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

在第23财年，我们继续将翻译毒理学分部(DTT)内的免疫毒理学研究计划集中在体外方法上。一项主要工作涉及使用体外人类全血培养系统调查个体间易感因素和环境风险因素如何影响对病毒感染的反应。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。到目前为止，我们已经筛选了200个个体样本，并正在分析体外数据，以检查年龄、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，性别和NK活性之间存在关联，男性外周血中的NK活性高于女性。目前正在分析免疫表型数据，以确定这种影响是由于男性NK细胞比女性更丰富或更活跃所致。这项研究的第二阶段是调查在体外暴露于已知免疫毒素(包括PAC)后对这些抗原的反应，以及暴露于这些环境介质可能如何影响病毒感染的易感性。PAC数据将与先前在PAC-MAP混合物研究中收集的小鼠体内数据相对照，并用于锚定人类相关性，并加强可以使用体外系统产生的数据。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，全血培养未被刺激或被抗CD3/CD28或病毒多肽库刺激。经抗CD3/CD28阳性对照后，地塞米松对NK细胞活性、细胞因子产生和T细胞活化均有抑制作用。重要的是，地塞米松治疗还以浓度相关的方式抑制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免疫抑制和对SARS-CoV-2多肽的反应变化。使用PAC苯并(A)芘的第二个概念验证研究正在进行中，在代谢酶存在的情况下暴露在这种化合物中导致到目前为止测量的免疫终点的有效抑制。BRT目前正致力于为该培养系统开发额外的体外工具，以促进体液抗体介导的免疫和T细胞驱动的免疫的询问。这个体外工具箱对于在用于识别可能调节免疫功能的化学物质的方法中提供直接的人类相关性将是至关重要的，并将减少在毒理学测试中使用动物。

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.