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  • In teleosts GR is expressed in almost every cell

    2021-09-23

    In teleosts, GR is expressed in almost every cell and regulates genes controlling development, metabolism, and immune response [17,18]. Several teleosts contain two GR genes, GR1 and GR2 [8]. Other groups have found only one GR in some species thus far, such as the Japanese flounder, brown trout, and zebrafish [19]. Furthermore, GR has been identified in a number of teleost species such as rainbow trout (Oncorhynchus mykiss) [5], common carp (Cyprinus carpio L) [20], and zebrafish (Danio rerio) [21]. Currently, there are limited studies focused on host-pathogen interactions in which an association between GR and immune response has been evaluated in macrophages after being challenged NADP/NADPH Quantitation Colorimetric Kit with highly pathogenic bacteria. Plecoglossus altivelis, commonly referred to as ayu, is an economically important fish in East Asia. Bacterial diseases caused by V. anguillarum have become widespread in this fish [22]. Innate immune response is considered to be the first line of host defense in opposing pathogenic infection in fish [23]. The innate immune response of teleost fishes occurs primarily in lymphoid organs such as head kidney and spleen, which produce NADP/NADPH Quantitation Colorimetric Kit and humoral parameters responsible for clearing a pathogen [24]. A deeper understanding of the regulation of the fish's innate immune response is currently required. In teleosts, transcription factors have been found to regulate the expression of immune genes. However, the intracellular signaling mechanisms of ayu monocytes/macrophages (MO/MФ) are still unknown. Given the important roles of transcription factors in the inflammatory response, studying and investigating their possible roles in pathological processes is a worthwhile endeavor. Therefore, the aim of this study was to evaluate the GR profile and modulation of stress- and innate immune-related genes in macrophages challenged with Vibrio anguillarum. Here, we determined the cDNA sequence of GR (PaGR) from ayu and analyzed the association between PaGR mRNA expression and protein levels after V. anguillarum infection. The subcellular localization of PaGR was analyzed. Moreover, the effect of PaGR on the expression of inflammatory cytokines, phagocytosis, bacterial killing, and apoptosis of MO/MФ by genomic or non-genomic pathways during infection were investigated.
    Materials and methods
    Results
    Discussion GR are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors. Previous studies have shown that GR are involved in the immune response [8,17]. However, the function of GR in an immune response after being challenged with pathogenic bacteria remains unclear. In the present study, we identified the cDNA sequence of a PaGR protein from ayu, a teleost fish. Multiple alignment of ayu GR with other teleost GR sequences revealed that the DBD, zinc finger regions and LBD were highly conserved. Phylogenetic analysis showed that PaGR was most closely related to rainbow trout GR2. In addition, GR gene and functions have been characterized in several teleosts, including zebrafish, rainbow trout, and Japanese ricefish. A study in zebrafish showed that activation of GR inhibits the expression of pro-inflammatory cytokines and decreases the migration of neutrophils [32]. In rainbow trout, GR signaling induced the expression of reactive oxygen species (ROS) via a non-genomic pathway [33]. In the present work, we identified a novel GR gene in ayu, and analyzed the subcellular localization of PaGR. Moreover, PaGR regulated inflammatory cytokine production, suppressed phagocytosis, promoted bacterial killing and apoptosis in MO/MΦ after live V. anguillarum infection. Taking together, these results suggest teleost GR play a crucial role in the immune system and can provide valuable insights into the role of nuclear hormone receptors in innate immunity from teleosts to mammals. In humans, GR cDNA was isolated by expression cloning in 1985, and GR is expressed virtually in all organs and tissue of mammals [3]. In the present study, PaGR was detected in all the tested tissues, including the main immune organs, the liver, head kidney, and spleen; the highest mRNA expression was detected in the muscle. Unlike PaGR, gilthead seabream GR mRNA is highly expressed in the heart and brain [34]. In channel catfish, expression of GR transcript is found in a number of immune tissues and is widely distributed in the gill and intestine [35]. Estuarine tapertail anchovy GR mRNA is strongly expressed in the liver and muscle [36]. The discrepancies in GR expression might result from both species variation and differences in immunological status, growth stage, genetic background, and environment. After V. anguillarum infection, PaGR expression was up-regulated in the liver, spleen, and head kidney, suggesting that PaGR expression was induced by infection. In gilthead seabream, GR transcripts were up-regulated in spleen, brain, and head kidney after LPS injection [34]. These tissues are important immune organs in teleosts and play a role in the immune response upon pathogen invasion [37]. Previous reports have shown that GR expression is up-regulated in neutrophils and B lymphocytes in both blood and spleen after LPS treatment [38]. Hence, both mRNA and protein levels of PaGR were also significantly up-regulated in MO/MΦ after infection with live V. anguillarum. Together, these results suggested that PaGR expression is closely related to the ayu immune response against pathogenic infections.