Modulatory function of calmodulin on phagocytosis and potential regulation mechanisms in the blood clam Tegillarca granosa.

Abstract Unlike vertebrate species, invertebrates lack antigen-antibody mediated immune response and mainly rely on haemocyte phagocytosis to fight against pathogen infection. Recently, studies conducted in model vertebrates demonstrated that the multifunctional protein calmodulin (CaM) plays an important role in regulating immune responses. However, the intrinsic relation between CaM and phagocytosis process remains poorly understood in invertebrate species such as bivalve mollusks. Therefore, in the present study, the immunomodulatory function of CaM on haemocyte phagocytosis was verified in the blood clam, Tegillarca granosa, using the CaM-specific inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7). Results obtained show that CaM inhibition significantly suppressed the phagocytic activity of haemocytes. In addition, CaM inhibition constrained intracellular Ca2+ elevation, hampered actin cytoskeleton assembly, suppressed calcineurin (CaN) activity, and disrupted NF-κB activation in haemocytes upon LPS induction. Furthermore, expression of seven selected genes from the actin cytoskeleton regulation- and immune-related pathways were significantly downregulated whereas those of CaM and CaN from the Ca2+-signaling pathway were significantly upregulated by in vitro incubation of haemocytes with W-7. For the first time, the present study demonstrated that CaM play an important role in phagocytosis modulation in bivalve species. In addition, the intracellular Ca2+ and downstream Ca2+-signaling-, actin cytoskeleton regulation-, and immune-related pathways offer candidate routes through which CaM modulates phagocytosis.