Abstract
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.
Highlights
The LabEx Milieu Interieur (MI) project is a clinical study aiming to provide the first description of both genetic and environmental determinants of immunologic variance within the general healthy population [1]
In a first step toward investigating such a phenomena in a high-value asset primary human cell collection comprising over three-hundred unique donor primary fibroblasts we report here the development of two standardized assays: 1. HSV-1(Herpes Simplex Virus type 1) infection: monitoring of the cell response were developed and tested by using cells harboring mutations in the TLR3 pathway
I:C (High molecular weight), a synthetic analog of double-stranded RNAs that binds to the TLR3 receptor, and LPS (LPS-EB Ultrapure E.coli 0111: BA), a component of the gram-negative bacterial membrane that binds to the TLR4 receptor were purchased from Invivogen
Summary
The LabEx Milieu Interieur (MI) project (www.milieuinterieur. com) is a clinical study aiming to provide the first description of both genetic and environmental determinants of immunologic variance within the general healthy population [1]. Fibroblasts actively organize and remodel ECM through the production of proteinases, and converse with nearby cells through paracrine, autocrine and other forms of communication [5]. As such fibroblasts are fundamental to tissue homeostasis and normal wound repair. The role of fibroblasts in the defense against pathogens and more generally in innate immunity has only recently emerged with the discovery of Toll-like receptors (TLRs; 6). Fibroblasts are emerging with a key role in local innate immunity evoked during response to pathogens as well as vaccines [11,12,13,14]
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