Developing druggable in vitro models for investigating itch in atopic dermatitis

Itch (pruritus) is a unique sensation that drives a desire to scratch. It can induce severe skin damage and critically affect psychological wellbeing (e.g., sleep-deprivation, body-image insecurity). To investigate the burden of itch, a survey was conducted with ~2500 women within the general German population, where more than one third claimed to suffer from itchy skin. Among participants affected by atopic dermatitis (AD, 6%), a relapse-remitting, inflammatory skin disease, 96.8% complained about chronic itch, making it a hallmark symptom. Some forms of itch (e.g., histaminergic, resulting from an insect bite) are well-understood and treatable, however, due to multifactorial mechanisms underlying AD itch, involving various cells (e.g., keratinocytes, Th2 cells), receptors, and mediators, treatment options are limited. Therefore, the aim of this PhD project was to develop physiologically relevant, fully human model systems for AD itch research and drug development. Human induced pluripotent stem cell-derived sensory neurons (iPSCSNs) were differentiated into a nociceptor-like phenotype and cultured with human primary skin cells to form deconstructed skin models. This included compartmentalized culture chips for examining neuronal innervation and an insert-based format with shared culture medium to observe the impact of mediators secreted by skin cells on the development of iPSCSNs. Using Ca²⁺-imaging in a direct contact 2.5D culture format, which mimics natural skin innervation and permits both paracrine exchange and juxtacrine signalling, iPSCSNs exhibited responses to pruritogens not seen in monotypic culture. Different AD/Th2-associated cytokines were used to stimulate the co-culture systems to resemble the inflamed AD lesional skin environment. It was found that TRPA1 and JAK1/2 inhibition reduced iPSCSN responses to the pruritogens thymic stromal lymphopoietin and interleukin-31, thus highlighting TRPA1 as a therapeutic target. In addition, other iPSCSN differentiation methods were explored and yielded functional TRPV1⁺, but TRPA1^-, cells, which could be used for investigating different itch/nociception mechanisms. A 3D model with an intact epidermal barrier would enable topical application testing. Therefore, skin surrogates were developed based on whole porcine skin extracellular matrix. Ultimately, the use of human extracellular matrix was also explored. There is a clear need for pharmacological advancements that benefit patients suffering from pruritic diseases, such as AD. The work described in this thesis creates further opportunities for examining the mechanisms underpinning itch and future drug development.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Mießner, Hendrik

Advisor dc:contributor.advisor

• Smith, Ewan

• 3D culture
• Atopic dermatitis
• Ca-imaging
• Co-culture
• Extracellular matrix
• Induced pluripotent stem cells
• iPSC
• Itch
• Microfluidics
• Patch-clamp electrophysiology
• Pruritus
• Sensory neurons
• Skin model
• Th2 cytokines
• TRPA1

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/95526019-4030-4fae-abd5-8af83425da33/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.101566

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/357079