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Microfluidic platform for the quantitative analysis of leukocyte migration signatures.

ABSTRACT: Leukocyte migration into tissues is characteristic of inflammation. It is usually measured in vitro as the average displacement of populations of cells towards a chemokine gradient, not acknowledging other patterns of cell migration. Here, we designed and validated a microfluidic migration platform to simultaneously analyse four qualitative migration patterns: chemoattraction, -repulsion, -kinesis and -inhibition, using single-cell quantitative metrics of direction, speed, persistence and fraction of cells responding. We find that established chemokines, complement component 5a and IL-8 induce chemoattraction and repulsion in equal proportions, resulting in the dispersal of cells. These migration signatures are characterized by high persistence and speed and are independent of the chemokine dose or receptor expression. Furthermore, we find that twice as many T lymphocytes migrate away than towards stromal cell-derived factor 1 and their directional migration patterns are not persistent. Overall, our platform helps discover migratory signature responses and uncovers an avenue for precise characterization of leukocyte migration and therapeutic modulators.

SUBMITTER: Boneschansker L

PROVIDER: S-EPMC4155519 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Microfluidic platform for the quantitative analysis of leukocyte migration signatures.

Boneschansker Leo L Yan Jun J Wong Elisabeth E Briscoe David M DM Irimia Daniel D

Nature communications 20140903

Leukocyte migration into tissues is characteristic of inflammation. It is usually measured in vitro as the average displacement of populations of cells towards a chemokine gradient, not acknowledging other patterns of cell migration. Here, we designed and validated a microfluidic migration platform to simultaneously analyse four qualitative migration patterns: chemoattraction, -repulsion, -kinesis and -inhibition, using single-cell quantitative metrics of direction, speed, persistence and fracti ...[more]

PMID: 25183261

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Project description:BackgroundBipolar disorder (BD) is a complex psychiatric phenotype with a high heritability and a multifactorial etiology. Multisite collaborative efforts using genome-wide association studies (GWAS) have identified only a portion of DNA sequence-based risk factors in BD. In addition to predisposing DNA sequence variants, epigenetic misregulation may play an etiological role in BD and account for monozygotic twin discordance, parental origin effects, and fluctuating course of BD. In this study, we investigated DNA methylation of the brain-derived neurotrophic factor (BDNF) gene in BD.MethodsFifty participants with BD were compared to the same number of age- and sex-matched controls for DNA methylation differences at BDNF promoters 3 and 5. DNA methylation reads were obtained using a mass spectrophotometer for 64 cytosine-guanine (CpG) sites in 36 CpG 'units' across three amplicons of BDNF promoters 3 and 5.Results and discussionMethylation fractions differed between BD participants and controls for 11 of 36 CpG units. Five CpG units, mostly in promoter 5, remained significant after false discovery rate correction (FDR) (p values ≤ 0.004) with medium to large effect sizes (Cohen's d ≥ 0.61). Several of the significant CpGs overlapped with or were immediately adjacent to transcription factor binding sites (TFBSs) - including two of the FDR-significant CpG units in promoter 5. For the CpGs in promoter 3, there was a positive and significant correlation between age at sample collection and DNA methylation fraction (rho = 0.56, p = 2.8 ×10(-5)) in BD cases, but not in controls. Statistically significant differences in mean methylation fraction at 5/36 CpG units (after FDR), some at or immediately adjacent to TFBSs, suggest possible relevance for the current findings to BD etiopathogenesis. The positive correlation between age and methylation seen in promoter 3 is consistent with age-related decline in BDNF expression previously reported. Future studies should provide more exhaustive epigenetic study of the BDNF locus to better characterize the relationship between BDNF methylation differences and BD.

Project description:To address the challenges of tracking the multitude of signaling molecules and metabolites that is the basis of biological complexity, we describe a strategy to expand the analytical techniques for dynamic systems biology. Using microfluidics, online desalting, and mass spectrometry technologies, we constructed and validated a platform well suited for sampling the cellular microenvironment with high temporal resolution. Our platform achieves success in: automated cellular stimulation and microenvironment control; reduced non-specific adsorption to polydimethylsiloxane due to surface passivation; real-time online sample collection; near real-time sample preparation for salt removal; and real-time online mass spectrometry. When compared against the benchmark of "in-culture" experiments combined with ultraperformance liquid chromatography-electrospray ionization-ion mobility-mass spectrometry (UPLC-ESI-IM-MS), our platform alleviates the volume challenge issues caused by dilution of autocrine and paracrine signaling and dramatically reduces sample preparation and data collection time, while reducing undesirable external influence from various manual methods of manipulating cells and media (e.g., cell centrifugation). To validate this system biologically, we focused on cellular responses of Jurkat T cells to microenvironmental stimuli. Application of these stimuli, in conjunction with the cell's metabolic processes, results in changes in consumption of nutrients and secretion of biomolecules (collectively, the exometabolome), which enable communication with other cells or tissues and elimination of waste. Naïve and experienced T-cell metabolism of cocaine is used as an exemplary system to confirm the platform's capability, highlight its potential for metabolite discovery applications, and explore immunological memory of T-cell drug exposure. Our platform proved capable of detecting metabolomic variations between naïve and experienced Jurkat T cells and highlights the dynamics of the exometabolome over time. Upregulation of the cocaine metabolite, benzoylecgonine, was noted in experienced T cells, indicating potential cellular memory of cocaine exposure. These metabolomics distinctions were absent from the analogous, traditional "in-culture" UPLC-ESI-IM-MS experiment, further demonstrating this platform's capabilities.

Dataset Information

Microfluidic platform for the quantitative analysis of leukocyte migration signatures.

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Microfluidic platform for the quantitative analysis of leukocyte migration signatures.

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