Complement C3 Regulates Inflammatory Response and Monocyte/Macrophage Phagocytosis of Streptococcus agalactiae in a Teleost Fish

The complement system is composed of a complex protein network and is pivotal to innate immunity. Complement 3 (C3) is a critical protein in the complement cascade and participates in complement activation and immune defense. In this study, C3 from Nile tilapia (Oreochromis niloticus) was cloned and its function in resisting pathogen infection was characterized. The full length of OnC3 open reading frame is 4974 bp, encoding 1657 aa, and the predicted protein mass weight is 185.93 kDa. The OnC3 amino acid sequence contains macroglobulin domains. The expression pattern of OnC3 mRNA in the tissues of healthy fish was detected, with the highest in the liver and the lowest in the muscle. After challenged with Streptococcus agalactiae and Aeromonas hydrophila, the expression of OnC3 mRNA was significantly up-regulated in the liver, spleen, and head kidney. Further, the recombinant OnC3 protein alleviated the inflammatory response and pathological damage of tissues after infected with S. agalactiae. Moreover, the OnC3 promoted the phagocytosis of monocytes/macrophages to S. agalactiae. The data obtained in this study provide a theoretical reference for in-depth understanding of C3 in host defense against bacterial infection and the immunomodulatory roles in teleost fish.


Introduction
Complement, a vital constituent of the innate immune system, is a complex protein network composed of more than 50 kinds of proteins and protein sub-fragments [1][2][3][4]. The activation of complement is achieved through the classical pathway, lectin pathway, as well as alternative pathway [3]. The classical pathway is activated by the binding of complement C1q to antigen-complexed IgM (CH3 domain) or IgG (CH2 domain) [5]. The lectin pathway is launched by the binding of mannose-binding lectin (MBL), collectins, and ficolins to carbohydrates present on the surface of pathogens [6]. In addition, the alternative pathway is activated by viruses, bacteria, and fungi, and directly activates C3 by passing C1, C2, and C4 [7]. Although the initiator molecules and components involved in the three activation pathways are different, they all center on proteolytic activation of C3. Then, the downstream C5-C9 proteins are activated, leading to the complement cascade effects and formation of the membrane-attack complex (MAC) [1,8]. After being activated, the complement system induces chemotaxis and cell activation by producing anaphylatoxins and mediates inflammation by promoting phagocytosis, degranulation, and cell lysis. The crucial immunological functions of the complement system include host

Tissue Distribution and Expression Pattern of OnC3
The expression of OnC3 transcripts in healthy Nile tilapia illustrated that OnC3 extensively expressed in various tissues. The OnC3 was expressed in the liver abundantly and sequentially decreased in the brain, head kidney, thymus, spleen, intestines, hind kidney, peripheral blood, and muscle. Significantly, the expression liver was about 62,000-fold of that in muscle (Figure 2A).
The expression patterns of OnC3 in vivo and in vitro were significantly changed S. agalactiae and A. hydrophila stimulation. In the liver, the OnC3 transcripts r massively and reached the maximum at 24 h after challenge. The expression of OnC 6-fold and 4-fold higher than the PBS group after challenged with S. agalactiae an hydrophila, respectively ( Figure 2B). A similar tendency was observed in the spleen, a expression of OnC3 was 10-fold and 7-fold higher after stimulated by these pathogenic bacteria ( Figure 2C). However, the transcripts of OnC3 in the head ki fluctuated between 2-fold and 4-fold higher than the PBS group after stimulation (F

Tissue Distribution and Expression Pattern of OnC3
The expression of OnC3 transcripts in healthy Nile tilapia illustrated that OnC3 was extensively expressed in various tissues. The OnC3 was expressed in the liver most abundantly and sequentially decreased in the brain, head kidney, thymus, spleen, skin, intestines, hind kidney, peripheral blood, and muscle. Significantly, the expression in the liver was about 62,000-fold of that in muscle (Figure 2A).
The expression patterns of OnC3 in vivo and in vitro were significantly changed after S. agalactiae and A. hydrophila stimulation. In the liver, the OnC3 transcripts raised massively and reached the maximum at 24 h after challenge. The expression of OnC3 was 6-fold and 4-fold higher than the PBS group after challenged with S. agalactiae and A. hydrophila, respectively ( Figure 2B). A similar tendency was observed in the spleen, as the expression of OnC3 was 10-fold and 7-fold higher after stimulated by these two pathogenic bacteria ( Figure 2C). However, the transcripts of OnC3 in the head kidney fluctuated between 2-fold and 4-fold higher than the PBS group after stimulation ( Figure 2D). In MO/Mø, the OnC3 expression was 7-fold and 4-fold higher than that of the PBS group at 24 h after challenge with S. agalactiae and A. hydrophila ( Figure 2E).
. Sci. 2022, 23, x FOR PEER REVIEW 2D). In MO/Mø, the OnC3 expression was 7-fold and 4-fold higher than that of t group at 24 h after challenge with S. agalactiae and A. hydrophila ( Figure 2E). (1 × 10 7 CFU/mL) challenged tissues (liver, spleen, and head kidney). The expression of On normalized to β-actin, and the multiple changes in OnC3 in each tissue were calculated d that in the PBS-immunized group. (E) Expression of OnC3 in the MO/Mø after stimulati inactivated S. agalactiae (1 × 10 7 CFU/mL) and A. hydrophila (1 × 10 7 CFU/mL). The er represent standard deviation (n = 3), and the significant differences are indicated by differen (a, b, c, d) (p < 0.05).

Effects of (r)OnC3 on Inflammatory Response In Vitro and In Vivo
The Ni-chelating affinity chromatography was applied, purifying the recom OnC3 protein. The SDS-PAGE was used to identify a band near 70 kDa, approach theoretical mass weight of (r)OnC3 ( Figure S2).
To detect the effects of (r)OnC3 on inflammatory response, the expression inflammatory cytokines IL-1β, IL-6, TNF-α and anti-inflammatory cytokines IL-TGF-β transcripts in the liver and spleen were detected. The results illustrate (r)OnC3 upregulated the transcripts of IL-1β, IL-6, TNF-α, and TGF-β in the liv spleen at 12 h after stimulation ( Figure 3A). (1 × 10 7 CFU/mL) challenged tissues (liver, spleen, and head kidney). The expression of OnC3 was normalized to β-actin, and the multiple changes in OnC3 in each tissue were calculated deciding that in the PBS-immunized group. (E) Expression of OnC3 in the MO/Mø after stimulation with inactivated S. agalactiae (1 × 10 7 CFU/mL) and A. hydrophila (1 × 10 7 CFU/mL). The error bars represent standard deviation (n = 3), and the significant differences are indicated by different letters (a, b, c, d) (p < 0.05).

Effects of (r)OnC3 on Inflammatory Response In Vitro and In Vivo
The Ni-chelating affinity chromatography was applied, purifying the recombinant OnC3 protein. The SDS-PAGE was used to identify a band near 70 kDa, approaching the theoretical mass weight of (r)OnC3 ( Figure S2).
To detect the effects of (r)OnC3 on inflammatory response, the expression of proinflammatory cytokines IL-1β, IL-6, TNF-α and anti-inflammatory cytokines IL-10 and TGF-β transcripts in the liver and spleen were detected. The results illustrated that (r)OnC3 upregulated the transcripts of IL-1β, IL-6, TNF-α, and TGF-β in the liver and spleen at 12 h after stimulation ( Figure 3A).
. Sci. 2022, 23, x FOR PEER REVIEW Figure 3. The effects of (r)OnC3 on tilapia inflammatory response in vivo. (A) The expressions of inflammatory cytokines IL-1β, IL-6, and TNF-α and anti-inflammatory cytok 10 and TGF-β in the liver and spleen of tilapia were detected at 12 h after treated with protein (50 µg/mL). The control groups were treated with 10 mM sterile PBS and Trx pro µg/mL). (B) (r)OnC3 promoted inflammatory infiltration in the liver and spleen by H&E (×400). (C) The injury score of the liver and spleen. The error bars represent standard devia 3), and the significant differences are indicated by different letters (a, b, c) (p < 0.05) and a (n.s. 0.05 < p, * 0.01 ≤ p < 0.05).
In MO/Mø, the expressions of all inflammatory cytokines were rema augmented after challenge with the (r)OnC3 protein. In the (r)OnC3 grou transcription of IL-1β, IL-6, TNF-α, and IL-10 was about 5-fold to 7-fold higher th Trx groups and PBS groups at 12 h, 24 h, and 48 h after stimulation ( Figure 4A-D expression of TGF-β in the (r)OnC3 group also was 5-fold higher than that in other at 12 h after stimulation ( Figure 4E). The mRNA expressions of inflammatory cytokines IL-1β, IL-6, and TNF-α and anti-inflammatory cytokines IL-10 and TGF-β in the liver and spleen of tilapia were detected at 12 h after treated with (r)OnC3 protein (50 µg/mL). The control groups were treated with 10 mM sterile PBS and Trx protein (50 µg/mL). (B) (r)OnC3 promoted inflammatory infiltration in the liver and spleen by H&E staining (×400). (C) The injury score of the liver and spleen. The error bars represent standard deviation (n = 3), and the significant differences are indicated by different letters (a, b, c) (p < 0.05) and asterisks (n.s. 0.05 < p, * 0.01 ≤ p < 0.05).
In MO/Mø, the expressions of all inflammatory cytokines were remarkably augmented after challenge with the (r)OnC3 protein. In the (r)OnC3 groups, the transcription of IL-1β, IL-6, TNF-α, and IL-10 was about 5-fold to 7-fold higher than the Trx groups and PBS groups at 12 h, 24 h, and 48 h after stimulation ( Figure 4A-D). The expression of TGF-β in the (r)OnC3 group also was 5-fold higher than that in other groups at 12 h after stimulation ( Figure 4E).

(r)OnC3 Regulates ROS Levels in MO/Mø
Before the experiment, the cells were loaded with the DCFH-DA fluorescent probe, as the fluorescence intensity of DCF can represent the content of ROS. The ROS in MO/Mø was assayed by flow cytometric analysis. The results showed that the DCF intensity in the (r)OnC3 group was 60.6%, which was apparently stronger than the PBS group (50.9%) and Trx group (51.4%) ( Figure 4F). The blank control group was 1.1%. Furthermore, the positive cell rate of the (r)OnC3 group was the highest ( Figure 4G).

(r)OnC3 Regulates Inflammation after S. agalactiae Infection
According to the results of the in vivo experiment, the transcripts of IL-1β, IL-6, TNFα, IL-10, and TGF-β in experimental groups were apparently higher than the PBS group

(r)OnC3 Regulates ROS Levels in MO/Mø
Before the experiment, the cells were loaded with the DCFH-DA fluorescent probe, as the fluorescence intensity of DCF can represent the content of ROS. The ROS in MO/Mø was assayed by flow cytometric analysis. The results showed that the DCF intensity in the (r)OnC3 group was 60.6%, which was apparently stronger than the PBS group (50.9%) and Trx group (51.4%) ( Figure 4F). The blank control group was 1.1%. Furthermore, the positive cell rate of the (r)OnC3 group was the highest ( Figure 4G).

(r)OnC3 Regulates Inflammation after S. agalactiae Infection
According to the results of the in vivo experiment, the transcripts of IL-1β, IL-6, TNF-α, IL-10, and TGF-β in experimental groups were apparently higher than the PBS group after 12 h of stimulation. However, in the S. agalactiae + (r)OnC3 group, the transcripts of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were lower than the S. agalactiae group and S. agalactiae + Trx group, while the expression of IL-10 and TGF-β was augmented compared to the S. agalactiae group and S. agalactiae + Trx group ( Figure 5A,B). after 12 h of stimulation. However, in the S. agalactiae + (r)OnC3 group, the transcripts of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were lower than the S. agalactiae group and S. agalactiae + Trx group, while the expression of IL-10 and TGF-β was augmented compared to the S. agalactiae group and S. agalactiae + Trx group ( Figure 5A,B).  The mRNA expressions of inflammatory cytokines IL-6, IL-8, and TNF-α and anti-inflammatory cytokines IL-10 and TGF-β were detected from Nile tilapia liver at 12 h after challenge with (r)OnC3 protein and S. agalactiae. The control group was treated with 10 mM sterile PBS, and the rest of the groups were treated with S. agalactiae (1 × 10 7 CFU/mL), S. agalactiae + Trx protein (50 µg/mL), or S. agalactiae + (r)OnC3 protein (50 µg/mL). (B) The same inflammatory factors were detected in spleen, and the experimental conditions were the same as those in panel A. (C) The same inflammatory factors were detected in MO/Mø, and the experimental conditions were the same as those in panel A. (D) (r)OnC3 inhibited inflammatory infiltration in the bacterial-infected liver and spleen by H&E staining (×400). The control group was challenged with PBS, and the positive groups were challenged with S. agalactiae and S. agalactiae with Trx protein, respectively. The experiment groups were challenged with S. agalactiae with (r)OnC3 protein.
Meanwhile, the expression of inflammation factors confirmed a similar changing tendency in MO/Mø ( Figure 5C). In the experimental groups, all detected inflammatory factors were remarkably increased at 12 h after stimulation, and there were no apparent divergences between the S. agalactiae group and S. agalactiae + Trx group. In the S. agalactiae + (r)OnC3 stimulated group, the expression of IL-1β, IL-6, and TNF-α was lower than the S. agalactiae and S. agalactiae + Trx stimulated group, while the expression of IL-10 and TGF-β was higher than the two groups.

(r)OnC3 Alleviates Inflammatory Response and Pathological Damage
The results of the histopathological analysis indicated that (r)OnC3 significantly induced the inflammatory infiltration of the liver and spleen ( Figure 3B) and greatly increased the injury score in healthy fish ( Figure 3C). When S. agalactiae were infected, the (r)OnC3 significantly alleviated the inflammatory response and injury score ( Figure 5D,E). The histopathological analysis by H&E staining indicated that the inflammatory infiltration and hemosiderosis could be detected in liver and spleen of S. agalactiae and the S. agalactiae + Trx group. However, the pathological injury was greatly relieved in the S. agalactiae + (r)OnC3 group ( Figure 5D). Additionally, the injury scores of the S. agalactiae + (r)OnC3 group were obviously lower in S. agalactiae and the S. agalactiae + Trx group ( Figure 5E).

(r)OnC3 Regulates Monocyte/Macrophage Phagocytosis
To further examine the effect of (r)OnC3 in phagocytosis, the expressions of phagocytosisrelated receptors (CD11b, CD18), enzymes (RhoA, Rock), as well as lysosome-related factors (EEA, M6PR, and vATPase) were detected by RT-qPCR. The results showed that in head kidney tissue, the transcripts of these factors in S. agalactiae + (r)OnC3 groups were significantly higher than in S. agalactiae, the S. agalactiae + Trx group, and the PBS group ( Figure 6A). Analogously, results in MO/Mø showed that the expression of these factors in the S. agalactiae + (r)OnC3 group was higher than S. agalactiae, the S. agalactiae + Trx group, and the PBS group ( Figure 6B). Furthermore, the assay of phagocytosis was developed with flow cytometric analysis ( Figure 6C). The MO/Mø without phagocytosis showed that the phagocytic rate was only 1.6%. The phagocytic rate of MO/Mø to S. agalactiae in the (r)OnC3 group was 61.5%, much higher than the 48.0% and 51.0% in the PBS and Trx groups ( Figure 6C,D).

Discussion
As a crucial constituent of innate immunity, the complement system has a long evolutionary history. Among them, C3, as an evolutionarily conserved core molecule, is ubiquitous in vertebrates and invertebrates [4,33]. C3 and its cleavage fragments bind to receptors on the cell surface, playing a variety of immunological roles, such as promoting phagocytosis and killing potential pathogens, or regulating the activation of the complement system for immune function [1][2][3]8]. Nonetheless, the specific immune mechanism of C3 is still unclear in early vertebrates. In this study, the molecular cloning and characterization of Nile tilapia C3 were performed. The role of OnC3 in defense against pathogenic bacteria from the perspective of phagocytosis and inflammatory responses was explored as well.
The results of this study revealed that OnC3 is composed of α and β chains, connected by disulfide bonds, and the putative cleavage site was located at 659-662 (RKKR). The OnC3 were predicted conserved with seven domains: A2M_N, A2M_N_2, ANATO, A2M, A2M_comp, A2M_recep, and C345C domains. Moreover, a highly conserved thioester region (GCGEQ) in OnC3 was also predicted. The phylogenetic analysis indicated that OnC3 clustered with C3 of other bony fish, and also has high similarity with mammals and invertebrates, which further reflects the high conservation of C3 in the evolutionary process [10,28,34].
In mammals, the liver is considered as the foremost organ of C3 synthesis [13]. Consistent with most reported teleosts, the expression of OnC3 was the highest in the liver tissue of Nile tilapia [25,28]. The brain tissue was second only to the liver. Coincidentally, recent studies have also shown that C3 has a greater relationship with the homeostasis of the central nervous system [35]. Furthermore, the expression of OnC3 mRNA in the head kidney, thymus, and spleen is also at a high level. After S. agalactiae and A. hydrophila infection, the expression levels of OnC3 mRNA in the liver, spleen, and head kidney were significantly up-regulated. These organs with high expression of OnC3 are important immune organs of Nile tilapia in antibacterial infection and are also suitable pathological models in vivo [2]. C3 is mainly synthesized in hepatocytes, and it is also synthesized in extrahepatic cells, including monocytes/macrophages, neutrophils, and fibroblasts [36]. Monocytes/macrophages are professional phagocytic cells and an important cellular model for studying phagocytosis and antibacterial immunity in vitro [37]. Similar to tissue challenge results in vivo, the transcription of OnC3 mRNA in head kidney MO/Mø remarkably rose after infected by the two pathogens as well. The S. agalactiae and A. hydrophila are currently the main pathogens of Nile tilapia, causing huge economic losses to the aquaculture industry [31,32]. These results indicated that the expression of OnC3 would increase after pathogen infection and that it is involved in the process of resisting pathogen infection, which further helps to explore the important mechanism of OnC3 in Nile tilapia resisting pathogen infection.
Complement components are present in an inactive state to prevent the negative effects of complement imbalances under normal circumstances. The imbalances of the complement activation are the important causes of tissue damage and end organ damage in autoimmune diseases, such as systemic lupus erythematosus and C3 glomerulopathy in humans [20,21,38]. To explore the effects of excess C3 on fish, healthy Nile tilapia were injected with the (r)OnC3 protein and the head kidney MO/Mø were stimulated with the (r)OnC3 protein. The expression of inflammatory factors (IL-1β, IL-6, TNF-α, IL-10, and TGF-β) were apparently ascended after injection of the (r)OnC3 protein in liver and spleen of healthy fish. The inflammatory factors and intracellular reactive oxygen species that increased significantly were also up-regulated in head kidney MO/Mø after (r)OnC3 challenge. Meanwhile, slight tissue damage was detected in liver and spleen by H&E staining. These results suggested the content and function of C3 in Nile tilapia is tightly regulated, preventing damage to the immune system due to the imbalance of complement [20].
The complement components will be activated and function in turn when stimulated [1,4]. After pathogenic bacteria invade the host, they can adapt to the host environment and proliferate rapidly and attack vital tissues such as spleen and liver through blood circulation [39]. The toxic substances released by these pathogenic bacteria can trigger an inflammatory response, resulting in different degrees of tissue damage [39,40]. The occurrence of inflammatory response is an obbligato process for the body to resist pathogenic infection. However, the damage caused by prolonged inflammatory response is also not negligible [41,42]. After infection with S. agalactiae, the liver and spleen of Nile tilapia were congested, with swelling and severe water accumulation [40,[43][44][45]. A large number of vacuoles appeared in the liver tissue, and a large number of cell necrosis and inflammatory cells appeared in liver and spleen. The corresponding symptoms are significantly relieved by injecting an appropriate amount of the (r)OnC3 protein. Necrotic cells were reduced in the liver, the vacuolation phenomenon was alleviated, and there was no significant inflammatory infiltrate in the spleen. Furthermore, we found that (r)OnC3 could regulate the inflammatory response by inhibiting the expression of pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α) and promoting the expression of anti-inflammatory cytokines (IL-10 and TGF-β), thereby slowing down the tissue damage caused by S. agalactiae. These results suggested that C3 plays a vital role in host immune defense and has antibacterial effects against pathogen infection in bony fishes.
Phagocytic cells are chief components of innate immunity and act as a bridge between innate and adaptive immunity [37]. In mammals, activation of C3 is capable of affecting the phagocytosis of phagocytic cells such as macrophages or neutrophils [4]. Recent studies on pearl oysters pointed out that disrupting the C3 gene could significantly reduce the phagocytosis of Vibrio alginolyticus by blood cells, which also showed the importance of C3 in invertebrate phagocytes [34]. Phagocytes are also the first line of defense in the innate immunity of bony fishes. Recent studies pointed out that C3a enhances the phagocytic activity of B cells through C3aR in grass carp [24,37]. The fragments of C3, such as C3b and iC3b, are extremely important in the complement system for opsonophagocytosis [15,16,46]. These fragments bind to complement receptors (CR) on the surface of macrophages, thereby affecting the phagocytic activity of macrophages, including CR1-CR4 [15]. CR3 (CD11b/CD18) is known as the principal β2 integrin, which plays a prominent role in recognizing bacteria. It can bind to inactivated C3b (iC3b) and activate phagocytosis [47,48]. Despite the signaling pathway(s) responsible for its cytokine response having not been scientifically elucidated, related studies also have reported that CR3-mediated phagocytosis requires the participation of RhoA and ROCK [49,50]. The Rho/ROCK signaling pathway acts on inducing cytoskeletal reorganization, cell migration, and stress fiber formation, thereby affecting tissue permeability and tissue contraction, and is also associated with phagosome formation [51][52][53]. In this study, (r)OnC3 significantly enhanced the phagocytosis of S. agalactiae by Nile tilapia head kidney MO/Mø. The expression of CR3-mediated phagocytosis-related genes was detected as well. Notably, CR3 (CD11b/CD18), RhoA, and ROCK were all up-regulated. Significant up-regulation also occurred in association with phagolysosome formation such as EEA, M6PR, and vATPase [54]. These data suggested that OnC3 can effectively promote the phagocytosis of monocytes/macrophages. Although the functional characteristics of C3 in inflammation and phagocytosis have been elucidated at the gene expression level, it is better to confirm the C3 function at the protein level. As the expression level between proteins and mRNAs does not always show a simple linear relationship, follow-up studies at the protein level need further exploration.
In conclusion, the current study clarified that C3 has a conserved structure and function of host defense in Nile tilapia. As schematically illustrated in Figure 7, OnC3 is involved in the processes of relieving inflammation, reducing tissue damage, and promoting MO/Mø phagocytosis. These data further support that OnC3 is related to antibacterial immunity. Overall, the findings of this research suggest that C3 plays an important role in innate immunity, which helps to further refine the function and mechanism of action of teleost C3. function of host defense in Nile tilapia. As schematically illustrated in Figure 7, OnC3 is involved in the processes of relieving inflammation, reducing tissue damage, and promoting MO/Mø phagocytosis. These data further support that OnC3 is related to antibacterial immunity. Overall, the findings of this research suggest that C3 plays an important role in innate immunity, which helps to further refine the function and mechanism of action of teleost C3. Figure 7. Complement C3 regulates inflammatory response and monocyte/macrophage phagocytosis to Streptococcus agalactiae in a teleost fish. The proposed model illustrates the mechanism by which OnC3 as a core molecule of the complement system effectively participates in non-specific cell immunity. After S. agalactiae infects fish, the C3 molecule is decomposed into C3a and C3b, and the inactivated iC3b can combine with CR3 (CD11b/CD18) to enhance the phagocytosis of monocytes/macrophages. The C3 molecule also modulates the inflammatory response to defend against S. agalactiae infection.

Animals and Sample Acquisition
Nile tilapia used in this study were obtained from the Guangdong Tilapia Breeding Farm [45,55]. The weight of fish was 70-100 g for tissue collection and 250-300 g for the isolation of Nile tilapia MO/Mø [45,55]. The Nile tilapia were cultured in a constant temperature semi-automatic circulatory system in the School of Life Sciences, South China Figure 7. Complement C3 regulates inflammatory response and monocyte/macrophage phagocytosis to Streptococcus agalactiae in a teleost fish. The proposed model illustrates the mechanism by which OnC3 as a core molecule of the complement system effectively participates in non-specific cell immunity. After S. agalactiae infects fish, the C3 molecule is decomposed into C3a and C3b, and the inactivated iC3b can combine with CR3 (CD11b/CD18) to enhance the phagocytosis of monocytes/macrophages. The C3 molecule also modulates the inflammatory response to defend against S. agalactiae infection.

Animals and Sample Acquisition
Nile tilapia used in this study were obtained from the Guangdong Tilapia Breeding Farm [45,55]. The weight of fish was 70-100 g for tissue collection and 250-300 g for the isolation of Nile tilapia MO/Mø [45,55]. The Nile tilapia were cultured in a constant temperature semi-automatic circulatory system in the School of Life Sciences, South China Normal University for at least three weeks. The water temperature was stabilized at about 25-26 • C, pH was controlled approximately at 7.0, and the visibility was dominated at 25-30 cm. The oxygen solubility, nitrite contents, along with hydrogen sulfide were detected and adjusted regularly. In all experimental processes, the regulation of the South China Normal University Institutional Animal Care and Use Committee (reference number 2019-02-0016), as well as the ethics of medical laboratory animals were strictly adhered [45,55].
Challenges and sampling methods refer to published articles, and the brief steps are as follows [56]. During the experiments, the Nile tilapia were anesthetized with 40 mg/L MS-222 (Aladdin, Los Angeles, CA, USA). In this study, liver, brain, head kidney, thymus, spleen, skin, intestine, hind kidney, peripheral blood, and muscle were collected for tissue distribution patterns while only liver, spleen, and head kidney were acquired in challenge experiments. The collected tissues of each group were quick-frozen in liquid nitrogen and then transferred to −80 • C for cryopreservation. The challenge groups were stimulated by intraperitoneal injection of 100 µL live S. agalactiae or A. hydrophila (1 × 10 7 CFU/mL), while the control group was treated with 100 µL 10 mM sterile PBS. Three Nile tilapia were selected for each group of experiments to obtain samples. The time of obtaining sample was 0 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h after challenge.

Total RNA Extraction
The total RNA of Nile tilapia tissue and cell samples were extracted with Trizol Reagent (Thermo, Waltham, MA, USA) according to the instructions. The tissue samples mixed with 200 µL Trizol were ground to homogeneity with an electric homogenizer, and then the Trizol was added to a final volume of 1 mL. The mixture was then placed on ice for 10 min and centrifuged at 12,000 rpm for 15 min at 4 • C. Then, the supernatant was mixed with 450 µL of chloroform in a new centrifuge tube. The above procedure was then repeated for standing and centrifugation. The upper organic phase was mixed with 600 µL of isopropanol in a new centrifuge tube. After standing and centrifugation, the bottom RNA pellet was collected. RNA samples were then washed with 75% ethanol solution twice. The resulting total RNA was confirmed by 1% agarose gel electrophoresis and the RNA concentration was determined using a Nanodrop 2000 spectrophotometer (Thermo, USA). All RNA samples were stored at −80 • C until use [57].

cDNA Synthesis
The cDNA template synthesis was performed with Hifair ® II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (YEASEN, Shanghai, China), and the brief steps were as follows. The reaction contained 1 µg of total RNA, 2.0 µL of 5 × gDNA digestion buffer, and 1.0 µL of gDNA digestion, and these were finally added to a total volume of 10 µL with RNase-free ddH 2 O. The mixture was maintained at 42 • C for 2 min. Then, 2 µL primescript RT enzyme mix, 1 µL RT primer mix, and 2.0 µL 5 × Hifair ® II (YEASEN, Shanghai, China) Buffer plus were mixed with the reaction solution from the first step, then RNase-free ddH 2 O was added to bring the total volume to 20 µL. After that, the mixture was maintained at 25 • C for 5 min, 42 • C for 30 min, and 85 • C for 5 min. Prepared cDNA templates were stored at −20 • C until use. The cDNA template was diluted to a total volume of 200 µL with RNase-free ddH 2 O before being used for RT-qPCR.

Amplification and Identification of OnC3
The predicted OnC3 sequence from NCBI (https://www.ncbi.nlm.nih.gov/ (accessed on 19 November 2020)) was acquired with XP_019203045.1. The sequencing primers were designed with Primer Premier 5 based on the predicted sequence and synthesized by BGI, China. All primers used in this study are listed in Table 1. The OnC3 ORF was confirmed by polymerase chain reaction (PCR). The PCR reaction volume was 25 µL, consisting of 12.5 µL LA Taq Mix (Takara, Japan), 7.5 µL ddH 2 O, 3 µL cDNA template, 1 µL forward primer, and 1 µL reverse primer. The PCR reaction process contained 5 steps, pre-denaturation (95 • C) for 5 min, denaturation (95 • C) for 1 min, annealing (50 • C) for 30 s, extension (72 • C) for 2 min, and final extension (72 • C) for 5 min. In those steps, denaturation, annealing, and extension were performed for 35 cycles. After amplification, the PCR product was detected with 1% agarose gel electrophoresis and sequenced by BGI, China.

Bioinformatics Analysis of OnC3
In this study, the multiple sequence alignment and identity of C3 were carried out with Blast X. The nucleotide sequence of OnC3 was translated into amino acid sequence with ExPASy tools (http://expasy.org/tools/ (accessed on 14 May 2021)) and the protein domain was analyzed with the Simple Modular Architecture Research Tool (SMART) (http: //smart.embl-heidelberg.de/ (accessed on 14 May 2021)). The signal peptide was predicted with SignalP 4.1 (http://www.cbs.dtu.dk/services-/SignalP/ (accessed on 14 May 2021)). The phylogenetic tree was constructed with MEGA 7 based on the neighbor-joining (NJ) algorithm. The structure of OnC3 was predicted by the server equipped with A100 GPU. The Github source code from AlphaFold2.0 (https://github.com/deepmind/alphafold/ (accessed on 23 December 2021)) [58].

Real-Time Quantitative PCR
The expression of OnC3 transcripts in the tissues was performed with QuantStudio 5 Real-time PCR system (ABI, Los Angeles, CA, USA). The volume of the reaction was 20 µL, including 10 µL Hieff ® qPCR SYBR Green Master Mix (YEASEN, China), 2 µL forward primers, 2 µL reverse primers, 3 µL cDNA template, and 4 µL ddH 2 O. The program was a two-step method comprising pre-denaturation (95 • C) for 5 min, denaturation (95 • C) for 10 s, and annealing/extension (60 • C) for 30 s. The denaturation and annealing/extension were performed for 40 cycles. The melt curve was acquired in the last stage of RT-qPCR. Nile tilapia β-actin was selected as the internal reference for calculating the relative expression of target genes based on the 2 −∆∆CT method [59]. In this research, all experiments were repeated three times.

Isolation of Nile Tilapia Head Kidney MO/Mø
The isolation of Nile tilapia head kidney MO/Mø was developed based on the references [60,61]. The head kidneys were obtained from healthy fish and mashed into tissue homogenate in sterile dishes with RPMI 1640 medium. The cell suspensions were obtained and gently loaded into the separation liquid, which consisted of 10 mL 54% percoll (Sigma, St. Louis. MO, USA) blanketed with 10 mL of 31% percoll. After that, the separation liquid was centrifuged at 4 • C, 500× g for 40 min, then the MO/Mø fraction of the 31-54% interface was collected and washed three times with RPMI 1640 medium. The cell concentration was adjusted to 5 × 10 6 cells/mL by the L-15 medium with 1% penicillin/streptomycin and 10% FBS. The isolated cells were cultured in 96-well plates (100 µL/well) (Thermo, USA) and incubated at 25 • C. After 3 days, the non-adherent cells were removed. The adherent Nile tilapia MO/Mø were resuspended and washed with 10 mL 1640 medium 3 times. The cells were diluted to 1 × 10 6 cells/mL and incubated in 96-well plates for experiments.
In order to explore the expression pattern of OnC3 in MO/Mø after bacterial challenge, the head kidney (1 × 10 6 cells/mL) cells were challenged with formalin inactivated S. agalactiae or A. hydrophila (1 × 10 7 CFU/mL) while the control was treated with sterile PBS (10 mM). After stimulation, the cell samples were collected at 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h.

Expression and Purification of (r)OnC3
The β chain of OnC3 was amplificated and inserted into the E. coli expression vector Trx-pET-32a (Trx). The recombinant Trx-OnC3 construct was transformed into E. coli. BL21 (DE3) competent cells and coated on LB medium plates consisting of 1% ampicillin. On the next day, the positive clones were selected, identified with PCR reaction, and sequenced by BGI, China. The cells were cultured with 250 mL LB-ampicillin medium until the OD value at 600 nm was 0.6-1.0. Then, the bacteria liquid was induced with 1 mM isopropyl β-D-thiogalactoside (IPTG) at 37 • C for 6 h. After that, the target recombinant protein was purified with His-Band Resin columns (Novagen, Darmstadt, Germany) according to the prot • Cols. Finally, the purified protein was detected by 10% SDS-PAGE. The Trx vector protein was prepared through the same methods for the follow-up experiments [45,55]. preparation and analysis were performed according to publications [45]. The obtained Nile tilapia liver and spleen were immediately fixed with 4% paraformaldehyde solution at 4 • C overnight. The fixed samples were sent to Servicebio Biological Technology Co., Ltd. (Wuhan, China), for histopathological analysis by H&E staining. Three Nile tilapia were randomly selected from each group for pathological statistical analysis. Scoring was performed by two blinded investigators. The liver and spleen injury score were recorded via a scale of 0 to 3 (normal 0, mild 1, moderate 2, severe 3) according to the inflammatory cell infiltration, cell necrosis, and tissue vacuolation [63,64].

Determination of ROS in MO/Mø
In this research, MO/Mø were isolated and cultured using the method in Section 4.7. The detection of reactive oxygen species (ROS) was carried out by the ROS Assay Kit of Beyotime Biotechnology [65]. The DCFH-DA was diluted with serum-free culture medium at 1:1000 to make the final concentration 10 µmol/L. After removing the cell culture medium, the cells (1 × 10 7 cells/mL) were treated with 200 µL DCFH-DA and incubated at 37 • C for 20 min. Cells were washed three times by serum-free cell culture medium. The MO/Mø in the experimental group was treated with 200 µL (r)OnC3 protein (50 µg/mL). The other groups were treated with 200 µL sterile PBS (10 mM) or Trx protein (50 µg/mL). After 30 min of stimulation at 37 • C, the DCF fluorescence was detected by FACS Aria III (BD, New York, NY, USA) flow cytometer with an argon-ion laser of 488 nm. A total of 10,000 individual cells were analyzed in each of the samples.

Detection of Phagocytosis-Related Receptors and Enzyme Expression
In Section 4.9, except for the liver and spleen, head kidney tissue was taken from all in vivo stimulation groups and stored at −80 • C according to the previous steps. Similarly, the cell samples were stimulated and collected as in Section 4.9. The mRNA expressions of phagocytosis-related receptors (CD11b, CD18), enzymes (RhoA, ROCK), and lysosomerelated factors (EEA, M6PR, and vATPase) in the head kidney and head kidney MO/Mø were detected at time of 6 h after challenge. The RNA extraction, cDNA synthesis, and RT-qPCR detection were performed according to the above sections.

Assay of Nile Tilapia Head Kidney MO/Mø Phagocytosis
Before experiments, the Nile tilapia head kidney MO/Mø and S. agalactiae (1 × 10 8 CFU/mL) labeled with fluorescein isothiocyanate (FITC) (Sigma, USA) were prepared while the forward scatter (FSC) and side scatter (SSC) were measured [56,57]. After that, 100 µL Trx (50 µg/mL) or (r)OnC3 (50 µg/mL) were added to 300 µL labeled bacteria and incubated avoid light for 1 h. The control group was treated with 100 µL sterile PBS (10 mM). Then, the suspensions were centrifuged, and the bacteria were resuspended with 300 µL PBS and 300 µL cell suspension (2 × 10 7 cells/mL) and incubated for 60 min with shaking. The solution above was centrifuged with over 3% BSA in PBS supplemented at 100× g, 4 • C for 10 min to eliminate the non-ingested bacteria. The extra fluorescence was quenched by mixing 1 µL trypan blue (0.4%). Finally, the cell suspension was detected by FACS Aria III (BD, USA) flow cytometer with a 488 nm argon-ion laser [61]. A total of 10,000 individual cells were analyzed in each of the samples.

Statistical Analysis
All data in the present research were repeated three times and shown as mean ± standard deviation (SD). Then, the data were analyzed by one-way ANOVA with SPSS 17.0. The statistical significance was indicated by letters and asterisks (* 0.01 ≤ p < 0.05, ** p < 0.01). The figures were made with GraphPad Prism 7 (GraphPad, San Diego, CA, USA).