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. 2020 Jan 11;23(2):100834. doi: 10.1016/j.isci.2020.100834

An Ancient BCR-like Signaling Promotes ICP Production and Hemocyte Phagocytosis in Oyster

Jiejie Sun 1,3, Lingling Wang 1,2,3,4,∗, Chuanyan Yang 1,3, Linsheng Song 1,2,3,5,∗∗
PMCID: PMC6994640  PMID: 31982779

Summary

BCR/TCR-based adaptive immune systems arise in the jawed vertebrates, and B cell receptors (BCRs) play an important role in the clonal selection of B cells and their differentiation into antibody-secreting plasma cells. The existence of BCR-like molecule and the activation mechanism of the downstream response are still not clear in invertebrates. In this study, an ancient BCR-like molecule (designated as CgIgR) with an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic tail was identified from the Pacific oyster Crassostrea gigas to investigate its involvement in immune response. CgIgR could bind different bacteria through five extracellular Ig domains and formed dimers. The activated CgIgR recruited CgSyk to promote CgERK phosphorylation. The CgIgR-mediated signaling promoted the production of immunoglobulin domain-containing proteins (CgICP-2 and CgLRRIG-1) through inducing CgH3K4me2. The produced CgICPs eventually facilitated hemocytes to phagocytize and eliminate V. splendidus. This study proposed that there was an ancient BCR-like molecule and BCR-like signaling in molluscs.

Subject Areas: Biological Sciences, Immunology, Cell Biology

Graphical Abstract

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Highlights

  • •

    An ancient BCR-like molecule (defined as CgIgR) was identified from C. gigas

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    We propose IgR-mediated signaling induces CgERK activity in oyster

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    IgR-mediated signaling induced CgH3K4me2 to promote the production of CgICPs

  • •

    CgICPs facilitated the hemocytes to phagocytize and eliminate V. splendidus

Biological Sciences; Immunology; Cell Biology

Introduction

Adaptive immunity involves a tightly regulated interplay between antigen-presenting cells and T/B cells, which facilitates pathogen-specific immunologic effector pathways, generation of immunologic memory, and regulation of immune homeostasis (Bonilla and Oettgen, 2010). B cell receptors (BCRs) are considered as the key molecules in the adaptive immunity, which can govern the initiation of transcriptional programs associated with B cell activation (Kwak et al., 2019) and then mediate the production of antibodies through plasma cells (Konigsberger et al., 2012, Mattila et al., 2013, Yang and Reth, 2010). BCRs comprise the membrane bound immunoglobulin (mIg) and the signal-transducing Igα/Igβ heterodimer, which function as the ligand-binding and signaling subunits, respectively (Monroe, 2006, Reth, 1989). The mIg recognizes various antigens via Ig domains and activates the membrane Igα and Igβ. The activated Igα and Igβ then form heterodimer to transduce signals through their immunoreceptor tyrosine-based activation motifs (ITAMs) (DeFranco, 1993, Papavasiliou et al., 1995, Teh and Neuberger, 1997). The clustering of Igα and Igβ initially stimulates the membrane-associated Src protein tyrosine kinases (PTKs) to phosphorylate the ITAM tyrosines of Igα and Igβ. The phosphorylated Igα and Igβ tyrosines then serve as membrane proximal binding sites for the tandem Src homology 2 (SH2) domains presented in spleen tyrosine kinase (Syk) (Rowley et al., 1995). This process allows Syk to bind BCRs and phosphorylate the neighboring ITAM tyrosines, thus amplifying the signaling output of the BCRs (Rolli et al., 2002). The signaling mediated by BCRs induces B cell activation, proliferation, differentiation, and eventually secretions of antibodies (Ollila and Vihinen, 2005, Werner et al., 2010). The antibodies are a class of Igs found only in vertebrates, which function in multiple biological processes such as specifically recognizing antigens, participating in neutralizing toxins, activating the complement pathway, and inducing opsonization (Panda and Ding, 2015). Although the ancestral cell lineage of Ig-producing B cells is still unknown, fish B cells are confirmed to represent the cell predecessors for amphibian, reptilian, avian, and mammalian B cells (Jirapongpairoj et al., 2017, Simon et al., 2019, Smith et al., 2019, Yu et al., 2018). However, there is still no report about BCR in fish, and the initiation mechanisms of B cells are far from well understood.

The immunological memory in invertebrates as well as the origin and evolution of immunoglobulins have been in controversy in the past decades (Chang et al., 2018, Torre et al., 2017). The memory of trained immunity is defined as a heightened response to a secondary infection (Netea et al., 2011). Although increasing evidences suggest that there exists trained immunity in invertebrates (Norouzitallab et al., 2016, Simoes and Dimopoulos, 2015), the underlying molecular and cellular mechanisms still need further investigation. As the key component of adaptive immunity, antibodies are assumed to have arisen in the jawed vertebrates (Smith et al., 2019), whereas their primitive ancestors and functions in invertebrate immune system are still largely unknown. So far, numerous immunoglobulin domain-containing proteins (ICPs) with one or more Ig-like domains have been identified in invertebrates (Dong et al., 2006, Hemani and Soller, 2012, Wang et al., 2018b). For instance, more than 190 ICPs were annotated in oyster Crassostrea gigas by screening the available genomic sequence (Zhang et al., 2015). Some of invertebrate ICPs are found to be alternatively spliced after immune stimuli, which is similar to that of antibodies in mammals (Parra et al., 2013). For example, Down syndrome cell adhesion molecule (Dscam) in Drosophila and mosquito could generate pathogen-splice form repertoires through alternative splicing upon immune challenge (Dong et al., 2006, Hemani and Soller, 2012). EsDscam in Chinese mitten crab Eriocheir sinensis potentially produced 30,600 isoforms due to the alternative splice of three Ig domains, which suggested that EsDscam owned specific recognition capability to different bacteria (Li et al., 2018). A cysteine-rich motif associated ICP (CgCAICP-1) was also reported to be spliced in C. gigas (Liu et al., 2018). These evidences suggest that the diversified ICPs are created by rearrangement and enable specific recognition and protection against bacteria (Kurtz and Armitage, 2006). In most invertebrates, circulating hemocytes are the main immunocytes responsible for recognition, phagocytosis, nodule formation, encapsulation, and effector synthesis (Christophides et al., 2002, Koiwai et al., 2018, Lau et al., 2017). Many ICPs in invertebrates are found to be expressed in hemocytes and function as pattern recognition receptors (PRRs) and opsonins. For example, a junctional adhesion molecule A (CgJAM-A-L) and CgCAICP-1 were found to be located on the hemocyte membrane. Both of them functioned as PRRs to recognize different bacteria and facilitated phagocytosis of oyster hemocytes (Liu et al., 2016b, Liu et al., 2018). However, the knowledge on the origin and evolution of BCR molecule as well as their possible ligand-binding mechanism and signaling cascades to regulate other ICP production in invertebrates is still very limited.

As aquaculture mollusk, oyster is of critically evolutional significance and economic importance and represents an attractive model for studying the immune function and evolution of immune system because it is a sessile and filter-feeder always exposed to tremendous pathogen challenge (Zhang et al., 2012). Hemocytes are important in the defense mechanisms of oyster (Moreau et al., 2015, Wang et al., 2018a), and many ICPs functioning as PRRs and opsonins are highly expressed in the oyster hemocytes. Four CgICPs (CgIgR, CgCAICP-1, CgICP-2, and CgLRRIG-1) were screened from the transcriptome data of oyster hemocytes after the successive V. splendidus and lipopolysaccharide (LPS) stimulations, which were suspected to be involved in the trained immunity. In the present study, an ancient BCR-like molecule (CgIgR) was identified from oyster, and its recognition and regulation mechanisms to induce CgICP production and phagocytosis toward V. splendidus were investigated with the objectives to comprehensively understand the function of immunoglobulin domain-containing proteins in the immune system of invertebrates and provide some clues for the origin of BCR-mediated antibody secretions and the evolution of adaptive immunity.

Results

The Phagocytic Rates and CgICP Transcripts Increased Significantly after the Immune Training with V. splendidus and LPS

The oysters were pre-stimulated with V. splendidus or LPS to train their immune responses. At the eighth day after the first stimulation, the oysters were stimulated again with V. splendidus and LPS for 6 h, respectively. Control oysters received a first injection with PBS and a second injection with V. splendidus. The hemocytes were collected to examine the phagocytic rates and the mRNA transcripts of CgICPs (Figure 1A). The phagocytic rates of hemocytes toward V. splendidus were apparently enhanced in V. splendidus and LPS training oysters, which were 1.58-fold and 1.52-fold (p < 0.05) higher than that in PBS training oysters (Figures 1B–1E). The flow cytometry assay also confirmed that the phagocytic rates of hemocytes toward V. splendidus increased significantly (1.73-fold and 1.79-fold of that in PBS training oysters, p < 0.05, respectively) in V. splendidus and LPS training oysters (Figure 1F). The mRNA transcripts of CgICPs (CgIgR, CgCAICP-1, CgICP-2, CgLRRIG-1) (Figure S1) increased significantly in V. splendidus training oysters (3.07-fold, p < 0.01; 2.69-fold, 2.35-fold, and 2.16-fold, p < 0.05) and LPS training oysters (3.56-fold, 2.93-fold, 2.71-fold, and 1.94-fold, p < 0.05), compared with that in PBS training oysters, respectively (Figures 1G–1N).

Figure 1.

Figure 1

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Hemocyte Phagocytosis and CgICPs Participated in Trained Immunity of Oysters

(A) Schematic overview of trained immunity methodology. The oysters were stimulated with V. splendidus or LPS. At the eighth day after the first stimulation, the oysters were re-stimulated with V. splendidus or LPS for 6 h.

(B–F) Hemocyte phagocytic rates detected by using the immunocytochemistry and flow cytometry in V. splendidus or LPS training oysters, respectively. (C) and (E) were the statistical analysis of (B) and (D), respectively. The hemocytes containing labeled bacteria were counted as phagocytosed cells (n = 3). Scale bar: 4 μm.

(G–N) The mRNA transcripts of CgIgR (G), CgCAICP-1 (I), CgICP-2 (K), and CgLRRIG-1 (M) detected by using qPCR in V. splendidus training group (n = 3). The mRNA transcripts of CgIgR (H), CgCAICP-1 (J), CgICP-2 (L), and CgLRRIG-1 (N) detected by using qPCR in LPS training group (n = 3). Data were representative of three independent experiments and shown as mean ± SD. *: p < 0.05, **: p < 0.01 (t test).

See also Figure S1.

No Significant Changes of Hemocyte Phagocytosis and CgICPs Transcripts Were Observed in CgIgR-RNAi Oysters after the Immune Training with V. splendidus and LPS

The phagocytosis of oyster hemocytes and mRNA expressions of CgICPs were examined after CgIgR was knocked down to study its possible function in training immunity. CgIgR-RNAi oysters were first stimulated with V. splendidus and LPS for immune training and stimulated with V. splendidus and LPS again at eighth day after the first stimulation as described above. Hemocytes in CgIgR-RNAi oysters were collected at 6 h after the second stimulation to examine the phagocytic rates and the mRNA transcripts of CgICPs (Figure 2A). There were no significant changes of hemocyte phagocytic rates toward V. splendidus and the mRNA transcripts of CgICPs (CgIgR, CgCAICP-1, CgICP-2, CgLRRIG-1) observed in V. splendidus and LPS training groups, compared with that in the PBS training group, respectively (Figures 2B–2K).

Figure 2.

Figure 2

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CgIgR Promoted Hemocyte Phagocytosis and CgICP Production in V. splendidus or LPS Immune Training Oysters

(A) Schematic overview of trained immunity methodology in CgIgR-RNAi oysters. CgIgR-RNAi oysters were stimulated with V. splendidus or LPS. At the eighth day after the first stimulation, the oysters were re-stimulated with V. splendidus or LPS for 6 h.

(B–E) Hemocyte phagocytic rates detected by using the flow cytometry (C) and immunocytochemistry (E) in CgIgR-RNAi oysters after the immune training with V. splendidus and LPS (n = 3). (B) and (D) were used as control for (C) and (E), respectively.

(F–K) The mRNA transcripts of CgCAICP-1 (F), CgICP-2 (H), and CgLRRIG-1 (J) in CgIgR-RNAi oysters detected after the immune training with V. splendidus. The mRNA transcripts of CgCAICP-1 (G), CgICP-2 (I), and CgLRRIG-1 (K) in CgIgR-RNAi oysters detected after the immune training with LPS (n = 3).

Data were representative of three independent experiments and shown as mean ± SD.

The Molecular Features of CgIgR and Its Potential Functions in Antibacterial Immunity

In the present study, CgIgR was screened from 190 ICPs in oyster C. gigas. There were five extracellular Ig domains, a TM domain, as well as a classical ITAM in its cytoplasmic tail in CgIgR (Figure S2). CgIgR was expressed in all the tested tissues with relatively higher expression level in hemocytes (12.9-fold of that in muscle, p < 0.05) (Figure 3A). The mRNA transcripts of CgIgR increased significantly from 6 to 48 h after V. splendidus and LPS stimulations and reached the highest level at 12 h (13.1-fold of that in the PBS group, p < 0.01) and 24 h (6.13-fold of that in the PBS group, p < 0.01) (Figures 3B and 3C), respectively.

Figure 3.

Figure 3

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The Potential Functions of CgIgR after V. splendidus and LPS Stimulations

(A–C) The tissue distribution of CgIgR (A) and its temporal expression patterns after V. splendidus (B) and LPS (C) stimulations (n = 3). PBS was used as control.

(D) The rTrx-his-5×Ig and Trx-his tag (control) expressed and purified from E. coli. Lane M, protein marker; Lane 1, rTrx-his-5×Ig and Trx-his tag of E. coli with recombinant vectors before induction with IPTG; Lanes 2 and 5, after IPTG induction; Lanes 3 and 6, purified rTrx-his-5×Ig and Trx-his tag.

(E and F) The binding activity of rTrx-his-5×Ig to different bacteria (E) and LPS (F) using anti-His antibody (n = 3).

(G) The molecular mass of native CgIgR detected with polyclonal antibody of anti-CgIgR (n = 3).

(H) Dimer of CgIgR detected with anti-CgIgR antibody in vivo treatment of hemocytes after V. splendidus stimulation with a cross-linker (BS3) by western blotting (n = 3).

(I and J) Purification of recombinant CgIgR (I) and the native PAGE of rCgIgR (J). Purified rCgIgR was analyzed using native PAGE.

(K) Subcellular localization of CgIgR in hemocytes (n = 3). Scale bar: 5 μm.

(L and M) The mRNA transcripts of CgCAICP-1, CgICP-2, and CgLRRIG-1 in CgIgR-RNAi oysters detected after V. splendidus (L) and LPS (M) stimulations (n = 3).

(N and O) The mRNA transcripts of CgCAICP-1, CgICP-2, and CgLRRIG-1 in CgIgR antibody-blockaded oysters detected after V. splendidus (N) and LPS (O) stimulations (n = 3).

Data were representative of three independent experiments. Error bars represented SD. *: p < 0.05, **: p < 0.01 (t test). Different letters: p < 0.05 (one-way ANOVA). See also Figure S2.

The five Ig domains of CgIgR with Trx-his tag (Trx-his-5×Ig) and Trx-his-tag were expressed and purified from E. coli (Figure 3D). After the recombinant Trx-his-5×Ig (rTrx-his-5×Ig) was incubated with G− bacteria (E. coli and V. splendidus) and G+ bacteria (S. aureus and M. luteus), positive bands were revealed by western blotting with anti-His tag mouse monoclonal antibody, whereas no bands were observed in Trx-his tag (control) groups (Figure 3E). The bands for G− bacteria were obviously thicker than those for G+ bacteria. The rTrx-his-5×Ig displayed relatively higher binding affinity toward LPS in a dose-dependent manner (Figure 3F), and the maximum binding parameter to bacteria (Bmax) was 0.83 (data not shown).

Western blotting assay of the oyster hemocytes with anti-CgIgR antibody revealed that there was a distinct band of 80 kDa (Figure 3G), indicating the high specificity of anti-CgIgR antibody. There were two bands of 80 and 160 kDa observed in the hemocyte sample by using cross-linking assay with CgIgR antibody after V. splendidus stimulation (Figure 3H). There was a distinct band about 80 kDa for rCgIgR revealed by SDS-PAGE assay (Figure 3I), and there was another band about 160 kDa for rCgIgR observed by using native PAGE (Figure 3J). The positive signals of CgIgR were observed in green fluorescence by using anti-CgIgR antibody, which were mainly distributed on the hemocyte membrane. The hemocyte nuclei stained with DAPI were in blue fluorescence (Figure 3K).

After CgIgR was silenced by RNAi, the mRNA expressions of CgICPs were investigated to reveal the potential immune function of CgIgR. The mRNA transcripts of CgIgR decreased significantly (0.31-fold of that in the EGFP group, p < 0.05) after CgIgR was silenced by dsRNA (Figure S3). In CgIgR-RNAi oysters, the mRNA transcripts of CgCAICP-1, CgICP-2, and CgLRRIG-1 decreased significantly after V. splendidus stimulation (0.48-fold, 0.60-fold, and 0.56-fold of that in the EGFP group, p < 0.05, respectively) (Figure 3L) and LPS stimulation (0.29-fold, 0.60-fold, and 0.42-fold of that in the EGFP group, p < 0.05, respectively) (Figure 3M). Meanwhile, the expressions of CgICPs were examined after CgIgR was blockaded by using CgIgR antibody. After the injection of CgIgR antibody, the mRNA transcripts of CgCAICP-1, CgICP-2, and CgLRRIG-1 in CgIgR antibody-blockaded oysters were down-regulated significantly at 6 h after V. splendidus stimulation (0.24-fold, p < 0.01; 0.63-fold and 0.55-fold, p < 0.05) (Figure 3N) and LPS stimulation (0.23-fold, p < 0.01; 0.57-fold and 0.55-fold, p < 0.05), compared with that in the control group, respectively (Figure 3O).

CgIgR Could Induce CgICP Production by Interacting with CgSyk after V. splendidus and LPS Stimulations

CgSyk was identified from oyster C. gigas with two src homology 2 (SH2) domains and a TyrKc domain (Figure 4A). It was expressed in all the tested tissues with relatively higher expressions in gills and hemocytes (3.34-fold and 3.89-fold of that in adductor muscle, p < 0.05, respectively) (Figure 4B). The mRNA transcripts of CgSyk in hemocytes increased significantly from 3 to 48 h after V. splendidus stimulation and peaked (17.6-fold of that in the PBS group, p < 0.01) at 12 h (Figure 4C). After LPS stimulation, CgSyk mRNA transcripts increased significantly from 6 to 48 h and reached the highest level (27.0-fold of that in the PBS group, p < 0.01) at 24 h (Figure 4D). The TyrKc domain of CgSyk was expressed and purified from E. coli (Figure 4E). Western blotting assay of the hemocyte sample with CgSyk antibody revealed that there was a distinct band of 72 kDa (Figure 4F). After V. splendidus and LPS stimulations, the bands of native CgSyk co-immunoprecipitated by CgIgR and CgIgR co-immunoprecipitated by CgSyk both became thicker (Figures 4G and 4H). The expression level of CgSyk was knocked down to 0.39-fold of that in the EGFP group (p < 0.05) (Figure S3). In CgSyk-RNAi oysters, the mRNA transcripts of CgCAICP-1, CgICP-2, and CgLRRIG-1 decreased significantly, which were 0.34-fold (p < 0.01), 0.52-fold (p < 0.05), and 0.64-fold (p < 0.05) after V. splendidus stimulation (Figure 4I) and 0.53-fold (p < 0.05), 0.40-fold (p < 0.01), and 0.42-fold (p < 0.05) after LPS stimulation, compared with that in the EGFP group, respectively (Figure 4J). In R406-injected oysters, the mRNA transcripts of CgCAICP-1, CgICP-2, and CgLRRIG-1 decreased significantly after V. splendidus stimulation (0.59-fold, 0.36-fold, and 0.43-fold of that in the DMSO-injected group, p < 0.05, respectively) (Figure 4K) and LPS stimulation (0.52-fold, 0.59-fold, and 0.55-fold of that in the DMSO-injected group, p < 0.05, respectively) (Figure 4L).

Figure 4.

Figure 4

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The Potential Functions of CgSyk after V. splendidus and LPS Stimulations

(A) The domain architecture of oyster CgSyk.

(B–D) The tissue distribution of CgSyk (B) and its temporal expression patterns after V. splendidus (C) and LPS (D) stimulations (n = 3).

(E) The rTyrKc domain of CgSyk was expressed and purified from E. coli. Lane M, protein marker; Lane 1, rTyrKc of E. coli with recombinant vectors before induction with IPTG; Lane 2, after IPTG induction; Lane 3, purified rTyrKc.

(F) The molecular mass of native CgSyk detected with polyclonal antibody of anti-CgSyk (n = 3).

(G and H) The interaction of CgIgR with CgSyk after V. splendidus (G) and LPS (H) stimulations (n = 3).

(I and J) The mRNA transcripts of CgCAICP-1, CgICP-2, and CgLRRIG-1 in CgSyk-RNAi detected after V. splendidus (I) and LPS (J) stimulations (n = 3).

(K and L) The mRNA transcripts of CgCAICP-1, CgICP-2, and CgLRRIG-1 in R406-injected oysters detected after V. splendidus (K) and LPS (L) stimulations (n = 3).

Data were representative of three independent experiments. Error bars represented SD. *: p < 0.05, **: p < 0.01 (t test). Different letters: p < 0.05 (one-way ANOVA). See also Figure S3.

IgR/Syk Pathway Induced CgERK Phosphorylation to Promote the Production of CgICPs after V. splendidus and LPS Stimulations

The phosphorylation of CgERK was examined to study the involvement of IgR/Syk pathway in regulating CgICP production. The bands of phospho-CgERK in CgIgR- and CgSyk-RNAi oysters became thinner, and the count values of these bands decreased significantly after V. splendidus (0.24-fold, p < 0.01; 0.27-fold, p < 0.05) (Figure 5A) and LPS stimulations (0.10-fold and 0.07-fold, p < 0.001) (Figure 5B), compared with that in EGFP-RNAi oysters, respectively. After CgIgR was blockaded by anti-CgIgR antibody, the bands of phospho-CgERK became thinner, and after V. splendidus and LPS stimulations, the count values of these bands were 0.17-fold (p < 0.05) and 0.12-fold (p < 0.01) of that in the pre-serum group, respectively (Figures 5C and 5D). The bands of phospho-CgERK in R406- and PD98059-injected oysters also became thinner, and the count values of these bands decreased significantly after V. splendidus (0.37-fold, p < 0.05; 0.08-fold, p < 0.001) (Figure 5E) and LPS stimulations (0.11-fold and 0.07-fold, p < 0.001), compared with that in the DMSO-injected group, respectively (Figure 5F).

Figure 5.

Figure 5

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CgIgR-CgSyk Pathway Promoted the Phosphorylation of CgERK to Induce CgICP Production

(A) The phospho-CgERK in CgIgR- or CgSyk-RNAi oysters detected after V. splendidus stimulation (n = 3). Histogram was statistical analysis of (A) after digitization with ImageJ.

(B) The phospho-CgERK in CgIgR- or CgSyk-RNAi oysters detected after LPS stimulation (n = 3). Histogram was statistical analysis of B after digitization with ImageJ.

(C and D) The phospho-CgERK in CgIgR antibody-blockaded oysters detected after V. splendidus and LPS stimulations (n = 3). The statistical analysis of C and D.

(E and F) The phospho-CgERK in R406- or PD98059-injected oysters detected after V. splendidus and LPS stimulations (n = 3). The statistical analysis of E and F.

(G and H) The mRNA transcripts of CgCAICP-1, CgICP-2, and CgLRRIG-1 in CgERK-RNAi oysters detected after V. splendidus (G) and LPS (H) stimulations (n = 3).

(I and J) The mRNA transcripts of CgCAICP-1, CgICP-2, and CgLRRIG-1 in PD98059-injected oysters detected after V. splendidus (I) and LPS (J) stimulations (n = 3).

Data were representative of three independent experiments. Error bars represented SD. *: p < 0.05, **: p < 0.01, ***: p < 0.001 (t test).

The mRNA transcripts of CgICPs were assessed by qRT-PCR after CgERK was knocked down to 0.26-fold of that in the EGFP-RNAi group (p < 0.05) (Figure S3). The mRNA transcripts of CgCAICP-1, CgICP-2, and CgLRRIG-1 in the CgERK-RNAi group decreased significantly after V. splendidus stimulation (0.57-fold, 0.66-fold, and 0.49-fold, compared with that in the EGFP-RNAi group, respectively, p < 0.05) (Figure 5G) and LPS stimulation (0.53-fold, 0.50-fold, and 0.65-fold, p < 0.05) (Figure 5H). PD98059 was used to inhibit ERK activity, and the mRNA expressions of CgICPs were examined to evaluate the function of CgERK in mediating CgICP production. In PD98059-injected oysters, the mRNA transcripts of CgCAICP-1, CgICP-2, and CgLRRIG-1 were down-regulated significantly after V. splendidus stimulation, which were 0.49-fold, 0.28-fold, and 0.22-fold (p < 0.05) of that in the DMSO-injected group, respectively (Figure 5I). Similarly, the mRNA transcripts of CgCAICP-1, CgICP-2, and CgLRRIG-1 decreased significantly in PD98059-injected oysters after LPS stimulation, which were 0.20-fold (p < 0.01), 0.26-fold (p < 0.05), and 0.22-fold (p < 0.01) of that in the DMSO-injected group, respectively (Figure 5J).

CgIgR Induced CgH3K4me2 to Promote the Production of CgICPs after V. splendidus and LPS Stimulations

After the oysters were stimulated with V. splendidus and LPS, the hemocytes were collected to detect the CgH3K4me2 proteins and the enrichment of CgH3K4me2 on CgICP promoters. The bands of CgH3K4me2 became thicker and the count values of these bands increased significantly (3.72-fold and 4.50-fold, p < 0.05) after V. splendidus and LPS stimulations, compared with that in the PBS group, respectively (Figures 6A and 6B). The values of CgH3K4me2 enrichment on CgICP-2 (2.92-fold and 3.43-fold, p < 0.05) and CgLRRIG-1 (2.95-fold and 2.70-fold, p < 0.05) promoters increased significantly after V. splendidus and LPS stimulations, compared with that in the PBS group, respectively (Figure 6C). The bands of CgH3K4me2 in CgIgR-RNAi oysters became thinner after V. splendidus and LPS stimulations, compared with that in the PBS group, respectively (Figures 6D and 6F). The values of CgH3K4me2 enrichment on CgICP-2 and CgLRRIG-1 promoters decreased significantly after V. splendidus stimulation (0.22-fold and 0.45-fold of that in the EGFP-RNAi group, p < 0.05, respectively) (Figure 6E) and LPS stimulation (0.37-fold and 0.36-fold of that in the EGFP-RNAi group, p < 0.05, respectively) (Figure 6G). In CgIgR antibody-blockaded oysters, the bands of CgH3K4me2 became thinner after V. splendidus and LPS stimulations, compared with that in the pre-serum group, respectively (Figures 6H and 6J). The enrichment values of CgH3K4me2 on CgICP-2 and CgLRRIG-1 promoters decreased significantly after V. splendidus stimulation (0.42-fold and 0.24-fold, p < 0.05) and LPS stimulation (0.26-fold and 0.29-fold, p < 0.05), compared with that in the pre-serum group, respectively (Figures 6I and 6K).

Figure 6.

Figure 6

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CgIgR Promoted CgH3K4me2 to Induce the mRNA Transcripts of CgICPs after V. splendidus and LPS Stimulations

(A and B) CgH3K4me2 after V. splendidus and LPS stimulations (A). (B) was the statistical analysis of CgH3K4me2 after digitization with ImageJ (n = 3).

(C) CgH3K4me2 enrichments for CgICP-2 and CgLRRIG-1 promoters after V. splendidus and LPS stimulations (n = 3).

(D and F) CgH3K4me2 in CgIgR-RNAi oysters after V. splendidus (D) and LPS (F) stimulations (n = 3).

(E and G) CgH3K4me2 enrichments on CgICP-2 and CgLRRIG-1 promoters in CgIgR-RNAi oysters after V. splendidus (E) and LPS (G) stimulations (n = 3).

(H and J) CgH3K4me2 in CgIgR-blockage oysters after V. splendidus (H) and LPS (J) stimulations (n = 3).

(I and K) The enrichments of CgH3K4me2 on CgICP-2 and CgLRRIG-1 promoters in CgIgR antibody-blockaded oysters after V. splendidus (J) and LPS (K) stimulations (n = 3).

Data were representative of three independent experiments. Error bars represented SD. *: p < 0.05 (t test).

The Phagocytic Rates of Hemocyte toward V. splendidus Decreased Significantly in CgICP-2- and CgLRRIG-1-RNAi Oysters

After CgICP-2 and CgLRRIG-1 were knocked down by RNAi, their mRNA transcripts decreased to 0.40-fold and 0.42-fold (p < 0.05) compared with that in the EGFP-RNAi group, respectively (Figure 7A). In CgICP-2- and CgLRRIG-1-RNAi oysters, the phagocytic rates of hemocytes toward V. splendidus were apparently reduced (0.46-fold and 0.59-fold of that in the EGFP-RNAi group, p < 0.05, respectively) (Figures 7B and 7C). The flow cytometry assay also confirmed that the rates of hemocyte phagocytosis toward V. splendidus in CgICP-2- and CgLRRIG-1-RNAi oysters decreased significantly (0.32-fold and 0.44-fold, p < 0.05), compared with that in the EGFP-RNAi group, respectively (Figure 7D). The hemocytes collected from CgICP-2- and CgLRRIG-1-RNAi oysters were incubated with FITC-labeled V. splendidus, and the co-localization of the phagocytized bacteria with lysosomes was detected by immunocytochemical analysis. The FITC-labeled V. splendidus was co-localized with lysosomes stained with LysoTracker red and the co-localization signals in CgICP-2-RNAi and CgLRRIG-1-RNAi oysters were all weakened, compared with that in the EGFP-RNAi group, respectively (Figure 7E).

Figure 7.

Figure 7

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CgICP-2 and CgLRRIG Promoted Hemocyte Phagocytosis and Degradation toward V. splendidus

(A) The mRNA expressions of CgICP-2- and CgLRRIG-1 in hemocytes after the injection of their specific dsRNA, respectively (n = 3).

(B–D) Hemocyte phagocytic rates toward V. splendidus detected by using the immunocytochemistry (B) and flow cytometry (D) in CgICP-2- or CgLRRIG-1-RNAi oysters (n = 3). (C) was the statistic analysis of (B). EGFP was used as the control. Scale bar: 5 μm.

(E) Co-localization of V. splendidus and lysosomes in CgICP-2- or CgLRRIG-1-RNAi oysters. EGFP was used as the control (n = 3). Scale bar: 5 μm.

Data were representative of three independent experiments. Error bars represented SD. *: p < 0.05 (t test).

Discussion

The BCRs, characterized by a complex hetero-oligomeric structure in which ligand binding and signal transduction are compartmentalized into distinct receptor subunits, are essential for the activation of B cells to induce the production of antibodies (Mattila et al., 2013, Yang and Reth, 2010). The BCR/TCR-based adaptive immune strategy is known to have evolved in jawed species and is mediated by B and T cell receptors. Jawless fish (agnathans) represent the most primitive living vertebrates, whereas BCRs and B cells have not been identified in these species (Parra et al., 2013). Although accumulating evidences suggest that invertebrate species could have some memory and specificity in their immune responses, there is still no report about B cells, BCRs, and Igs in invertebrates. In the present study, an ancient BCR-like molecule (defined as CgIgR) was identified from oyster, and its involvement in immune recognition, regulation of CgICP production, hemocyte phagocytosis, as well as the trained immunity was investigated.

Innate immunity, known as the non-specific immunity or in-born immunity, is an important component of the host defense against a wide variety of pathogens, serving as the front line and providing immediate response in preventing infection. The innate immune responses exhibit memory characteristics after the first encounter with the pathogen (Netea et al., 2016, Saz-Leal et al., 2018, Uehara et al., 2018). For instance, the human monocytes or macrophages exposed continuously to certain pathogen-associated molecular patterns (PAMPs) for a week displayed strong immune responses to defend against pathogen invasion (Bekkering et al., 2016). This induction of non-specific memory in innate immune cells is termed as trained immunity (Moorlag et al., 2018). As invertebrates lack bona fide B/T cells and antibodies, they depend, to a large extent, on their innate system to recognize and clear invading pathogens. Recently, the trained immunity has also been found in invertebrates. After a primary immunization, mosquito exhibited greater resistance to a subsequent infection with the same pathogen (Simoes and Dimopoulos, 2015). The resistance of brine shrimp Artemia against V. campbellii invasion was significantly increased when it encountered the homologous bacteria (Norouzitallab et al., 2016). Similarly, in shrimp Litopenaeus vannamei, the hemocyte phagocytosis against V. alginolyticus was enhanced when the shrimps were re-stimulated with V. alginolyticus (Lin et al., 2013). Apart from this, Dscam in Drosophila and mosquito also had alternative splicing upon immune stimulus, similar to that of mammalian antibodies (Dong et al., 2006, Hemani and Soller, 2012). In the present study, hemocyte phagocytosis increased significantly in V. splendidus and LPS training oysters, which was consistent with the previous reports in oysters (Zhang et al., 2014) and scallops (Wang et al., 2013), indicating the presence of training immunity in mollusks. Ig isotypes have been identified in cartilaginous and teleost fishes (Bengten and Wilson, 2015), whereas there is no report about immunoglobulins in invertebrates to date (Parra et al., 2013). The analysis of transcriptome data indicated that the expression levels of four CgICPs (CgIgR, CgCAICP-1, CgICP-2, CgLRRIG-1) were higher in hemocytes of V. splendidus training oysters, compared with that in the PBS training group. Further qRT-PCR analysis also confirmed that the transcripts of CgIgR, CgCAICP-1, CgICP-2, and CgLRRIG-1 increased significantly in hemocytes of V. splendidus training oysters. These results indicated that CgIgR, CgCAICP-1, CgICP-2, and CgLRRIG-1 were involved in the trained immunity induced by V. splendidus or LPS. It was reported that CgCAICP-1 could generate diverse isoforms and mediate hemocyte phagocytosis to different bacteria (Liu et al., 2018). CgLRRIG-1 might function as a PRR to recognize different bacteria and induce the production of tumor necrosis factor 1 (CgTNF-1) and interleukin 17-5 (CgIL17-5) (Wang et al., 2017b). These results suggested that CgICPs in oysters might display some similar functions with that of the antibodies in mammals. After CgIgR was knocked down by RNAi, the mRNA transcripts of CgCAICP-1, CgICP-2, and CgLRRIG-1, as well as hemocyte phagocytosis in V. splendidus or LPS training oysters, decreased significantly. These results collectively suggested that CgIgR participated in the trained immunity in oyster by regulating CgICP expressions and hemocyte phagocytosis.

The essential component for BCR/TCR-based adaptive immunity, including T cells, B cells, Igs, and major histocompatibility complex (MHC), has been so far identified in cartilaginous and teleost fishes (Bengten and Wilson, 2015). The accumulating evidence indicates that annelids and mollusks have evolved specialized immune cells (Koiwai et al., 2018, Wang et al., 2017a). In most invertebrates, hemocytes play important roles in mediating the immune responses to defend against pathogen invasion, and the granulocytes are the main immunocompetent hemocytes (Christophides et al., 2002, Wang et al., 2017a). The recognition is the key initiation step of the immune response, which is mediated by the PRRs on the surface of immune cells to recognize self and non-self. Some ICPs with TM domain, such as EsDscam in crabs, CgCAICP-1 and CgSiglec-1 in oysters, are found to be highly expressed in hemocytes and function as PRRs to recognize invading bacteria (Li et al., 2018, Liu et al., 2016a, Liu et al., 2018). In the present study, an ancient BCR-like molecule CgIgR was identified from C. gigas, which possessed five extracellular Ig domains, a TM domain, and a cytoplasmic tail. The cytoplasmic tail of CgIgR contained a sequence (EGDYTELGQCDPETPYEKL) that was consistent with the classical ITAM sequence (D/ExxYxxL/Ixx(6-12)YxxL/I) in mammalian BCR Igα/Igβ (Monroe, 2006). CgIgR protein was found to be located on the membrane of oyster hemocytes, similar to many other invertebrate ICPs with TM domain (Li et al., 2018, Liu et al., 2016a, Liu et al., 2018). The Ig domain mediates a variety of functions, including pathogen recognition, cell adhesion, and regulation of immune system (Teichmann and Chothia, 2000). Invertebrate ICPs can recognize bacteria and polysaccharides through their Ig domains (Liu et al., 2018). In the present study, the Ig domains of CgIgR displayed binding activities to various bacteria with higher binding activity to G− bacteria and LPS. These results indicated that the Ig domains could endow CgIgR with recognition and binding activity toward invading bacteria. It was worth noting that CgIgR could form dimers in response against V. splendidus stimulation, which was similar to mammalian BCR Igα/Igβ complex. After recognizing antigens, mammalian mIg could interact with BCR Igα/Igβ to form BCR Igα/Igβ complex, which could further transduce signals via their intracellular ITAM (DeFranco, 1993, Monroe, 2006, Reth, 1989), and finally led to the activation of B cells. The recognition capability of extracellular Ig domains and the presence of the classical ITAM in the cytoplasmic tail of CgIgR encouraged us to suspect that invertebrates might have evolved the similar recognition and regulation mechanism of the hemocyte surface receptor as the vertebrate BCRs even if they lacked BCR/TCR-based adaptive immunity.

BCR Igα/Igβ complex occurs through binding of cognate antigen to induce downstream signal transduction, which eventually promotes B cell activation and differentiation (De et al., 2017, Monroe, 2006, Panda and Ding, 2015). Upon ligand binding, the activated BCR Igα/Igβ complex recruits Syk to activate the downstream signaling cascades, including the MEK-ERK1/2 and PLC-NF-κB pathways (Ivashkiv, 2009, Niiro and Clark, 2002, Yang et al., 2015). In invertebrates, the research about ICP functions is mainly focused on cell phagocytosis (Dong et al., 2006, Li et al., 2018, Liu et al., 2018), whereas the signaling mediated by those ICPs has not been reported. In the present study, the activated CgIgR with a classical ITAM in the cytoplasmic tail was found to interact with CgSyk, demonstrating that the signaling mediated by the recognition receptors with ITAM was relatively conserved in vertebrates and invertebrates. In mammals, Syk recruited by the membrane receptors with ITAM participates in the activation of ERK. For example, BCR Igα/Igβ complex and membrane receptor Dectin-1 could recruit Syk to induce ERK phosphorylation (Drummond and Brown, 2013, Monroe, 2006). In the present study, CgIgR was found to interact with CgSyk through its intracellular ITAM to promote CgERK phosphorylation in oyster, which was similar to BCR Igα/Igβ in inducing Syk-ERK pathway in mammals. These results suggested that there existed an IgR-Syk-ERK signaling pathway in the primitive mollusks, which was similar to BCR Igα/Igβ-mediated Syk-ERK signaling in the mammals.

The activation of BCR Igα/Igβ complex can activate B cells to differentiate into plasmocytes to promote the generation of antibodies in mammals (Mattila et al., 2013, Netea et al., 2016, Yang and Reth, 2010). It has been reported that CgCAICP-1 displays binding activity to different bacteria and functions as an opsonin in mediating hemocyte phagocytosis against bacteria (Liu et al., 2018), and CgLRRIG-1 is also able to recognize various bacteria (Wang et al., 2017b). In the present study, CgIgR could form dimers after recognizing bacteria and activate CgSyk and CgERK to induce the production of CgICPs, which might function like mammalian BCR Igα/Igβ complex to transduce signaling to intracellular adaptor to induce the secretions of antibodies (Mattila et al., 2013, Yang and Reth, 2010). All these results suggested that membrane receptor CgIgR in molluscs might be the primitive ancestors of the mammalian BCR Igα/Igβ complex and could activate CgSyk and CgERK to generate CgICPs.

The epigenetic modulation is an important characteristic of the immune protection against pathogen infection, and it plays crucial roles in trained immunity. As a kind of epigenetic modification, histone methylation mainly promotes gene transcription through enrichment on gene promoters (Soares et al., 2017). In murine RAW264.7 cells and bone marrow-derived macrophages (BMDMs), H3K4me1, H3K4me2, and H3K4me3 increased after LPS stimulation and the histone methylation in particular H3K4me2 played a critical role in regulating the expressions of IL-6 and TNF-α after LPS stimulation (Zhao et al., 2018). The activated C-type lectin Dectin-1 could promote histone methylation, leading to immune training of monocytes (Quintin et al., 2012) and productions of IL-1β, IL-6, and TNF-α (Saz-Leal et al., 2018). In invertebrates, the study about epigenetic modulation in immunity is still in its infancy, and there is only one report in Caenorhabditis elegans about the enhanced monomethylation of Histone H1 variant HIS-24 and the association with daf-21 promoter after Bacillus thuringiensis stimulation (Studencka et al., 2012). In the present study, the level of CgH3K4me2 and its enrichment on the promoters of CgICP-2 and CgLRRIG-1 were found to increase significantly after V. splendidus and LPS stimulations. These results indicated that the methylation of CgH3K4 could be induced by CgIgR and participated in the immune response by inducing the expression of CgICPs in oyster.

Phagocytosis is a major mechanism used to remove pathogens and cell debris, and the phagocytized pathogens are degraded by lysosomes (Krokowski et al., 2018, Li et al., 2016). The phagocytized pathogens form phagosomes in macrophages, and the phagosomes subsequently fuse with intracellular granules to form the phagolysosome. In the phagolysosome, microbial killing is achieved by a combination of non-oxidative and oxidative mechanisms (Pluddemann et al., 2011, Stuart and Ezekowitz, 2005). In the present study, the co-localization of phagocytized V. splendidus and lysosomes was observed in hemocytes, which suggested that the phagocytized V. splendidus could be degraded by lysosomes in oyster hemocytes. After CgICP-2 and CgLRRIG-1 were silenced by RNAi, the hemocyte phagocytic rates toward V. splendidus were reduced and the co-localization of V. splendidus with lysosomes was less observed, indicating that CgICPs might act as cell surface receptors and opsonins to participate in hemocyte phagocytosis and regulate the degradation of bacteria. The above results demonstrated that CgIgR-mediated signaling could induce the expressions of CgCAICP-1, CgICP-2, and CgLRRIG-1 to promote the hemocyte phagocytosis and clearance of bacteria, which acted as the similar signaling mediated by BCR Igα/Igβ in promoting antibody secretions to induce bacterial elimination (Niiro and Clark, 2002, Teh and Neuberger, 1997).

In conclusion, an ancient BCR-like molecule CgIgR was identified in oyster C. gigas, which was involved in the trained immunity induced by V. splendidus and LPS through promoting the transcriptions of CgICPs and hemocyte phagocytosis. CgIgR with five Ig domains could serve as a hemocyte membrane receptor to recognize different bacteria. The activated CgIgR formed dimers and then interacted with CgSyk through its classical ITAM in cytoplasmic tail. The association of CgIgR with CgSyk could promote CgERK phosphorylation and induce the dimethylation at CgH3K4, which eventually induced the production of CgCAICP-1, CgICP-2, and CgLRRIG-1. The produced CgICPs could initiate the hemocyte phagocytosis toward V. splendidus, and the phagocytized V. splendidus were finally degraded in hemocyte phagolysosomes (Figure 8). It was suggested that CgIgR in oyster might be one of ancient molecules of mammalian BCR Igα/Igβ complex, and CgIgR-mediated signaling in inducing CgICP production was similar to that of BCR Igα/Igβ-mediated signaling in promoting antibody secretions. The results demonstrated an ancient BCR-like signaling (CgIgR-mediated signaling) in inducing CgICP secretions and elucidated the function of CgIgR in trained immunity and the role of CgICPs in degrading bacteria, indicating the existence, evolution, and functions of ancient BCR-like molecule in molluscs. Although significant disparities are evident between jawed vertebrate immune system and invertebrate immune system, the study of BCR-like signaling will probably unveil the conserved structural and functional aspects of B cell biology among these animals having been subjected to very similar selective pressures.

Figure 8.

Figure 8

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CgIgR-Mediated Pathway Induced CgICP Production to Promote Hemocyte Phagocytosis and Degradation of V. splendidus

Upon recognizing V. splendidus and LPS, CgIgR formed dimers and transferred signals to intracellular CgSyk. The activated CgSyk induced CgERK phosphorylation, which then promoted the enrichments of CgH3K4me2 on CgICP promoters to induce the production of CgICPs. CgCAICP-1, CgICP-2, and CgLRRIG-1 all containing a TM domain could locate on hemocyte membrane to recognize V. splendidus and promote hemocyte phagocytosis toward V. splendidus, which were then degraded by lysosomes in hemocytes. CgIgR upon recognizing V. splendidus and LPS could activate Syk-ERK pathway to induce CgICP production, which eventually promoted hemocytes to phagocytize and eliminate the invading bacteria.

Limitations of the Study

The study clearly demonstrated an ancient BCR-like signaling (CgIgR-mediated signaling) in inducing CgICP secretions and the phagocytosis and degradation of bacteria. An ancient BCR-like molecule was identified in oyster, but potential memory cells like B/T cells are still not found in oyster and other invertebrates. CgIgR is defined as one of ancient BCR-like molecules found in oyster, and there might exist other ancient BCR-like molecules in oyster, which still need further investigation in the future. Multiple CgICPs were found to participate in trained immunity of oyster, and CgIgR could regulate CgICP secretions and hemocyte phagocytosis in the successive V. splendidus and LPS stimulations. However, the involvement of CgIgR-mediated signaling in oyster trained immunity still needs to be further investigated.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.

Acknowledgments

We are grateful to all the laboratory members for their technical advice and helpful discussions. This research was supported by National Key R&D Program (2018YFD0900606), grants (Nos. U1706204, 41961124009) from National Science Foundation of China, Key R&D Program of Liaoning Province (2017203004, 2017203001), earmarked fund (CARS-49) from Modern Agro-industry Technology Research System, the Fund for Outstanding Talents and Innovative Team of Agricultural Scientific Research, AoShan Talents Cultivation Program Supported by Qingdao National Laboratory for Marine Science and Technology (No. 2017ASTCP-OS13), Dalian High Level Talent Innovation Support Program (2015R020), Liaoning Climbing Scholar, the Distinguished Professor of Liaoning, and the Research Foundation for Talented Scholars in Dalian Ocean University.

Author Contributions

Study concept and design: J.S., L.W., L.S.; acquisition of data: J.S., L.W., L.S.; analysis and interpretation of data: J.S., L.W., C.Y., L.S.; drafting of the manuscript and preparation of figures: J.S., L.W., L.S.; critical revision of the manuscript: J.S., L.W., L.S.; obtained funding: L.W., L.S.; administrative, technical, or other material support: J.S., L.W., C.Y., L.S.; study supervision: J.S., L.W., L.S.

Declaration of Interests

The authors declare no competing interests.

Published: February 21, 2020

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.isci.2020.100834.

Contributor Information

Lingling Wang, Email: wanglingling@dlou.edu.cn.

Linsheng Song, Email: lshsong@dlou.edu.cn.

Supplemental Information

Document S1. Transparent Methods, Figures S1–S3, and Table S1
mmc1.pdf (1.1MB, pdf)

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Supplementary Materials

Document S1. Transparent Methods, Figures S1–S3, and Table S1
mmc1.pdf (1.1MB, pdf)

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