Mucosal Immune Surveillance at the Taste Papillae

项目摘要 味蕾不断暴露在口腔和食源性微生物中，但这三种微生物背后的途径-- 味觉细胞、口腔微生物群和口腔上皮免疫细胞之间的相互作用方式尚未得到 仔细研究过了。虽然口腔中数百种微生物中的绝大多数是 无论对宿主是无害的还是有益的，口腔微生物群的失调都会导致味觉障碍。 在新冠肺炎、病毒性肝炎、流感和念珠菌病的病例中观察到与感染相关的味觉丧失， 举几个例子。粘膜，如肠道和扁桃体中的粘膜，具有次级淋巴组织。 粘膜相关淋巴组织(MALT)由特化上皮覆盖，称为滤泡相关 上皮细胞(FAE)。FAE含有被称为微折叠细胞(M细胞)的免疫监视细胞，它可以通过细胞糖 并将它们呈现给底层生发中心的免疫细胞，从而产生一种 适当的免疫反应。因此，M细胞是粘膜免疫和M调节失调的中心角色。 众所周知，细胞途径会导致感染。利用单细胞RNAseq，我们发现甜味受体 Von Ebner腺(VDC)的细胞(STRC)和导管细胞(VDC)，这是一种与味觉有关的小唾液腺 乳头，表达几个M细胞标志基因，包括M细胞所需的转录因子SPIB 发展和再生。使用RNAScope和双重标记证实了这些发现 用STRC等味觉细胞类型标记基因进行免疫组织化学染色。RANKL的管理，一种增长 M细胞再生所需的因子导致味觉乳头中M细胞标记基因的显著上调 来自野生型(WT)，但不是Spib基因敲除小鼠。我们假设STRC和VDC参与免疫 以与M细胞相同的方式在味觉乳头进行监视，这一途径中的扰动可能 会导致感染、发炎和味觉丧失。我们将通过彻底检查 应用定量聚合酶链式反应、RNAseq和RT-PCR检测味觉乳头中M细胞标志基因的表达 组织学技术，并通过确定RANKL在特定情况下触发M细胞增殖的能力 无病原体(SPF)和无菌(GF)WT小鼠和SPF SPIB条件性基因敲除小鼠(SpibCKO小鼠)和 品尝从他们那里培养出来的有机化合物。我们还将确定免疫细胞募集的变化对口味的影响 在上述所有三个品系/条件下的乳突。STRCs和VDC在体内和体内的能力 将通过定量荧光摄取来测量味觉有机物对跨细胞腔微生物的影响 标签纳米颗粒和绿色荧光蛋白在大肠杆菌中的表达。口味的变化 SpibCKO小鼠的敏感度将通过Gustometer中的简单访问品尝测试来确定。我们的研究 代表着对味觉生物学和免疫之间的交集的一次新的尝试，并有可能 加深对味觉乳头粘膜免疫监测的认识。成功完成这项工作 这项研究承诺帮助设计新的策略来治疗与感染、肥胖和衰老相关的味觉丧失。

Project Summary Taste buds are continually exposed to oral and food borne microbes, but the pathways underlying the three- way interactions between taste cells, the oral microbiome and oral epithelial immune cells have not been studied in sufficient detail. While the vast majority of the hundreds of microbial species in the oral cavity are either innocuous or beneficial to the host, dysregulation of the oral microbiome can cause taste disorders. Infection associated taste loss is observed in the cases of COVID-19, viral hepatitis, influenza, and candidiasis, to name a few examples. Mucosae such as those in the gut and tonsils possess secondary lymphoid tissues called mucosae associated lymphoid tissue (MALT) over laid by specialized epithelia call the follicle associated epithelium (FAE). FAE contain immune surveillance cells called microfold cells (M cells) that transcytose luminal microbes and present them to immune cells in the underlying germinal centers, that generate an appropriate immune response. Thus, M cells are central players in mucosal immunity, and dysregulation of M cell pathways are known to cause infection. Using single cell RNASeq, we discovered that sweet taste receptor cells (STRCs) and duct cells of the von Ebner gland (VDCs), a minor salivary gland associated with taste papillae, express several M cell marker genes, including Spib, a transcription factor required for M cell development and regeneration. These findings were confirmed using RNAScope and double label immunohistochemistry with STRC and other taste cell type marker genes. Administration of RANKL, a growth factor required for M cell regeneration led to dramatic upregulation of M cell marker genes in taste papillae from wild type (WT) but not Spib knock out mice. We hypothesize that STRCs and VDCs participate in immune surveillance at the taste papillae in the same manner as M cells, and that perturbances in this pathway might lead to infection, inflammation and taste loss. We will test this hypothesis by thoroughly examining the expression of M cell marker genes in taste papillae using quantitative polymerase chain reaction, RNASeq and histological techniques, and by determining the ability of the RANKL to trigger M cell proliferation in specific pathogen free (SPF) and germ free (GF) WT mice and SPF Spib conditional knockout mice (SpibCKO mice) and taste organoids cultured from them. We will also determine the changes in immune cell recruitment to the taste papillae in all three mouse strains/conditions described above. The ability of STRCs and VDCs in vivo and in taste organoids to transcytose luminal microbes will be measured by quantifying the uptake of fluorescently tagged nanoparticles and green fluorescent protein expressing Escherichia coli. The changes in taste sensitivity in SpibCKO mice will be determined using brief access taste test in a gustometer. Our study represents a novel foray into the intersection between taste biology and immunity and has the potential to deepen the understanding of mucosal immune surveillance at taste papillae. Successful completion of this study promises to help design novel strategies to treat taste loss associated with infection, obesity and aging.