COMPARATIVE SENSITIVITY AND RELIABILITY OF A MULTI-ANALYTES PLATFORM AND A MULTIPLEX FLOW CYTOMETRY IN THE QUANTITATION OF CIRCULATING CYTOKINES LEVEL IN A MOUSE MODEL OF ENDOTOXEMIA

Scientific Poster
Comparative Sensitivity and Reliability of a Multi-Analytes Platform and a Multiplex Flow Cytometry in the Quantitation of Circulating Cytokines Level in a Mouse Model of Endotoxemia - SciPos113

Abstract

The objective of the study was to compare two different multiplex assay formats to quantify the release of the 7 circulating Th1/Th2/Th17 cytokines in plasma of lipopolysaccharide (LPS)-challenged mice.

Female C57BL6 mice (6-9 months old) were treated orally with vehicle (0.5% methyl cellulose in water) or dexamethasone (DEX, 5 mg/kg), followed by LPS (0.2 mg/kg) via intravenous injection (IV). Blood samples were collected at multiple time points after LPS challenge and were analyzed with the Rodent MAP V3.0 Antigens assay and the Mouse Cytokine Panels A & B assay (Myriad-RBM; Luminex platform), then were compared to the Mouse Th1/Th2/Th17 cytokines (Becton-Dickinson: BD CBA) assay on a BD Accuri C6 flow cytometer platform.

Per assays with Myriad-RBM Luminex platform, tumor necrosis factor α (TNF-α), interferon γ (IFN-γ), and interleukins (IL) 2, 4, 6, and 10 were shown to be the early phase responders and IL-17 to be a sustained responder. (Table 1) Pre-treatment with DEX reduced expression of the 7 circulating Th1/Th2/Th17 cytokines. (Table 2 and 3) Plasma levels of IL-4 were detected with the Mouse Cytokine Panels A and B assay but were below the lower limit of quantification (LLOQ) of the Rodent MAP V3.0 Antigen assay.

The 2- and 4- hour plasma samples were quantified with the BD CBA cytokine assay. IL-2 and IL-4 were below the lower limit of detection (LLOD) and IL-17A was below the LLOQ. The other 4 cytokines (TNF-α, IFN-γ, IL-6 and IL-10) were detected within the defined concentration ranges. IL-6 was the only cytokine that should be quantitated in the diluted plasma. Variations in concentrations of cytokines were consistent and acceptable in diluted 2-hour plasma. (Table 5)

Variations were observed between the two different multiplex assay platforms in terms of cytokine concentrations, time course effects of LPS, and magnitudes of DEX inhibition. Reproducible circulating IL-6 was obtained for plasma samples of the LPS treated mice with the assays from both Myriad-RBM and BD Biosciences. The BD CBA cytokine assay was not as sensitive as the Myriad-RBM assays in detecting and quantitating circulating IL-2 and IL-10 and IL-17A levels in the LPS-treated mice but was more reliable in measuring circulating IL-4 and TNF-α and IFN-γ levels. Index Terms: LPS, Inflammatory cytokine assay, Th1/Th2/Th17 cytokines, dexamethasone

Authors

Alain Stricker-Krongrad, PhD; Jason Liu, PhD; Guy Bouchard, DVM; Miao Zhong, PhD
Sinclair Research Center, LLC

Introduction

LPS administration induces an acute inflammation that is associated with increases of a number of inflammatory cytokines in the peripheral blood of treated animals. DEX inhibits the effect of LPS on cytokine synthesis. IL-2, IFN-γ, TNF-α, IL-4, IL-6, IL-10, and IL-17A become elevated in response to inflammation. These cytokines are also key regulators of immune responses4 . IL-2, IFN-γ, and TNF-α are associated with the Th1 response. IL-4, IL-6 and IL-10 are associated with the Th2 response. IL-6 and IL-17A are associated with the Th17 response. Hence, measuring the expression profiles of these cytokines is important in monitoring the polarization of the immune response and the results should therefore be independent of the quantitation methods. To evaluate this effect, the 7 circulating Th1/Th2/Th17 cytokines were quantified in plasma of LPS (+/- dexamethasone versus vehicle) treated mice with two different multiplex platforms (Myriad-RBM and BD Biosciences).

Results

Note: The plasma samples were analyzed with Myriad-RBM Luminex platform for concentrations of the circulating Th1/Th2/Th17 cytokines. The 4-hour time point samples were analyzed with the Mouse Cytokine Panel A & B, while samples of other time points were analyzed with the Rodent MAP V3.0 Antigen assay. Numbers in red indicated below the lower limit of quantification.

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References

  1. Dinges MM & Schlievert PM (2001) Infection & Immunity 69:1256.
  2. Baumgarten G, et al., (2001) J. Infectious Diseases 183:1617.
  3. Pini M, et al., (2013) Cytokine 61:71.
  4. Raphael I, et al., (2015) Cytokine 74:5

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