Temperature synchronisation of circadian rhythms in primary human airway epithelial cells from children

Abstract

Introduction: Cellular circadian rhythms regulate immune pathways and inflammatory responses that mediate human disease such as asthma. Circadian rhythms in the lung may also contribute to exacerbations of chronic diseases such as asthma by regulating observed rhythms in mucus production, bronchial reactivity, airway inflammation and airway resistance. Primary human airway epithelial cells (AECs) are commonly used to model human lung diseases, such as asthma, with circadian symptoms, but a method for synchronising circadian rhythms in AECs has not been developed, and the presence of circadian rhythms in human AECs remains uninvestigated.

Methods: We used temperature cycling to synchronise circadian rhythms in undifferentiated and differentiated primary human AECs. Reverse transcriptase-quantitative PCR was used to measure expression of the core circadian clock genes ARNTL, CLOCK, CRY1, CRY2, NR1D1, NR1D2, PER1 and PER2.

Results: Following temperature synchronisation, the core circadian genes ARNTL, CRY1, CRY2, NR1D1, NR1D2, PER1 and PER2 maintained endogenous 24-hour rhythms under constant conditions. Following serum shock, the core circadian genes ARNTL, NR1D1 and NR1D2 demonstrated rhythmic expression. Following temperature synchronisation, CXCL8 demonstrated rhythmic circadian expression.

Conclusions: Temperature synchronised circadian rhythms in AECs differentiated at an air-liquid interface can serve as a model to investigate circadian rhythms in pulmonary diseases.

Keywords: airway epithelium; asthma mechanisms; paediatric asthma; paediatric lung disaese; respiratory infection; sleep apnoea.

Figure 1

RT-qPCR analysis of core circadian gene expression normalised to GAPDH in undifferentiated airway epithelial cells following 6 days of temperature cycling. Individual biological replicates plotted as fold expression change relative to all time points with line plot of mean expression at each time point and SD error bars. Zeitgeber time 0=end of 6 days temperature synchronisation. RT-qPCR, reverse transcriptase-quantitative PCR.

Figure 2

RT-qPCR analysis of core circadian gene expression normalised to GAPDH in differentiated airway epithelial cells following 6 days of temperature cycling. Individual biological replicates plotted as fold expression change relative to all time points with line plot of mean expression at each time point and SD error bars. Zeitgeber time 0=end of 6 days temperature synchronisation. RT-qPCR, reverse transcriptase-quantitative PCR.

Figure 3

RT-qPCR analysis of core circadian gene expression normalised to GAPDH in differentiated airway epithelial cells following serum shock. Individual biological replicates plotted as fold expression change relative to all timepoints with line plot of mean expression at each time point and SD error bars. Zeitgeber time 0=serum shock. RT-qPCR, reverse transcriptase-quantitative PCR.

Figure 4

RT-qPCR analysis of core circadian gene expression normalised to GAPDH in differentiated airway epithelial cells under constant temperature conditions. Individual biological replicates plotted as fold expression change relative to all timepoints with line plot of mean expression at each time point and SD error bars. Zeitgeber time 0=media change and start of sampling. RT-qPCR, reverse transcriptase-quantitative PCR.

Figure 5

RT-qPCR analysis of IL6, CXCL5, CCL5, CXCL8, TP63 and TUBB4A normalised to GAPDH in differentiated airway epithelial cells following 6 days of temperature cycling. Individual biological replicates plotted as fold expression change relative to all timepoints with line plot of mean expression at each time point and SD error bars. Zeitgeber time 0=end of 6 days temperature synchronisation. RT-qPCR, reverse transcriptase-quantitative PCR.