Label-Free Identification of Persistent Particles in Association with Primary Immune Cells by Imaging Flow Cytometry.

The frequency of human exposure to persistent particles via consumer products, air pollution, and work environments is a modern-day hazard and an active area of research. Particle density and crystallinity, which often dictate their persistence in biological systems, are associated with strong light absorption and reflectance. These attributes allow several persistent particle types to be identified without the use of additional labels using laser light-based techniques such as microscopy, flow cytometry, and imaging flow cytometry. This form of identification allows the direct analysis of environmental persistent particles in association with biological samples after in vivo studies and real-life exposures. Microscopy and imaging flow cytometry have progressed with computing capabilities and fully quantitative imaging techniques can now plausibly detail the interactions and effects of micron and nano-sized particles with primary cells and tissues. This chapter summarises studies which have utilized the strong light absorption and reflectance characteristics of particles for their detection in biological specimens. This is followed by the description of methods for the analysis of whole blood samples and the use of imaging flow cytometry to identify particles in association with primary peripheral blood phagocytic cells, using brightfield and darkfield parameters.

Imaging flow cytometry methods for quantitative analysis of label-free crystalline silica particle interactions with immune cells.

Exposure to respirable fractions of crystalline silica quartz dust particles is associated with silicosis, cancer and the development of autoimmune conditions. Early cellular interactions are not well understood, partly due to a lack of suitable technological methods. Improved techniques are needed to better quantify and study high-level respirable crystalline silica exposure in human populations. Techniques that can be applied to complex biological matrices are pivotal to understanding particle-cell interactions and the impact of particles within real, biologically complex environments. In this study, we investigated whether imaging flow cytometry could be used to assess the interactions between cells and crystalline silica when present within complex biological matrices. Using the respirable-size fine quartz crystalline silica dust Min-u-sil® 5, we first validated previous reports that, whilst associating with cells, crystalline silica particles can be detected solely through their differential light scattering profile using conventional flow cytometry. This same property reliably identified crystalline silica in association with primary monocytic cells in vitro using an imaging flow cytometry assay, where darkfield intensity measurements were able to detect crystalline silica concentrations as low as 2.5 µg/mL. Finally, we ultilised fresh whole blood as an exemplary complex biological matrix to test the technique. Even after the increased sample processing required to analyse cells within whole blood, imaging flow cytometry was capable of detecting and assessing silica-association to cells. As expected, in fresh whole blood exposed to crystalline silica, neutrophils and cells of the monocyte/macrophage lineage phagocytosed the particles. In addition to the use of this technique in in vitro exposure models, this method has the potential to be applied directly to ex vivo diagnostic studies and research models, where the identification of crystalline silica association with cells in complex biological matrices such as bronchial lavage fluids, alongside additional functional and phenotypic cellular readouts, is required.

Silicon Forms a Rich Diversity of Aliphatic Polyol Complexes in Aqueous Solution.

A detailed examination of aqueous Si complexation by alditols and aldonic acids was conducted using high-sensitivity 29Si NMR spectroscopy of isotopically enriched solutions combined with theoretical modeling. Contrary to previous thinking, we have established that aliphatic polyols do not require a threo pair of hydroxy groups to form hypercoordinated Si complexes, although formation constants may be orders of magnitude higher if they are present. Thirteen distinctly different molecular assemblages containing 4-, 5-, or 6-coordinate Si centers have been identified, with significant concentrations of 5-coordinate Si bis-ligand complex being detected even under biologically relevant solution conditions.

Ultrasmall silica nanoparticles directly ligate the T cell receptor complex.

The impact of ultrasmall nanoparticles (<10-nm diameter) on the immune system is poorly understood. Recently, ultrasmall silica nanoparticles (USSN), which have gained increasing attention for therapeutic applications, were shown to stimulate T lymphocytes directly and at relatively low-exposure doses. Delineating underlying mechanisms and associated cell signaling will hasten therapeutic translation and is reported herein. Using competitive binding assays and molecular modeling, we established that the T cell receptor (TCR):CD3 complex is required for USSN-induced T cell activation, and that direct receptor complex-particle interactions are permitted both sterically and electrostatically. Activation is not limited to αß TCR-bearing T cells since those with γδ TCR showed similar responses, implying that USSN mediate their effect by binding to extracellular domains of the flanking CD3 regions of the TCR complex. We confirmed that USSN initiated the signaling pathway immediately downstream of the TCR with rapid phosphorylation of both ζ-chain-associated protein 70 and linker for activation of T cells protein. However, T cell proliferation or IL-2 secretion were only triggered by USSN when costimulatory anti-CD28 or phorbate esters were present, demonstrating that the specific impact of USSN is in initiation of the primary, nuclear factor of activated T cells-pathway signaling from the TCR complex. Hence, we have established that USSN are partial agonists for the TCR complex because of induction of the primary T cell activation signal. Their ability to bind the TCR complex rapidly, and then to dissolve into benign orthosilicic acid, makes them an appealing option for therapies targeted at transient TCR:CD3 receptor binding.

Non-Functionalized Ultrasmall Silica Nanoparticles Directly and Size-Selectively Activate T Cells.

Sub-micron-sized silica nanoparticles, even as small as 10-20 nm in diameter, are well-known for their activation of mononuclear phagocytes. In contrast, the cellular impact of those <10 nm [ i.e., ultrasmall silica nanoparticles (USSN)] is not well-established for any cell type despite anticipated human exposure. Here, we synthesized discrete populations of USSN with volume median diameters between 1.8 to 16 nm and investigated their impact on the mixed cell population of human primary peripheral mononuclear cells. USSN 1.8-7.6 nm in diameter, optimally 3.6-5.1 nm in diameter, induced dose-dependent CD4 and CD8 T-cell activation in terms of cell surface CD25 and CD69 up-regulation at concentrations above 150 µM Sitotal (∼500 nM particles). Induced activation with only ∼2.4 µM particles was (a) equivalent to that observed with typical positive control levels of Staphylococcal enterotoxin B (SEB) and (b) evident in antigen presenting cell-deplete cultures as well as in a pure T-cell line (Jurkat) culture. In the primary mixed-cell population, USSN induced IFN-γ secretion but failed to induce T-cell proliferation or the secretion of IL-2, IL-10, or IL-4. Collectively, these data indicate that USSN initiate activation, with Th1 polarization, of T cells via direct particle-cell interaction. Finally, similarly sized iron hydroxide particles did not induce the expression of T-cell activation markers, indicating some selectivity of the ultrasmall particle type. Given that humans may be exposed to ultrasmall particles and that these materials have emerging bioclinical applications, their off-target immunomodulatory effects via direct T-cell activation should be carefully considered.

Imaging flow cytometry assays for quantifying pigment grade titanium dioxide particle internalization and interactions with immune cells in whole blood.

Pigment grade titanium dioxide is composed of sub-micron sized particles, including a nanofraction, and is widely utilized in food, cosmetic, pharmaceutical, and biomedical industries. Oral exposure to pigment grade titanium dioxide results in at least some material entering the circulation in humans, although subsequent interactions with blood immune cells are unknown. Pigment grade titanium dioxide is employed for its strong light scattering properties, and this work exploited that attribute to determine whether single cell-particle associations could be determined in immune cells of human whole blood at "real life" concentrations. In vitro assays, initially using isolated peripheral blood mononuclear cells, identified titanium dioxide associated with the surface of, and within, immune cells by darkfield reflectance in imaging flow cytometry. This was confirmed at the population level by side scatter measurements using conventional flow cytometry. Next, it was demonstrated that imaging flow cytometry could quantify titanium dioxide particle-bearing cells, within the immune cell populations of fresh whole blood, down to titanium dioxide levels of 10 parts per billion, which is in the range anticipated for human blood following titanium dioxide ingestion. Moreover, surface association and internal localization of titanium dioxide particles could be discriminated in the assays. Overall, results showed that in addition to the anticipated activity of blood monocytes internalizing titanium dioxide particles, neutrophil internalization and cell membrane adhesion also occurred, the latter for both phagocytic and nonphagocytic cell types. What happens in vivo and whether this contributes to activation of one or more of these different cells types in blood merits further attention. © 2017 International Society for Advancement of Cytometry.