Auguste M , Melillo D , Corteggio A , Marino R , Canesi L , Pinsino A , Italiani P , Boraschi D .

	FIGURE 1. Schematic representation of experimental immune assessments using the mussel Mytilus galloprovincialis. Studies can be performed at the whole animal level with in vivo experiments or on isolated haemocytes and haemolymph with in vitro assays.
	FIGURE 2. Immune responses and defensive reactions in Ciona robusta. Following exposure to inflammatory molecules (LPS), immunocompetent haemocytes are activated, and granular haemocytes release antimicrobial peptides, the cytokines TNFα and IL-17, phenoloxidase and complement components. These molecules participate to cellular defence activities, such as phagocytosis, encapsulation and wound healing, and recruit other immune cells to the sites of inflammation. Exposure to toxicants activates a detoxifying system (thiol-containing molecules) through the direct release of metallothioneins, GSH and phytochelatins by granular haemocytes. Exposure to inflammatory/toxic agents modulates the expression of immune-related genes and activates haematopoiesis.
	FIGURE 3. Immune defensive reactivity of sea urchin to external stimuli and toxicants. Schematic depiction of the immune functions, molecules, and signalling pathways activated during the sea urchin defensive response to threats and toxicants. Upper left: sea urchin anatomy. SRCR: Scavenger Receptor Cysteine-Rich; TLR: Toll-like receptor; NLR NOD-like receptor.
	FIGURE 4. Immunotoxicity evaluation in human cells. The most used in vitro models/systems are based on cell lines or primary cells (e.g., leukocytes isolated from whole blood). They encompass conventional 2D cultures, 3D co-cultures and the more innovative Organs-on-ChiP arrays. The whole blood can be considered as a “ready-to-use” system. 2D cultures with monocytic cell lines (e.g., THP-1 or U937) or with the more reliable primary cells (e.g., monocytes, NK or DC) are mainly used to measure the inflammatory reactivity of innate immune cells. Conversely, the Whole Blood Assay (WBA; based on the use of anticoagulated whole blood) allows us to study the global reactivity of blood cells. Overall, the immunotoxicity is evaluated by measuring several biological and functional endpoints through different evaluation techniques. The main techniques and endpoints are shown.

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