An important component of the innate immune system, macrophage cells respond to viruses, bacteria, and other insults through membrane proteins called Toll-like Receptors (TLRs). Specifically, TLR4 surface receptors identify lipopolysaccharide (LPS) on Gram-negative bacterial cells (e.g., Francisella tularensis, Yersinia pestis), triggering an intracellular signaling cascade that instructs the cell to mount an innate immune response through the secretion of cytokines, chemokines, interferons and other inflammation effectors. Our group is interested in measuring and monitoring processes important to the TLR4 signaling pathway, such as cytosolic protein expression level variations and protein phosphorylation events, with single-cell resolution in an effort to obtain a systems level understanding of the response.

In particular, we are developing and employing microfluidic devices and methods to tease apart the inherently stochastic, dynamic cellular response – a feat unattainable with conventional bench top proteomic methods. Significant advantages arise from the use of microfluidic tools for bioanalytical questions such as proteomic profiling of the TLR4 pathway. Multiple cell processing steps such as sample addition, LPS dosage, dilution, mixing, metering, multiple reagent addition for fixation, permeabilization and fluorescent labeling as well as selective washing may be performed rapidly, in serial or parallel through fine microfluidic control. Such an automated cell preparation module can be integrated with subsequent quantitative fluorescence-based measurements – thereby streamlining assays composed of multiple steps. In such microfluidic formats, small sample volumes (< 10 µL) are routinely manipulated with negligible sample loss and substantial reduction in contamination – a compelling requirement while working with bacterial and viral pathogens. Further, rapid preparation of macrophage cells and quick subsequent analyses allow high-throughput measurements of large sets of variables and thus enable a quantitative systems level view.

To monitor TLR4 signaling, we have developed combinatorial assays to challenge macrophage cells (RAW264.7) with an E. coli LPS under a range of dosage conditions (i.e., times from 0 to 60 min; concentrations from 0 to 32 ug/mL). The small lengths associated with channels in our cell preparation module (100 um x 30 um cross-sectional area) enable rapid diffusive mixing of challenge within the small volume cell culture chamber. After challenge, populations of cells are prepared for flow cytometry in the same cell preparation module. The module consists of functions capable of fixing the phophorylated state in time and space, permeabilizing the cell plasma membrane, and selectively labeling the phosphorylated protein with a fluorescently labeled phospho-specific antibody. All three processing steps require introduction and routing of aqueous reagents. Therefore key components in the microfluidic platform are in situ photopolymerized porous acrylate monoliths that act as size-exclusion filters and passive valves – allowing bulk pressure-driven fluid to flow through the monoliths, while retaining cells in specific locations on the chip. Batch processing of cell cultures composed of 103 cells in volumes on the order of 100 nL are processed in the preparation module. Further the surface of the fused silica microfluidic channels and chambers is selectively treated to reduce cell loss via surface adhesion. Development of the in-line cell preparation module is underway and is important for streamlining cell challenge, preparation, and subsequent analysis of macrophage cells under TLR4 stimulating conditions.

To date, we have developed on-chip flow cytometry assays for monitoring the phosphorylation event of p38 (p38-P), a key protein in the MyD88 pathway of the TLR4 response in a macrophage cell. Immunofluorescence staining of macrophage cells for p38 and p38-P allows ratiometric comparison of the endogenous and phosphorylated protein states. To complement the p38 phosphorylation assays, we have also developed assays to monitor the phosphorylation of ERK1 and ERK2, also of the MyD88 pathways and act upstream of the p50 homodimer. Approximately 106 macrophage cells are challenged with 1µg/mL of E. coli LPS in a total volume of 1mL. Conventional flow cytometry, validated with western blots, show significant increases in p38 phosphorylation at 30 min post-LPS exposure, with subsequent reduction in the levels of phosphorylated p38 by 60 min. Initial results from the on-chip flow cytometry assays report a similar trend, with the compelling advantage of using only 10uL of fluid roughly 0.1% of that required for conventional flow cytometry.

Current efforts focus on providing robust, quantitative measurements of protein expression levels and states as related to the TLR4 signaling pathway in statistically significant macrophage populations. Incorporation of cell dosing and preparation functions with an on-chip flow cytometry capability provides the opportunity for ready analysis of multiple parameters in a single low volume cell culture with single cell resolution. The presentation will describe flow cytometric based assay development and results, as well as microfluidic design of the streamlined assay, in our efforts to establish a systems level understanding of the TLR4 signaling pathway.

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