Collectin-K1 Plays a Role in the Clearance of Streptococcus agalactiae in Nile Tilapia (Oreochromis niloticus).

Collectin-K1 (CL-K1) is a multifunctional C-type lectin that has been identified as playing a crucial role in innate immunity. It can bind to carbohydrates on pathogens, leading to direct neutralization, agglutination, and/or opsonization, thereby inhibiting pathogenic infection. In this study, we investigated a homolog of CL-K1 (OnCL-K1) in Nile tilapia (Oreochromis niloticus) and its role in promoting the clearance of the pathogen Streptococcus agalactiae (S. agalactiae) and enhancing the antibacterial ability of the fish. Our analysis of bacterial load displayed that OnCL-K1 substantially reduced the amount of S. agalactiae in tissues of the liver, spleen, anterior kidney, and brain in Nile tilapia. Furthermore, examination of tissue sections revealed that OnCL-K1 effectively alleviated tissue damage and inflammatory response in the liver, anterior kidney, spleen, and brain tissue of tilapia following S. agalactiae infection. Additionally, OnCL-K1 was found to decrease the expression of the pro-inflammatory factor IL-6 and migration inhibitor MIF, while increasing the expression of anti-inflammatory factor IL-10 and chemokine IL-8 in the spleen, anterior kidney, and brain tissues of tilapia. Moreover, statistical analysis of survival rates demonstrated that OnCL-K1 significantly improved the survival rate of tilapia after infection, with a survival rate of 90%. Collectively, our findings suggest that OnCL-K1 plays a vital role in the innate immune defense of resisting bacterial infection in Nile tilapia. It promotes the removal of bacterial pathogens from the host, inhibits pathogen proliferation in vivo, reduces damage to host tissues caused by pathogens, and improves the survival rate of the host.

CL-K1 Promotes Complement Activation and Regulates Opsonophagocytosis of Macrophages with CD93 Interaction in a Primitive Vertebrate.

Collectin is a crucial component of the innate immune system and plays a vital role in the initial line of defense against pathogen infection. In mammals, collectin kidney 1 (CL-K1) is a soluble collectin that has recently been identified to have significant functions in host defense. However, the evolutionary origins of immune defense of CL-K1 and its mechanism in clearance of pathogenic microorganisms remain unclear, especially in early vertebrates. In this study, the Oreochromis niloticus CL-K1 (OnCL-K1) protein was purified and identified, which was capable of binding to two important pathogens of tilapia, Streptococcus agalactiae and Aeromonas hydrophila. Interestingly, OnCL-K1 exhibited direct bactericidal activity by binding to lipoteichoic acid or LPS on cell walls, disrupting the permeability and integrity of the bacterial membrane in vitro. Upon bacterial challenge, OnCL-K1 significantly inhibited the proliferation of pathogenic bacteria, reduced the inflammatory response, and improved the survival of tilapia. Further research revealed that OnCL-K1 could associate with OnMASPs to initiate and regulate the lectin complement pathway. Additionally, OnCD93 reduced the complement-mediated hemolysis by competing with OnMASPs for binding to OnCL-K1. More importantly, OnCL-K1 could facilitate phagocytosis by collaborating with cell surface CD93 in a lectin pathway-independent manner. Moreover, OnCL-K1 also promoted the formation of phagolysosomes, which degraded and killed ingested bacteria. Therefore, this study reveals the antibacterial response mechanism of CL-K1 in primitive vertebrates, including promoting complement activation, enhancing opsonophagocytosis, and killing of macrophages, as well as its internal links, all of which provide (to our knowledge) new insights into the understanding of the evolutionary origins and regulatory roles of the collectins in innate immunity.

Long-chain pentraxin 3 possesses agglutination activity and plays a role in host defense against bacterial infection in Oreochromis niloticus.

Pentraxin 3 (PTX3) is a soluble pattern recognition molecule in the innate immune system that has multiple functions. It is involved in resisting pathogen infection. However, the functions of PTX3 in teleost fish are not well understood. In this study, we identified and characterized PTX3 in Nile tilapia (Oreochromis niloticus) (OnPTX3). The open reading frame of OnPTX3 was found to be 1305 bp, encoding 434 aa. We conducted spatial mRNA expression analysis and found that the expression of OnPTX3 was significantly increased after infection with Streptococcus agalactiae and Aeromonas hydrophila, both in vivo and in vitro. We also observed that recombinant OnPTX3 protein could bind and agglutinate bacterial pathogen. Furthermore, OnPTX3 enhanced the phagocytosis of bacteria (S. agalactiae and A. hydrophila) by head kidney macrophages. Additionally, OnPTX3 was found to influence the expression of inflammatory cytokines, suggesting its involvement in the regulation of the inflammatory response. Moreover, OnPTX3 was shown to promote complement-mediated hemolysis and possess antibacterial activity. In conclusion, our research demonstrates that OnPTX3 has bacterial binding and agglutination activities, enhances phagocytosis, and regulates inflammation. It plays a crucial role in the defense of Nile tilapia against pathogenic bacteria, providing valuable insights for the prevention and control of aquatic diseases in the future.

Mannose-binding lectin suppresses macrophage proliferation through TGF-ß1 signaling pathway in Nile tilapia.

Mannose-binding lectin (MBL) is a multifunctional pattern recognition molecule, which not only mediates the recognition of pathogenic microorganisms and their products, playing an important role in innate immune defense, but also participates in adaptive immune responses of mammalian. However, it's related immune mechanism remains limited, especially the regulation of cell proliferation in early vertebrates. In this study, OnMBL was found to bind to kidney macrophages (MФ) from Nile tilapia (Oreochromis niloticus). Interestingly, OnMBL was able to reduce the proliferation of activated-MФ by regulating the cell cycle, arresting a large number of cells in the G0/G1 phase, and increasing the probability of apoptosis. More importantly, we found that the inhibition of cell proliferation by OnMBL was closely related to the evolutionarily conserved canonical transforming growth factor-beta 1 (TGF-ß1) signaling pathway. Mechanistically, OnMBL could significantly increase the expression of TGF-ß1, activate and regulate the downstream Smad-dependent pathway to reduce the MФ proliferation, thereby maintaining cellular homeostasis in the body's internal environment. This study represents the first description regarding the regulatory mechanisms of the MBL on cell proliferation in teleost fish, which provides a novel perspective on the understanding of the multiple function and evolutionary origins of C-type lectins in the immune system.

C9 regulates the complement-mediated cell lysis in association with CD59 to resist bacterial infection in a primary animal.

Complement component 9 (C9), as an essential component of terminal membrane attack complex of complement system, plays an important role in innate immune defense. However, the function and regulatory mechanism of C9 in the antimicrobial immune response of teleost fish remain unclear. In this study, the open reading frame of Nile tilapia (Oreochromis niloticus) C9 (OnC9) gene was amplified. The mRNA and protein expression of OnC9 were significantly changed upon infection with Streptococcus agalactiae and Aeromonas hydrophila in vivo and in vitro. Upon bacterial challenge, the OnC9 knockdown could lead to rapid proliferation of the pathogenic bacteria, ultimately resulting in tilapia death. However, the phenotype was rescued by re-injection of OnC9, which restored the healthy status of the knockdown tilapia. Further, the OnC9 was an essential component in complement-mediated cell lysis and associated with OnCD59 to regulate the efficiency of lysis. Overall, this study indicates that OnC9 is involved in host defense against bacterial infection, and provides a valuable reference for further exploration of the molecular regulatory mechanism of C9 in innate immune defense in a primary animal.

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.

Functional Characterization of Serum Amyloid P Component (SAP) in Host Defense against Bacterial Infection in a Primary Vertebrate.

Serum amyloid P component (SAP), an ancient short pentraxin of the pentraxin family, plays an essential role in resistance to bacterial infection. In this study, the expression and functional characterization of SAP (OnSAP) in Nile tilapia (Oreochromis niloticus), a primary vertebrate, are investigated. The open reading frame of OnSAP is 645 bp of a nucleotide sequence encoding a polypeptide of 214 amino acids. As a calcium-binding protein, the structure and relative motif of OnSAP is highly similar to those of humans, containing amino acid residues Asn, Glu, Gln and Asp. In healthy fish, OnSAP mRNA is extensively distributed in all eleven tissues examined, with the highest level in spleen. The mRNA expression of OnSAP was significantly up-regulated after being challenged with gram-positive bacterium Streptococcus agalactiae and gram-negative bacterium Aeromonas hydrophila in vivo. In addition, recombinant OnSAP ((r)OnSAP) protein had capacities of binding S. agalactiae or A. hydrophila in the presence of Ca2+. Further, (r)OnSAP helped monocytes/macrophages to efficiently phagocytize bacteria. Moreover, the (r)OnSAP was able to enhance the complement-mediated lysis of the chicken red blood cells. Collectively, the evidence of SAP in tilapia, based on the results including its evolutionary conserved protein structure, bacterial binding and agglutination, opsonophagocytosis of macrophage and hemolysis enhancement, enriches a better understanding of the biological functions of the pentraxin family.

Affinity-Driven Site-Specific High Mannose Modification Determines the Structural Polymerization and Function of Tetrameric IgM in a Primitive Vertebrate.

Teleost tetramer IgM is the predominant Ig in the immune system and plays essential roles in host defense against microbial infection. Due to variable disulfide polymerization of the monomeric subunits, tetrameric IgM possesses considerable structural diversity. Previous work indicated that the teleost IgM H chain was fully occupied with complex-type N-glycans. However, after challenge with trinitrophenyl (TNP) Ag, the complex N-glycans in the Asn-509 site of Oreochromis niloticus IgM H chain transformed into high mannose. This study, therefore, was conducted to examine the functional roles of the affinity-related high-mannose modification in tilapia IgM. The TNP-specific IgM Ab affinity maturation was revealed in tilapia over the response. A positive correlation between TNP-specific IgM affinity and its disulfide polymerization level of isomeric structure was demonstrated. Mass spectrometric analysis indicated that the relationship between IgM affinity and disulfide polymerization was associated with the Asn-509 site-specific high-mannose modification. Furthermore, the increase of high mannose content promoted the combination of IgM and mannose receptor (MR) on the surface of phagocytes. Moreover, the increased interaction of IgM and MR amplified the phagocytic ability of phagocytes to Streptococcus agalactiae. To our knowledge, this study demonstrates that site-specific high-mannose modification associates with IgM Ab affinity and its structural disulfide polymerization and amplifies the phagocytosis of phagocytes by the combination of IgM and MR. The present study provides evidence for understanding the association of IgM structure and function during the evolution of the immune system.

Expression and functional characterization of the mannose receptor (MR) from Nile tilapia (Oreochromis niloticus) in response to bacterial infection.

Mannose receptor (MR) as a member of the pattern recognition receptors (PRRs) plays an important role in the immune response. In mammals, the role of MR in the regulation of phagocytosis is clarified; however, its contribution to opsonize phagocytosis remains unclear in bony fish. In this study, the expression pattern of Nile tilapia mannose receptor gene (OnMR) was investigated and its regulation of the phagocytosis of monocytes/macrophages to pathogenic bacteria was identified. The full-length of OnMR open reading frame is 4314 bp, encoding a peptide containing 1437 amino acid residues. The deduced amino acid sequence revealed that OnMR contained a cysteine-rich domain, a fibronectin type II domain, multiple C-type lectin-like domains, a transmembrane domain and a short cytoplasmic domain. Tissue distribution analysis showed the OnMR transcripts was widely distribute in the ten detected tissues, and highly expressed in head kidney, hind kidney, intestine and spleen. After S. agalactiae and A. hydrophila infection, the expression of OnMR in head kidney and spleen increased significantly. Moreover, the expression of OnMR in MO/Mø were also upregulated post the infection of bacteria and mannose solutions in vitro. This suggested that MR, as a mannose receptor on macrophage surface, could respond strongly to the stimulation of pathogenic bacteria. In addition, the (r)OnMR protein could effectively bind and agglutinate S. agalactiae and A. hydrophila, and regulate the phagocytic ability of monocytes/macrophages to pathogenic bacteria. These results suggest that OnMR is involved in response against bacterial infection in Nile tilapia, and this study will help us better understand the function of MR in teleost fish.

An l-rhamnose-binding lectin from Nile tilapia (Oreochromis niloticus) possesses agglutination activity and regulates inflammation, phagocytosis and respiratory burst of monocytes/macrophages.

Rhamnose-binding lectins (RBLs), a Ca2+-independent lectin family, are widely present in vertebrates and invertebrates, which involve in the innate immune response. However, the functional characterization and related regulation mechanisms of RBLs remain unclear in teleost fish. In this study, an l-rhamnose-binding lectin-like (OnRBL-L) was identified and functionally characterized from Nile tilapia (Oreochromis niloticus). The open reading frame of OnRBL-L is 678 bp encoding 225 aa. The sequence of OnRBL-L has relatively conservative characteristic peptide motifs, including YGR, DPC, and KYL-motif. Expression analysis showed that OnRBL-L was abundantly distributed in intestine tissue, and widely existed in all detected tissues. Meanwhile, the expression of OnRBL-L increased significantly in vivo (liver, spleen, head kidney, intestine, gills and peripheral blood) and in vitro (monocytes/macrophages) following challenges with two important tilapia pathogenic bacteria Streptococcus agalactiae and Aeromonas hydrophila. In addition, the recombinant OnRBL-L was found to bind and agglutinate S. agalactiae and A. hydrophila. Furthermore, OnRBL-L could participate in non-specific cellular immune defense, including reducing the expression of pro-inflammatory factors (IL-6、IL-8 and TNF-α), and enhancement of the phagocytosis and respiratory burst of MO/MФ. Overall, our results provide new insights into the understanding of RBL as an important pattern recognition molecule and regulator in non-specific cell immunity in an early vertebrate.

Hemopexin as an acute phase protein regulates the inflammatory response against bacterial infection of Nile tilapia (Oreochromis niloticus).

Hemopexin, a high affinity heme-binding protein is widely involved in variety physiological and pathological processes. It is an important acute phase response protein, and is important in regulating the inflammatory response. In this study, the open reading frame of Nile tilapia hemopexin (OnHpx) gene was amplified. The expression pattern of OnHpx in natural and bacterial challenged tilapia tissues were analyzed through RT-qPCR. The results indicated the OnHpx was most abundant in liver, and increased significantly in liver, spleen, head kidney and peripheral blood after bacterial challenge. Furthermore, the OnHpx mRNA was also significantly up-regulated in monocytes/macrophages and hepatocytes under the stimulation of S. agalactiae or A. hydrophila. In addition, the recombinant OnHpx protein could effectively reduce the bacteria proliferation and alleviate the inflammatory reaction caused by bacteria. Moreover, the (r)OnHpx also regulated the respiratory burst of monocytes/macrophages and played an important role in the antioxidant process. To our knowledge, these results provide the first evidence on the antibacterial and anti-inflammatory response mechanism of Hpx in early vertebrates. This brings new insights about the understanding of the evolutionary origins and ancient roles of the Hpx in the innate immune defense.

Mannose-Binding Lectin Possesses Agglutination Activity and Promotes Opsonophagocytosis of Macrophages with Calreticulin Interaction in an Early Vertebrate.

The innate immune system is an ancient defense system in the process of biological evolution, which can quickly and efficiently resist pathogen infection. In mammals, mannose-binding lectin (MBL) is a key molecule in the innate immune and plays an essential role in the first line of host defense against pathogenic bacteria. However, the evolutionary origins and ancient roles of immune defense of MBL and its mechanism in clearance of microbial pathogens are still unclear, especially in early vertebrates. In this study, Oreochromis niloticus MBL (OnMBL) was successfully isolated and purified from the serum of Nile tilapia (O. niloticus). The OnMBL was able to bind and agglutinate with two important pathogens of tilapia, Streptococcus agalactiae and Aeromonas hydrophila Interestingly, the OnMBL was able to significantly inhibit the proliferation of pathogenic bacteria and reduce the inflammatory response. Upon bacterial challenge, the downregulation of OnMBL expression by RNA interference could lead to rapid proliferation of the pathogenic bacteria, ultimately resulting in tilapia death. However, the phenotype was rescued by reinjection of the OnMBL, which restored the healthy status of the knockdown tilapia. Moreover, a mechanistic analysis revealed that the OnMBL could clear pathogenic bacteria by collaborating with cell-surface calreticulin to facilitate phagocytosis in a complement activation-independent manner. To our knowledge, these results provide the first evidence on the antibacterial response mechanism of MBL performing evolutionary conserved function to promote opsonophagocytosis of macrophages in early vertebrates and reveals new insights into the understanding of the evolutionary origins and ancient roles basis of the C-type lectins in the innate immune defense.

MAp34 Regulates the Non-specific Cell Immunity of Monocytes/Macrophages and Inhibits the Lectin Pathway of Complement Activation in a Teleost Fish.

The lectin pathway of the complement system is one of the main components of innate immunity, which plays a pivotal role in the defense against infectious microorganisms and maintains immune homeostasis. However, its control mechanisms remain unclear in teleost fish. In this study, we described the identification and functional characterization of a mannose-binding lectin associated protein MAp34 (OnMAp34) from Nile tilapia (Oreochromis niloticus) at molecular, cellular, and protein levels. The open reading frame (ORF) of OnMAp34 is 918 bp of nucleotide sequence encoding a polypeptide of 305 amino acids. The deduced amino acid sequence has three characteristic structures, including two C1r/C1s-Uegf-BMP domains (CUB) and one epidermal growth factor domain (EGF). Expression analysis revealed that the OnMAp34 was highly expressed in the liver and widely existed in other examined tissues. In addition, the mRNA and protein expression levels of OnMAp34 were remarkably altered upon infection with Streptococcus agalactiae and Aeromonas hydrophila in vivo and in vitro. Further, we found that the OnMAp34 could participate in the non-specific cellular immune defense, including the regulation of inflammation, migration, and enhancement of phagocytosis of monocytes/macrophages. Moreover, the OnMAp34 could compete with OnMASPs to combine OnMBL and inhibit the lectin pathway of complement activation. Overall, our results provide new insights into the understanding of MAp34 as a potent regulator in the lectin complement pathway and non-specific cell immunity in an early vertebrate.

Relative content detection of oligomannose modification of IgM heavy chain induced by TNP-antigen in an early vertebrate through nanoLC-MS/MS.

N-glycan modification is reported to be important in regulating the structure and function of immunoglobulins in mammals. While, the study on teleost immunoglobulin glycosylation is still limitted. In this study, we constructed a TNP-antigen driven model, and detected the site-specific N-glycans of PBS-immunized and TNP-specific Oreochromis niloticus serum IgM through 18O-labeling and nanoLC-MS/MS. These methods are widely used for peptide enrichment and protein modification identification, but rarely used in detecting the level of N-glycosylation in teleost Igs that driven by specific antigen. The results revealed that there are four N-glycosylation sites in O.niloticus IgM heavy chain, namely, the Asn-315 site in the CH2 domain, the Asn-338 site in the CH3 domain, and the Asn-509 and Asn-551 sites in the CH4 domain, All of the four residues were efficiently N-glycosylated. After immunized with TNP-antigen, the signal strength of oligomannose in the TNP-specific IgM in primary mass spectrometry was significantly higher than that in the PBS-immunized IgM. Notably, the TNP-specific IgM had an Asn-509 site fully occupied with oligomannose, while only a small amount of oligomannose was found in the PBS-immunized IgM of this site. N-glycans in other sites were mainly complex-type with a low content of fucosylation and sialylated. The oligomannose in TNP-specific IgM was further verified to be essential for the binding of IgM and MBL. These results demonstrated that the TNP-antigen induced the site-specific oligomannose modification of O.niloticus IgM heavy chain, and played an important role in the interaction of IgM and MBL, which provided insights into the evolutionary understanding of the IgM oligomannose modification and function.

A microfibril-associated glycoprotein 4 (MFAP4) from Nile tilapia (Oreochromis niloticus) possesses agglutination and opsonization ability to bacterial pathogens.

Microfibril-associated glycoprotein 4 (MFAP4), a pattern recognition-like molecule with a fibrinogen-like domain (FBG), has the ability to combine and agglutinate pathogens, playing an essential role in the first line of innate immune defense. In this study, the sequence of Nile tilapia (Oreochromis niloticus) microfibril-associated glycoprotein 4 (OnMFAP4) open reading frame (ORF) was amplified and identified. The ORF of OnMFAP4 is 720 bp of nucleotides and codes for 239 amino acids. Spatial mRNA encoding analysis indicated that OnMFAP4 was highly produced in liver, intestine and head kidney in healthy tilapia, and with the lowest expression in muscle. After challenges with Streptococcus agalactiae (S. agalactiae) and Aeromonas hydrophila (A. hydrophila), the expression of OnMFAP4 mRNA was prominently produced in the liver, spleen and head kidney. The up-regulation of OnMFAP4 expression was also presented in head kidney monocytes/macrophages (MO/MΦ) and hepatocytes. Recombinant OnMFAP4 ((r)OnMFAP4) could bind and agglutinate both bacterial pathogens. Moreover, (r)OnMFAP4 could take part in the modulation of inflammation and phagocytosis. In conclusion, this study revealed that OnMFAP4 might take effect in host defense against bacterial infection in Nile tilapia, with agglutination and opsonization capability to bacterial pathogens.

A Siglec-1-like lectin from Nile tilapia (Oreochromis niloticus) possesses functions of agglutination and mediation of macrophage phagocytic activity.

Siglec-1, one of the sialic acid-binding immunoglobulin-type lectins, is closely related to the recognition of host-pathogen and cell-cell interactions in the adaptive and innate immune systems. In this communication, a Siglec-1-like gene (OnSiglec-1-like) from Nile tilapia (Oreochromis niloticus) was analyzed. Relative expression revealed that the OnSiglec-1-like was expressed in all tested tissues, and the highest expression was found in the anterior kidney. Upon Streptococcus agalactiae (S. agalactiae) infection, the expression of OnSiglec-1-like was up-regulated in anterior kidney and spleen significantly in vivo. Additionally, the same phenomenon was observed in anterior kidney leukocytes upon LPS and S. agalactiae challenges as well in vitro. Western-blotting and ELISA analyses revealed that recombinant OnSiglec-1-like protein possessed high binding activity to LTA, LPS and S. agalactiae. Further, the recombinant OnSiglec-1-like was able to agglutinate S. agalactiae. Moreover, with the digestion of specific sialidase, the phagocytic ability of macrophages to S. agalactiae was greatly enhanced. Taken together, these results indicated that the Siglec-1-like possesses conserved functions of agglutination and promotion of macrophage phagocytic activity in Nile tilapia.

Expression and functional analysis of Nile tilapia transferrin receptors (TfRs) in host resistance to pathogenic bacteria and iron ion metabolism.

Transferrin receptors (TfRs) play an essential role in iron-withholding strategy, and are involved in immune response against bacterial infection. In this study, the transferrin receptor 1 (OnTfR1) and transferrin receptor 2 (OnTfR2) genes are identified and characterized in Nile tilapia (Oreochromis niloticus). The open reading frames of OnTfR1 and OnTfR2 are 2220 and 2343 bp of nucleotide sequence, encoding 739 and 780 amino acids, respectively. The deduced proteins of OnTfR1 and OnTfR2 are highly homologous to those of other species, containing three conserved TfR superfamily domains (PA TfR domain, M28 TfR domain and TfR dimer domain). Expression analyses of OnTfRs in the healthy tilapia reveal that the OnTfR1 and OnTfR2 transcripts are the most abundant in the liver. The in vivo studies show that the expressions of OnTfRs are significantly up-regulate in liver and spleen, following infections of Streptococcus agalactiae and Aeromonas hydrophila. In addition, the in vitro studies reveal that the up-regulations of OnTfR expressions are also significant in monocytes/macrophages and hepatocytes upon the stimulations of S. agalactiae and A. hydrophila. Moreover, the iron ion (Fe3+) could significantly increase the expressions of OnTfRs in monocytes/macrophages and hepatocytes. Taken together, the present study indicates that OnTfRs may be involved in host defense against bacterial infection and possess the function of combining or transporting iron ions in Nile tilapia.

Effects of Cell Differentiation on the Phagocytic Activities of IgM+ B Cells in a Teleost Fish.

Teleost B cells have phagocytic activities for ingesting particulate antigens, such as bacteria, in addition to the functional secretion of immunoglobulins (Igs). In the present study, the phagocytic activities of IgM+ B cells under various differentiational conditions residing in peripheral blood leukocytes were investigated in a teleost fish Nile tilapia (Oreochromis niloticus). The IgM+ B cells were recognized as IgMlo or IgMhi subsets based on their membrane IgM (mIgM) levels. The mIgM, secreted IgM (sIgM), major histocompatibility complex class II and reactive oxygen species were detected. Expressions of transcription factors (Pax5 and Blimp-1) and B cell signaling molecules (CD79a, CD79b, BLNK, and LYN) suggested that IgMlo B cells were resembling as plasma-like cells and IgMhi resembling as naïve/mature B cells, respectively. Analysis of phagocytic activities demonstrated that both IgMlo and IgMhi B cells have a similar phagocytic ability (phagocytosis percentage); however, the phagocytic capacity [phagocytic index and the mean fluorescence intensity (MFI)] of IgMhi B cells was significantly higher than that of IgMlo B cells. Taken together, the results indicated that B cell differentiation may cause the decrease of phagocytic capacity but not phagocytic ability of phagocytic IgM+ B cells in teleost. The finding may provide an evolutionary evidence for understanding the greater specialization of the B cell in more sophisticated adaptive humoral immunity, by decreasing phagocytic activity in order to contribute its function more specifically into antibody-secreting.

Molecular and functional characterization of a mannose-binding lectin/ficolin-associated protein (MAp44) from Nile tilapia (Oreochromis niloticus) involved in the immune response to bacterial infection.

The lectin pathway of the complement system has a pivotal role in the defense against infectious organisms. Mannose-binding lectin/ficolin-associated protein (MAp44), a multifunctional complement regulator, regulates the complement activation by competing with MASP-1, MASP-2 and MASP-3 for MBL and ficolin binding sites. In this study, we described the identification and functional characterization of a MAp44 homologue (OnMAp44) from Nile tilapia (Oreochromis niloticus) at molecular, cellular and protein levels. The open reading frame (ORF) of OnMAp44 is 1140 bp of nucleotide sequence encoding a polypeptide of 379 amino acids. The deduced amino acids sequence has four characteristic structures, including two C1r/C1s-Uegf-BMP domains (CUB), one epidermal growth factor domain (EGF) and one complement control protein domains (CCP). Expression analysis revealed that the OnMAp44 was highly expressed in liver, and widely existed in other examined tissues. In addition, the OnMAp44 expression was significantly up-regulated in spleen and head kidney following challenges with Streptococcus agalactiae and Aeromonas hydrophila. The up-regulations of OnMAp44 mRNA and protein expression were also observed in hepatocytes and monocytes/macrophages in vitro stimulation with S. agalactiae and A. hydrophila. Recombinant OnMAp44 protein was able to participate in the regulation of inflammation and migration reaction. Taken together, the results indicated that OnMAp44 was likely to involve in the immune response to bacterial infection in Nile tilapia.

Expression and functional characterization of a mannose-binding lectin-associated serine protease-1 (MASP-1) from Nile tilapia (Oreochromis niloticus) in host defense against bacterial infection.

Mannose-binding lectin-associated serine protease-1 (MASP-1), a multifunctional serine protease, plays an important role in innate immunity which is capable of activating the lectin pathway of the complement system and also triggering coagulation cascade system. In this study, a MASP-1 homolog (OnMASP-1) was identified from Nile tilapia (Oreochromis niloticus) and characterized at expression and inflammation functional levels. The open reading frame (ORF) of OnMASP-1 is 2187 bp of nucleotide sequence encoding a polypeptide of 728 amino acids. The deduced amino acid sequence has 6 characteristic structures, including two C1r/C1s-Uegf-BMP domains (CUB), one epidermal growth factor domain (EGF), two complement control protein domains (CCP) and a catalytic serine protease domain (SP). Expression analysis revealed that the OnMASP-1 was highly expressed in the liver, and widely exhibited in other tissues containing intestine, spleen and kidney. In addition, the OnMASP-1 expression was significantly up-regulated in spleen and head kidney following challenges with Streptococcus agalactiae and Aeromonas hydrophila. The up-regulations of OnMASP-1 mRNA and protein expression were also demonstrated in hepatocytes and monocytes/macrophages in vitro stimulation with S. agalactiae and A. hydrophila. Recombinant OnMASP-1 protein was likely to participate in the regulation of inflammatory and migration reaction by monocytes/macrophages. These results indicated that OnMASP-1, playing an important role in innate immunity, was likely to involve in host defense against bacterial infection in Nile tilapia.