Laetitia Pele

Origin and fate of dietary nanoparticles and microparticles in the gastrointestinal tract

Humans have evolved with oral exposure to dietary microparticles and nanoparticles as a normal occurrence but the ever-growing exploitation of nanotechnology is likely to increase exposure further, both qualitatively and quantitatively. Moreover, unlike the situation with respirable particles, relatively little is known about gastrointestinal intake and handling of nanoparticles. With a long term interest in gut exposure and responses to dietary microparticles, our group is now applying its expertise to nanoparticles in the gastrointestinal tract. Here we aim to address (i) the current challenges associated with the characterisation of particle-host or particle-cell interactions, (ii) the origin and mechanisms of uptake of particles in the gastrointestinal tract, especially via the Peyers patch and (iii) potential cellular effects of nanoparticles in the generation of reactive oxygen species and inflammasome activation, or microparticles in their adjuvant activity in pro-inflammatory signalling and immune responsiveness.

An endogenous nanomineral chaperones luminal antigen and peptidoglycan to intestinal immune cells

In humans and other mammals, it is known that calcium and phosphate ions are secreted from the distal small intestine into the lumen. However, why this secretion occurs is unclear. Here, we show that the process leads to the formation of amorphous magnesium-substituted calcium phosphate nanoparticles that trap soluble macromolecules, such as bacterial peptidoglycan and orally-fed protein antigens, in the lumen and transport them to immune cells of the intestinal tissue. The macromolecule-containing nanoparticles utilize epithelial M cells to enter Peyer’s patches - small areas of the intestine concentrated with particle-scavenging immune cells. In wild type mice, intestinal immune cells containing these naturally-formed nanoparticles expressed the immune tolerance-associated molecule ‘programmed death-ligand 1 (PD-L1)’, whereas in NOD1/2 double knock-out mice, which cannot recognize peptidoglycan, PD-L1 was undetected. Our results explain a role for constitutively formed calcium phosphate nanoparticles in the gut lumen and how this helps to shape intestinal immune homeostasis.

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 Crohns 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.

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.

Artefactual nanoparticle activation of the inflammasome platform: in vitro evidence with a nano-formed calcium phosphate

AIM To determine whether in vitro experimental conditions dictate cellular activation of the inflammasome by apatitic calcium phosphate nanoparticles. MATERIAL & METHODS The responses of blood-derived primary human cells to in situ-formed apatite were investigated under different experimental conditions to assess the effect of aseptic culture, cell rest and duration of particle exposure. Cell death and particle uptake were assessed, while IL-1β and caspase 1 responses, with and without lipopolysaccharide prestimulation, were evaluated as markers of inflammasome activation. RESULTS Under carefully addressed experimental conditions, apatitic nanoparticles did not induce cell death or engage the inflammasome platform, although both could be triggered through artefacts of experimentation. CONCLUSION In vitro studies often predict that engineered nanoparticles, such as synthetic apatite, are candidates for inflammasome activation and, hence, are toxic. However, the experimental setting must be very carefully considered as it may promote false-positive outcomes.

Synthetic mimetics of the endogenous gastrointestinal nanomineral: Silent constructs that trap macromolecules for intracellular delivery

Amorphous magnesium-substituted calcium phosphate (AMCP) nanoparticles (75-150 nm) form constitutively in large numbers in the mammalian gut. Collective evidence indicates that they trap and deliver luminal macromolecules to mucosal antigen presenting cells (APCs) and facilitate gut immune homeostasis. Here, we report on a synthetic mimetic of the endogenous AMCP and show that it has marked capacity to trap macromolecules during formation. Macromolecular capture into AMCP involved incorporation as shown by STEM tomography of the synthetic AMCP particle with 5 nm ultra-fine iron (III) oxohydroxide. In vitro, organic cargo-loaded synthetic AMCP was taken up by APCs and tracked to lysosomal compartments. The AMCP itself did not regulate any gene, or modify any gene regulation by its cargo, based upon whole genome transcriptomic analyses. We conclude that synthetic AMCP can efficiently trap macromolecules and deliver them to APCs in a silent fashion, and may thus represent a new platform for antigen delivery.

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.

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.

Dietary Mineral Nanoparticles are a normal occurrence in the human gastrointestinal tract: The case for calcium phosphate

Gastrointestinal exposure to nanoparticles and microparticles appears to be a normal occurrence and, likely, is something that humans have faced throughout evolution. In fact mechanisms have evolved to utilise, beneficially, at least one dietary nanoparticle, namely ferritin. This is the storage form of iron and is ingested in both the meat-based and plant-based aspects of the diet. Additionally, however, we propose that beneficial nano/microparticles may actively from in situ in the gut lumen. Of special note is calcium phosphate. The secretion of calcium and phosphate ions in the succus entericus (gut secretion fluid) leads to co-precipitation of particles. These could trap organic luminal molecules and then cross the epithelial barrier, especially at the M cell portal, as an entire conjugate. Cellular dissolution of the conjugate would release calcium ions and phosphate ions as well as the organic molecules (typically antigens) being carried and thus allow the immune system to survey the luminal contents. Our group is working to prove this hypothesis. Additionally, however, the M cell portal will be exposed to man-made particles such as silicates and titanium dioxide that enter the diet through ingestion of processed foods, pharmaceuticals, nutraceuticals and toothpaste. These particles are scavenged- probably via the mechanism intended for endogenously forming calcium phosphate- and can be seen to accumulate in the cells (macrophages) at the base of human intestinal lymphoid aggregates. They are likely to also adsorb to their surface luminal organic molecules, which they may subsequently release following cellular uptake. But unlike calcium phosphate, these man-made exogenous particles will not dissolve in the cell lysosome. So whether they then provide aggressive, persistent signals for cellular responses remains to be elucidated. Again, our group has a particular interest in the idea that certain genetically-susceptible individuals may have pro-inflammatory responses to these exogenous particles.