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Mucosal immunity in mucosa-associated lymphoid tissues (MALTs) plays crucial roles in resisting infection by pathogens, including parasites, bacteria and viruses. However, the mucosal immune response in the MALTs of large yellow croaker (Larimichthys crocea) upon parasitic infection remains largely unknown. In this study, we investigated the role of B cells and T cells in the MALTs of large yellow croaker following Cryptocaryon irritans infection. Upon C. irritans infection, the total IgM and IgT antibody levels were significantly increased in the skin mucus and gill mucus. Notably, parasite-specific IgM antibody level was increased in the serum, skin and gill mucus following parasitic infection, while the level of parasite-specific IgT antibody was exclusively increased in MALTs. Moreover, parasitic infection induced both local and systemic aggregation and proliferation of IgM + B cells, suggesting that the increased levels of IgM in mucus may be derived from both systemic and mucosal immune tissues. In addition, we observed significant aggregation and proliferation of T cells in the gill, head kidney and spleen, suggesting that T cells may also be involved in the systemic and mucosal immune responses upon parasitic infection. Overall, our findings provided further insights into the role of immunoglobulins against pathogenic infection, and the simultaneous aggregation and proliferation of both B cells and T cells at mucosal surfaces suggested potential interactions between these two major lymphocyte populations during parasitic infection., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

As the predominant immunoglobulin (Ig) isotype, IgM plays a crucial role in the acquired immunity of vertebrates. There is only one Igμ gene in mammals, except cattle, while the number of Igμ gene varies among teleost fish. In the current study, we found two functional Igμ genes (Igμ1 and Igμ2) and a pseudo Cμ gene (ψIgμ) in large yellow croaker (Larimichthys crocea). Both Igμ1 and Igμ2 genes possessed two transcript variants, which encoded the heavy chains of secreted (sIgM1 and sIgM2) and membrane-bound IgM1 and IgM2 (mIgM1 and mIgM2), respectively. Both the heavy chains of sIgM1 and sIgM2 consisted of a variable Ig domain, four constant Ig domains (CH1, CH2, CH3 and CH4) and a secretory tail, while those of mIgM1 and mIgM2 consisted of a variable Ig domain, three constant Ig domains (CH1, CH2 and CH3), a transmembrane domain and a short cytoplasmic tail. Cysteine residues that are necessary for the formation of intrachain and interchain disulfide bonds and tryptophan residues that are important for the folding of the Ig superfamily domain were well conserved in large yellow croaker IgM1 and IgM2. Interestingly, large yellow croaker IgM2 had an extra cysteine (C94) in the CH1 domain compared with IgM1, which may cause the structural difference between IgM1 and IgM2. A liquid chromatography-tandem mass spectrometry analysis revealed that both IgM1 and IgM2 were present at the protein level in large yellow croaker serum. Both the Igμ1 and Igμ2 genes were mainly expressed in systemic immune tissues, such as head kidney and spleen, but the expression level of Igμ2 was much lower than that of Igμ1. After Pseudomonas plecoglossicida infection, the expression levels of Igμ1 and Igμ2 in both the spleen and head kidney were significantly upregulated, with a higher upregulation of Igμ2 than that of Igμ1. These results suggested that Igμ1 and Igμ2 may play a differential role in the immune response of large yellow croaker against bacterial infection., Competing Interests: Declaration of competing interest The authors declare that they have no commercial interests or personal relationships in the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

As an inflammatory cytokine of the interleukin-20 (IL-20) subfamily, IL-20 has various functions in immune defenses, inflammatory diseases, tissue regeneration, cancer, and metabolism. Although the characteristics and functions of mammalian IL-20 have been clarified, those of fish IL-20 remain unclear. In this study, the IL-20 gene from the snakehead Channa argus (shIL-20) was cloned and functionally characterized. Similar to the IL-20 homologues of other species, the shIL-20 has a five exon/four intron structure in the coding region. The open reading frame of shIL-20 consists of 528 base pairs and encodes 175 amino acids (aa), including a signal peptide (aa 1-24) and a mature peptide (aa 25-175). The mature shIL-20 protein has six conserved cysteine residues, which occur in the IL-20 proteins of all species analyzed, and an additional cysteine residue (Cys-82) found only in the IL-20 proteins of several teleosts. The modeled tertiary structure of shIL-20 is similar with that of Homo sapiens IL-20. The shIL-20 was expressed constitutively in all the tissues analyzed, and its transcription was induced in the spleen and head kidney by Aeromonas schubertii and Nocardia seriolae in vivo and in head kidney leukocytes (HKLs) by lipoteichoic acid, lipopolysaccharide, and polyinosinic-polycytidylic acid in vitro. The recombinant shIL-20 protein induced the transcription of tumor necrosis factor α1 (TNF-α1), TNF-α2, IL-1β, and endogenous shIL-20, and promoted the proliferation of HKLs. In conclusion, these findings demonstrate that shIL-20 participates in the immune response to bacterial invasion and promotes leukocyte proliferation, offering new insights into the functions of fish IL-20 during pathogen invasion., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Interleukin-21 (IL-21), a crucial immune regulatory molecule, belongs to the common γ-chain family of type I cytokines, and exerts pleiotropic effects on multiple immune cell types in mammals. However, the characteristics and functions of fish IL-21 remain unclear. To further investigate the molecular mechanism of IL-21 in teleosts, we first cloned and identified the IL-21 gene (designated shIL-21) of the snakehead (Channa argus). The full-length open reading frame of shIL-21 is 438 bp in length, and encodes a predicted protein of 145 amino acid residues. A sequence analysis showed that shIL-21 has the typical structural characteristics of other IL-21 proteins, containing four α-helices and four conserved cysteine residues. In a phylogenetic analysis, shIL-21 clustered within a subgroup of IL-21 proteins from other teleost species and shared its closest evolutionary relationship with that of Lates calcarifer. The expression analysis showed that shIL-21 was ubiquitously expressed in all the healthy snakehead tissues tested, albeit at different levels. After infection with Nocardia seriolae or Aeromonas schubertii, the relative expression of shIL-21 was mainly upregulated in the head kidney and spleen in vivo. Similarly, after stimulation with the three pathogen analogues lipoteichoic acid, lipopolysaccharides, and polyinosinic-polycytidylic acid, the expression of shIL-21 was also induced in head kidney leukocytes in vitro. A recombinant shIL-21 protein was expressed and purified, and promoted the proliferation of head kidney leukocytes, induced the expression of genes encoding critical signaling molecules in the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) pathway, including JAK1, JAK3, STAT1, and STAT3, and induced the expression of endogenous shIL-21 and genes encoding several key proinflammatory cytokines (tumor necrosis factor-α, interferon-γ, and IL-1β). Taken together, these preliminary findings suggest that shIL-21 is involved in the immune defense against bacterial infection, in leukocyte proliferation, and in the activation of the JAK-STAT pathway. They thus extend the functional studies of IL-21 in teleosts., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

ATG12, a core autophagy protein, forms a conjugate with ATG5 to promote the formation of autophagosome membrane, and plays an important role in antiviral immunity. However, little is known about the function of ATG12 in fish. Here, we cloned the open reading frame (ORF) of large yellow croaker (Larimichthys crocea) ATG12 (LcATG12), which is 354 nucleotides long and encodes a protein of 117 amino acids. The deduced LcATG12 possesses a conserved APG12 domain (residues 31 to 117), and shares 91.45% identities with ATG12 in orange-spotted grouper (Epinephelus coioides). LcATG12 was constitutively expressed in all examined tissues, with the highest level in intestine. Its transcript was also detected in primary head kidney granulocytes (PKG), primary head kidney macrophages (PKM), primary head kidney lymphocytes (PKL), and large yellow croaker head kidney (LYCK) cell line, and was significantly up-regulated by poly(I:C). LcATG12 was regularly distributed in both cytoplasm and nucleus of LYCK and epithelioma papulosum cyprinid (EPC) cells. Overexpression of LcATG12 in EPC cells significantly inhibited the replication of spring viremia of carp virus (SVCV). Further studies reveled that LcATG12 could induce the occurrence of autophagy in LYCK cells. Furthermore, overexpression of LcATG12 in LYCK cells increased the expression levels of large yellow croaker type I interferons (IFNs, IFNc, IFNd, and IFNh), IFN regulatory factors (IRF3 and IRF7), and IFN-stimulated genes (PKR, Mx, and Viperin). All these data indicated that LcATG12 plays a role in the antiviral immunity possibly by inducing both autophagy and type I IFN response in large yellow croaker., (Copyright © 2021 Elsevier Ltd. All rights reserved.)