MDSC Polarization and Helicobacter-Induced Gastric Metaplasia

摘要 慢性幽门螺杆菌感染和由此引起的炎症诱导WT小体胃化生 小鼠，而不是在小鼠中Gli1为空，表明这种癌前病变需要诱导 典型的刺猬信号。此外，我们报告了一组表达表面的髓系细胞 髓系抑制细胞(MDSCs)表达的标志和T细胞抑制功能 Schlafen4(Slfn4)，Gli1转录因子的直接靶标，是一种已知的髓系分化因子。 由于MDSCs是未成熟的细胞，我们的研究共同证明了这种髓系细胞的成熟 亚群需要Gli1，并产生促炎细胞因子，创造胃微环境 有利于化生和肿瘤性转化。最近，我们分析了人类 SLFN4的同源物，包括SLFN5和SLFN12L。我们报道了SLFN5在胃中的表达高峰 组织出现在患有肠化生的受试者中，这些受试者在大约10年后发展为胃 癌症。因此，我们认为在肿瘤发生之前，髓系细胞对MDSCs的极化 转化可能预测谁更有可能患上胃癌，因此可能提供 治疗目标，以防止未来的转化。我们使用RNA-Seq和纳米串微阵列来鉴定 幽门螺杆菌感染小鼠胃SLFn4+-MDSCs的转录产物和microRNAs的表达 并发现miR130b与SLFn4+细胞在化生小鼠胃中共定位。类似的结果是 在化生组织中观察到SLFN12L提示miR130b可能识别出 胃化生。我们的初步结果表明，SLFn4和miR130b是发挥T细胞所必需的 压制。除了RNA-Seq鉴定的1型干扰素调节基因外，这些SLFn4+-MDSCs还 表达多种肿瘤坏死因子超家族配体(TNFsf)和ALARMIN IL-1a。因此，我们将 检验损伤胃上皮细胞碎片激活损伤激活分子的假说 模式(DAMP)信号、IFNA的产生和对MDSCs的极化，这些都有助于化生 表型。目标1，我们将定义潮湿信号如何诱发SLFn4+-MDSC的极化。在目标2中，我们将 定义SLFN4对MDSC功能的贡献。在目标3中，我们将定义SLFN4+-MDSCs对 幽门螺杆菌诱导的化生。在目标1中，我们将结合使用流式细胞术、T细胞抑制 鉴定产生干扰素的细胞群和基因靶点的检测和转染研究 对这种潮湿激活的细胞因子做出反应。在目标2中，我们将使用质谱分析和下拉分析 鉴定SLFN4和SLFN12L相互作用蛋白。在目标3中，我们将有条件地从Gli1-中删除slfn4- 表达细胞以确定它们在幽门螺杆菌感染后观察到的化生变化中的作用。 完成这些目标将导致对这一MDSC亚群的更好理解，这可能 可作为生物标记物和小分子抑制剂的靶点。

Abstract Chronic Helicobacter infection and the resulting inflammation induces gastric metaplasia in the corpus of WT mice but not in mice null for Gli1, demonstrating that this pre-neoplastic lesion requires the induction of canonical Hedgehog signaling. Moreover, we reported that a subset of myeloid cells expressing surface markers and T cell suppressor function indicative of myeloid-derived suppressor cells (MDSCs) express Schlafen4 (Slfn4), a direct target of the Gli1 transcription factor and a known myeloid differentiation factor. Since MDSCs are immature cells, collectively our studies demonstrate that maturation of this myeloid cell subpopulation requires Gli1 and produces proinflammatory cytokines creating a gastric microenvironment favorable for metaplasia and neoplastic transformation. More recently, we have analyzed the human homologs of Slfn4, which include SLFN5 and SLFN12L. We reported that peak expression of SLFN5 in gastric tissue occurred in human subjects with intestinal metaplasia who about a decade later developed gastric cancer. We therefore considered that the polarization of myeloid cells to MDSCs prior to neoplastic transformation might predict who is more likely to develop gastric cancer and as such could provide a therapeutic target to prevent future transformation. We used RNA-Seq and Nanostring microarrays to identify transcripts and microRNAs expressed in Slfn4+-MDSCs from the stomachs of a Helicobacter-infected mouse and found that miR130b co-localized with Slfn4+ cells in the metaplastic mouse stomach. A similar result was observed for SLFN12L in the metaplastic human stomach suggesting that miR130b might identify patients with gastric metaplasia. Our preliminary results demonstrated that Slfn4 and miR130b are required to exert T-cell suppression. In addition to type 1 interferon-regulated genes identified by RNA-Seq, these Slfn4+-MDSCs also expressed several tumor necrosis factor superfamily ligands (TNFsf) and the alarmin IL-1a. Therefore we will test the hypothesis that debris from damaged gastric epithelial cells activates Damage-activated molecular pattern (DAMP) signaling, production of IFNa and polarization to MDSCs, which contribute to a metaplastic phenotype. Aim 1, we will define how DAMP signals induce polarization of Slfn4+-MDSC. In Aim 2, we will define how Slfn4 contributes to MDSC function. In Aim 3, we will define the contribution of Slfn4+-MDSCs to Helicobacter-induced metaplasia. In Aim 1, we will use a combination of flow cytometry, T cell suppression assays and transfection studies to identify the cell populations producing IFNa and the gene targets responding to this DAMP-activated cytokine. In Aim 2, we will used mass spectrometry and pull down assays to identify Slfn4 and SLFN12L interacting proteins. In Aim 3, we will conditionally delete Slfn4 from Gli1- expressing cells to define their contribution to the metaplastic changes observed after Helicobacter infection. Completion of these aims will result in a better understanding of this MDSC subpopulation that can potentially be used as a biomarker and target of small molecule inhibitors.