Sviluppo di un modello in-vitro per lo studio dell'infiammazione granulomatosa nei pazienti con sarcoidosi ed immunodeficienti

Sarcoidosis is a rare multisystemic inflammatory disease with unknown etiology, characterized by the formation of non-caseating granulomas. Disease induction and maintenance mechanisms are still unclear, aided by the lack of in-vitro models able to properly recapitulate the disease. Recent studies also demonstrated the relevance of innate immunity, proving complete granulomas formation (Granulomatous Lymphocytic Interstitial Lung Disease: GLILD) in patients with Common Variable Immunodeficiency (CVID). As macrophages were identified as microenvironmental influencers in sarcoidosis whereas their role in GLILD is currently unknown and considering evidence indicating the importance of models’ 3D structure, the aim of the study was to develop a 3D in-vitro model helpful to clarify macrophage involvement in granuloma formation and persistence. Moreover, as the role of mTOR pathway was recently highlighted in sarcoidosis, its involvement in multinucleated giant cells (MGC) formation was also investigated. Therefore, monocytes from THP-1 monocytic cell line, sarcoidosis, CVID, GLILD patients and healthy donors were stimulated in 2D for 7 days with GM-CSF and for 7 days with IL-4 to obtain MGC. Cells underwent immunofluorescence staining and 80 images per sample were analyzed by Fiji to evaluate the fusion index (number of MGC nuclei /total number of nuclei), MGC % and area. By Western Blot P-S6/S6 and P-AKT/AKT were evaluated as markers of mTORC1 pathway activation while CD301 due its involvement in IL-4-induced MGC formation. The 3D model was developed by human femoral bone fragments decellularization and then recellularized with THP-1 or healthy macrophages and cultured as above. 3D digital reconstructions were performed by confocal microscopy. Data showed a statistically higher MGC% and fusion index in sarcoidosis and CVID + GLILD samples in respect to the non-granulomatous controls. Moreover, the smallest cell area was found in sarcoidosis MGC, whereas the biggest one in CVID + GLILD. Interestingly, a modulation of the pathway in granulomatous patients treated cells with respect to healthy donors was found, straightening the idea that the mTORC1 pathway is implicated in granuloma formation. Moreover, the surface molecule CD301 was significantly increased after IL-4 stimulation, mostly in granulomatous patients’ samples suggesting a role behind the higher intrinsic tendency of sarcoidosis and CVID + GLILD macrophages to fuse into MGC. Preliminary results suggest that THP-1 and primary macrophages were able to adhere and recellularized the bone-based 3D scaffold, paving the way for MGC studies in the 3D environment. Furthermore, a potential implication of neoplastic B cells was found in MGC formation. The development of a fluidic system, together with the results obtained so far, will help to clarify the mechanisms behind granuloma formation both in sarcoidosis and CVID + GLILD. Nevertheless, this approach may be exploited in the future to create patient-specific models in order to design patient-tailored therapies.