Immunopathogenesis of SARS-CoV-2 infection

该项目的目的是在小鼠模型中验证特异性趋化因子和趋化因子受体驱动SARS-CoV-2感染的免疫病理或免疫控制的假设。这一目标的动机是趋化因子调节白细胞运输和covid-19疾病的发病机制涉及依赖白细胞流入的肺部严重免疫病理。我们使用C57BL/6背景的huACE2转基因小鼠感染了武汉病毒株，非huACE2小鼠感染了MA-10小鼠适应型病毒株。我们的目标是首先测试WT LD50剂量，筛选增加生存率的受体敲除，因为任何阳性结果都具有转化潜力。筛选阴性的受体将在LDzero上重新筛选，寻找模型中的保护性趋化因子受体。根据小鼠的其他传染病模型，将对女性和男性进行检测，预计在冠状病毒疾病的临床进展和组织病理学方面存在差异，并在感染SARS-CoV和SARS-CoV-2的人类中观察到性别差异。在所有后续实验中，将通过咽拭子、血液和粪便中的病毒恢复来评估临床疾病进展；感染前及感染后多个时间间隔的血清抗体滴度、血象、血液化学及血清细胞因子；以及身体外观，体重减轻，进展到死亡和感染后一个月的存活。感染后最迟一个月，动物将通过二氧化碳和颈椎脱臼安乐死。根据我们的经验，为了观察生存的显著差异，每个实验需要20只小鼠/队列，由于实验之间的生存差异，我们需要重复实验3次。这些数字应该足以观察到其他临床数据的显著差异。由于预计这些对照之间的差异较小，因此需要较少的未感染小鼠。在本报告期内，我们的ko筛选结果揭示了Ackr1缺乏的保护作用和Ccr2， Ccr5和Cxcl10缺乏的有害作用。

The aim of this project is to test the hypothesis that specific chemokines and chemokine receptors drive either immunopathology or immune control of SARS-CoV-2 infection in a mouse model. The motivation for this aim is that chemokines regulate leukocyte trafficking and covid-19 disease pathogenesis involves severe immunopathology in the lung dependent on leukocyte influx. We are using huACE2 transgenic mice on a C57BL/6 background infected with the Wuhan strain of the virus as well as non-huACE2 mice infected with the MA-10 mouse adapted strain of the virus. Our goal is to test a WT LD50 dose first, screening for receptor knockouts that increase survival, since any positive results will have translational potential. Receptors that screen negative will be rescreened at an LDzero looking for protective chemokine receptors in the model. Both females and males will be tested and are expected to have differences in clinical progression of coronaviral disease and histopathology, based on other infectious disease models in mice, and differences between the sexes observed in humans infected with SARS-CoV and SARS-CoV-2. In all subsequent experiments, clinical disease progression will be assessed by virus recovery from throat swabs, blood and stool; serum antibody titers, hemograms, blood chemistries and serum cytokines before and at multiple intervals after infection; as well as physical appearance, weight loss, and progression to moribundity and survival for a month after infection. Animals will be euthanized by CO2 and cervical dislocation at the latest one-month post-infection. In our experience, to observe significant differences in survival we will need 20 mice/cohort in each experiment and repeat the experiment 3 times due to the variance in survival between experiments. These numbers should be sufficient to observe significant differences in the other clinical data as well. Fewer non-infected mice will be needed since there is expected to be less variance among these controls. In this reporting period, the results of our ko screen has revealed protective effects of Ackr1 deficiency and harmful effects of Ccr2, Ccr5 and Cxcl10 deficiency. A secondary goal is to characterize the local immune response in cultured human lung explants infected with SARS-CoV-2. We have succeeded in collaboration with L Margolis from NICHD in establishing this system and demonstrating productive infection in epithelial cells. In FY23, we reported that atypical chemokine receptor 1 (Ackr1, also known as Duffy antigen receptor for chemokines/DARC) was the most highly upregulated chemokine receptor in infected lung, where it localized to endothelial cells of veins and arterioles. In a screen of 7 leukocyte chemoattractant or chemoattractant receptor knockout mouse lines, Ackr1-/- mice were unique in having lower mortality after SARS-CoV-2 infection, particularly in males. ACKR1 is a non-signaling chemokine receptor that in addition to endothelium is also expressed on erythrocytes and Purkinje cells of the cerebellum. It binds promiscuously to both inflammatory CC and CXC chemokines and has been reported to control chemokine availability which may influence the shape of chemotactic gradients and the ability of leukocytes to extravasate and produce immunopathology. Of note, erythrocyte ACKR1 deficiency is fixed in sub-Saharan African populations where COVID-19 has been reported to result in low mortality compared to worldwide data. Our data suggest the possibility of a causal contribution of ACKR1 deficiency to low sub-Saharan COVID-19 mortality and identify ACKR1 as a possible drug target in the disease.