Abstract
Invertebrates generally lack adaptive immunity and compensate for this with highly efficient innate immune machineries such as phagocytosis by hemocytes to eradicate invading pathogens. However, how extrinsically cued hemocytes marshal internal signals to accomplish phagocytosis is not yet fully understood. To this end, we established a facile magnetic cell sorting method to enrich professional phagocytes from hemocytes of the Hong Kong oyster (Crassostrea hongkongensis), an ecologically and commercially valuable marine invertebrate. Transcriptomic analysis on presorted cells shows that phagocytes maintain a remarkable array of differentially expressed genes that distinguish them from non-phagocytes, including 352 significantly upregulated genes and 479 downregulated genes. Pathway annotations reveal that focal adhesion and extracellular matrix–receptor interactions were the most conspicuously enriched pathways in phagocytes. Phagocytosis rate dramatically declined in the presence of an FAK inhibitor, confirming importance of the focal adhesion pathway in regulating phagocytosis. In addition, we also found that heparan sulfate proteoglycan (HSPG) families were lineage-specifically expanded in C. hongkongensis and abundantly expressed in phagocytes. Efficiency of phagocytosis and hemocytes aggregation was markedly reduced upon blockage of endogenous synthesis of HSPGs, thus implicating these proteins as key surface receptors in pathogen recognition and initiation of phagocytosis.
Highlights
Phagocytes are crucial executors in innate host defense against invading microbial pathogens, including bacteria and fungi [1, 2]
A cell sorting assay based on magnetic beads was used to separate phagocytes and non-phagocytes from total hemocytes, in which magnetic-enriched cells were considered as phagocytes, and non-enriched cells were non-phagocytes (Figure 1)
A series of RNA-seq libraries were constructed from phagocytes, non-phagocytes, and hemocytes, each group consisting of three biological replicates
Summary
Phagocytes are crucial executors in innate host defense against invading microbial pathogens, including bacteria and fungi [1, 2]. Impairment of phagocytic functions is often associated with microbial infections and could bring adverse consequences to pathogen replication, immune evasion, and host mortality [6]. B lymphocytes were demonstrated to possess potent phagocytic and bactericidal capacities, implying incomplete hemocytopoiesis in the lower vertebrates [7, 8]. Emerging evidences suggest that circulating hemocytes are indispensable to innate immune response, nutrition, wound healing, detoxification, and even shell mineralization [11, 12]. Surveillance and elimination of pathogens depend heavily on phagocytic capacities of hemocytes, whose efficiency in containing and killing pathogens is intimately tied to disease resistance of individual hosts [13]
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