Nuclear translocation kinetics of NF-kappaB in macrophages challenged with pathogens in a microfluidic platform.
We have developed a microfluidic platform for real-time imaging of host-pathogen interactions and cellular signaling events. Host cells are immobilized in a controlled environment for optical interrogation of the kinetics and stochasticity of immune response to pathogenic challenges. Here, we have quantitatively measured activation of the toll-like receptor 4 (TLR4) pathway in RAW264.7 murine macrophage-like cells. This was achieved by measuring the cytoplasm-to-nucleus translocation kinetics of a green fluorescent protein fusion construct to the NF-kappaB transcription factor subunit RelA (GFP-RelA). Translocation kinetics in response to live bacteria and purified lipopolysaccharide (LPS) challenges were measured, and this work presents the first demonstration of live imaging of host cell infection on a microfluidic platform with quantitative analysis of an early (<0.5 h from infection) immune signaling event. Our data show that a 1,000x increase in the LPS dose led to a ~10x increase in a host cell activation metric we developed in order to describe NF-kappaB translocation kinetics. Using this metric, live bacteria challenges were assigned an equivalent LPS dose as a first step towards comparing NF-kappaB translocation kinetics between TLR4-only pathway signaling (activated by LPS) and multiple pathway signaling (activated by whole bacteria). The device also contains a unique architecture for capturing and fluidically isolating single host cells for the purpose of differentiating between primary and secondary immune signaling.
James
Conrad D
CD
Sandia National Laboratories, P.O. Box 5800, Albuquerque, NM 87185, USA. cdjame@sandia.gov
Moorman
Matthew W
MW
Carson
Bryan D
BD
Branda
Catherine S
CS
Lantz
Jeffrey W
JW
Manginell
Ronald P
RP
Martino
Anthony
A
Singh
Anup K
AK
D000199:Actins / Q000737:chemistry / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003587:Cytomegalovirus / Q000235:genetics; D004867:Equipment Design; D004926:Escherichia coli / Q000472:pathogenicity / Q000502:physiology; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D054884:Host-Pathogen Interactions; D006801:Humans; D007700:Kinetics; D008070:Lipopolysaccharides / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000378:metabolism* / Q000382:microbiology; D051379:Mice; D044085:Microfluidics / Q000295:instrumentation* / Q000379:methods; D055616:Microtechnology; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012235:Rhodamines / Q000737:chemistry; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology; D013696:Temperature; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D051996:Transcription Factor RelA / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D000199:Actins; D005456:Fluorescent Dyes; D008070:Lipopolysaccharides; D016328:NF-kappa B; C498144:Rela protein, mouse; D012235:Rhodamines; D051197:Toll-Like Receptor 4; D051996:Transcription Factor RelA; D049452:Green Fluorescent Proteins; D019793:Fluorescein