Chronic obstructive pulmonary disease (COPD) is a progressive lung disease that is commonly considered to be a potent driver of non-small cell lung cancer (NSCLC) development and related mortality. A growing body of evidence supports a role of the immune system, mainly played by alveolar macrophages (AMs), in key axes regulating the development of COPD or NSCLC phenotypes in response to harmful agents. MicroRNAs (miRNAs) are small non-coding RNAs that influence most biological processes and interfere with several regulatory pathways. The purpose of this study was to assess miRNA expression patterns in patients with COPD, NSCLC, and ever- or never-smoker controls to explore their involvement in smoking-related diseases. Bronchoalveolar lavage (BAL) specimens were collected from a prospective cohort of 43 sex-matched subjects to determine the expressions of hsa-miR-223-5p, 16-5p, 20a-5p, -17-5p, 34a-5p and 106a-5p by RT-PCR. In addition, a bioinformatic analysis of miRNA target genes linked to cancer was performed. Distinct and common miRNA expression levels were identified in each pathological group, suggesting their possible role as an index of NSCLC or COPD microenvironment. Moreover, we identified miRNA targets linked to carcinogenesis using in silico analysis. In conclusion, this study identified miRNA signatures in AMs, allowing us to understand the molecular mechanisms underlying smoking-related conditions and potentially providing new insights for diagnosis or pharmacological treatment.

Cigarette smoke (CS) is a driver of many respiratory diseases, including chronic obstructive pulmonary disease (COPD) and non-small cell lung cancer (NSCLC). Tobacco causes oxidative stress, impaired phagocytosis of alveolar macrophages (AMs), and alterations in gene expression in the lungs of smokers. MicroRNAs (miRNAs) are small non-coding RNAs that influence several regulatory pathways. Previously, we monitored the expressions of hsa-miR-223-5p, 16-5p, 20a-5p, -17-5p, 34a-5p, and 106a-5p in AMs derived from the bronchoalveolar lavage (BAL) of subjects with NSCLC, COPD, and smoker and non-smoker control groups. Here, we investigated the capability of CS conditionate media to modulate the abovementioned miRNAs in primary AMs obtained in the same 43 sex-matched subjects. The expressions of has-miR-34a-5p, 17-5p, 16-5p, 106a-5p, 223-5p, and 20a-5p were assessed before and after in vitro CS exposure by RT-PCR. In addition, a comprehensive bioinformatic analysis of miRNAs KEGGS and PPI linked to inflammation was performed. Distinct and common miRNA expression profiles were identified in response to CS, suggesting their possible role in smoking-related diseases. It is worth noting that, following exposure to CS, the expression levels of hsa-miR-34a-5p and 17-5p in both smokers and non-smokers, 106a-5p in non-smokers, and 20a-5p in smokers, shifted towards those found in individuals with COPD, suggesting them as a risk factor in developing this lung condition. Moreover, CS-focused sub-analysis identified miRNA which exhibited CS-dependent pattern and modulated mRNA involved in the immune system or AMs property regulation. In conclusion, our study uncovered miRNA signatures in AMs exposed to CS, indicating that CS might modify epigenetic patterns that contribute to macrophage activation and lung disease onset and progression.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease that is commonly considered to be a potent driver of non-small cell lung cancer (NSCLC) development and related mortality. A growing body of evidence supports the role of the immune system, mainly played by alveolar macrophages (AMs), as key axes regulating the development of COPD or NSCLC phenotypes in response to harmful agents. MicroRNAs (miRNAs) are small non-coding RNAs that influence most biological processes and interfere with several regulatory pathways. The purpose of this study was to assess miRNA expression patterns in patients with COPD, NSCLC, and ever- or never-smoker controls to explore theirinvolvment in smoking-related diseases. Bronchoalveolar lavage (BAL) specimens were collected from a prospective cohort of 43 sex-matched subjects to determine expression of hsa-miR-223-5p, 16-5p, 20a-5p, -17-5p, 34a-5p and 106a-5p by RT-PCR. In addition, bioinformatic analysis of miRNA target genes linked to cancer was performed. Distinct and common miRNA expression levels were identified in each pathological group suggesting their possible role as an index of NSCLC or COPD microenvironment. Moreover, we identified miRNA targets linked to carcinogenesis using in silico analysis. In conclusion, this study identified miRNA signatures in AMs, allowing us to understand the molecular mechanisms underlying smoking-related conditions and potentially providing new insight for diagnosis or pharmacological treatment.

Background: Cigarette smoke (CS) is a major risk factor for chronic lung conditions. Oxidative stress and mitochondrial dysfunction play a crucial role in CS-induced pulmonary injury. 3,5-Diiodothyronine (T2) affects energy metabolism, having mitochondria as a major target. However, the underlying mechanisms of T2 related to lung diseases are poorly understood. Aims: To investigate the protective action of T2 on CS-induced mitochondrial dysfunction in an in vitro model of human epithelial alveolar cells. Methods: ATP synthesis and cytochrome c oxidase (COX) activity, as a marker of mitochondrial function, was assessed in A549 cells pretreated with T2 and exposed to CS using a bioluminescence assay and an Oroboros 2k-Oxygraph system, respectively. An evaluation of the oxidative status was conducted by assessing superoxide radical production, superoxide dismutase (SOD) activity, and H₂O₂ levels. Moreover, we investigated the mitochondrial mass via Mito-Tracker Green (MTG) staining and flow cytometry analysis. Results: CS significantly reduced ATP production. T2 pretreatment was found to prevent CS-induced impairments in ATP synthesis, enhancing COX activity. Additionally, the 2 h T2 pretreatment of CS-exposed cells mitigated CS-induced oxidative stress, thereby enhancing SOD activity and reducing the superoxide anion and H₂O₂ levels. Finally, MTG labeling was correlated with CS-induced mitochondrial mass gain, which is associated with cell senescence. Unexpectedly, T2 was not able to significantly prevent this mass increment, probably due to its rapid mode of action. Conclusions: Our results provide new insights into the protective effects of T2 against CS-induced mitochondrial damage.

Background: Cigarette smoke (CS) is a socially acceptable addiction and a major risk factor for chronic lung conditions. Oxidative stress and mitochondrial dysfunction are thought to play an important role in CS-induced pulmonary injury. 3,5-diiodothyronine (T2) affects energy metabolism, having mitochondria as a major target. However, T2 has been relatively understudied in relation to lung diseases. Aims: to investigate T2 ability to mitigate CS-mitochondrial dysfunction in an in vitro model of human epithelial alveolar cells. Methods: ATP synthesis, as a marker of mitochondrial activity, was assessed in A549 cells exposed to CS using a bioluminescence assay. Evaluation of oxidative status was conducted through examination of superoxide radical production, H2O2 levels, and Mito-Tracker Green (MTG) FACS measurements. Moreover, we are currently evaluating superoxide dismutase (SOD) activity and cytochrome c oxidase (COX) capacity using an Oroboros O2k-FluoRespirometer. Results: CS significantly affected ATP production while pretreatment for 2h with 10μM T2 mitigates these effects (p <0.01). Additionally, T2 reduced the levels of superoxide and H2O2 induced by CS exposure (p <0.05). Finally, MTG labelling correlated with CS-induced redox imbalance (p <0.0001), whereas T2 pretreatment showed restoring properties (p <0.05). Conclusions: Our results provide new insights into the protective effects of T2 against CS-induced mitochondrial damage. Ongoing evaluations of the correlation between CS and SOD/ COX activity could pave the way for future studies focused on T2 as a novel promising strategy for CS-related diseases.