| In mammalian immune system, interleukin-10(IL-10) is considered as an important regulatory cytokine with pleiotropic effects. IL-10 produced by various myeloid and lymphoid cells modulates growth, maturation, proliferation, differentiation and activation of many immune cells including T lymphocytes, B lymphocytes, monocytes/macrophages and dentritic cells. IL-10 regulates both innate and adaptive immune responses and interacts with other cytokines to maintain immune homeostasis.Teleost IL-10 gene has been preliminarily isolated from several fishes such as zebrafish, goldfish, common carp and rainbow trout. However, the immune function, receptor signaling and regulatory mechanisms of fish IL-10 are largely unknown. In this study, we not only confirmed the classical immunoinhibitory function of grass carp(Ctenopharyngodon idllus) IL-10(gcIL-10), but also illustrated the regulatory mechanism and relationship of gcIL-10 and grass carp transforming growth factor beta 1(gcTGF-β1) which is another potent anti-inflammatory cytokine in grass carp as we reported previously, expanding our understanding of the role of IL-10 in fish immunity. In order to study the molecular mechanisms for the bioactivity of gcIL-10, its receptor subunits, IL-10 receptor 1(IL-10R1) and IL-10 receptor 2(IL-10R2) were identified, and gcIL-10-activated STAT3 signaling was characterized. Further study elucidated the transcriptional mechanisms of gcIL-10 in response to LPS and interferon γ(IFN-γ) challenges. The detail results are shown as follows:1. Bioactivity identification and immunostimulatory function of recombinant gcIL-10:(1) The full-length cDNA of gcIL-10 was isolated using homology-based cloning techniques and the conserved domains in this sequence were characterized with bioinformatics tools.(2) Tissue distribution of gcIL-10 transcript was analyzed with real-time quantitative PCR(qPCR). The highest mRNA levels of gcIL-10 were detected in immune related tissues, such as head kidney, kidney and gill, implying gcIL-10 may participate in immune responses.(3) Recombinant gcIL-10 protein(rgcIL-10) was acquired by prokaryotic expression system and affinity purification, and this high-purity protein was used to study immune function of gcIL-10.(4) CCK-8 assay indicated that rgcIL-10 could stimulate cell viability of grass carp peripheral blood lymphocytes(PBLs) to a similar extent with the cells challenged by recombinant gcTGF-β1(rgcTGF-β1).(5) Immunoneutralization assay of gcIL-10 found that rgcTGF-β1 regulated the cell viability possibly through a gc IL-10-denpendent way, revealing regulatory mechanism of TGF-β1-mediatied effect on PBLs cell viability.(6) This notion was further supported by qPCR and CI-ELISA results in which rgcTGF-β1 induced gcIL-10 mRNA expression and secretion in grass carp PBLs, indicating that rgcTGF-β1 stimulated PBLs cell viability by inducing gcIL-10 production. Taken together, results in this section show the immunostimulatory effect of IL-10 in teleost immunity, especially explicating functional connection between IL-10 and TGF-β1 in the immunostimulatory response.2. Functional characterization and relationship of gcIL-10 and gcTGF-β1 in inflammatory regulation:(1) Both rgcIL-10 and rgcTGF-β1 inhibited LPS-stimulated mRNA expression of several pro-inflammatory factors TNF-α, IL-1β, iNOS and IL-8 in grass carp head kidney monocytes/macrophages.(2) To the contrary, gcIL-10 polyclonal antibody(anti-gcIL-10 pAb) and gcTGF-β1 monoclonal antibody(anti-gcTGF-β1 mAb) could amplify LPS-induced mRNA levels of these pro-inflammatory factors, suggesting that endogenous gcIL-10 and gcTGF-β1 actually regulated the expression of pro-inflammatory factors.(3) Both gcIL-10 and gcTGF-β1 prevented LPS-induced IκBα protein degradation, thereby impairing NF-κB activation. This unveiled the regulatory mechanism of gcIL-10 and gcTGF-β1 in controlling LPS-induced pro-inflammatory factors expression.(4) Anti-gcIL-10 pAb and anti-gcTGF-β1 mAb were unable to change the inhibitory effects of rgcTGF-β1 and rgcIL-10 on pro-inflammatory factors expression, respectively, implying gcTGF-β1 and gcIL-10 independently execute their regulatory functions in LPS-stimulated monocytes/macrophages. These results represent the inhibitory function, molecular mechanism and parallel correlation between IL-10 and TGF-β1 in teleost inflammatory response, enhancing our knowledge on inflammatory regulation in fish.3. Identification of grass carp IL-10 receptor subunits and signaling pathway:(1) Full-length cDNA sequences of grass carp IL-10 receptor 1(gcIL-10R1), cytokine receptor family member 4(gcCRFB4) and cytokine receptor family member 5(gcCRFB5) were obtained using homology-based cloning techniques and bioinformatics methods were used to analyze these sequences.(2) Expression analysis of gcIL-10R1, gcCRFB4 and gcCRFB5 by qPCR indicated gcIL-10R1 mRNA was relatively high expressed in the immune related tissues, such as gill, thymus, spleen and head kidney. However, gcCRFB4 and gcCRFB5 were widely expressed in all selected tissues, showing similar expression pattern with mammalian counterparts.(3) GcIL-10R1, gcCRFB4 and gcCRFB5 eukaryotic expression plasmids were constructed and transfected into CIK cells(a cell stain from grass carp kidney). By detecting luciferase-based STAT3 activity, the results demonstrated that rgcIL-10 could activate STAT3 and gcIL-10R1 played a key role in gcIL-10 signal transduction. Moreover, the results suggested that CRFB4 but not CRFB5 was fish IL-10R2 for the first time.(4) Grass carp suppressor of cytokine signaling 3(gcSOCS3) promoter region was isolated and gcSOCS3 promoter-regulated or STAT3-muted gcSOCS3 promoter-regulated luciferase reporter plasmid was constructed and transfected into CIK cells. By detecting luciferase activity, it was found that the stimulation of gcIL-10 on gcSOCS3 promoter activity was mediated by STAT3.(5) In grass carp head kidney leukocytes(HKLs), Western blotting and qPCR assays showed that rgcIL-10 induced phosphorylation of gcSTAT3 and modulated SOCS3 transcript in a STAT3-dependent manner. Therefore, these findings clarify gcIL-10 downstream IL-10R1/CRFB4/STAT3 pathway and its target gene SOCS3 in grass carp.4. The transcriptional regulation of grass carp IL-10:(1) LPS stimulated gcIL-10 mRNA expression through MAPK/ERK signaling in grass carp monocytes/macrophages.(2) In the same cell model, recombinant grass carp IFN-γ(rgcIFN-γ) inhibited gcIL-10 gene expression via MAPK/ERK pathway.(3) LPS increased gcIL-10 promoter activity. In contrast, rgcIFN-γ decreased the promoter activity. These results imply that different stimuli regulate gcIL-10 expression by distinct signaling pathways.In conclusion, this study described the immunoregulatory role of gcIL-10, including a new finding on its immunostimulatory bioactivity on PBL cell viability and its classical inhibitory function in inflammation, and clarified the regulatory mechanisms and correlation of two main anti-inflammatory cytokines IL-10 and TGF-β1 in fish. What’s more, gcIL-10 receptor subunits(ligand-binding subunit IL-10R1 and accessory subunit CRFB4), its signaling pathways and target gene SOCS3 were identified. Finally, the regulatory mechanisms for grass carp IL-10 production under different stimuli were studied. These findings not only shed light on the immune function, regulatory mechanisms and signaling pathway of teleost IL-10, but also provide novel understanding of fish immune regulation which was based upon interaction among different cytokines. |
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