Journal: bioRxiv

Article Title: Spatiotemporal NF-κB dynamics encodes the position, amplitude and duration of local immune inputs

doi: 10.1101/2021.11.30.470463

Figure Lengend Snippet: (A) Diagram illustrating a model of immune signaling following a local infectious signal. A macrophage detects a pathogen, and releases TNF to alert nearby cells. Receiving cells interpret this signal and regulate NF-κB dynamics. (B) Simplified NF-κB network used for mathematical modeling. (C) Microfluidic device designed to study the infection scenario represented in (A). Either TNF (blue dot) or a single macrophage was loaded in the source chamber. The macrophage was stimulated with LPS and washed with fresh medium. When the valve is opened, TNF or macrophage secretions diffuse into the receiving chamber and activate fibroblasts. (D) Diffusion of signals in the receiving chamber is tested with fluorescent dye. (E) Kinetics of dye fluorescence are plotted at various distances from the source chamber. (F) NF-κB activation at each location derived from simulation. Cell activation probability was calculated at various distance from source. Each point represents a single cell with the regulatory network in (C). (G) Simulation shows the release of TNF from source chamber elicits wave-like NF-κB propagation in the receiving cell population. (H) Microfluidic experiments exhibited wave propagation of NF-κB activation after release of TNF (100 ng/ml) from a source chamber. Fluorescently tagged NF-κB shuttles into the nucleus upon activation. Yellow arrows indicate examples of activated cells in each microscopy image. Graphs on the right of each image illustrate the fraction of activated cells at different distances. Circles and error bars indicate the mean and standard deviation in ten replicates. (I) NF-κB responses in near (0 – 0.4 mm), mid (0.8 – 1.2 mm), and far (1.8 – 2.2 mm) regions. Red lines show the mean of all cell responses in each region (n > 300), while thin lines represent 20 random responses. Purple bars indicate the delay time between the initiation of TNF diffusion and onset of local NF-κB activation, while blue bars indicate the activation time. (J) Activation probability at various distance calculated for three different doses of TNF (10, 30 and 100 ng/ml). Each dose had four replicates.

Article Snippet: The patterns for our diffusion microfluidic chip were designed with AutoCAD (Autodesk, Inc.) and KLayout.

Techniques: Infection, Diffusion-based Assay, Fluorescence, Activation Assay, Derivative Assay, Microscopy, Standard Deviation

Journal: bioRxiv

Article Title: Spatiotemporal NF-κB dynamics encodes the position, amplitude and duration of local immune inputs

doi: 10.1101/2021.11.30.470463

Figure Lengend Snippet: (A) Downstream expressions from three different cytokine secretion durations (2, 4, and 8 h) are simulated with our 2-D model. Expression at each grid point is calculated using the corresponding NF-κB dynamics as an input to a gene expression function. Blue indicates genes with rapid degradation (early), while red represents genes with slow degradation (late). (B) Diagram illustrates the schematics of spatially-resolved downstream measurement using a custom microfluidic chip. (C) Cells from three different regions are collected 4 h after releasing TNF from source chamber (30 or 100 ng/ml). After sequencing, the pairwise distance between samples are compared in MDS plot for 1,094 significantly upregulated genes. (D) Using RT-qPCR, the kinetics of A20 (early), CASP4, and RANTES (late) genes are measured for different locations in 100 ng/ml sample. (E) Upregulated genes from sequencing data were clustered using correlation method. The functional characteristics of genes in each cluster were analyzed through enrichment of GO terms and TF (transcription factor) motifs. The numbers to the right of the bar indicate the p -value.

Article Snippet: The patterns for our diffusion microfluidic chip were designed with AutoCAD (Autodesk, Inc.) and KLayout.

Techniques: Expressing, Sequencing, Quantitative RT-PCR, Functional Assay