Variables to be controlled in the assessment of blood innate immune responses to Toll-like receptor stimulation

Abstract

Variability in TLR function influences susceptibility to infectious as well as immune-mediated diseases. Given the outbred nature of humans, identifying functional Toll-like receptor variability and its role in clinical disease requires such analysis to be conducted in large, often multi-centered cohorts. Yet the technically complex measurements involved in innate immune analysis benefit from centralized processing of samples. Centralization requires shipping of samples or prolonged storage, possibly even cryopreservation. Deviation from standard operating procedures (SOP) for sample procurement, storage and processing may alter the final innate immune read out. We here set out to define the impact of variables most likely to be encountered during large, multi-site studies: (i) the source of the sample, (ii) time between sample procurement to processing, and (iii) processing of fresh vs. cryopreserved samples. We found that all of these variables exert a profound impact on the final innate response to TLR stimulation. Specific innate responses appeared to be affected in response to specific TLR stimuli by a particular variable under study, proving it impossible to provide global generalizations. Based on our studies and other published work on this topic, we propose a minimal list of variables that have to be met for samples to be comparable within and across studies: a) timing between procurement and processing cannot vary by more than 10%; b) all samples have to be stored the same; and c) the source of samples needs to be the same. In summary, for innate immune analysis scrupulous adherence to standard operating procedures is paramount.

Copyright © 2011 Elsevier B.V. All rights reserved.

Figures

Figure 1. An example of intracellular cytokine staining illustrating the extent of change in cytokine expression after TLR stimulation in adult whole blood monocytes with delayed stimulation

An overlay is used to compare the freshly stimulated sample (0 h; black) with the sample stimulated after a delay (grey) of either 24 h (top) or 48 h (bottom) row. A) shows TNF-α vs. IL-6 intracellular expression; B) shows TNF-α vs. IL-12/23p40 intracellular expression after stimulation with either a TLR2/1 ligand (PAM, 10 μg/ml), or TLR7/8 ligand (R848; 10 μM).

Figure 2. Delay of stimulation impacts whole blood monocyte cytokine responses to TLR stimulation in a stimulus- and cytokine-dependent manner

Whole blood samples from healthy adults (n = 3) were stimulated for 6 hours with nothing (Unstim), a TLR2/1 ligand (PAM, 10 μg/ml), a TLR4 ligand (LPS, 100 ng/ml), or a TLR7/8 ligand (R848; 10 μM) either fresh (0) or a delay of 4–48 hours. The intracellular expression of TNF-α, IL-6 and IL-12/23p40 by monocytes was determined by polychromatic flow cytometry. (A) shows the percent of cells expressing TNF-α (top), IL-6 (middle), or IL-12/23p40 (bottom); B) shows the corresponding mean fluorescence intensity values. Means for each population were derived using the FlowJo software; error bars indicate SEM.

Figure 3. Bulk measurement of secreted cytokine production in whole blood after processing delay in response to TLR stimulation

Cytokines secreted by 100 μl of adult whole blood (n = 3) into tissue culture media containing the indicated TLR ligands were measured by Luminex’s xMAP cytokine assays. The cultures were stimulated for 24 h at 37°C, 5% CO2, after which the supernatants were harvested and frozen at −80°C until time of assay. The y-axis represents the mean cytokine concentration in pg/ml; x-axis indicates the delay in stimulation measured in hours; error bars indicate SEM.

Figure 4. Qualitative summary of the impact of cryopreservation on TLR stimulation

PBMC samples – either fresh or after cryopreservation – from the same healthy adults (n = 3) were stimulated for 24 h with nothing (Unstim), a TLR2/1 ligand (PAM, 10 μg/ml), a TLR4 ligand (LPS, 100 ng/ml), or a TLR7/8 ligand (R848; 10 uM). The culture supernatants were harvested to measure the bulk cytokine production using Luminex platform.

Figure 5. Qualitative contrast of the impact of delivery mode on the cord blood responses to TLR stimulation

Mononuclear cells obtained from cord blood from either unlabored elective Caesarian sections (CSD) (n = 10) or from labored normal spontaneous vaginal deliveries (NSVD) (n = 8) were stimulated overnight with nothing (Unstim), a TLR2/1 ligand (PAM, 10 μg/ml), a TLR4 ligand (LPS, 100 ng/ml), or a TLR7/8 ligand (R848; 10 μM). The culture supernatants were harvested after 24 h to measure the bulk cytokine production using Luminex platform