Mucosal Immune Surveillance at the Taste Papillae

项目摘要 味蕾不断暴露于口腔和食物传播的微生物，但这三种微生物的潜在途径- 味觉细胞、口腔微生物组和口腔上皮免疫细胞之间的相互作用方式尚未被证实。 研究得足够详细。虽然口腔中的数百种微生物中的绝大多数是 口腔微生物组的失调对宿主无害或有益，可引起味觉障碍。 在COVID-19、病毒性肝炎、流感和念珠菌病的病例中观察到感染相关的味觉丧失， 举几个例子。粘膜如消化道和扁桃体中的粘膜具有次级淋巴组织 称为粘膜相关淋巴组织（MALT），其上覆盖着称为滤泡相关淋巴组织的特化上皮细胞。 上皮（FAE）。FAE含有称为微折叠细胞（M细胞）的免疫监视细胞， 并将它们呈递给下面的生发中心的免疫细胞， 适当的免疫反应。因此，M细胞是粘膜免疫的中心参与者，并且M细胞的调节异常 已知细胞途径会引起感染。使用单细胞RNASeq，我们发现甜味受体 细胞（STRC）和vonEbner腺（VDCs）的导管细胞，VDCs是一种与味觉相关的小唾液腺 乳头，表达几种M细胞标记基因，包括Spib，M细胞所需的转录因子 发展和再生。这些发现证实了使用RNAScope和双标记 用STRC和其他味细胞类型标记基因进行免疫组织化学分析。给予RANKL，一种生长因子 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）而不是Spib敲除小鼠。我们假设STRC和VDC参与免疫调节， 在味觉乳头的监视在相同的方式作为M细胞，并在这一途径的扰动可能 导致感染、炎症和味觉丧失。我们将通过彻底检查 使用定量聚合酶链反应，RNASeq和 组织学技术，并通过确定RANKL在特定的细胞中触发M细胞增殖的能力， 无病原体（SPF）和无菌（GF）WT小鼠和SPF Spib条件性敲除小鼠（SpibCKO小鼠），以及 品尝从中培养的类器官。我们还将确定免疫细胞募集的变化来品尝 上述所有三种小鼠品系/条件下的乳头。STRC和VDC在体内和体内的能力 将通过定量荧光物质的摄取来测量类味觉器官对转胞吞管腔微生物的作用。 标记的纳米颗粒和表达绿色荧光蛋白的大肠杆菌。口味的变化 SpibCKO小鼠中的敏感性将使用味觉计中的短暂接触味觉测试来确定。我们的研究 代表了味觉生物学和免疫之间的交叉点的新尝试，并有可能 加深对味觉乳头粘膜免疫监视的认识。成功完成本 这项研究有望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.