The Microbiome and the Human Immune Response in Atopic Dermatitis : Exploring Microbial Targets for Personalized Treatment

Atopic dermatitis (AD) is a common chronic inflammatory skin disease. An impaired skin barrier and altered immune mechanisms are considered the two major players in AD inflammation. Alterations in the microbiome are an established finding in AD, but its role in the pathogenesis is still poorly understood. In this thesis we primarily aimed to characterize the microbial composition of the skin, nose and gut in pediatric patients with mild to severe AD. Our second aim was to estimate the prevalence of Staphylococcus (S.) aureus in patients with AD and to study the humoral immune response against S. aureus. Additionally, we designed a clinical study to test the effect of a new endolysin-based therapy that specifically targets S. aureus in AD. The results of the research described in this thesis show the relevance of the microbiome, in particular S. aureus in AD. The thesis describes associations between S. aureus, but also other microbes on the skin and in the nose with AD disease severity. Thereby, S. aureus seems to evoke immune responses via different mechanisms, as a directly stimulating antigen and as an allergen. The results of this thesis may contribute to the development of treatment strategies that target the microbiome in AD. Further prospective cohort studies and experimental research are needed to clarify the role of the microbiome in AD

Use of fluorescence expression tools for the comparative analysis of the interactions of Mycoplasma mycoides and Mycoplasma bovis with bovine cells

Background: Fluorescence expression systems adapted to the analysis of host-mycoplasma interactions were recently developed. The aim of this work was to apply them to study the colonisation and persistence capabilities of the bovine pathogens Mycoplasma mycoides subsp. mycoides (Mmm) and Mycoplasma bovis in bovine cells. Methods. Mini-transposons affording high-level expression of GFP2, mCherry, mKO2 and mNeonGreen were used to mark Mmm and M. bovis strains. The resulting fluorescent mutants were characterised by epifluorescence microscopy and fluorimetry and the sites of transposon insertion were identified by sequencing. Interactions of mNeonGreen mutants with bovine cells were analysed using flow cytometry and confocal microscopy. Results: The production, selection and characterisation of fluorescent clones were straightforward and compatible with the production of fluorescent mutant banks. M. bovis presented much higher adhesion, invasion and proliferation capacities than Mmm in culture with non-phagocytic cells and showed increased resistance to elimination by macrophages. Conclusion: The remarkable differences in the invasion and persistence capabilities of Mmm and M. bovis observed here are in agreement with the pathogenesis of the infections caused by these mycoplasmas. mNeonGreen fluorescent mutants have proven extremely useful for analysis of mycoplasma-host cell interactions. Furthermore, the fluorescence expression tools used in this study offer innumerable perspectives for the functional analysis of a wide range of mycoplasma species both in vitro and in vivo