Periodic Reporting for period 2 - DISSection (Doctoral Industrial School on Human Skin models for Staphylococcal infections)
Reporting period: 2018-11-01 to 2020-10-31
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.
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.
ESR1 and ESR2 spent the first 18 months at UMCU, Utrecht, and ESR3 and ESR4 at GSK, Siena. In January 2019 the students swapped between the 2 sites where they spent the remaining 18 months.
ESR1 studied interactions of antigen-presenting cells with S. aureus. She obtained 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. She demonstrated that S. aureus strains harbour 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; 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 setup experimental conditions to demonstrate an important mechanism of protection against bacteria: how purified complement factors can mediate opsonophagocytosis in absence of other serum factors. This study has important implications: 1) improve our understanding of opsonophagocytosis (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 established experimental conditions for extracting T cells from fresh human skin and analyze their response to S. aureus ex vivo. She identified CD4+, CD8+ and γδTCR+ T cells in human skin cell suspension. 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 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 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. 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 time; 2) give insight into the pathogenicity of S. aureus in skin and soft tissue infection progression; 3) suggests a role of antigens in skin infection which could be targeted by a S. aureus vaccine.
The students had the opportunity to work in very different environments. At UMCU they were more exposed to a basic science environment, while at GSK they were immersed in an applied science culture. The two different experiences had complementary features and the students learnt both aspects and exploited in the best way possible this program in which they had the opportunity to spend half of their PhD in academia and half in industry.
ESR4 submitted a paper to PNAS; the manuscript “Virulence gene expression of Staphylococcus aureus in human skin” will be soon submitted to J Infect Dis.
ESR1 and ESR2 published “Human three-dimensional models for studying skin pathogens” in the book series of Curr Top Microbiol Immunol (2020).
ESR2 submitted “Use of flow cytometry to evaluate phagocytosis of Staphylococcus aureus by human neutrophils” to Front Immunol; she is preparing two manuscripts to be submitted in 2021.
ESR1 published a study on the recognition of S. aureus WTA with the MGL receptor in Cell Microbiol (2019), and a review article on the role of C-type lectin receptors in anti-bacterial host defense, including S. aureus, in Front Cell Infect Microbiol (2020)
ESR3 submitted a paper on the recognition and activation of S. aureus WTA by Langerhans cells.
ESR1 and ESR3 are preparing a manuscript on skin-resident memory CD4+ T cells to be submitted to Front Immunol.
ESR1 studied interactions of antigen-presenting cells with S. aureus. She obtained 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. She demonstrated that S. aureus strains harbour 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; 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 setup experimental conditions to demonstrate an important mechanism of protection against bacteria: how purified complement factors can mediate opsonophagocytosis in absence of other serum factors. This study has important implications: 1) improve our understanding of opsonophagocytosis (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 established experimental conditions for extracting T cells from fresh human skin and analyze their response to S. aureus ex vivo. She identified CD4+, CD8+ and γδTCR+ T cells in human skin cell suspension. 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 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 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. 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 time; 2) give insight into the pathogenicity of S. aureus in skin and soft tissue infection progression; 3) suggests a role of antigens in skin infection which could be targeted by a S. aureus vaccine.
The students had the opportunity to work in very different environments. At UMCU they were more exposed to a basic science environment, while at GSK they were immersed in an applied science culture. The two different experiences had complementary features and the students learnt both aspects and exploited in the best way possible this program in which they had the opportunity to spend half of their PhD in academia and half in industry.
ESR4 submitted a paper to PNAS; the manuscript “Virulence gene expression of Staphylococcus aureus in human skin” will be soon submitted to J Infect Dis.
ESR1 and ESR2 published “Human three-dimensional models for studying skin pathogens” in the book series of Curr Top Microbiol Immunol (2020).
ESR2 submitted “Use of flow cytometry to evaluate phagocytosis of Staphylococcus aureus by human neutrophils” to Front Immunol; she is preparing two manuscripts to be submitted in 2021.
ESR1 published a study on the recognition of S. aureus WTA with the MGL receptor in Cell Microbiol (2019), and a review article on the role of C-type lectin receptors in anti-bacterial host defense, including S. aureus, in Front Cell Infect Microbiol (2020)
ESR3 submitted a paper on the recognition and activation of S. aureus WTA by Langerhans cells.
ESR1 and ESR3 are preparing a manuscript on skin-resident memory CD4+ T cells to be submitted to Front Immunol.
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.