Abstract Rationale Leukocyte trafficking, inflammation, and immune responses are tightly regulated by the circadian rhythm. Core clock genes such as Rev-erbα maintain a 24-hour rhythm that optimizes immune surveillance and defense. Immune cell populations, including neutrophils, monocytes, and lymphocytes, exhibit diurnal variations influencing chronic inflammatory diseases like asthma, where disease severity and airway inflammation fluctuate with time of day. We hypothesize that the circadian clock within myeloid cells is critical for maintaining immune homeostasis and modulating pulmonary immune responses to allergen exposure. Methods Bone marrow cells from C57BL/6 wild-type (WT) mice were differentiated into bone marrow-derived macrophages (BMDMs) and treated with house dust mite (HDM; 50 µg) for 24 hours. Changes in inflammatory cytokine and circadian clock gene/protein expression were assessed by qRT-PCR, immunoblotting, and immunofluorescence. To evaluate whether inflammation and circadian dysregulation could be modulated, BMDMs were treated with the REV-ERBα agonist GSK4112 (20 µM) or antagonist SR8278 (20 µM). Ongoing studies extend these findings to human PBMCs from healthy and asthmatic donors differentiated into macrophages to examine HDM-induced inflammation and circadian disruption in vitro. Results HDM exposure increased expression of inflammatory cytokine genes (Il1β, Il6, Tnfα, Tgf-β1, Ccl5) and altered macrophage polarization markers, elevating M1 markers (Gpr18, Cd38) and M2 marker (Arg1) while reducing iNOS protein, indicating a mixed activation phenotype. HDM also disrupted clock gene expression, increasing BMAL1 and CRY1 proteins but reducing CLOCK, REV-ERBα, and PER2, along with decreased transcripts of Clock, Bmal1, Cry1, and Rev-erbβ. Pharmacologic activation of REV-ERBα with GSK4112 attenuated Il6, Tnfα, and Tgf-β1 expression following HDM exposure, whereas inhibition with SR8278 further amplified HDM-induced Il6, Il1β, Tgf-β1, and Ccl5 expression. These results demonstrate that REV-ERBα is a key regulator of macrophage inflammatory and circadian responses to allergens. Conclusions HDM-treated BMDMs exhibit heightened inflammatory gene expression and altered macrophage polarization accompanied by circadian clock disruption. Activation of REV-ERBα mitigated these effects, while antagonism produced the opposite outcome. These findings highlight the critical role of the circadian clock in myeloid-cell-mediated inflammation during allergic asthma. Ongoing studies using myeloid-specific Rev-erbα knockout mice exposed to aeroallergens aim to define the mechanisms linking circadian disruption to asthma pathophysiology and explore potential chronotherapeutic strategies for disease modulation. This abstract is funded by: NIH R01 HL142543
Sundar et al. (Fri,) studied this question.