PO:06:089 | Heat-not-burn cigarettes induce protein post-translational modifications (PTM): possible role in the pathogenesis of rheumatoid arthritis

Claudia Ciancarella¹, Federica Maria Ucci¹, Valeria Manganelli², Elena Fasciolo², Roberta Priori¹, Fulvia Ceccarelli¹, Manuel Sergi³, Camilla Montesano³, Francesco Bartolini³, Maurizio Sorice², Cristiano Alessandri¹, Fabrizio Conti¹ | ¹Rheumatology Unit, Department of Internal Medicine, Anesthesiology and Cardiovascular Sciences, La Sapienza, Roma, Italy; ²Department of Experimental Medicine, La Sapienza University of Rome, Italy; ³Department of Chemistry, La Sapienza University Of Rome, Italy

Aim of the work. Rheumatoid arthritis (RA) is an autoimmune inflammatory disease in which a complex interaction of genetic, environmental, and hormonal factors leads to breakdown of tolerance, resulting in autoantibody production, synovial inflammation and articular damage. Cigarette smoke plays a central role in the development of ACPA positive RA, especially in people who present HLA-DRB*0104 allele. The role of post-translational modifications (PTM), such as citrullination and carbamylation, is central. Cigarette smoke damages the epithelium of the respiratory tract, leading to an increased rate of necrosis, apoptosis and NET-osis, all associated to an increase in proteins citrullination. Moreover, nanoparticles found in tobacco smoke lead to PAD-IV activation, favouring citrullination. Finally, cigarette smoke is rich in thiocyanate which participates in protein carbamylation. Recently, tobacco companies developed new products, such as electronic and heat-not-burn cigarettes (HNBC), which have become increasingly popular in recent years. To date, no studies on these products have been conducted in the rheumatology field. We evaluated whether HNBC is associated with an increase in PTM and the effects on cell viability and mechanism of cell death, such as apoptosis.

Methods. We used HNBC IQOS and burning cigarettes (BC) Malboro Red. CSE was prepared by a method modified by Chen et al. by bubbling a cigarette in 10 mL of cell growing medium (DMEM) at a constant rate until fully consumed.Bronchial epithelial cells (BEAS-2B), were treated with extracts from BC and HNBC undiluted (100%), diluted to 50% and 25% and incubated at 37°C for 4 hours. Evaluation of citrullinated and carbamylated proteins was performed by Western Blot.Furthermore, incubated BEAS-2B were stained with FITC-conjugated annexin V (AV) and propidium iodide (PI) before being analysed by flow cytometry to evaluate apoptosis.

Results. Densitometric analysis revealed that the levels of both citrullinated and carbamylated proteins were significantly higher in BEAS-2B cells treated with extracts from BC and HNBC than in the untreated control cells (Figure 1A). Representative dot plots of propidium iodide (PI)-Annexin V by flow cytometry are shown (Figure 1B). Interestingly, following treatment with either BC or HNBC, the levels of apoptosis observed in BEAS-2B cells were significantly increased compared to untreated control cells, with apoptosis rates varying according to the treatment concentration.

Conclusions. Our results show that the extract of HNBC and BC increases citrullination, carbamylation and influences cell viability, causing an augmentation of apoptosis. This is the first study to show the effects of HNBC on post-translational modifications and cell death mechanisms. The impact of HNBC CSE on key cellular mechanisms in the pathogenesis of autoimmune diseases raises alarm about the safety of these smoking alternatives in rheumatology.These data allow us to speculate that HNBC cigarettes, like BC,could lead to the development of overt disease in genetically susceptible individuals.