Department of Mechanical Engineering, Virginia Tech, Blacksburg, Virginia, USA.
Virginia Western Community College, Roanoke, Virginia, USA.
mSphere. 2021 Feb 24;6(1):e01012-20. doi: 10.1128/mSphere.01012-20.
Neutrophils, the first line of defense against pathogens, are critical in the host response to acute and chronic infections. In Gram-negative pathogens, the bacterial outer membrane (OM) is a key mediator of pathogen detection; nonetheless, the effects of variations in its molecular structure on the neutrophil migratory response to bacteria remain largely unknown. Here, we developed a quantitative microfluidic assay that precludes physical contact between bacteria and neutrophils while maintaining chemical communication, thus allowing investigation of both transient and steady-state responses of neutrophils to a library of serovar Typhimurium OM-related mutants at single-cell resolution. Using single-cell quantitative metrics, we found that transient neutrophil chemokinesis is highly gradated based upon OM structure, while transient and steady-state chemotaxis responses differ little between mutants. Based on our finding of a lack of correlation between chemokinesis and chemotaxis, we define "stimulation score" as a metric that comprehensively describes the neutrophil response to pathogens. Complemented with a killing assay, our results provide insight into how OM modifications affect neutrophil recruitment and pathogen survival. Altogether, our platform enables the discovery of transient and steady-state migratory responses and provides a new path for quantitative interrogation of cell decision-making processes in a variety of host-pathogen interactions. Our findings provide insights into the previously unexplored effects of envelope defects on fundamental innate immune cell behavior, which advance the knowledge in pathogen-host cell biology and potentially inspire the rational design of attenuated strains for vaccines or immunotherapeutic strains for cancer therapy. Furthermore, the microfluidic assay platform and analytical tools reported herein enable high-throughput, sensitive, and quantitative screening of microbial strains' immunogenicity This approach could be particularly beneficial for rapid screening of engineered microbial strains (e.g., vaccine candidates) as the quantitative ranking of the overall strength of the neutrophil response, reported by "stimulation score," agrees with cytokine response trends reported in the literature.
中性粒细胞是抵御病原体的第一道防线，在宿主对急性和慢性感染的反应中至关重要。在革兰氏阴性病原体中，细菌外膜（OM）是病原体检测的关键介质；然而，其分子结构的变化对中性粒细胞迁移反应的影响在很大程度上仍然未知。在这里，我们开发了一种定量微流控分析，该分析在保持化学通讯的同时，避免了细菌和中性粒细胞之间的物理接触，从而可以在单细胞分辨率下研究中性粒细胞对沙门氏菌属 Typhimurium OM 相关突变体库的瞬时和稳态反应。使用单细胞定量指标，我们发现，基于 OM 结构，中性粒细胞的短暂趋化运动呈高度梯度分布，而突变体之间的瞬时和稳态趋化运动差异很小。根据我们发现趋化运动和趋化运动之间缺乏相关性，我们将“刺激评分”定义为一个综合描述中性粒细胞对病原体反应的指标。结合杀伤测定，我们的结果提供了关于 OM 修饰如何影响中性粒细胞募集和病原体存活的见解。总之，我们的平台能够发现瞬态和稳态迁移反应，并为定量研究各种宿主-病原体相互作用中的细胞决策过程提供了新的途径。我们的发现提供了对包膜缺陷对基本先天免疫细胞行为的以前未探索的影响的深入了解，这推进了病原体-宿主细胞生物学的知识，并可能为疫苗的减毒菌株或癌症治疗的免疫治疗菌株的合理设计提供启发。此外，本文报道的微流控分析平台和分析工具可实现微生物菌株免疫原性的高通量、敏感和定量筛选。这种方法对于快速筛选工程化微生物菌株（例如，疫苗候选物）特别有益，因为“刺激评分”报告的中性粒细胞反应总体强度的定量排序与文献中报告的细胞因子反应趋势一致。
