Oysters as sentinel bivalves with vital ecological importance are widely distributed in marine environments. Their innate immune systems are the sensitive targets of environmental pollutants. As the central component of innate immunity, hemocytes are endowed with specialized endolysosomal or phagolysosomal systems for particle internalization and metal detoxification. They consist of three cellular subtypes (agranulocytes, semigranulocytes, and granulocytes) with heterogeneous functions in an estuarine oyster Crassostrea hongkongensis. Their different roles in immune system against foreign particles and metal regulation and detoxification remain largely unknown. In this study, silver nanoparticles (AgNPs) and copper (Cu) were chosen as the representative nanoparticles and metal ions.

Two aggregated-induced emission (AIE) fluorogens, AIE-AgNPs (visualizing AgNPs) and TEZ-TPE-1 (visualizing Ag⁺), were simultaneously employed to monitor the intracellular accumulation, dissolution, and distribution of AgNPs and Ag⁺ in oyster hemocyte subpopulations, which were directly associated with a variety of cytotoxic effects. The most affected cell subtype by AgNPs was the granulocytes, followed by semigranulocytes, whereas agranulocytes were not affected following exposure to AgNPs. Interestingly, AgNPs induced the granule formation in semigranulocytes and further increased the proportion of granulocytes, whereas their ionic counterparts had no such effects on hemocyte composition, indicating the different detoxification mechanisms for nanoparticulate and ionic form. Following AgNP exposure, the dissolved Ag ions were accumulated in lysosomes. Furthermore, AgNP exposure induced the production of reactive oxygen species (ROS) and impeded the lysosome function and phagocytosis in granulocytes, with impaired immunity system in oysters. In addition, mitochondria and lysosomes are recognized as the major intracellular targets of AgNPs. The function and morphology of mitochondria and lysosomes were affected by AgNPs and their ionic counterparts with equivalent intracellular Ag⁺ contents. In addition, both types of Ag species could affect mitochondria–lysosome interactions, including mitochondria–lysosome contacts (MLCs) and mitophagy. This study demonstrated that Ag ions mainly contributed to AgNP toxicity, and MLCs exerted critical roles as upstream mediators of mitochondrial dynamics and functions, which can cast light on the mechanisms of immune cell cytotoxicity of animals caused by AgNPs.

Cu is hyperaccumulated in oyster hemocytes and an essential trace metal indispensable for diverse innate immune functions. The roles of Cu in oyster immune defense are still unclear. In this study, Cu exposure enhanced the phagocytosis of zymosan by increasing the number and length of filopodia, as well as mitochondrial ROS (mitoROS) production mainly in granulocytes, followed by semigranulocytes and agranulocytes. The intracellular calcium level increased to promote the phagosome–lysosome fusion after Cu exposure. The enhancement of phagosomal acidification and mitochondrion–phagosome juxtaposition were also found in granulocytes after Cu exposure. These results indicated that Cu could regulate the phagolysosomal system to enhance the antimicrobial ability of oyster hemocytes with the assistance of mitoROS. Furthermore, Cu(I) and Cu(II) were predominately located in lysosomes, and degranulation may provide a mechanism for exposing Cu to bacteria to prevent their survival and proliferation. Specifically, The newly formed Cu(I) arising from lysosomal Cu(II) moved to lysosomes and mitochondria in activated hemocytes to induce strong immune responses. The ability of the transformation of Cu(I) from Cu(II) followed granulocytes > semigranlocytes > agranulocytes, indicating that granulocytes played important roles in immune functions of oysters. In addition, Cu caused an increased formation of TNTs with elongation to facilitate the intercellular communication. Surprisingly, directional mitochondria transfer was induced under Cu stress from low to high phagocytic subpopulations via TNTs, which provided an enhanced energy and phagocytic function of granular hemocytes. Despite its low proportion, agranulocytes played a vital role in intercellular communication, and granulocytes were impaired in cell-to-cell communication under Cu exposure. Elevated glutathione (GSH) contents and heat-shock protein (Hsp) levels, and activated cell cycle were found to maintain cellular homeostasis and function under Cu exposure. Cu(I) was predominately sequestrated in lysosomes of granulocytes to reduce toxic effects of intracellular labile Cu(I) via Cu transport-related proteins (CTR1, ATP7, and COMMD1). Cu exposure increased the membrane protein expressions (MYOF, RalA, RalBP1, and cadherins) and lipid transporter activity, which could induce TNT formation. Interestingly, Cu(I) could directly move along TNTs or was carried by lysosomes relocating from high Cu(I) cells to low Cu(I) cells, reducing the burden caused by overloaded Cu(I). Cu also activated lysosomal signaling pathway, promoting intercellular lysosomal trafficking dependent on increased hydrolase activity and ATP-dependent activity. ATP7A existed in TNTs to facilitate the efflux of intracellular Cu(I).

In conclusion, three subtypes of oyster hemocytes were used as a unique cellular model to study the immune cell responses upon AgNPs and Cu exposure. They displayed different accumulation and dissolution abilities of AgNPs and transformation abilities of Cu(I)/Cu(II). TNTs and MLCs were first examined in detail in oyster hemocytes. This study explored the cellular responses of oyster hemocytes under exposure to AgNPs and Cu, which can help to understand the potential toxicity and fate of nanoparticles and metals in marine animals.