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

Abstract

How myeloid cells evolved from invertebrate to vertebrate is still a mystery. Here we collected circulating hemocytes from a marine invertebrate-Penaeus vannamei via gradient centrifugation and identified prohemocyte, monocytic hemocyte and granulocyte as three major types of cells in shrimp hemolymph by single-cell mRNA sequencing. Additional pseudotime trajectory analysis revealed that shrimp monocytic hemocytes and granulocytes were differentiated from a common progenitor which was similar with that of human myeloid cells. More interestingly, we identified that MH2, a terminal differentiated monocytic hemocyte, was a macrophage-like phagocytic cell which could engulf fluorescein labelled Vibrio parahaemolyticus and shared nine marker genes including inflammasome components Nlrp3 and Casp1 with human macrophage. After that, we compared our classification with traditional shrimp hemocytes classification and found that hyalinocyte included both prohemocyte and monocytic hemocyte while semi-granulocyte included both monocytic hemocyte and granulocyte. In general, our results redefine shrimp hemocyte classification based on functional marker genes and unveil evolutionary trace of myeloid cells in marine invertebrate.

Single-cell RNA-seq uncovered hemocyte functional subtypes and their differentiational characteristics and connectivity with morphological subpopulations in Litopenaeus vannamei

“…Under ammonia stress, hemocytes of Penaeus monodon were divided into seven main cell groups ( 124 ). Following stimulation by hemocyte activating factors, hemocytes of L. vannamei were classified into prohemocytes, granulocytes, monocytic hemocytes, transitional cells and germ-like cells ( 125 ). The difference in hemocyte classification results of scRNA-seq might be due to species specificity.…”

“…In healthy M. japonicus hemocytes, the cell type that mainly exerts phagocytosis was also not clearly identified ( 73 ). Nevertheless, the hemocytes from treated L. vannamei and P. monodon exhibited cell clusters that exerted phagocytosis ( 124 , 125 ), suggesting that the phagocytic cell types might be differentiated from a particular type of hemocytes, which also indicated that different treatments would lead to the differences of cell clustering.…”

“…This conclusion is supported by previous studies that found TGase is mainly expressed in hyalinocytes, and semi-granulocytes take the lead in responding to foreign pathogens and AMPs are synthesized and secreted in granulocytes ( 36 , 52 , 129 ). Moreover, based on previously reported marker genes for different morphological subpopulations, recent scRNA-seq analysis of shrimp hemocyte also inferred that hyalinocytes are the main cell type that exert cellular agglutination and granule-containing hemocytes mainly perform the immune defense functions against pathogens ( 73 , 124 , 125 ). However, we still lack direct evidence to develop the connectivity between CLT + cluster and Crustin + cluster with semi-granulocytes and granulocytes.…”

“…defined that the prohemocytes of L. vannamei hemocytes exhibiting high expression of TGase were classified as early differentiated cell types, while they defined monocytic hemocyte and granulocytes with high expression of molecules such as AMP were classified as late differentiated cell types ( 125 ). Taken together, these evidence suggested that there are two developmental lineages of shrimp hemocytes that differentiate and mature from hyalinocytes into different granule-containing hemocytes, and semi-granulocytes and granulocytes still show some differentiation association ( 96 , 102 , 107 , 125 ). However, the differentiation relationship between granulocytes and semi-granulocytes still cannot be determined, since we are unable to arbitrarily judge how the CTL + cluster and Crustin + cluster correspond to granulocytes and semi-granulocytes.…”

“…This subcluster was in an oligopotent state and might be critical for the self-renewal of TGase + cluster. Previous studies have also discovered a small number of hemocytes undergoing the cell cycle in crustacean hemolymph ( 9 , 125 , 133 ). In parallel, we identified subclusters of hemocyte clusters in transient intermediate states.…”