Abstract
Hemocytes play unequivocally central roles in host immune defense of bivalve mollusks, though the exact mechanisms underlying their functional differentiation are only partially understood. To this end, granulocytes and hyalinocytes were sorted via flow cytometry from hemocytes of the Pacific oyster Crassostrea gigas, and consequently quantitative transcriptomic analysis revealed a striking array of differentially expressed genes (DEGs), which were globally upregulated in granulocytes, dedicating to functional differentiation among oyster hemocytes. Our network of DEGs illustrated actively engaged signaling pathways, with Cdc42/Cdc42l being a core regulator of pathway network, which was validated by a dramatically reduced capacity for hemocyte phagocytosis in the presence of Cdc42 inhibitors. Additionally, a number of transcription factors were identified among DEGs, including ELK, HELT, and Fos, which were predominantly expressed in granulocytes. The AP-1 transcription factor Fos was confirmed to facilitate functional differentiation of hemocytes in an assay on binding to target genes by the AP-1 binding site, consistent with downstream phagocytosis and ROS production. Importantly, Cdc42/Cdc42l were also regulated by the expression of Fos, providing a possible regulatory mechanism-guided hemocyte functional differentiation. Findings in this study have bridged a knowledge gap on the mechanistic underpinnings of functional differentiation of hemocytes in a marine invertebrate C. gigas, which promise to facilitate research on the evolution of immune defense and functional differentiation of phagocyte in higher-order and more recent phyla.
Highlights
Marine invertebrates are intrinsically useful reductionist models for investigating host defense primarily based on innate immunity
Hemolymph from 10 oysters was analyzed by flow cytometry, in which two cell populations with distinguishable complexity and continuous size were found (Figure 1A)
A representative section with low SSC constituted an agranular population, corresponding to hyalinocytes. Another representative section with high SSC was a high-granularity population, corresponding to granulocytes. These two subpopulations of oyster hemocytes were sorted for RNA extraction and transcriptomic analysis, which were confirmed based on morphological features under a light microscopy (Figure 1B)
Summary
Marine invertebrates are intrinsically useful reductionist models for investigating host defense primarily based on innate immunity. An open circulatory system is populated by hemocytes, which patrols between hemal sinus and soft tissues. These immunologically plastic cells excel at performing a diverse range of cellular functions including phagocytosis of invading pathogens, encapsulation of bulky invaders, enzymatic digestion and transport of nutrients, and biosynthesis and secretion of humoral factors [1,2,3]. Spontaneous mitosis of hemocytes increases during circulation in hemolymph vessels, sinuses, and soft tissues [14,15,16] This raises the possibility of observing plasticity during various stages of hemocyte maturation, rather than categorizing cells into distinct subtypes [17,18,19]
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