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

Highlights

• CaM inhibition constrains intracellular Ca²⁺ elevation and actin cytoskeleton assemble.

• CaM inhibition suppresses calcineurin (CaN) activity and disrupts NF-κB activation.

• CaM inhibition alters expression of Ca²⁺-, cytoskeleton -, and immune-related genes.

• CaM modulates phagocytosis via regulating intracellular Ca²⁺ and downstream pathways.

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.

Introduction

Calmodulin (CaM) is a multifunctional intracellular calcium transducer found ubiquitously in eukaryotes (Chin and Means, 2000; Kirberger et al., 2017), playing crucial roles in regulating various physiological processes such as ion-channel activity (Saimi and Kung, 2002), secretion (Ashby and Tepikin, 2002; Richard et al., 2007), cellular metabolism (Cheung, 1980; Marcelo et al., 2016), muscle contraction (Jiang et al., 2010), cell proliferation and differentiation (Zayzafoon, 2006; Li et al., 2011), calcareous exoskeleton formation (Li et al., 2004), polyspermy blocking (Guo et al., 2017), and transcription factor activation (Bouché et al., 2002; Finkler et al., 2007). In recent years, it has been shown that CaM also modulates immunity in human and mice (Racioppi and Means, 2008; Ichinose et al., 2011). For instance, the formation of Ca²⁺/CaM complex in vivo can activate calcineurin (CaN) and thereby T cells in mice (Feske et al., 2003). In addition, CaM can regulate intracellular Ca²⁺ content and subsequently the process of phagocytosis through activating immune-related receptors such as the Fc gamma receptors (FcγRs) in mice (Nunes and Demaurex, 2010). Although similar immune modulatory effect of CaM has also been reported in few invertebrate species such as the Chinese mitten crab Eriocheir sinensis and the sea cucumber Stichopus monotuberculatus (Li et al., 2014; Chen et al., 2015), to the best of our knowledge, the immune modulatory function of CaM awaits exploration in bivalve mollusks.

Devoid of antigen-antibody mediated immune response, the immunity of bivalve mollusks varies greatly from that of the vertebrate species (Song et al., 2010; Wang et al., 2018). It is widely accepted that phagocytosis via haemocytes is one of the most important immune responses to fight against pathogen infection in bivalve mollusks (Song et al., 2010; Wang et al., 2018). According to previous studies, the process of phagocytosis of bivalve species is closely related to intracellular Ca²⁺ (Clapham, 2007; Rao and Hogan, 2010; Guan et al., 2019), cytoskeleton assembling (May and Machesky, 2001), Ca²⁺ and immune-related signaling pathways (Feske et al., 2003; Guan et al., 2019), and the complicated interactions between these factors (Stuart and Ezekowitz, 2005). For example, it has been demonstrated that the phagocytic activity of haemocytes can be significantly upregulated by the elevation of intracellular Ca²⁺ in the blood clam Tegillarca granosa (Shi et al., 2018; Guan et al., 2019). Similar activation impact of Ca²⁺ on phagocytosis was also detected in the Pacific oyster C. gigas (Aton et al., 2006) and the black tiger shrimp Penaeus monodon (Xian et al., 2010). In addition, the main molecules of Ca²⁺ signaling pathway, such as calmodulin kinase II (CaMKII) and calmodulin kinase II (CaMKKII), activated by intracellular Ca²⁺, also play important roles in regulating phagocytosis through interacting with immune-related pathways such as the NF-κB signaling pathway (Ling et al., 2013; Kim et al., 2014). It is also well known that phagocytosis is a process mediated by actin cytoskeleton (May and Machesky, 2001; Rougerie et al., 2013). For instance, both the formation of phagocytosis-related F-actin-rich membrane structures (i.e. lamellipodia and phagocytic cups) and the intracellular transportation of engulfed foreign particles depend on the remodeling of actin cytoskeleton (mainly the formation of F-actin) in haemocytes (Rougerie et al., 2013; Su et al., 2018).

The phagocytosis-related factors mentioned above could be directly or indirectly affected by CaM (Chin and Means, 2000; Finkler et al., 2007; Ling et al., 2013). For instance, as the sensor and regulation protein for intracellular calcium, CaM is known to regulate the intracellular concentration of Ca²⁺ (Means and Dedman, 1980; Jiang et al., 2010). In addition, it has been suggested that both Ca²⁺ and downstream components of the Ca²⁺-signaling pathway such as the CaMKII are critical regulatory molecules in the assemble of actin cytoskeleton (Sanabria et al., 2009; Hoffman et al., 2013). Therefore, it is highly probable that CaM modulate haemocytes phagocytosis in bivalve mollusks through affecting these phagocytosis-related factors, suggesting a potential mechanism underpinning the phagocytosis-regulation function of CaM. However, empirical data are necessitated to support this inference. In addition, the intrinsic relation between CaM, phagocytosis, and the potential affecting factors mentioned above remains elusive in bivalve species.

The blood clam, T. granosa, is a typical bivalve species widely distributed along the Indo-Pacific region (Shao et al., 2016; Shi et al., 2018). As a traditional aquaculture species and a key component of the intertidal ecosystem, blood clam is not only important for the aquaculture industry but also plays essential ecological roles in the intertidal ecosystem (Han et al., 2019; Zhou et al., 2020). Inhabiting coastal mudflat, blood clams are frequently challenged by various pathogens and environmental stressors (Shi et al., 2018; Guan et al., 2019; Tang et al., 2020). Therefore, holding a robust immune response is crucial for the survival of this important bivalve species. Under this circumstance, a better understanding of the regulation mechanism of phagocytosis could contribute positively to both aquaculture industry and the knowledge of immunity in bivalve mollusks. Therefore, in the present study, the modulatory function of CaM on phagocytosis was verified with CaM-specific inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) in blood clam. In addition, the impacts of CaM inhibition on the intracellular Ca²⁺, actin cytoskeleton assembling, calcineurin (CaN) activity, and NF-κB activation status in the haemocytes were also analyzed. Furthermore, the expression of genes from Ca²⁺-signaling, cytoskeleton regulation-, and immune-related pathways upon CaM inhibition were investigated.