Expression Levels Of Immune-related Genes Research Articles

Evaluation of the dietary taurine requirement for early juvenile mud crab Scylla paramamosain

As a conditionally essential amino acid (CEAA), taurine plays an important role in the growth, development, and physiological metabolism of aquatic animals; however, its demand level in the mud crab Scylla paramamosain is still poorly understood. A 60-day growth trial was conducted to investigate the effects of different taurine levels (0% (T0.0), 0.2% (T0.2), 0.4% (T0.4), 0.8% (T0.8), and 1.6% (T1.6)) added to diet on the survival, growth, body composition, immunity, antioxidant and osmoregulatory capacity of early juvenile S. paramamosain (initial body weight 8.69 ± 0.19 mg). The results showed that dietary taurine levels >0.4% (inclusive) significantly increased the survival of early juvenile mud crabs. The growth performance (final body weight (FBW), weight gain (WG), specific growth rate (SGR)) of early juvenile mud crabs increased significantly with increasing dietary taurine levels, reaching a maximum in the T0.8 group, and the target of rapamycin (tor) gene transcription levels also peaked in the T0.8 group. The body composition of mud crabs was not affected by dietary treatments. Further, dietary taurine significantly increased digestive enzyme (trypsin, lipase, and amylase) activities and transcript levels of appetite-related genes (neuropeptide F1 (npf1) and neuropeptide F2 (npf2)) in early juvenile mud crabs. In addition, dietary taurine increased the expression levels of immune-related genes (prophenoloxidase (proPO), prophenoloxidase activating factor 1 (ppaf1), prophenoloxidase activating factor 2 (ppaf2), crustin antimicrobial peptide (crustin), lysozyme (lzm)) and antioxidant capacity (total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and catalase (CAT) activities) in early juvenile mud crabs. Finally, the results of the acute salinity challenge showed that the early juvenile mud crabs' survival in the T0.8 group was significantly higher than that of the other groups, while significantly higher Na+/K+-ATPase (NKA) activity and Na+/K+-ATPase α-subunit (nka-α) and Na+/K+/2Cl−-cotransporter (nkcc) gene expression levels were also detected in the T0.8 group. Together with growth performance, immunity, antioxidant capacity and osmoregulatory capacity, the present study concluded that the appropriate dietary taurine level for early juvenile mud crabs was 1.08%–1.33%.

RNA interference confirmed a close association of CYP, ApD, and UCH with the function of CpG ODNs in Litopenaeus vannamei

CpG oligonucleotides (CpG ODNs) have been demonstrated to enhance the innate immune response in various organisms, including Litopenaeus vannamei, a species of significant economic importance. However, the precise mechanism or the genetic factors of CpG ODNs underlying function in L. vannamei remained elusive. Our previous experiment has identified five potential marker genes, including cytochrome P450 (CYP), gamma-butyrobetaine dioxygenase (γ-BDO), apolipoprotein D (ApD), prostaglandin reductase 1 (PGR1), and ubiquitin carboxyl-terminal hydrolase (UCH). In this study, RNA interference (RNAi) was employed to validate the relevance of these five genes in responding the stimulation of CpG ODNs. Briefly, shrimps were fed CpG ODNs and subsequently injected dsRNA targeting these five genes for interference. Finally, the non-specific immune parameters of shrimps, including the expression of immune-related genes and antioxidant capacity of hepatopancreas, as well as the mortality of shrimps after pathogen attack were assessed. The tissue distribution analysis revealed a high abundance of CYP in the hepatopancreas, γ-BDO in the hemocytes, ApD in the eyestalk, PGR1 in the hemocytes, and UCH in the gonad. After blocking CYP, ApD, and UCH through RNAi, a significant down-regulation of the mRNA expression levels of immune-related genes, including ALF, TNF-α, Toll, and TOR, was observed. Additionally, a notable decline in the activity of antioxidant enzymes, such as CAT, GPX, GST, and SOD, was also observed. Moreover, the mortality rates of shrimps in the CYP, ApD, and UCH inhibited groups significantly increased compared to other treatment groups following exposure to Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VpAHPND) and white spot syndrome virus (WSSV). These results collectively suggest a close association of CYP, ApD, and UCH with the function of CpG ODNs in shrimps which will provide significant theoretical insights into comprehending the mechanism of CpG ODNs and optimizing their practical application in shrimp's aquaculture.

Systemic immune response of rainbow trout exposed to Flavobacterium psychrophilum infection

Bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum is one of the most serious bacterial diseases leading to significant economic loss for rainbow trout (Oncorhynchus mykiss) aquaculture. However, little is known about the systemic immune response of rainbow trout against F. psychrophilum infection. This study investigated the immune response of rainbow trout to F. psychrophilum infection using multiple experiments, including bacterial load detection, phagocyte activity assessment, enzyme activity evaluation, and gene expression profiling. Results showed that the spleen index and intestinal pathogen load reached a peak at 3 days post-infection, with strong pro-inflammatory gene expression observed in rainbow trout. Leukocytes RBA and PKA were significantly elevated in the spleen, blood and intestine at 7 days post-infection. Heat map analysis demonstrated that the spleen had a more substantial pro-inflammatory response compared to the intestine post-infection and exhibited higher expression levels of immune-related genes, including IgM, il1β, il6, cd4, cd8a, cd8b, c1q, chathelicidin, inos, and lysozyme. Both Th1 and Th2 polarized responses in the spleen were activated, with Th2 (il4/13a, gata3) (FC > 4) being more intense than Th1 (tnfα, t-bet) (FC > 2). Tight junction proteins exhibited down-regulation followed by up-regulation post-infection. Collectively, the results of this study expand our current understanding of the immune response of rainbow trout post F. psychrophilum infection but also provide new avenues for investigation in salmonid aquaculture.

A nanocarrier immersion vaccine encoding surface immunogenic protein confers cross-immunoprotection against Streptococcus agalactiae and Streptococcus iniae infection in tilapia

Streptococcosis is a highly contagious aquatic bacterial disease that poses a significant threat to tilapia. Vaccination is a well-known effective measure to prevent and control fish bacterial diseases. Among the various immunization methods, immersion vaccination is simple and can be widely used in aquaculture. Besides, nanocarrier delivery technology has been reported as an effective solution to improve the immune effect of immersion vaccine. In this study, the surface immunogenic protein (Sip) was proved to be conserved and potential to provide cross-immunoprotection for both Streptococcus agalactiae (S. agalactiae) and Streptococcus iniae (S. iniae) by multiple sequences alignment and Western blotting analysis. On this basis, we expressed and obtained the recombinant protein rSip and connected it with functionalized carbon nanotubes (CNT) to construct the nanocarrier vaccine system CNT-rSip. After immersion immunization, the immune effect of CNT-rSip against above two streptococcus infections was evaluated in tilapia based on some aspects including the serum specific antibody level, non-specific enzyme activities, immune-related genes expression and relative percent survival (RPS) after bacteria challenge. The results showed that compared with control group, CNT-rSip significantly (P < 0.05) increased the serum antibody levels, related enzyme activities including acid phosphatase, alkaline phosphatase, lysozyme and total antioxidant capacity activities, as well as the expression levels of immune-related genes from 2 to 4 weeks post immunization (wpi), and all these indexes peaked at 3 wpi. Besides, the above indexes of CNT-rSip were higher than those of rSip group with different extend during the experiment. Furthermore, the challenge test indicated that CNT-rSip provided cross-immunoprotection against S. agalactiae and S. iniae infection with RPS of 75 % and 72.41 %, respectively, which were much higher than those of other groups. Our study indicated that the nanocarrier immersion vaccine CNT-rSip could significantly improve the antibody titer and confer cross-immuneprotection against S. agalactiae and S. iniae infection in tilapia.

Efficacy of a formalin-inactivated vaccine against Lactococcus garvieae infection in golden pompano Trachinotus ovatus

Lactococcus garvieae is one of the primary pathogens causing the severe disease to golden pompano (Trachinotus ovatus). Vaccination is the most effective measure of preventing the occurrence and spread of infectious diseases. However, no commercially available vaccine protects T. ovatus against L. garvieae infection. In the present study, the formalin-inactivated L. garvieae vaccines, consisting of bacterin alone (aqueous vaccine) and combined with oil adjuvant Montanide™ ISA 763 A VG (vaccine+763A), were prepared and administered to T. ovatus by intraperitoneal injection. The protective efficacy was evaluated by testing the relative percent survival (RPS), serum enzyme activity, and expression levels of immune-related genes in the tissues of T. ovatus after immunization. The results indicated that the relative percentage survival (RPS) values of aqueous vaccine and vaccine+763A were 80% and 90.8% at 8 weeks post-vaccination. The level of antibody titer, lysozyme activities, superoxide dismutase, and catalase in the two vaccinated fish groups increased significantly compared to that in the control group. Moreover, the expression levels of immune-related genes IgM, TNF-α, IL-8, MHCI-α, MHCII, CD4, CD8α, and IL-1β in all the tested tissues from T. ovatus were significantly enhanced after inoculation with aqueous vaccine and vaccine+763A. These findings demonstrated that the developed vaccine combined with oil adjuvant ISA 763 A VG improved immune responses and provided good protective efficacy against L. garvieae infection in the golden pompano culture.

Unraveling The Effects of DICER1 Overexpression on Immune-Related Genes Expression in Mesenchymal Stromal/Stem Cells: Insights for Therapeutic Applications.

The immunoregulatory properties of mesenchymal stromal/stem cells (MSCs) bring a promise for the treatment of inflammatory diseases. However, their ability to suppress the immune system is unstable. To enhance their effectiveness against immune responses, it may be necessary to manipulate MSCs. Although some dsRNA transcripts come from invading viruses, the majority of dsRNA has an endogenous origin and is known as endo-siRNA. DICER1 is a ribonuclease protein that can generate small RNAs to modulate gene expression at the post-transcriptional level. We aimed to evaluate the expression of several immune-related genes at mRNA and protein levels in MSCs overexpressing DICER1 exogenously. In this comparative transcriptomic experimental study, the adipose-derived MSCs (Ad-MSCs) were transfected using the pCAGGS-Flag-hsDicer vector for the DICER1 overexpression. Following the RNA extraction, mRNA expression level of DICER1 and several inflammatory cytokines were examined. We performed a relative real-time polymerase chain reaction (PCR) assay and transcriptome analysis between two groups including DICER1- transfected MSCs and control MSCs. Moreover, media from the transfected MSCs were evaluated for various interferon response factors by ELISA. The overexpression of DICER1 is associated with a significant increase in the mRNA expression level of COX-2, DDX-58, IFIH1, MYD88, RNase L, TLR3/4, and TDO2 genes and a downregulation of the TSG-6 gene in MSCs. Moreover, the expression levels of IL-1, 6, 8, 17, 18, CCL2, INF-γ, TGF-β, and TNF-α were higher in the DICER1-transfected MSCs group. It seems that the ectopic expression of DICER1 in Ad-MSCs is linked to alterations in the expression level of immune-related genes. It is suggested that the manipulation of immune-related pathways in MSCs via the Dicer1 overexpression could facilitate the development of MSCs with distinct immunoregulatory phenotypes.

Mitfa deficiency promotes immune vigor and potentiates antitumor effects in zebrafish

The mitfa gene is a well-known transcription factor associated with microphthalmia and is essential for early melanophore development. However, little is known about how mitfa affects the immune system. Here, we generated a novel mitfa knock-out zebrafish line using the CRISPR/Cas9 system. The mitfa−/− zebrafish exhibited reduced melanin levels compared to the nacre mutant. We investigated the impact on the immune system after exposure to Edwardsiella tarda and bifenazate in zebrafish larvae, and observed that the macrophage numbers were reduced in both treated groups. Remarkably, the expression levels of immune-related genes exhibited significant increases after bacterial challenge or bifenazate exposure in the mitfa−/− zebrafish, except for tlr4 and rela. Furthermore, we conducted xenograft experiments using mouse B16 melanoma cells. Notably, the cancer cells didn't show a high cell migration ratio, implying that the immune system was highly activated after the loss of mifta. Taken together, our findings suggest that mitfa−/− zebrafish serve as a valuable model for investigating the relationship between the immune system and melanocytes, providing new insights into the role of mitfa in immune responses.

Exploration of Key Immune-Related Transcriptomes Associated with Doxorubicin-Induced Cardiotoxicity in Patients with Breast Cancer

Based on a few studies, heart failure patients with breast cancer were assessed to find potential biomarkers for doxorubicin-induced cardiotoxicity. However, key immune-related transcriptional markers linked to doxorubicin-induced cardiotoxicity in breast cancer patients have not been thoroughly investigated. We used GSE40447, GSE76314, and TCGA BRCA cohorts to perform this study. Then, we performed various bioinformatics approaches to identify the key immune-related transcriptional markers and their association with doxorubicin-induced cardiotoxicity in patients with breast cancer. We found 255 upregulated genes and 286 downregulated genes in patients with doxorubicin-induced heart failure in breast cancer. We discovered that in patients with breast cancer comorbidity doxorubicin-induced cardiotoxicity, the 58 immunological genes are elevated (such as CPA3, VSIG4, GATA2, RFX2, IL3RA, and LRP1), and the 60 genes are significantly suppressed (such as MS4A1, FCRL1, CD200, FCRLA, FCRL2, and CD79A). Furthermore, we revealed that the immune-related differentially expressed genes (DEGs) are substantially associated with the enrichment of KEGG pathways, including B-cell receptor signaling pathway, primary immunodeficiency, chemokine signaling pathway, hematopoietic cell lineage, cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, MAPK signaling pathway, focal adhesion, dilated cardiomyopathy, cell adhesion molecule, etc. Moreover, we discovered that the doxorubicin-induced immune-related genes are crucially involved in the protein–protein interaction and gene clusters. The immune-related genes, including IFIT5, XCL1, SPIB, BTLA, MS4A1, CD19, TCL1A, CD83, CD200, FCRLA, CD79A, BIRC3, and IGF2R are significantly associated with a poor survival prognosis of breast cancer patients and showed diagnostic efficacy in patients with breast cancer and heart failure. Molecular docking revealed that the survival-associated genes interact with the doxorubicin with appreciable binding affinity. Finally, we validated the expression level of immune-related genes in breast cancer patients-derived cardiomyocytes with doxorubicin-induced cardiotoxicity and found that the level of RAD9A, HSPA1B, GATA2, IGF2R, CD200, ERCC8, and BCL11A genes are consistently dysregulated. Our findings offered a basis for understanding the mechanism and pathogenesis of the cardiotoxicity caused by doxorubicin in breast cancer patients and predicted the interaction of immune-related potential biomarkers with doxorubicin.

Effectiveness of dietary heat-killed Bacillus subtilis harboring plasmid containing 60 copies of CpG-ODN 1668 against Vibrio harveyi in Penaeus vannamei.

The cost of the purification process hinders the extensive use of cytosine phosphate guanosine-oligodeoxynucleotides (CpG-ODNs) for shrimp culture. Therefore, this study used a shuttle vector plasmid to carry 60 copies of CpG-ODN 1668 (pAD43-25_60CpG), which can replicate in Escherichia coli and Bacillus subtilis strain RIK1285. The first experiment used a reverse gavage procedure to deliver a substance (PBS [CK], pAD43-25 [P0], and pAD43-25_60CpG [P60], respectively) directly into the anterior midgut of Penaeus vannamei and transcriptome sequence analysis with a reference genome was performed to examine the expression of well-known immune-related genes. The results showed that the expression levels of immune-related genes in P60 group were significantly increased, particularly those associated with AMPs. In addition, using RT‒qPCR, the expression levels of AMP genes (LvALF, LvPEN-2, and LvPEN-3) in the P60 group may vary depending on the tissue and time point. The second experiment used dietary supplementation with three kinds of heat-killed B. subtilis (HKBS, HKBS-P0, and HKBS-P60) in 28 days of feeding experiments. The results showed that dietary supplementation with HKBS-P60 did not significantly improve shrimp growth performance and survival. However, on days 14 and 28 of the feeding regimens, alkaline phosphatase (AKP) and acid phosphatase (ACP) activity were considerably higher than in other treatments. In addition, following infection with Vibrio harveyi, AKP and ACP activity in the HKBS-P60 group was significantly higher than in other treatments, particularly at the early stage of bacterial infection. Moreover, HKBS-P60 was found to be better protected against V. harveyi infection with lower cumulative mortality (60%) compared to HKBS (90%) and HKBS-P0 (100%) at 7 days after infection. Overall, these findings confirmed that P60 could increase immunological responses in the shrimp midgut, and HKBS-P60 could be used as an effective tool to enhance the immune response and disease resistance in shrimp.

Long-Term Application of a Synbiotic Chitosan and Acinetobacter KU011TH Mixture on the Growth Performance, Health Status, and Disease Resistance of Hybrid Catfish (Clarias gariepinus × C. macrocephalus) during Winter.

The effects of potential synbiotic chitosan and Acinetobacter KU011TH mixture on growth performance, immune response, and A. hydrophila resistance were investigated for the first time. The control group was fed a basal diet (A), and group B was given the formula B diet that was supplemented with chitosan at 20 mL/kg diet via top dressing. The other synbiotic groups, C, D, and E, were top-dressed with the target probiotics at 1 × 108, 1 × 109, and 1 × 1010 CFU/kg diet, respectively, and coated with the same concentration of chitosan. Fish were continuously fed the five different feeds for 16 weeks during winter. At the end of the trial, the growth parameters of the test groups did not significantly differ from those of the control (p > 0.05). All the symbiotic-chitosan treatments significantly increased various hematological and serum immune parameters. Moreover, the expression levels of immune-related genes were strongly elevated in the head kidney and spleen, whereas upregulated expression was observed in the liver and whole blood (p < 0.05). Survival analysis indicated that fish in groups B and C showed significantly higher survival (84.33 ± 2.21 and 79.50 ± 6.34%) than those in groups A, D and E (55.33 ± 8.82%-74.00 ± 6.50) (p < 0.05) after injection with A. hydrophila for 14 days.

Immune-related transcripts, microbiota and vector competence differ in dengue-2 virus-infected geographically distinct Aedes aegypti populations

BackgroundVector competence in Aedes aegypti is influenced by various factors. Crucial new control methods can be developed by recognizing which factors affect virus and mosquito interactions.MethodsIn the present study we used three geographically distinct Ae. aegypti populations and compared their susceptibility to infection by dengue virus serotype 2 (DENV-2). To identify any differences among the three mosquito populations, we evaluated expression levels of immune-related genes and assessed the presence of microbiota that might contribute to the uniqueness in their vector competence.ResultsBased on the results from the DENV-2 competence study, we categorized the three geographically distinct Ae. aegypti populations into a refractory population (Vilas do Atlântico), a susceptible population (Vero) and a susceptible but low transmission population (California). The immune-related transcripts were highly expressed in the California population but not in the refractory population. However, the Rel-1 gene was upregulated in the Vilas do Atlântico population following ingestion of a non-infectious blood meal, suggesting the gene’s involvement in non-viral responses, such as response to microbiota. Screening of the bacteria, fungi and flaviviruses revealed differences between populations, and any of these could be one of the factors that interfere with the vector competence.ConclusionsThe results reveal potential factors that might impact the virus and mosquito interaction, as well as influence the Ae. aegypti refractory phenotype.Graphical

Effect of continuous dietary administration of the Saccharomyces cerevisiae yeast, Δmnn9, on Pacific oyster (Crassostrea gigas) juveniles: Immunological and histopathological findings after Vibrio coralliilyticus challenge

Yeast cells and their cell wall components, mainly β-glucans, are strong immunostimulants in bivalves, however, it remains unclear whether long-term administration of yeast contributes to prolonged activity of bivalve immune systems. Understanding these innate immune mechanisms is essential to establishing feeding strategies and disease prevention in hatcheries and nurseries. The present study examined the immunostimulatory effect of continuous dietary supply of the Saccharomyces cerevisiae mnn9 mutant (∆mnn9), which exhibits elevated exposure of β-glucans on the cell-wall surface, on Crassostrea gigas juveniles, and its protective effect against Vibrio coralliilyticus infections. In trial 1 the expression levels of immune-related genes were determined in oysters fed the ∆mnn9-containing diet 50A50Y (50% algae, 50% ∆mnn9; based on dry weight), compared to those fed the 100% algae-based diet (100A), after 6, 9, 12, 18, 24 h of feeding. In trial 2: the feeding period was expanded to 8 days, where the algal diet (100A) was provided every day, but substituted with 50A50Y for 0, 1, 2, 4, and 8 days (treatments 0D (control), 1D, 2D, 4D, and 8D, respectively) just before a Vibrio challenge, which was monitored for 7 days. Gene expression was measured before and 24-h after the Vibrio challenge. Histological analysis was performed on oysters on day 7 post-Vibrio challenge. Results showed that 50A50Y induced upregulation of interleukin 17–5 (CgIL17–5; 6 h), lysozyme (CgLys; 6 and 9 h), β-glucan-binding protein (CgβGBP; 18 h), and superoxide dismutase (CgSOD; 18 and 24 h) genes in juveniles (trial 1). Before the challenge, trial 2 demonstrated overexpression of CgSOD in animals from 1D, CgLys in those from 1D, 2D, and 4D, but not in oysters from 8D, compared to those from 0D. However, higher CgIL17–5 expression levels were observed in animals from 2D, 4D, and 8D, suggesting that inflammatory genes can remain inducible after 8 days of ∆mnn9 feeding. After the Vibrio challenge, a significantly higher survival was observed in animals from 1D (35.8%), 2D (45.0%), 4D (49.2%), but not 8D (25.8%), compared to the survival of animals from 0D (16.7%). These findings suggest that regulatory feedback mechanisms occurring during continuous ∆mnn9 feeding might lead to host tolerance or homeostasis. Also, we showed that V. coralliilyticus promotes overexpression of CgβGBP, C-type lectin receptor (CgCLec-3), myeloid differentiation factor 88 (CgMyD88), nuclear factor-kappa B (CgNFκB), CgLys, and CgSOD genes in oysters at 24-h post-exposure. Finally, this study provides insights into histopathological manifestations in the digestive tract, muscles, and gills of Vibrio-infected oysters.

Characterization of fliR-deletion mutant ΔfliR from Vibrio alginolyticus and the evaluation as a live attenuated vaccine.

Vibrio alginolyticus is the common pathogen affecting various species of marine organisms. It has been demonstrated that fliR is a necessary virulence factor to adhere and infect their hosts for pathogenic bacteria. Frequent disease outbreaks in aquaculture have highlighted the necessity of developing effective vaccines. In the present study, in order to investigate the function of fliR in V.alginolyticus, the fliR deletion mutant ΔfliR was constructed and its biological properties were evaluated, additionally, the differences in gene expression levels between wild-type and ΔfliR were analyzed by transcriptomics. Finally, ΔfliR was used as a live attenuated vaccine to immunize grouper via the intraperitoneal route to evaluate its protective effect. Results show that fliR gene of V. alginolyticus was identified as being 783 bp in length, encoding 260 amino acids, and showing significant similarity to homologs of other Vibrio species. The fliR-deletion mutant ΔfliR of V. alginolyticus was successfully constructed, and its biological phenotype analysis showed no significant differences in growth capacity and extracellular enzyme activity compared to the wild-type. However, a substantial reduction of motility ability was detected in ΔfliR. Transcriptomic analysis revealed that the absence of fliR gene is responsible for a significantly decreased expression of flagellar genes, including flaA, flaB, fliS, flhB and fliM. The fliR-deletion mainly affects the related pathways involved in cell motility, membrane transport, signal transduction, carbohydrate metabolism, and amino acid metabolism in V. alginolyticus. The efficacy of ΔfliR as a candidate of live attenuated vaccine were evaluated by intraperitoneal injection in grouper. The ΔfliR provided the RPS (Relative protection rate) of 67.2% against V. alginolyticus in groupers. The ΔfliR efficiently stimulated antibody production with specific IgM still detected at 42d post-vaccination, and significantly elevated the activity of antioxidant enzymes like Catalase (CAT), Superoxide dismutase (SOD), and lactate dehydrogenase (LDH) in the serum. The higher expression levels of immune-related genes were observed in the immune tissues of inoculated grouper compared to the control. In conclusion, ΔfliR effectively improved the immunity of inoculated fish. The results suggest that ΔfliR is an effective live attenuated vaccine against vibriosis in in grouper.

An ecdysteroid-regulated 16-kDa protein homolog participates in the immune response of the crayfish Procambarus clarkii

An ecdysteroid-regulated 16-kDa protein homolog (named Pc-E16), encoding 150 amino acid residues with a conserved MD-2-related lipid-recognition domain, was first identified in Procambarus clarkii. Phylogenetic analyses indicated similarity between Pc-E16 and 16-kDa proteins from Aplysia californica and insects. Recombinant Pc-E16 protein was successfully expressed in BL21 (DE3) Escherichia coli cells, and polyclonal antibodies against purified Pc-E16 proteins were prepared. In comparison with other tissues, Pc-E16 was highly expressed in the intestine; real-time PCR and Western blotting results indicated that Pc-E16 expression was significantly induced by lipopolysaccharides in hepatopancreas and hemocytes. Pc-E16-mediated signaling pathways were investigated by digital gene expression analysis following RNA interference targeting Pc-E16. A total of 6103 differentially expressed genes (DEGs) were identified, of which 3318 were up- and 2785 were downregulated. Many DEGs were involved in binding and catalytic activity. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that DEGs were clustered into 225 pathways, and 15 significantly enriched pathways were identified at the immune system level. In addition, the expression level of Pc-E16 in hemocytes and hepatopancreas was obviously downregulated at 48 h after dsRNA injection, and Pc-E16-RNAi treatment affected the expression levels of immune-related genes. Altogether, our results suggest that Pc-E16 is involved in the innate immune response of P. clarkii.

Immune regulation mechanism of Saposhnikoviae Radix polysaccharide based on zebrafish model

The immunomodulatory effect of Saposhnikoviae Radix polysaccharide(SRP) was evaluated based on the zebrafish mo-del, and its mechanism was explored by transcriptome sequencing and real-time fluorescence-based quantitative PCR(RT-qPCR). The immune-compromised model was induced by navelbine in the immunofluorescence-labeled transgenic zebrafish Tg(lyz: DsRed), and the effect of SRP on the density and distribution of macrophages in zebrafish was evaluated. The effect of SRP on the numbers of macrophages and neutrophils in wild-type AB zebrafish was detected by neutral red and Sudan black B staining. The content of NO in zebrafish was detected by DAF-FM DA fluorescence probe. The content of IL-1β and IL-6 in zebrafish was detected by ELISA. The differentially expressed genes(DEGs) of zebrafish in the blank control group, the model group, and the SRP treatment group were analyzed by transcriptome sequencing. The immune regulation mechanism was analyzed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment, and the expression levels of key genes were verified by RT-qPCR. The results showed that SRP could significantly increase the density of immune cells in zebrafish, increase the number of macrophages and neutrophils, and reduce the content of NO, IL-1β, and IL-6 in immune-compromised zebrafish. The results of transcriptome sequencing analysis showed that SRP could affect the expression level of immune-related genes on Toll-like receptor pathway and herpes simplex infection pathway to affect the release of downstream cytokines and interferon, thereby completing the activation process of T cells and playing a role in regulating the immune activity of the body.

Construction and efficacy of Aeromonas veronii mutant Δhcp as a live attenuated vaccine for the largemouth bass (Micropterus salmoides)

Aeromonas veronii is a human and animal co-pathogenic bacterium that could have a significant negative impact on both human health and aquaculture. In this study, a mutant strain of A. veronii with deletion of the hemolysin co-regulated protein (hcp) gene was constructed (Δhcp-AV). Compared with the wild strain, Δhcp-AV showed significantly reduced growth capacity and biofilm formation ability. Motility tests showed that the hcp gene had no significant effect on the swimming and swarming ability. In addition, the pathogenicity was also reduced. To evaluate the efficacy of Δhcp-AV as a live attenuated vaccine for prevention of Aeromonas veronii infection, we compared the immune response of largemouth bass (Micropterus salmoides) after immunization with 500 μL of 1.47 × 105 CFU/mL of Δhcp-AV and 4 × 108 CFU/mL of inactivated A. veronii. Obvious increases of serum immune related enzyme activity were observed in immunization groups. Expression levels of immune-related genes in Δhcp-AV group were up-regulated, and higher than those in inactivated A. veronii group. After challenging with live A. veronii, the relative percent survival (RPS) was 100% in Δhcp- AV group, whereas the RPS was 76.67% in inactivated A. veronii group. Our data suggest that the live attenuated vaccine Δhcp- AV could elicit a stronger immune response and provide a higher RPS than inactivated A. veronii. These data suggest that hcp gene is an important virulence factor of A. veronii, and the live attenuated vaccine Δhcp-AV is safe and effective for prevention A. veronii infection in M. salmoides farming.

Abstract P6-14-11: Exosome-mediated Circ-CRSP1 promotes tumor proliferation and metastasis through stabilizing ELAVL1 protein and suppresses anti-tumor immune response in regulating the progression of Triple-Negative Breast Cancer

Abstract Background: Triple-negative breast cancer (TNBC) is one of the most malignant subtype of breast cancer. Lacking targetable molecular drivers, chemotherapy is almost the only effective systematic treatment for TNBC. Compared with other types of breast cancer, TNBC shows a higher long-term recurrence rate and worse prognostic outcome, and its main cause of death is tumor metastasis. Therefore, there is an urgent clinical need to explore underlying molecular mechanisms and find novel targets for therapeutic intervention, as well as biomarkers for early clinical diagnosis. Materials &amp; Methods: To explore the signal pathways and target molecules related to tumor immunity by bioinformatics analysis of tissue samples [TNBC (n = 55), Her2 (n = 39), Luminal B (n = 30), Luminal A (n = 29), healthy (n = 11)]. Co-culture experiment was conducted to study the effect of TNBC-derived exosome-mediated molecules on the proliferation and migration of breast cancer. qRT-PCR assay was used to confirm the expression level of Circ-CRSP1 in tumor tissues, breast cancer cells, exosomes of breast cancer cells and serum exosomes of breast cancer patients. Overexpression and knockout experiments investigated the role of Circ-CRSP1 in promoting the proliferation and migration of TNBC cells. Protein molecular docking technique was used to predict the possible targeting binding protein ELAVL1, and molecular experiments and nude mice model were used to study the mechanism of Circ-CRSP1-ELAVL1 complex in tumor immunity. Results: TNBC-derived exosome-mediated molecular transmission promotes the proliferation and migration of breast cancer, and may promote distant metastasis of breast cancer by changing the tumor microenvironment (TME) before metastasis. Bioinformatics analysis of TNBC tumor samples confirmed that TNBC is strongly associated with tumor immune-related biological processes and signal pathways. Circ-CRSP1 is highly expressed in TNBC tumor tissues, TNBC cells, exosome derived from TNBC cells and serum exosomes of TNBC patients, and is related to the poor clinical prognosis of TNBC patients. Overexpression of Circ-CRSP1 enhanced the proliferation and migration ability of TNBC cells, and the expression levels of EMT and cell-cycle related genes altered. Circ-CRSP1 binds to protein ELAVL1 at the physical level. The target gene of Circ-CRSP1-ELAVL1 complex is involved in the immune response, and the overexpression of Circ-CRSP1 significantly down-regulates the expression level of immune-related genes. Conclusion: Exosome-mediated Circ-CRSP1 promotes the transport of ELAVL1 from nucleus to cytoplasm through targeted binding with ELAVL1, and then regulates cytoplasmic proliferation and metastasis and inhibits the stability and translation of anti-tumor immune-related gene mRNA, thus finally promotes the proliferation, invasion and metastasis of TNBC. This study can further understand the mechanism of circRNA in the growth and metastasis of TNBC from the theoretical level, further explore the feasibility of Circ-CRSP1 as a molecular target for gene therapy, and provide theoretical basis and pre-clinical data for Circ-CRSP1 in serum exosomes as a high risk screening index and prognostic marker for TNBC patients. Citation Format: Sujin Yang, Jinhai Tang. Exosome-mediated Circ-CRSP1 promotes tumor proliferation and metastasis through stabilizing ELAVL1 protein and suppresses anti-tumor immune response in regulating the progression of Triple-Negative Breast Cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-11.

Effects of Different LED Light Spectra on Growth and Immunity of the Japanese Eel (Anguilla japonica) and Giant Mottled Eel (A. marmorata).

Indoor recirculating aquaculture systems make light control possible and enable the usage of specific coloured lights to promote the growth and immunity of aquaculture species. Five different LED wavelengths (white light [460 nm], red light [622 nm], green light [517 nm], blue light [467 nm], and the dark) were used in this study to evaluate growth and immunity in the glass eel stage of two high-valued anguillid species, Japanese eel (Anguilla japonica) and giant mottled eel (A. marmorata). There were no significant differences in growth of the Japanese eel among the groups after 12 weeks of feeding (p > 0.05); the survival rate of each group was over 95%. The giant mottled eel showed better growth in total length and body weight in the red light and dark groups (p < 0.05). Expression levels of immune-related genes were not significantly different between each group of the Japanese eel and the giant mottled eel (p > 0.05). The growth of the Japanese glass eel was not significantly sensitive to different LED wavelengths, while the giant mottled glass eel showed better growth under red light and dark environments. Neither eel species showed significant differences in innate immunity under different LED wavelengths.

Growth Performance, Nutritional Quality, and Immune-Related Gene Expression of the Chinese Mitten Crab (Eriocheir sinensis) in Pond Ecosystem as Influenced by Stocking Density

This study aimed to evaluate the influence of stocking density on growth performance, nutritional quality, and immune-related gene expression of Eriocheir sinensis and water quality in pond ecosystems. E. sinensis were randomly divided into five density groups (G1: 1.2 inds/m2; G2: 1.5 inds/m2; G3: 1.8 inds/m2; G4: 2.1 inds/m2; G5: 2.4 inds/m2) based on their breeding densities, and three replicates were set up for each density group, for a total of fifteen ponds. After 169 days of breeding, the muscle, hepatopancreatic tissue, and hemolymph of E. sinensis, randomly selected from each group, were collected for analysis. Body weight (BW), carapace length, and carapace width increased with breeding time, and the BW of E. sinensis in G3 was higher than that in the other groups. In the muscle and hepatopancreas, the crude lipid content in the G3 group was higher than that in the other groups (p &gt; 0.05). In the muscle tissue, the contents of total, essential, and delicious amino acids in the G3 group were higher than those in the other groups. The expression levels of immune-related genes in G5 were higher than those in the other groups. The value of pH decreased and NO2-N increased significantly in the G5 group and pH, as a synergetic factor of culture density, affected the growth performance of E. sinensis. In conclusion, stocking density has a certain impact on the growth performance, nutrition quality, and immunity of E. sinensis and water quality in pond ecosystems, and a stocking density of 1.8 inds/m2 can be considered optimal for rearing in pond ecosystems of the Poyang Lake region.

Tachykinin-related peptides modulate immune-gene expression in the mealworm beetle Tenebrio molitor L.

Tachykinins (TKs) are a group of conserved neuropeptides. In insects, tachykinin-related peptides (TRPs) are important modulators of several functions such as nociception and lipid metabolism. Recently, it has become clear that TRPs also play a role in regulating the insect immune system. Here, we report a transcriptomic analysis of changes in the expression levels of immune-related genes in the storage pest Tenebrio molitor after treatment with Tenmo-TRP-7. We tested two concentrations (10–8 and 10–6 M) at two time points, 6 and 24 h post-injection. We found significant changes in the transcript levels of a wide spectrum of immune-related genes. Some changes were observed 6 h after the injection of Tenmo-TRP-7, especially in relation to its putative anti-apoptotic action. Interestingly, 24 h after the injection of 10–8 M Tenmo-TRP-7, most changes were related to the regulation of the cellular response. Applying 10–6 M Tenmo-TRP-7 resulted in the downregulation of genes associated with humoral responses. Injecting Tenmo-TRP-7 did not affect beetle survival but led to a reduction in haemolymph lysozyme-like antibacterial activity, consistent with the transcriptomic data. The results confirmed the immunomodulatory role of TRP and shed new light on the functional homology between TRPs and TKs.