Multi-omics approach to investigate the immune-cholangiocyte spatial relationship to stratify liver diseases

促炎细胞因子IL-17（白细胞介素-17）的参与与多种慢性炎症性疾病有关，损害伤口愈合并促进纤维化进展。IL 17水平的增加已经在广泛的慢性肝病中被鉴定，例如非酒精性脂肪肝病（NAFLD）、原发性胆汁性胆管炎（PBC）和原发性硬化性胆管炎（PSC）。据估计，慢性肝病影响全球8亿人，没有有效的抗纤维化治疗。众所周知，IL 17通过活化成纤维细胞促进纤维形成。然而，IL 17对上皮细胞命运和再生的作用尚不清楚，特别是最近的工作表明IL 17信号传导引发上皮细胞增殖以响应损伤，这与我们实验室的初步数据相吻合，该数据显示IL 17信号传导引发肝干细胞活化以响应肝损伤。我们以前已经表明，肝脏中的胆管细胞成为再生的肝干细胞，以响应损伤，但控制这一点的确切机制仍有待研究。此外，再生胆管细胞的出现往往与再生小生境周围的瘢痕沉积相关，再生小生境具有增殖上皮细胞、免疫细胞、成纤维细胞和瘢痕的混合物。目前还不清楚肝脏中IL 17细胞和信号的出现是一种保护机制还是人类疾病预后不良的指标。事实上，涉及全面阻断IL 17的治疗，如苏金单抗，正在进行银屑病的1/2期临床试验，然而，由于慢性肝病和病理学的广泛性，我们想知道是否“一种治疗适合所有人”？在这里，我们的目标是分析已发表的、公开可用的单细胞RNA（scRNA）测序数据集，这些数据集涵盖一系列具有不同病因的肝病，如肝硬化、NAFLD、PSC，并分析IL 17是否对再生胆管细胞有影响。此外，我们的目标是在空间上分析每种疾病对人类IL 17介导的免疫浸润的影响。这将在一系列肝脏疾病中对IL 17和胆管细胞的作用进行分层，并在转录和空间上对不同疾病中的胆管细胞和免疫细胞进行分类，从而回答IL 17是否使再生胆管细胞致病？为了补充来自临床样品的scRNA测序数据集的发现，我们将使用在胆管细胞中有条件地缺失IL 17受体的小鼠模型（Krt 19 CreERT 2 tdTomatoloxSTOPloxIL 17 Raflox）来抑制胆管细胞上的IL 17信号传导。在存在或不存在胆管细胞IL 17信号传导的情况下，将对转基因小鼠使用胆管或肝细胞损伤模型。将进行scRNA测序以研究胆管细胞上IL 17信号传导的缺乏是否改变肝脏中的免疫和再生景观。此外，该数据集将用于与公开可用的人类数据集进行匹配，以识别人类疾病中匹配的胆管细胞群。将对分离的鼠胆管细胞进行定性和定量蛋白质组学分析，以识别致病性与再生性蛋白质特征。 胆管细胞特异性IL 17抑制策略将显示IL 17信号传导是否响应于不同的损伤机制而不同（即胆道与肝脏），基于疾病病因学对IL 17靶向治疗进行分层，并补充泛IL 17阻断策略。我们的目标是努力实现总体目标，了解IL 17信号在慢性肝病中的作用-朋友还是敌人？更重要的是，导致基于病因的慢性肝病的潜在靶向治疗。

The involvement of proinflammatory cytokine IL17 (Interleukin-17) has been linked to multiple chronic inflammatory diseases, impairing wound healing and promoting fibrosis progression. The increase in IL17 level has been identified in a wide range of chronic liver diseases such as Non-Alcoholic Fatty Liver Disease (NAFLD), Primary Biliary Cholangitis (PBC), and Primary Sclerosing Cholangitis (PSC). Chronic liver disease is estimated to affect 800 million people worldwide with no effective anti-fibrotic treatments. It is well known that IL17 promotes fibrogenesis through the activation of fibroblasts. However, the role of IL17 on epithelial cell fate and regeneration is less clear, especially with recent work suggesting that IL17 signalling primes epithelial proliferation in response to injury, fitting with preliminary data in our lab showing that IL17 signalling primes liver stem cell activation in response to liver injury. We have shown previously that cholangiocytes in the liver become regenerative liver stem cells in response to injury but the exact mechanisms which control this remains to be investigated. Furthermore, the emergence of the regenerative cholangiocytes tends to correlate with scar deposition around the regenerative niche which has a mixture of proliferating epithelial cells, immune cells, fibroblasts, and scar. It is unclear whether the emergence of IL17 cells and signalling in the liver is a protective mechanism or an indicator of poor prognosis in human diseases. Indeed, treatments involving pan-blockade of IL17 such as Secukinumab are on Phase 1/2 clinical trials for psoriasis, however, with the wide spectrum of chronic liver diseases and pathology, we wonder whether "one treatment fits all"?Here, we aim to analyse published, publicly available single cell RNA (scRNA) sequencing datasets across a spectrum of liver diseases with variable aetiology such as cirrhosis, NAFLD, PSC and analyse whether IL17 has implications on regenerative cholangiocytes. Furthermore, we aim to spatially analyse the implications each disease has on IL17-mediated immune infiltrates in humans. This will stratify the role of IL17 and cholangiocytes across a spectrum of liver diseases and characterise the cholangiocytes and immune cells in different diseases transcriptionally and spatially, answering whether IL17 makes regenerative cholangiocytes pathogenic? To complement the findings from scRNA sequencing datasets from clinical samples, we will use a mouse model where IL17 receptor is conditionally deleted in the cholangiocytes (Krt19CreERT2tdTomatoloxSTOPloxIL17Raflox) to inhibit IL17 signalling on cholangiocytes. Models of biliary or hepatocyte damage will be used on the transgenic mice in the presence or absence of cholangiocyte IL17 signalling. scRNA sequencing will be performed to investigate whether the absence of IL17 signalling on cholangiocytes alters the immune and regenerative landscape in the liver. Furthermore, this dataset will be used to match with the human datasets available publicly to identify matching cholangiocyte populations in human diseases. Qualitative & quantitative proteomics analysis will be performed on isolated murine cholangiocytes to identify pathogenic vs regenerative protein signatures. The cholangiocyte specific IL17 inhibition strategy will show whether IL17 signalling differs in response to different injury mechanisms (i.e biliary vs hepatic), stratifying IL17 targeting treatment based on disease aetiology and complementing the pan-IL17 blockade strategies.Dissecting the cell specific effect of IL17 on tissue regeneration and fibrosis, we aim to work towards the overarching aim to understand the role of IL17 signalling in chronic liver diseases - a friend or foe? More importantly, leading to potential targeted treatments for chronic liver disease based on aetiology.