Biology subjects

Connelly, J. T.

Publications and source records attributed to Connelly, J. T..

4 recordsLinked to original sources

bioRxiv2025

Development of human skin equivalents with inducible ceramide depletion for in vitro modelling of lipid deficiency

The lipid composition of the epidermis plays a critical role in the skins barrier function, and defects in lipid synthesis or assembly can cause a spectrum of skin diseases, ranging from dry skin to severe ichthyoses. The aim of this study was to develop an in vitro model of human skin with tuneable lipid deficiency. Human N/TERT keratinocytes were engineered to express doxycycline-inducible short hairpin RNAs targeting ceramide synthase 3 (CerS3), which is essential for synthesis of ultra long chain ceramides and skin barrier function. We show that 3D human skin equivalents (HSEs) with induced knockdown of CerS3 display normal stratification and terminal differentiation but have reduced Nile Red staining for polar lipids. Further analysis of the lipidome by mass spectrometry confirmed a significant reduction in specific classes of ceramides and ceramide chain length in the CerS3 depleted HSEs. We also show that CerS3 knockdown is reversible upon removal of doxycycline and can be used to study recovery and repair of epidermal lipids. Together, these findings provide an overall strategy for genetically tuning the lipid composition within human skin models and establish a new in vitro model of ceramide deficiency.

bioengineering↗

bioRxiv2024

Replicating dynamic immune responses at single-cell resolution within a microfluidic human skin equivalent

The intricate immunological functions of human skin involve the interplay between multiple different cell types as well as dynamic trafficking of leukocytes in and out the tissue, both of which are extremely challenging to replicate in vitro. To enable in vitro investigation of human skin immunology, we developed a microfluidic human skin equivalent (HSE) that supports the delivery of circulating immune cells via a vascular microchannel embedded within the dermis of a full-thickness construct. We demonstrated that stimulation of keratinocyte inflammation with lipopolysaccharide and nigericin promoted rapid monocyte recruitment out of the vascular channel and into the epidermal layer within 24 hours, followed by a second wave of monocyte migration into the dermis over a period of six days. Single-cell transcriptomic analysis of the tissue-resident and recruited cell populations revealed dynamic and cell-specific patterns of gene expression that were characteristic of acute activation and resolution of an inflammatory immune response. Moreover, comparison of the gene signatures of the monocyte-derived cells to in vivo populations provided molecular level validation of the model and indicated a differentiation trajectory of the monocytes through to mature dermal macrophages. To extend the microfluidic platform to additional applications, we also modelled age-associated immune dysfunction by the inclusion of senescent fibroblasts, which promoted increased monocyte recruitment into the HSE, replicating previous in vivo human studies. Thus, the microfluidic HSE presented here replicates key aspects of dynamic inflammatory immune responses within the skin and represents a tractable experimental tool for interrogating mechanisms of human skin immunology.

bioengineering↗

bioRxiv2023

A biomechanical switch promotes lysosomal remodelling and exocytosis in keloid fibroblasts

Keloids are a severe form of scarring for which the underlying mechanisms are poorly understood, and treatment options are limited or inconsistent. While biomechanical forces are potential drivers of keloid scarring, the direct cellular responses to mechanical cues have yet to be defined. The aim of this study was to examine the distinct responses of normal dermal fibroblasts (NDFs) and keloid-derived fibroblasts (KDFs) to changes in extracellular matrix (ECM) stiffness. When cultured on hydrogels mimicking the elasticity of normal or scarred skin, KDFs displayed greater stiffness-dependent increases in cell spreading, F-actin stress fibre formation, and focal adhesion assembly. Elevated acto-myosin contractility in KDFs disrupted the normal mechanical regulation of ECM remodelling, leading to constitutive collagen and fibronectin deposition. Transcriptional profiling identified mechanically-regulated pathways in NDFs and KDFs, including the actin cytoskeleton, Hippo signalling, and autophagy. Further analysis of the autophagy pathway revealed that autophagic flux was intact in both fibroblast populations and depended on acto-myosin contractility. However, KDFs displayed marked changes in lysosome organisation and an increase in lysosomal exocytosis, which was mediated by acto-myosin contractility. Together, these findings demonstrate that KDFs possess an intrinsic increase in cytoskeletal tension, which heightens the response to ECM mechanics and promotes lysosomal exocytosis.

cell biology↗

bioRxiv2019

The desmosomal cadherin Desmoglein-3 regulates YAP and phospho-YAP in keratinocyte responses to mechanical forces

Desmoglein 3 (Dsg3), plays a crucial role in cell-cell adhesion and tissue integrity. Increasing evidence suggests that Dsg3 acts as a regulator of cellular mechanotransduction, but little is known about its direct role in mechanical force transmission. The present study investigated the impact of cyclic strain and substrate stiffness on Dsg3 expression and its role in mechanotransduction. A direct comparison was made with E-cadherin, a well-characterized mechanosensor, in human keratinocytes. Exposure of oral and skin keratinocytes to equiaxial cyclic strain promoted changes in expression and localization of junction assembly proteins. Knockdown of Dsg3 by siRNA blocked strain-induced junctional remodeling of E-cadherin and Myosin IIa. Importantly, the study demonstrated that Dsg3 regulates the expression and localization of YAP, a mechanosensor and an effector of the Hippo pathway. Furthermore, we showed that Dsg3 forms a complex with phospho-YAP and sequestered it to the plasma membrane, while Dsg3 depletion had an impact on both YAP and phospho-YAP in their response to mechanical forces, increasing the sensitivity of keratinocytes to both strain-or substrate rigidity-induced nuclear relocalization of YAP and phospho-YAP. We showed that PKP1 seemed to be the key in such a complex formation since its silencing resulted in Dsg3 disruption at the junctions with concomitant loss of pYAP in the peripheral cytoplasm. Finally, we demonstrated that this Dsg3/YAP pathway has an influence on the expression of YAP1 target genes as well as cell proliferation in keratinocytes. Together, these findings provide evidence of a novel role for Dsg3 in keratinocyte mechanotransduction.

cell biology↗