Untersuchung Nanopartikel-induzierter toxischer und proinflammatorischer Effekte in einem neuen in vitro Modell des gesunden und entzündlichen Darms
Zusammenfassung der Projektergebnisse
The scope of the project was to develop and improve in vitro models of the healthy and inflamed intestine, and to apply these models for toxicity testing of food-relevant nanoparticles (NP). A first aim included the development of a coculture model using intestinal epithelial cells and mucin secreting cells to study the influence of a mucin layer on NP toxicity. This, however, proved impossible, as none of the available cell systems harmonised in co-culture conditions. As the host group had a strong, not only scientific, but also clinical background in inflammatory bowel diseases, I had the unique opportunity to receive human colonic tissue from surgical resections. These could be used as an ideal ex vivo model to test NP uptake and pro-inflammatory effects in the most relevant model system available. It was found that the secreted mucosal layer indeed acts as a reliable barrier, even against small particles the size of 50 nm. Moreover, short term incubation with the food-relevant NP SiO2, TiO2 and ZnO did not induce pro-inflammatory effects in the tissue samples, when the commonly accepted marker IL-8 was tested. The ex vivo model was found useful for short term incubation and fully reflects the complexity of the human intestinal mucosal tissue, however it requires further optimisation to be utilised in long term studies. To model intestinal inflammation, human blood-derived neutrophils were used to induce inflammatory effects in differentiated intestinal epithelial cells (Caco-2). As neutrophils don't attach easily to synthetic surfaces, in existing co-culture models they are added to the apical side of the cellular layer, where they settle. This however is of limited use when NP are applied, as the neutrophils might directly interfere with the NP, which might lead to false outcomes when measuring effects on the cell line of interest. To fix the neutrophils and provide them with a matrix, collagen was successfully used. The neutrophils could be fixed in this collagen layer on the basal side of the cell layer. This allowed the cells to actively migrate towards a cytokine gradient in the direction of the Caco-2 cells, and at the same time, spatial separation of neutrophils and NP could be maintained. Two ways of neutrophil activation could be established, one leading to oxidative stress and direct damage of the cellular layer, the other inducing rather inflammatory effects in the Caco-2 cell layer. The new co-culture model was described in detail. Additionally, it was found that all NP did either not or only mildly damage the cellular system, however, ZnO was shown to induce additional pro-inflammatory effects. To analyse the influence of cell surface mucins on NP toxicity in more detail, gastric cells (MKN7) with a transient knock-down as well as permanent depletion of MUC1 were used. These cells were optimised in the host group and could therefore be used to study NP toxicity. The absence of the MUC1 protein resulted in highly increased uptake of green fluorescent model particles of different sizes. Moreover, it was found that NP appeared to bind to MUC1 and led to its shedding from the cell, which can be assumed to be the mechanism that prevents the particles from entering the cells. This protection is lost when MUC1 is knocked down. However, not all particles induced this effect. Upon incubation with TiO2 no MUC1 shedding was found, and mRNA levels of IL-8 appeared even lower in knock-down cells, compared to control cells. On the contrary, SiO2 significantly increased IL-8 mRNA levels in cells lacking MUC1. Taken together, when advanced and differentiated tissue or cell models were applied, adverse effects of a selection of food-relevant nanomaterials generally appeared low. However, there is a constant need to improve existing in vitro/ex vivo models and to establish new models, to increase food safety and further contribute to the 3R principle to reduce, refine and replace animal experiments.
Projektbezogene Publikationen (Auswahl)
- Distinctive toxicity of TiO2 rutile/anatase mixed phase nanoparticles on Caco-2 cells. Chem Res Toxicol. 2012a Mar 19;25(3):646-55
Gerloff K, Fenoglio I, Carella E, Kolling J, Albrecht C, Boots AW, Förster I, Schins RP
(Siehe online unter https://doi.org/10.1021/tx200334k) - Genotoxicity of carbon nanotubes. Book chapter in: (Donaldson K, Poland C, Duffin R, Eds.) The Inhalation Toxicology of Carbon Nanotubes. Cambridge University Press, UK, 2012, pp.150-173
Schins RPF, Albrecht C, Gerloff K, Van Berlo D
- Neutrophil induced inflammation increases nanoparticle toxicity in nd nd th intestinal epithelial cells. Australasian Society for Immunology (ASI) 42 Annual Scientific Meeting. 2 -6 December 2012, Melbourne, Australia
Gerloff K, Lourie R, Kerwick A-M, Florin TH, McGuckin MA
- Development of a new model of intestinal inflammation to test nd th th nanotoxicity in vitro. The Society of Toxicology (SOT) 52 Annual Meeting. 10 -14 March 2013, San Antonio, USA
Gerloff K, Lourie R, Kerwick A-M, Florin TH, McGuckin M
- Influence of simulated gastrointestinal conditions on particle-induced cytotoxicity and interleukin-8 regulation in differentiated and undifferentiated Caco-2 cells. Nanotoxicol 2013 Jun;7(4):353-66
Gerloff K, Pereira DI, Faria N, Boots AW, Kolling J, Förster I, Albrecht C, Powell JJ, Schins RP
(Siehe online unter https://doi.org/10.3109/17435390.2012.662249) - Modulation of inflammatory signaling by MUC1 and MUC13 in gastrointestinal epithelial cells. Mucosal Immunol 2013 May;6(3):557-68
Sheng Y, Triyana S, Wang R, Das I, Gerloff K, Florin TH, Sutton P, McGuckin MA
- Nanoparticles in our food – a health risk for the intestine? Invited seminar, 7 March 2013, Diamantina Immunology group meeting, Brisbane, Australia
Gerloff K
