Heat-Not-Burn Tobacco Aerosols Induce Immune Dysregulation and Barrier Disruption Comparable to Conventional...

Heat-not-burn (HnB) tobacco products are marketed as a safer alternative to conventional cigarettes (CC), yet their health effects remain controversial, largely based on industry-funded studies. We aimed to assess lung function in HnB and CC users and examine the mechanistic effects of HnB exposure using animal and in vitro models.

Spirometry data from 650 non-smokers (NS), 293 CC smokers, and 49 HnB users enrolled in the Bialystok-PLUS cohort were analyzed. To investigate underlying mechanisms, a whole-body mouse exposure model was performed with HnB aerosol or 3R4F cigarette smoke, assessing lung transcriptomic profiles (by means of RNA sequencing), lung inflammation (by flow cytometry and histochemical staining), and barrier integrity (by using immunofluorescent staining). Moreover, an in vitro ALI-culture model of human airway epithelial cells was used to validate the mouse experiments. The effect of HnB and CC was assessed through cytotoxic effect (MTT and LDH), inflammatory responses (cytokine release by ELISA), and tight junction proteins (immunofluorescence staining).

Spirometric analysis (FEV1, FVC, VC) revealed no significant overall differences in lung function among HnB users, CC smokers, and non-smokers (NS) in the unadjusted comparison. However, subgroup analyses showed that younger (< 45 years) HnB users and those with shorter smoking histories (< 20 years) already exhibited significantly lower FEV1, FVC, and VC compared to NS and CC smokers. After adjustment for age and smoking-related covariates, HnB users displayed modest but significant reductions in static FEV1 and FVC relative to NS, while no adjusted differences were detected between HnB and CC groups, indicating comparable baseline ventilatory function. Both exposures in mice increased eosinophils and B cell counts by ~2-fold, while decreasing ST2⁺ CD4⁺ T cells over 80%, and induced epithelial apoptosis. HnB aerosol selectively reduced basophils by almost 50%. Both HnB and CC exposure in mice caused significant epithelial damage and impaired airway barrier function, evidenced by a ~60% reduction in ZO-1. Transcriptomic analysis revealed that CC smoke triggered a robust inflammatory gene response, whereas HnB aerosol predominantly altered metabolic pathways. In vitro, barrier function was impaired under both conditions, but cytokine secretion increased only after CC smoke exposure.

Despite different molecular impacts, HnB aerosol exposure induces a level of airway epithelial injury and immune cell infiltration comparable to CC smoke. Our findings challenged the narrative of HnB products being a harmless alternative and highlight their potential to cause significant lung pathology.