Mytilus galloprovincialis releases immunologically functional haemocytes to the intervalvar space in response to tissue injury and infection.

Haemocytes of Mytilus galloprovincialis represent the main component of the internal self-defence system. Although haemocytes from haemolymph are usually studied to analyse these animals' immune response, the presence of haemocytes in the intervalvar liquid, which is essentially sea water, led us to characterize them. Several functional (ROS production, phagocytosis, gene expression, travel velocity and distance) and morphological (area, size and granularity) assays were performed by applying different stimuli to the mussels (waterborne infection, shell injury and their combination). Our results revealed that intervalvar liquid haemocytes share common characteristics with haemolymph haemocytes (for instance, the cell morphology and the cell population structure divided in three main groups) but also show significant differences in size (usually smaller in the intervalvar liquid), mobility (commonly faster in the intervalvar liquid), ROS production (higher in non-stimulated intervalvar liquid cells) and gene expression (IL17, Myd88 and CathL are over expressed in liquid intervalvar cells compared to haemolymph cells). Moreover, differences were observed when mussels were subjected to the mentioned treatments. These free intervalvar haemocytes could constitute the first line of defence as external sentinels extending the immunological alert system outside of the mussel body.

Immune-responsive gene 1 (IRG1) and dimethyl itaconate are involved in the mussel immune response.

Immune-responsive gene 1 (irg1) is a gene that is well-conserved among different taxa and is highly expressed in the mussel Mytilus galloprovincialis at the constitutive level. The expression of this gene increases after a bacterial infection, primarily in haemocytes. irg1 catalyses the production of itaconic acid from cis-aconitic acid in the Krebs cycle. Recently, itaconate has been revealed as an immune metabolite involved in macrophage polarization. In this work, we studied the effects of exogenous dimethyl itaconate (DI) on mussels in vitro and in vivo at relevant previously described endogenous concentrations and in mussels infected with Vibrio splendidus. DI did not have adverse effects on the haemocytes viability, apoptotic cells, proliferation and phagocytic activity; however, haemocyte size, velocity and accumulated distance were decreased. The antibacterial activity of DI in vitro and in vivo was observed with high concentrations of DI, that is, 30 and 50 mM, respectively. Furthermore, DI inhibited total ROS, increased mitochondrial ROS and modulated antioxidant genes, such as SOD and CAT, related to Nrf2 activation. In this research, we have demonstrated some important pathways in haemocytes in which itaconate can be involved after its production in a bacterial infection.