Search

Interleukin-17 (IL-17) represents a class of proinflammatory cytokines involved in chronic inflammatory and degenerative disorders. Prior to this study, it was predicted that an IL-17 homolog could be targeted by Mc-novel_miR_145 to participate in the immune response of Mytilus coruscus. This study employed a variety of molecular and cell biology research methods to explore the association between Mc-novel_miR_145 and IL-17 homolog and their immunomodulatory effects. The bioinformatics prediction confirmed the affiliation of the IL-17 homolog with the mussel IL-17 family, followed by quantitative real-time PCR assays (qPCR) to demonstrate that McIL-17-3 was highly expressed in immune-associated tissues and responded to bacterial challenges. Results from luciferase reporter assays confirmed the potential of McIL-17-3 to activate downstream NF-κb and its targeting by Mc-novel_miR_145 in HEK293 cells. The study also produced McIL-17-3 antiserum and found that Mc-novel_miR_145 negatively regulates McIL-17-3 via western blotting and qPCR assays. Furthermore, flow cytometry analysis indicated that Mc-novel_miR_145 negatively regulated McIL-17-3 to alleviate LPS-induced apoptosis. Collectively, the current results showed that McIL-17-3 played an important role in molluscan immune defense against bacterial attack. Furthermore, McIL-17-3 was negatively regulated by Mc-novel_miR_145 to participate in LPS-induced apoptosis. Our findings provide new insights into noncoding RNA regulation in invertebrate models. [ABSTRACT FROM AUTHOR]

Amyloodinium ocellatum (AO) infection in silver pomfret (Pampus argenteus) causes extensive mortality. Insufficient information exists on the molecular immune response of silver pomfret to AO infestation, so herein we simulated the process of silver pomfret being infected by AO. Translucent trophosomes were observed on the gills of AO‐infected fish. Transcriptome profiling was performed to investigate the effects of AO infection on the gill, kidney complex and spleen. Overall, 404,412,298 clean reads were obtained, assembling into 96,341 unigenes, which were annotated against public databases. In total, 2730 differentially expressed genes were detected, and few energy‐ and immune‐related genes were further assessed using RT‐qPCR. Moreover, activities of three immune‐related (SOD, AKP and ACP) and three energy‐related (PKM, LDH and GCK) enzymes were determined. AO infection activated the immune system and increased interleukin‐1 beta and immunoglobulin M heavy chain levels. Besides, the PPAR signalling pathway was highly enriched, which played a role in improving immunity and maintaining homeostasis. AO infection also caused dyspnoea, leading to extensive lactic acid accumulation, potentially contributing towards a strong immune response in the host. Our data improved our understanding regarding the immune response mechanisms through which fish coped with parasitic infections and may help prevent high fish mortality in aquaculture. [ABSTRACT FROM AUTHOR]

Background: Carrageenan is a traditional ingredient that has been widely used in the food industry. In the present study, we propose a hypothesis that carrageenan is a conditional inflammatory agent. When the intestinal tract is in an "unhealthy" state such as that during bacterial infection or acute inflammation, carrageenan can synergistically enhance the inflammatory response.Methods: BALB/C mice received κ-carrageenan via intragastric administration prior to the induction of oxazolone colitis. Weight changes, survival rate, histologic change, secretion of inflammatory cytokines, ratio of regulatory T cells (Tregs) in peripheral blood, and expression of genes and proteins involved in inflammation and cell proliferation in the colonic mucosa were examined.Results: Intragastric administration of κ-carrageenan to BALB/c mice prior to the induction of oxazolone colitis resulted in an aggravation of body weight loss, a decrease in the survival ratio, aggravation of colonic inflammation, and decrease in the ratio of CD4 + CD25+/CD4+. The secretion of interleukin-4 (IL-4), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) also significantly increased after κ-carrageenan administration. κ-Carrageenan, together with oxazolone, suppressed the expression of forkhead box p3 (FOXp3) and increased the expression of Toll-like receptor 4 (TLR4), Nuclear factor-κB (NF-κB), and proliferating cell nuclear antigen in the colonic mucosa. These results were confirmed by qRT-PCR and western blot analyses at the molecular and protein levels, respectively.Conclusions: κ-Carrageenan aggravated oxazolone-induced intestinal inflammation in BALB/c mice. This effect is associated with an activation of the TLR4-NF-κB pathway, a decreased ratio of Tregs, and the induction of Th2-dependent immune responses. [ABSTRACT FROM AUTHOR]

Mytilus shows great immune resistance to various bacteria from the living waters, indicating a complex immune recognition mechanism against various microbes. Peptidoglycan recognition proteins (PGRPs) play an important role in the defense against invading microbes via the recognition of the immunogenic substance peptidoglycan (PGN). Therefore, eight PGRPs were identified from the gill transcriptome of Mytilus coruscus. The sequence features, expression pattern in various organs and larval development stages, and microbes induced expression profiles of these Mytilus PGRPs were determined. Our data revealed the constitutive expression of PGRPs in various organs with relative higher expression level in immune-related organs. The expression of PGRPs is developmentally regulated, and most PGRPs are undetectable in larvae stages. The expression level of most PGRPs was significantly increased with in vivo microbial challenges, showing strong response to Gram-positive strain in gill and digestive gland, strong response to Gram-negative strain in hemocytes, and relative weaker response to fungus in the three tested organs. In addition, the function analysis of the representative recombinant expressed PGRP (rMcPGRP-2) confirmed the antimicrobial and agglutination activities, showing the immune-related importance of PGRP in Mytilus. Our work suggests that Mytilus PGRPs can act as pattern recognition receptors to recognize the invading microorganisms and the antimicrobial effectors during the innate immune response of Mytilus. • Eight PGRP genes were identified from the gill transcriptome of M. coruscus. • The expression profiles of the eight Mytilus PGRP genes were determined. • McPGRPs showed different responses to different in vivo microbial challenges. • Recombinant McPGRP-2 showed classical PGRP functions. [ABSTRACT FROM AUTHOR]

Histones and their N-terminal or C-terminal derived peptides have been studied in vertebrates and presented as potential antimicrobial agents playing important roles in the innate immune defenses. Although histones and their derived peptides had been reported as components of innate immunity in invertebrates, the knowledge about the histone derived antimicrobial peptides (HDAPs) in invertebrates are still limited. Using a peptidomic technique, a set of peptide fragments derived from the histones was identified in this study from the serum of microbes challenged Mytilus coruscus. Among the 85 identified histone-derived-peptides with high confidence, 5 HDAPs were chemically synthesized and the antimicrobial activities were verified, showing strong growth inhibition against Gram-positive bacteria, Gram-negative bacteria, and fungus. The gene expression level of the precursor histones matched by representative HDAPs were further tested using q-PCR, and the results showed a significant upregulation of the histone gene expression levels in hemocytes, gill, and mantle of the mussel after immune stress. In addition, three identified HDAPs were selected for preparation of specific antibodies, and the corresponding histones and their derived C-terminal fragments were detected by Western blotting in the blood cell and serum of immune challenged mussel, respectively, indicating the existence of HDAPs in M. coruscus. Our findings revealed the immune function of histones in Mytilus , and confirmed the existence of HDAPs in the mussel. The identified Mytilus HDAPs represent a new source of immune effector with antimicrobial function in the innate immune system, and thus provide promising candidates for the treatment of microbial infections in aquaculture and medicine. • Abundant HDAPs were identified from Mytilus coruscus serum. • Synthesized Mytilus HDAPs showed strong growth inhibition against bacteria and fungus. • The histone gene expression level of Mytilus histones showed significant upregulation in tissues of challenged mussel. • Western blotting with specific antibody confirmed the existence of HDAPs in M. coruscus serum. [ABSTRACT FROM AUTHOR]

Nocardia seriolae , a widespread opportunistic pathogen, has the ability to infect various economically farmed fishes, leading to prolonged disease courses and high mortality rates. In our current study, we focused on the silver pomfret (Pampus argenteus) and examined the liver, spleen, and kidney tissues at three specific N. seriolae infection phases: control (0 day), L group (infection for 5 days), and H group (infection for 15 days). Utilizing HE staining, we observed typical granulomatous nodules displaying stratified concentric layers, including caseous necrotic cells, epithelioid cells and multinucleated giant cells, fibroblasts and fibrocyte envelopes. The TUNEL assay revealed a significant presence of apoptotic cells concentrated within the renal nodules. Following the infection, there was a noticeable increase in AKP, ACP, and LZM levels in all tissues, while LPS exhibited a consistent decline. Through combined analysis of WGCNA and KEGG pathways derived from the tissue transcriptome, we identified several highly enriched pathways, such as the PI3K-Akt signaling pathway and p53 signaling pathway, in all three tissues. Key DEGs from these pathways underwent further investigation through RT-qPCR. In comparison to the control group, the L group exhibited up-regulation of genes associated with biosynthesis catabolism, whereas genes related to immune response, apoptosis, and ion transport displayed down-regulation. Interestingly, the H group showcased a stark reversal of this trend. To validate these DEGs expressions in vitro, we established a Pampus argenteus spleen cell line (PaS). By evaluating the invasion metrics of NS into PaS, we determined an optimal invasion threshold of 105 CFU/ml. The TUNEL assay demonstrated a notable presence of apoptotic signals in the 5 h and 7 h treatment groups. JC-1 assays indicated a gradual decline in membrane potential starting from the 3-h mark. As the duration of treatment increased, RT-qPCR and IHC findings highlighted an initial up-regulation followed by a subsequent down-regulation of genes associated with biosynthesis catabolism. In contrast, genes related to innate immunity displayed up-regulation, whereas genes related to adaptive immunity and ion transport exhibited an initial surge followed by a gradual decrease. Additionally, genes inhibiting apoptosis showed down-regulation. In conclusion, these findings provide valuable insights into the apoptosis mediated by N. seriolae in piscine hosts. These effects are potentially attributed to the increased expression of ion transport-related genes. Moreover, our study sheds light on the intricate molecular mechanisms underlying piscine immunological responses throughout the entire infection timeline, offering promising opportunities for disease control. • N. seriolae infection caused quintessential granulomatous nodules. • Many apoptotic cells centrally located within the renal nodules. • The weakening of metabolism declined acquired immunity after N. seriolae infection. • We constructed the spleen tissue cell line of silver pomfret. • The apoptosis caused by N. seriolae might be related to ion transport. [ABSTRACT FROM AUTHOR]

Photobacterium damselae subsp. damselae (PDD) is a dangerous bacterial disease that affects silver pomfret (Pampus argenteus) and its main virulence factors are pore-forming toxins. In this study, a highly pathogenic strain was obtained from naturally infected silver pomfret and was introduced into healthy fish for experimentation. Liver, gill, kidney and spleen samples were collected at 0, 24, 48, 72 and 96 h. Histopathological evaluations of these tissues, via HE staining, showed multi-organ bleeding, inflammatory infiltration and histopathocyte vacuolation as the main pathological changes. The levels of AKP, ACP, LZM, SOD, GSH and CAT in serum also displayed an increase following infection. Transcriptome sequencing of liver, kidney and spleen was performed using samples 96 h post-infection. The analysis of the resulting 770,878,120 clean reads collectively assembled into 89,310 unigenes. The WGCNA analysis demonstrated enrichment of phagosome and cytokine-cytokine receptor interaction in the liver, drug metabolism-cytochrome P450 in the kidney, and MAPK signaling pathway and other signaling pathways in the spleen. DEGs related to immune and immune-related pathways, such as Phagosome, Autophagy-animal and Glycerolipid metabolism, were significantly enriched. Selected key genes from these KEGG pathways were examined using RT-qPCR. The expression level of autophagy related genes (pi3k, bcl2) increased after infection, while the gene associated with MAPK and apoptosis (Caspase 9, mapk4) significantly increased after 72 h infection. The expression of phagosome related genes (atp6v1f, ctsl) increased initially, but later decreased. The genes related to innate immunity (ccl4, il15) were up-regulated, while those related to acquired immunity (mhc2a, mhc2b) showed a downward trend. Lysosome-related genes (gilta, giltb, cftr, c-type lectin, m6pra and m6prb) were up-regulated in liver and spleen and down-regulated in kidney and gill. The data obtained illustrated that PDD activated the immune defenses of the fish, increasing metabolic expenditure, but that the pore-forming toxins released by PDD could destroy the phagosome membrane and help the bacteria escape, which could disturb antigen presentation and inhibit the activation of the acquired immunity. This study reveals the potential immune escape mechanism of PDD in the host for the first time and has the potential to aid in the development of anti-PDD drugs. • PDD infection caused multi-organ bleeding, inflammatory infiltration in fish tissues. • Immune relating enzyme activities increased after PDD infection. • PDD activated the immune defenses and increased metabolic expenditure. • Pore-forming toxins of PDD might destroy phagosome and help PDD escape. • This immune escape might disturb antigen presentation and inhibit acquired immunity. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLR s) are vital pattern recognition receptors that play a critical role in the innate immune response against pathogenic attack. Among the bacteria commonly found in the culture process of silver pomfret, Photobacterium damselae subsp. Damselae (PDD, gram-negative) and Nocardia seriolae (NS, gram-positive), can cause large-scale mortality in this fish species. However, there is currently no research on the role of TLR s in mediating the immune response of silver pomfret to these two bacterial infections. Therefore, in this study, we identified nine PaTLRs family members, including several fish-specific TLRs (TLR14 and TLR21). Phylogenetic analysis revealed that these PaTLRs genes could be classified into five subfamilies, namely TLR1 , TLR3 , TLR5 , TLR7 , and TLR11 , indicating their evolutionary conservation. To further explore the interactions of TLR genes with immune-related mediators, protein and protein interaction network (PPI) results were generated to explain the association of TLR genes with TNF receptor-associated factor 6 (TRAF6) and other relevant genes in the MyD88 -dependent pathway and NF-κb signaling pathway. Subsequently, RT-qPCR was conducted to verify the expression patterns of the nine TLR genes in the gills, skin, kidney, liver, and spleen of healthy fish, with most of the TLRs showing high expression levels in the spleen. Following infection with PDD and NS, these PaTLRs exhibited different expression patterns in the spleen, with PaTLR2 , PaTLR3 , PaTLR5 , PaTLR7 , PaTLR9 , and PaTLR14 being significantly up-regulated. Furthermore, when spleen cells were treated with bacterial compositions, the majority of PaTLR s expression was up-regulated in response to Lipopolysaccharide (LPS) and lipophosphorylcholic acid (LTA) treatment, except for PaTLR21. Finally, changes in the expression levels of TLR -interacting genes were also observed under the stimulation of bacteria and bacterial compositions. The results of this study provide a preliminary reference for further understanding the mechanism of the innate immune response of the TLR gene family in silver pomfret and offer theoretical support for addressing the disease problems encountered during large-scale fish breeding. • We identified and characterized all 10 TLR genes in Pampus argenteus. • PaTLRs were classified into six subfamilies, revealing evolutionary conservation. • PaTLRs were widely expressed in the immune-related tissues, especially the spleen. • Most PaTLRs in spleen response to Gram-positive/negative bacterial infection. [ABSTRACT FROM AUTHOR]

Abstract Formaldehyde can effectively control ectoparasites in silver pomfret (Pampus argenteus). However, there is limited information on the effects of formaldehyde treatment at a molecular level in fishes. In the present study, transcriptome profiling was conducted to investigate the effects of formaldehyde treatment (80 mg/L, bath for 1 h every day for three consecutive days) on the liver and kidney tissues of silver pomfret. A total of 617959982 clean reads were obtained and assembled into 265760 unigenes with an N50 length of 1507 bp, and the assembled unigenes were all annotated by alignment with public databases. A total of 2204 differentially expressed genes (DEGs) were detected in the liver and kidney tissues, and they included 7 detoxification- related genes and 9 immune-related genes, such as CYP450 , GST , MHC I & II , and CCR. In addition, 1440 DEGs were mapped to terms in the GO database, and 1064 DEGs were mapped to the KEGG database. The expression of 4 detoxification-related genes and 6 immune-related genes in three days formaldehyde treatment were analyzed using RT-qPCR, and the antioxidant enzyme levels were also determined. The results indicate differential expression of detoxification- and immune-related genes during the three days formaldehyde treatment. Our data could provide a reference for the treatment of parasites to avoid high mortality and help in understanding the molecular activity in fishes after formaldehyde exposure. Highlights • RNA-seq was performed to investigate the responses of detoxification- and immune related genes to formaldehyde exposure. • The formaldehyde treatment of parasites was reported to avoid high mortality in aquaculture. • The treatment was assessed to be safe by mortality of fish, transcriptome level and antioxidant enzymes. [ABSTRACT FROM AUTHOR]

Cryptorchidism irritant (CI) infection is a major problem in the culturing process of silver pomfret (Pampus argenteus), which can result in rapid and massive death. However, there is limited information available on the immune response of silver pomfret infected by CI. To address this gap, we sampled naturally infected fish and observed milky white translucent oval CI trophozoites on the gills, body surface, and fin rays. Histological analysis showed that CI infection led to vacuolation of epithelial cells and a decrease in blood cells in the gills. We also performed transcriptome profiling of the gill, kidney complex, and spleen, generating 399,616,194 clean reads that assembled into 101,228 unigenes, which were annotated based on public databases. We detected 14,369 differentially expressed genes, and selected several key immune-related genes for further validation using RT-qPCR. The Graft-versus-host pathway and Allograft rejection pathway were enriched in the gills, leading to inflammation and ulceration. CI infection activated the immune system, increasing levels of interleukin-1 beta and MHC class II antigen, and also activated innate and acquired immune genes in silver pomfret. Furthermore, we measured the activities of five immune-related enzymes (SOD, AKP, CAT, CSH and ACP), which all increased to varying degrees after CI infection. Our findings enhance our understanding of the immune response of fish to parasitic infection and may contribute to the development of strategies to prevent high mortality in CI-stimulated fish in aquaculture. • CI infection caused damage to the gill tissue and the shedding of chloride secreting cells. • CI infection induced innate and acquired immune and apoptosis of silver pomfret. • CI infection could cause the accumulation of peroxides. [ABSTRACT FROM AUTHOR]

Apelin, a pleiotropic cytokine with various biological activities, plays important roles in both infectious and noninfectious inflammatory diseases in mammals by regulating immune responses. However, the effect of apelin on the host defenses against microbial infections in teleosts is still unknown. Therefore, this study aimed to characterize the molecular features of apelin in Larimichthys crocea (designated LCapelin) and to investigate the roles of LCapelin in bacterial infection in vivo. The complete open reading frame (ORF) of LCapelin , which encodes a 77-amino acid precursor, was cloned and identified. In healthy fish, LCapelin was constitutively expressed in all 10 tissues examined, and the highest expression level was observed in muscle tissue. Following a challenge with Vibrio parahemolyticus , the mRNA expression of LCapelin was significantly increased at 4 hpi in the spleen. However, in the liver, the expression of LCapelin decreased initially but gradually returned to normal. Furthermore, pretreatment with the synthetic peptide LCapelin-13 prior to Vibrio parahaemolyticus challenge significantly ameliorated clinical signs, inhibited the expression of inflammatory cytokines (TNF-α, IL-1β, and IL-6) in the spleen and liver, decreased the proliferation of splenocytes and melanomacrophage centers (MMCs) in the spleen, and promoted bacterial clearance. Taken together, our results show that LCapelin participates in host defense against V. parahaemolyticus infection in L. crocea and may have potential application in the development of new strategies to combat infectious diseases in teleosts. • LCapelin expression responded to Vibro parahaemolyticus challenge in spleen and liver. • LCapelin significantly ameliorated the clinical signs of experimental vibriosis. • LCapelin inhibited Vibrio parahaemolyticus induced-expression of inflammatory cytokines. • LCapelin promoted bacterial clearance in vivo. [ABSTRACT FROM AUTHOR]

Mytilus can survive in heavily anthropized coastal waters despite being constantly surrounded by abundant microorganisms, indicating an environmental adaptation and therefore an important research significance for this species. However, there is a qualitative and quantitative lack of knowledge of the molecular mechanisms involved in the physiology and immune response of Mytilus tissues, especially in tissues other than hemocytes. For filling this gap, we have studied the transcriptome of gill and gonad of M. coruscus exposed to distinct stimuli, including antibiotics pretreatment and subsequent different bacteria challenges at two time points. A total of 36 cDNA libraries were sequenced and 245.01 Gb clean data was acquired with an average of 5.91 GB and Q30 > 94% for each library. The filtered high-quality reads were assembled into 91,850 transcripts, which after clustering resulted in 59,206 unigenes. Overall, 19,783 differentially expressed genes (DEGs) were detected from the pair-wise comparisons among the gill samples, and 12,655 DEGs among the gonad samples. Comparing with the gonad, the gill showed strong immune-related responses towards bacteria challenge, with abundant immune-related signaling pathway being disturbed, highlighting the important roles of gill in mussel immune system and the tissue specificity of mussel immune responses. The gonad showed that the DEGs induced by bacteria were mainly enriched in regulatory pathways related to cell growth and proliferation. In addition, both gill and gonad presented rapid activation towards Micrococcus luteus infection comparing with that of Vibrio parahemolyticus , indicating a more sensitivity of mussel to Gram-positive bacteria. Our data revealed different immune responses in different mussel tissues under different bacteria challenges, and thus provided clues for exploring the mechanism of mussel immune system. • The transcriptome of M. coruscus gill and gonad were analyzed under distinct stimuli. • Antibiotics pretreatment of mussel provided a clean background for bacteria challenge. • The gill showed a stronger immune response to bacteria challenge than that of gonad. • Both gill and gonad showed rapid immune responses towards Gram-positive bacterium. • Gram-negative bacterium challenge induced the immune response with fall-rise pattern. [ABSTRACT FROM AUTHOR]