Immune response induced by multi-wall carbon nanotubes: in vivo and in vitro studies on the medicinal leech. Risposta immunitatia indotta da nanotubi di carbonio a parete multipla: studi in vivo e in vitro sulla sanguisuga medicinale.

Since the production and use of engineered nanomaterials (NMs) is steadily increasing, the development of new reliable methods to analyze NMs effects are critical. Here we propose the medicinal leech as alternative animal model to study multi-walled carbon nanotubes (MWCNTs) effects by means of in vivo (environmental dispersion and MWCNT supplemented Matrigel injection) and in vitro studies on leeches macrophages. Our results show that water dispersed MWCNTs evoke a massive migration of CD45+/CD68+ monocyte-macrophages cells and the production of molecules involved in innate immune response, such as the pro-inflammatory cytokine IL-18 and amyloid fibrils. Ultrastructural analysis of MWCNTs-supplemented biomatrix revealed that in leech macrophages MWCNTs are internalized both actively (phagocytosis) and passively (membrane piercing). Finally, MWCNT in vitro treatment cause the decrease of cell proliferation rate and the increase of both ROS production and apoptotic rate. Moreover, in this study we identified and characterized in Hirudo two inflammatory markers striking similar to vertebrate’s MIF and GMFG, showing that these molecules are involved in macrophage response to MWCNTs. Our combined experimental approaches, not only attest the ability of MWCNTs in inducing a potent inflammatory response, but also confirm the medicinal leech as a good alternative model that can be successfully used to study, both in vivo and in vitro, the possible harmful effects of any nanomaterial.