal development of follicles in the PP begins first in the jejunum during the middle of gestation and then in the ileum during late gestation. B-cells proliferate rapidly in the ileal PP follicle; up to five percent of these cells survive while the majority dies by apoptosis, perhaps driven by the influence of environmental antigen and/or self-antigen. The surviving cells migrate from the ileal PP and populate the peripheral B-cell compartment. By adolescence, the ileal PP has involuted but the function of jejunal PP, compatible with a role as secondary lymphoid organ, continues throughout life. In this review, we focus on the development of PP as a site of B-cell repertoire generation, positive and negative B-cell selection, and the differences between ileal PP and jejunal PP.