A MULTILEVEL APPROACH TO DEFINE THE ROLE OF IMMUNE AND METABOLIC CUES ON THE MACROPHAGE/FIBROBLAST INTERPLAY DURING FIBROSIS DEVELOPMENT

Alterations in tissue homeostasis occur during inflammation, oxygen deprivation, and remodeling, but they are generally reversible processes. When the cause of injury is severe, persistent or repetitive, acute inflammation could become chronic and dysregulated wound-healing could lead to fibrosis. Dynamic interplay through immune and non-immune cells, such as macrophages and tissue fibroblasts, is crucial to determinate pathological outcome. Macrophages (Mφ) are highly plastic cells, able to assume different functional phenotypes depending on the microenvironment. Fibroblasts (Fb) are heterogeneous cells that can be activated into myofibroblasts, which are the main sources of extracellular matrix (ECM) components. In inflamed tissues and fibrotic scars macrophages accumulate in areas of hypoxia, which is known to impact on macrophages activation. In these contexts the interplay of macrophages with fibroblasts likely affects their biology. In this study, we establish an in vitro direct contact co-culture model with macrophages and fibroblasts in order to elucidate cell-to-cell interactions. The system set-up take into account different microenvironment alterations, including T-cells cytokine secretion and oxygen deprivation, in order to mimic adaptive immune contribution and metabolic switch during inflammation and fibrosis. To investigate the relevance of different microenvironmental cues on Mφ and Fb activation, we stimulated the co-cultures with pro-inflammatory stimuli (LPS+IFNor with pro-fibrotic cytokine (IL-4) or with 1% of oxygen tension (to mimic severe hypoxia). Model parameters will be extracted from molecular profiling approaches investigating 44 different combinations of Mφand Fb polarized into inflammatory (MI and FbI) and fibrotic (MF and FbF) settings under normoxic and hypoxic conditions, in single cell cultures and direct-contact co-cultures. A multi-level strategy let us to compare different samples, to discriminate the effect of single variable on the system and to combine up to four variables together. We found that, when cells are in direct-contact in hypoxic environment, resting and pro-inflammatory Mφ and Fb assume or maintain an enriched inflammatory signature whereas pro-fibrotic macrophages inhibit the acquisition of a pro-inflammatory phenotype of fibroblasts. Indeed, single influencing factor alone is not able to induce differences in resulting phenotype but when immune stimuli, hypoxia and co-cultivation are combined together, for a long period of time, they change Mφ and Fb transcriptional landscape. Implementation of these findings with functional assays is essential to deeper investigate this crosstalk in chronic inflammation and fibrosis to translate candidate genes into predictive biomarkers.