Shrimp Plasma CREG Is a Hemocyte Activation Factor

Abstract

Cytokines are a class of immunoregulatory proteins that are secreted by cells. Although vertebrate cytokine, especially mammalian cytokine has been well studied for the past decades. Much less attention has been paid to invertebrate so that only some cytokines have been identified in invertebrates. We have chosen Peaneus vannamei as a model to explore novel invertebrate cytokines. To achieve this, we previously purified shrimp plasma low abundance proteins and identified more than 400 proteins with proteomics analyses. In this study, a cellular repressor of E1A-stimulated gene (CREG)-like protein, which is highly conserved from Drosophila melanogaster to Homo sapiens, was further characterized in shrimp plasma. We found that shrimp plasma CREG was a glycoprotein which was strongly induced in hemolymph at 8 h post-LPS injection. Further function experiment unveiled that recombinant shrimp CREG protein injection significantly increased phagocytic hemocyte and lysosome-high hemocyte proportion in hemolymph. After that, hemocytes from rEGFP- and rCREG-protein injected shrimps were subjected to transcriptome analyses, which revealed that shrimp CREG protein could comprehensively promote hemocyte maturation and activation. Taken together, our data clearly indicated that shrimp plasma CREG protein is a novel hemocyte activation factor, which is probably a conserved myeloid cell lineage activation factor from invertebrate to vertebrate.

Single-cell RNA sequencing analysis of shrimp immune cells identifies macrophage-like phagocytes

“…The rCREG-treated samples had the highest unique molecular identifiers (UMIs) and detected genes compared with that of the other two groups. This observation is consistent with our previous conclusion that CREG is a hemocyte activation factor ( Huang et al, 2021 ).…”

Myeloid cell evolution uncovered by shrimp immune cell analysis at single-cell resolution

“…Previously we identified an LPS-induced shrimp plasma protein-CREG. Injection of recombinant CREG (rCREG) could effectively activate shrimp hemocyte compared with recombinant EGFP (rEGFP) injected group(Huang et al, 2021). Encouraged by this observation, we did single-cell RNA-seq for rCREG treated shrimp hemocytes to further explore its function.…”

“…The recombinant EGFP and CREG were purified as previously described (Huang et al, 2021). Sixty shrimps were equally divided into three groups.…”

“…Shrimp phagocytic cells were labelled as previously described (Huang et al, 2021). In brief, FITC labelled Vibrio parahaemolyticus (VP) (2×10 6 particles/g) were injected into shrimps.…”

“…Moreover, its circulating plasma contains more than 400 various proteins, many of which are human homologs(Luo, Chen, & Wang, 2022; Tao et al, 2019); its circulating hemocytes contain proliferating cells, phagocytic cells and effector secreting cells(Lin & Soderhall, 2011), which are functionally similar with human peripheral myeloid cells. With these advantages, we systemically explored shrimp plasma proteins and identified that CREG is a shrimp hemocyte activation factor(Huang, Yang, & Wang, 2021; Tao et al, 2019). In this study, we further explored CREG effect via single-cell sequencing of shrimp hemocytes and redefined shrimp hemocyte classification according to their functional marker genes distribution.…”