Bacterial Engulfment Mechanism Is Strongly Conserved in Evolution Between Earthworm and Human Immune Cells

Abstract

Invertebrates, including earthworms, are applied to study the evolutionarily conserved cellular immune processes. Earthworm immunocytes (so-called coelomocytes) are functionally similar to vertebrate myeloid cells and form the first line of defense against invading pathogens. Hereby, we compared the engulfment mechanisms of THP-1 human monocytic cells, differentiated THP-1 (macrophage-like) cells, and Eisenia andrei coelomocytes towards Escherichia coli and Staphylococcus aureus bacteria applying various endocytosis inhibitors [amantadine, 5-(N-ethyl-N-isopropyl) amiloride, colchicine, cytochalasin B, cytochalasin D, methyl-ß-cyclodextrin, and nystatin]. Subsequently, we investigated the messenger RNA (mRNA) expressions of immune receptor-related molecules (TLR, MyD88, BPI) and the colocalization of lysosomes with engulfed bacteria following uptake inhibition in every cell type. Actin depolymerization by cytochalasin B and D has strongly inhibited the endocytosis of both bacterial strains in the studied cell types, suggesting the conserved role of actin-dependent phagocytosis. Decreased numbers of colocalized lysosomes/bacteria supported these findings. In THP-1 cells TLR expression was increased upon cytochalasin D pretreatment, while this inhibitor caused a dropped LBP/BPI expression in differentiated THP-1 cells and coelomocytes. The obtained data reveal further insights into the evolution of phagocytes in eukaryotes. Earthworm and human phagocytes possess analogous mechanisms for bacterial internalization.

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

“…These cells had round nuclei with a highly vacuolated cytoplasm similar to that in vertebrate macrophages ( Figure 3C ). To further compare these cells with vertebrate macrophage, we performed a phagocytosis inhibition assay using an actin polymerization inhibitor—cytochalasin D ( Kokhanyuk et al, 2021 )—that effectively suppressed the shrimp hemocyte phagocytosis rate ( Figure 3D ). To characterize the R1 cells, we quantified CHIT1 , Lyz1 , and NAGA expression in R1 and R2 using qPCR.…”

“…Phagocytic hemocytes (R1) were collected, stained with Hoechst 33342 (Beyotime, Shanghai, China), and observed using an LSM800 confocal microscope (Zeiss, Germany). The phagocytosis inhibition assay was performed according to a previously described method with some modifications ( Kokhanyuk et al, 2021 ). In brief, each shrimp was injected with either FITC-VP (2×10 6 particles/g) or FITC-VP (2×10 6 particles/g)+cytochalasin D (5 μM/g).…”

“…Among these cells, the phagocytes have been identified in all metazoans ( Musser et al, 2021 ), although they appear to be different in invertebrates and vertebrates. Some recently performed functional assays suggest that the bacterial engulfment in phagocytes may be conserved from invertebrates to vertebrates ( Kokhanyuk et al, 2021 ). In this study, we attempted to analyze shrimp hemocytes via single-cell sequencing and redefined their classification according to their functional marker gene distribution.…”

Neutrophils and aquatic pathogens

“…The coelomocytes display TLRs and may be cytotoxic 38 based on the production coelomic cytolytic factor (CCF) from the coelomic fluid of earthworms 39 . Functional comparative physiological and behavioural studies on phagocytes (isolated from humans and earthworms), when exposed to Escherichia coli and Staphylococcus aureus bacteria, have added to the notion that the neutrophils carry ancient traits seen early in evolution 40 . Another annelid group is the leeches, worms which are mainly known as blood sucking parasites on vertebrates, although we know that a range of species are non‐parasitic.…”

“… 39 Functional comparative physiological and behavioural studies on phagocytes (isolated from humans and earthworms), when exposed to Escherichia coli and Staphylococcus aureus bacteria, have added to the notion that the neutrophils carry ancient traits seen early in evolution. 40 Another annelid group is the leeches, worms which are mainly known as blood sucking parasites on vertebrates, although we know that a range of species are non‐parasitic. Members of this invertebrate group possess a circulatory system with the presence of relatively well‐characterized haemocytes.…”