Host- and Microbe-Dependent Dietary Lipid Metabolism in the Control of Allergy, Inflammation, and Immunity

The intestine is the largest immune organ in the body, provides the first line of defense against pathogens, and prevents excessive immune reactions to harmless or beneficial non-self-materials, such as food and intestinal bacteria. Allergic and inflammatory diseases in the intestine occur as a result of dysregulation of immunological homeostasis mediated by intestinal immunity. Several lines of evidence suggest that gut environmental factors, including nutrition and intestinal bacteria, play important roles in controlling host immune responses and maintaining homeostasis. Among nutritional factors, ω3 and ω6 essential polyunsaturated fatty acids (PUFAs) profoundly influence the host immune system. Recent advances in lipidomics technology have led to the identification of lipid mediators derived from ω3- and ω6-PUFAs. In particular, lipid metabolites from ω3-PUFAs (e.g., eicosapentaenoic acid and docosahexaenoic acid) have recently been shown to exert anti-allergic and anti-inflammatory responses; these metabolites include resolvins, protectins, and maresins. Furthermore, a new class of anti-allergic and anti-inflammatory lipid metabolites of 17,18-epoxyeicosatetraenoic acid has recently been identified in the control of allergic and inflammatory diseases in the gut and skin. Although these lipid metabolites were found to be endogenously generated in the host, accumulating evidence indicates that intestinal bacteria also participate in lipid metabolism and thus generate bioactive unique lipid mediators. In this review, we discuss the production machinery of lipid metabolites in the host and intestinal bacteria and the roles of these metabolites in the regulation of host immunity

Persistent colonization of non-lymphoid tissue-resident macrophages by Stenotrophomonas maltophilia

Accumulating evidence has revealed that lymphoid tissue-resident commensal bacteria (e.g. Alcaligenes spp.) survive within dendritic cells. We extended our previous study by investigating microbes that persistently colonize colonic macrophages. 16S rRNA-based metagenome analysis using DNA purified from murine colonic macrophages revealed the presence of Stenotrophomonas maltophilia. The in situ intracellular colonization by S. maltophilia was recapitulated in vitro by using bone marrow-derived macrophages (BMDMs). Co-culture of BMDMs with clinically isolated S. maltophilia led to increased mitochondrial respiration and robust IL-10 production. We further identified a 25-kDa protein encoded by the gene assigned as smlt2713 (recently renamed as SMLT_RS12935) and secreted by S. maltophilia as the factor responsible for enhanced IL-10 production by BMDMs. IL-10 production is critical for maintenance of the symbiotic condition, because intracellular colonization by S. maltophilia was impaired in IL-10-deficient BMDMs, and smlt2713-deficient S. maltophilia failed to persistently colonize IL-10-competent BMDMs. These findings indicate a novel commensal network between colonic macrophages and S. maltophilia that is mediated by IL-10 and smlt2713

Id2-, RORγt-, and LTβR-independent initiation of lymphoid organogenesis in ocular immunity

The eye is protected by the ocular immunosurveillance system. We show that tear duct–associated lymphoid tissue (TALT) is located in the mouse lacrimal sac and shares immunological characteristics with mucosa-associated lymphoid tissues (MALTs), including the presence of M cells and immunocompetent cells for antigen uptake and subsequent generation of mucosal immune responses against ocularly encountered antigens and bacteria such as Pseudomonas aeruginosa. Initiation of TALT genesis began postnatally; it occurred even in germ-free conditions and was independent of signaling through organogenesis regulators, including inhibitor of DNA binding/differentiation 2, retinoic acid–related orphan receptor γt, lymphotoxin (LT) α1β2–LTβR, and lymphoid chemokines (CCL19, CCL21, and CXCL13). Thus, TALT shares immunological features with MALT but has a distinct tissue genesis mechanism and plays a key role in ocular immunity

Enteroendocrine cells are specifically marked by cell surface expression of claudin-4 in mouse small intestine.

Enteroendocrine cells are solitary epithelial cells scattered throughout the gastrointestinal tract and produce various types of hormones, constituting one of the largest endocrine systems in the body. The study of these rare epithelial cells has been hampered by the difficulty in isolating them because of the lack of specific cell surface markers. Here, we report that enteroendocrine cells selectively express a tight junction membrane protein, claudin-4 (Cld4), and are efficiently isolated with the use of an antibody specific for the Cld4 extracellular domain and flow cytometry. Sorted Cld4+ epithelial cells in the small intestine exclusively expressed a chromogranin A gene (Chga) and other enteroendocrine cell-related genes (Ffar1, Ffar4, Gpr119), and the population was divided into two subpopulations based on the activity of binding to Ulex europaeus agglutinin-1 (UEA-1). A Cld4+UEA-1- cell population almost exclusively expressed glucose-dependent insulinotropic polypeptide gene (Gip), thus representing K cells, whereas a Cld4+UEA-1+ cell population expressed other gut hormone genes, including glucagon-like peptide 1 (Gcg), pancreatic polypeptide-like peptide with N-terminal tyrosine amide (Pyy), cholecystokinin (Cck), secretin (Sct), and tryptophan hydroxylase 1 (Tph1). In addition, we found that orally administered luminal antigens were taken up by the solitary Cld4+ cells in the small intestinal villi, raising the possibility that enteroendocrine cells might also play a role in initiation of mucosal immunity. Our results provide a useful tool for the cellular and functional characterization of enteroendocrine cells

Obesity Suppresses Cell-Competition-Mediated Apical Elimination of RasV12-Transformed Cells from Epithelial Tissues

Recent studies have revealed that newly emerging transformed cells are often eliminated from epithelial tissues via cell competition with the surrounding normal epithelial cells. This cancer preventive phenomenon is termed epithelial defense against cancer (EDAC). However, it remains largely unknown whether and how EDAC is diminished during carcino-genesis. In this study, using a cell competition mouse model, we show that high-fat diet (HFD) feeding substantially attenuates the frequency of apical elimination of RasV12-transformed cells from intestinal and pancreatic epithelia. This process involves both lipid metabolism and chronic inflammation. Furthermore, aspirin treatment significantly facilitates eradication of transformed cells from the epithelial tissues in HFD-fed mice. Thus, our work demonstrates that obesity can profoundly influence competitive interaction between normal and transformed cells, providing insights into cell competition and cancer preventive medicine

Claudin 4 in pancreatic β cells is involved in regulating the functional state of adult islets

The functional state (FS) of adult pancreatic islets is regulated by a large array of regulatory molecules including numerous transcription factors. Whether any islet structural molecules play such a role has not been well understood. Here, multiple technologies including bioinformatics analyses were used to explore such molecules. The tight junction family molecule claudin 4 (Cldn4) was the highest enriched amongst over 140 structural genes analysed. Cldn4 expression was ~75‐fold higher in adult islets than in exocrine tissues and was mostly up‐regulated during functional maturation of developing islet cells. Cldn4 was progressively down‐regulated in functionally compromised, dedifferentiating insulin‐secreting β cells and in db/db type 2 diabetic islets. Furthermore, the genetic deletion of Cldn4 impaired significantly the FS without apparently affecting pancreas morphology, islet architectural structure and cellular distribution, and secretion of enteroendocrine hormones. Thus, we suggest a previously unidentified role for Cldn4 in regulating the FS of islets, with implications in translational research for better diabetes therapie

Claudin 4 in pancreatic β cells is involved in regulating the functional state of adult islets

The functional state (FS) of adult pancreatic islets is regulated by a large array of regulatory molecules including numerous transcription factors. Whether any islet structural molecules play such a role has not been well understood. Here, multiple technologies including bioinformatics analyses were used to explore such molecules. The tight junction family molecule claudin 4 (Cldn4) was the highest enriched amongst over 140 structural genes analysed. Cldn4 expression was ~75‐fold higher in adult islets than in exocrine tissues and was mostly up‐regulated during functional maturation of developing islet cells. Cldn4 was progressively down‐regulated in functionally compromised, dedifferentiating insulin‐secreting β cells and in db/db type 2 diabetic islets. Furthermore, the genetic deletion of Cldn4 impaired significantly the FS without apparently affecting pancreas morphology, islet architectural structure and cellular distribution, and secretion of enteroendocrine hormones. Thus, we suggest a previously unidentified role for Cldn4 in regulating the FS of islets, with implications in translational research for better diabetes therapies

Cld4⁺ epithelial cells isolated by a cell-sorter with anti-Cld4 antibody exclusively express the genes related to enteroendocrine cells.

<p>(<i>A</i>) Multicolor FACS analysis of small intestine single-cell suspension from WT and <i>Cldn4</i>^−/− mice. The profile of EpCAM expression in a PI⁻CD45⁻Ter119⁻ viable nonhematopoietic cell gate is shown (a−c). In the EpCAM⁺ cell gate, FACS profiles of biotinylated anti-Cld4 antibody (HKH-189) are indicated for WT and <i>Cldn4</i>^−/− mice (d). Percentages of positive staining and negative staining are shown. (<i>B</i>) Cld4⁺ and Cld4⁻ fractions of EpCAM⁺ cells were sorted separately with FACSAria and examined for the transcripts of claudin-4 (<i>Cldn4</i>), claudin-3 (<i>Cldn3</i>), ZO-1 (<i>Tjp1</i>), chromogranin A (<i>Chga</i>), Gpr40 (<i>Ffar1</i>), Gpr119 (<i>Gpr119</i>), and Gpr120 (<i>Ffar4</i>) with quantitative RT-PCR. These results are representative of at least three independent experiments. *<i>P</i> < 0.05 and **<i>P</i> < 0.01, Student <i>t</i> test.</p

Transepithelial passage of luminal dextran (10-kDa) via Cld4⁺ enteroendocrine cells.

<p>Eight-week-old to 10<b>-</b>week<b>-</b>old WT mice were administered oral rhodamine or fluorescein<b>-</b>conjugated 10-kDa dextran and euthanized 30 minutes later. (<i>A</i>) The small intestines were immunostained with rabbit anti-chromogranin A (green) and rat anti-Cld4 (HKH-189) (blue) antibodies. Characteristic rhodamine<b>-</b>labeled cylindrical columns (red) in association with a Cld4⁺ chromogranin A⁺ cells (closed arrowheads) and a Cld4⁻ chromogranin A⁻ cells (arrows) are indicated. Bar, 20 µm. Asterisks (*) indicate luminal space. (B, C) Single cell suspensions of the small intestinal cells from control mice and those orally administered with fluorescein-dextran 30 minutes before were multi-color analyzed with FACS. The expression profiles of fluorescein-dextran in Cld4^– (dark blue boxes) and Cld4⁺ (red boxes) cells (B) and in Cld4^–UEA-1⁺ (orange boxes), Cld4⁺UEA-1⁺ (yellow boxes), Cld4^–UEA-1^– (light blue boxes) and Cld4⁺UEA-1^– (pink boxes) cells (C) of a PI^– CD45^– Ter119^– EpCAM⁺ cell gate are indicated. Black and green lines in the histograms indicate the profiles of control and dextran-administered mice, respectively. Percentages of fluorescence-positive populations are shown. The results are representative of at least three independent experiments.</p

Cld4 expression and UEA<i>-</i>1 reactivity define the distinct enteroendocrine cell populations.

<p>(<i>A</i>) FACS profiles of biotinylated anti-Cld4 antibody (HKH-189) staining and UEA-1 binding in CD45⁻Ter119⁻PI⁻EpCAM⁺ intestinal epithelial cells from <i>Cldn4</i>^+/+ and <i>Cldn4</i>^−/− mice are indicated. (<i>B</i>, <i>C</i>) Each epithelial cell population of four quadrant fractions in (<i>A</i>) of <i>Cldn4</i>^+/+ mice was sorted separately with FACSAria and examined for the expressions of the indicated genes with quantitative RT<b>-</b>PCR. The mean relative transcripts and standard errors are shown. The results are representative of at least three independent experiments. *<i>P</i> < 0.01 and **<i>P</i> < 0.001, ANOVA.</p