Mucus Properties and Goblet Cell Quantification in Mouse,Rat and Human Ileal Peyer's Patches

Peyer''s patches (PPs) are collections of lymphoid follicles in the small intestine, responsible for scanning the intestinal content for foreign antigens such as soluble molecules, particulate matter as well as intact bacteria and viruses. The immune cells of the patch are separated from the intestinal lumen by a single layer of epithelial cells, the follicle-associated epithelium (FAE). This epithelium covers the dome of the follicle and contains enterocyte-like cells and M cells, which are particularly specialized in taking up antigens from the gut. However, the presence and number of goblet cells as well as the presence of mucus on top of the FAE is controversial. When mouse ileal PPs were mounted in a horizontal Ussing-type chamber, we could observe a continuous mucus layer at mounting and new, easily removable mucus was released from the villi on the patch upon stimulation. Confocal imaging using fluorescent beads revealed a penetrable mucus layer covering the domes. Furthermore, immunostaining of FAE from mice, rats and humans with a specific antibody against the main component of intestinal mucus, the MUC2 mucin, clearly identify mucin-containing goblet cells. Transmission electron micrographs further support the identification of mucus releasing goblet cells on the domes of PPs in these species.     

Phenotypic Plasticity of Mouse Spermatogonial Stem Cells

Background

Spermatogonial stem cells (SSCs) continuously undergo self-renewal division to support spermatogenesis. SSCs are thought to have a fixed phenotype, and development of a germ cell transplantation technique facilitated their characterization and prospective isolation in a deterministic manner; however, our in vitro SSC culture experiments indicated heterogeneity of cultured cells and suggested that they might not follow deterministic fate commitment in vitro.

Methodology and Principal Findings

In this study, we report phenotypic plasticity of SSCs. Although c-kit tyrosine kinase receptor (Kit) is not expressed in SSCs in vivo, it was upregulated when SSCs were cultured on laminin in vitro. Both Kit⁻ and Kit⁺ cells in culture showed comparable levels of SSC activity after germ cell transplantation. Unlike differentiating spermatogonia that depend on Kit for survival and proliferation, Kit expressed on SSCs did not play any role in SSC self-renewal. Moreover, Kit expression on SSCs changed dynamically once proliferation began after germ cell transplantation in vivo.

Conclusions/Significance

These results indicate that SSCs can change their phenotype according to their microenvironment and stochastically express Kit. Our results also suggest that activated and non-activated SSCs show distinct phenotypes.     

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer''s patch (PP). Common dendritic cell progenitors (CDPs, lin^- c-kit^lo CD115⁺ Flt3⁺) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar^-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1⁺ and recipients being CD45.2⁺. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar^-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.     

Cells Derived from the Coelomic Epithelium Contribute to Multiple Gastrointestinal Tissues in Mouse Embryos

Gut mesodermal tissues originate from the splanchnopleural mesenchyme. However, the embryonic gastrointestinal coelomic epithelium gives rise to mesenchymal cells, whose significance and fate are little known. Our aim was to investigate the contribution of coelomic epithelium-derived cells to the intestinal development. We have used the transgenic mouse model mWt1/IRES/GFP-Cre (Wt1^cre) crossed with the Rosa26R-EYFP reporter mouse. In the gastrointestinal duct Wt1, the Wilms’ tumor suppressor gene, is specific and dynamically expressed in the coelomic epithelium. In the embryos obtained from the crossbreeding, the Wt1-expressing cell lineage produces the yellow fluorescent protein (YFP) allowing for colocalization with differentiation markers through confocal microscopy and flow cytometry. Wt1^cre-YFP cells were very abundant throughout the intestine during midgestation, declining in neonates. Wt1^cre-YFP cells were also transiently observed within the mucosa, being apparently released into the intestinal lumen. YFP was detected in cells contributing to intestinal vascularization (endothelium, pericytes and smooth muscle), visceral musculature (circular, longitudinal and submucosal) as well as in Cajal and Cajal-like interstitial cells. Wt1^cre-YFP mesenchymal cells expressed FGF9, a critical growth factor for intestinal development, as well as PDGFRα, mainly within developing villi. Thus, a cell population derived from the coelomic epithelium incorporates to the gut mesenchyme and contribute to a variety of intestinal tissues, probably playing also a signaling role. Our results support the origin of interstitial cells of Cajal and visceral circular muscle from a common progenitor expressing anoctamin-1 and SMCα-actin. Coelomic-derived cells contribute to the differentiation of at least a part of the interstitial cells of Cajal.     

Unique Properties of a Cytotoxic CD4+CD8+ Intraepithelial T-Cell Line Established from the Mouse Intestinal Epithelium

Growth factor-dependent gut intraepithelial lymphocyte (IEL) cell lines were established from a long-term in vitro culture of BALB/c IEL with syngeneic irradiated spleen cells in the presence of concanavalin A-stimulated spleen supernatant fluids. The cell lines were preferentially consisted of very limited thymoindependent subsets of IEL; i.e., Thy-1⁺ CD5^–TCRαβ⁺ CD4⁺CD8 α⁺β^– (double-positive; DP) IEL and Thy-1⁺ CD5^– TCRαβ⁺ CD4^–CD8α⁺β^– (CD8 single-positive; CD8 SP) IEL. The CD8 SP IEL cell line had cytotoxic activities and was triggered to proliferate by T-cell receptor (TCR)-directed stimuli. The DP IEL cell line expressed high levels of the CD3-TCRαβ, exhibited cytotoxic activity in redirected lysis assays, and had perforin in the cytoplasm, indicating the functional maturity of this cell line. However, the DP IEL cell line did not proliferate in response to TCRαβ-directed stimuli, which indicated that TCRαβ-mediated signalling was able to initiate cytotoxic function but not to induce proliferation of the DP IEL cell line. Although both cell lines were shown to have functional competence, they expressed J11d antigen which marks immaturity in thymocyte differentiation pathways. These results indicate that the established thymoindependent DP and CD8 SP IEL cell lines have unique properties distinct from DP thymocytes and CD8 SP peripheral T cells. Together with a recent report on freshly isolated DP IEL (10), the unique properties of the DP IEL cell line seems to support the notion that DP IEL may undergo a unique maturation process in the gut microenvironment.     

Kidney Proximal Tubule Cells Originate from Approximately Four Progenitor Cells and Make Distinct Patches in Mouse Aggregation Chimeras

Giant lysosomal granules of bg^J/bg^J mutant mice were used as a marker to investigate the histological composition of kidney proximal tubule cells. Embryos of the bg^J/bg^J genotype and those of the +/+ genotype were aggregated, and lysosomes of their proximal tubule cells were histochemically stained with the β-glucuronidase activity. Tubules composed of bg^J/bg^J -type epithelial cells alone, tubules composed of +/+-type epithelial cells alone and tubules containing both types of epithelial cells were observed in cross sections. When the long straight portion of proximal tubules was reconstructed from serial sections, most of the tubules were of the mixed type, and distinct patches of bg^J/bg^J -type or +/+-type epithelial cells were detectable. These patches appeared to extend to the longitudinal rather than the circumferential direction. This suggests the clonal proliferation followed the mixing of embryonic progenitor cells for proximal tubule cells. Estimation from proportions of bg^J/bg^J -type proximal tubules in total examined proximal tubules gave a pool size of approximately four primordial precursor cells for proximal tubules in both right and left kindeys. The fact that the proportion of bg^J/bg^J -type components was comparable between the right and left kidneys of each chimera suggests that all proximal tubule cells in both right and left kidneys may originate from common primordial precursor cells.     

Genetic Ablation of Afadin Causes Mislocalization and Deformation of Paneth Cells in the Mouse Small Intestinal Epithelium

Afadin is an actin filament-binding protein that acts cooperatively in cell adhesion with the cell adhesion molecule nectin, and in directional cell movement with the small G protein Rap1 in a nectin-independent manner. We studied the role of afadin in the organization of the small intestinal epithelium using afadin conditional gene knockout (cKO) mice. Afadin was localized at adherens junctions of all types of epithelial cells throughout the crypt-villus axis. Paneth cells were localized at the base of the crypt in control mice, but not confined there, and migrated into the villi in afadin-cKO mice. The distribution of other types of epithelial cells did not change significantly in the mutant mice. The Paneth cells remaining in the crypt exhibited abnormal shapes, were buried between adjacent cells, and did not face the lumen. In these cells, the formation of adherens junctions and tight junctions was impaired. Rap1 and EphB3 were highly expressed in control Paneth cells but markedly down-regulated in the afadin-deficient Paneth cells. Taken together, the results indicate that afadin plays a role in the restricted localization of Paneth cells at the base of the crypt by maintaining their adhesion to adjacent crypt cells and inhibiting their movement toward the top of villi.     

The Fine Structure of the Epithelial Cells of the Mouse Prostate : II. Ventral Lobe Epithelium

The fine structure of the epithelial cells of one component of the prostatic complex of the mouse—the ventral lobe—has been investigated by electron microscopy. This organ is composed of small tubules, lined by tall simple cuboidal epithelium, surrounded by smooth muscle and connective tissue. Electron micrographs of the epithelial cells of the ventral lobe show these to be limited by a cell membrane, which appears as a continuous dense line. The nucleus occupies the basal portion of the cell and the nuclear envelope consists of two membranes. The cytoplasmic matrix is of moderately low density. The endoplasmic reticulum consists of elongated, circular, and oval profiles representing the cavities of this system bounded by rough surfaced membranes. The Golgi apparatus appears localized in a region between the apical border and the nucleus, and is composed of the usual elements found in secretory cells (3, 9). At the base of the cells, a basement membrane is visible in close contact with the outer aspect of the cell membrane. A space of varying width, which seems to be occupied by connective tissue, separates the epithelial cells from the surrounding smooth muscle fibers and the blood vessels. Bodies with the appearance of portions of the cytoplasm, mitochondria, or profiles of the endoplasmic reticulum can be seen in the lumina of the acini and on the bases of these pictures and others of the apical region the mechanism of secretion by these cells is discussed. The fine structural organization of these cells is compared with that of another component of the mouse prostate—the coagulating gland.     

The Role of Secretory Immunoglobulin A in the Natural Sensing of Commensal Bacteria by Mouse Peyer's Patch Dendritic Cells

The mammalian gastrointestinal (GI) tract harbors a diverse population of commensal species collectively known as the microbiota, which interact continuously with the host. From very early in life, secretory IgA (SIgA) is found in association with intestinal bacteria. It is considered that this helps to ensure self-limiting growth of the microbiota and hence participates in symbiosis. However, the importance of this association in contributing to the mechanisms ensuring natural host-microorganism communication is in need of further investigation. In the present work, we examined the possible role of SIgA in the transport of commensal bacteria across the GI epithelium. Using an intestinal loop mouse model and fluorescently labeled bacteria, we found that entry of commensal bacteria in Peyer''s patches (PP) via the M cell pathway was mediated by their association with SIgA. Preassociation of bacteria with nonspecific SIgA increased their dynamics of entry and restored the reduced transport observed in germ-free mice known to have a marked reduction in intestinal SIgA production. Selective SIgA-mediated targeting of bacteria is restricted to the tolerogenic CD11c⁺CD11b⁺CD8⁻ dendritic cell subset located in the subepithelial dome region of PPs, confirming that the host is not ignorant of its resident commensals. In conclusion, our work supports the concept that SIgA-mediated monitoring of commensal bacteria targeting dendritic cells in the subepithelial dome region of PPs represents a mechanism whereby the host mucosal immune system controls the continuous dialogue between the host and commensal bacteria.     

The Fine Structure of the Epithelial Cells of the Mouse Prostate : I. Coagulating Gland Epithelium

The fine structure of the epithelial cells of the anterior lobe, or coagulating gland, of the mouse prostate has been investigated by electron microscopy. This organ is composed of small tubules, lined by tall, simple cuboidal epithelium surrounded by connective tissue and smooth muscle. The epithelial cells are limited by a distinct plasma membrane, which covers minute projections of the cytoplasm into the lumen. The cell membranes of adjacent cells are separated by a narrow layer of structureless material of low density. The cavities of the endoplasmic reticulum are greatly dilated, and the cytoplasmic matrix is reduced to narrow strands, in which the various organelles are visible. The content of the cavities of the endoplasmic reticulum appears as structureless material of lesser density than the cytoplasmic matrix. Material which may be interpreted as secretion products can be seen in the lumina of the tubules. The possible nature of the material inside the cisternal spaces and the secretory mechanisms in these cells is discussed.     

Selective adherence of IgA to murine Peyer's patch M cells: evidence for a novel IgA receptor

M cells represent the primary route by which mucosal Ags are transported across the intestinal epithelium and delivered to underlying gut-associated lymphoid tissues. In rodents and rabbits, Peyer's patch M cells selectively bind and endocytose secretory IgA (SIgA) Abs. Neither the nature of the M cell IgR nor the domains of SIgA involved in this interaction are known. Using a mouse ligated ileal loop assay, we found that monoclonal IgA Abs with or without secretory component, but not IgG or IgM Abs, bound to the apical surfaces of Peyer's patch M cells, indicating that the receptor is specific for the IgA isotype. Human serum IgA and colostral SIgA also bound to mouse M cells. The asialoglycoprotein receptor or other lectin-like receptors were not detected on the apical surfaces of M cells. We used recombinant human IgA1 and human IgA2 Abs and domain swapped IgA/IgG chimeras to determine that both domains Calpha1 and Calpha2 are required for IgA adherence to mouse Peyer's patch M cells. This distinguishes the M cell IgA receptor from CD89 (FcalphaI), which binds domains Calpha2-Calpha3. Finally, we observed by immunofluorescence microscopy that some M cells in the human ileum are coated with IgA. Together these data suggest that mouse, and possibly human, M cells express an IgA-specific receptor on their apical surfaces that mediates the transepithelial transport of SIgA from the intestinal lumen to underlying gut-associated organized lymphoid tissues.     

Analysis of the Specificity of IgA Antibodies Produced in the Mouse Small Intestine

Intestinal microbiota controls multiple aspects of body homeostasis. The microbiota composition changes easily in response to internal or external factors, which may result in dysbiosis and associated inflammatory reactions. Thus, maintaining the microbiota composition by the host immune system is crucial, and one of the main mechanisms for microbiota control is production of immunoglobulin A (IgA) at mucosal surfaces. The molecular mechanisms regulating the interactions between the immune system and microbiota remain obscure. A panel of hybridoma cell lines was constructed to produce monoclonal IgA antibodies specific to various commensal bacteria present in intestinal microbiota. The panel can be used to further understand the mechanisms whereby the adaptive immune system controls the microbiota composition.     

Sphingosine 1-phosphate regulates the egress of IgA plasmablasts from Peyer's patches for intestinal IgA responses

It is well established that Peyer's patches (PPs) are sites for the differentiation of IgA plasma cell precursors, but molecular and cellular mechanisms in their trafficking remain to be elucidated. In this study, we show that alterations in type 1 sphingosine 1-phosphate (S1P) receptor expression during B cell differentiation in the PPs control the emigration of IgA plasma cell precursors. Type 1 S1P receptor expression decreased during the differentiation of IgM(+)B220(+) B cells to IgA(+)B220(+) B cells, but recovered on IgA(+)B220(-) plasmablasts for their emigration from the PPs. Thus, IgA(+)B220(-) plasmablasts migrated in response to S1P in vitro. Additionally, IgA(+) plasmablasts selectively accumulated in lymphatic regions of PPs when S1P-mediated signaling was disrupted by FTY720 treatment. This accumulation of IgA(+) plasmablasts in the PPs led to their reduction in the intestinal lamina propria and simultaneous impairment of Ag-specific intestinal IgA production against orally administered Ag. These findings suggest that S1P regulates the retention and emigration of PP B cells and plays key roles in the induction of intestinal IgA production.     

Loss of the V-ATPase B1 Subunit Isoform Expressed in Non-Neuronal Cells of the Mouse Olfactory Epithelium Impairs Olfactory Function

The vacuolar proton-pumping ATPase (V-ATPase) is the main mediator of intracellular organelle acidification and also regulates transmembrane proton (H⁺) secretion, which is necessary for an array of physiological functions fulfilled by organs such as the kidney, male reproductive tract, lung, bone, and ear. In this study we characterize expression of the V-ATPase in the main olfactory epithelium of the mouse, as well as a functional role for the V-ATPase in odor detection. We report that the V-ATPase localizes to the apical membrane microvilli of olfactory sustentacular cells and to the basolateral membrane of microvillar cells. Plasma membrane V-ATPases containing the B1 subunit isoform are not detected in olfactory sensory neurons or in the olfactory bulb. This precise localization of expression affords the opportunity to ascertain the functional relevance of V-ATPase expression upon innate, odor-evoked behaviors in B1-deficient mice. This animal model exhibits diminished innate avoidance behavior (revealed as a decrease in freezing time and an increase in the number of sniffs in the presence of trimethyl-thiazoline) and diminished innate appetitive behavior (a decrease in time spent investigating the urine of the opposite sex). We conclude that V-ATPase-mediated H⁺ secretion in the olfactory epithelium is required for optimal olfactory function.