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Front Immunol
2022 Jan 01;13:927880. doi: 10.3389/fimmu.2022.927880.
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Evolutionarily Ancient Caspase-9 Sensitizes Immune Effector Coelomocytes to Cadmium-Induced Cell Death in the Sea Cucumber, Holothuria leucospilota.
Li X
,
Chen T
,
Wu X
,
Li Z
,
Zhang X
,
Jiang X
,
Luo P
,
Hu C
,
Wong NK
,
Ren C
.
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Heavy-metal pollution has increasingly jeopardized the habitats of marine organisms including the sea cucumber, a seafloor scavenger vital to seawater bio-decontamination, ocean de-acidification and coral-reef protection. Normal physiology including immune functions of sea cucumbers is toxicologically modulated by marine metal pollutants such as cadmium (Cd). The processes underpinning Cd's toxic effects on immune systems in the sea cucumber, Holothuria leucospilota, are still poorly understood. To this end, we cloned and characterized a full-length caspase-9 (Hl-CASP9) cDNA in the sea cucumber, Holothuria leucospilota. Hl-CASP9 mRNA levels evolved dynamically during embryonic development. Coelomocytes, a type of phagocytic immune effectors central to H. leucospilota immunity, were found to express Hl-CASP9 mRNA most abundantly. Hl-CASP9 protein structurally resembles caspases-2 and -9 in both invertebrate and vertebrate species, comprising a CARD domain and a CASc domain. Remarkably, Hl-CASP9 was transcriptionally sensitive to abiotic oxidative stress inducers including hydrogen peroxide (H2O2), nitric oxide (•NO) and cadmium (Cd), but insensitive to immunostimulants including lipopolysaccharide (LPS), and poly(I:C). Overexpression of Hl-CASP9 augmented mitochondria-dependent apoptosis in HEK293T cells, while knock-down of Hl-CASP9 blunted Cd-induced coelomocyte apoptosis in vivo. Overall, we illustrate that an evolutionarily ancient caspase-9-dependent pathway exists to sensitize coelomocytes to premature cell death precipitated by heavy metal pollutants, with important implications for negative modulation of organismal immune response in marine invertebrates.
Figure 1. Functional domains of Hl-CASP9 and its evolutionary relationships with other caspases. (A) Structural domains of Hl-CASP9 predicted by using the SMART and ScanProsite programs. (B) Phylogenetic analysis of caspases among various species by using Neighbor-Joining method with bootstrap value of 1,000.
Figure 2. Analysis on amino acid sequence alignment, and organization of domains and 3-D structures of caspase-9. (A) Amino acid sequence alignment of caspase-2 and caspase-9 in various species. Conserved amino acid residues are boxed in dark blue, and similarities in amino acid residues are highlighted in light blue. (B) Comparison on 3-D structures of CASc domains in caspase-9 among three species. Positions of the P20 and P10 subunits are boxed. (C) Protein structural organization of caspase-9 among various species.
Figure 3. Spatial and temporal expression patterns of Hl-CASP9 mRNA in developing embryos and adult tissues. (A) Typical embryonic and larval development of H. leucospilota. Numbers indicate time lapsed post-fertilization. (B) Anatomy of H. leucospilota. (C)
Hl-CASP9 mRNA expression profiles in developing embryos and larvae of H. leucospilota. (D)
Hl-CASP9 mRNA expression profiles in different adult tissues of H. leucospilota.
Figure 4. Transcriptional expression patterns of Hl-CASP9 following challenges of immunostimulants or oxidative stress inducers. (A) Time-course study on the expression of Hl-CASP9 mRNA in coelomocytes treated with LPS (10 μg/mL). (B) Time-course study on the expression of Hl-CASP9 mRNA in coelomocytes treated with poly(I:C) (10 μg/mL). (C) Time-course study on the expression of Hl-CASP9 mRNA in coelomocytes treated with inactivated V. harveyi (107 cells/mL). (D) Time-course study on the expression of Hl-CASP9 mRNA in coelomocytes treated with H2O2 (2 μM). (E) Time-course study on the expression of Hl-CASP9 mRNA in coelomocytes treated with NOC-18 (1 μM). (F) Time-course study on the expression of Hl-CASP9 mRNA in coelomocytes treated with CdCl2 (20 μM).
Figure 5. Formation of cellular ROS and associated mitochondrial stress in CdCl2-treated coelomocytes. (A)
In vitro dose-dependent effects of CdCl2 (at 3 h) on ROS formation levels in primary coelomocytes as detected by the fluorescent DCF. Scale bar: 50 μm. (B) Quantification of DCF fluorescence intensities as reported in (A). (C) Effects of Hl-CASP9 overexpression on apoptosis in HEK293T cells, as analyzed by flow cytometry. HEK293T cells were treated with or without CCCP (100 μM). Comparisons are made between group with vector alone (HEK293T cells transfected with pcDNA3.1(+) blank plasmid) and group overexpressing Hl-CASP9 (HEK293T cells transfected with pcDNA3.1(+)/Hl-CASP9 recombinant plasmid). (D) Comparison on average apoptosis rates in HEK293T cells treated with or without CCCP in different groups. (E) Changes in mitochondrial membrane potential (Δψm) in CdCl2-treated primary coelomocytes with or without dsRNA (including dsGFP and dsCASP9) as assessed by JC-1 (2.5 μM) in flow cytometry. For comparison, coelomocytes were treated with or without CCCP (100 μM) or CdCl2 (20 μM). (F) Changes of Δψm in coelomocytes in response to CdCl2 were quantified by JC-1 intensity ratio for λ
ex 543 nm/488 nm. (G)
In vitro effects of CdCl2 (20 μM) on mitochondrial superoxide formation in primary coelomocytes with or without dsRNA (including dsGFP and dsCASP9), as visualized by MitoSOX (2.5 μM) in confocal imaging. Scale bar: 15 μm. (H) Quantification of MitoSOX fluorescence intensities in coelomocytes. Data were analyzed by using ImageJ.
Figure 6. Coelomocyte apoptosis analysis by flow cytometry following Hl-CASP9 knock-down in vivo.
(A) Coelomocytes treated with the indicated stimulants including H2O2 (2 μM), NOC-18 (1 μM) or CdCl2 (20 μM). Effects are compared among the “-dsRNA” group (sea cucumber injected of RNase-free saline solution), “+dsGFP” group (sea cucumber injected of dsGFP) and “+dsCASP9” group (sea cucumber injected of dsCASP9). (B) Statistical analysis on mean rates of early apoptosis in coelomocytes exposed to H2O2 (2 μM) for 24 h in different groups. (C) Statistical analysis on mean rates of early apoptosis in coelomocytes exposed to NOC-18 (1 μM) for 24 h in different groups. (D) Statistical analysis on mean rates of early apoptosis in coelomocytes exposed to CdCl2 (20 μM) for 24 h in different groups.
Figure 7. Conceptualization of Cd-induced mitochondria-dependent apoptosis in sea cucumber coelomocytes.
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