Regulation of the lung fibroblast immune response by the core clock protein BMAL1

Circadian rhythms are daily oscillations in biological processes, which peak and trough once every 24-hours. Within each cell, circadian rhythms are controlled by a system of positive and negative transcriptional-translational feedback loops known as the endogenous “molecular clock” comprised of the core clock components. The core clock protein BMAL1 is central to this time-keeping system as its deletion leads to disruption of the molecular clock and associated outputs. Shift-work and erratic sleeping/eating patterns, both of which are prevalent in modern society, leads to circadian misalignment and disruption and is associated with a host of chronic inflammatory diseases. The lung is an important immunological organ that displays significant circadian rhythmicity in function, while numerous respiratory pathologies have time-of-day variation in disease severity. Lung fibroblasts stomal cells abundant throughout tissues and are integral coordinators of immune cell recruitment to the lung through chemokine secretion. We are just beginning to appreciate that the molecular clock plays a crucial role in mediating immune cell function through control of intracellular metabolic pathways, allowing coordination of time-of-day responses in numerous tissues including the lung. Circadian rhythms direct the recruitment of immune cells to the lung, which in turn impacts the immune response to infection and survival. Although fibroblasts display robust circadian rhythms, the contribution of the fibroblast molecular clock to lung-specific migration of immune cells and recruitment remains to be established. 

A key aim in this thesis is to evaluate the effect of Bmal1 deletion on the lung fibroblast response to inflammatory mediators. Bmal1 deletion in immortalised lung fibroblasts enhanced glycolytic flux, chemokine, and growth factor expression upon IL-1β stimulation, indicating that Bmal1 acts as an anti-inflammatory break in IL-1β signalling in lung fibroblasts. This is a novel role for Bmal1 in the lung fibroblast immune response. 

Glycolysis and NF-κB signalling are crucial mediators of the immune response to infection and have been widely investigated in classical innate immune cells such as macrophages. Herein, the importance of glycolysis and NF-κB signalling are investigated for the first time in IL-1β-activated lung fibroblasts. This thesis explores the possible mechanism behind increased inflammatory responses in IL-1β-activated Bmal1-/- immortalised lung fibroblasts. Bmal1-/- immortalised lung fibroblasts are dependent on glycolysis and NF-κB signalling for enhanced expression of the chemokines Ccl5 and Cxcl5, indicating that the molecular clock, metabolism, and immune signalling are interconnected in lung fibroblasts to coordinate immune cell recruitment. 

Time-of-day immune responses in the lung are coordinated by peripheral synchronisers to ensure that immune cells are recruited to the lung when we are most likely to encounter an infection. Here, the ability of lung fibroblasts to coordinate time-of-day responses to IL-1β is investigated for the first time. IL-1β-stimulated immortalised Bmal1+/+ lung fibroblasts that are synchronised in vitro have increased CXCL5 production at dusk compared to dawn, which is also observed in whole lung samples from wild-type mice administered intranasal LPS at dusk compared to dawn. The importance of the molecular clock in lung fibroblasts immune cell recruitment is also established, whereby loss of Bmal1 in immortalised lung fibroblasts increases Cxcl5 expression and neutrophil recruitment. Inhibition of NF-κB reduces Cxcl5 expression and neutrophil recruitment to levels observed for Bmal1+/+ immortalised lung fibroblasts. Collectively, these results demonstrate that Bmal1 represses glycolysis and NF-κB activity in lung fibroblasts to control chemokine expression and immune cell recruitment during an inflammatory response. This is a new mechanism by which Bmal1 regulates intracellular signalling and metabolism in lung fibroblasts to control the immune response to infection.