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.

Pro-inflammatory adjuvant properties of pigment-grade titanium dioxide particles are augmented by a genotype that potentiates interleukin 1ß processing.

BACKGROUND: Pigment-grade titanium dioxide (TiO2) particles are an additive to some foods (E171 on ingredients lists), toothpastes, and pharma-/nutraceuticals and are absorbed, to some extent, in the human intestinal tract. TiO2 can act as a modest adjuvant in the secretion of the pro-inflammatory cytokine interleukin 1ß (IL-1ß) when triggered by common intestinal bacterial fragments, such as lipopolysaccharide (LPS) and/or peptidoglycan. Given the variance in human genotypes, which includes variance in genes related to IL-1ß secretion, we investigated whether TiO2 particles might, in fact, be more potent pro-inflammatory adjuvants in cells that are genetically susceptible to IL-1ß-related inflammation. METHODS: We studied bone marrow-derived macrophages from mice with a mutation in the nucleotide-binding oligomerisation domain-containing 2 gene (Nod2 m/m), which exhibit heightened secretion of IL-1ß in response to the peptidoglycan fragment muramyl dipeptide (MDP). To ensure relevance to human exposure, TiO2 was food-grade anatase (119 ± 45 nm mean diameter ± standard deviation). We used a short 'pulse and chase' format: pulsing with LPS and chasing with TiO2 +/- MDP or peptidoglycan. RESULTS: IL-1ß secretion was not stimulated in LPS-pulsed bone marrow-derived macrophages, or by chasing with MDP, and only very modestly so by chasing with peptidoglycan. In all cases, however, IL-1ß secretion was augmented by chasing with TiO2 in a dose-dependent fashion (5-100 µg/mL). When co-administered with MDP or peptidoglycan, IL-1ß secretion was further enhanced for the Nod2 m/m genotype. Tumour necrosis factor α was triggered by LPS priming, and more so for the Nod2 m/m genotype. This was enhanced by chasing with TiO2, MDP, or peptidoglycan, but there was no additive effect between the bacterial fragments and TiO2. CONCLUSION: Here, the doses of TiO2 that augmented bacterial fragment-induced IL-1ß secretion were relatively high. In vivo, however, selected intestinal cells appear to be loaded with TiO2, so such high concentrations may be 'exposure-relevant' for localised regions of the intestine where both TiO2 and bacterial fragment uptake occurs. Moreover, this effect is enhanced in cells from Nod2 m/m mice indicating that genotype can dictate inflammatory signalling in response to (nano)particle exposure. In vivo studies are now merited.

Intestinal APCs of the endogenous nanomineral pathway fail to express PD-L1 in Crohn's disease.

Crohn's disease is a chronic inflammatory condition most commonly affecting the ileum and colon. The aetiology of Crohn's disease is complex and may include defects in peptidoglycan recognition, and/or failures in the establishment of intestinal tolerance. We have recently described a novel constitutive endogenous delivery system for the translocation of nanomineral-antigen-peptidoglycan (NAP) conjugates to antigen presenting cells (APCs) in intestinal lymphoid patches. In mice NAP conjugate delivery to APCs results in high surface expression of the immuno-modulatory molecule programmed death receptor ligand 1 (PD-L1). Here we report that NAP conjugate positive APCs in human ileal tissues from individuals with ulcerative colitis and intestinal carcinomas, also have high expression of PD-L1. However, NAP-conjugate positive APCs in intestinal tissue from patients with Crohn's disease show selective failure in PD-L1 expression. Therefore, in Crohn's disease intestinal antigen taken up by lymphoid patch APCs will be presented without PD-L1 induced tolerogenic signalling, perhaps initiating disease.

Immuno-inhibitory PD-L1 can be induced by a peptidoglycan/NOD2 mediated pathway in primary monocytic cells and is deficient in Crohn's patients with homozygous NOD2 mutations.

Peptidoglycan (PGN) is a ubiquitous bacterial membrane product that, despite its well known pro-inflammatory properties, has also been invoked in immuno-tolerance of the gastrointestinal tract. PGN-induced mucosal IL-10 secretion and downregulation of Toll like receptors are potential mechanisms of action in the gut but there are few data on tolerogenic adaptive immune responses and PGN. Here, using blood-derived mononuclear cells, we showed that PGN induced marked cell surface expression of PD-L1 but not PD-L2 or CD80/CD86, and specifically in the CD14(+) monocytic fraction. This was reproduced at the gene level with rapid induction (<4 h) and, unlike for LPS stimulation, was still sustained at 24 h. Using transfected and native muramyl dipeptide (MDP), which is a cleavage product of PGN and a specific NOD2 agonist, in assays with wild type cells or those from patients with Crohn's disease carrying the Leu1007 frameshift mutation of NOD2, we showed that (i) both NOD2 dependent and independent signalling (appearing TLR2 mediated) occurred for PGN upregulation of PD-L1 (ii) upregulation is lost in response to MDP in patients with the homozygous mutation and (iii) PD-L1 upregulation was unaffected in patients with heterozygous mutations as previously reported for cytokine responses to MDP. The uptake of PGN and its cleavage products by the intestinal mucosa is well recognised and further work should consider PD-L1 upregulation as one potential mechanism of the commensal flora-driven intestinal immuno-tolerance. Indeed, recent work has shown that loss of PD-L1 signalling in the gut breaks CD8(+) T cell tolerance to self antigen and leads to severe autoimmune enteritis.

MHC class I molecules with Superenhanced CD8 binding properties bypass the requirement for cognate TCR recognition and nonspecifically activate CTLs.

CD8(+) CTLs are essential for effective immune defense against intracellular microbes and neoplasia. CTLs recognize short peptide fragments presented in association with MHC class I (MHCI) molecules on the surface of infected or dysregulated cells. Ag recognition involves the binding of both TCR and CD8 coreceptor to a single ligand (peptide MHCI [pMHCI]). The TCR/pMHCI interaction confers Ag specificity, whereas the pMHCI/CD8 interaction mediates enhanced sensitivity to Ag. Striking biophysical differences exist between the TCR/pMHCI and pMHCI/CD8 interactions; indeed, the pMHCI/CD8 interaction can be >100-fold weaker than the cognate TCR/pMHCI interaction. In this study, we show that increasing the strength of the pMHCI/CD8 interaction by approximately 15-fold results in nonspecific, cognate Ag-independent pMHCI tetramer binding at the cell surface. Furthermore, pMHCI molecules with superenhanced affinity for CD8 activate CTLs in the absence of a specific TCR/pMHCI interaction to elicit a full range of effector functions, including cytokine/chemokine release, degranulation and proliferation. Thus, the low solution binding affinity of the pMHCI/CD8 interaction is essential for the maintenance of CTL Ag specificity.