Transcriptomic and Quantitative Proteomic Analyses Provide Insights Into the Phagocytic Killing of Hemocytes in the Oyster Crassostrea gigas

Shuai Jiang,Conghui Liu,Limei Qiu,Zhao Lv,Lingling Wang,Zhihao Jia,Linsheng Song,Jiachao Xu,Mengqiang Wang

doi:10.3389/fimmu.2018.01280

Abstract

As invertebrates lack an adaptive immune system, they depend to a large extent on their innate immune system to recognize and clear invading pathogens. Although phagocytes play pivotal roles in invertebrate innate immunity, the molecular mechanisms underlying this killing remain unclear. Cells of this type from the Pacific oyster Crassostrea gigas were classified efficiently in this study via fluorescence-activated cell sorting (FACS) based on their phagocytosis of FITC-labeled latex beads. Transcriptomic and quantitative proteomic analyses revealed a series of differentially expressed genes (DEGs) and proteins present in phagocytes; of the 352 significantly high expressed proteins identified here within the phagocyte proteome, 262 corresponding genes were similarly high expressed in the transcriptome, while 140 of 205 significantly low expressed proteins within the proteome were transcriptionally low expressed. A pathway crosstalk network analysis of these significantly high expressed proteins revealed that phagocytes were highly activated in a number of antimicrobial-related biological processes, including oxidation–reduction and lysosomal proteolysis processes. A number of DEGs, including oxidase, lysosomal protease, and immune receptors, were also validated in this study using quantitative PCR, while seven lysosomal cysteine proteases, referred to as cathepsin Ls, were significantly high expressed in phagocytes. Results show that the expression level of cathepsin L protein in phagocytes [mean fluorescence intensity (MFI): 327 ± 51] was significantly higher (p < 0.01) than that in non-phagocytic hemocytes (MFI: 83 ± 26), while the cathepsin L protein was colocalized with the phagocytosed Vibrio splendidus in oyster hemocytes during this process. The results of this study collectively suggest that oyster phagocytes possess both potent oxidative killing and microbial disintegration capacities; these findings provide important insights into hemocyte phagocytic killing as a component of C. gigas innate immunity.

Highlights

The process of phagocytosis has been shown to play essential roles in pathogen defense and immune surveillance [1, 2]
To obtain comprehensive insights into phagocytic killing in the Pacific oyster, C. gigas, an alternative strategy based on the phagocytosis of FITC-labeled latex beads was used to differentiate these cells from non-phagocytic hemocytes via fluorescence-activated cell sorting (FACS)
Macrophages produce a large variety of cytokines, including tumor necrosis factor-α, interferon-β, and interleukin-6 following pathogen engulfment, and these cytokines activate both innate and adaptive immune responses to bacterial infection [40, 41]

Summary

Introduction

The process of phagocytosis has been shown to play essential roles in pathogen defense and immune surveillance [1, 2]. Previous work has shown that both dendritic cells (DCs) and macrophages phagocytose and enclose bacteria in membrane-bound phagosomes (preferentially phagolysosomes) before killing these invading pathogens via the activity of reactive oxygen species (ROS) or lysosomal hydrolases [5, 6]. An increasing body of evidence suggests that phagocytes play important roles in invertebrate innate immune responses [11]: in the Pacific white shrimp Litopenaeus vannamei, for example, hyalinocytes and granulocytes both phagocytose fungi and bacteria as ROS production increases [12], while phagocytes in the Hawaiian bobtail squid Euprymna scolopes degrade engulfed bacteria via the lysosomal proteolytic pathway [13]. Previous research has shown a significant reduction in hemocyte phagocytosis in the blue mussel Mytilus edulis following cadmium exposure; this finding highlights the regulation of hemocyte phagocytic activity in innate immunity [14]. A number of immune-related genes involved in hemocyte phagocytosis in M. edulis were significantly upregulated following Vibrio splendidus challenge [15]

Methods

Results

Conclusion