Unveiling Interindividual Variability of Human Fibroblast Innate Immune Response Using Robust Cell-Based Protocols.
Unveiling Interindividual Variability of Human Fibroblast Innate Immune Response Using Robust Cell-Based Protocols.
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DOI：
10.3389/fimmu.2020.569331
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发表时间：
2020
影响因子：
7.3
通讯作者：
David-Watine B
中科院分区：
文献类型：
--
作者：
Chansard A;Dubrulle N;Poujol de Molliens M;Falanga PB;Stephen T;Hasan M;van Zandbergen G;Aulner N;Shorte SL;David-Watine B
LabEx Milieu Interieur (MI) 项目是一项临床研究，重点关注 1,000 名健康献血者血液样本中的基线和诱导免疫反应的详细特征。对这些样本的分析为健康群体中免疫变异的遗传和环境决定因素的开创性研究奠定了基础。在当前的研究中，我们开发了体外方法，能够对 MI 队列衍生的原代成纤维细胞反应进行标准化量化。我们的结果表明，体外人类供体群体成纤维细胞对不同 MAMP 类似物刺激的反应可以表征个体供体免疫表型变异性。结果为此类研究的新实验框架提供了概念验证基础。从 MI 研究队列中选出的 1,000 名健康个体中，选出了 323 名，建立了原代成纤维细胞系生物库。为了研究原代人真皮成纤维细胞中先天免疫反应的供体间变异性，我们选择测量 TLR3 和 TLR4 反应途径，这两种受体都在成纤维细胞中表达和先前研究过。我们使用分别刺激 TLR3 和 TLR4 途径的纯化 MAMPS 类似物、poly I:C 和 LPS，建立了用于标准化原代成纤维细胞激活的高通量自动化兼容方法。进而将这些结果与使用 HSV-1 病毒感染的刺激方法进行比较。我们的“仅添加”方案最大限度地减少了高通量自动化系统的可变性，促进从细胞铺板到刺激到细胞上清液回收和荧光标记的整个过程自动化。通过自动高内涵成像显微镜上的高通量采集自动获取图像。在这些方法条件下，标准化图像采集提供了细胞反应的量化，允许以低系统噪声和高生物信号保真度测量生物变异性。最适合对原代人类细胞反应的免疫表型进行自动分析，我们的方法和实验框架（如本文所述）与高通量筛选方案（例如化学基因组筛选所需的方案）高度兼容。在来自参加 MI 临床研究的供体的原代成纤维细胞的背景下，我们的结果为断言研究与人类临床队列相关的免疫表型特征的效用开辟了道路。
The LabEx Milieu Interieur (MI) project is a clinical study centered on the detailed characterization of the baseline and induced immune responses in blood samples from 1,000 healthy donors. Analyses of these samples has lay ground for seminal studies on the genetic and environmental determinants of immunologic variance in a healthy cohort population. In the current study we developed in vitro methods enabling standardized quantification of MI-cohort-derived primary fibroblasts responses. Our results show that in vitro human donor cohort fibroblast responses to stimulation by different MAMPs analogs allows to characterize individual donor immune-phenotype variability. The results provide proof-of-concept foundation to a new experimental framework for such studies. A bio-bank of primary fibroblast lines was generated from 323 out of 1,000 healthy individuals selected from the MI-study cohort. To study inter-donor variability of innate immune response in primary human dermal fibroblasts we chose to measure the TLR3 and TLR4 response pathways, both receptors being expressed and previously studied in fibroblasts. We established high-throughput automation compatible methods for standardized primary fibroblast cell activation, using purified MAMPS analogs, poly I:C and LPS that stimulate TLR3 and TLR4 pathways respectively. These results were in turn compared with a stimulation method using infection by HSV-1 virus. Our “Add-only” protocol minimizes high-throughput automation system variability facilitating whole process automation from cell plating through stimulation to recovery of cell supernatants, and fluorescent labeling. Images were acquired automatically by high-throughput acquisition on an automated high-content imaging microscope. Under these methodological conditions standardized image acquisition provided for quantification of cellular responses allowing biological variability to be measured with low system noise and high biological signal fidelity. Optimal for automated analysis of immuno-phenotype of primary human cell responses our method and experimental framework as reported here is highly compatible to high-throughput screening protocols like those necessary for chemo-genomic screening. In context of primary fibroblasts derived from donors enrolled to the MI-clinical-study our results open the way to assert the utility of studying immune-phenotype characteristics relevant to a human clinical cohort.
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影响因子：
13.6
作者：
Freund A;Orjalo AV;Desprez PY;Campisi J
通讯作者：
Campisi J
DOI：
10.1084/jem.20161451
发表时间：
2017-05-01
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Rodero MP;Decalf J;Bondet V;Hunt D;Rice GI;Werneke S;McGlasson SL;Alyanakian MA;Bader-Meunier B;Barnerias C;Bellon N;Belot A;Bodemer C;Briggs TA;Desguerre I;Frémond ML;Hully M;van den Maagdenberg AMJM;Melki I;Meyts I;Musset L;Pelzer N;Quartier P;Terwindt GM;Wardlaw J;Wiseman S;Rieux-Laucat F;Rose Y;Neven B;Hertel C;Hayday A;Albert ML;Rozenberg F;Crow YJ;Duffy D
通讯作者：
Duffy D
影响因子：
4.8
作者：
Salyer, Alex C. D.;David, Sunil A.
通讯作者：
David, Sunil A.
影响因子：
3.5
作者：
Kandasamy, S.;Kerr, D. E.
通讯作者：
Kerr, D. E.
年龄，性别和遗传学在塑造人类免疫反应对微生物挑战的转录变化中的独特作用。
DOI：
10.1073/pnas.1714765115
发表时间：
2018-01-16
影响因子：
11.1
作者：
Piasecka B;Duffy D;Urrutia A;Quach H;Patin E;Posseme C;Bergstedt J;Charbit B;Rouilly V;MacPherson CR;Hasan M;Albaud B;Gentien D;Fellay J;Albert ML;Quintana-Murci L;Milieu Intérieur Consortium
通讯作者：
Milieu Intérieur Consortium