Smoking affects epigenetic ageing of lung bronchoalveolar lavage cells in Multiple Sclerosis

Background: A compelling body of evidence implicates cigarette smoking and lung inflammation in Multiple Sclerosis (MS) susceptibility and progression. Previous studies have reported epigenetic age (DNAm age) acceleration in blood immune cells and in glial cells of people with MS (pwMS) compared to healthy controls (HC).Objectives: We aimed to examine biological ageing in lung immune cells in the context of MS and smoking. Methods: We analyzed age acceleration residuals in lung bronchoalveolar lavage (BAL) cells, constituted of mainly alveolar macrophages, from 17 pwMS and 22 HC in relation to smoking using eight DNA methylation-based clocks, namely AltumAge, Horvath, GrimAge, PhenoAge, Zhang, SkinBlood, Hannum, Monocyte clock as well as two RNA-based clocks, which capture different aspects of biological ageing.Results: After adjustment for covariates, five epigenetic clocks showed significant differences between the groups. Four of them, Horvath (P-adj = 0.028), GrimAge (P-adj = 4.28 x 10(-7)), SkinBlood (P-adj = 0.001) and Zhang (P-adj = 0.02), uncovered the sole effect of smoking on ageing estimates, irrespective of the clinical group. The Horvath, SkinBlood and Zhang clocks showed a negative impact of smoking while GrimAge detected smoking-associated age acceleration in BAL cells. On the contrary, the AltumAge clock revealed differences between pwMS and HC and indicated that, in the absence of smoking, BAL cells of pwMS were epigenetically 5.4 years older compared to HC (P-adj = 0.028). Smoking further affected epigenetic ageing in BAL cells of pwMS specifically as non-smoking pwMS exhibited a 10.2-year AltumAge acceleration compared to pwMS smokers (P-adj = 0.0049). Of note, blood-derived monocytes did not show any MS-specific or smoking-related AltumAge differences. The difference be-tween BAL cells of pwMS smokers and non-smokers was attributable to the differential methylation of 114 AltumAge-CpGs (P-adj < 0.05) affecting genes involved in innate immune processes such as cytokine production, defense response and cell motility. These changes functionally translated into transcriptional differences in BAL cells between pwMS smokers and non-smokers.Conclusions: BAL cells of pwMS display inflammation-related and smoking-dependent changes associated to epigenetic ageing captured by the AltumAge clock. Future studies examining potential confounders, such as the distribution of distinct BAL myeloid cell types in pwMS compared to control individuals in relation to smoking may clarify the varying performance and DNAm age estimations among epigenetic clocks.