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.

Genetic analysis and functional study of novel CFTR variants in Chinese children with cystic fibrosis.

OBJECTIVES: To describe the clinical characteristics of Chinese cystic fibrosis (CF) patients and to investigate the variants of CFTR and their potential pathogenicity. STUDY DESIGN: Chinese patients with potential CF diagnosis were studied. Clinical data were reviewed retrospectively from medical records. Whole exome sequencing and genetic evaluation were conducted to explore potential gene variants. The disruption of the variants to protein structure and function was explored and validated using in vitro experiments and in silico analysis. RESULTS: Four patients were recruited to the study, three of them were diagnosed as CF, and one was diagnosed as CFTR-related disorder. The age at symptom onset for the patients in this study ranged from newborn to 6 years, while the age at diagnosis varied from 3 to 11 years. All four patients exhibited bilateral diffuse bronchiectasis with Pseudomonas aeruginosa infections, and three of them had malnutrition. Finger clubbing was observed in three patients, two of whom displayed mixed ventilatory dysfunction. The CFTR variants spectrum of Chinese children with CF differs from that of Caucasian. A total of six variants were identified, two of which were first reported (c.1219G > T [p.Glu407*] and c.1367delT [p.Ala457Leufs*12]). The nonsense variants c.1219G > T, c.1657C > T and c.2551C > T and the frameshift variant c.1367delT were predicted to introduce premature stop codon and produce shorten CFTR protein, which was also first validated by in vitro truncation assay in this study. The missense variant c.1810A > C was predicted to disrupt the function of the nucleotide-binding domain 1 (NBD1) in the CFTR protein. The splicing variant c.1766 + 5G > T caused skipping of exon 13 and damaged the integrity of CFTR protein. CONCLUSIONS: Our study expands the spectrum of phenotypes and genotypes for CF of Chinese origin, which differs significantly from that of Caucasian. Genetic analysis and counseling are crucial and deserve extensive popularization for the diagnosis ofCF in patients of Chinese origin.

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.

ß-Glucans in particulate and solubilized forms elicit varied immunomodulatory and apoptosis effects in teleost macrophages in a dosedependent manner.

ß-Glucans are a group of heterogeneous glucose polymers that possess immunomodulatory activities. The complex nature of their structures, uncertainty regarding the doses, and variable immune effects pose a challenge to comprehensive understanding. In this study, we investigated the immune responses and apoptosis effects in Nile tilapia (Oreochromis niloticus) head kidney macrophages (MФ) upon exposure to two ß-Glucans (Paramylon and Laminarin) at low and high doses. Our results demonstrate that Paramylon elicits more robust immune responses than Laminarin, albeit with a dose-limiting effect. We also observed that the high-dose Paramylon induces apoptosis, whereas no such effect was detected in Laminarin treatment. Mechanistically, high-dose Paramylon activates the intrinsic apoptosis pathway, with significantly up-regulation of intrinsic apoptosis-related genes and impaired mitochondrial function. On the other hand, Laminarin triggers metabolic reprogramming in MФ, resulting in the enrichment of the metabolite α-Ketoglutarate, which protects the MФ from apoptosis. Overall, our findings highlight the importance of identifying the optimal dose range for ß-Glucans, based on sources or structures, to achieve maximal immunomodulatory effects. These results have important implications for the design and optimization of ß-Glucans-based drugs or adjuvants in immunotherapies.

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.

Major histocompatibility complex class IIα and IIß of pufferfish (Takifugu obscurus): Identification and functional characterization in response to bacterial challenge.

Classical major histocompatibility complex (MHC) class II molecules play an essential role in immune system. In this study, MHC IIα (Pf-MHC IIα) and MHC IIß (Pf-MHC IIß) homology genes from pufferfish (Takifugu obscurus) were cloned and their functional characterization in response to bacterial challenge was identified. The nucleotide sequences of the open reading frames (ORFs) of pufferfish Pf-MHC IIα and Pf-MHC IIß were 708 bp and 750 bp, encoding 235 aa and 249 aa, respectively. The structure of Pf-MHC IIα or Pf-MHC IIß contained a signal peptide, an α1/ß1 domain, an α2/ß2 domain, a transmembrane region and a cytoplasmic region. Multiple sequence alignment and phylogenetic analysis showed that Pf-MHC IIα and Pf-MHC IIß molecules had the highest similarity with Fugu rubripes (Takifugu rubripes). Cellular localization analysis indicated that the distribution of Pf-MHC IIα and Pf-MHC IIß was in the lymphocyte membrane and cytoplasm. qRT-PCR results showed that Pf-MHC IIα and Pf-MHC IIß expressed relatively high in skin, gills and gut. In addition, after stimulation challenge in vitro (lipopolysaccharide, or polyinosinic: polycytidylic acid) and in vivo (A. hydrophila), the mRNA expressions of Pf-MHC IIα and Pf-MHC IIß were significantly up-regulated in lymphocytes and in tissues of skin, gills, gut and head kidney. Moreover, Pf-MHC IIα or Pf-MHC IIß neutralization reduced the ability of A. hydrophila to induce the expressions of lymphocyte cytokines (TNF-α, IL-1ß and IL-10). Overall, it is speculated that Pf-MHC IIα and Pf-MHC IIß may play an important role in the host response against A. hydrophila in pufferfish.

Identification and Functional Analysis of Tartrate-Resistant Acid Phosphatase Type 5b (TRAP5b) in Oreochromis niloticus.

Tartrate-resistant acid phosphatase type 5 (TRAP5) is an enzyme that is highly expressed in activated macrophages and osteoclasts and plays important biological functions in mammalian immune defense systems. In the study, we investigated the functions of tartrate-resistant acid phosphatase type 5b from Oreochromis niloticus (OnTRAP5b). The OnTRAP5b gene has an open reading frame of 975 bp, which encodes a mature peptide consisting of 302 amino acids with a molecular weight of 33.448 kDa. The OnTRAP5b protein contains a metallophosphatase domain with metal binding and active sites. Phylogenetic analysis revealed that OnTRAP5b is clustered with TRAP5b of teleost fish and shares a high amino acid sequence similarity with other TRAP5b in teleost fish (61.73-98.15%). Tissues expression analysis showed that OnTRAP5b was most abundant in the liver and was also widely expressed in other tissues. Upon challenge with Streptococcus agalactiae and Aeromonas hydrophila in vivo and in vitro, the expression of OnTRAP5b was significantly up-regulated. Additionally, the purified recombinant OnTRAP5b ((r)OnTRAP5) protein exhibited optimal phosphatase activity at pH 5.0 and an ideal temperature of 50 °C. The Vmax, Km, and kcat of purified (r)OnTRAP5b were found to be 0.484 µmol × min-1 × mg-1, 2.112 mM, and 0.27 s-1 with respect to pNPP as a substrate, respectively. Its phosphatase activity was differentially affected by metal ions (K+, Na+, Mg2+, Ca2+, Mn2+, Cu2+, Zn2+, and Fe3+) and inhibitors (sodium tartrate, sodium fluoride, and EDTA). Furthermore, (r)OnTRAP5b was found to promote the expression of inflammatory-related genes in head kidney macrophages and induce reactive oxygen expression and phagocytosis. Moreover, OnTRAP5b overexpression and knockdown had a significant effect on bacterial proliferation in vivo. When taken together, our findings suggest that OnTRAP5b plays a significant role in the immune response against bacterial infection in Nile tilapia.

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.

In vivo DNA methylation editing in zebrafish.

The CRISPR/dCas9-based epigenome editing technique has driven much attention. Fused with a catalytic domain from Dnmt or Tet protein, the CRISPR/dCas9-DnmtCD or -TetCD systems possess the targeted DNA methylation editing ability and have established a series of in vitro and in vivo disease models. However, no publication has been reported on zebrafish (Danio rerio), an important animal model in biomedicine. The present study demonstrated that CRISPR/dCas9-Dnmt7 and -Tet2 catalytic domain fusions could site-specifically edit genomic DNA methylation in vivo in zebrafish and may serve as an efficient toolkit for DNA methylation editing in the zebrafish model.

Preparation of the monoclonal antibody against Nile tilapia Igλ and study on the Igλ+ B cell subset in Nile tilapia.

Immunoglobulins (Igs) are important effector molecules that mediate humoral immunity. A typical Ig consists of two heavy and two light chains. In teleosts, three Ig heavy chain isotypes (Igµ, Igδ and Igτ) and three Ig light chain isotypes (Igκ, Igλ and Igσ) have been identified. Compared to the heavy chains, teleost Ig light chains have been poorly studied due to the lack of antibodies. In this study, a mouse anti-Nile tilapia Igλ monoclonal antibody (mAb) was prepared, which could specifically recognize Igλ in serum and Igλ+ B cells in tissues. Further, the composition of IgM+ and Igλ+ B cell subsets was analyzed using this antibody and a mouse anti-tilapia IgM heavy chain mAb. The ratio of IgM+Igλ+ B cells to total IgM+ B cells in head kidney and peripheral blood was about 30%, while that in spleen was about 50%; the ratio of IgM-Igλ+ B cells to total Igλ+ B cells in head kidney and peripheral blood was about 45%, while that in spleen was about 25%. The IgM-Igλ+ B cells was speculated to be IgT+ B cells. Finally, we detected an increase in the level of specific antibodies against the surface antigen-Sip of Streptococcus agalactiae in serum after S. agalactiae infection, indicating that mouse anti-tilapia Igλ mAb can be used to detect the antibody level after immunization of Nile tilapia, which lays a foundation for the evaluation of immunization effect of tilapia vaccine.

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.

Teleost fish IgM+ plasma-like cells possess IgM-secreting, phagocytic, and antigen-presenting capacities.

Plasma cells are terminally differentiated antibody-secreting B lymphocytes that contribute to humoral immunity by producing large numbers of antibodies. Increasing evidence suggests that teleost fish B cells share certain characteristics with mammalian B1 B cells, including antibody-secreting, phagocytic, and antigen-presenting capacities. However, the difference between mature B cells and plasma cells remains unclear. In this study, we found that, based on their light-scattering characteristics, tilapia anterior kidney (AK) leukocytes can be categorized into two IgM+ B-cell subsets: the lymphoid (L) gate and granulocyte-monocyte/macrophage (G-M) subsets. G-M gate cells are more numerous than L-gate cells and have higher mean fluorescence, but lower forward scatter and side scatter. We analyzed the morphological and ultrastructural features of sorted IgM+ cells and found that L-gate IgM+ cells have a high nucleus-cytoplasm ratio and lymphocyte-like morphology, whereas G-M gate IgM+ cells have a small nucleus, more abundant endoplasmic reticulum, and a larger number of mitochondria, and have a plasma cell-like or macrophage-like morphology. To further characterize the cell types, we examined the specific patterns of expression of B-cell- and T-cell-related genes. We found that B-cell-specific genes were expressed by both L-gate and G-M gate IgM+ cells, and that G-M gate IgM+ cells secreted extremely high levels of IgM. However, T-cell-related genes were highly expressed only in L-gate IgM- cells. These results suggest that G-M gate IgM+ cells are similar to plasma-like cells, with high antibody-secreting capacity. Given that G-M gate cells include the granulocyte, monocyte, and macrophage cell types, but not B cells, monocyte/macrophage markers were used to investigate the cell types further. A macrophage receptor with a collagenous structure was frequently observed, and macrophage-expressed gene-1 was highly expressed, in the G-M gate IgM+ cells. Phagocytic capacity, as determined by ingestion of beads or bacteria, was significantly higher in G-M gate IgM+ cells than in L-gate IgM+ cells, as was antigen-processing capacity. Our findings show that tilapia AK leukocytes can be divided into two IgM+ B-cell subsets and that G-M gate IgM+ cells resemble plasma-like cells, having high antibody-secreting, phagocytic, and antigen-presenting capacities. Thus, this study increases our understanding of the functions of teleost fish plasma-like cells.

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.

Functional characterization of complement factor H in host defense against bacterial pathogen in Nile tilapia (Oreochromis niloticus).

Complement factor H (CFH), a multifunctional soluble complement regulatory protein, can bind to a variety of pathogens and play a crucial role in host innate immune defense. To explore the functional characteristics of CFH (OnCFH) in Nile tilapia (Oreochromis niloticus), we cloned and characterized the open reading frame (ORF) of OnCFH in this study. The full-length of OnCFH ORF is 1359 bp, encoding 452 aa for a 48.85 kDa peptide, and its predicted structure containing six short complement-like repeats (SCRs). The analysis of tissue distribution showed that OnCFH was constitutively expressed in all tested tissues, with the highest in the liver. Upon Streptococcus agalactiae and Aeromonas hydrophila stimuli in vivo and in vitro, OnCFH mRNA transcript was significantly upregulated in head kidney tissue as well as head kidney monocytes/macrophages. Further, the recombinant OnCFH protein ((r)OnCFH) could bind to pathogenic bacteria in a dose-dependent. Moreover, it got involved in the regulation of inflammation as well as phagocytosis of monocytes/macrophages. The knockdown of OnCFH remarkably decreased the amount of bacteria in the head kidney. In summary, our data demonstrated that OnCFH could participate in the immune response of Nile tilapia against bacterial infection.

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.

Molecular cloning, characterization and expression analysis of CXCR3a and CXCR3b from Nile tilapia (Oreochromis niloticus).

The CXC chemokine receptors (CXCRs) are members of the seven transmembrane (7-TM) G-protein-coupled receptor superfamily that involves innate and adaptive immune systems. In this study, CXCR3a and CXCR3b from Nile tilapia (Oreochromis niloticus) were cloned and identified, designated as OnCXCR3a and OnCXCR3b. The open reading frames of OnCXCR3a and OnCXCR3b were 1074 and 1080 bp, encoding the predicted proteins of 357 and 359 amino acids, respectively. Multiple alignment analysis of OnCXCR3a- and OnCXCR3b-deduced protein sequences with the mammalian and bird sequences indicated the presence of typical structural features of chemokine receptors, including a 7-TM domain and conserved motifs. Quantitative real-time PCR analysis revealed that OnCXCR3a and OnCXCR3b were constitutively expressed in a wide range of tissues. When stimulated with Streptococcus agalactiae, Aeromonas hydrophila, polyinosinic:polycytidylic acid and lipopolysaccharide in vivo or in vitro on leukocytes, the mRNA levels of OnCXCR3a and OnCXCR3b were significantly upregulated. Overall, these results indicated that OnCXCR3s might be involved in host immune responses in Nile tilapia.

Toxicological damages on copper exposure to IgM+ B cells of Nile tilapia (Oreochromis niloticus) and mitigation of its adverse effects by ß-glucan administration.

Present investigation was carried out to study toxicological damages of copper exposure and mitigation of its adverse effects with ß-glucan administration in IgM+ B cells which processes multiple roles similar to macrophages in Nile tilapia (Oreochromis niloticus). IgM+ B cells were pretreated with ß-glucan (25 µg/mL) for 24 h before exposed to cupric oxide nanoparticles (CuO NPs) or cupric chloride (Cu ions) at the doses of 0, 5, 10, and 20 µg/mL for 24 h, respectively. Our results demonstrated that ß-glucan increased reduced glutathione (GSH) to against oxidative damage from CuO NPs and Cu ions exposure in IgM+ B cells. The apoptosis process through mitochondrial signaling pathway was depressed in IgM+ B cells since the mitochondrial membrane potential (ΔΨm) was protected from copper exposure by ß-glucan treatment. Furthermore, the inhibition on phagocytic abilities of IgM+ B cells caused by copper exposure could be enhanced with ß-glucan treatment via evaluation of microspheres and bioparticles uptake and LPS-induced NO production. Importantly, ß-glucan might participate in immunomodulation in IgM+ B cells through B cell antigen receptor (BCR) to suppress toxicological effect derived from copper exposure. Taken together, this study provides more information on the toxicological damages in IgM+ B cells upon copper exposure and explains the molecular mechanism to reverse adverse effects caused by copper exposure with ß-glucan administration.