Intestinal Microparticles and Inflammatory Bowel Diseases

Microparticles (MPs) are defined as nonbiological undegradable particles of the gastrointestinal lumen that are of suitable size and shape (typically 0.2–1.0 µm diameter) for uptake by mucosal phagocytes.1 MPs can be classified into 2 groups, endogenous and exogenous, based on their source of origin. The endogenous MPs are mainly composed of calcium phosphate deriving from the co-precipitation of Ca2+ and ions secreted by the intestinal mucosa and, a smaller portion, from food intake.2,–4 In the human intestine, the endogenous MPs, together with luminal constituents (namely bacterial lipopolysaccharide [LPS]), are scavenged by M cells of Peyer’s patches and passed to underlying phagocytic cells for processing.5 The exogenous MPs are mainly composed of aluminosilicates and titanium dioxide, which are common additives to food, pharmaceuticals, and toothpaste. Research has shown that in the United Kingdom, the typical intake of titanium dioxide and aluminosilicates are up to 1012 particles and 5 × 1012 particles per day per person, respectively.6 The exogenous MPs are highly resistant to chemical breakdown, so they survive both gastrointestinal digestion and cellular processing after ingestion and uptake into the mucosa.1 These MPs may “hijack”(capture) the putative route for endogenous MP uptake1 and use the some pathways for macrophage uptake. Located in the base of the human Peyer’s patches in the terminal ileum, there is a population of cells that have been termed “pigment cells.”7,8 The pigment in fact is micron-sized cellular lysosomes that are full of small, dense submicron particles that do not allow transmission of light and thus appear black under regular light microscopy.8 Studies seeking to characterize these intracellular pigments7,8 showed that the pigments are composed of aluminosilicates, titanium dioxide, and a small percentage of non–aluminium containing silicates, such as silica (SiO2) or magnesium trisilicate (talc), indicating that they have been derived mainly from dietary intake.6,9 In contrast, the mechanism of translocation of calcium phosphate MPs into the intestinal mucosa is yet to be defined. This has mainly been due to technical challenges in preserving and observing freshly precipitated and thus labile mineral structures in situ.1 Here, we briefly overview MP detection techniques, the clinical relevance of MPs in inflammatory bowel diseases (IBD) (focusing on Crohn’s disease [CD]), and conflicting results of MP research to date. As the pigment cells within Peyer’s patch were found to be a storage site of MPs in human intestine, Thoree et al10 studied the phenotype of exogenous MP-containing pigment cells through the use of a series of antibodies against CD68, MAC387, CD14, CD11b, CD15, CD1a, S100, HLA-DR, CD86, and Cathepsin D. This work demonstrated that the majority of pigment cells were CD68 positive with a minority staining of MAC387, indicating that the MP-containing pigment cells are mainly mature macrophages of low metabolic and immunological activity. Therefore, MP-containing pigment cells located in the Peyer’s patch of the human intestine can be detected by immunohistochemical staining by using the primary antibody against CD68, which may indirectly reflect the distribution and qualitative abundance of MPs. The exact quantity and subcellular distribution of MPs, however, cannot be measured with this method, which limits its application in MP research. An electron microscope uses an electron beam to illuminate a specimen and produce a magnified image with much greater resolving power than a light microscope. It can achieve better than 50 pm resolution11and magnifications of up to about ×10,000,000. Electron microscopes are used to investigate the ultrastructure of a wide range of biological and inorganic specimens, including microorganisms, cells, large molecules, biopsy samples, metals, and crystals. There are 4 types of electron microscopes, namely transmission electron microscopes (TEM), scanning electron microscopes, reflection electron microscopes, and scanning transmission electron microscopes. Powell et al8 characterized the inorganic MPs in pigment cells of gut-associated lymphoid tissue using TEM. Due to the high magnification of TEM and the fact that the majority of MPs in gut-associated lymphoid tissue contain metal elements, such as aluminum and/or titanium, TEM is able to very clearly visualize the spatial location of MPs at the subcellular level (Fig. 1). This technique, however, cannot provide any information about the composition, structure, and quantity of the MPs. In addition, the sample processing for a TEM test is quite complicated and may also change the MPs distribution or cause nonspecific background signals. MPs in pigment cell detected by TEM.8 Left: The cell nucleus (N) and distinct intravesicular areas of MPs (e.g., P) are apparent (original magnification ×8200). The extracellular material appears to be mainly collagen (C) and also shown is probably a plasma cell (top right). Right: High power (original magnification ×25,000), individual particles, largely within the vesicular areas, are better resolved. X-ray microanalysis is the chemical identification and quantitative analysis of very small amounts of chemical substances (generally less than 10 mg or 1 mL) or small surfaces of material through the use of an x-ray beam. It is the well-established multi-elemental bulk analysis method: (energy-dispersive) x-ray fluorescence spectrometry.12 In this method, radiation from bremsstrahlung tubes or radioactive sources is used to induce emission of element-specific radiation by sample atom. To yield detectable count rates in the range of 103 to104 counts per second, the sample surface area is usually required to be 1 to 2 cm2. X-ray microanalysis may reduce the spot size down to 70 µm. Obviously, this is not suitable for determining the spatial distribution, chemical characteristics, and quantity of MPs at cellular or subcellular levels in the human intestine, although it was previously reported to be applied for chemical characterization of samples derived from human intestine.8 To yield detectable count rates from a very small sample area, for example, 100 µm2, x-ray sources that are typically 106 times more intense are required, which can only be achieved through the use of synchrotron radiation. Synchrotron radiation-induced x-ray microfluorescence (SXRF) spectrometry is the microscopic analogue of the aforementioned energy-dispersive x-ray fluorescence spectrometry with highly intense, collimated, and polarized x-rays,12 of which the beam size could be as small as only a few micrometers. So this technique should be a very suitable tool for determining the spatial distribution, chemical characteristics, and quantity of MPs at cellular or subcellular levels in the human intestine. In addition, it also has the following advantages:12,–14 (1) it is a nondestructive technique for collecting data on multiple metals simultaneously without destroying the sample because it deposits much less power on the sample than other microbeam methods, such as electron microprobe analysis and particle-induced x-ray emission; (2) it requires minimal sample preparation; (3) it is capable of performing sensitive elemental mapping with proven accuracy and reliability; (4) it is suitable for trace determination of heavier elements (atomic number Z >20–25); (5) it achieves nearly back ground-free spectra and minimum detection limits (ppm). Morishita et al15 reported a method for detecting titanium dioxide particles in frozen tissue sections using SXRF analysis. Using the x-ray fluorescence end station of the Very Sensitive Elemental and Structural Probe Employing Radiation from a Synchrotron (VESPERS) micro-probe beam line at the Canadian Light Source,16 we have also conducted several experiments for quantitative mapping of titanium- and/or calcium-containing MPs at the cellular and subcellular level in samples of human intestines.17,18 Our results indicate that SXRF microprobe techniques are capable of quantitatively mapping titanium- and calcium-containing MPs in normal and IBD-affected human intestine at the cellular or subcellular level (Fig. 2). See Table 1 for a summary of the previously described methods. A–E, Quantitative mapping of titanium or calcium at a cellular or subcellular level by using the beam sized 4 μm. Comparison of the 4 Methods Used to Detect MPs Comparison of the 4 Methods Used to Detect MPs The contribution of dietary MPs to the pathogenesis of IBD is supported by the following evidence: (1) Inflammasomes are a group of protein complexes that recognize a diverse set of inflammation-inducing stimuli that include pathogen-associated molecular patterns and damage-associated molecular patterns and that control the production of important pro-inflammatory cytokines, such as interleukin-1β (IL-1β) and IL-18.19,20 The diverse functions of these complexes in IBD have begun to be revealed. Small particles, in particular the particles in the range of 0.5 to 1 µm in diameter, induce the highest amount of IL-1β in dendritic cells.21,–24 Becker, et al25 recently reported that incubation of cells with TiO2 resulted in the assembly of NLRP3, one of the major inflammasome members, with caspase-1, and this inflammasome assembly correlated with secretion of IL-1β. Although dietary MPs alone have limited effects on basic macrophage functions, their ability to act as adjuvants could aggravate ongoing inflammatory responses toward bacterial antigens in the gastrointestinal tract.26 (2) The consumption of diets rich in processed foods containing large quantities of MPs closely parallels the epidemiology of IBD.1,27 (3) The finding, in a double-blind pilot study, that intervention with a low MP diet was efficacious in inducing disease response in ileal CD.28 And (4) the association between inflammatory response and MPs has been observed in other organs, such as in podoconiosis,29 a disease of the lymph vessels of the lower extremities caused by chronic exposure to irritant soil particles. Results from other studies, however, conflict with these findings. MPs alone have been found to be incapable of stimulating an inflammatory response in intestinal tissues.26,30,31 The quantity of MP-containing pigment cells has not been observed to be associated with the severity of IBD,10 and subsequent prospective clinical trial has failed to confirm the treatment efficacy of a low MP diet in patients with CD.32 MPs may be simply taken up by macrophages after inflammation-induced intestinal barrier damage. The reasons for these conflicting results are unclear and lead to uncertainty regarding the exact role that MPs play in immunopathogenic mechanisms in IBD. In fact, the in vivo mineral toxicity has been found to be dependent on many factors, including the shape,33 rate of particle dissolution in vivo,34 crystalline structure,35 particle size,36 microtopographies,37,38 surface interaction with other elements and molecules,39 and interaction with microorganisms.40 Studies30,31,41 have shown that exogenous MPs (either titanium dioxide or aluminosilicates) can absorb luminal constituent (namely, bacterial LPS) in the presence of sufficient quantities of calcium ions to form LPS–calcium–MP conjugates, which still fall well within the MP range and are avidly taken up by phagocytic cells.41 Functionally, a small (1.5- to 4-fold) increase in IL-1β secretion was observed when LPS and dietary MPs were combined in the presence of excess calcium.41 This implies that conjugates, not MPs alone, stimulate the inflammatory response in the human intestine and that only intestinal MPs with certain characteristics may contribute to IBD immunopathogenesis. To elucidate the functional characteristics of the MPs will require methods capable of characterizing the spatial distribution, structure, composition, and quantity of MPs at the subcellular level. So far, none of the methods other than SXRF described in the literature to date have this capability. Moreover, MPs detected in the pigment cells by transmission electronic microscope, x-ray microanalysis, and CD68 immunohistochemical staining may not represent the functional MPs, as the CD68+ MP-containing cells are of low metabolic and immunological activity and probably represent inert “storage sites” for some of the particles that traverse the patch.10 Therefore, it is possible that methodological differences have led to different conclusions of MP role (adjuvant versus primary). Morishita et al15 have reported that SXRF is a very useful tool for quantitating and characterizing the MPs at the cellular or subcellular level in frozen fresh tissue samples. The preliminary results from our ongoing studies also showed that SXRF microprobe is a method capable of subcellular MP characterization and quantitation.17,18 The precise characterization and quantitation of MPs at the cellular or subcellular level in normal and diseased human intestinal tissues is critical for elucidating the role of MPs and their underlying immunopathogenic mechanism in IBD. Results from studies in this area will provide very important information for an improved understanding of the association between MPs and the inflammatory response in IBD and for developing new strategies for prevention and treatment of the disease. Further studies should be focused on the characteristics of functional MPs in IBD samples and normal control as well by the use of SXRF microprobe.

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