Investigation into the mechanisms of Stevens-Johnson syndrome and toxic epidermal necrolysis

Nwikue, GB
(2019) Investigation into the mechanisms of Stevens-Johnson syndrome and toxic epidermal necrolysis. PhD thesis, University of Liverpool.

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Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) are life-threatening immune-mediated blistering cutaneous reactions with mortality rates ranging from 10-30%. The pathophysiological mechanisms of SJS/TEN are complex and not fully understood. However, fulminant keratinocyte death and inflammation have been suggested as the major players in the mechanisms of disease. To further understand the mechanisms of SJS/TEN, we utilised both clinical samples and in vitro human immortalised keratinocyte cell line (HaCaT) cell death models to investigate mediators and putative biomarkers of SJS/TEN. HaCaT cells are phenotypically similar to primary keratinocytes and represent a suitable in vitro model for dermatologic studies. A previous study had suggested that serum profiles of high mobility group box-1 (HMGB1) protein, a known of marker of cell death and inflammation distinguished SJS/TEN from other cutaneous phenotypes (drug rash with eosinophilia and systemic symptoms, DRESS and maculopapular exanthema, MPE). To further elucidate the putative role of HMGB1 in SJS/TEN, serum, blister-fluid and skin biopsy samples from SJS/TEN were analysed for HMGB1 profile and expression. HMGB1 levels in serum were elevated compared to tolerant controls with even higher levels detected in blister-fluid compared to serum from a Spanish SJS/TEN cohort (p<0.001, n=12). In addition, HMGB1 skin epidermal expression was diminished in SJS/TEN compared to both MPE and healthy control. Nitric oxide (NO) is implicated in the destruction of the epidermis in SJS/TEN. The mechanisms of NO mediated keratinocyte death and its effect on extracellular HMGB1 levels was investigated using a NO-donating compound (NOC-18). NOC-18 treated HaCaT cells were killed via both apoptosis and necrosis and extracellular HMGB1 release was higher (p<0.05, n=3) in comparison to untreated control. Apoptosis is regulated by caspases while necroptosis is regulated by receptor interacting protein kinase-1 (RIPK1), RIPK3 and mixed lineage kinase domain-like (MLKL) protein. Tumour necrosis factor-α (TNF-α) is implicated in SJS/TEN pathology. To determine the mechanisms of TNF-α induced HaCaT keratinocyte death and assess their impact on extracellular HMGB1 profiles, HaCaT cells were treated with TNF-α. This showed that TNF-α caused keratinocyte necroptosis and apoptosis, and that necroptosis contributed significantly more to HMGB1 levels. RIPK3 is thought to play a role in SJS/TEN pathology. Triggering necroptosis in cells is problematic and generally results in a mixture of apoptosis and necroptosis. Constructs of wild-type human RIPK3, MLKL and Bak were made and stable HaCaT cell lines generated. Interestingly, RIPK3 activated both caspase-dependent apoptosis and MLKL-dependent necroptosis. Also, MLKL-induced extracellular HMGB1 levels were significantly higher than both Bak and RIPK3. Together, these results suggest that TNF-α, RIPK3 and HMGB1 might be involved in the mechanisms of SJS/TEN and could potentially be used as putative biomarkers of SJS/TEN. Further preclinical and clinical studies are needed to confirm the mechanistic roles of these proteins in the pathogenesis of SJS/TEN.

Item Type: Thesis (PhD)
Divisions: Faculty of Health and Life Sciences > Institute of Life Courses and Medical Sciences > School of Medicine
Depositing User: Symplectic Admin
Date Deposited: 06 Jan 2020 11:35
Last Modified: 02 Apr 2021 07:22
DOI: 10.17638/03056025