Mechanisms of immunopathology of COVID-19/ARDS, and strategies to mitigate detrimental inflammatory responses

COVID-19/ARDS 的免疫病理学机制以及减轻有害炎症反应的策略

• 批准号: 10692246
• 负责人: Sonja Best
• 金额: $ 20.47万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10692246
• 关键词: 2019-nCoV; ACE2; Acute; Affect; Age; Alveolar; Animals; Attention; Biological Products; Body Weight decreased; Brain; Bypass; CCL2 gene; CCL7 gene; COVID-19; COVID-19 pandemic; COVID-19 treatment; COVID-19/ARDS; CXCL10 gene; Cells; Central Nervous System Diseases; Cessation of life; Clinical; Colchicine; Combined Modality Therapy; Complement; Coronavirus; Data; Disease; Disease Resistance; Dose; Early treatment; Employment; Equilibrium; Event; Genes; Genetic; Genetic Variation; Genetically Engineered Mouse; Goals; Host Defense Mechanism; House mice; Human; Image; Immune; Immune mediated destruction; Immune response; Immunity; Inbred BALB C Mice; Infection; Inflammatory; Inflammatory Infiltrate; Inflammatory Response; Influenza; Inosine; Interferon-beta; Interferons; Interleukin-10; Interleukin-6; Intervention; K-18 conjugate; Laboratories; Long COVID; Lung; Lung infections; Mediating; Metabolic; Methods; Modeling; Molecular; Morbidity - disease rate; Mouse Strains; Mus; Myeloid Cells; Natural Immunity; Neutrophil Infiltration; Nose; Organ; Oseltamivir; Pathogenesis; Pathologic; Pathology; Play; Predisposition; Process; Protocols documentation; Publications; RANTES; Recovery of Function; Residual state; Resistance; Role; Route; SARS-CoV-2 infection; Sex Bias; Sex Differences; Signal Transduction; Stains; Structure; Structure of parenchyma of lung; T-Lymphocyte; Testing; Thrombus; Time; Tissues; Transgenes; Transgenic Mice; Transgenic Model; Tumor-infiltrating immune cells; Vaccination; Viral; Viral Pneumonia; Virus; Virus Replication; Work; Zileuton; adaptive immune response; adaptive immunity; base; brain fog; cell type; clinical effect; cytokine; disease phenotype; efficacy testing; human disease; human model; immunopathology; inflammatory marker; influenza infection; inorganic phosphate; lung imaging; lung lobe; male; mortality; mouse model; multiplexed imaging; neutralizing monoclonal antibodies; pneumocyte; post SARS-CoV-2 infection; preservation; prevent; repaired; response; sample fixation; severe COVID-19; sex; success; surfactant; targeted treatment; therapeutic development; tool; transcriptome sequencing

The SARS-CoV-2 global pandemic has a continuing demand for greater understanding of the mechanisms of disease and the development of therapeutics to complement success in vaccination. Several mouse models have emerged that can be used, albeit with limitations, as models of severe SARS-Cov2 infection. Therefore we have initiated two major efforts to employ mouse models that can be utilized in the efforts to understand dysregulation of innate and adaptive immunity associated with severe viral pneumonia. The ultimate goal is to define these processes as they relate to SARS-CoV-2 to identify points of intervention that can be targeted therapeutically. The first major initiative is to develop mouse models of SARS-CoV-2 infection that model human disease. To achieve this, we have partnered with Jackson Laboratories to genetically engineer mice that express a humanized ACE2 gene to enable virus replication in tissues. We have tested 4 separate strategies to humanize ACE2 at the endogenous locus, or as a transgene. We have also tested mouse backgrounds for susceptibility. The ultimate goal will be to fully characterize the host responses as they relate to pathology in these models, and then utilize the models for testing biologics that block various events in inflammatory cascades. During this year, we finalized establishing 12 models of SARS-CoV-2 infection in 10 strains of mice. In humans, the range of disease phenotypes observed is extreme, from asymptomatic to critical, and is dependent on age, sex, genetics and metabolic status. The 10 strains of mice used included the 8 founder strains of the Collaborative Cross (B6, A/J, 129SJ, NZO, NOD, PWK, CAST and WSB) with additional strains Balb/c and DSB. Together, these strains represent over 90% of the genetic diversity within Mus musculus and have enabled us to model distinct disease phenotypes including a) sensitive mice with high sustained virus replication in lung and CNS (B6,A/J), b) resistant mouse strains associated with lower peak virus titer and earlier control of replication in the lung with no or low dissemination to other organs (PWK, NZO), and c) sex bias where resistance is independent of virus titer in the lung suggesting a sex-differences in host response (CAST, NOD, WSB). Cytokine analysis in the BAL revealed that resistance to disease in males was associated with high IFNb expression at 3dpi. Cytokine profiles generally modeled human responses with lethality associated with sustained high IP-10, as well as increasing MCP3, TNFa, IL-10, RANTES, IFNg and IL1b. Taken together, these mouse models represent a powerful tool to understand mechanisms of immune-mediated control and pathology following SARS-CoV-2 infection. The work is available as a pre-print and is being finalized for publication to include RNAseq data from lungs and brain of all models. We have developed a protocol for safe fixation and subsequent multiplex imaging of the lungs of SAR-COv2 infected animals. Remarkably, in the K18-hACE2 transgenic mouse model of SARS-CoV-2 lethality, lungs on day two after infection showed almost no inflammatory infiltrate and no evidence of type 1 interferon signaling, whereas the influenza-infected animals showed robust innate immune cell infiltrates and interferon signaling at this time point. This reinforces existing evidence that coronaviruses potently suppress type 1 interferon responses and markedly change the inflammatory process. We are pursuing these observations across a more complete time course, using more markers to identify cell types and cell states, to better understand how these changes in innate immunity affect later adaptive responses and also if the discoordination of viral spread and innate immunity plays a special role in pathogenesis. The second major initiative is the employment of a lethal influenza infection as a model for severe viral pneumonia.. Ongoing studies involve (i) tests of interventions in the lethal influenza model that might have clinical utility and (ii) molecular, cell, and tissue level studies aimed at better understanding the underlying mechanism(s) of tissue damage and why interventions that constrain viral replication or innate immunity often fail after an early point in infection but well before death of the host. Using a severe influenza infection model that bypasses early nasopharyngeal replication and leads to rapid deep lung infection, we found that only very early treatment with the anti-viral oseltamivir phosphate (Tamiflu) could prevent death. Among 50 single or combined treatments covering many of the agents tested or used clinically for COVID-19 treatment (anti-IL-6, PANAM-G3, PMX205, inosine Pranobex, anti-PSGL1, ruxolitinib, inbrutinib, acalabrutinib, dypridamole, baricitinib, colchicine, silvelestat, AZD5059, anti-IL-6R, anti-CCL2, and Zileuton among others), none reduced weight loss or led to survival of any of the infected animals, and several worsened disease. These findings argue that either (i) multiple damaging activities are involved and blunting only one is insufficient for a clinical effect, and/or (ii) that irreversible tissue damage occurs early and once this occurs, interfering independently with viral replication or host immunity does not play a major role in preventing eventual death. Imaging of whole lung lobes using our IBEX method for multiplex staining showed that in this influenza infection model, there was early infiltration by neutrophils, extensive spread of the virus, loss of pro-surfactant and associated type 2 pneumocytes, alveolar disruption, and myeloid cell bronchiolar plugging, followed by later arrival of T cells in concert with marked loss of viable lung tissue. While some treatments modified the balance and extent of cell infiltrates, none prevented the damage and parenchymal loss. From these data, we developed the hypothesis that the infected animals rapidly pass a tipping point with respect to residual functional pulmonary capacity and that after this point, interference with inflammatory processes alone is insufficient to rescue the animals. This led to a change in strategy based on combining arrest of further damage and promoting recovery of functional lung structures. Preliminary data indicate that the combination of low dose Tamiflu administered late in the course of infection in combination with one of two additional treatments that either promote pneumocyte replication and alveolar repair or limit further immune destruction can rescue mice from death. These findings prompted testing in the K18-hACE2 transgenic model of SARS-Cov2 infection. Using anti-SARS-Cov2 neutralizing mAb as a substitute for Tamiflu, we ascertained a dose that resulted in 50% survival of animals when administered several days after infection. Initial studies have provided intriguing data. Because CNS disease is a major part of the pathology in the K18-hACE2 model when using nasal infection, our findings may have applicability to the brain fog of long COVID and this is a new direction of study, while at the same time, we are modifying the route of infection to tracheal instillation to better mimic the influenza model and provide a suitable test of the efficacy of the two additional interventions that proved successful in the lethal influenza model.

SARS-CoV-2 全球大流行不断需要更好地了解疾病机制和开发治疗方法，以补充疫苗接种的成功。已经出现了几种小鼠模型，尽管存在局限性，但可以用作严重 SARS-Cov2 感染的模型。因此，我们发起了两项重大努力，以利用小鼠模型来了解与严重病毒性肺炎相关的先天性和适应性免疫失调。最终目标是定义与 SARS-CoV-2 相关的这些过程，以确定可作为治疗目标的干预点。 第一个重大举措是开发模拟人类疾病的 SARS-CoV-2 感染小鼠模型。为了实现这一目标，我们与 Jackson Laboratories 合作，对小鼠进行基因改造，使其表达人源化 ACE2 基因，从而使病毒能够在组织中复制。我们测试了 4 种不同的策略，在内源基因座上人源化 ACE2，或作为转基因。我们还测试了小鼠背景的敏感性。最终目标是充分表征与这些模型中的病理学相关的宿主反应，然后利用这些模型来测试阻止炎症级联中各种事件的生物制剂。 今年，我们最终在 10 种小鼠中建立了 12 个 SARS-CoV-2 感染模型。在人类中，观察到的疾病表型范围很广，从无症状到危重，并且取决于年龄、性别、遗传和代谢状态。使用的 10 种小鼠包括 Collaborative Cross 的 8 个创始品系（B6、A/J、129SJ、NZO、NOD、PWK、CAST 和 WSB）以及其他品系 Balb/c 和 DSB。这些品系代表了小家鼠内超过 90% 的遗传多样性，使我们能够模拟不同的疾病表型，包括 a) 在肺部和中枢神经系统中具有高度持续病毒复制的敏感小鼠 (B6,A/J)，b) 与较低峰值病毒滴度和早期控制肺部复制相关的抗性小鼠品系，而没有或很少传播到其他器官（PWK、NZO），以及 c) 性别偏见，其中耐药性与肺部病毒滴度无关，表明宿主反应存在性别差异（CAST、NOD、WSB）。 BAL 中的细胞因子分析表明，雄性对疾病的抵抗力与 3dpi 时的高 IFNb 表达相关。细胞因子谱通常模拟了与持续高 IP-10 以及增加的 MCP3、TNFa、IL-10、RANTES、IFNg 和 IL1b 相关的人类反应和致死率。总而言之，这些小鼠模型是了解 SARS-CoV-2 感染后免疫介导控制和病理机制的强大工具。该工作已作为预印本提供，并正在最终确定出版，其中包括来自所有模型的肺部和大脑的 RNAseq 数据。 我们开发了一种对 SAR-COv2 感染动物的肺部进行安全固定和后续多重成像的方案。值得注意的是，在 SARS-CoV-2 致死性的 K18-hACE2 转基因小鼠模型中，感染后第二天的肺部几乎没有显示炎症浸润，也没有 1 型干扰素信号传导的证据，而流感感染的动物在这个时间点显示出强大的先天免疫细胞浸润和干扰素信号传导。这强化了现有证据，即冠状病毒可有效抑制 1 型干扰素反应并显着改变炎症过程。我们正在更完整的时间过程中进行这些观察，使用更多标记来识别细胞类型和细胞状态，以更好地了解先天免疫的这些变化如何影响后来的适应性反应，以及病毒传播和先天免疫的不协调是否在发病机制中发挥特殊作用。 第二个主要举措是采用致命流感感染作为严重病毒性肺炎的模型。正在进行的研究包括（i）可能具有临床实用性的致命流感模型中的干预措施测试，以及（ii）分子、细胞和组织水平研究，旨在更好地了解组织损伤的潜在机制以及为什么限制病毒复制或先天免疫的干预措施经常出现 在感染早期但在宿主死亡之前就失败了。使用绕过早期鼻咽复制并导致快速深部肺部感染的严重流感感染模型，我们发现只有非常早期使用抗病毒磷酸奥司他韦（达菲）治疗才能预防死亡。在 50 种单一或联合治疗中，涵盖了临床测试或用于 COVID-19 治疗的许多药物（抗 IL-6、PANAM-G3、PMX205、肌苷 Pranobex、抗 PSGL1、ruxolitinib、inbrutinib、acalabrutinib、dypridamole、baricitinib、colchicine、silvelestat、AZD5059、anti-IL-6R、 抗 CCL2 和齐留通等），但都没有减少体重减轻或导致任何受感染动物的生存，并且一些疾病恶化。这些发现认为，要么（i）涉及多种破坏性活动，而仅减弱一种破坏性不足以产生临床效果，和/或（ii）不可逆的组织损伤很早就发生，一旦发生这种情况，独立干扰病毒复制或宿主免疫并不会在预防最终死亡方面发挥主要作用。 使用我们的 IBEX 多重染色方法对整个肺叶进行成像表明，在这种流感感染模型中，中性粒细胞早期浸润，病毒广泛传播，前表面活性剂和相关 2 型肺细胞损失，肺泡破裂和骨髓细胞细支气管堵塞，随后 T 细胞较晚到达，同时活肺组织显着损失。虽然一些治疗方法改变了细胞浸润的平衡和程度，但没有一种方法可以阻止损伤和实质损失。根据这些数据，我们提出了这样的假设：受感染的动物迅速通过残余功能性肺容量的临界点，并且在此之后，仅干扰炎症过程不足以拯救动物。这导致了基于阻止进一步损伤和促进功能性肺结构恢复相结合的策略的改变。初步数据表明，在感染过程后期给予低剂量达菲与另外两种促进肺细胞复制和肺泡修复或限制进一步免疫破坏的治疗方法之一相结合，可以使小鼠免于死亡。这些发现促使在 SARS-Cov2 感染的 K18-hACE2 转基因模型中进行测试。使用抗 SARS-Cov2 中和单克隆抗体作为达菲的替代品，我们确定了感染几天后给予动物 50% 存活率的剂量。初步研究提供了有趣的数据。由于中枢神经系统疾病是K18-hACE2模型中使用鼻腔感染时病理学的主要部分，我们的研究结果可能适用于长期COVID的脑雾，这是一个新的研究方向，同时，我们正在修改气管滴注的感染途径，以更好地模拟流感模型，并为在致命性中被证明成功的另外两种干预措施的功效提供合适的测试。 流感模型。