In humans, a key and systemic function of the liver is to synthesize the majority of serum proteins, including complement proteins, fibrinogen, clotting factors and protease inhibitors. These proteins are involved in the regulation of systemic homeostasis and some also act as immune effectors. Amphioxus has extensive coelomic cavities and a closed circulatory system but lacks free-circulating blood cells. To date, many immune effectors have been identified in the humoral fluid of amphioxus, most of which originate from the hepatic cecum. This suggests that the hepatic cecum is essential for the humoral defensive system. Here we focus on the immune function of the humoral defensive molecules synthesized in the hepatic cecum.
The human complement system is composed of more than 30 distinct plasma proteins and membrane-associated proteins, and ~ 90% of the plasma complement components are synthesized in the liver (Sarma and Ward 2011). Complement, which can be activated by pathogens either directly or indirectly with the aid of antibodies, initiates a cascade of cleavage and activation events leading to the formation of membrane attack complexes or the recruitment of immune cells (Merle et al. 2015a). The amphioxus genome contains multiple copies of a number of complement-related genes, such as 50 C1q-like, 41 ficolin-like, two MASP, two C3, three Bf/C2, five C6-like and 427 CCP (complement control protein)-containing genes. In this regard, the complement system of amphioxus seems more complex and diverse than that of vertebrates including mammals (Huang et al. 2008). The vertebrate complement system has three activation pathways, i.e., the classical, alternative and lectin pathways, as well as two terminal pathways, i.e., the cytolytic and opsonic pathways. In all the three activation pathways, C3-convertase cleaves and activates C3, triggering a cascade of further cleavage and activation events. Amphioxus complement can be activated through alternative (C3 autohydrolysis), lectin (ficolin-MASPs activating C3) as well as classical-like pathway (C1q-MASPs activating C3) (Gao et al. 2014, 2017; Huang et al. 2011a; Li et al. 2008). Several key molecules involved in the complement activation pathways have been characterized, such as C1q-like, ficolin, MASP1/3, C3, Bf/C2 and properdin (Endo et al. 2003; Gao et al. 2013, 2014, 2017; He et al. 2008; Huang et al. 2011a; Suzuki et al. 2002). They are all predominantly synthesized in the hepatic cecum except that the properdin gene is ubiquitously expressed, similar to the expression profile of vertebrate properdin (Cortes et al. 2012). In addition, a conserved microRNA (miR-92d) has been shown to be involved in the regulation of amphioxus complement pathway by targeting C3, the center molecule of complement system (Yang et al. 2013). The expression level of miR-92d in the hepatic cecum is higher than in any other tissues, and is down-regulated by challenge with bacteria (Yang et al. 2013). These findings indicate that the hepatic cecum plays a central role in the regulation of the complement system.
In mammals, the terminal cytolytic pathway of complement needs both C5 and four C6-like proteins (C6, C7, C8 and C9) to assemble the membrane attack complex that performs cytolytic effects against the targeted cells. Furthermore, the resulting fragmentary molecules of complement activation, such as C3a, C5a and C3b, are capable of killing bacteria directly or enhancing macrophage phagocytosis of pathogenic microbes (Merle et al. 2015b). Amphioxus has five C6-like proteins each of which has the membrane attack complex/perforin domain required for membrane perforation, but all lack the key domains responsible for interaction with C5 (Suzuki et al. 2002). Although complement-mediated cytolysis has been observed in amphioxus (Li et al. 2008; Zhang et al. 2003), the cytotoxic pore-forming mechanism is unclear and deserves further study. Amphioxus C3 can be cleaved into C3a and C3b (Huang et al. 2011a). The C3a fragment is directly bactericidal and capable of enhancing macrophage phagocytosis of bacteria (Gao et al. 2013). The presence of both the cytolytic and opsonic pathways indicates that amphioxus has a functional prototypic complement system (Fig. 3).
Figure 3. Amphioxus complement systems are activated and amplified by the formation of C3 convertases through the C1q-mediated, lectin and alternative pathways. C3 can be cleaved by C3 convertases to form C3a and C3b. The C3a fragment has bactericidal activity and is capable of enhancing phagocyte phagocytosis against bacteria. The C3b fragment can bind covalently to cell surface carbohydrates via its reactive thioester. Amphioxus has five C6-like proteins, but it is unclear whether they participate in the formation of the membrane attack complex (MAC). A solid arrow indicates that the pathway was supported by experimental data; a dashed arrow indicates that no experimental support is present
Invertebrate phenoloxidase (PO), homologous to vertebrate tyrosinase, is a multi-function oxidase involved in sclerotization of the cuticle, defensive encapsulation, melanization of foreign microorganisms, and wound healing (Lemaitre and Hoffmann 2007). Intermediates produced in the melanization process can kill bacteria directly. PO normally exists as proPO zymogen. Upon recognition of the invasive microorganism by PRRs (e.g., PGRP and C-type lectins), a complex protease cascade is soon initiated to cleave proPO, resulting in the generation of active PO (Lu et al. 2014). In amphioxus, PO activity is detected in the humoral fluid, which is increased significantly after bacterial stimulation, suggesting that PO plays a crucial role in the humoral defensive system (Pang et al. 2009). Amphioxus PO is a tyrosinase-type enzyme (Pang et al. 2005), which is consistent with the fact that it has a close phylogenetic relationship with vertebrate tyrosinase. The tyrosinase-like gene is expressed in muscle, epidermis, hepatic cecum and some other tissues (Pang et al. 2013). However, the proteases and modulators of PO activation pathway remain to be identified in amphioxus.
In the absence of free-circulating immune cells, amphioxus needs an efficient mechanism to prevent the dissemination of pathogens in the humoral fluid. This is seemingly achieved by lectin or lectin-like proteins. Amphioxus humoral fluid possesses bacterial-agglutinating activity towards both Gram-negative and Gram-positive bacteria (Pang et al. 2012). Over 1200 genes in the amphioxus genome, contain the C-type lectin domain (CTLD) (Huang et al. 2008). Hereafter, the CTLD refers to the carbohydrate recognition domain (CRD) of C-type lectins (a family of Ca2+-dependent lectins). CTLDs usually have sugar-binding motifs (mostly EPN/QPD + WND). In amphioxus, CTLDs have various sugar-binding motifs, such as EP(N/S/K/E/D) and QP(D/S/N), hinting that C-type lectins have diverse sugar-binding specificities in amphioxus. Half of these CTLD-containing proteins consist solely of a CRD domain. It has been demonstrated that in amphioxus, a C-type lectin consisting of a signal peptide and a single CRD, termed AmphiCTL1, can agglutinate Gram-positive bacteria and yeast cells, but has little binding activity toward Gram-negative bacteria. AmphiCTL1 can directly kill Staphylococcus aureus and Saccharomyces cerevisiae. Interestingly, the AmphiCTL1 gene is mainly expressed in the hepatic cecum and is dramatically up-regulated when challenged with S. aureus, S. cerevisiae and zymosan (Yu et al. 2007a). In addition, the C-type lectins with multiple domain combinations are in most cases associated with collagen, CCP and EGF domains. A C-type lectin containing EGF and low-density lipoprotein receptor domains has been identified in B. japonicum (BjCTL). This is a typical Ca2+-dependent carbohydrate-binding protein capable of binding to and agglutinating both Gram-negative and Gram-positive bacteria, though it is only slightly expressed in the hepatic cecum (Qu et al. 2016). Given the abundant presence of C-type lectins with diversified structures in the genome, amphioxus may have a pre-prepared defense network against almost all possible invading microorganisms.
Intelectin (a type of galactofuranose-binding lectin) and galectin (a type of galactoside-binding lectin) are two important groups of pattern recognition molecules capable of regulating immune and inflammatory responses (Boscher et al. 2011; Yan et al. 2013a). On the one hand, there are 22 intelectin homologs identified in amphioxus two of which, i.e., AmphiITLN71469 and AmphiITLN239631, have been characterized (Yan et al. 2012, 2013b). AmphiITLN71469 can strongly agglutinate Gram-positive bacteria in a Ca2+-dependent manner, but has lower agglutination activity towards Gram-negative bacteria, whereas AmphiITLN239631 can agglutinate both Gram-positive and Gram-negative bacteria in a Ca2+-independent manner, but its bacterial binding and agglutinating activity are both lower than that of AmphiITLN71469. On the other hand, amphioxus has two forms of galectins, i.e., dual-CRD tandem galectin (Gal-L) and its alternatively spliced mono-CRD isoform (Gal-S), both having β-galactoside binding activity (Yu et al. 2007b). The Gal-L gene is mainly expressed in the hepatic cecum, whereas the Gal-S gene is ubiquitously expressed in all the tissues. Bacterial or fungal (S. cerevisiae) stimulation can induce up-regulation of Gal-L expression. Like some mammalian galectins, amphioxus Gal-L and Gal-S are present both intracellularly and extracellularly, hinting that they are involved in the inflammatory response by cross-linking β-galactoside glycoconjugates or glycoprotein receptors on cell surfaces to mediate cell-cell or cell-matrix interactions.
Apextrins are a group of proteins capable of interacting with the major bacterial cell wall polymer, peptidoglycan (PGN). Amphioxus has nine apextrin-like genes, and two of them have been characterized in B. japonicum (BjALP1 and BjALP2) (Huang et al. 2014). Both BjALP1 and BjALP2 can interact with bacterial PGN and the minimal PGN motif muramyl dipeptide. BjALP1 is a secreted effector that agglutinates Gram-positive bacteria, but not Gram-negative bacteria. Neutralization of secreted BjALP1 by anti-BjALP1 monoclonal antibodies can cause serious damage to the gut epithelium and rapid death of the host animal after bacterial infection. The BjALP1 gene is mainly expressed in the hepatic cecum, intestine, gill and skin, and its expression in the hepatic cecum and intestine shows thousand-fold up-regulation after bacterial infection. BjALP2 is an intracellular sensor associated with the NF-κB signaling pathway and its expression shows hundred-fold up-regulation after bacterial infection.
Many well-known microbicidal proteins have been identified in amphioxus, e.g., PGN recognition protein (PGRP) (Yao et al. 2012), lysozymes (Liu et al. 2006; Xu et al. 2014), chitotriosidase (Xu and Zhang 2012), defensin (Teng et al. 2012), and apolipoprotein A-I (Wang et al. 2019), most of which are predominantly synthesized in the hepatic cecum (Table 1). Amphioxus has 17-18 PGRPs, all of which have Zn2+ binding and amidase active sites (Huang et al. 2011b). A short PGRP (PGRP-S) possessing a domain combination of CBD-PGRP has been identified in B. japonicum (Yao et al. 2012). The PGRP-S can bind to E. coli, S. aureus and Pichia pastoris, and displays enzymatic activity of amidase which is capable of hydrolyzing PGN. The bactericidal activity of PGRP-S against E. coli and S. aureus is mainly due to the PGRP domain, whereas the anti-P. pastoris activity relies on the CBD domain. Notably, this domain combination of CBD-PGRP has been found only in amphioxus, which might result from domain shuffling when a great expansion of amphioxus immune gene repertoire occurred, possibly broadening its recognition spectrum.
Proteins Tissue expression Activity (IC50) * Reference Alanine aminotransaminase Mainly in hepatic cecum G-: E. coli (< 1 μmol/L) Jing and Zhang (2011) AmphiCTL1 Mainly in hepatic cecum G+: S. aureus (Not determined)
F: S. cerevisiae (Not determined)
Yu et al. (2007a) Apolipoprotein A-I Mainly in hepatic cecum G-: Aeromonas hydrophila (1.47 μmol/L), Vibrio vulnificus (2.15 μmol/L), Pseudomonas aeruginosa (1.54 μmol/L) Wang et al. (2019) Big defensin Hepatic cecum, muscle, gill and intestine G+: S. aureus (~5 μmol/L)
G-: A. hydrophila (> 20 μmol/L)
Teng et al. (2012) BjAMP Mainly in hepatic cecum, intestine, etc. G-: E. coli (3.2 μmol/L), Vibrio anguillarum (6.3 μmol/L)
G+: S. aureus (6.3 μmol/L), Micrococcus luteus (0.8 μmol/L)
Liu et al. (2015a) C3a Cleaved from C3 G-: E. coli (> 6.6 μmol/L), V. anguillarum (> 6.6 μmol/L)
G+: S. aureus (6.6 μmol/L), Bacillus subtilis (1.6 μmol/L)
Gao et al. (2013) Chitotriosidase-like protein Mainly in hepatic cecum F: Candida albicans (Not determined) Xu and Zhang (2012) Creatine kinase Not determined G-: E. coli (~10 μmol/L) An et al. (2009) Fibrinogen-related protein Mainly in hepatic cecum and intestine G-: E. coli (~3 μmol/L)
G+: S. aureus (< 0.5 μmol/L)
Fan et al. (2008) Lysozymes g-type in hepatic cecum, i-type in gill, c-type in hepatic cecum, intestine and gill Micrococcus lysodeikticus cell wall degradation activity of the three type lysozymes is in an order of i-type > c-type > g-type Xu et al. (2014) Miple Mainly in ovary G-: A. hydrophila (0.25 μmol/L), E. coli (1 μmol/L)
G+: S. aureus (0.25 μmol/L), B. subtilis (1 μmol/L)
Gao et al. (2018) PGRP-S Mainly in hepatic cecum and muscle G-: E. coli (~7 μmol/L)
G+: S. aureus (~7 μmol/L)
F: Pichia pastoris (~7 μmol/L)
Yao et al. (2012) Ribosomal protein S15 High expression in hepatic cecum G-: E. coli (0.5 μmol/L), A. hydrophila (0.5 μmol/L)
G+: S. aureus (0.5 μmol/L), M. luteus (0.5 μmol/L)
Qu et al. (2020a) Ribosomal protein S23 High expression in hepatic cecum and ovary G-: E. coli (4.2 μmol/L), A. hydrophila (4.2 μmol/L)
G: S. aureus (3 μmol/L), M. luteus (3 μmol/L)
Ma et al. (2020) Tachylectin-related protein Mainly in hepatic cecum and intestine G-: E. coli (~7 μmol/L) Ju et al. (2009) Transferrin-like protein Mainly in hepatic cecum, intestine and ovary G-: E. coli (~8 μmol/L) Liu et al. (2009) *The antimicrobial activities against different types of microbes are annotated as below: G -Gram-negative bacteria, G+ Gram-positive bacteria, F fungi. The IC50 is the concentration of protein needed to inhibit microbe growth by 50%
Table 1. Antimicrobial proteins identified in amphioxus
Lysozymes are a class of enzymes that are able to catalyze hydrolysis of the β-(1, 4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in PGN, and are produced in virtually all organisms, ranging from bacteriophages to humans. Most organisms are able to produce different types of lysozymes and/or multiple forms of the same lysozyme. It is presumed that different types/forms of lysozymes may have different or complementary functions. In amphioxus, lysozyme activity has been detected in the humoral fluid, and the lysozyme activity of humoral fluid increases significantly after exposure to bacteria (Pang et al. 2006, 2010, 2012). Peculiarly, amphioxus possesses three types of lysozymes, i.e., c-, g- and i-type lysozymes (Xu et al. 2014). The c- and g-type lysozymes are predominantly synthesized in the hepatic cecum, whereas the i-type lysozyme is primarily generated in the gill. All the three types of lysozyme have the capacity to degrade the Micrococcus lysodeikticus cell wall, but their levels of activity differ from each other in the order i-type > c-type > g-type.
In addition to these known bactericidal proteins, some well-known molecules are found to possess newly discovered antimicrobial functions. For example, alanine aminotransaminase (ALT) is an acute phase protein primarily synthesized in the liver and is regarded as an index for clinical diagnosis of liver function in humans. It has been revealed by Jing and Zhang (2011) that ALT has antibacterial activity. Amphioxus ALT can bind to LPS, and cause the Gram-negative bacterium E. coli to lyse. In contrast, it can neither bind nor kill Gram-positive bacterium S. aureus. LPS stimulation can induce a 40-fold increase in the expression of the amphioxus ALT gene, suggesting the important role of ALT in antibacterial defense. Recently, two ribosomal proteins of B. japonicum, BjRPS15 and BjRPS23, have been shown capable of killing both Gram-negative and Gram-positive bacteria (Ma et al. 2020; Qu et al. 2020a). The functional characteristics of BjRPS15 and BjRPS23 are similar in that both can interact with bacterial membranes via LPS and LTA, and cause membrane depolarization. They can also stimulate production of intracellular ROS in bacteria. Their expression in the hepatic cecum is significantly higher than in other tissues, and markedly up-regulated following challenge with bacteria, LPS or LTA. Moreover, both can be detected extracellularly in the humoral fluid, suggesting that they are "moonlighting" proteins, functioning not only as house-keeping proteins but also as antibacterial effectors.
Antimicrobial peptides (AMPs), usually composed of fewer than 100 amino acids, are endogenous antibiotics that are widely distributed in nature as ancient components of innate immunity. Most AMPs are cationic and amphipathic molecules that interact with microbial membranes and are capable of killing microbial cells, either by disrupting membrane integrity or by interacting with certain intracellular targets (Bahar and Ren 2013). More than 3000 AMPs have been recorded in the AMP database, only two of which are from amphioxus (Wang et al. 2016). This may be due to the fact that the study of AMPs in amphioxus is still in its infancy. The precursors of AMPs generally contain signal sequences and proregions, and these regions tend to be more conserved than mature AMPs. This advantage has been successfully employed to search for novel AMPs from databases within the same lineages of amphibians and fish (Juretic et al. 2011; Tessera et al. 2012). In the amphioxus B. japonicum, an AMP named BjAMP1 has been identified using the signal sequence of the jawless hagfish, HFIAP-1, a known AMP of the cathelicidin family. The mature peptide of BjAMP1, i.e., the C-terminal 21 residues, can directly kill a broad spectrum of microbes via a membrane active mechanism (Liu et al. 2015a). The other AMP known in amphioxus is big defensin, termed BjBD, identified by Teng et al. (2012). The BjBD gene is constitutively expressed in most of the tissues examined, and remarkably up-regulated following challenge with LPS, LTA, A. hydrophila and S. aureus. Moreover, recombinant BjBD is able to inhibit the growth of S. aureus, E. coli and A. hydrophila.
Besides the antibacterial molecules listed above, there are several other molecules, such as fibrinogen-related protein (Fan et al. 2008), transferrin (Liu et al. 2009), Gram-negative bacteria-binding proteins (Jin et al. 2012), alpha-2 macroglobulin (Pathirana et al. 2016), avidin (Guo et al. 2017), Miple (Gao et al. 2018), HSP5 and HSP90α (Yao et al. 2019), that play roles in the defense system of amphioxus. These molecules are primarily produced in the hepatic cecum. They either display bactericidal activity or bacteria-binding/agglutinating activity or both.
Lectin and lectin-like protein
Antimicrobial protein and peptide
Other immune-relevant effectors
Acute phase response (APR) is a physiological process occurring rapidly after the onset of infection, inflammation and trauma. One of the most prominent consequences of APR is the change in concentrations of a number of plasma proteins, collectively known as acute phase proteins (APPs). APRs function in a variety of defense-related activities, thereby protecting the body by preventing microbial growth, limiting tissue damage, and helping to restore metabolic homeostasis. In humans, the liver is the primary organ for APP production, which involves a range of processes to enhance inflammation and limit excessive inflammation (Strnad et al. 2017). The amphioxus hepatic cecum, like the vertebrate liver, plays a central role during APR (Wang and Zhang 2011). At least 58 vertebrate (zebrafish) liver-specific genes are expressed in a tissue-specific manner in the hepatic cecum of amphioxus, and 52 out of the 58 genes display similar expression profiles in both amphioxus hepatic cecum and zebrafish liver in response to LPS challenge, suggesting that these genes are commonly involved in APR in both animals. Liver-specific transcription factors such as HNF-1, HNF-4 and C/EBP are known to control the expression of APR-related genes (Armendariz and Krauss 2009; Babeu and Boudreau 2014; van der Krieken et al. 2015). Notably, in both amphioxus and zebrafish, the majority of the 52 APR-related genes possess binding sites for one or more of the above-mentioned transcription factors in the promoter sequences, suggesting that these APR-associated factors forms a similar network in both species for regulating the expression of APR-related genes (Wang and Zhang 2011).
The hepatic cecum secretes APPs into the humoral fluid and these usually have direct effector functions, including the clearance of pathogens and the regulation of inflammatory responses (Table 2). For example, the classical APPs, including alanine aminotransaminase (Jing and Zhang 2011), transferrin (Liu et al. 2009), alpha-2 macroglobulin (Liang et al. 2011), plasminogen (Liu and Zhang 2009), tachylectin-related protein (Ju et al. 2009), fibrinogen-related protein (Fan et al. 2008), and complement components C3 and Bf (Pan et al. 2011), all exhibit functional conservation in amphioxus and vertebrates. Most of the hepatic cecum-secreted APPs have antimicrobial activity or pro-inflammatory function, but BjIM1 has anti-inflammatory function. It is clear that the hepatic cecum-secreted APPs are important for maintaining systemic homeostasis of amphioxus. Consequently, the hepatic cecum can be regarded as a key integrator of immunity, similar to the vertebrate liver.
Proteins Examples Immune function Complement proteins C1q, Bf/C2 and C3 Induce bacterial cell lysis, and enhance phagocytosis Lectins C-type lectins, ficolins, intelectins and apextrin 1 Agglutinate bacteria, and active complement Antimicrobial proteins PGRP, lysozymes, chitotriosidase, defensin, apolipoprotein A-I, alanine aminotransaminase, fibrinogen-related protein, AMPs, etc. Kill or inhibit the growth of microbes Iron-binding proteins Ferritin and transferrin Act to reduce free iron in the humoral fluid, and antimicrobial functions Inflammatory modulators TGF-β and BjIM1 Anti-inflammatory functions
Table 2. Humoral protein synthesis by the hepatic cecum during the acute phase response in amphioxus
In summary, recent findings regarding the function of the hepatic cecum in immune surveillance, clearance of pathogens and acute phase response, all indicate that this organ is a crucial conductor of immune and inflammatory process in amphioxus. The similarities in liver/hepatic cecum-specific genes and immune functions between amphioxus and vertebrates supports the notion that the amphioxus hepatic cecum is the precursor of the vertebrate liver, acting as a key integrator of immunity.