COVID-19 and lung disease: developing therapy through immune cell re-programming (Ref: 3949)

SARS-CoV-2呼吸道感染可导致轻度或重度疾病（COVID-19）。年龄是一个关键的风险因素，此外还有男性和高血压和糖尿病等合并症。同样的风险也是其他严重、不可治愈和致命的肺部疾病的关键，如肺纤维化（PF）和慢性阻塞性肺疾病（COPD）。新出现的数据表明，COVID-19可导致肺瘢痕形成（即PF），患有PF的患者因COVID-19死亡率更高。PF已经占英国所有死亡人数的1%，而COPD是全球第三大杀手。（超过500，000名参与者的遗传，生物标志物和健康数据资源）揭示了PF和COPD中免疫细胞数量和端粒磨损的有趣关联（接受发表在《柳叶刀呼吸医学》上）;我们正在我们的联合Exeter-Bristol患者队列中随访其中的一些发现。免疫细胞库也是COVID-19的一个主要研究焦点。巨噬细胞在防御病原体方面发挥着关键作用，但也可能导致疾病发展-尤其是通过过度产生炎性细胞因子（在PF和COVID-19中均可见）-这与线粒体功能有关。线粒体是细胞活动的关键调节器，作为病毒感染的细胞内信号“枢纽”，并通过炎性小体介导细胞对炎症的反应;它们也是SARS-CoV蛋白的靶点。有效的免疫力随着年龄的增长而下降，导致免疫衰老和慢性低度炎症。我们最近的两个MRC奖项（总计> 150万英镑）专注于COPD和PF背景下的线粒体。我们有令人兴奋的数据显示，Whiteman教授开发的新型药物可以产生生理剂量的靶向硫化氢（H2S），可以改善实验系统中的促炎和促纤维化反应。线粒体是一种控制先天免疫活性的新型药物靶点。林赛教授最近的工作进一步关注了长链非编码RNA（lncRNA）在炎症反应调节中的作用，而Scotton博士小组的初步数据表明，过继转移人类可以加剧小鼠模型中的纤维化疾病。我们的假设是，我们的新型H2S药物可以改变线粒体功能障碍，以改变细胞代谢和转录组，减少过度活化并促进组织修复。这些过程将与PF和COVID-19中的功能中断有关。目的有三：1）比较PF患者单核细胞源性巨噬细胞的lncRNA/mRNA转录组和表型，使用RNA-Seq、生物信息学和标准体外测定，（例如细胞因子ELISA、流式细胞术、趋化性和吞噬作用）：a）炎性刺激物，包括热灭活的SARS-CoV-2病毒颗粒和刺突蛋白，和B）巨噬细胞极化，例如M（LPS），M（IL 4）。2）在用LPS、IL-4或SARS-CoV-2/刺突蛋白刺激的人中，探究H2S药物补充对巨噬细胞功能和lncRNA水平的功能后果。诱导的多能干细胞衍生的肺泡也将用于增加与肺的相关性。3）在PF的实验性体内模型中确定在过继转移到免疫受损的NOD/SCID小鼠中后年轻人与老年人的纤维化潜力，以及我们的H2S药物的治疗益处。最终，我们希望将这些新药从实验室转化为临床应用。方法学：RNASeq; qRT-PCR; ELISA法;表型/功能的流式细胞术; Seahorse细胞外通量分析; PF的实验小鼠模型;肺胶原积聚的荧光标记测量的体内成像系统（IVIS）跟踪和离体微型计算机断层扫描

SARS-CoV-2 infection of the respiratory tract can lead to mild or severe disease (COVID-19).Ageing is a crucial risk factor, alongside male sex and co-morbidities such as hypertension and diabetes. The same risks are key in other serious, incurable and fatal lung diseases such as pulmonary fibrosis (PF) and chronic obstructive pulmonary disease (COPD). Emerging data suggest that COVID-19 can lead to lung scarring (i.e. PF), and patients who have PF have greater mortality from COVID-19. PF already accounts for 1% of all UK deaths, while COPD is the 3rd biggest killer globally.Our recent analysis using the UK Biobank (a resource of genetic, biomarker and health data for over 500,000 participants) has revealed intriguing associations with immune cell numbers and also telomere attrition in PF and COPD (accepted for publication in the Lancet Respiratory Medicine); we are following up some of these findings in our joint Exeter-Bristol patient cohorts. Immune cell repertoires are also a major research focus in COVID-19.Macrophages play a key role in defence in against pathogens, but can also contribute to disease development - not least via the overproduction of inflammatory cytokines (seen in both PF and COVID-19) - and this is linked to mitochondrial function. Mitochondria are key regulators of cellular activity, act as intracellular signalling 'hubs' for viral infection, and mediate the cellular response to inflammation through the inflammasome; they are also targets for SARS-CoV proteins.Effective immunity deteriorates with age, leading to immunosenescence and chronic low-grade inflammation. Our two recent MRC awards (totalling >£1.5m) focus on mitochondria in the context of COPD and PF. We have exciting data showing that novel drugs developed by Prof Whiteman, which generate physiological doses of mitochondrial-targeted hydrogen sulfide (H2S), can ameliorate pro-inflammatory and pro-fibrotic responses in experimental systems. mitochondria are a novel drug target to control innate immune activity.Recent work from Prof Lindsay has further focused on the role of long non-coding RNA (lncRNA) in the regulation of inflammatory responses in while preliminary data from Dr Scotton's group has demonstrated that adoptive transfer of human can exacerbate fibrotic disease in murine models. Our hypothesis is that mitochondrial dysfunction in can be modified by our novel H2S drugs to alter cellular metabolism and transcriptome, reducing hyper-activation and facilitating tissue repair. These processes will be relevant for disrupted function in both PF and COVID-19. There are 3 Aims:1) Characterise the lncRNA/mRNA transcriptome and phenotype of monocyte-derived macrophages from patients with PF versus age-matched (60 year old) and young controls using RNA-Seq, bioinformatics, and standard in vitro assays (e.g. cytokine ELISA, flow cytometry, chemotaxis, and phagocytosis) during the response to: a) inflammatory stimuli, including heat-inactivated SARS-CoV-2 viral particles and Spike protein, and b) macrophage polarisation e.g. M(LPS), M(IL4).2) Interrogate the functional consequence of H2S drug supplementation on macrophage function and lncRNA levels in human stimulation with LPS, IL-4 or SARS-CoV-2/Spike protein. Induced pluripotent stem cell-derived alveolar will also be used to increase relevance to the lung.3) Determine the fibrogenic potential of young vs-old human following adoptive transfer into immunocompromised NOD/SCID mice in an experimental in vivo model of PF, and the therapeutic benefit of our H2S drugs. Ultimately, we would like to translate these novel drugs from bench to bedside. Methodology: RNASeq; qRT-PCR; ELISA; flow cytometry of phenotype/function; Seahorse extracellular flux analysis; experimental mouse model of PF; In Vivo Imaging System (IVIS) tracking of fluorescently-tagged measurement of lung collagen accumulation and ex vivo micro- Computed Tomography