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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - DISSection (Doctoral Industrial School on Human Skin models for Staphylococcal infections)

Teaser

Summary

Staphylococcus aureus is a leading pathogen in surgical site and skin infections which are associated with an enormous unmet medical need. There are no licensed vaccines on the market despite the significant efforts by public and private initiatives. Indeed, vaccines tested in clinical trials in the last two decades have failed to show efficacy. Lack of known correlates of protection and reliable animal models are main reasons behind these failures.
Skin is not only a major target of S. aureus infections of different severity, which often recur, but also the entry site for systemic infection and bacterial dissemination to distant sites. Since human skin is remarkably different from mouse skin, and S. aureus expresses several human-specific virulence factors, three-dimensional organotypic human skin models would represent a valid alternative to animal infection models. However, at present such models have not been established yet for S. aureus vaccine research.
The aim of this project is to study the role of staphylococcal virulence factors in the pathogenesis and immune evasion within the human skin. To this end, the project aims at characterizing how expression of virulence factors is affected by the interaction with the skin and conversely, how skin resident immune cells respond to the pathogen.
DISSection is a multidisciplinary project where four PhD students contribute to the study of S. aureus infection of human skin from different angles.

Work performed

Two students (ESR1 and ESR2) have spent the first half of the program (18 months) at UMCU, Utrecht, and the other two (ESR3 and ESR4) at GSK, Siena. At the beginning of January 2019 the students will swap between the two sites where they will spend the remaining 18 months.
During the first 18 months, ESR1 has studied interactions of antigen-presenting cells with S. aureus. She has obtained important data supporting the identification of pattern-recognition receptors on Langerhans cells and dermal dendritic cells that sense S. aureus and assessed the cellular responses. She obtained experimental evidence that a major structural component of the S. aureus cell wall plays an important role in these interactions. Furthermore, she demonstrated that S. aureus strains harbor different modifications of this structure, which dictates the molecular interaction with specific antigen-presenting cells. These data are important both from the basic and applied science point of view as they provide: 1) increased knowledge on staphylococcal strains diversity; 2) understanding how different staphylococcal strains interact with immune cells at the molecular level and evade their response; 3) important implications for vaccine development.
ESR2 has setup experimental conditions to demonstrate a very important mechanism of protection against bacteria: how purified complement factors can mediate opsonophagocytosis in absence of other serum factors. The finding of this study has general important implications: 1) improve our understanding of opsonophagocytosis in general (a critical protective mechanism against extracellular bacteria such as S. aureus); 2) potential exploitation for developing more reliable opsonophagocytic assays, which could have implications for evaluation of vaccine-induced antibody responses; 3) understand if individual purified complement factors can mediate opsonophagocytosis of S. aureus in absence of other serum factors and in human skin.
ESR3 has established experimental conditions for extracting T cells from fresh human skin and analyze their response to S. aureus ex vivo. She was able to identify CD4+, CD8+ and Î³Î´TCR+ T cells in human skin cell suspension. Moreover, she showed that almost all CD3+ T cells expressed CD45RO, CLA and CD69, indicating that these cells are skin resident memory T cells (Tsrm). CD4+ T cell proliferation was observed after 4 days of stimulation with heat-killed S. aureus USA300, suggesting the presence of skin resident S. aureus-specific CD4+ Tsrm cells in the skin of healthy donors. A large panel of cytokines (27) produced in response to HK-S. aureus USA300 by human skin cells was analyzed, too. Notably IL-17A, IL-22 and IFN-g production was observed. Human skin contains by estimation twice as many T cells as compared to peripheral blood. Still, the protective role of skin resident memory T cells against invading pathogens remains largely unknown. This study has the following major outcomes: 1) sheds new light on T cell responses against S. aureus; 2) suggests a potentially important role of skin resident T cells against invading staphylococci; 3) sets the stage to study local T cell responses following vaccination and other therapeutic interventions.
ESR4 has determined the expression level of a large panel of S. aureus virulence factors in human skin by high-throughput real-time PCR. A shift in S. aureus transcriptional response was observed both upon adhesion and infection of skin tissues. The increased expression of some of the transcripts, such as the gene encoding -toxin (hla), was expected due its known role in human skin infection. Instead, the increased expression of other genes, was not anticipated. These data suggest a potential role of other genes, in addition to hla, in human skin infection. Therefore, this study has the following implications: 1) shows for the first time in the literature how staphylococcal gene expression is affected by the interaction with human skin over

Final results

Establishment of relevant models for studying S. aureus pathogenesis and for conducting vaccine research is a recognized scientific priority. Given that the skin is a major target of staphylococcal infection in humans, the model that we are pursuing with this project appears particularly suitable for achieving that goal. The research conducted so far puts the basis for using human skin models to study staphylococcal skin infection and the role of the local immune response against the pathogen. These data have a significant potential to help identifying novel medical interventions against S. aureus which affects millions of patients worldwide.