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.

Size- and Morphology-Controlled Preparation of Surface-Modified Water-Dispersible Fullerene Nanoparticles for Bioapplications.

In this study, we investigated water-dispersible surface modification for size- and shape-controlled fullerene nanoparticles (C60P) based on a condensation reaction with di-amino alkane. This modification provided for water dispersibility of C60P and the capability for secondary modification as well. The resultant C60P particles have several useful physical properties: water-dispersibility for ease of injection; fluorescence for detection and quantification; and a characteristic morphology to assist identification. These properties will widely extend the applications of these particles, especially into the biological fields of bioimaging and drug delivery.

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.

Sox8 is essential for M cell maturation to accelerate IgA response at the early stage after weaning in mice.

Microfold (M) cells residing in the follicle-associated epithelium (FAE) of the gut-associated lymphoid tissue are specialized for antigen uptake to initiate mucosal immune responses. The molecular machinery and biological significance of M cell differentiation, however, remain to be fully elucidated. Here, we demonstrate that Sox8, a member of the SRY-related HMG box transcription factor family, is specifically expressed by M cells in the intestinal epithelium. The expression of Sox8 requires activation of RANKL-RelB signaling. Chromatin immunoprecipitation and luciferase assays revealed that Sox8 directly binds the promoter region of Gp2 to increase Gp2 expression, which is the hallmark of functionally mature M cells. Furthermore, genetic deletion of Sox8 causes a marked decrease in the number of mature M cells, resulting in reduced antigen uptake in Peyer's patches. Consequently, juvenile Sox8-deficient mice showed attenuated germinal center reactions and antigen-specific IgA responses. These findings indicate that Sox8 plays an essential role in the development of M cells to establish mucosal immune responses.

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.

GP2-expressing cells in the conjunctiva and tear ducts of mice: identification of a novel type of cells in the squamous stratified epithelium.

GP2 is a membrane-associated secretory protein originally identified in zymogen granules of pancreatic acinar cells. Recently, this glycoprotein has attracted attention as a marker substance of M cells of Peyer's patches and for its involvement in the selective uptake of pathological bacteria via M cells. When we stained the conjunctiva and tear ducts of mice using a GP2 antibody, all goblet cells in the squamous stratified epithelium of the conjunctiva were intensely immunolabeled, while goblet cells in the intestine and airway were devoid of the immunoreactivity, indicating that the conjunctiva contains a special type of goblet cell. Further immunostaining for GP-2 labeled dispersed cells of peculiar shapes within the stratified squamous epithelium in the lacrimal canaliculi, lacrimal sac, and nasolacrimal duct. The GP2-immunoreactive cells in the tear duct projected arched or branched processes toward the basement membrane. Electron-microscopically, immunogold particles for GP2 outlined the basolateral plasma membrane of both the conjuntival goblet cells and the peculiarly shaped cells in the tear duct. Intracellularly, GP2 products of the goblet cells were localized around secretory granules in the apical cytoplasm and those of the tear duct cells inside the vesicles. The luminal contents close to apical plasma membrane were heavily labeled with immunogold particles, suggesting an exocytosis-based targeting of GP2 to the plasma membrane and its release into the lumen. The possible function of GP2 in tear ducts is discussed in relation to a defense system against invasive microoranisms and antigens.