SUMMARY The type 2 cytokine-high asthma endotype (T2H) is characterized by IL-13-driven mucus obstruction of the airways. To further investigate this incompletely understood pathobiology, we characterize IL-13 effects on human airway epithelial cell cultures using single-cell RNA sequencing, finding that IL-13 generates a distinctive transcriptional state for each cell type. Specifically, we discover a mucus secretory program induced by IL-13 in all cell types which converts both mucus and defense secretory cells into a metaplastic state with emergent mucin production and secretion, while leading to ER stress and cell death in ciliated cells. The IL-13-remodeled epithelium secretes a pathologic, mucin-imbalanced, and innate immunity-depleted proteome that arrests mucociliary motion. Signatures of IL-13-induced cellular remodeling are mirrored by transcriptional signatures characteristic of the nasal airway epithelium within T2H versus T2-low asthmatic children. Our results reveal the epithelium-wide scope of T2H asthma and present candidate therapeutic targets for restoring normal epithelial function.

The technique of differential dynamic microscopy is extended here, showing that it can provide a powerful and objective method of video analysis for optical microscopy videos of in vitro samples of live human bronchial epithelial ciliated cells. These cells are multiciliated, with motile cilia that play key physiological roles. It is shown that the ciliary beat frequency can be recovered to match conventional analysis, but in a fully automated fashion. Furthermore, it is shown that the properties of spatial and temporal coherence of cilia beat can be recovered and distinguished, and that if a collective traveling wave (the metachronal wave) is present, this has a distinct signature and its wavelength and direction can be measured.

Personalized approaches for systematically assessing ciliary beat dynamics and for drug testing would improve the challenging task of diagnosing and treating respiratory disorders. In this pilot study, we show how multiscale differential dynamic microscopy (multi-DDM) can be used to characterize collective ciliary beating in a non-biased automated manner. We use multi-DDM to assess the efficacy of different CFTR-modulating drugs in human airway epithelial cells derived from subjects with cystic fibrosis (ΔF508/ΔF508 and ∆F508/-) based on ciliary beat frequency and coordination. Similar to clinical observations, drug efficacy is variable across donors, even within the same genotype. We show how our assay can quantitatively identify the most efficient drugs for restoring ciliary beating for each individual donor. Multi-DDM provides insight into ciliary beating responses following treatment with drugs, and has application in the broader context of respiratory disease and for drug screening.

Coordinated motion of cilia is a fascinating and vital aspect of very diverse forms of eukaryotic life, enabling swimming and propulsion of fluid across cellular epithelia. There are many questions still unresolved, and broadly they fall into two classes. (i) The mechanism of how cilia physically transmit forces onto each other. It is not known for many systems if the forces are mainly of hydrodynamical origin, or if elastic forces within the cytoskeleton are important. (ii) In those systems where we know that forces are purely hydrodynamical, we do not have a framework for linking our understanding of how each cilium behaves in isolation to the collective properties of two or more cilia. In this work, we take biological data of cilia dynamics from a variety of organisms as an input for an analytical and numerical study. We calculate the relative importance of external flows versus internal cilia flows on cilia coupling. This study contributes to both the open questions outlined above: firstly, we show that it is, in general, incorrect to infer cilium–cilium coupling strength on the basis of experiments with external flows, and secondly, we show a framework to recapitulate the dynamics of single cilia (the waveform) showing classes that correspond to biological systems with the same physiological activity (swimming by propulsion, versus forming collective waves). This article is part of the Theo Murphy meeting issue ‘Unity and diversity of cilia in locomotion and transport’.