Enabling risk-based testing through characterization of environmentally induced immune dysregulation and susceptibility to the SARS-CoV2 virus and COVID-19

在23财年，我们继续将翻译毒理学（DTT）部门的免疫毒理学研究项目的重点放在体外方法上。主要的工作包括研究个体间易感性因素和环境风险因素如何影响对病毒感染的反应，使用体外人全血培养系统。终点包括淋巴细胞毒性、细胞因子释放和自然杀伤细胞活性。迄今为止，我们已经筛选了200个个体样本，并正在分析体外数据，以研究年龄、性别和种族等内在因素如何影响外周血白细胞对流感和SARS-CoV-2抗原的反应。初步结果表明性别与NK活性之间存在关联，男性外周血中NK活性高于女性。正在分析免疫表型数据，以确定这种影响是否由于男性NK细胞丰度较高或NK细胞活性高于女性。本研究的第二阶段是研究在体外暴露于已知免疫毒物（包括PACs）后对这些抗原的反应，以及暴露于这些环境因子如何影响对病毒感染的易感性。PAC数据将与之前在PAC- map混合物研究中收集的小鼠体内数据进行参考，并用于确定人类相关性并加强使用体外系统可以生成的数据。作为概念的证明，在地塞米松（一种已知的免疫抑制药物）存在的情况下，全血培养不受刺激或受抗cd3 /CD28或病毒肽池的刺激。DEX治疗导致NK活性、细胞因子产生和t细胞活化受到阳性对照抗cd3 /CD28刺激的抑制。重要的是，DEX治疗还以浓度相关的方式抑制了SARS-CoV-2抗原池刺激的细胞因子的产生。这项工作表明，体外免疫毒性平台能够检测对SARS-CoV-2肽反应的免疫抑制和改变。使用PAC苯并(A)芘的第二项概念验证研究正在进行中，在代谢酶存在的情况下暴露于这种化合物导致迄今测量的免疫终点受到有效抑制。BRT目前正在为该培养系统开发额外的体外工具，以促进对体液抗体介导免疫和t细胞驱动免疫的研究。这个体外工具箱将至关重要，它将为鉴定有可能调节免疫功能的化学物质的方法提供直接的人类相关性，并将减少在毒理学试验中使用动物。

In FY 23 we continued to focus the immunotoxicology research program within the the Division of Translation Toxicology (DTT) on in vitro approaches. A major effort involved investigating how interindividual susceptibility factors and environmental risk factors impact the response to viral infection, using an in vitro human whole blood culture system. Endpoints include lymphocytotoxicity, cytokine release, and Natural Killer cell activity. We have screened >200 individual samples to date and are analyzing in vitro data to examine how intrinsic factors such as age, gender and ethnicity influence the response of peripheral blood leukocytes to influenza and SARS-CoV-2 antigens. Preliminary results demonstrate an association between sex and NK activity with males having higher NK activity in peripheral blood than females. Immunophenotyping data are being analyzed to determine if this effect is due to males having higher abundance of NK cells or more active NK cells than females. A second phase of this study is investigating the responses to these antigens following in vitro exposure to known immunotoxicants, including PACs and how exposure to these environmental agents may affect susceptibility to viral infection. The PAC data will be referenced against mouse in vivo data previously collected in the PAC-MAP mixtures studies and serve to anchor human relevance and strengthen the data that can be generated using the in vitro system. As proof of concept, whole blood cultures were unstimulated or stimulated with anti-CD3/CD28 or viral peptide pools in the presence of dexamethasone, a known immunosuppressive drug. DEX treatment resulted in inhibition of NK activity, cytokine production, and T-cell activation following stimulation with the positive control anti-CD3/CD28. Importantly, DEX treatment also inhibited the production of cytokines stimulated by SARS-CoV-2 antigen pool in a concentration related manner. This work demonstrated that the in vitro immunotoxicity platform was capable of detecting immune suppression and alterations in responses to SARS-CoV-2 peptides. A second proof of concept study using the PAC Benzo(a)pyrene is ongoing and exposure to this compound in the present of metabolizing enzymes resulted in potent suppression in the immune endpoints measured to date. BRT is currently working on development of additional in vitro tools for this culture system that will facilitate interrogation of humoral antibody mediated immunity and T-cell driven immunity. This in vitro toolbox will be critically important to provide direct human relevance in the methods used to identify chemicals that have the potential to modulate immune function, and will reduce the use of animals in toxicology testing.