Final Progress Report
Proposal No. IBD-0040R
Principal Investigator: Subrata Ghosh, M.D.
Applicant Organization: Imperial College School of Science, Technology and Medicine (London, United Kingdom)
Project Title: Do dietary microparticles modulate macrophage activation and phagocytosis in Crohn's disease?
Period of Award: September 1, 2003 - April 30, 2005
A. Summary of project aims
The aim of this project was to test the following hypothesis:
Dietary microparticles contribute to the aetiology of Crohn’s disease by modulating macrophage function.
Specifically….
1. Is microparticle uptake by macrophages different in Crohn’s disease compared with healthy controls?
2. Are microparticles pro-inflammatory when phagocytosed by macrophages derived from human peripheral blood or do these cause macrophage inactivation?
3. Does the uptake of aluminium or titanium-containing particles by healthy macrophages alter their phagocytic function for inert particles or apoptotic cells?
4. What is the effect of inorganic microparticle uptake on expression of key receptors and cytokines?
5. Do isolated lamina propria macrophages from resected intestine behave similarly to peripheral blood derived monocytes when exposed to dietary microparticles?
B. Accomplishments towards meeting those aims
Differentiation of macrophages from peripheral blood monocytes. We developed a system for the culture of human macrophages by culture of peripheral blood monocytes for nine days in the presence of 10% human serum and 20ng/ml M-CSF. These cells displayed typical macrophage morphology and expressed MHC class I and II, low CD14, no CD80, low CD86, high CD68, and CD206. Macrophages, developed from patients with active Crohn’s, showed very similar phenotypes (not shown) (1,2).
Aluminium silicates show toxicity at high doses. Before carrying out more complicated experiments, we determined the toxicity profile of the two common food additives used in this study - aluminium silicate (AlSi) and titanium dioxide (TiO2) (3-5). Macrophages from active Crohn’s patients and healthy controls were cultured for 24 hours with a range of TiO2 and AlSi concentrations. Cells were then analyzed for adverse responses by measuring the number of apoptotic (annexin V+) and dead (propidium iodide+) cells by flow cytometry (Fig. 1a). All concentrations of TiO2 were very well tolerated by macrophages from both groups, but AlSi was found to induce cell death at concentrations >10 µg/ml. This adverse effect was confirmed by measurement of inflammatory cytokines, IL-8 and TNFα, by ELISA (Fig. 1b). Incubation in the presence of AlSi (>10µg/ml) induced relatively large increases in IL-8 and TNFα. As a positive control, macrophages were stimulated with 10 ng/ml LPS for 24 hours. Macrophages from active Crohn’s patients and healthy controls showed similar responses (not shown).
Macrophages, developed from Crohn’s patients and healthy controls, show similar microparticle uptake kinetics. Uptake of microparticles by macrophages was clearly observable under polarised light microscopy (Fig. 2a). Uptake also induced changes in cellular granularity that could be monitored by flow cytometry measuring side scattered light (SSC). Using this method, we were able to measure the amount of microparticle uptake following 24 hours incubation with a range of microparticle doses. This allowed a direct comparison between the endocytic activity of macrophages from active Crohn’s disease patients or healthy controls (Fig. 2b). The results indicated macrophages from both groups could capture and internalize microparticles with similar efficiency.
Microparticles do not significantly affect cytokine secretion from healthy control macrophages unless delivered in the presence of a bacterial stimulus (LPS). We next examined the effect of microparticle uptake on macrophage cytokine responses in the presence or absence of a bacterial stimulus (LPS) since phagocytosis is known to affect cellular responses (6-8). Healthy control cells were treated for 24 hours with 5 µg/ml AlSi or TiO2 +/- 10ng/ml LPS and supernatants were harvested and analyzed for the presence of two inflammatory cytokines (IL-8 and TNFα; Fig.3a and b), and two anti-inflammatory cytokines (IL-10 and TGFβ; Fig. 3c and d).
We observed considerable heterogeneity in the cytokine responses of healthy individuals and thus chose to examine responses in ten donors. Overall, the presence of AlSi or TiO2 alone had no effect on cytokine production (inflammatory or anti-inflammatory). However, in the presence of LPS, microparticles displayed significant adjuvant activity, enhancing IL-8, TNFα, and IL-10 production, whilst inhibiting TGFβ secretion. In each case, TiO2 appeared to display the most potent adjuvant activity. We then obtained macrophages from three patients with active Crohn’s disease and one healthy control and repeated the experiment described above (Fig. 4).
In all cases, microparticles were unable to induce or inhibit cytokine secretion when administered alone. As observed in healthy controls, microparticles act as adjuvants in the presence of 10ng/ml LPS, enhancing IL-8 and TNFα (IL-10 and TGFβ data not shown). However, we found that responses to LPS alone were significantly enhanced in macrophages from active Crohn’s patients. We believe this observation may reflect changes to monocyte development and increased bacterial sensitivity in those with active Crohn’s and does not relate to microparticle activity (9). Taking this into account, microparticles appeared relatively inert on their own, but acted as adjuvants in the presence of LPS and this was the same in cells from healthy controls or Crohn’s patients (4).
Dietary microparticles inhibit macrophage phagocytic function. Macrophages are extremely efficient phagocytic cells whose main role is the clearance of apoptotic cells and debris (10-12). We thus sought to investigate the effect of dietary microparticle uptake on this important cellular activity. To begin with, we analyzed phagocytic activity by incubating cells with 2 μM fluorescent latex beads and quantifying by fluorescence microscopy (Fig. 5a).
Further experiments showed us that flow cytometric analysis yielded a much more detailed measure of phagocytic activity. Using a histogram to analyze fluorescence intensity allowed quantification of cells internalizing 1, 2, 3, 4, 5+ beads (Fig. 5b shaded histogram). Following incubation with 5 µg/ml of microparticles for 24 hours, the ability of macrophages to phagocytose and accumulate five or more beads was severely diminished – although their ability to take up one or two beads remained more or less intact (Fig. 5b - bold line). These changes led to measurable differences in the mean fluorescence intensity (MFI) of cells cultured with fluorescent latex beads. These data were used to calculate the reduction of phagocytic activity expressed as a percentage of the MFI of cells cultured in the absence of fluorescent beads (100%). Standardizing the data in this way allowed us to compare the amounts of phagocytosis in macrophages from healthy controls and active Crohn’s patients (Fig. 5c). Shaded areas on the chart represent toxic concentrations of microparticles. Although both types of microparticle partially inhibited phagocytic activity (~20-30% reduction), we could observe no significant differences between cells from Crohn’s patients and healthy controls. Additional experiments in which cells were treated with microparticles +LPS showed similar results, although the overall level of phagocytosis was lower in these cells (not shown). Similar results were also obtained using CFSE-labelled, apoptotic, Jurkat T cells instead of latex beads (not shown).
Dietary microparticles have no effect on T cell stimulatory capacity at non-toxic concentrations. In addition to their role in the clearance of apoptotic cells, macrophages express MHC and co-stimulatory molecules allowing them to efficiently stimulate naïve T lymphocytes (13,14). Microparticle-treated macrophages were used as stimulators in a five-day mixed lymphocyte reaction using allogeneic CD4+ T cells as responders and proliferation was measured by tritiated thymidine incorporation (Fig. 6). Microparticles had no significant effect on macrophage stimulatory capacity unless given at doses greater than 5 μg/ml (toxic). This was true for cells from both healthy controls and active Crohn’s patients. Additional experiments, in which cells were treated with microparticles +LPS, showed similar results although the overall level of stimulation was slightly higher (not shown).
C. Future Applications
Titanium dioxide microparticles mediate their adjuvant activity by binding biomolecules to their surface. Although dietary microparticles had minimal effects on macrophage function (cytokine response, phagocytosis, stimulatory capacity), we did see adjuvant activity in the presence of LPS. Since many microparticles possess charged surfaces, we hypothesized that LPS might be binding to the surface of the microparticles, thus enhancing its stimulatory effects. Using FITC-labelled LPS, we were able to show a substantial amount of binding at the surface of TiO2 particles (not shown), particularly in the presence of calcium ions (4).
Drugs, such as infliximab, can be coated onto the surface of dietary microparticles to enhance their efficacy in Crohn’s disease. Inorganic dietary microparticles, such as titanium dioxide, target and accumulate inside macrophages at important sites of immunity called Peyer’s patches (3,15,16). Macrophages play an important role in the development of Crohn’s-associated inflammation by responding to bacterial antigens and necrotic cells and by releasing large concentrations of inflammatory cytokines (such as TNFα). We hypothesised that if TiO2 particles could enhance inflammatory responses to LPS by binding molecules to their surface, these particles might also enhance the efficacy of anti-inflammatory drugs such as infliximab (an anti-TNFα therapy). We found that we could efficiently coat the surface of TiO2 particles with infliximab even in the absence of calcium (Fig. 7a).
When these complexes were used to inhibit macrophage responses to LPS, they were found to be more efficient at inhibiting down-stream cytokine release of IL-8 than the same concentration of infliximab only (Fig. 7b). These observations are preliminary, but highlight the potential of particulate-mediated delivery of drugs to inaccessible cells and tissues of the gastrointestinal tract.
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D. Lay Summary
A number of researchers have demonstrated the presence of inorganic particles within cells of the gastrointestinal tract (macrophages) and pinpointed the source as the diet. The present work builds on recent studies conducted by the co-investigator, Dr Powell, in which exogenous microparticles (titanium) and freshly formed endogenous particles (calcium phosphate) were shown to enhance bacterial LPS stimulation of gut immune cells from Crohn’s patients. In this study, we have shown that two common types of dietary microparticle have minimal effects on human macrophage functions, such as cytokine secretion, phagocytosis and T cell stimulatory activity. However, in the presence of a bacterial stimulus like LPS, dietary microparticles have significant adjuvant activity – enhancing inflammatory cytokine responses. This effect is the likely result of LPS binding at the surface of microparticles leading to more efficient capture and sensing by macrophages.
We believe that microparticles entering the healthy gut have minimal effects on macrophage function, are non-inflammatory, and are dealt with by the same homeostatic mechanisms that also ensure we do not mount aggressive immune responses towards commensal or ‘healthy’ bacteria. However, in individuals with ongoing gastrointestinal inflammation, the presence of dietary microparticles could enhance immune responses to bacterial antigens – possibly by aiding their transport across the gut wall and subsequent entry into macrophages.
Finally, we have utilised the fact that microparticles are targeted to gut macrophages by using them to enhance the delivery and efficacy of the therapeutic agent, infliximab.