Final Report Summary - MAST CELLS IN ECZEMA (Study of the role of mast cells in the modulation of immune and inflammatory responses associated with eczema: novel genetic approaches from mouse systems and relevance to the human disease.)
This project has brought together a young researcher from Belgium, Thomas Marichal, with two experts on complementary aspects of the immunological mechanisms of allergic diseases: Prof. Stephen J. Galli, world-leading expert in the biology of mast cells and basophils, at Stanford University, CA, USA, and Prof. Fabrice Bureau at the University of Liege, Belgium, to work on the very active and timely field of the cellular mechanisms of eczema.
Eczema, or atopic dermatitis (AD), is a pruritic inflammatory skin disease affecting up to 20% of children in Europe and other developed countries. Its prevalence has dramatically increased over the last decades, significantly impacting the burden of health care costs and patients’ quality of life. Yet to date, treatments remain mostly symptomatic and increasing our knowledge of cellular and molecular mechanisms involved in AD represents an urgent need for the development of novel and more effective therapeutic approaches. AD manifestations are commonly characterized by pruritus (itching), skin inflammatory lesions associated with cellular infiltration and histopathological changes, and atopy. Indeed, about 80% patients suffering from AD exhibit increased serum levels of antigen-specific and total type E Immunoglobulins (Ig) and are more susceptible to develop other atopic disorders later in life, such as allergic rhinitis and allergic asthma. The sequential development of AD followed by allergic rhinitis and then allergic asthma is called “the atopic march”.
Pathophysiologically, AD is an immunological disorder arising from complex interactions among genetic and environmental factors and recent clinical and experimental studies have abundantly illustrated the role of defective skin barrier function as a primary contributor to AD pathogenesis. The current paradigm suggests that an abnormal skin microenvironment, a consequence of skin barrier dysfunction, endows dendritic cells (DCs) with the capacity to induce dominant type 2 T cell-mediated immune responses, including the development of IgE antibodies recognizing environmental antigens that have entered the skin. In turn, skin-homing type 2 T cells, IgE-binding DCs and other immune cells present in the skin (including mast cells [MCs], basophils, eosinophils, and macrophages), are thought to release numerous pro-inflammatory cytokines and chemokines that are responsible for the cutaneous inflammatory response, macroscopic eczematous lesions, and pruritus.
Our initial project aimed to characterized the contribution of MCs in immune and inflammatory responses associated with AD. We planned to use a clinically relevant mouse model of inducible AD in combination with two complementary genetic approaches employing MC-deficient mice. First, we developed and characterized a new model of AD using a clinically relevant allergen (house dust, mite, HDM). Such models consist of skin tape stripping followed by HDM patching on the skin for 3 days. This antigen exposure was repeated 3 times at 2 week intervals, and the pathology was assessed three days after the last patching period. We showed, using C57BL/6 WT mice, that this model caused a spongiotic dermatitis (like that seen in human AD) characterized by epidermal hyperplasia and spongiosis, dermal infiltration of inflammatory and immune cells (such as neutrophils and T cells), increased numbers of skin MCs, as well as increased total and antigen-specific IgE in the serum. We then applied this model to two types of mutant mice that are deficient in MCs. The results of these experiments clearly showed that the biological responses were not significantly different between the two types of MC-deficient mice and the corresponding MC-sufficient controls, suggesting that, in this model, MCs do not substantially contribute to the disease manifestations analyzed.
We therefore took advantage of another model of skin inflammation and AD-like lesions that was developed in Dr. Stephen Galli's laboratory and that initially was thought to be influenced by MCs. Briefly, a protein called RAB guanine nucleotide exchange factor 1 (RABGEF1, also known as Rabex-5)), was shown to negatively regulate MC activation and mediator production in vitro and, in addition, mice globally deficient in Rabgef1 develop morbidity and severe skin inflammation associated with marked increases in skin MC numbers, suggesting that over-reactive skin MCs may contribute to the observed skin pathology in vivo (Tam SY et al., 2004, Nature Immunology, 5:844:52). In order to identify the extent to which various cell type(s) might contribute to skin inflammation when RABGEF1 is absent, Dr. Marichal attempted to delete Rabgef1 gene specifically in three major skin cell types, namely MCs, myeloid cells and keratinocytes; thus, Rabgef1 floxed (fl/fl) mice were crossed with mice expressing the Cre-recombinase under the influence of the promoter of Mcpt5 (to delete RABGEF1 in skin MCs), LysZ (to delete RABGEF1 in myeloid cells) or K14 (to delete RABGEF1 in keratinocytes), respectively. Unexpectedly, Mcpt5-Cre;Rabgef1fl/fl and Lysz-Cre;Rabgef1fl/fl mice appeared normal without phenotypic abnormalities. However, K14-Cre;Rabgef1fl/fl mice (which lacked RABGEF1 only in keratinocytes) rapidly developed morbidity and skin inflammation resembling AD, and died between 1 and 8 weeks of age. Mice surviving 6-8 weeks also displayed signs of systemic inflammation associated with elevated levels of serum IgE. In contrast to the initial hypothesis that RabGEF1 in MCs may be important for the development of AD skin lesions in this model, these results suggested that RabGEF1 in keratinocytes plays a crucial role in preventing the development of skin inflammation in vivo.
Further experiments have been performed in order to determine the mechanisms by which keratinocyte-restricted RabGEF1 expression plays an essential role in maintaining skin immune homeostasis in vivo. In an effort to add a translational approach to the research project as initially proposed, we also analysed genome-wide expression profiles of human lesional and non lesional AD skin biopsies. This project will reveal new insights into immune functions of the skin that are fundamental for maintaining skin immune homeostasis and to clarify how dysregulation of such skin homeostasis can contribute to AD-like skin inflammation in mice and humans (Marichal et al., submitted). Ultimately, pursuing such work may enable the discovery of novel therapeutic targets for the prevention or treatment of AD and perhaps other atopic disorders.
In addition to this project (and also during his first year of postdoctoral training funded by a BAEF fellowship), Dr. Marichal has been involved in tasks and activities that have considerably strengthened his training, including:
- the publication of 2 original co-first-authored articles, in Immunity and in the Journal of Allergy and Clinical Immunology. These reflect his undertaking, during his first year at Stanford (funded by a Belgian American Educational Foundation fellowship), another project completely relevant to his field of expertise, namely Th2 and allergic responses, about the role of Th2 responses and IgE in host defense against venoms from the honeybee and the Russell's viper. These findings provide the first experimental evidence that IgE antibodies, rather than contributing to deleterious reactions in allergies to venoms (and many other types of allergens), can have a beneficial role in host defense against venoms.
- Dr. Marichal's application and selection as a "Promodoc Ambassador"; Promodoc is a European Union-supported initiative to increase awareness of doctoral-level study in Europe. Dr. Marichal is in contact with US students and serves as a resource in order to attract prospective students to Europe.
- multiple scientific distinctions, such as the successful participation in the IFReC-SIgN 1st Winter School on Advanced Immunology and the 2014 Lindau Nobel Laureates meeting, and receiving the 2014 ADF/ECARF European Allergy Research Award and the 2015 ACTERIA Early Career Research Prize in Allergology, which recognizes the most promising young researcher in this field in Europe.
- Last but not least, Dr. Marichal has been invited to academic institutions (among others, SIgN, Singapore; IFReC, Osaka University, Japan, NIBIO, Japan) and international conferences (EAACI, Barcelona 2015, ECI, Vienna 2015) to give seminars, lectures and present his results.