Intestinal single cell analyses and population differences in innate immunity in Crohn's disease drive treatment response and clinical heterogeneity: towards Precision IBD

克罗恩病是一种慢性的、典型的进行性炎症，最常累及末端回肠。的 在200多个相关的遗传基因座中，最显著的三个克罗恩关联包括NOD2、IL23R和 PTGER4.我们提供了来自回肠组织和血液的单细胞RNASeq(ScRNASeq)数据，涉及超过100,000人 转录本和确定治疗难治性患者表达炎性单核细胞的亚群 吞噬细胞(信息MNP)模块。该模块包括炎性巨噬细胞、活化的成纤维细胞、成熟细胞 树突状细胞，以及活化的T细胞和产生免疫球蛋白的浆母细胞。在目标1中，我们将验证蛋白质 并提纯炎性单核巨噬细胞(inf.MNP)模块通过回肠组织的CITE-SEQ研究 和外周血液。我们将改进现有的细胞团分类，并提供更精细的蛋白质 通过CITE-SEQ进行验证，其中将对同一 获取其scRNASeq数据的单元格。通过这样做，这些新的CITE-SEQ研究将a)完善和验证 基于转录组的患者结局定义，b)细化关键细胞簇定义，用于相对不常见的 细胞(成纤维细胞、树突状细胞)，c)改善血液到组织的适应性映射(T、B和浆细胞) 免疫，以及d)优化PTGER4和IL23R基因表达，假设在治疗非霍奇金淋巴瘤中发挥主要作用 回应(目标3)。在目标2中，我们将定义由以下因素驱动的巨噬细胞-成纤维细胞的早期干扰 NOD2缺乏症，建立抗肿瘤坏死因子应答机制。NOD2是胞内对胞壁糖胺的感受器 二肽(MDP)，细菌肽多聚糖的最小生物活性成分。我们观察到了共同表达 NOD2阳性细胞表达CD14(血单核细胞标记物)，单个细胞高水平胶原蛋白； 这一关键发现提供了新的假设，即功能丧失的NOD2致病性部分是由故障驱动的 分化为居住巨噬细胞，有利于更多多能间质类型的细胞。在目标3中，我们寻求 利用IL23R和IL23R的细胞特异性基因表达加速精确克罗恩病的进展 PTGER4优先考虑抗肿瘤坏死因子难治患者的细胞和分子挽救机制。尽管 可观的反IL12/23救助，基本上没有反应。利用这一未得到满足的医疗需求，我们 将通过分析检测表达IL23R的免疫细胞与抗IL12/23临床应答的相关性 多个现有的和新收集的批量RNA数据集。我们假设炎性巨噬细胞 表达IL23R的细胞串扰介导对抗IL12/23阻断的反应，但通过 PTGER4诱导的原位成熟树突状细胞分化异常。抗IL-12/23抗体的比较分析 应答者与非应答者，将突出难治细胞和通路，以确定目标的优先顺序 确定优先顺序。这一建议结合了三大关联信号、尖端单细胞方法、 与当前未得到满足的医疗需求的领域，以促进对人类克隆氏病的机制基础的理解 疾病。

Crohn's disease is a chronic, typically progressive inflammation most commonly affecting the terminal ileum. Of the over 200 associated genetic loci, the three most significant Crohn's associations include NOD2, IL23R and PTGER4. We present single cell RNASeq (scRNASeq) data from ileal tissues and blood involving over 100,000 transcriptomes and define a subset of treatment refractory patients expressing an inflammatory mononuclear phagocyte (inf. MNP) module. This module includes inflammatory macrophages, activated fibroblasts, mature dendritic cells, as well as activated T cells and IgG producing plasmablasts. In Aim 1, we will protein validate and refine the inflammatory mononuclear phagocyte (inf. MNP) module through CITE-seq studies of ileal tissues and peripheral blood. We will improve on present cell cluster classifications and provide a more fine-scale protein validation through CITE-seq, where quantitative surface protein measurements will be performed on the same cells for which scRNASeq data are obtained. By so doing, these new CITE-seq studies will a) refine and validate transcriptome-based patient outcome definitions, b) refine key cell cluster definitions for relatively uncommon cells (fibroblasts, dendritic cells), c) improve blood to tissue mappings of adaptive (T, B, and plasma cells) immunity, and d) refine PTGER4 and IL23R gene expression, hypothesized to play major roles in treatment non- response (Aim 3). In Aim 2, we will define early perturbations in macrophage-fibroblast cross-talk driven by NOD2-deficiency and establish anti-TNF responsive mechanisms. NOD2 is an intracellular sensor of muramyl dipeptide (MDP), the minimal bioactive component of bacterial peptidoglycan. We have observed co-expression of NOD2 with cells expressing CD14 (blood monocyte marker) and high levels of collagens in individual cells; this key finding informs the novel hypothesis that loss-of-function NOD2 pathogenicity is partly driven by a failure to differentiate into residential macrophages, favoring more pluripotent stromal-type cells. In Aim 3, we seek to accelerate progress towards precision Crohn's disease by leveraging cell-specific gene expression of IL23R and PTGER4 to prioritize cellular and molecular salvage mechanisms in anti-TNF refractory patients. Despite appreciable anti-IL12/23 salvage, substantial non-response remains. Leveraging this unmet medical need, we will test for correlation of IL23R-expressing immune cells to anti-IL12/23 clinical response through analyses of multiple existing and newly-collected bulk RNA datasets. We hypothesize that inflammatory macrophage to IL23R-expressing cell cross talk mediates response to anti-IL12/23 blockade, but leaves pathogenicity via aberrant in situ mature dendritic cell differentiation via PTGER4. These comparative analysis of anti-IL12/23 responders vs. non-responders, will highlight refractory cells and pathways to be prioritized for target prioritization. This proposal combines the three major association signals, cutting-edge single cell approaches, with current areas of unmet medical needs to advance understanding of the mechanistic basis for human Crohn's disease.