From a 3D-model of particle-induced granuloma-like structure to a simple macrophage bioassay predicting granulomagenic and fibrotic activity of particles

Macrophages orchestrate reactive particle segregation, compact aggregates of immune cells and non-immune cells and promote fibrosis-surrounding granulomas. We developed a simple 3D in-vitro model that mimics granuloma formation and categorizes granuloma-inducing inorganic particles. Macrophage cell line (MHS) pre-exposed for 24h to 10µg/mL of granuloma-inducing (Carbon nanotubes, CNT) or not (Carbon black,CB) particles are cocultured with fibroblasts and epithelial cells (respectively MLG and LA4 cell lines) on 0,3% agarose coated wells. Fluorescent dyes and confocal microscopy showed that these cells in presence of CNT but not CB were organized in layered compact cellular aggregates comparable to granulomas after 7 days. The supernatant collected at 24hours (but also at 78hours and 7days) contains significantly elevated levels of the pro-fibrotic mediator TIMP1 (metallopeptidase inhibitor 1) only in granuloma-inducing conditions (CNT). The levels of other pro-granulomagenic and fibrotic mediators (such as matrix metalloproteinase 1, MMP-1; Osteopontin, OPN or the chemokine CCL2) were not increased. Our data suggest that macrophages combined to structural cells respond to granuloma-inducing particles by releasing TIMP-1 and organizing in vitro granuloma-like spheroids. This model was further simplified, as MHS macrophages alone were sufficient for the specific release of TIMP-1 in response to granulomagenic particles. Quantification of macrophage-produced TIMP-1 is a novel and simple tool for predicting and assessing granuloma-inducing new material and airborne dust particles.