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The crosstalk between the estrogen receptor (ER) and NF-κB signalling pathways has merged in vertebrates and plays a key role in the control of genes involved in inflammation, cell proliferation and apoptosis. However, such crosstalk between the endocrine and immune systems needs to be explored in lower invertebrates. In this study, we identified a 2856-bp homologue of the estrogen receptor from Hong Kong oyster (ChER), containing a 5' untranslated region (UTR) of 234 bp, a 3' UTR of 387 bp, and an open reading frame (ORF) of 2235 bp. We observed that overexpression of ChER suppressed ChRel-dependent NF-kappaB (NF-κB) activation in the HEK293T (human embryonic kidney 293T) cell line, and depletion of ChER in vivo resulted in upregulation of two NF-κB-responsive marker genes, namely, TNF-α and IL-17, which confirmed its potential role in controlling NF-κB signalling. Furthermore, an EMSA (electrophoretic mobility shift assay) showed that ChER could negatively regulate the binding of ChRel to NF-κB probe-responsive elements. Serial domain requirement analysis showed that both region C (DNA-binding domain) and region E (ligand-binding domain) of ChER were essential for mediating the crosstalk underlying ChER-dependent NF-κB suppression. In conclusion, we demonstrate for the first time the negative regulatory role of the ER in NF-κB signalling in oysters, strongly indicating the presence of complex crosstalk between the endocrine and immune systems in lower marine molluscs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

It is increasingly appreciated that neuroendocrine-immune interactions hold the key to understand the complex immune system. In this study, we explored the role of a reproductive regulation-related hormone, GnRH, in the regulation of immunity in Hong Kong oysters. We found that vibrio bacterial strains injection increased the expression of ChGnRH. Moreover, ChGnRH neuropeptide promotes the phagocytic ability and bacterial clearance effect of hemocytes which regarded to be the central immune organ. The content of cAMP after incubation with ChGnRH peptide was increased, which could be blocked by adenylyl cyclase inhibitor SQ 22,536. Furthermore, the stimulated effect of ChGnRH peptide on the phagocytosis and bacterial clearance was also blocked by SQ 22,536, H89 and enzastaurin, strongly demonstrating that cAMP dependent PKA and PKC signaling pathway was involved in ChGnRH mediated immune regulation. In conclusion, this study confirms the presence of neuroendocrine-immune regulatory system in marine invertebrates, which contributes to understand the complexity of oyster immune defense system., (Copyright © 2019. Published by Elsevier Ltd.)

NEMO (NF-κB essential modulator) is one of the important regulatory subunits of the IκB kinase (IκK) complex that controls the activation of the NF-κB signaling pathway. Here, we have identified the homolog of NEMO from the pacific oyster Crassostrea gigas. CgNEMO harbors the conserved the IκK binding region, NEMO ubiquitin binding domain and Zinc finger domain. In terms of tissue distribution, CgNEMO is expressed in various tissues with an observed highest expression in the hemocytes. Furthermore, infection by two related Vibrio strains significantly increased CgNEMO expression in the hemocytes. Cell culture based luciferase reporter assays showed that CgNEMO activates the NF-κB reporter in a dose-pendent manner. Moreover, CgNEMO was also found to counter the IkB-dependent inhibitory effect on NF-κB activation, providing a plausible mechanism of NF-κB activation by CgNEMO. Meanwhile, site-directed mutagenesis demonstrated that the putative ubiquitination site K535 is required for the activation of NF-κB, implying that ubiquitination of NEMO may be involved in regulating its activity. Finally, RNAi mediated knockdown of CgNEMO in vivo significantly compromised the bacterial induction of key cytokines TNF-α and IL-17, strongly suggesting a role for CgNEMO in acute immune defense in oyster. In conclusion, this study provides new insights into our understanding about the evolution of NEMO mediated NF-κB activation and the induction of cytokine. Our findings may provide valuable information about diseases control and management in oyster aquaculture., (Copyright © 2018. Published by Elsevier Ltd.)

In bivalve mollusks, circulating hemocyte mediated phagocytosis is one of the primary ways to eliminate invading microbes. Here, we have identified one CgLRFN (leucine-rich repeat and fibronectin type-III domain-containing protein) in the Crassostrea gigas as a novel transmembrane LRR (Leucine-rich repeat) domain containing protein in C. gigas, homologous to the jawless fish VLR protein, that plays an important role in recognizing bacteria and promoting hemocytic phagocytosis. Tissue distribution analysis of CgLRFN in Pacific oyster showed that it is widely expressed in various tissues like the gills, adductor muscles, digestive glands, gonads, heart and in the hemocytes. Furthermore, infection of Pacific oysters with two marine Vibrio strains V. alginolyticus and V. parahaemolyticus was found to significantly increase CgLRFN expression in the hemocytes. Analysis of subcellular localization showed that CgLRFN is primarily localized in the cell membrane. Additionally, CgLRFN was found to be able to bind both the bacterial strains, indicating its possible role as a cell surface receptor. Flow cytometry analysis revealed that CgLRFN coated bacteria was phagocytosed by oyster hemocytes at a significantly higher rate compared to the uncoated bacteria. Finally, RNAi mediated knockdown of CgLRFN in vivo resulted in reduced clearance of both the bacterial strains from the oyster hemolymph. Overall, our study demonstrates that CgLRFN acts as a pattern recognition receptor for Vibrio spp. and promotes hemocytic phagocytosis in the Pacific oyster, which is critical for understanding the mechanism of bacterial infection in lower invertebrates, and also contributes to disease management of this economically and ecologically important marine mollusk., (Copyright © 2017 Elsevier Ltd. All rights reserved.)