Asthma exacerbations often begin and increase in severity at night. Though animal models have shown molecular circadian rhythm involvement in immune and inflammatory responses, little is known about how circadian rhythms impact responses in humans or diseases such as asthma. BMAL1/ARNTL, CRY1, NR1D1, and PER2 are the genes that form the “cellular clock” by which cells tell time. Our hypothesis is that core circadian gene expression is maintained in an expected, rhythmic manner in epithelial cells from donors with asthma. We use an ex vivo model with human airway epithelial cells cultured at an air-liquid interface in a temperature cycled incubator to mimic the epithelia of the human airway. After temperature cycling to synchronize cellular circadian cycles, RNA collection occurs every four hours over a 48-hour period. After RNA isolation, I perform reverse-transcriptase quantitative polymerase chain (RT-qPCR) on a planned eight donor lines (4 healthy/4 asthmatic) to measure the gene expression of the four clock forming genes. In three asthmatic donor lines, I have found that core circadian rhythmicity is maintained in asthmatic epithelial cells and resembles the circadian rhythm expression in eight healthy donor lines previously analyzed. Shown through a preliminary study conducted by the lab, genes linked to asthma in the IL-17 signaling pathway have altered circadian rhythms of gene expression. In the future, I will use qPCR to study immune and inflammatory genes to confirm the altered rhythmicity across a wider scope of donor lines. In addition, I will analyze gene expression in different subsets of asthma to investigate whether altered circadian regulation contributes to asthma subtypes, such as T2-low which has been linked to IL-17 signaling pathway dysregulation. Investigating the differences in asthma-related circadian gene expression is essential to the development of chronotherapeutics – therapies that take into account time of day.

The immune system and inflammatory responses to viral infections are regulated by molecular circadian rhythms in mouse models. Mice infected with influenza just prior to their active phase have a mortality rate four times higher than mice infected just prior to their rest phase. As a result, circadian rhythms are hypothesized to regulate viral replication and early immune responses in airway epithelia during viral infections. Prior work has shown circadian cycles regulate gene expression in human epithelial cells. However, the influence of time of infection on viral replication in human airway epithelia has not yet been explored. We hypothesized that circadian-synchronized human airway epithelial cells would demonstrate differential viral replication and immune responses when infected at two different times of day. To address this gap, we differentiated primary epithelial cells from healthy children at an air-liquid interface to create an ex vivo cellular model of the human airway. Airway epithelial cells underwent circadian synchronization using temperature cycled incubators and were exposed on the apical surface to human rhinovirus-16 at time 0 and 12 hours during a circadian cycle. The RNA from seven total cell lines was sequenced and viral genome copy number was quantified at hour 96 following infection using GeneSig qPCR. Infection at hour 12 led to two-fold higher viral genome copy number 96 hours after infection as compared to hour 0. Infection late in the circadian phase (time 12) leads to increased viral replication at the airway epithelium and may explain the difference in mortality in mouse models of viral infection. Ongoing work is investigating immune responses based on time of infection. In the future, we will investigate changes in circadian regulation of viral infection in airway epithelia from healthy children and children with airway diseases such as asthma.