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.

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.

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.

Transformer-based ozone multivariate prediction considering interpretable and priori knowledge: A case study of Beijing, China.

Ozone pollution is the focus of current environmental governance in China and high-quality prediction of ozone concentration is the prerequisite to effective policymaking. The studied ozone pollution time series exhibits distinct seasonality and secular trends and is associated with various factors. This study developed an interpretable hybrid model by combining STL decomposition and the Transformer (STL-Transformer) with the prior information of ozone time series and global multi-source information as prediction basis. The STL decomposition decomposes ozone time series into trend, seasonal, and remainder components. Then, the three components, along with other air quality and meteorological data, are integrated into the input sequence of the Transformer. The experiment results show that the STL-Transformer outperforms the other five state-of-the-art models, including the standard Transformer. Specially, the univariate forecasting for ozone relies on mimicking the patterns and trends that have occurred in the past. In contrast, multivariate forecasting can effectively capture complex relationships and dependencies involving multiple variables. The method successfully grasps the prior and global multi-source information and simultaneously improves the interpretability of ozone prediction with high precision. This study provides new insights for air pollution forecasting and has reliable theoretical value and practical significance for environmental governance.

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.

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.

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.

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.

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.

Purification and functional characterization of serum transferrin from Nile tilapia (Oreochromis niloticus).

Transferrin (TF), an iron-binding multifunctional protein, could participate in the iron-withholding strategy, an effective antimicrobial defense mechanism in innate immunity, and is involved in host defense against pathogenic infection. In this study, a TF homologue (OnTF) was purified from serum of Nile tilapia (Oreochromis niloticus) through a two-step affinity chromatography, and characterized its antibacterial function and the role in inflammatory response. The identification by mass spectrometry showed that peptide sequence of the purified OnTF was highly consistent with its amino acids sequence, containing two conserved iron binding lobes: N-lobe and C-lobe. The native OnTF was able to bond iron ions, and possessed capability to inhibit the growth of both bacterial pathogens (Streptococcus agalactiae and Aeromonas hydrophila) in vitro. Upon infections of S. agalactiae and A. hydrophila, the expression of OnTF protein was significantly up-regulated in vivo and in vitro. In addition, the OnTF participated in the regulation of inflammation, migration, and enhancement of phagocytosis and respiratory burst activity in head kidney macrophages/monocytes. Taken together, the results of this study indicated that OnTF is likely to involve in innate immunity to play a role in host defense against bacterial infection in Nile tilapia.

Identification and characterization of a B-type mannose-binding lectin from Nile tilapia (Oreochromis niloticus) in response to bacterial infection.

Lectins are a group of carbohydrate-binding proteins, which play an important role in innate immune system against pathogen infection. In this study, a B-type mannose-binding lectin (OnBML) was identified from Nile tilapia (Oreochromis niloticus), and characterized at expression patterns against bacterial infection and capability to promote phagocytosis by macrophages. The open reading frame of OnBML is 354 bp of nucleotide sequence encoding polypeptides of 117 amino acids. The deduced protein is highly homologous to other teleost BMLs, containing two repeats of the conserved mannose-binding motif QXDXNXVXY. Expression of OnBML was widely exhibited in all examined tissues, with the most abundance in spleen and following gill, peripheral blood, and head kidney. The OnBML expressions were significantly up-regulated following two major bacterial infections including a Gram-positive bacterium (Streptococcus agalactiae) and a Gram-negative bacterium (Aeromonas hydrophila) in vivo and in vitro. Recombinant OnBML protein possessed capacities of mannose-binding and calcium-dependent agglutination to S. agalactiae and A. hydrophila, and promoted the phagocytosis by macrophages. Taken together, the present study indicated that OnBML is likely to get involved in host defense against bacterial infection in Nile tilapia.

B cell receptor accessory molecule CD79 gets involved in response against Streptococcus agalactiae infection and BCR signaling in Nile tilapia (Oreochromis niloticus).

CD79, composed of two distinct chains called CD79a and CD79b, is a transmembrane protein that forms a B cell antigen receptor with membrane immunoglobulin, and generates a signal following antigen recognition by the B cell receptor. In this study, the CD79a (OnCD79a) and CD79b (OnCD79b) were cloned and identified from Nile tilapia (Oreochromis niloticus). The cDNA of ORF for OnCD79a and OnCD79b are 669 and 627 bp, coding 222 and 208 amino acids, respectively. The deduced protein analysis showed that both CD79a andCD79b contain an immunoreceptor tyrosine-based activation motif in their intracellular tails that used to propagate a signal in a B cell. Expression analysis revealed that both CD79a and CD79b expressed at high levels in immune tissues, such as anterior kidney and spleen, and in IgM+ B cells. Upon Streptococcus agalactiae (S. agalactiae) infection, the expressions of OnCD79a and OnCD79b were significantly up-regulated in anterior kidney and spleen. The significant up-regulations of OnCD79a and OnCD79b were also detected in leukocytes after in vitro challenge with S. agalactiae. Further, stimulations of LPS and anti-OnIgM monoclonal antibody induced significant up-regulations of OnCD79a and OnCD79b in leukocytes. Taken together, the results of this study indicated that CD79 molecule, playing roles in BCR signaling, was likely to get involved in host defense against bacterial infection in Nile tilapia.

Functional characterization of a mannose-binding lectin (MBL) from Nile tilapia (Oreochromis niloticus) in non-specific cell immunity and apoptosis in monocytes/macrophages.

Mannose-binding lectin (MBL), a soluble pattern recognition receptor, is able to recognize antigen and participate in non-specific cell immunity, such as regulation of inflammation, migration, opsonization, phagocytosis and killing, which plays an important role in innate immunity. In this study, we have investigated the contributing mechanisms and effects of MBL on the cell immunity of Nile tilapia (Oreochromis niloticus) monocytes/macrophages. The mRNA expression level of OnMBL was significantly up-regulated in monocytes/macrophages after in vitro bacterial infection (Streptococcus agalactiae and Aeromonas hydrophila). Recombinant OnMBL ((r)OnMBL) protein could participate in the regulation of inflammation, migration, and enhancement of phagocytosis and respiratory burst activity in monocytes/macrophages. Moreover, the (r)OnMBL could induce the apoptosis of monocytes/macrophages. Taken together, the results of this study indicated that OnMBL is likely to involve in immune regulation, which may play an important role in host defense of innate immunity 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.

Expression and functional characterization of transferrin in Nile tilapia (Oreochromis niloticus) in response to bacterial infection.

Transferrin (TF), an iron-binding glycoprotein, plays an important role in host defense against pathogenic infection, which inhibits the growth and proliferation of pathogens, deprives iron from invading pathogens, and activates anti-microbial responses in macrophages. In this study, a TF homologue (OnTF) was identified from Nile tilapia (Oreochromis niloticus) and characterized at expression pattern against bacterial infection and capability binding bacterial pathogens. The open reading frame of OnTF is 2118 bp of nucleotide sequence encoding polypeptides of 705 amino acids. The deduced protein is highly homology to the other species, containing two conserved iron binding lobes: N-lobe and C-lobe. Expression analysis revealed that the OnTF was extremely highly expressed in liver tissue; however, much weakly exhibited in other examined tissues including spleen and head kidney. The OnTF expression was significantly up-regulated in the liver, spleen and head kidney following infection of a Gram-positive bacterial pathogen (Streptococcus agalactiae) and a Gram-negative bacterial pathogen (Aeromonas hydrophila). The up-regulation of OnTF expression was also demonstrated in hepatocytes and macrophages in vitro stimulated with S. agalactiae and A. hydrophila. In addition, recombinant OnTF ((r)OnTF) protein possessed capability to bind both S. agalactiae and A. hydrophila in vitro. Taken together, the present study indicated that OnTF might be involved in host defense against bacterial infection in Nile tilapia.

Spleen tyrosine kinase from Nile tilapia (Oreochromis niloticus): Molecular characterization, expression pattern upon bacterial infection and the potential role in BCR signaling and inflammatory response.

Spleen tyrosine kinase (SYK), a member of non-receptor tyrosine kinase family, plays an important role in immune responses against pathogen infection, which is capable of activating B cells signaling pathway and regulating inflammatory response. In this study, Nile tilapia (Oreochromis niloticus) ortholog (OnSYK) was identified and characterized at expression pattern against bacterial infection, function in B cells activation pathway and inflammatory response. The cDNA of OnSYK ORF contained 1851 bp of nucleotide sequence encoding polypeptides of 616 amino acids. The deduced OnSYK protein was highly homologous to other species SYK, containing two SH2 domains and a TyrKc domain. Spatial mRNA expression analysis revealed that OnSYK had wide tissue distribution and was highly expressed in the liver. After challenge of Streptococcus agalactiae (S. agalactiae) in vivo, mRNA expression of OnSYK was significantly up-regulated in the head kidney, spleen and liver. The up-regulation of OnSYK transcript was also displayed in the head kidney and spleen leukocytes stimulation with S. agalactiae and LPS in vitro, which was confirmed at protein level in the head kidney leukocytes by FACS analysis. In addition, after induction with mouse anti-OnIgM monoclonal antibody in vitro, the expressions of OnSYK and its downstream molecules (OnLYN, OnBLNK and OnAP-1) were significantly up-regulated in the head kidney leukocytes, and pharmacological inhibition of SYK activity with inhibitor (P505-15) significantly attenuated the expressions of OnLYN, OnBLNK and OnAP-1. Moreover, upon LPS challenge, the expressions of OnSYK, OnTNF-α, OnIL-6 and OnAP-1 were also up-regulated in the head kidney monocytes/macrophages. After treatment with SYK inhibitor (BAY 61-3606), the expressions of OnTNF-α, OnIL-6 and OnAP-1 were inhibited in the LPS-challenged head kidney monocytes/macrophages. Taken together, the results of this study indicated that OnSYK, playing potential roles in BCR signaling and inflammatory response, was likely to get involved in host defense against bacterial infection in Nile tilapia.

Identification and characterization of a mannose-binding lectin from Nile tilapia (Oreochromis niloticus).

Mannose-binding lectin (MBL) is a pattern recognition protein that plays an important role in innate immunity capable of activating the lectin pathway of the complement system. In this study, a MBL homologue (OnMBL) was identified from Nile tilapia (Oreochromis niloticus) and characterized at expression and agglutination functional levels. The open reading frame of OnMBL is 687 bp of nucleotide sequence encoding polypeptides of 228 amino acids. The deduced amino acid sequence is highly homology to teleost and similar to mammalian MBL, containing a canonical collagen-like region, a carbohydrate recognition domain and a neck region. Expression analysis revealed that the OnMBL was highly expressed in the liver, and also exhibited in other tissues including hind kidney, intestines, head kidney and spleen. In addition, the OnMBL expression was significantly up-regulated in spleen and head kidney following challenges with a Gram-positive bacterial pathogen (Streptococcus agalactiae) and a Gram-negative bacterial pathogen (Aeromonas hydrophila). Recombinant OnMBL ((r)OnMBL) protein was able to agglutinate both S. agalactiae and A. Hydrophila in vitro. Taken together, the results of this study indicated that OnMBL, possessing apparent agglutination ability to bacterial pathogens, might be involved in host defense against bacterial infection in Nile tilapia.

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.

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.