Cellular in vitro models are useful in infection research to analyze host-pathogen interactions. Epithelial models are particularly suitable for reproducing the infection of human pathogenic organisms.
We are developing flexible 3-dimensional infection models that not only include structural components of human skin but also immune cells and reporter systems for the activation of immune signaling pathways.
In infection research, in vitro models are well suited for studying initial processes in the colonization of epithelial surfaces by pathogens, in particular adhesion and invasion processes.
Immunocompetent models are used to study innate immune responses to invading fungi and viruses.
In addition, these models can be used effectively for identifying ways to modulate such host responses. In the screening for new active substances [1], effects on human cells can be recorded and thus an initial assessment of the toxic and protective effects of the potential drug can be made at the same time.
In humans, it is the innate immune system that determines whether an infection occurs after the invasion of pathogens. The innate defense mechanisms against pathogens are often strongly dependent on different cell types and the 3-dimensional tissue structure.
At Fraunhofer IGB, we therefore develop 3D skin infection models that contain not only the epithelial cells but also structural components such as fibroblasts and collagen as well as immunologically relevant components such as various immune cell types. Such models are used to study mechanisms of host-pathogen interaction, in particular infection processes in fungi (Candida albicans) and viruses (herpes simplex virus, HSV-1) and the defense mechanisms against these pathogens are analyzed.
In healthy humans, the skin is resistant to symptomatic infections by microorganisms that, like Candida albicans, naturally colonize the skin. Skin infections in general occurring rarely are primarily superficial. Deeper invasion into subepithelial tissue that would allow C. albicans to access the blood stream leading to systemic spread doesn't normally occur in humans with intact skin.
However, in vitro skin models consisting of keratinocytes as an epidermal layer and fibroblasts embedded in collagen as a dermal layer are penetrated and destroyed very quickly by C. albicans [2]. This is not surprising since these skin models do not contain any components of the immune system. Hence, we developed skin models that also include immune cells. In order to design reproducible skin models that are independent of donor-based differences in primary cells, we built them from immortalized keratinocytes and fibroblasts. Cells known as T cells (T-lymphocytes) that provide immune response to C. albicans in humans as well (Fig. 1) were integrated into the models as immune cells. In the presence of T cells, invasion of C. albicans is significantly reduced and even stopped during the observation period. This means that the system shows at least partial immunocompetence in vitro.
Using next-generation sequencing, this partial immunocompetent system was comprehensively studied in dual RNA sequence analyses in the presence and absence of C. albicans. We discovered that none of the individual cell types alone achieved an effective defense against C. albicans infection. Instead, cytokine-mediated communication between the different cell types seems to be necessary to trigger an effective antimicrobial response. One of the key molecules identified by us in these analyses is the immune receptor TLR2 required for recognition of the pathogen. This receptor induces a signal cascade that finally stops the fungal invasion.
These results underscore the role of immune receptors as important sensors and regulators of the immune system that help the body to decide when and how the body´s own defense mechanisms must be activated.
1. Burger-Kentischer, A.; Finkelmeier, D.; Keller, P.; Bauer, J.; Eickhoff, H.; Kleymann, G.; Abu Rayyan, W.; Singh, A.; Schroppel, K.; Lemuth, K.; Wiesmuller, K. H.; Rupp, S. (2011) A screening assay based on host-pathogen interaction models identifies a set of novel antifungal benzimidazole derivatives, Antimicrobial agents and chemotherapy 55 (10): 4789-4801. doi:10.1128/AAC.01657-10
2. Dieterich, C.; Schandar, M.; Noll, M.; Johannes, F.J.; Brunner, H.; Graeve, T.; Rupp, S. (2002) In vitro reconstructed human epithelia reveal contributions of Candida albicans EFG1 and CPH1 to adhesion and invasion, Microbiology 148 (Pt 2): 497-506
3. Burger-Kentischer, A.; Abele, I. S.; Finkelmeier, D.; Wiesmuller, K. H.; Rupp, S. (2010) A new cell-based innate immune receptor assay for the examination of receptor activity, ligand specificity, signalling pathways and the detection of pyrogens, Journal of immunological methods 358 (1-2): 93-103. doi:10.1016/j.jim.2010.03.020
In addition, these models will be increasingly used in the future for the identification and validation of immunomodulatory substances for the control of infections and immunological diseases. For this purpose, reporter systems for the activation of receptors of the innate immune system (PRRs) were introduced into different cell types of a 3D skin model. These 3D reporter skin models make it possible to measure the activation and inhibition of central signaling pathways of the innate immune system in the three-dimensional tissue context.
Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB