RANKL regulates differentiation of microfold cells in mouse nasopharynx-associated lymphoid tissue (NALT).

Murine nasopharynx-associated lymphoid tissue (NALT), located at the base of the nasal cavity, serves as a major site for the induction of mucosal immune responses against airway antigens. The follicle-associated epithelium (FAE) covering the luminal surface of NALT is characterized by the presence of microfold cells (M cells), which take up and transport luminal antigens to lymphocytes. Glycoprotein 2 (GP2) has recently been identified as a reliable marker for M cells in Peyer's patches of the intestine. However, the expression of GP2 and other functional molecules in the M cells of NALT has not yet been examined. We have immunohistochemically detected GP2-expressing cells in the FAE of NALT and the simultaneous expression of other intestinal M-cell markers, namely Tnfaip2, CCL9, and Spi-B. These cells have been further identified as M cells because of their higher uptake capacity of luminal microbeads. Electron microscopic observations have shown that GP2-expressing cells on the FAE display morphological features typical of M cells: they possess short microvilli and microfolds on the luminal surface and are closely associated with intraepithelial lymphocytes. We have also found that the receptor activator of nuclear factor kappa-B ligand (RANKL) is expressed by stromal cells underneath the FAE, which provides its receptor RANK. The administration of RANKL markedly increases the number of GP2(+)Tnfaip2(+) cells on the NALT FAE and that of intestinal M cells. These results suggest that GP2(+)Tnfaip2(+) cells in NALT are equivalent to intestinal M cells, and that RANKL-RANK signaling induces their differentiation.

Inhibition of RANKL and Sema4D improves residual ridge resorption in mice.

Residual ridge resorption (RRR) is a chronic and progressive bone resorption following tooth loss. It causes deterioration of the oral environments and leads to the pathogenesis of various systemic diseases. However, the molecular mechanisms and risk factors for RRR progression are still unclear and controversial. In this study, we developed a tooth extraction model using mice for analyzing long-term morphological and gene expression changes in the alveolar bone. We further applied ovariectomy to this model to elucidate the effects of osteoporosis on RRR progression. As a result, the alveolar bone loss was biphasic and consisted of rapid loss in the early stages and subsequently slow and sustained bone loss over a long period. Histological analysis indicated that ovariectomy prolonged the activation of osteoclasts in the alveolar bone. Furthermore, the expressions of Tnfsf11 and Sema4d kept increasing for a long time in OVX mice. Administration of neutralization antibodies for receptor activator of NF-κB ligand (RANKL) effectively suppressed RRR. Similarly, inhibition of Semaphorin 4D (Sema4D) also improved alveolar bone loss. This study demonstrated that reduced ovarian function may be a risk factor for RRR and that RANKL and Sema4D suppression are potential treatments.

Osteoprotegerin-dependent M cell self-regulation balances gut infection and immunity.

Microfold cells (M cells) are responsible for antigen uptake to initiate immune responses in the gut-associated lymphoid tissue (GALT). Receptor activator of nuclear factor-κB ligand (RANKL) is essential for M cell differentiation. Follicle-associated epithelium (FAE) covers the GALT and is continuously exposed to RANKL from stromal cells underneath the FAE, yet only a subset of FAE cells undergoes differentiation into M cells. Here, we show that M cells express osteoprotegerin (OPG), a soluble inhibitor of RANKL, which suppresses the differentiation of adjacent FAE cells into M cells. Notably, OPG deficiency increases M cell number in the GALT and enhances commensal bacterium-specific immunoglobulin production, resulting in the amelioration of disease symptoms in mice with experimental colitis. By contrast, OPG-deficient mice are highly susceptible to Salmonella infection. Thus, OPG-dependent self-regulation of M cell differentiation is essential for the balance between the infectious risk and the ability to perform immunosurveillance at the mucosal surface.

Airway M Cells Arise in the Lower Airway Due to RANKL Signaling and Reside in the Bronchiolar Epithelium Associated With iBALT in Murine Models of Respiratory Disease.

Microfold (M) cells residing in the follicle-associated epithelium of mucosa-associated lymphoid tissues are specialized for sampling luminal antigens to initiate mucosal immune responses. In the past decade, glycoprotein 2 (GP2) and Tnfaip2 were identified as reliable markers for M cells in the Peyer's patches of the intestine. Furthermore, RANKL-RANK signaling, as well as the canonical and non-canonical NFκB pathways downstream, is essential for M-cell differentiation from the intestinal stem cells. However, the molecular characterization and differentiation mechanisms of M cells in the lower respiratory tract, where organized lymphoid tissues exist rarely, remain to be fully elucidated. Therefore, this study aimed to explore M cells in the lower respiratory tract in terms of their specific molecular markers, differentiation mechanism, and functions. Immunofluorescence analysis revealed a small number of M cells expressing GP2, Tnfaip2, and RANK is present in the lower respiratory tract of healthy mice. The intraperitoneal administration of RANKL in mice effectively induced M cells, which have a high capacity to take up luminal substrates, in the lower respiratory epithelium. The airway M cells associated with lymphoid follicles were frequently detected in the pathologically induced bronchus-associated lymphoid tissue (iBALT) in the murine models of autoimmune disease as well as pulmonary emphysema. These findings demonstrate that RANKL is a common inducer of M cells in the airway and digestive tracts and that M cells are associated with the respiratory disease. We also established a two-dimensional culture method for airway M cells from the tracheal epithelium in the presence of RANKL successfully. This model may be useful for functional studies of M cells in the sampling of antigens at airway mucosal surfaces.

Developmental changes in primary cilia in the mouse tooth germ and oral cavity.

The primary cilium, a sensory apparatus, functions as both a chemical and mechanical sensor to receive environmental stimuli. The present study focused on the primary cilia in the epithelialmesenchymal interaction during tooth development. We examined the localization and direction of projection of primary cilia in the tooth germ and oral cavity of mice by immunohistochemical observation. Adenylyl cyclase 3 (ACIII)-immunolabeled cilia were visible in the inner/outer enamel epithelium of molars at the fetal stage and then conspicuously developed in the odontoblast layer postnatally. The primary cilia in ameloblasts and odontoblasts-shown by the double staining of acetylated tubulin and γ-tubulin-were regularly arranged from postnatal Day12, projecting apart from each other. The periodontal ligament possessed ACIII-positive cilia, which gathered on both sides of the dentin/cement and alveolar bone in postnatal days. In the oral cavity, numerous long primary cilia immunoreactive for ACIII were condensed at subepithelial stromal cells in the oral processes in fetuses, while postnatally a small number of short cilia were dispersed throughout the stroma of the oral cavity. These findings suggest that the primary cilia showing stage- and regionspecific morphology are involved in the epithelial-mesenchymal interaction during tooth development via mechano- and/or chemoreception for growth factors.

A systematic analysis for localization of predominant growth factors and their receptors involved in murine tooth germ differentiation using in situ hybridization technique.

Tooth development is regulated by various growth factors and their receptors. However, the overall mechanism of growth factor-mediated odontogenesis remains to be elucidated. The present study examined expression sites and intensities of major growth factors and receptors in the tooth germ of murine fetuses and neonates. Signals of TGF-ß and CTGF in fetuses were released from the enamel epithelium, while their neonatal signals arose in odontoblasts. Moreover, BMP/Smad signaling may affect the differentiation of ameloblasts, in contrast to PDGFα whose signals may cause odontoblast differentiation. Growth factors associated with the formation of the periodontium were IGF1, IGF2, IGFBP3, CTGF, and PDGFα. Concerning cusp formation, the enamel knot selectively expressed FGF4, BMP2, and BMP4 with an expression of PDGFα in the enamel-free area. It is concluded that many molecules play critical roles in the epithelium-mesenchyme interaction of tooth germ differentiation, and their expressions are precisely controlled.

A novel type of cells expressing GP2 in the respiratory epithelium of the paranasal sinuses in mice.

GP2, a GPI-anchored glycoprotein, is a useful marker of M cells in Peyer's patches. Our immunostaining of the paranasal sinuses in mice detected a condensed distribution of GP2-immunoreactive cells within the epithelium, apart from lymphoid tissues. In the paranasal sinuses, the cells exhibited a unique morphology characterized by a slender neck portion and huge terminal bulb, quite different from M cells. Electron microscopically, the GP2 immunoreactivity centered on the luminal plasma membrane of the terminal bulb, being less intense in the baso-lateral plasma membrane and not visible at all in the cytoplasm. The cells frequently came in contact with nerve fibers containing small synaptic vesicles. These nerve fibers contained neither CGRP nor substance P-indicators of sensory neurons; moreover, no signal molecules used for a sensory function were expressed in the GP2-immunoreactive cells, implying that these nerves are efferent in nature. A weak but significant stainability in PAS reaction and an intense GP2 immunoreactivity for typical goblet cells in the tunica conjunctiva suggest that the GP2-expressing cells in paranasal sinuses are in the lineage of goblet cells.