Phd in EXPERIMENTAL MEDICINE

Thesis title: Role of IL-23/IL-17 axis in the development of atherosclerotic plaque

Atherosclerosis is the main cause of death in industrialized countries and, more recently, it has also started to affect developing countries. The immune system plays a key role in the disease progression. Both innate and adaptive immunity cells are involved in the inflammatory response. Macrophages, belonging to the innate immunity, are plastic cells and their phenotype can be influenced by the surrounding microenvironment. Indeed, these cells can polarize into two types of macrophages: one characterized by a proinflammatory phenotype, called M1, which promotes inflammation by releasing proinflammatory cytokines, and one characterized by an anti-inflammatory phenotype, M2, which releases anti-inflammatory cytokines. In human atherosclerotic plaques the localization of M1 and M2 is different. Indeed, M1 macrophages are aggregated on the shoulders of the vulnerable plaques while M2 macrophages are present in the stable region away from the lipid core. Cytokines can drive macrophages’ phenotype and functions. IL-23 is a proinflammatory cytokine involved in various inflammatory and autoimmune diseases, which may also play a role in the development of atherosclerosis. IL-23 binds to its receptor IL-23R and IL-12Rβ1 and activates the JAK/STAT signaling pathway via STAT3, releasing cytokines such as IL-17A, IL-22 and IL-6. Our study focused on investigating the role of IL-23 in atherosclerosis. In particular, we analysed the role of IL-23 in macrophage polarization by Real-Time PCR, immunoenzymatic assay, flow cytometry and immunofluorescence. The analysis was carried out both on macrophages derived from THP-1 cell line models and on macrophages derived from monocytes obtained from healthy donors. Results regarding macrophages derived from human leukemia THP-1 cell line showed that IL-23 promotes the expression of the pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α in M0 macrophages, suggesting the induction of macrophage activation. It also led to an increase in the expression of IL-10 in all populations studied. In inflammatory conditions, after the stimulation with lipopolysaccharide (LPS), IL-23 increased the expression of IL-1β, IL-6 and TNF-α and IL-10 levels in all macrophage phenotypes, suggesting a synergic action with LPS. Experiments on macrophages derived from healthy donors’ monocytes did not give the same results. Indeed, IL-23 induced the expression and release of IL-6 in all subpopulations examined, while the expression of IL-1β, IL-6, TNF-α and IL-10 only increased in M1 macrophages in pro-inflammatory conditions. Furthermore, IL-23 induced M1 macrophages migration suggesting a chemotactic function. Another surprising phenomenon observed was that all macrophages produced IL-17A, with resting macrophages representing the main source of production. IL-23 also induced an increase in the expression of IL-22 and IL-10 in resting macrophages and, in association with LPS and IFN-γ, it potentiated the expression and production of IL-17A and IL-10, suggesting a synergic action between LPS and IL-23. Indeed, the stimulation with IL-23 increased both CD86 and CD206 surface markers, although, CD86 appeared to be prevalent. The immunofluorescence analysis showed that all macrophages produce IL-17A and that IL-23 increases its production, while the immunohistochemistry analysis confirms the presence of macrophages and IL-17A in atherosclerotic plaques and their co-localization.