3Rs-Centre Utrecht Life Sciences

Faculteit Dierengeneeskunde

In a series of interviews with researchers of Utrecht University, Utrecht Medical Center or other ULS-partners, we want to give more insight into current developments in the replacement, reduction and refinement of laboratory animal experimentation within these research facilities. Take a look at former interviews here.

May 2016
Novel co-culture model to study cross-talk between human intestinal epithelial cells and immune cells could reduce future animal use

Life-threatening allergic reactions are more common today than 10 years ago, especially food allergies. Prebiotic non-digestible oligosaccharides, used as supplementation in infant formula, are suggested to provide protection to allergic symptoms in early life. To study underlying mechanisms of prebiotics in reducing food allergy responses, animal models are still invaluable tools. However, Dr. Linette Willemsen, Assistant Professor of the Division of Pharmacology in the group of Prof. Garssen at Utrecht University, developed a novel in vitro co-culture model to study these mechanisms which can be used as a screening tool to reduce animal use.

Nowadays, food allergy occurs in 6-8% of young children and in 1-4% of adults1. Cow's milk, hen's egg, peanut, wheat, soy and fish are responsible for more than 90% of food allergies and are therefore called culprit food. Symptoms occur in the skin, gastro-intestinal and/or respiratory tract. In worst cases, even anaphylactic shock and death can occur upon oral ingestion of the culprit food. Around 30% of children affected with the skin disease atopic dermatitis have an underlying food allergy2.

Specific prebiotic non-digestible oligosaccharides selectively support the growth of beneficial bacteria, such as bifidobacteria and lactobacilli. These dietary supplements have shown to be effective in the prevention of atopic dermatitis in infants. Anti-allergic effects by prebiotics are still not completely defined, but presumably cross-talk between the intestinal epithelial cells and immune cells plays an important role. This cross-talk is important because beyond providing a barrier, the intestinal epithelium mediates immunomodulatory effects.  It can surveille the intestinal content, since epithelial cells amongst others express Toll-like receptors (TLRs) and glycan receptors that can recognize bacterial and dietary components. They can modify the underlying immune cells via secretion of immune mediators like cytokines and galectins.

Before turning to the well-known mouse food allergy models, Dr. Willemsen and coworkers developed a novel in vitro co-culture model to study this cross-talk between intestinal epithelial cells and immune cells. Moreover, the in vitro model could be used as a screening tool for candidate prebiotic oligosaccharides and probiotics and at the same time reducing the number of animals used. 

Mimicking the intestinal environment: co-culture model

In the co-culture model, intestinal epithelial cells are cultured with a diverse subset of immune cells, Peripheral Blood Mononuclear Cells (PBMC) from healthy adult human donors provided by Sanquin.
In the transwell co-culture model, intestinal epithelial cells, which are human colon adenocarcinoma cells (HT-29 or T84), are cultured on filters and PBMC are added to the basolateral compartment (Figure 1).

Figure 1. Transwell co-culture model to study the cross-talk between human  intestinal epithelial cells (HT-29 or T84) and immune cells (PBMC). PBMC consist mainly of a mixture of T- and B-lymphocytes (yellow, dark green and blue cells) and monocytes (light green cells).

The model is unique in the way that it includes a variety of immune cells of the innate as well as the adaptive immune system, mimicking some aspects of a real-life intestinal environment. Moreover, the model includes a structural component: the intestinal epithelium, being the barrier system between the mucosal immune system and the external milieu.

Polarizing towards Th1-immune responses

Linette Willemsen and coworkers showed a modulatory role of intestinal epithelial cells on immune responses, upon different stimuli applied to the apical membrane, like bacterial DNA or other TLR agonists4. As a result, with bacterial DNA added to the apical membrane, a Th1-polarized regulatory (IL-10 high) type of immune response was raised. This is a desired type of immune response when allergic reactions are to be prevented. Furthermore, oligosaccharides could further enhance the effect of bacterial DNA on Th1 immune polarization. It was revealed that galectin-9 was increased and this contributed to the immune polarizing effect, suggesting an important allergy-suppressing role of galectin-96.

Translation from in vitro to in vivo

The in vitro results were compared with in vivo results from mice. In a murine model for cow’s milk allergy, mice were sensitized orally to cow’s milk protein whey, while receiving a dietary supplement of Bifidobacterium breve and oligosaccharides5.
Results showed that dietary supplementation in these mice increased galectin-9 levels in the intestine and serum, while intestinal Th1- and regulatory T cell markers were increased and the acute hypersensitivity response was reduced. Furthermore, in vivo results shown in mice were consistent with results shown in infants, after analyzing serum samples of a finalized cohort study5.

Reducing future animal use

Because of these promising results, the co-culture model is already in use as a screening tool of the generic immunomodulatory effect of different candidate prebiotic oligosaccharides to predict the in vivo situation. The co-culture model is a novel tool and can be used to select the most efficient candidates to prevent or treat food allergy. In the future this may reduce the numbers of animals, since the co-culture mode can predict the efficacy of certain dietary components beforehand. Unfortunately, it cannot replace the animal models since these are still needed to study the basic mechanisms by which a selected dietary component can reduce allergy at a multi-organ level in an allergen specific manner.

Dr. Willemsen and coworkers also want to develop and apply allergen immunotherapy in the future, when more insights into the working mechanism of prebiotics are available. Therefore other in vitro models are being developed, focusing on the cross-talk between epithelial cells and dendritic cells and allergen specific models relevant for the treatment of allergy. For this purpose, PBMC of allergic donors are used in which allergen specific immune activation can be provoked and the actions of dietary components on this response can be studied.

1: Ozol D. and Mete E. (2008), Asthma and food allergy, Curr Opin Pulm Med. 14 (1): 9-12.
2: Bergman M. et al. (2013), Evaluation of Food Allergy in Patients with Atopic Dermatitis, J Allergy Clin Immunol: in Practice 1:22-8.
3: Shandilya U.K. et al. (2015), Probiotic Dahi containing Lactobacillus acidophilus and Bifidobacterium bifidum modulates immunoglobulin levels and cytokines expression in whey proteins sensitized mice, J. Sci. Food Agric., online publication.
4: de Kivit S. et al. (2011), Apical TLR ligation of intestinal epithelial cells drives a Th1- polarized regulatory or inflammatory type effector reponses in vitro, Immunobiol 216: 518-527.
5: de Kivit S. et al. (2012), Galectin-9 induced by dietary synbiotics is involved in suppression of allergic symptoms in mice and humans, Allergy 67: 343-352. 
6. de Kivit S. et al. (2013), Intestinal epithelium-derived galectin-9 is involved in the immunomodulating effects of nondigestible oligosaccharides, J Innate Immun, 5(6):625-38.