CD161 contributes to prenatal immune suppression of IFNγ-producing PLZF+ T cells.

BACKGROUND: While the human fetal immune system defaults to a program of tolerance, there is concurrent need for protective immunity to meet the antigenic challenges encountered after birth. Activation of T cells in utero is associated with the fetal inflammatory response with broad implications for the health of the fetus and of the pregnancy. However, the characteristics of the fetal effector T cells that contribute to this process are largely unknown. METHODS: We analyzed primary human fetal lymphoid and mucosal tissues and performed phenotypic, functional, and transcriptional analysis to identify T cells with pro-inflammatory potential. The frequency and function of fetal-specific effector T cells was assessed in the cord blood of infants with localized and systemic inflammatory pathologies and compared to healthy term controls. RESULTS: We identified a transcriptionally distinct population of CD4+ T cells characterized by expression of the transcription factor Promyelocytic Leukemia Zinc Finger (PLZF). PLZF+ CD4+ T cells were specifically enriched in the fetal intestine, possessed an effector memory phenotype, and rapidly produced pro-inflammatory cytokines. Engagement of the C-type lectin CD161 on these cells inhibited TCR-dependent production of IFNγ in a fetal-specific manner. IFNγ-producing PLZF+ CD4+ T cells were enriched in the cord blood of infants with gastroschisis, a natural model of chronic inflammation originating from the intestine, as well as in preterm birth, suggesting these cells contribute to fetal systemic immune activation. CONCLUSION: Our work reveals a fetal-specific program of protective immunity whose dysregulation is associated with fetal and neonatal inflammatory pathologies.

Comparative analysis of the merino sheep and Iberian red deer abomasum during prenatal development.

The aim of this study is to describe differences in the ontogenesis of the abomasum in sheep (domestic ruminant) and deer (wild ruminant). Histomorphometric and immunohistochemical analysis were carried out on 50 embryos and fetuses of the sheep and 50 red deer from the first prenatal stages until birth. To compare similar periods of gestation in both species, we calculate the percentages of gestation. The appearance of the abomasum was earlier in the red deer (22% gestation) than in the sheep (25% gestation). Throughout development the epithelium happened sequentially, being of the types pseudostratified to simple cylindrical. This important modification was earlier in the red deer than the sheep. At 46% gestation in red deer and 50% in sheep, gastric pits were observed on the surface of abomasal folds. Our studies suggest a close link between the initial formation of these pseudoglandular structures and the clear separation of lamina propria and submucosa separated by de muscularis mucosae. At 54% gestation in red deer and at 60% in sheep, in the bottom of these pits the first outlines of glands were distinguishable. Finally, the presence of neuroendocrine and glial cells were detected in deer at earlier stages than in sheep.

Gmnc Is a Master Regulator of the Multiciliated Cell Differentiation Program.

Multiciliated cells (MCCs) differentiate hundreds of motile cilia that generate mechanical force required to drive fluid movement over epithelia [1, 2]. For example, metachronal beating of MCC cilia in the mammalian airways clears mucus that traps inhaled pathogens and pollutants. Consequently, abnormalities in MCC differentiation or ciliary motility have been linked to an expanding spectrum of human airway diseases [3­6]. The current view posits that MCC precursors are singled out by the inhibition of Notch signaling. MCC precursors then support an explosive production of basal bodies, which migrate to the apical surface, dock with the plasma membrane, and seed the growth of multiple motile cilia. At the center of this elaborate differentiation program resides the coiled-coil-containing protein Multicilin, which transcriptionally activates genes for basal body production and the gene for FoxJ1, the master regulator for basal body docking, cilia formation, and motility [7, 8]. Here, using genetic analysis in the zebrafish embryo, we discovered that Gmnc is a novel determinant of the MCC fate. Like Multicilin, Gmnc is a coiled-coil-containing protein of the Geminin family. We show that Gmnc functions downstream of Notch signaling, but upstream of Multicilin in the developmental pathway controlling MCC specification. Moreover, we find that loss of Gmnc in Xenopus embryos also causes loss of MCC differentiation and that overexpression of the protein is sufficient to induce supernumerary MCCs. Together, our data identify Gmnc as an evolutionarily conserved master regulator functioning at the top of the hierarchy of transcription factors involved in MCC differentiation.

TMPRSS13 deficiency impairs stratum corneum formation and epidermal barrier acquisition.

Membrane-anchored serine proteases serve as important regulators of multiple developmental and homoeostatic processes in mammals. TMPRSS13 (transmembrane protease, serine 13; also known as mosaic serine protease large-form, MSPL) is a membrane-anchored serine protease with unknown biological functions. In the present study, we used mice with the Tmprss13 gene disrupted by a ß-galactosidase-neomycin fusion gene insertion to study the expression and function of the membrane-anchored serine protease. High levels of Tmprss13 expression were found in the epithelia of the oral cavity, upper digestive tract and skin. Compatible with this expression pattern, Tmprss13-deficient mice displayed abnormal skin development, leading to a compromised barrier function, as measured by the transepidermal fluid loss rate of newborn mice. The present study provides the first biological function for the transmembrane serine protease TMPRSS13.

The development and function of mucosal lymphoid tissues: a balancing act with micro-organisms.

Mucosal surfaces are constantly exposed to environmental antigens, colonized by commensal organisms and used by pathogens as points of entry. As a result, the immune system has devoted the bulk of its resources to mucosal sites to maintain symbiosis with commensal organisms, prevent pathogen entry, and avoid unnecessary inflammatory responses to innocuous antigens. These functions are facilitated by a variety of mucosal lymphoid organs that develop during embryogenesis in the absence of microbial stimulation as well as ectopic lymphoid tissues that develop in adults following microbial exposure or inflammation. Each of these lymphoid organs samples antigens from different mucosal sites and contributes to immune homeostasis, commensal containment, and immunity to pathogens. Here we discuss the mechanisms, mostly based on mouse studies, that control the development of mucosal lymphoid organs and how the various lymphoid tissues cooperate to maintain the integrity of the mucosal barrier.

Analysis of the expression of p53 during the morphogenesis of the gastroesophageal mucosa of Gallus gallus domesticus (Linnaeus, 1758).

Ontogenesis comprises a series of events including cell proliferation and apoptosis and resulting in the normal development of the embryo. Protein p53 has been described as being involved in the development of several animal species. The aim of this study was to analyze the expression of protein p53 during the morphogenesis of the gastroesophageal mucosa of Gallus gallus domesticus and to correlate it with the histogenesis of structures present in this tissue. We used 24 embryos (at 12-20 days of incubation) and the thymus of two chickens. Immunohistochemical analysis was performed with the ABC indirect method. The expression of p53 in the gastroesophageal mucosa increased during the formation of the organ, mainly at the stages during which tissue remodeling and cell differentiation began. In the esophagus at stages 42 and 45, we observed immunoreactive (IR) cells in the surface epithelium and in early esophageal glands. In the proventriculus at stages 39-45, IR cells were present in the epithelial mucosa and rarely in the proventricular glands. In the gizzard after stage 42, we found IR cells mainly in the medial and basal epithelial layers of the mucosa and especially within the intercellular spaces that appeared at this phase and formed the tubular gland ducts. Thus, protein p53 occurs at key stages of development: in the esophagus during the remodeling of esophageal glands, in the proventriculus during the differentiation of the epithelium of the mucosa and in the gizzard during the formation of tubular glands.

[Morphological features of the esophagogastric transition zone at fetuses and newborns].

Morphological research of the esophagogastric transition mucosa at 35 fetuses and newborns was done. The esophagogastric transition was lined by high columnar epithelium and mucos glands. At fetuses of 22-24 week gestational age studied zone didn't have any glands. Histochemical features of the epithelium, particularly MUC5AC positive staining, corresponded to cardial type of the Barrett esophagus, defined at adults. We have revealed that mucosa of the esophagogastric transition has gastric origin and arises before birth. We found out the islets of columnar epithelium on the surface of the laminated pavement epithelium, indicated about its uneven development up to the birth. The sites of immature epithelium could be considered as transformation zones both of laminated pavement epithelium or columnar one.

Morphology and physiological changes of genital skin and mucosa.

The morphology and physiology of both the vulva and vagina undergo characteristic age-related changes over a lifetime. At birth, these tissues exhibit the effects of residual maternal estrogens. During puberty, the vulva and vagina mature under the influence of adrenal and gonadal steroid hormones. During the reproductive years, the vagina responds to ovarian steroid hormone cycling, and both tissues adapt to the needs of pregnancy and delivery. Following menopause, the vulva and vagina atrophy. A rise in the prevalence of incontinence among older women increases the risk of vulvar and perineal dermatitis. This chapter covers the morphology and physiology of the genital area from infancy to old age.

Tgm2/Gh, Gbx1 and TGF-beta are involved in retinoic acid-induced transdifferentiation from epidermis to mucosal epithelium.

We previously demonstrated that retinoic acid (RA) induces epidermis to transdifferentiate to mucosal epithelium with goblet cells in chick embryonic cultured skin. To characterize the molecular mechanism of this transdifferentiation process, we used rat embryonic cultured skin and immunohistochemistry to confirm that RA-induced epidermal transdifferentiation accompanies the expression of markers of esophagus epithelium. Because Gbx1, TG2/Gh (transglutaminase2) and TGF-beta2 are reported individually to be induced by RA in cultures of chick embryonic skin, mouse epidermal cells and human hair follicles respectively, here, we investigated whether cooperative interplay of Gbx1, TG2/Gh and TGF-beta2 is required for the transdifferentiation of epidermal cells to mucosal cells. We have shown that expression of Gbx1, TG2/Gh and TGF-beta proteins were all upregulated in RA-induced transdifferentiated skin and that the former two were expressed in the epidermis, while TGF-beta was expressed in the dermis. Inhibitors of the TGF-beta signal pathway partially inhibited transdifferentiation. Overexpression of both hTG2/Gh and mGbx1 together in the epidermis by electroporation resulted in cuboidal cells in the upper cell layers of the epidermis without keratinized layers, although epidermal keratinization was observed in skin by overexpression of either of them. Labeling DNA with BrdU indicated that RA directly transdifferentiated transient amplifying epidermal cells, not stem cells, to mucosal cells. This study showed that coexpression of TG/2 and Gbx1 in the epidermis was required for esophagus-like mucosal transdifferentiation, and that increase in TGF-beta2 expression by RA in the dermis was essential to induce transdifferentiation through epithelial-mesenchymal interaction.

The presence of a vocal ligament in fetuses: a histochemical and ultrastructural study.

Although it is currently believed that the vocal ligament of humans undergoes considerable development postnatally, there is no consensus as to the age at which it first emerges. In the newborn infant, the lamina propria has been described as containing a sparse collection of relatively unorganized fibres. In this study we obtained larynges from autopsy of human fetuses aged 7-9 months and used light and electron microscopy to study the collagenous and elastic system fibres in the lamina propria of the vocal fold. Collagen fibres were viewed using the Picrosirius polarization method and elastic system fibres were stained using Weigert's resorcin-fuchsin after oxidation with oxone. The histochemical and electron microscopic observations were consistent, showing collagen populations with an asymmetric distribution across different compartments of the lamina propria. In the central region, the collagen appeared as thin, weakly birefringent, greenish fibres when viewed using the Picrosirius polarization method, whereas the superficial and deep regions contained thick collagen fibres that displayed a strong red or yellow birefringence. These findings suggest that the thin fibres in the central region consist mainly of type III collagen, whereas type I collagen predominates in the superficial and deep regions, as has been reported in studies of adult vocal folds. Similarly, elastic system fibres showed a differential distribution throughout the lamina propria. Their distribution pattern was complementary to that of collagen fibres, with a much greater density of elastic fibres apparent in the central region than in the superficial and deep regions. This distribution of collagen and elastic fibres in the fetal vocal fold mirrors that classically described for the adult vocal ligament, suggesting that a vocal ligament has already begun to develop by the time of birth. The apparently high level of organization of connective tissue components in the newborn is in contrast to current hypotheses that argue that the mechanical stimuli of phonation are essential to the determination of the layered structure of the lamina propria and suggests that genetic factors may play a more significant role in the development of the vocal ligament than previously believed.

Cell density of the lamina propria of neonatal vocal folds.

OBJECTIVES: We undertook (1) to measure the cell density within the lamina propria of the neonatal vocal folds and (2) to examine changes in cell density in the lamina propria with increasing gestational age of the neonatal vocal folds. METHODS: Intact neonatal larynges were obtained from fresh cadaveric specimens. Hematoxylin and eosin-stained slides were used to visualize the laryngeal structures, and photomicrographs of the vocal folds were taken at 100x magnification. The cell density of the lamina propria was calculated by counting the cells within each of five 100-microm2 regions within the study area, and the totals were then averaged for each area. RESULTS: A total of 62 sections from 14 larynges with gestational ages of 19 to 36 weeks were examined. Histologic analysis revealed a uniform appearance of the vocal fold without apparent layers. The cell density of the lamina propria was 30 or more cells per 100 microm2 for 51.2% of larynges with less than 27 weeks of gestation. However, only 14.3% of the larynges with 27 or more weeks of gestation had an average cell density of 30 or more cells per region (p < 0.005). CONCLUSIONS: As described by previous studies, the lamina propria of the neonatal vocal folds is a hypercellular monolayer. The process of vocal fold maturation appears to occur earlier than previously thought, with decreasing cell density in the lamina propria by 27 weeks' gestation.

[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.

Identification of novel ciliogenesis factors using a new in vivo model for mucociliary epithelial development.

Mucociliary epithelia are essential for homeostasis of many organs and consist of mucus-secreting goblet cells and ciliated cells. Here, we present the ciliated epidermis of Xenopus embryos as a facile model system for in vivo molecular studies of mucociliary epithelial development. Using an in situ hybridization-based approach, we identified numerous genes expressed differentially in mucus-secreting cells or in ciliated cells. Focusing on genes expressed in ciliated cells, we have identified new candidate ciliogenesis factors, including several not present in the current ciliome. We find that TTC25-GFP is localized to the base of cilia and to ciliary axonemes, and disruption of TTC25 function disrupts ciliogenesis. Mig12-GFP localizes very strongly to the base of cilia and confocal imaging of this construct allows for simple visualization of the planar polarity of basal bodies that underlies polarized ciliary beating. Knockdown of Mig12 disrupts ciliogenesis. Finally, we show that ciliogenesis factors identified in the Xenopus epidermis are required in the midline to facilitate neural tube closure. These results provide further evidence of a requirement for cilia in neural tube morphogenesis and suggest that genes identified in the Xenopus epidermis play broad roles in ciliogenesis. The suites of genes identified here will provide a foundation for future studies, and may also contribute to our understanding of pathological changes in mucociliary epithelia that accompany diseases such as asthma.

Ultrastructural dynamics of the human endometrium from 14 to 22 weeks of gestation.

In order to elucidate the ultrastructural dynamics of endometrium differentiation, uterine samples of fetuses aged 14 to 22 weeks of gestation (WG) were analyzed. Samples were processed for light (LM), transmission (TEM) and field-emission scanning electron microscopy (FE-SEM). Initial stratification of the uterine wall occurred at 14 WG: endometrial, myometrial, and perimetrial primordia were identified. At this age, the endometrial epithelium was simple columnar to pseudostratified and consisted of microvillous cells. Blood capillaries developed mainly in the stroma and between the myometrium and perimetrium primordia. At 18-20 WG the endometrial epithelium became clearly pseudostratified, with active ciliogenesis and a predominance of microvillous cells. Primordia of tubular glands were present at 20 WG. Microvillous cells still predominated in the endometrial epithelium at 21-22 WG and showed morphological features of apoptosis. The endometrial stroma at this stage was organizing into a thick lamina propria provided with subepithelial capillary plexuses. However, the stroma was formed by still undifferentiated mesenchymal cells during the whole period of study. Our data showed that the epithelial differentiation and distribution in the uterus occur in the human fetus in a similar way as in the adult. The above events are likely the expression of an early developmental patterning and related to future reproductive processes, such as the regulation of gamete passage and blastocyst implantation. Because the structure of the adult uterus is determined by the degree of paramesonephric duct fusion, septum absorption, and differentiation of the uterine primordial layers, our study may contribute toward clarifying to normal urogenital development.

Expression of mucosal chemokines TECK/CCL25 and MEC/CCL28 during fetal development of the ovine mucosal immune system.

CCL25/TECK and CCL28/MEC are CC chemokines primarily expressed in thymic dendritic cells and mucosal epithelial cells. The cognate receptors of CCL25 and CCL28, CCR9 and CCR10, respectively, are mainly expressed on T and B lymphocytes. In human, mouse and pig, CCL25 and CCL28 play a key role in the segregation and the compartmentalization of the mucosal immune system through recruitment of immune cells to specific locations. However, little is known about their role in the ontogeny of the mucosal immune system during fetal development. In the present paper, we report the cloning and the sequencing of ovine CCL25, CCL28, CCR9 and CCR10 and the subsequent assessment of their mRNA expression by q-polymerase chain reaction in several tissues, including thymus, gut-associated lymphoid tissue and mammary gland, from young and adult sheep and in the fetal lamb during the development of the immune system. CCL25 mRNA was highly expressed in thymus and gut while CCL28 mRNA was more expressed in large intestine, trachea, tonsils and mammary gland, especially at the end of gestation. These results are consistent with observations in other species suggesting similar roles for these chemokines in sheep. In fetuses, mRNA of CCL25, CCL28 and their receptors are expressed early in the thymus and mucosal tissues, including the small intestine and the nasal mucosa. Furthermore, their expression increased towards the end of gestation. Consequently, we hypothesize that CCL25 and CCL28 play an important role in the lymphocyte colonization of fetal tissues, enabling the development of a functional immune system.

The histological findings in the gastroesophageal junction of fetuses.

The development of the esophageal and gastric mucosa in the gastroesophageal junction was studied in 61 fetuses of 13-41 weeks of the gestational age. During the 13th-15th week, the esophageal multilayered epithelium was covered by a continuous layer of columnar mucous ciliated cells which were present only focally till the 25th week and disappeared later. Before the 15th week, the gastric mucosa was formed by pits only. The glands started as proliferating tubules in the basal parts of the pits in the 15th week. Further, they differentiated into oxyntic glands. The mucosa of the corpus was fully developed in the 27th week. The cardiac mucosa was absent in all the 10 fetuses examined between the 27th and 41st week of gestation. This supports the view that the gastric cardiac mucosa is not a physiological structure but that it results from glandular metaplasia of the distal esophageal mucosa due to gastroesophageal reflux.

Extracellular matrix protein expression during mouse detrusor development.

BACKGROUND/PURPOSE: Extracellular matrix proteins are implicated in regulating cell proliferation and differentiation. The authors systematically analysed the expression of elastin; collagen types I, III, IV; laminin; and fibronectin during mouse detrusor muscle development, a period during which downregulation of detrusor proliferation and increasing smooth muscle differentiation is known to occur. METHODS: Embryonic days 14 (E14) and 18 (E18), and postnatal day 1 (D1) and week 6 (6wk) were examined, a period spanning the inception of the bladder to postnatal maturity. Immunohistochemistry of whole bladders was used to immunolocalise protein expression, and Western blot of dissected detrusor layers was used to semiquantify soluble protein expression. RESULTS: All proteins were detected at all 4 stages. Statistically significant increases were documented for elastin (E14 to 6wk), collagen type I (E18 to 6wk), collagen type III (D1 to 6wk) and laminin (E14 to 6wk). Fibronectin levels were relatively high up to D1, after which levels declined significantly. Collagen type IV levels decreased significantly (E18 to 6wk). CONCLUSIONS: The authors postulate that changing levels of laminin and fibronectin have opposing effects on the transition from proliferating primitive mesenchymal cells to differentiated detrusor muscle. Furthermore, changes in collagen type III and elastin may be important for bladder compliance.

Microvascularization of the mucocutaneous junction of the eyelid in fetuses and neonates.

The aim of this study was to specify the microvascularization of the junctional region between the integuments of the superficial surface of the free margin of the eyelid and the palpebral conjunctival mucosa. The study was carried out using histological or transparified slices of upper and lower eyelids taken from fetuses and neonates, in which the vascular system was injected with agarized China ink. The mucocutaneous junction of the eyelid is located at the posterior border of the free margin and extends to its deep surface. It has vascular similarities to the oral cavity and the nasal pyramid. Under a thickened avascular epithelium, there is a papillary network composed of characteristic loops that are less raised than in the lips but more developed than in the nose. The superficial and deep vascular reticular networks are comparable in fundamental arrangement to those of other junctional zones. Thus, the palpebral mucocutaneous junction shows cutaneous-type microvascularization, just like the other junctional zones of the head.

Ex vivo development of functional human lymph node and bronchus-associated lymphoid tissue.

We introduce a novel in vivo model of human mucosal immunity, based on the implantation of human fetal bronchial mucosa and autologous peribronchial lymph node (PLN) in the severe combined immunodeficiency (SCID) mouse. In the SCID host, human fetal bronchi implanted alone retain macrophages and mast cells but lose T cells. In contrast, fetal bronchi co-implanted with PLN contain, in addition to macrophages and mast cells, numerous T cells and B cells, often clustered in intramucosal bronchus-associated lymphoid tissue (BALT). Functionally, bronchus-PLN cografts are able to mount robust alphabeta and gammadelta T-cell-mediated immune responses to Pseudomonas aeruginosa and 3,4-epoxy-3-methyl-1-butyl-diphosphate challenges. No other autologous lymphoid organ (bone marrow, thymus, liver) allows for BALT development in co-implanted bronchi, which suggests special ontogenetic and functional relations between extramucosal PLN and intramucosal BALT. Overall, the bronchus-PLN cograft appears as a promising model for human bronchial immune development and function. Our study is the first to document long-term ex vivo maintenance of functional human lymph nodes as native appendices to mucosal tissue. Our results, therefore, suggest a simple strategy for developing similar experimental models of human immune function in other mucosae.

Foxp4: a novel member of the Foxp subfamily of winged-helix genes co-expressed with Foxp1 and Foxp2 in pulmonary and gut tissues.

In this study, we describe the isolation and characterization of Foxp4, a new member of the Foxp subfamily of winged-helix transcription factors. The full-length mouse Foxp4 cDNA encodes a 685-amino-acid protein that is similar to Foxp1 and Foxp2. Foxp4 gene expression is observed primarily in pulmonary, neural, and gut tissues during embryonic development. To compare the protein expression patterns of Foxp4 to Foxp1 and Foxp2, specific polyclonal antisera to each of these proteins was used in immunohistochemical analysis of mouse embryonic tissues. All three proteins are expressed in lung epithelium with Foxp1 and Foxp4 expressed in both proximal and distal airway epithelium while Foxp2 is expressed primarily in distal epithelium. Foxp1 protein expression is also observed in the mesenchyme and vascular endothelial cells of the lung. At embryonic day 12.5, Foxp1 and Foxp2 are expressed in both the mucosal and epithelial layers of the intestine. However, Foxp2 is expressed only in the outer mucosal layer of the intestine and stomach later in development. Finally, Foxp4 is expressed exclusively in the epithelial cells of the developing intestine, where, in late development, it is expressed in a gradient along the longitudinal axis of the villi.