Use of the cellular cultures and flow cytometry for the study of chronic lung diseases: a translational research project

Abstract Eosinophils are pleiotropic granulocytes implicated in host defense against parasites and modulation of Th2 inflammatory responses. Their involvement is relevant in chronic lung diseases, such as asthma and COPD, where they contribute to the release of pro-inflammatory mediators and tissue damage. Recent studies described two distinct eosinophilic subtypes: resident eosinophils (rEos), involved in tissue homeostasis, and inflammatory eosinophils (iEos), recruited in response to pathogenic stimuli in inflammatory sites. These two subtypes can be distinguished by the expression of CD62L, being elevated (CD62Lhigh) in residents and reduced (CD62Llow) in inflammatory eosinophils. This thesis aimed to optimize an experimental model of eosinophil maturation by differentiating EoL-1 cells with butyric acid (BA), and to investigate the effects of IL-5, LPS, and macrophage-derived supernatants (M0, M1, and M2) on eosinophil differentiation toward rEos or iEos phenotypes. In parallel, it aimed to develop and validate a human whole-blood assay, for the flow cytometry characterization of eosinophil subsets in asthma and COPD. Methods Commercial EoL-1 cell line was stimulated with different concentration of BA at different time-points and subsequently exposed to IL-5, LPS, or supernatants from polarized macrophages (M0, M1, M2). Morphological, phenotypic and molecular characteristics were assessed by microscopy, flow cytometry, and RT-PCR. In parallel, a whole-blood flow cytometry assay was developed and validated for the identification of rEos and iEos in human peripheral blood. Results The stimulation with BA, 500 μM for 120 hours, was the most effective in inducing differentiation of EoL-1 cells, as confirmed by the microscopic analysis that showed nuclear remodeling, increased cell dimension and granularity. Flow cytometry analysis confirmed that BA-treated EoL-1 cells expressed a significant increase of eosinophils population markers, such as Siglec-8 and CCR3, and of functional molecules, such as CD62L and IL5Rα. At the same time, gene expression analysis confirmed the presence of transcripts characteristic of mature eosinophils, such as GATA1, ECP and CCR3. IL-5 and LPS did not further enhance maturation but selectively modulated inflammatory mediators (IL-5, IL-8, IL-13). Exposure to macrophage-polarized supernatants influenced eosinophil differentiation: M0, M1 and M2 supernatants reduced CD62L and IL5Rα, increased CCR3, and enhanced pro-inflammatory gene expression, thus driving cells toward an iEos-like phenotype. In human peripheral blood, the validated flow cytometry assay showed low intra- and inter-sample variability and stability of samples up to 4 hours at room temperature. Evaluation of eosinophils in peripheral blood demonstrated significantly higher iEos and lower rEos proportions in both asthmatic and COPD patients compared to controls, while CCR3 and IL5Rα levels remained unchanged. Conclusion In conclusion, the results of this thesis indicate that treatment with butyric acid represents an effective experimental strategy for inducing the maturation of the EoL-1 cells. The integration of butyric acid treatment with specific inflammatory stimuli derived from differentiated macrophages (M0, M1, and M2) drove the cells toward a pro-inflammatory phenotype. Finally, a whole-blood assay for discriminating eosinophils subsets in human blood samples was validated. The findings obtained indicate that inflammatory eosinophils were increased in asthma and COPD supporting their contribution in airway inflammation. Therefore, the findings of this thesis provide valuable insights into the cellular and molecular mechanisms regulating eosinophil function in chronic inflammatory lung diseases such as asthma and COPD, while also offering new opportunities for identifying potential therapeutic targets and developing innovative intervention strategies.