Investigating gastric inflammation and preneoplastic progression driven by Helicobacter pylori infection

项目摘要 许多癌症归因于慢性炎症，慢性炎症可导致突变并激活致癌信号。 小路。一个重要的例子是胃癌，它是全球第四大癌症死亡原因。至少80% 的胃癌病例被归因于幽门螺杆菌(HP)的胃部感染，这种细菌会导致 终生慢性炎症，不能根除感染。在某些人中，这种炎症可能会导致 胃萎缩、化生(一种正常细胞类型转化为另一种细胞类型)、异型增生(存在异常 细胞)，最后是癌症，但幽门螺杆菌引发这种级联反应的具体机制(S)还不是很清楚 明白了。通过他莫昔芬诱导的小鼠模型可重现类似的癌前病变 活性KRAS在胃主细胞(KRAS+小鼠)中的表达。我发现KRAS+小鼠的Hp感染 疾病加重：与Hp-KRAS+小鼠相比，Hp+KRAS+小鼠的化生轨迹和 加速发育不良。Hp+KRAS+小鼠也有“变异”凹坑细胞(表面粘液细胞)的扩张 表达与化生和癌症相关的基因，如粘蛋白Muc4。根据惠普的假设 通过引发慢性炎症而致癌，HP+KRAS+小鼠患有严重的炎症，其特征是10- 与Hp-KRAS阳性小鼠相比，T细胞数量增加了一倍。在这个提案中，我将调查惠普(S)通过 加重KRAS+小鼠的疾病，更广泛的目标是更好地了解Hp感染及其与 慢性炎症会导致癌症。我推测幽门螺杆菌通过调节炎症反应来预防幽门螺杆菌 从胃中清除，这种有害的免疫反应会导致化生和异型增生。在目标1中，我 将有针对性地耗尽CD_4~+和CD_8α~+T细胞亚群，以评估免疫紊乱是否影响 化生、异型增生和Hp定植。我还将评估在HP+KRAS+中T细胞是否进入胃 老鼠或局部繁殖。最后，我将测试与HP-相比，HP+人类样本是否增加了T细胞。 样本。在目标2中，我将研究调节炎症的候选Hp毒力因子：CAG IV型 分泌系统和两种调节T细胞的蛋白质，VacA毒素和转肽酶GGT。我会决定 毒力因子突变是否能在KRAS+中诱导相同的炎症和疾病进展表型 像野生型幽门螺杆菌一样的老鼠。在目标3中，我将测试变异的凹陷细胞是否来自胃祖细胞，并将 评估HP+KRAS+MUC4-小鼠的疾病表型以确定Muc4表达是否驱动PIT细胞 转型。我还将在+/-HP，+/-KRAS小鼠中进行空间单细胞RNA测序，以发现 变异的PIT细胞与胃第一腺中表达Muc4的细胞有共同的基因表达特征，a 形态独特的腺体，被认为是修复性细胞谱系的来源。综合起来，结果是 这些研究将对幽门螺杆菌感染和炎症如何导致胃癌前病变提供新的理解 进展，并可能揭示胃免疫治疗的新靶点。这些发现也可能适用于其他 与细菌感染和/或炎症有关的癌症，如结直肠癌。

Project Summary Many cancers are attributed to chronic inflammation, which can cause mutations and activate oncogenic signaling pathways. An important example is gastric cancer, the fourth-leading cause of cancer death worldwide. At least 80% of gastric cancer cases are attributed to stomach infection with the bacterium Helicobacter pylori (Hp), which causes lifelong chronic inflammation that does not eradicate the infection. In some individuals, this inflammation can cause gastric atrophy, metaplasia (conversion of one normal cell type to another), dysplasia (presence of abnormal cells) and finally cancer, but the specific mechanism(s) through which Hp triggers this cascade are not well understood. Similar preneoplastic changes are recapitulated in a mouse model through tamoxifen-induced expression of active KRAS in the chief cells of the stomach (KRAS+ mice). I found that Hp infection of KRAS+ mice exacerbated disease: compared to Hp-KRAS+ mice, Hp+KRAS+ mice had an altered trajectory of metaplasia and accelerated dysplasia. Hp+KRAS+ mice also had expansion of “variant” pit cells (surface mucous cells) that expressed metaplasia- and cancer-related genes like the mucin Muc4. In accordance with the hypothesis that Hp causes cancer through eliciting chronic inflammation, Hp+KRAS+ mice had severe inflammation marked by a ten- fold increase in T cells vs. Hp-KRAS+ mice. In this proposal I will investigate the mechanism(s) through which Hp worsens disease in KRAS+ mice, with a broader goal of better understanding how Hp infection and its associated chronic inflammation cause cancer. I hypothesize that Hp modulates the inflammatory response to prevent Hp eradication from the stomach, and this deleterious immune response leads to metaplasia and dysplasia. In Aim 1 I will perform targeted depletion of CD4+ and CD8α+ T cell subsets to assess whether immune perturbation impacts metaplasia, dysplasia and Hp colonization. I will also assess whether T cells traffic to the stomach in Hp+KRAS+ mice or proliferate locally. Finally, I will test whether Hp+ human samples have increased T cells relative to Hp- samples. In Aim 2 I will investigate candidate Hp virulence factors that modulate inflammation: the cag type IV secretion system and two proteins that modulate T cells, the toxin VacA and the transpeptidase gGT. I will determine whether virulence factor mutants can elicit the same inflammatory and disease progression phenotypes in KRAS+ mice as wild-type Hp does. In Aim 3 I will test whether variant pit cells arise from gastric progenitor cells, and will assess disease phenotypes in Hp+KRAS+MUC4- mice to determine whether Muc4 expression drives pit cell transformation. I will also perform spatial single-cell RNA sequencing in +/- Hp, +/- KRAS mice to discover whether variant pit cells share a gene expression signature with Muc4-expressing cells in the “first gland” of the stomach, a morphologically distinct gland that is believed to be a source of reparative cell lineages. Taken together, the results of these studies will provide new understanding of how Hp infection and inflammation cause gastric preneoplastic progression and may reveal new targets for gastric immunotherapy. These findings may also be applicable to other cancers associated with bacterial infection and/or inflammation, like colorectal cancer.