MDSC Polarization and Helicobacter-Induced Gastric Metaplasia

摘要 慢性螺杆菌感染及其引起的炎症诱导WT胃体化生 小鼠，而不是在小鼠的Gli 1无效，表明这种癌前病变需要诱导 典型的Hedgehog信号此外，我们还报道了一个表达表面活性剂的髓样细胞亚群， 指示髓源性抑制细胞（MDSC）表达的标志物和T细胞抑制功能 Schlafen 4（Slfn 4），Gli 1转录因子的直接靶点，也是一种已知的髓样分化因子。 由于MDSC是未成熟的细胞，我们的研究表明，这种髓样细胞的成熟 亚群需要Gli 1并产生促炎细胞因子，从而创造胃微环境 有利于化生和肿瘤转化。最近，我们分析了人类 Slfn 4的同源物，其包括SLFN 5和SLFN 12 L。我们报道了SLFN 5在胃粘膜中的高峰表达， 组织发生在患有肠上皮化生的人类受试者中， 癌因此，我们认为髓系细胞向MDSC的极化先于肿瘤发生， 转化可能预测谁更有可能患胃癌，因此可以提供一个 治疗目标，以防止未来的转变。我们使用RNA-Seq和Nanostring微阵列来识别 来自螺杆菌感染小鼠胃的Slfn 4 +-MDSC中表达的转录物和microRNA 并发现miR 130 b与Slfn 4+细胞共定位于化生小鼠胃中。了类似的结果 在化生的人胃中观察到SLFN 12 L，这表明miR 130 b可能鉴定出患有 胃上皮化生我们的初步结果表明，Slfn 4和miR 130 b是发挥T细胞功能所必需的。 镇压除了通过RNA-Seq鉴定的1型干扰素调节基因外，这些Slfn 4 +-MDSC还 表达几种肿瘤坏死因子超家族配体（TNF sf）和Alarmin IL-1a。因此我们将 测试来自受损胃上皮细胞的碎片激活损伤激活分子的假设， 模式（DAMP）信号传导、IFNa的产生和向MDSC的极化，这有助于化生。 表型目的1：明确DAMP信号如何诱导Slfn 4 +-MDSC的极化。在目标2中，我们将 定义Slfn 4如何有助于MDSC功能。在目标3中，我们将定义Slfn 4 +-MDSC对以下的贡献： 螺杆菌引起的化生。在目标1中，我们将使用流式细胞术、T细胞抑制 鉴定产生IFNa的细胞群和基因靶点的测定和转染研究 对这种DAMP激活的细胞因子产生反应。在目标2中，我们将使用质谱法和下拉分析法 以鉴定Slfn 4和SLFN 12 L相互作用蛋白。在目标3中，我们将有条件地从Gli 1中删除Slfn 4。 表达细胞，以确定它们对螺杆菌感染后观察到的化生变化的贡献。 这些目标的完成将使我们更好地了解MDSC亚群， 可用作小分子抑制剂的生物标志物和靶点。

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.