Patients with allergic asthma exhibit distinct temporal variations in symptoms and disease severity, which oscillate across the 24 h day. We have previously shown that the molecular circadian clock plays a critical role in temporally gating the response to allergen challenge in mice. Club cells, a subset of pulmonary bronchial epithelial cells, are implicated in the gating of time-dependent responses to type-1 inflammatory stimuli and trafficking of neutrophils to the lung. Using a club cell specific Bmal1 knockout mouse (CCSP-Bmal1 KO) we found that this is also true in the setting of chronic type-2 pulmonary inflammation, where eosinophil and T-helper 2 cell trafficking patterns were abolished in mice lacking a functional circadian clock within the lung. Notably, we find that circadian patterns in airway permeability are lost in CCSP-Bmal1 KO mice, rendering the barrier equally permissible to trafficking cells at all times of day.

Allergic asthma is promoted by type 2 inflammation involving cytokines such as IL-4, IL-5, and IL-13, with group 2 innate lymphoid cells (ILC2s) playing a key pathogenic role. Here, we identify T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) as a negative regulator of ILC2 function. Tim-3 expression is upregulated in activated pulmonary ILC2s, and engagement with Tim-3 agonists inhibits ILC2 activation, proliferation, and type 2 cytokine production via the Nemo Like Kinase (NLK) signaling pathway and suppression of mitochondrial metabolism. In vivo, Tim-3 agonists alleviate airway hyperreactivity (AHR) and inflammation in both IL-33- and Alternaria alternata-induced AHR models, while ILC2-specific Tim-3 deletion exacerbates AHR. These results are confirmed in human ILC2s and humanized mice, supporting the translational relevance. Our findings establish Tim-3 as an inhibitory checkpoint for ILC2s and suggest its potential as a therapeutic target in allergic asthma and other ILC2-mediated diseases.

Group 2 innate lymphoid cells (ILC2s) are tissue-resident immune cells that play a central role in type 2 immunity. Beyond cytokine signaling, they integrate inputs from lipids, nutrients, neuroendocrine mediators, and local metabolic cues, establishing cellular metabolism as a key regulator of their function. Immunometabolism provides a framework to understand how ILC2s adapt to diverse tissue environments such as the lung, adipose tissue, gut, skin, and brain, each defined by distinct nutrient availability, oxygen tension, and inflammatory conditions. Unlike many immune cells that primarily rely on glycolysis, ILC2s dynamically balance glycolysis, fatty acid oxidation (FAO), and oxidative phosphorylation (OXPHOS) depending on activation state and tissue context. Lipids not only serve as energy substrates but also regulate membrane organization, lipid raft–dependent signaling, and the generation of bioactive mediators, including eicosanoids, oxysterols, and sphingolipids. Emerging evidence linking cholesterol biosynthesis, steroid metabolism, and sphingolipid signaling to ILC2 function underscores the importance of lipid-dependent immune regulation. Dysregulation of these pathways contributes to chronic inflammatory diseases such as asthma, metabolic disorders, and fibrosis. Targeting metabolic pathways and checkpoints may therefore offer new strategies to modulate ILC2-driven pathology. This review summarizes current insights into metabolic programs governing ILC2 activation, survival, and plasticity and highlights emerging therapeutic opportunities.

Rationale: Asthma displays temporally variable symptoms which worsen overnight, corresponding with a nocturnal increase in airway eosinophils. The molecular clock within the club cell of the bronchial epithelium is a key driver of lung rhythmic processes, however, it’s role in chronic allergic airways disease (AAD) is not known. Elucidating the role of the club cell clock in regulating rhythmic inflammation in AAD could lead to new therapeutic advances. Objectives: To investigate the club cell molecular clock regulation of leukocyte trafficking in chronic AAD. Methods: ccsp - bmal1 KO mice (which lack a functional clock in club cells) and littermate control mice underwent a 5-week chronic house dust mite (HDM) model of AAD, following which leukocyte populations and cytokines from blood, lung and airway compartments were quantified in a 24-hour time-course. Airway epithelial cells were cultured and transepithelial electrical resistance measured to explore circadian variability in barrier permeability and impact of pharmacological modulation of the clock. Main Results: Leukocyte populations accumulate in the blood, lung and airways of HDM exposed mice in a time-of-day dependent manner, with time of peak accumulation dependent on cell type. This temporal gating of leukocyte accumulation is controlled and coordinated by the club cell circadian clock, which also regulates airway barrier integrity. Targeting REVERBa (a component of the molecular circadian clock), was effective at modifying airway barrier permeability achieving reduced transepithelial leukocyte migration. Conclusions: The club cell clock gates leukocyte trafficking signals and airway barrier integrity by time of day in chronic allergic airway inflammation.

Background: The club cell (CC) circadian clock has been identified as a key local regulator in the lung, potentially driving many of the inflammatory responses experienced by patients with asthma. The CC circadian clock is driven by BMAL1 expression, which promotes the expression of Reverba, a potential novel target for improving airway barrier function. Methods: Airway epithelial cells (AECs) were isolated from male C57BL/6, wild type (WT) and CCSP-BMAL1 knock out (KO) mice (lacking a functional CC clock). Cells were grown in air liquid interface cell culture to produce 2-D lung organoids which were then examined for epithelial barrier function, using transepithelial electrical resistance (TEER) measurements, every 4hrs over 96hrs, at baseline, +/- IL13, +/- Reverba ligand SR9009. Results: TEER values from WT AECs show a daily pattern of rhythmic fluctuations in tightness, suggesting that tight junction function is naturally rhythmic in the lung. This rhythmicity was abolished when BMAL1 was knocked out of the CCs in the lung. Furthermore, IL13 exposure dampens the rhythmicity in the airway barrier and lowers its integrity. Finally, when dosed with SR9009, a Reverba ligand, barrier integrity is improved especially in KO cells. erj;64/suppl_68/PA2659/F1 F1 F1 Perspectives: The circadian clock mechanism, particularly in CCs, plays an important role in maintaining airway barrier integrity. In the presence of IL13, barrier integrity worsens, but can be improved by targeting Reverba.