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

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 responses . 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 responses 8 . 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 aging 9 . Video LinkThe video component of this article can be found at

Multiparameter Single Cell Profiling of Airway Inflammatory Cells

“…In addition, CyTOF analysis can be combined with other recent advances in technology, such as intracellular signaling pathways with high-resolution digital imaging (49) and transcriptional profiling (7). These quantitative assays are feasible to perform in sputum samples and open a new avenue for multidimensional profiling of functional cells in relevant patient cohorts to investigate cellular mechanisms and allow more individualized diagnosis and targeted therapeutic options for refractory patients.…”

CyTOF supports efficient detection of immune cell subsets from small samples

“…Further, CyTOF analysis can be combined with other recent advances in technology, such as intracellular signaling pathways with high-resolution digital imaging (Qian and Montgomery, 2012) and transcriptional profiling (Blankley et al, 2014), both quantitative assays that are feasible to perform from < 5 ml of blood. Such in depth phenotyping and functional assessments from small samples ushers in new efficiency for translational studies.…”

Imaging Immunosenescence

“…To design a labeling panel, markers should be tested singly and in combination to build a panel with specific signal after subtraction of isotype staining, consistent detection across the channels, and faithful quantitation of each marker (6). For transcription factors that translocate into the nucleus, it is better to use a small molecule dye such as Alexa 647 or FITC.…”

“…Imagestream has been employed to elucidate many cellular functions, e.g. to quantify nuclear translocation of transcription factors (4), bacterial phagocytosis and oxidative burst (5), as well as differences in signaling in studies of aging subjects and disease cohorts (6–8). Imagestream’s advantages include quantitation, reproducibility, feasibility with limited samples, and ability to promote in depth examination of cellular mechanisms between cohorts of samples.…”