IFN-γ Manipulates NOD1-Mediated Interaction of Autophagy and Edwardsiella piscicida to Augment Intracellular Clearance in Fish.

Edwardsiella piscicida is an intracellular pathogenic bacterium accounting for significant losses in farmed fish. Currently, cellular and molecular mechanisms underlying E. piscicida-host cross-talk remain obscure. In this study, we revealed that E. piscicida could increase microtubule-associated protein L chain 3 (LC3) puncta in grass carp (Ctenopharyngodon idella) monocytes/macrophages and a carp cell line, Epithelioma papulosum cyprini The autophagic response was confirmed by detecting the colocalization of E. piscicida with LC3-positive autophagosomes and LysoTracker-probed lysosomes in the cells. Moreover, we unveiled the autophagic machinery targeting E. piscicida by which the nucleotide-binding oligomerization domain receptor 1 (NOD1) functioned as an intracellular sensor to interact and recruit autophagy-related gene (ATG) 16L1 to the bacteria. Meanwhile, E. piscicida decreased the mRNA and protein levels of NOD1 and ATG16L1 in an estrogen-related receptor-α-dependent manner, suggesting a possible mechanism for this bacterium escaping autophagy. Subsequently, we examined the effects of various E. piscicida virulence factors on NOD1 expression and found that two of them, EVPC and ESCB, could reduce NOD1 protein expression via ubiquitin-dependent proteasomal degradation. Furthermore, an intrinsic regulator IFN-γ was found to enhance the colocalization of E. piscicida with NOD1 or autophagosomes, suggesting its involvement in the interaction between autophagy and E. piscicida Along this line, a short-time treatment of IFN-γ caused intracellular E. piscicida clearance through an autophagy-dependent mechanism. Collectively, our works demonstrated NOD1-mediated autophagy-E. piscicida dialogues and uncovered the molecular mechanism involving autophagy against intracellular bacteria in fish.

Functional characterization of grass carp (Ctenopharyngodon idella) interleukin-2 in head kidney leukocytes.

Interleukin (IL)-2 belongs to the four-helix bundle cytokine family and plays key roles in growth, survival, activation-induced cell death and differentiation of the immune cells. In cyprinid fish, only common carp interleukin-2 (il2) has been cloned because of relatively low sequence homology between carp Il-2 and its homologs in other fish species. In the present study, the coding sequence of grass carp Il-2 (gcIl-2) was cloned and its identity was verified via bioinformatic analysis. Tissue distribution study showed that grass carp il2 (gcil2) mRNA was expressed in thymus, head kidney and gill with relatively high levels. Recombinant gcIl-2 (rgcIl-2) protein was subsequently prepared by using a prokaryotic expression system followed by a refolding method. The purified rgcIl-2 displayed an ability to stimulate the cell proliferation along with an increased mRNA expression of cd4l but not cd8a, igm or mcsfr in grass carp head kidney leukocytes (HKLs), suggesting the possible involvement of gcIl-2 in T helper (Th) cell proliferation. In the same cell model, rgcIl-2 significantly enhanced mRNA expression of some cytotoxic molecules including perforin-like protein 2, granzyme B-like and Fas ligand, indicating the modulation of cytotoxic cells by gcIl-2 in grass carp HKLs. Besides, gene expression of regulatory T (Treg) cell- and Th1/2 cell-related cytokines or transcription factors was detected in grass carp HKLs treated by rgcIl-2. Results showed that rgcIL2 treatment increased the mRNA expression of foxp3, cd25l, ifng2, il12p35, tbet, tnfa, il2, il4/13a, il4/13b and gata3l in HKLs, implying the regulatory roles of Il-2 in the expression of these immune genes and its possible involvement in differentiation of Treg and Th1/2 cells. These observations together with the related studies in other fishes suggest the existence of cytotoxic cells, Treg and Th1/2 subpopulations in fish species and the functional roles of Il-2 in these cells.

Characterization of a new il-4/13 homologue in grass carp (Ctenopharyngodon idella) and its cooperation with M-CSF to promote macrophage proliferation.

In this study, a new il-4/13 cDNA was isolated from grass carp (Ctenopharyngodon idella) using homologous cloning. The phylogenetic tree and sequence alignment of the deduced amino acid (aa) sequence showed that it was closer to grass carp il-4/13b (gcil-4/13b) than other homologues and therefore named gcil-4/13b-like (gcil-4/13bl). It has 399-nt coding sequence (CDS) which is less than gcil-4/13b (408 nt). In addition, the cloned gcil-4/13bl gene is approximately 1600 bp in length and has a conserved genetic structure consisting of four exons and three introns. Compared to gcil-4/13b gene, it has a variety of nucleotides variation across the CDS and contains a longer intron 3, suggesting that it is a new gcil-4/13 gene. The gcil-4/13bl transcripts were ubiquitously expressed in almost all selected tissues, and there was almost only gcil-4/13bl detected in brain and head kidney (HK). Recombinant grass carp (rgc) Il-4/13bl was prepared by using Escherichia coli (E. coli) Rosetta-gami 2 (DE3). The functional study demonstrated that rgcIl-4/13bl significantly upregulated arginase-2 gene expression and arginase activity, whilst downregulated nitric oxide (NO) production as well as the transcript levels of inducible nitric oxide synthesase (inos) and ifn-γ in freshly isolated grass carp HK monocytes/macrophages (M0/Mϕ). These data suggested that the newly cloned il-4/13bl had the conserved functions to activate M2-type but antagonize M1-type macrophages. Furthermore, rgcIl-4/13bl was able to drive the proliferation of M0/Mϕ which were pre-treated by rgcM-csf, indicating the involvement of gcIl-4/13bl in the proliferation of macrophages. Here we not only identified a new il-4/13-encoding gene in grass carp, but also for the first time revealed a novel function of fish Il-4/13 combined with M-csf engaging in M0/Mϕ proliferation.

Functional characterization of three fish-specific interleukin-23 isoforms as regulators of Th17 signature cytokine expression in grass carp head kidney leukocytes.

Mammalian Interleukin (IL)-23 is a heterodimeric cytokine with an IL-23-specific P19 subunit and a P40 subunit shared with IL-12, and plays a key role in the regulation of cell differentiation as well as inflammation. We previously demonstrated the existence of three soluble fish Interleukin (Il)-23 isoforms consist of a single P19 and one of three P40 isoforms (P40a/b/c) in grass carp. In the present study, three recombinant grass carp Il-23 (rgcIl-23) isoforms were prepared by linking gcP19 and gcP40a/b/c in a prokaryotic expression system, and then their functional properties were verified in grass carp head kidney leukocytes (HKLs). All three rgcIl-23 isoforms showed the bioactivities to divergently upregulate the mRNA expression of Th17 signature cytokines (il17a/f1, il21, il22 and il26) as well as Il-23 receptor (il23r) in HKLs. Moreover, they also promoted gcIl-17a/f1 secretion in a dose-dependent manner, strengthening their roles in Th17-like response. Furthermore, induction of il17a/f1 and il23r transcription by rgcIl-23 was blocked by a STAT3 inhibitor in grass carp HKLs, suggesting the involvement of STAT3 signaling in these inductions. Taken together, we for the first time identified the bioactivities of fish Il-23 isoforms and particularly revealed the existence of Il-23/Il-17a/f1 axis in fish, thereby advancing our understanding of Th17-like responses in fish immunity.

Functional characterization of grass carp runt-related transcription factor 3: Involvement in TGF-ß1-mediated c-Myc transcription in fish cells.

In mammals, both runt-related transcription factor 3 (RUNX3) and c-Myc are the downstream effectors of transforming growth factor-ß1 (TGF-ß1) signaling to mediate various cellular responses. However, information of their interaction especially in fish is lacking. In the present study, grass carp (Ctenopharyngodon idella) runx3 (gcrunx3) cDNA was cloned and identified. Interestingly, opposing effects of recombinant grass carp TGF-ß1 (rgcTGF-ß1) on c-myc and runx3 mRNA expression were observed in grass carp periphery blood lymphocytes (PBLs). Parallelly, Runx3 protein levels were enhanced by rgcTGF-ß1 in the cells. These findings prompted us to examine whether Runx3 can mediate the inhibition of TGF-ß1 on c-myc expression in fish cells. In line with this, overexpression of grass carp Runx3 and Runx3 DN (a dominant-negative form of Runx3) in grass carp kidney cell line (CIK) cells decreased and increased c-myc transcript levels, respectively. Particularly, the regulation of Runx3 and Runx3 DN on c-myc mRNA expression was direct since they were presented in the nucleus without any stimulation. In addition, rgcTGF-ß1 alone suppressed c-myc mRNA expression in CIK cells as in PBLs. Moreover, this inhibitory effect was also observed when grass carp Runx3 and Runx3 DN were overexpressed. These results strengthened the role of TGF-ß1 signaling in controlling c-myc transcription. Taken together, TGF-ß1-mediated c-myc expression was affected at least in part by Runx3, thereby firstly exploring the functional role of Runx3 in TGF-ß1 down-regulation on c-myc mRNA expression in fish.

Lipopolysaccharide-induced autophagy participates in the control of pro-inflammatory cytokine release in grass carp head kidney leukocytes.

Microtubule-associated protein 1 light chain 3B (LC3B) is a known marker of autophagy in mammals. In the present study, we isolated and identified grass carp LC3B (gcLC3B) cDNA and found its inductive expression in response to bacterial infection in vivo. To assess the occurrence of autophagy in immune response, the role of gcLC3B as an autophagy marker in grass carp was characterized. Accordingly, grass carp kidney cells (CIKs) with stable expression of GFP-gcLC3B were established and GFP-gcLC3B puncta were counted under a confocal fluorescence microscope. Results showed that starvation, a conventional inducer of autophagy, significantly enhanced GFP-gcLC3B puncta number, indicating the existence of gcLC3B-linked autophagy in fish cells. Moreover, a commercial antibody recognizing gcLC3B and 3-methyladenine (3-MA) were validated in grass carp CIKs, and used to evaluate autophagy in grass carp head kidney leukocytes (HKLs) in response to LPS. Western blotting assay showed that LPS significantly induced the conversion of gcLC3B protein, providing the evidence for autophagy induced by LPS in fish immune cells. Importantly, autophagy inhibition by 3-MA enhanced grass carp IL-1ß and TNF-α secretion, indicating the involvement of autophagy in pro-inflammatory cytokine production. Besides, 3-MA could amplify LPS-induced IL-1ß and TNF-α release, implying that autophagic induction may drive a mechanism for controlling inflammatory response in fish. Thus, our data highlight the role of autophagy in fish immunity and provide new insight into the mechanism for the regulation of inflammation in fish.

Dual-parallel inhibition of IL-10 and TGF-ß1 controls LPS-induced inflammatory response via NF-κB signaling in grass carp monocytes/macrophages.

In fish, the knowledge on the regulation of inflammatory responses is limited. In the present study, LPS rapidly increased the mRNA levels of grass carp pro-inflammatory factors, including tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), inducible nitric oxides synthase (iNOS) and IL-8 in monocytes/macrophages, indicating the occurrence of innate inflammatory responses in fish as seen in mammals. Intriguingly, the gene expression and protein secretion of grass carp IL-10 (gcIL-10) and TGF-ß1 (gcTGF-ß1) were induced by LPS in the same cell model, promoting us to clarify their roles in regulating inflammatory response. Results revealed that grass carp IL-10 polyclonal antibody (anti-gcIL-10 pAb) and grass carp TGF-ß1 monoclonal antibody (anti-gcTGF-ß1 mAb) could amplify the stimulation of LPS on the mRNA levels of tnfα, il1ß, inos and il8, suggesting the inhibitory tone of endogenous IL-10 and TGF-ß1 in LPS-challenged immune responses. This notion was further supported by the fact that recombinant grass carp IL-10 (rgcIL-10) and recombinant grass carp TGF-ß1 (rgcTGF-ß1) attenuated LPS-stimulated tnfα, il1ß, inos and il8 gene expression in monocytes/macrophages. Further study revealed that rgcIL-10 and rgcTGF-ß1 impaired NF-κB activation by blocking LPS-induced grass carp IκBα (gcIκBα) protein degradation in the cells. In addition, the correlation between gcIL-10 and gcTGF-ß1 in this regulation was examined by immunoneutralization, unveiling that anti-gcTGF-ß1 mAb and anti-gcIL-10 pAb were unable to alter the inhibitory effects of rgcIL-10 and rgcTGF-ß1 on pro-inflammatory factors expression in grass carp monocytes/macrophages, respectively. This dual and parallel effect of gcIL-10 and gcTGF-ß1 strengthened their importance in controlling inflammatory responses. Taken together, our findings shed a light on the functional role, regulatory mechanism and relationship of fish IL-10 and TGF-ß1 in regulating inflammatory response.

Identification and functional characterization of grass carp IL-17A/F1: An evaluation of the immunoregulatory role of teleost IL-17A/F1.

In mammals, IL-17A and IL-17F are hallmark cytokines of Th17 cells which act significant roles in eradicating extracellular pathogens. IL-17A and IL-17F homologs nominated as IL-17A/F1-3 have been revealed in fish and their functions remain largely undefined. Here we identified and characterized grass carp IL-17A/F1 (gcIL-17A/F1) in fish immune system. In this regard, both tissue distribution and inductive expression of gcIL-17A/F1 indicated its possible involvement in immune response. Moreover, recombinant gcIL-17A/F1 (rgcIL-17A/F1) was prepared and displayed an ability to enhance pro-inflammatory cytokines (IL-1ß, TNF-α and IL-6) mRNA expression in head kidney leukocytes. It is suggestive of that gcIL-17A/F1 may act as a proinflammatory cytokine in fish immunity. Besides, rgcIL-17A/F1 induced gene expression and protein release of grass carp chemokine CXCL-8 (gcCXCL-8) in head kidney cells (HKCs), probably via NF-κB, p38 and Erk1/2 pathways. In particular, culture medium from the HKCs treated by rgcIL-17A/F1 could stimulate peripheral blood leukocytes migration and immunoneutralization of endogenous gcCXCL-8 could partially attenuate this stimulation, suggesting that rgcIL-17A/F1 could recruit immune cells through producing gcCXCL-8 as mammalian IL-17 A and F. Taken together, we not only identified the pro-inflammatory role of gcIL-17A/F1 in host defense, but also provided the basis for clarifying Th17 cells in teleost.

In vitro and in vivo acute toxicity of fenpyroximate to flounder Paralichthys olivaceus and its gill cell line FG.

Fenpyroximate, an acaricide, is widely used in the prevention of acarids (mites) in plants. In this study, the in vitro and in vivo acute toxicity of fenpyroximate was examined using the marine flounder Paralichthys olivaceus and its gill cell line (FG). The 48h-IC(50) (95% confidence limits) values of fenpyroximate in the FG cells were 890 (790-990)nM, 950 (881-1019)nM and 1250 (1159-1341)nM, and 96h-IC(50) (95% confidence limits) were 480 (388-572)nM, 490 (454-526)nM and 510 (469-551)nM, for methyl thiazolyl tetrazolium (MTT) assay, neutral red (NR) uptake and cell protein assay, respectively. The 48h- and 96h-LC(50) (95% confidence limits) values of fenpyroximate in living flounders were 28.84 (14.28-58.26)nM and 11.74 (6.06-22.8)nM, respectively. This indicated that fenpyroximate was highly toxic to both flounders and FG cells. Moreover, comparisons of the ratios of average 48h-IC(50) to 48h-LC(50) and average 96h-IC(50) to 96h-LC(50) showed that the length of exposure time did not significantly affect the correlation between the FG cells and living flounders in the acute toxicity estimation of fenpyroximate provided the selected exposure time is the same. Thus, we suggest that FG cells could be a good bioassay system in rapid estimation of the corresponding LC(50) values of pollutants to living fish, instead of whole living fish. Histopathological examinations showed that liver and gill were the major target organs of fenpyroximate, especially the damage of gill tissues may account much for the high lethality of exposed flounders. Consistent with the histopathological observations, analysis of the activities of two key detoxification metabolism-related enzymes, glutathione S-transferase (GST) and cytochrome P4501A1 (CYP1A1)-dependent ethoxyresorufin-O-deethylase (EROD), in the liver and gill tissues of exposed flounders indicated that liver has much higher detoxification capacity than gills, and this contributes to the higher tolerance of liver to the toxicity of fenpyroximate in the exposed flounders. Fenpyroximate can initially induce a quick and significant increase of the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in all the exposed FG cells, and liver and gill tissues of exposed flounders. Upon continuation of the exposure the enzyme activities were inhibited, implying the occurrence of oxidative stress in the exposed fish cells and the possible interruption of the mitochondrial respiratory chain which involves redox reactions by fenpyroximate.