Food antigens suppress small intestinal tumorigenesis.

Food components suppressing small intestinal tumorigenesis are not well-defined partly because of the rarity of this tumor type compared to colorectal tumors. Using Apcmin/+ mice, a mouse model for intestinal tumorigenesis, and antigen-free diet, we report here that food antigens serve this function in the small intestine. By depleting Peyer's patches (PPs), immune inductive sites in the small intestine, we found that PPs have a role in the suppression of small intestinal tumors and are important for the induction of small intestinal T cells by food antigens. On the follicle-associated epithelium (FAE) of PPs, microfold (M) cells pass food antigens from lumen to the dendritic cells to induce T cells. Single-cell RNA-seq (scRNA-seq) analysis of immune cells in PPs revealed a significant impact of food antigens on the induction of the PP T cells and the antigen presentation capacity of dendritic cells. These data demonstrate the role of food antigens in the suppression of small intestinal tumorigenesis by PP-mediated immune cell induction.

A circulating subset of iNKT cells mediates antitumor and antiviral immunity.

Invariant natural killer T (iNKT) cells are a group of innate-like T lymphocytes that recognize lipid antigens. They are supposed to be tissue resident and important for systemic and local immune regulation. To investigate the heterogeneity of iNKT cells, we recharacterized iNKT cells in the thymus and peripheral tissues. iNKT cells in the thymus were divided into three subpopulations by the expression of the natural killer cell receptor CD244 and the chemokine receptor CXCR6 and designated as C0 (CD244-CXCR6-), C1 (CD244-CXCR6+), or C2 (CD244+CXCR6+) iNKT cells. The development and maturation of C2 iNKT cells from C0 iNKT cells strictly depended on IL-15 produced by thymic epithelial cells. C2 iNKT cells expressed high levels of IFN-γ and granzymes and exhibited more NK cell-like features, whereas C1 iNKT cells showed more T cell-like characteristics. C2 iNKT cells were influenced by the microbiome and aging and suppressed the expression of the autoimmune regulator AIRE in the thymus. In peripheral tissues, C2 iNKT cells were circulating that were distinct from conventional tissue-resident C1 iNKT cells. Functionally, C2 iNKT cells protected mice from the tumor metastasis of melanoma cells by enhancing antitumor immunity and promoted antiviral immune responses against influenza virus infection. Furthermore, we identified human CD244+CXCR6+ iNKT cells with high cytotoxic properties as a counterpart of mouse C2 iNKT cells. Thus, this study reveals a circulating subset of iNKT cells with NK cell-like properties distinct from conventional tissue-resident iNKT cells.

Bacterial cancer therapy in autochthonous colorectal cancer affects tumor growth and metabolic landscape.

Bacterial cancer therapy (BCT) shows great promise for treatment of solid tumors, yet basic mechanisms of bacterial-induced tumor suppression remain undefined. Attenuated strains of Salmonella enterica serovar Typhimurium (STm) have commonly been used in mouse models of BCT in xenograft and orthotopic transplant cancer models. We aimed to better understand the tumor epithelium-targeted mechanisms of BCT by using autochthonous mouse models of intestinal cancer and tumor organoid cultures to assess the effectiveness and consequences of oral treatment with aromatase A-deficient STm (STmΔaroA). STmΔaroA delivered by oral gavage significantly reduced tumor burden and tumor load in both a colitis-associated colorectal cancer (CAC) model and in a spontaneous Apcmin/+ intestinal cancer model. STmΔaroA colonization of tumors caused alterations in transcription of mRNAs associated with tumor stemness, epithelial-mesenchymal transition, and cell cycle. Metabolomic analysis of tumors demonstrated alteration in the metabolic environment of STmΔaroA-treated tumors, suggesting that STmΔaroA imposes metabolic competition on the tumor. Use of tumor organoid cultures in vitro recapitulated effects seen on tumor stemness, mesenchymal markers, and altered metabolome. Furthermore, live STmΔaroA was required, demonstrating active mechanisms including metabolite usage. We have demonstrated that oral BCT is efficacious in autochthonous intestinal cancer models, that BCT imposes metabolic competition, and that BCT has direct effects on the tumor epithelium affecting tumor stem cells.

Asymmetric distribution of TLR3 leads to a polarized immune response in human intestinal epithelial cells.

Intestinal epithelial cells (IECs) act as a physical barrier separating the commensal-containing intestinal tract from the sterile interior. These cells have found a complex balance allowing them to be prepared for pathogen attacks while still tolerating the presence of bacterial or viral stimuli present in the lumen of the gut. Using primary human IECs, we probed the mechanisms that allow for such a tolerance. We discovered that viral infections emanating from the basolateral side of IECs elicit a stronger intrinsic immune response in comparison to lumenal apical infections. We determined that this asymmetric immune response is driven by the clathrin-sorting adaptor AP-1B, which mediates the polarized sorting of Toll-like receptor 3 (TLR3) towards the basolateral side of IECs. Mice and human IECs lacking AP-1B showed an exacerbated immune response following apical stimulation. Together, these results suggest a model where the cellular polarity program plays an integral role in the ability of IECs to partially tolerate apical commensals while remaining fully responsive to invasive basolateral pathogens.

Intestinal M cells: Tireless samplers of enteric microbiota.

Mucosal immune responses in the inductive lymphoid tissues of the intestine begin with uptake of particulate antigens, including components of the gut microbiota by specialized antigen sampling M cells. M cells represent a distinct lineage of enterocytes that arise from crypt stem cells in response to the cytokine receptor of NF-κB ligand (RANKL). Full differentiation of M cells requires the transcription factor Spi-B to yield mature M cells that express multiple receptors for bacteria including glycoprotein 2. M cell differentiation can be recapitulated in vitro using three-dimensional enteroid cultures of primary intestinal stem cells supplemented with RANKL. This article summarizes the current knowledge about the genesis of intestinal M cells and highlights some of the remaining unanswered questions about this enigmatic cell type.

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.

Development of intestinal M cells and follicle-associated epithelium is regulated by TRAF6-mediated NF-κB signaling.

M cells are located in the follicle-associated epithelium (FAE) that covers Peyer's patches (PPs) and are responsible for the uptake of intestinal antigens. The differentiation of M cells is initiated by receptor activator of NF-κB. However, the intracellular pathways involved in M cell differentiation are still elusive. In this study, we demonstrate that the NF-κB pathway activated by RANK is essential for M cell differentiation using in vitro organoid culture. Overexpression of NF-κB transcription factors enhances the expression of M cell-associated molecules but is not sufficient to complete M cell differentiation. Furthermore, we evaluated the requirement for tumor necrosis factor receptor-associated factor 6 (TRAF6). Conditional deletion of TRAF6 in the intestinal epithelium causes a complete loss of M cells in PPs, resulting in impaired antigen uptake into PPs. In addition, the expression of FAE-associated genes is almost silenced in TRAF6-deficient mice. This study thus demonstrates the crucial role of TRAF6-mediated NF-κB signaling in the development of M cells and FAE.

IL-22BP dictates characteristics of Peyer's patch follicle-associated epithelium for antigen uptake.

Interleukin-22 (IL-22) acts protectively and harmfully on intestinal tissue depending on the situation; therefore, IL-22 signaling needs to be tightly regulated. IL-22 binding protein (IL-22BP) binds IL-22 to inhibit IL-22 signaling. It is expressed in intestinal and lymphoid tissues, although its precise distribution and roles have remained unclear. In this study, we show that IL-22BP is highly expressed by CD11b+CD8α- dendritic cells in the subepithelial dome region of Peyer's patches (PPs). We found that IL-22BP blocks IL-22 signaling in the follicle-associated epithelium (FAE) covering PPs, indicating that IL-22BP plays a role in regulating the characteristics of the FAE. As expected, FAE of IL-22BP-deficient (Il22ra2-/-) mice exhibited altered properties such as the enhanced expression of mucus and antimicrobial proteins as well as prominent fucosylation, which are normally suppressed in FAE. Additionally, Il22ra2-/- mice exhibited the decreased uptake of bacterial antigens into PPs without affecting M cell function. Our present study thus demonstrates that IL-22BP promotes bacterial uptake into PPs by influencing FAE gene expression and function.

Mast Cells Are Crucial for Induction of Group 2 Innate Lymphoid Cells and Clearance of Helminth Infections.

Mast cells are important for eradication of intestinal nematodes; however, their precise mechanisms of action have remained elusive, especially in the early phase of infection. We found that Spi-B-deficient mice had increased numbers of mast cells and rapidly expelled the Heligmosomoides polygyrus (Hp) nematode. This was accompanied by induction of interleukin-13 (IL-13)-producing group 2 innate lymphoid cells (ILC2) and goblet cell hyperplasia. Immediately after Hp infection, mast cells were rapidly activated to produce IL-33 in response to ATP released from apoptotic intestinal epithelial cells. In vivo inhibition of the P2X7 ATP receptor rendered the Spi-B-deficient mice susceptible to Hp, concomitant with elimination of mast cell activation and IL-13-producing ILC2 induction. These results uncover a previously unknown role for mast cells in innate immunity in that activation of mast cells by ATP orchestrates the development of a protective type 2 immune response, in part by producing IL-33, which contributes to ILC2 activation.

The Ets transcription factor Spi-B is essential for the differentiation of intestinal microfold cells.

Intestinal microfold cells (M cells) are an enigmatic lineage of intestinal epithelial cells that initiate mucosal immune responses through the uptake and transcytosis of luminal antigens. The mechanisms of M-cell differentiation are poorly understood, as the rarity of these cells has hampered analysis. Exogenous administration of the cytokine RANKL can synchronously activate M-cell differentiation in mice. Here we show the Ets transcription factor Spi-B was induced early during M-cell differentiation. Absence of Spi-B silenced the expression of various M-cell markers and prevented the differentiation of M cells in mice. The activation of T cells via an oral route was substantially impaired in the intestine of Spi-B-deficient (Spib(-/-)) mice. Our study demonstrates that commitment to the intestinal M-cell lineage requires Spi-B as a candidate master regulator.

Orally administered prion protein is incorporated by m cells and spreads into lymphoid tissues with macrophages in prion protein knockout mice.

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases. Infection by the oral route is assumed to be important, although its pathogenesis is not understood. Using prion protein (PrP) knockout mice, we investigated the sequence of events during the invasion of orally administered PrPs through the intestinal mucosa and the spread into lymphoid tissues and the peripheral nervous system. Orally administered PrPs were incorporated by intestinal epitheliocytes in the follicle-associated epithelium and villi within 1 hour. PrP-positive cells accumulated in the subfollicle region of Peyer's patches a few hours thereafter. PrP-positive cells spread toward the mesenteric lymph nodes and spleen after the accumulation of PrPs in the Peyer's patches. The number of PrP molecules in the mesenteric lymph nodes and spleen peaked at 2 days and 6 days after inoculation, respectively. The epitheliocytes in the follicle-associated epithelium incorporating PrPs were annexin V-positive microfold cells and PrP-positive cells in Peyer's patches and spleen were CD11b-positive and CD14-positive macrophages. Additionally, PrP-positive cells in Peyer's patches and spleen were detected in the vicinity of peripheral nerve fibers in the early stages of infection. These results indicate that orally delivered PrPs were incorporated by microfold cells promptly after challenge and that macrophages might act as a transporter of incorporated PrPs from the Peyer's patches to other lymphoid tissues and the peripheral nervous system.

Characterization of newly established bovine intestinal epithelial cell line.

Membranous epithelial cells (M cells) of the follicle-associated epithelium in Peyer's patches have a high capacity for transcytosis of several viruses and microorganisms. Here, we report that we have successfully established a bovine intestinal epithelial cell line (BIE cells) and developed an in vitro M cell model. BIE cells have a cobblestone morphology and microvilli-like structures, and strongly express cell-to-cell junctional proteins and cytokeratin, which is a specific intermediate filament protein of epithelial cells. After co-culture with murine intestinal lymphocytes or treatment with supernatant from bovine PBMC cultured with IL-2, BIE cells acquired the ability of transcytosis. Therefore, BIE cells have typical characteristics of bovine intestinal epithelial cells and also have the ability to differentiate into an M cell like linage. In addition, our results indicate that contact between immune cells and epithelial cells may not be absolutely required for the differentiation of M cells. We think that BIE cells will be useful for studying the transport mechanisms of various pathogens and also the evaluation of drug delivery via M cells.

Expression of inflammatory-related factors in porcine anterior pituitary-derived cell line.

Recent studies have shown that undifferentiated stem cells act as immunomodulators. To investigate the immunomodulatory function of the progenitor cells of the anterior pituitary gland, we attempted to establish a stem/progenitor cell line from the porcine anterior pituitary gland, and to detail its inflammatory cytokine expression. A cloned cell line from the porcine anterior pituitary gland was established and was designated as the porcine anterior pituitary-derived cell line (PAPC). PAPC expressed the mRNA of Nanog and Oct-4, and showed positive immunoreactivity for beta-catenin and Hes1 in its nucleus. PAPC grew stably by repeated passage and rapidly in the EGF and bFGF containing medium. RT-PCR showed that PAPC expressed mRNA of IL-1alpha, IL-6, IL-12, IL-15, IL-18 and TLR4. PAPC expressed S100alpha and IL-18 protein, which was localized in the marginal epithelial cells of Rathke's pouch. These results suggest that PAPC is a stem/progenitor cell and may regulate anterior pituitary cell function through an immuno-endocrine pathway.

Immunohistochemical characterization of cell types expressing the cellular prion protein in the small intestine of cattle and mice.

The gastrointestinal tract is thought to be the main site of entry for the pathological isoform of the prion protein (PrP(Sc)). Prion diseases are believed to result from a conformational change of the cellular prion protein (PrP(c)) to PrP(Sc). Therefore, PrP(c) expression is a prerequisite for the infection and spread of the disease to the central nervous system. However, the distribution of PrP(c) in the gut is still a matter of controversy. We therefore investigated the localization of PrP(c) in the bovine and murine small intestine. In cattle, most PrP(c) positive epithelial cells were detected in the duodenum, while a few positive cells were found in the jejunum. PrP(c) was expressed in serotonin producing cells. In bovine Peyer's patches, PrP(c) was distributed in extrafollicular areas, but not in the germinal centre of the jejunum and ileum. PrP(c) was expressed in myeloid lineage cells such as myeloid dendritic cells and macrophages. In mice, PrP(c) was expressed in some epithelial cells throughout the small intestine as well as in cells such as follicular dendritic cell in the germinal centre of Peyer's patches. In this study, we demonstrate that there are a number of differences in the localization of PrP(c) between the murine and bovine small intestines.

Apoptotic process of porcine intestinal M cells.

Membranous (M) cells of the follicle-associated epithelium (FAE) are believed to sample antigens from the gut lumen. However, the origin, differentiation mechanism, and cell death of M cells are still a matter of controversy. Therefore, we investigated the process of M cell differentiation and determined their fate in the intestine of three-way crossbred female pigs. We used anti-cytokeratin 18 and anti-PCNA antibodies to distinguish M cells and proliferative cells and performed immunohistochemistry, enzyme histochemistry, and scanning electron microscopy on fresh ileal Peyer's patches. Cell migration and apoptotic cells were detected by BrdU labeling and the TUNEL method, respectively. The turnover of the FAE was similar to that of the villi. M cells were mostly observed from the FAE crypt to the FAE periphery, but not in the FAE apex. As proliferative M cells (cytokeratin 18(+)/PCNA(+) cells) have previously been detected in the FAE crypt, porcine M cells may be directly derived from intestinal epithelial stem cells and committed as a distinct cell lineage in the crypts. M cells from the FAE periphery were unstained or only weakly stained for alkaline phosphatase, whereas cytokeratin 18(+)/alkaline phosphatase(+) cells lying near to the FAE apex showed a columnar shape similar to that of adjacent enterocytes. These data suggest that the committed M cells differentiate to mature M cells by contact with lymphocytes at the FAE periphery, and that they trans-differentiate to enterocytes and are finally excluded near the FAE apex.