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Quantitative Multiplex Analysis of Low-level Cytokine Expression  

Sep 01 2015

Author: Robert Keith, Munmun Banerjee, Debra MacIvor, Min Zhu, Barbara Nikolajczyk, Caroline M. Apovian, Xiao Qiang on behalf of Merck KGaA. The life science business of Merck operates as MilliporeSigma in the U.S. and Canada

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Cytokines are immunomodulatory polypeptides that play key roles in both adaptive and innate immune responses. A generic term, ‘cytokines’ includes interleukins (acting as mediators among multiple immune cell types), chemokines (responsible for immune cell migration), lymphokines (produced by activated lymphocytes), adipokines (produced by adipocytes) and myokines (produced by muscle cells). As regulators of the immune system, cytokines act at the recognition, activation, and/or effector phases of an immune response, modulating the development and functional activities of the subtypes of T cells, B cells and myeloid cells.

The significant roles of cytokines in normal inflammatory responses and immune cell development and activation drive their involvement in a variety of disease types. Even low levels of the chronic inflammation mediated by cytokines are involved in many clinical and subclinical disease states. According to the CDC (Centers for Disease Control and Prevention, FASTSTATS - Leading Causes of Death. cdc.gov. 2011), low-level chronic inflammation contributes to at least seven of the 10 leading causes of mortality in the US, including cardiovascular disease, stroke, Alzheimer’s disease, diabetes and cancer.

Consequently, research involving cytokines plays a significant role in achieving a more detailed understanding of the immune system and its multi-faceted response to most antigens, especially those responses that make up the inflammatory process. In order to develop a more robust understanding of pro-inflammatory cytokine networks, it is critical to be able to quantitate multiple cytok ines simultaneously.

This article describes development and use of a 21-plex assay for simultaneously detecting cytokines associated with Th1, Th2, and Th17 cells, based on the Luminex xMAP® technology and the MILLIPLEX® MAP Human High Sensitivity T Cell Panel (EMD Millipore Billerica, MA).  This panel is available in a premixed-bead format as either a 21-plex or a 13-plex kit, with the latter including only the Th1 and Th2 cell markers. Although these standard panels measure a defined group of cytokines, each panel is customisable to enable the user to choose any number of analytes within the panel to meet specific research needs.

The article also presents data from collaboration with Barbara Nikolajczyk, PhD and Min Zhu, PhD of Boston University School of Medicine, who are studying the association between obesity and inflammation due to the overexpression of cytokines that support immune cell differentiation/activation.

Methods
For serum samples, blood was allowed to clot for 30 minutes before centrifugation for 10 minutes at 1000 x g. The serum was removed and either assayed immediately or stored at -20°C. Plasma samples, with EDTA anticoagulant, were centrifuged at 1000 x g within 30 minutes of blood collection. Plasma was removed and assayed immediately or stored at -20°C. Frozen samples were thawed completely, vortexed, and centrifuged prior to use, to remove particulates. Neat samples were added directly into the assay plate. Sepsis samples were obtained from Discovery Life Sciences, Los Osos, CA. 
For the obesity study, human peripheral blood mononuclear cells (PBMCs) were isolated from four groups of subjects (n=8 per group) defined as Healthy Subjects (Lean) with BMI<25, Non-Diabetic Obese Subjects (ND) with BMI 30-35 and HbA1c<5.7%, Prediabetic Subjects (PD) with BMI 30-35 and HbA1A1c 5.7-6.5% who had never taken metformin and Prediabetic with Metformin Subjects (PD+Met) with BMI 30-35 and A1c 5.6-6.5 who were treated with metformin (500 mg 2x daily) as standard-of-care. The PBMCs were stimulated with plate-bound CD3 and soluble CD28 (2 µg/mL) at 1 x 106 cells/mL for
40 hours, as described previously [1]. 
Serum, plasma and PBMC supernatants were analysed using the MILLIPLEX® MAP kit, according to the kit protocol (HSTCMAG--28SK).

Results 
The standard curves and standard concentrations for the cell panel are shown in Figure 1 and Table 1, respectively. The minimum detectable concentrations (Table 2) indicate the sensitivity for most assays to be less than 1 pg/mL. The standard curves show a broad linear range of detection for all the analytes in the panel (Figure 1).

Cross-reactivity
Potential analyte cross-reactivity within the assays was determined with a single standard cross-reactivity test. Each individual standard was tested in the presence of multiplexed beads and detection antibodies. All standards had less than five percent cross-reactivity with the other assays (data not shown).
Stability: Freeze/Thaw and Heat Stress No analyte in the panel was affected (10% less than control) by up to three freeze-thaw cycles of the samples (Figure 2). Only Fractalkine and MIP1β concentrations were affected (40-50% less than control) by temperature stress on the serum samples (24 hours RT or
2 hours at 37°C, Figure 3).

Precision
Intra-assay precision (%CV) was determined from eight duplicates of the standard controls (Table 3A) and inter assay precision (%CV) was determined from twelve independent replicates of the standard controls (Table 3B).

Recovery
Assay accuracy was determined as the percentage of the observed concentration of known amount of standard spiked into serum matrix. The percent recoveries were between 96% and 106% for all assays (data not shown).

Sensitivity Comparison
The sensitivity of the cell panel was compared to the sensitivity of two high sensitivity multiplexed Luminex® assay kits from different suppliers. The MILLIPLEX® MAP assay was found to have, overall, higher sensitivity than the competitor kits (Figure 4).

Sample Detection
Initial sample testing compared the percent sample detection between the MILLIPLEX® MAP Human High Sensitivity T Cell Panel and a high sensitivity multiplex assay kit from a different supplier. Both normal serum samples (n=9) and serum samples from sepsis patients (n=16) were tested. The MILLIPLEX® MAP Human High Sensitivity T Cell Panel detected analytes at a similar or greater frequency than did the other kit (Table 4).
The panel was further validated in a study done in collaboration with Dr Nikolajczyk and Dr Zhu. To investigate immune cell function in obese and prediabetic subjects, four groups of subjects (n=8 per group, as described in Methods section and in Table 5) were recruited following informed consent. The study design was cross-sectional.
T cells from subjects in the Lean, ND, PD and PD+Met groups were stimulated in the context of PBMCs with plate bound CD3 and soluble CD28 (Figures 5 and 6). Figure 5 shows cytokine secretion patterns indicative of Th1, B cell and myeloid immune cell function, with a statistically significant decrease in levels of the anti-inflammatory cytokine, IL-10. These results are consistent with our demonstration that T cell stimulation (αCD3/αCD28) induces TNFα production by CD14+ myeloid cells [2].
While Figure 6 does not show a significant decrease in pro-inflammatory cytokine levels in prediabetic subjects taking metformin, there is a statistically significant increase in the anti-inflammatory cytokine IL-10. This IL-10 boosting action of metformin is consistent with the demonstration that metformin decreases function of the inflammasome in insulin resistant individuals [3].

Conclusions
Low levels of chronic inflammation are involved in many clinical and subclinical disease states. Consequently, research investigating low levels of cytokine expression plays a significant role in achieving a deeper understanding of the immune system and its multi-faceted response to both antigens and physiological perturbation, especially those responses that make up the immune cell-mediated inflammatory process. 
The MILLIPLEX® MAP Human High Sensitivity T Cell Magnetic Bead Panel provides researchers with an analytically validated ‘must-have’ assay, not only to study low-level cytokine expression, but also to quantify multiple cytokine secretion levels simultaneously and in a biologically relevant context.
References
1. Jagannathan-Bogdan M, McDonnell ME, Shin H, Rehman Q, Hasturk H, Apovian CM, Nikolajczyk BS. Elevated proinflammatory cytokine production by a skewed T cell compartment requires monocytes and promotes inflammation in type 2 diabetes. J Immunol. 2011 Jan 15;186(2):1162-72.

2. Ip BC, Nikolajczyk BS et al., Obesity. 2015 (in press).

3. Lee HM, Kim JJ, Kim HJ, Shong M, Ku BJ, Jo EK. Upregulated NLRP3 inflammasome activation in patients with type 2 diabetes. Diabetes. 2013 Jan;62(1):194-204.

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