Reproducibility of LPS-Induced ex vivo Cytokine Response of Healthy Volunteers Using a Whole Blood Assay

Abstract

Introduction

Lipopolysaccharide (LPS) stimulation of human whole blood ex vivo has been widely used to investigate human innate immune responses. However, there are uncertainties regarding the reproducibility and reliability of this assay.

Methods

In this prospective, single-center study, cytokine responses (interleukin 8, interferon-α, interferon-γ, interleukin 10, interleukin 1-β, interleukin 6, and tumor necrosis factor-α) to ex vivo whole blood LPS stimulation were assessed in 12 healthy volunteers. Cytokine levels were measured at 0, 2, and 4 h using a multiplex immunoassay (Luminex ^®). Stimulation was repeated after six weeks. We examined reproducibility across technical and biological replicates at baseline and between repeated experiments after 6 weeks based on the area under the curve (AUC) of the individual cytokines using Pearson’s correlation coefficient and the mean coefficient of variation.

Results

The lowest mean coefficients of variation were observed for the technical replicates (5.4 to 9.2%), followed by the biological replicates (8.1 to 24.8%), and the repeated experiments after 6 weeks (17 to 31.2%). Between the baseline and 6-week AUCs, the following Pearson correlation coefficients R were observed: interleukin 10, 0.97; interferon-α, 0.84; interleukin 1-β, 0.83; interleukin 8, 0.79; interleukin 6, 0.73; interferon-γ, 0.73; and tumor necrosis factor-α, 0.63.

Discussion

The level of agreement between the baseline and week-6 cytokine response to ex vivo LPS stimulation was high across the seven cytokines analyzed. While interleukin 10 exhibited the lowest level of variability over time, tumor necrosis factor-α showed the highest variability in repeated experiments, which should be considered in the design and interpretation of future studies.

Keywords:

• LPS
• lipopolysaccharide
• inflammation
• endotoxin

Disclosure

The authors report no conflicts of interest in this work.

Additional information

Funding

This research was funded by the Austrian Science Fund (FWF) [grant KLI 1143-B, AZI_IMQ_LPS]. For open access purposes, the authors have applied a CC BY public copyright license to any author-accepted manuscript version arising from this submission.