Oxidised IL-33 drives COPD epithelial pathogenesis via ST2-independent RAGE/EGFR signalling complex.

BACKGROUND: Epithelial damage, repair and remodelling are critical features of chronic airway diseases including chronic obstructive pulmonary disease (COPD). Interleukin (IL)-33 released from damaged airway epithelia causes inflammation via its receptor, serum stimulation-2 (ST2). Oxidation of IL-33 to a non-ST2-binding form (IL-33ox) is thought to limit its activity. We investigated whether IL-33ox has functional activities that are independent of ST2 in the airway epithelium. METHODS: In vitro epithelial damage assays and three-dimensional, air-liquid interface (ALI) cell culture models of healthy and COPD epithelia were used to elucidate the functional role of IL-33ox. Transcriptomic changes occurring in healthy ALI cultures treated with IL-33ox and COPD ALI cultures treated with an IL-33-neutralising antibody were assessed with bulk and single-cell RNA sequencing analysis. RESULTS: We demonstrate that IL-33ox forms a complex with receptor for advanced glycation end products (RAGE) and epidermal growth factor receptor (EGFR) expressed on airway epithelium. Activation of this alternative, ST2-independent pathway impaired epithelial wound closure and induced airway epithelial remodelling in vitro. IL-33ox increased the proportion of mucus-producing cells and reduced epithelial defence functions, mimicking pathogenic traits of COPD. Neutralisation of the IL-33ox pathway reversed these deleterious traits in COPD epithelia. Gene signatures defining the pathogenic effects of IL-33ox were enriched in airway epithelia from patients with severe COPD. CONCLUSIONS: Our study reveals for the first time that IL-33, RAGE and EGFR act together in an ST2-independent pathway in the airway epithelium and govern abnormal epithelial remodelling and muco-obstructive features in COPD.

Dynamic regulation of human epidermal differentiation by adhesive and mechanical forces.

The interfollicular epidermis is the multilayered epithelium that forms the outer layer of the skin. It is maintained by stem cells that are attached to a basement membrane, which lies on top of the underlying connective tissue, the dermis. Cells undergo terminal differentiation as they detach from the basement membrane and move toward the outer epidermal surface. Over time, many of the molecular regulators of this process have been identified. It is now is clear that these pathways also receive critical input from the physical properties of the tissue. In this review, we describe how changes in these factors regulate differentiation and how new insights from single cell RNA sequencing could provide validation or challenge to the existing experimental models.

A reductionist approach to determine the effect of cell-cell contact on human epidermal stem cell differentiation.

The balance between stem cell renewal and differentiation is determined by the interplay between intrinsic cellular controls and extrinsic factors presented by the microenvironment, or 'niche'. Previous studies on cultured human epidermis have utilised suspension culture and restricted cell spreading to investigate regulation of differentiation in single keratinocytes. However, keratinocytes are typically adherent to neighbouring cells in vivo. We therefore developed experimental models to investigate the combined effects of cell-ECM adhesion and cell-cell contact. We utilized lipid-modified oligonucleotides to form clusters of keratinocytes which were subsequently placed in suspension to induce terminal differentiation. In this experimental model cell-cell contact had no effect on suspension-induced differentiation of keratinocytes. We next developed a high-throughput platform for robust geometrical confinement of keratinocytes to hexagonal ECM-coated islands permitting direct cell-cell contact between single cells. As in the case of circular islands, differentiation was stimulated on the smallest single hexagonal islands. However, the percentage of involucrin-positive cells on small bowtie islands was significantly lower than on single islands, demonstrating that cell-cell contact reduced differentiation in response to decreased substrate adhesion. None of the small bowtie islands contained two involucrin-positive cells. Rather, if one cell was involucrin-positive the other was involucrin-negative. This suggests that there is intrinsic asymmetry in the effect of cell-cell contact in decreasing differentiation. Thus, our reductionist approaches provide new insights into the effect of the niche on keratinocyte differentiation. STATEMENT OF SIGNIFICANCE: Stem cell behaviour is regulated by a combination of external signals, including the nature of the adhesive substrate and cell-cell interactions. An understanding of how different signals are integrated creates the possibility of developing new biomaterials to promote tissue regeneration and broaden our understanding of skin diseases such as eczema and psoriasis, in which stem cell proliferation and differentiation are perturbed. In this study we have applied two methods to engineer intercellular adhesion of human epidermal stem cells, one involving lipid-modified DNA and the other involving hexagonal micropatterns. We show that the effect of cell-cell adhesion depends on cell-substrate adhesion and uncover evidence that two cells in equivalent environments can nevertheless behave differently.

Developmental cell programs are co-opted in inflammatory skin disease.

The skin confers biophysical and immunological protection through a complex cellular network established early in embryonic development. We profiled the transcriptomes of more than 500,000 single cells from developing human fetal skin, healthy adult skin, and adult skin with atopic dermatitis and psoriasis. We leveraged these datasets to compare cell states across development, homeostasis, and disease. Our analysis revealed an enrichment of innate immune cells in skin during the first trimester and clonal expansion of disease-associated lymphocytes in atopic dermatitis and psoriasis. We uncovered and validated in situ a reemergence of prenatal vascular endothelial cell and macrophage cellular programs in atopic dermatitis and psoriasis lesional skin. These data illustrate the dynamism of cutaneous immunity and provide opportunities for targeting pathological developmental programs in inflammatory skin diseases.

Delta-like 1-mediated cis-inhibition of Jagged1/2 signalling inhibits differentiation of human epidermal cells in culture.

Epidermal homeostasis depends on a balance between self-renewal of stem cells and terminal differentiation of their progeny. Notch signalling is known to play a role in epidermal  stem cell patterning and differentiation. However, the molecular mechanisms are incompletely understood. Here we demonstrate dynamic patterns of Notch ligand and receptor expression in cultured human epidermis. Notch2 and 3 act together to promote differentiation, while Notch1 decreases stem cell proliferation. The Notch ligand Jagged1 triggers differentiation when presented on an adhesive substrate or on polystyrene beads and over-rides the differentiation inhibitory effect of cell spreading. In contrast, Delta-like 1 (Dll1) overexpression abrogates the pro-differentiation effect of Jagged1 in a cell autonomous fashion. We conclude that Dll1 expression by stem cells not only stimulates differentiation of neighbouring cells in trans, but also inhibits differentiation cell autonomously. These results highlight the distinct roles of different Notch receptors and ligands in controlling epidermal homeostasis.