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A pilot study: Investigating beneficial effects of fever on EBS (Magin 4)

Project lead Prof Thomas M Magin, Dr Katrin Rietscher
Organisation University of Leipzig, Leipzig, GERMANY
Project budget EUR 12,000.00
Start date / Duration 01. Apr 2020 / 24 months
Funder(s) / Co-Funder(s) Others, DEBRA Austria
Other funder(s) DEBRA Sweden
Research area EB genetics, epigenetics & biology

Project details

Short lay summary

Epidermolysis bullosa simplex (EBS) is a rare genetic skin fragility disorder affecting approximately 1 person per 25,000 live births. 75% of EBS cases are caused by mutations in KRT5 or KRT14, which in severe cases triggers collapse of the keratin cytoskeleton into cytoplasmic protein aggregates and renders the skin fragile to mild trauma.  Surprisingly, several EBS patients report significant improvement of their condition rapidly following onset of fever, which can last for weeks thereafter. We hypothesize that pro-inflammatory cytokines upregulated during fever directly or indirectly via immune cells, promote restoration of stress-resilient keratin networks through several pathways. Here, we examined a potential direct effect of fever-associated cytokines on the keratin cytoskeleton and cell adhesion in EBS keratinocytes.

Scientific summary

Due to the fact that fever episodes improve the EBS condition, we hypothesized that pyrogenic cytokines such as interleukin-1β (IL-1β), IL-6 or tumor necrosis factor α (TNFα) may have a detrimental or beneficial impact on the keratin cytoskeleton, either directly or indirectly. How cytokines may affect keratin filament organization is still unknown. One could hypothesize that (1) cytokine induction alters the isotype composition of the keratin cytoskeleton (K5, K14, K6, K16 and K17), which is a major determinant of adhesive strength, (2) pyrogenic cytokines activate stress/inflammatory pathways leading to altered post-translational modifications of keratins and thereby affecting their properties and organization or (3) stimulation by fever-induced cytokines may have an impact on keratin cytoskeleton indirectly via immune cells or other unknown mechanisms. 

Based on the observation that a child suffering from severe EBS (K14.R125C mutation) shows tremendous improvement immediately upon fever onset and for weeks thereafter, we analyzed the response of immortalized patient-derived K14.R125C keratinocytes in comparison to normal human keratinocytes (NHK) to the exposure of IL-1β, IL-6 and TNFα by immunofluorescence microscopy and epithelial sheet assays to assess potential effects on keratins and their associated desmosomes. We found that both cell lines can be stimulated by IL-1β, IL-6 and TNFα, which was shown by the activation of the appropriate signaling pathways using immunoblot analyses. Immunofluorescence analysis and epithelial sheet assays showed that all three tested cytokines showed neither a detrimental nor a beneficial effect on EBS K14.R125C keratinocytes.

Strategic relevance

We are aware that additional cytokines and chemokines and combinations therefrom may be relevant. In addition, an improved skin condition of EBS patients during fever episodes may be also due to invading immune cells into the skin in response to inflammatory signals, which could be analyzed by co-cultures of keratinocytes together with immune cells such as innate immune and T-cells. As keratinocytes not only sense but also secrete cytokines and this essentially determines the fate of inflammatory responses, it will be of future interest to evaluate and compare secreted cytokines from EBS keratinocytes in comparison to NHK by ELISA.

What did this project achieve?

To get a first insight into underlying mechanisms, this pilot project focused on the three cytokines IL-1β, IL-6 and TNFα and their potential impact on keratin filament organization in EBS K14.R125C keratinocytes in comparison to NHK. In addition, heat stress response of the same set of keratinocytes was analyzed to discriminate between heat- and fever-induced stresses. Epithelial sheet assays revealed that upon elevated temperature (as observed during fever episodes), EBS K14.R125C keratinocytes as well as NHK were unable to sustain a stress-resilient cytoskeleton. Elevated temperature aggravated keratin aggregation in K14.R125C keratinocytes revealed by immunofluorescence analyses, supporting the view, that elevated temperature may not be the trigger to improve the EBS condition. Stimulation of EBS K14.R125C keratinocytes and NHK with different concentrations and time points of IL-1β, IL-6 or TNFα lead to the optimal conditions to activate the appropriate signaling pathways. Using these optimized conditions, we analyzed the direct impact of these pyrogenic cytokines on the keratin cytoskeleton by immunofluorescence and epithelial sheet assays.

These analyzes revealed that these cytokines had neither a detrimental nor beneficial impact on the keratin cytoskeleton. This strongly suggests that the positive effect of fever on EBS skin conditions may be not due to a direct impact of IL-1β, IL-6 or TNFα on keratinocytes. One could speculate that fever-induced cytokines may have an impact on the keratin cytoskeleton indirectly via immune cells, which was not assessed by this pilot study due to the limitation of resources. We predict that biopsy and serum samples during the course of a fever episode from those EBS patients that report a beneficial effect of fever will be highly informative, in comparison to EBS patients who do not show this and to healthy individuals suffering from fever of similar nature.

In view of the challenge hat such a study imposes, mouse models for fever would provide a comprehensive insight not only in cytokine networks but into innate and adaptive immune cells that may be involved in the positive response of EBS skin to fever. We suggest that this should form the basis for future studies including EBS individuals.


Epidermolysis bullosa simplex
Magin 4
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