Cellular mechanisms by which human keratinocytes secrete IL-33 and TSLP in response to Staphylococcus aureus

  • Yasmine Alshammari

Student thesis: Phd

Abstract

Atopic Dermatitis (AD) is a common inflammatory skin disease, characterised by epidermal barrier disruption and a type-2 cytokine response. Staphylococcus aureus (S. aureus) preferentially colonises AD skin and induces the release of IL-33 and TSLP. The aim of this thesis was to investigate the cellular mechanisms by which soluble staphylococcal virulence factors (FSA) induce type 2 cytokines. Release of IL-33 and TSLP by Normal Human Epidermal Keratinocytes (NHEK) exposed to the filtered supernatant of S. aureus (FSA) were assayed by ELISA. Cytotoxicity was assessed by flow cytometry. The role of serine proteases and Protease Activated Receptors (PAR1 & PAR2) were investigated using activating peptides, protease inhibitors and small interfering RNA (siRNA). Potential synergy with keratinocyte growth factors and their receptors were studied using recombinant proteins and neutralising monoclonal antibodies. FSA-induced IL-33 and TSLP release by NHEK were blocked by protease inhibitors. Additionally, FSA contained inherent chymotrypsin-like activity, and FSA co-culture with NHEK induced the secretion of kallikrein (KLK) 5, 7 and 14 and additional trypsin-like activity. This correlation with IL-33 release suggested that proteases were involved in triggering cutaneous type 2 responses. However, rKLKs and PAR2 peptides did not promote type 2 responses, and PAR2 silencing by siRNA did not suppress FSA-induced cytokine release. In contrast, high dose insulin synergised with FSA to release IL-33 and TSLP but did not act via its cognitive insulin receptor or through the related insulin-like growth factor-1 receptor, suggesting an alternative mechanism. Separately, Epidermal Growth Factor receptor was shown to play a role in release of IL-33 since rEGF suppressed, and the EGF neutralising monoclonal antibody cetuximab augmented release of IL-33 from NHEK. FSA-induced KLKs 5, 7 and 14 do not directly promote the rapid secretion of epidermal IL-33 and TSLP from human keratinocytes. Similarly, PAR2 is not involved in this process. In contrast, the EGF pathway appears to regulate FSA-induced IL-33 release. A better understanding of the mechanisms by which staphylococcal virulence factors and the EGFR pathway synergise to promote type 2 responses may lead to novel therapies for AD.
Date of Award31 Dec 2021
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorPeter Arkwright (Supervisor), Joanne Pennock (Supervisor) & Guoqing Xia (Supervisor)

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