Corrigendum: Unveiling Interindividual Variability of Human Fibroblast Innate Immune Response Using Robust Cell-Based Protocols.

[This corrects the article DOI: 10.3389/fimmu.2020.569331.].

Unveiling Interindividual Variability of Human Fibroblast Innate Immune Response Using Robust Cell-Based Protocols.

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.

Selection of ovine PrP high-producer subclones from a transfected epithelial cell line.

The hallmarks of prion diseases are the conversion of the normal prion into an abnormal protease resistant isoform and its brain accumulation. Purification of the native abnormal prion isoform for biochemical and biophysical studies has been hampered by poor recovery from brain tissue. An epithelial cell transfected with the ovine VRQ allele prion, called Rov9, has been used to select prion high-producer cells by flow cytometry. The representative clone 4 described here produced 6.2 microg of cellular prion protein per mg of total protein extract, representing 8- to 10-fold the amount produced by the Rov9 parental cells. After exposure to the scrapie agent (PG128/98), clone 4 produced 2.6 microg of abnormal isoform per mg of total protein. When infected clone 4 cell cultures were treated with tunicamycin, 80% of the abnormal isoform was deglycosylated. The infectivity of the prions produced in clone 4 cultures was confirmed in a mouse bioassay. Such high-producer clones represent new tools for producing large amounts of glycosylated and/or non-glycosylated PrP(Sc) and for a powerful screening of clinical samples' infectivity.