Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Summary

We describe use of ImageStream technology (www.amnis.com), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

Abstract

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses^1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses³. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs⁴ and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity⁵. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus^6,7.

Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-κB, which initiates transcription of numerous genes that are critical for immune responses⁸. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-κB pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging⁹.

Protocol

1. Isolation and Stimulation of Immune Cells

1. Obtain 10-50 ml heparinized blood from healthy volunteers after written informed consent under the guidelines of the local Institutional Review Board. For studies of aging or disease pathogenesis, exclusion and inclusion criteria for each volunteer or patient must be explicitly determined.
2. Isolate human peripheral blood mononuclear cells (PBMCs) using CPT tubes or Ficoll-Paque Plus according to the manufacturer's instructions (GE Healthcare, NJ) and conduct experiments on the day of isolation⁷ (Reagents, Table 1).
3. Incubate PBMCs (3 x 10⁶/tube) in 1.5 ml ....

Discussion

The critical steps in use of ImageStream for translational studies are the selection of relevant comparison groups and optimization and validation of antibody specificity. Differences between subject groups in the labeled target will be readily apparent through histograms and cell images. In combination with a thorough analysis of changes in relevant receptors and signaling pathways, these data will provide valuable insight into mechanisms that underlie immune dysregulation in specialized cohorts. The technique will b.......