![]() 3 4 While the core circadian machinery is conserved evolutionarily, translating molecular studies from animal to human samples is critical to advance our understanding of the relationship between circadian rhythms and human disease. As circadian rhythms in mice have been shown to gate lung inflammatory responses to viruses such as influenza, and those to allergy-mediated house dust mites, our work suggests that cultured human AEC demonstrate similar molecular pathways. These results confirm the presence of endogenous, self-sustaining rhythms in core circadian genes. To develop a model for future ex vivo studies of human lung and airway diseases, we temperature synchronised human AECs in ALI culture and measured rhythmic expression in core circadian genes over 48 hours.Ĭycling incubator temperature leads to synchronisation of circadian rhythms in differentiated primary human AECs, with sustained rhythms for at least 48 hours under subsequent constant temperature. 6 Use of temperature as a synchronising signal mimics in vivo temperature rhythms and follows existing ex vivo protocols for viral infections performed at 34☌. Alternatively, temperature has also been used in immortalised cell lines in vitro to synchronise circadian rhythms but has not be investigated in the organotypic AEC-ALI model. 5 Prior work has investigated glucocorticoid or serum shock synchronisation of circadian rhythms in immortalised lung cells and mouse AECs however, such methods limit the investigation of innate inflammatory responses due to induction of widespread transcriptional changes by serum shock and glucocorticoid administration. We developed a model of synchronised circadian rhythms in human airway epithelial cells (AECs) differentiated at an air–liquid interface (ALI) to investigate whether human AECs demonstrate similar circadian rhythms to those seen in mice models.ĭifferentiated primary human AECs are used as an organotypic ex vivo model to perform molecular characterisation of the human airway since ALI cultures demonstrate all the major cell types, basal-apical organisation and intact host inflammatory responses to viral infections exhibited in vivo. 3 4 Investigation of molecular circadian rhythms in humans is limited by the need for repeated measurements within 24 hours. 1 In mice, circadian genes in airway epithelia regulate cyclical expression of inflammatory genes and susceptibility to viral infections such as influenza A. #CLOCKWORK 3D CLOCK SERIES#1 2 A series of transcription/translation feedback loops in core circadian genes such as ARNTL (encoding BMAL1), CLOCK, PER1-3, CRY1-2 and NR1D1/REV-ERBα generate molecular circadian rhythms. ![]() 1 For example, circadian rhythms in the lung may contribute to exacerbations of chronic diseases such as asthma by regulating observed rhythms in mucus production, bronchial reactivity, airway inflammation and airway resistance. Circadian rhythms are hypothesised to play an important role in complex functions exhibited at cellular, tissue, organ and organism levels. ![]()
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