Expression Levels Of Immune-related Genes Research Articles

CqsA inhibits the virulence of Vibrio harveyi to the pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatus)

Vibrio harveyi is an opportunistic Gram-negative pathogen that threatens future development of global aquaculture as well as human health. In this study, to gain insight into virulence regulation of cqsA in V. harveyi, a ΔcqsA mutant strain and complementation of the ΔcqsA mutant (C-cqsA) were constructed and phenotypically characterized. Deletion of cqsA led to enhanced mobility, iron absorption, resistance to H2O2, and biofilm formation ability, and reduced the ability to secrete extracellular proteases. In addition, pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatu) challege experiment was conducted to observe the changes in virulence at the absence of cqsA in V. harveyi. The results showed that the cumulative mortalities of the pearl gentian grouper challenged with the ΔcqsA+ mutant by intraperitoneal injection reached 91.67%, which was significantly higher than that of wild-type (WT+) (41.67%) and C-cqsA (41.67%) strains. Deletion of cqsA also resulted in a significant increase in the ability of V. harveyi to colonize the spleen and liver of the pearl gentian grouper simultaneously. Meanwhile, the early expression levels of immune-related genes of the pearl gentian grouper in the ΔcqsA+ mutant group, including TNF-α, IL-1β, MHC-Iα, IL-16, and IgM were up-regulated post-challenge, indicating that the ΔcqsA+ mutant may hijack the immune signaling pathway and cause early overexpression of some host immune-related genes. The expression levels of the V. harveyi virulence-associated genes flaA, flaC, fliA, fliS, cheR, lafA, aphA, and fur were up-regulated, while vhh and hapR were down-regulated. Collectively, these findings point to broad regulatory functions of cqsA in V. harveyi cells, which likely underlie an important role in pathogenicity.

Enrichment effects of fermented by-product of Shochu distillery on Brachionus plicatilis sp. rotifer and larviculture performance in Japanese flounder (Paralichthys olivaceus)

The effective use of distillery by-products of the shochu sweet potato has long been considered as a rich source of protein, fiber, amino acids, fatty acids, probiotics with a great potential as a functional feed for animals. This study applied fermented by-product of Shochu distillery (FBPSD) and salmon roe emulsion oil for rotifer enrichment to evaluate its effects on the: (i) nutritional composition of rotifers and (ii) feeding responses of Paralichthys olivaceus larvae when fed with enriched rotifers. The salmon roe oil enrichment was set as a control diet and FBPSD was supplemented at different doses (1, 5 and 10 % based on the salmon roe oil amount). The results showed that the rotifers supplemented with FBPSD at 5 and 10 % significantly enhanced protein, carbohydrate, and HUFA content. Similarly, the activity and expression profiles of targeted enzymes including trypsin, chymotrypsin, lipase and amylase effectively responded at early and middle-metamorphosis of the larvae to 5 and 10 % enrichment regiment. These two treatments were also found to be better supported in energy metabolism evidenced by Cytochrome-C-Oxidase (cox) and ATPase-pk (atp) gene expression in the larvae. Moreover, the expression levels of immune-related genes (IL-10, IL-1β, TNF-α, P2Y6) and antioxidant-related genes (SOD, CAT) were significantly improved at 5 and 10 % of FBPSD. Cessation of feeding and delay in growth during the metamorphic process was also molecularly demonstrated. Larvae suffered low survival (24.5–25.3 %) in control and 1 % supplementation of FBPSD, but significantly improved at 5 and 10 % (36.4–38.7%). However, the growth performance of larval fish was not remarkable after 40 days of culture. The results of this study revealed that FBPSD could be considered as a potential supplementary enrichment for live feed in fish larviculture, especially at 5 and 10 % rotifer enrichment dosage.

Proteomic Analyses Detect Higher Expression of C-Type Lectins in Imidacloprid-Resistant Colorado Potato Beetle Leptinotarsa decemlineata Say.

Simple SummarySurveillance and determining the mechanisms of pesticide resistance are key components of resistance management. Mechanisms can be investigated using biochemical, genomic, proteomic and other modern analytical techniques. In the present study, proteomic analyses of Colorado potato beetle (CPB), one of the most adaptable insect pests to both plant toxins and synthetic insecticides, were applied to identify protein differences in insecticide-susceptible and resistant strains. Proteins identified in abdominal and midgut tissues based on separating by 2-dimensional (2-D) gels and mass spectrometry were associated with insect innate immunity. A database search found that the highest match was a C-type lectin (CTL), which is a component in the insect’s innate immune system. The 2-D gel spot identified as a CTL was greater in the insecticide-resistant CPB strain, but the CTL spot size was increased by exposure to imidacloprid in the susceptible strain. This is a novel finding, which suggests that CTLs and insect immunity may respond to certain toxins as well as to pathogens. There may also be a potential application for pest management if insect immunity is targeted.The Colorado potato beetle (CPB) is one of the most adaptable insect pests to both plant toxins and synthetic insecticides. Resistance in CPB is reported for over 50 classes of insecticides, and mechanisms of insecticide-resistance include enhanced detoxification enzymes, ABC transporters and target site mutations. Adaptation to insecticides is also associated with changes in behaviour, energy metabolism and other physiological processes seemingly unrelated to resistance but partially explained through genomic analyses. In the present study, in place of genomics, we applied 2-dimensional (2-D) gel and mass spectrometry to investigate protein differences in abdominal and midgut tissue of insecticide-susceptible (S) and -resistant (R) CPB. The proteomic analyses measured constitutive differences in several proteins, but the highest match was identified as a C-type lectin (CTL), a component of innate immunity in insects. The constitutive expression of the CTL was greater in the multi-resistant (LI) strain, and the same spot was measured in both midgut and abdominal tissue. Exposure to the neonicotinoid insecticide, imidacloprid, increased the CTL spot found in the midgut but not in the abdominal tissue of the laboratory (Lab) strain. No increase in protein levels in the midgut tissue was observed in the LI or a field strain (NB) tolerant to neonicotinoids. With the exception of biopesticides, such as Bacillus thuringiensis (Bt), no previous studies have documented differences in the immune response by CTLs in insects exposed to synthetic insecticides or the fitness costs associated with expression levels of immune-related genes in insecticide-resistant strains. This study demonstrates again how CPB has been successful at adapting to insecticides, plant defenses as well as pathogens.

Oral immunization with surface immunogenic protein from Streptococcus agalactiae expressed in Lactococcus lactis induces protective immune responses of tilapia (Oreochromis niloticus)

Streptococcus agalactiae infection has become a serious bacterial disease of tilapia for its damage. In order to effectively prevent the outbreak of streptococcicosis, a recombinant Lactococcus lactis expressing surface immunogenicity protein (Sip) of S. agalactiae was constructed as probiotic vector oral vaccine in this study. The immunogenicity of the recombinant L. lactis was evaluated through detecting the level of specific serum IgM antibody, expression level of immune-related genes and relative percent survival (RPS). The results showed that the recombinant L. lactis could express a 45.5 kDa protein consistent with the expected size of Sip. The recombinant Sip mainly expressed in the form of inclusion body and the concentration of purified Sip could reach 7.65 mg / mL. Nile tilapia were orally immunized with different concentrations (2.24 × 109, 2.24 × 1010 and 2.24 × 1011 CFU / mL) of recombinant L. lactis (NZ9000-pNZ8148-sip). The analysis results of specific serum antibody showed that the level of antibody of tilapia which were vaccinated with medium and high concentration of recombinant L. lactis (2.24 × 1010 CFU / mL and 2.24 × 1011 CFU / mL) were significantly higher than that of control group. The relative percentage of survival (RPS) of tilapia vaccinated with medium concentration of NZ9000-pNZ8148-sip reached 61.6 % at 21 days post-immunization. Quantitative real-time PCR (qRT-PCR) results revealed that the immune-related genes of IgT, IgM, CD8a and C3 were significantly upregulated expression in thymus, liver, spleen and hindgut. In conclusion, the recombinant L. lactis vaccine induced both humoral and cellular responses of tilapia. This study demonstrates the potential of using L. lactis as a delivery system to develop an oral vaccine against S. agalactiae.

Dietary supplementation of black mulberry (Morus nigra) syrup improves the growth performance, innate immune response, antioxidant status, gene expression responses, and disease resistance of Nile tilapia (Oreochromis niloticus)

The present study investigated the effects of black mulberry (Morus nigra) syrup supplementation on growth performance, hematological, serum biochemical, innate immune parameters, immune and antioxidant related gene expression responses, and disease resistance of Nile tilapia, Oreochromis niloticus. Five isonitrogenous and isoenergetic diets were formulated to contain black mulberry syrup at levels of 0%, 0.75%, 1.5%, 2.0%, and 3.0%. Fish were fed experimental diets for 60 days. Dietary black mulberry syrup increased activities of serum lysozyme, myeloperoxidase, superoxide dismutase and catalase, and increased the expression levels of immune-related genes (interleukin 1, beta, tumor necrosis factor, immunoglobulin M, interferon gamma and heat shock protein 70) in the spleen and antioxidant-related genes (superoxide dismutase, catalase and glutathione peroxidase) in the liver of fish fed especially with 1.5%, 2.0%, and 3.0% black mulberry syrup supplemented diets. Furthermore, at 2.0% incorporation level, growth performance increased.The findings of the present study indicate that Nile tilapia fed with diet containing 2.0% black mulberry might be adequate to improve the growth performance, innate immune parameters, antioxidant related gene expression responses, and disease resistance against Aeromonas veronii.

Potential influence of jaggery-based biofloc technology at different C:N ratios on water quality, growth performance, innate immunity, immune-related genes expression profiles, and disease resistance against Aeromonas hydrophila in Nile tilapia (Oreochromis niloticus)

Biofloc technology is increasingly becoming the most promising aquaculture tool especially in places where water is scarce and the land is very expensive. The dynamics of water quality, as well as plankton and microbial abundance, are collectively necessary for successful fish farming. The prospective use of jaggery as a potential carbon source and its influence on water quality, growth performance, innate immunity, serum bactericidal capacity, and disease resistance to Aeromonas hydrophila was investigated in Oreochromis niloticus. A completely randomized design was used in triplicates, where the control group was reared in a water system with no carbon source, while T1, T2, and T3 groups were raised in biofloc systems at C:N ratios of C:N12, C:N15, and C:N20, respectively. Water specimens were collected daily and fortnightly, while blood, serum, and head kidneys were collected at 75 days of experimental period for further analysis. TAN, nitrite, and ammonia values were considerably reduced, while the TSS values elevated significantly in all treated groups compared to the control. Jaggery-based biofloc system (JB-BFT) has a pronounced effect on hematological and growth performance parameters rather than control. Similarly, serum antioxidants, lysozyme, protease, antiprotease and bactericidal capacity were significantly increased (p < 0.05) in the treated groups in a dose-dependent manner. LYZ, TNF-α, and IL-1β genes were upregulated in proportion to C:N ratios with the highest fold in C:N20. Furthermore, fish treated with JB-BFT presented lower cumulative mortalities and better relative levels of production (RLP) after experimental challenge with A. hydrophila compared to control. In conclusion, JB-BFT has a robust influence on Nile tilapia (O. niloticus) innate immunity through favorable innovation of various immune-cells and enzymes as well as upregulating the expression levels of immune-related genes. This study offers jaggery as a new carbon source with unique properties that satisfy all considerations of biofloc technology in an eco-friendly manner.

Curcumin supplement diet: Enhanced growth and down‐regulated expression of pro‐inflammatory cytokines in Labeo rohita fingerlings

The present study was conducted to determine the effect of curcumin supplement diet (CSD) on growth, non-specific immunity and pro-inflammatory cytokine gene expression in Labeo rohita fingerlings. 60-day (0–45 days pre-challenge and 15 days post challenge; Aeromonas hydrophila) experiment was conducted for with four treatment groups: T1 (0.5% curcumin level), T2 (1% curcumin level), T3 (1.5% curcumin level), T4 (2% curcumin level) and a control group (0% curcumin level). Kidney tissue samples were collected for RT-PCR from all the treatment groups at 15 days of interval. After 45 days of feeding, a sub-sample of fish (15 fish from each replicate) were challenged with a virulent strain of A. hydrophila. The mRNA expression levels of immune-related genes, viz. tumour necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and Toll-like receptor-22 (TLR-22), were evaluated along with nitro blue tetrazolium (NBT) assay and lysozyme activity. TNF-α mRNA expression levels were significantly lowered in CSD treatment with the lowest level observed in the T2 treatment group, followed by T1 during pre-challenging period respectively. The IL-1β and TLR-22 expression levels also followed the similar trend in different treatment groups. Similarly, NBT and serum lysozyme activities were significantly different among treatment groups. Fish fed with 1.5% level curcumin diet (T3) had significantly highest relative percentage survival (63.64%) compared to the rest of the treatment groups. The results suggested that the diet supplemented with 1.5% curcumin level provides protective effect against A. hydrophila in L. rohita by increasing immunity and reducing the activity of pro-inflammatory genes.

Bioinformatics profiling integrating a four immune-related long non-coding RNAs signature as a prognostic model for papillary renal cell carcinoma.

Background: Papillary renal cell carcinoma (pRCC) was the 2nd most common subtype, accounting for approximately 15% incidence of renal cell carcinoma (RCC). Immune related long non-coding RNAs (IR-lncRs) plentiful in immune cells and immune microenvironment (IME) are potential in evaluating prognosis and assessing the effects of immunotherapy. A completed and meaningful IR-lncRs analysis based on abundant pRCC gene samples from The Cancer Genome Atlas (TCGA) will provide insight in this field.Results: 17 IR-lncRs were selected by Pearson correlation analysis of immune score and the lncRNA expression level, and 5 sIRlncRs were significantly correlated with the OS of pRCC patients. 4 sIRlncRs (AP001267.3, AC026471.3, SNHG16 and ADAMTS9-AS1) with the most remarkable prognostic values were identified to establish the IRRS model and the OS of the low-risk group was longer than that in the high-risk group. The IRRS was certified as an independent prognosis factor and correlated with the OS. The high-risk group and low-risk group showed significantly different distributions and immune status through PCA and GSEA. In addition, we further found the expression levels of SNHG16 was remarkably enhanced in female patients with more advanced T-stages, but ADAMTS9-AS1 showed the opposite results.Conclusion: The IRRS model based on the identified 4 sIRlncRs showed the significant values on forecasting prognoses of pRCC patients, with the longer OS in the low-risk group.Methods: We integrated the expression profiles of LncRNA and overall survival (OS) in the 322 pRCC patients based on the TCGA dataset. The immune scores calculated on account of the expression level of immune-related genes were used to verify the most relevant IR-lncRs. Survival-related IR-lncRs (sIRlncRs) were estimated by COX regression analysis in pRCC patients. The high-risk group and low-risk group were identified by the median immune-related risk score (IRRS) model established by the screened sIRlncRs. Functional annotation was displayed by gene set enrichment analysis (GSEA) and principal component analysis (PCA), and the immune composition and purity of the tumor were evaluated through microenvironment cell count records. The expression levels of sIRlncRs of pRCC samples were verified by real-time quantitative PCR.

Differences in immune-related gene expressions and tumor-infiltrating lymphocytes according to chemotherapeutic response in ovarian high-grade serous carcinoma

BackgroundHigh-grade serous carcinoma (HGSC) of the ovary is the most common subtype of epithelial ovarian cancer (EOC) and has an overall poor prognosis. There is increasing awareness of the importance of immune cell populations and tumor-infiltrating lymphocytes (TILs) in various immune pathways in the tumor microenvironment. The present study evaluated immune-related gene expressions and TIL levels, as well as associated chemotherapeutic responses, to elucidate the correlation between gene expression and TIL levels in HGSC.Materials and methodsFresh tissue samples from 12 HGSC patients were included in this study. Depending on their response to adjuvant chemotherapy, the patients were divided into two groups: chemosensitive (CS) or chemoresistant (CR). The expression levels of 770 genes were analyzed using the nCounter® PanCancer Immune Profiling Panel of the NanoString nCounter® Analysis System. Quantitative real-time polymerase chain reaction (qPCR) was performed to validate the NanoString data obtained. The TIL levels in representative sections were examined via hematoxylin and eosin staining. Gene and TIL levels were subsequently correlated with the chemotherapeutic response.ResultsSeveral genes were differentially expressed in the two study groups. Eleven representative genes were selected for further evaluation. Of those, 9 genes (IRF1, CXCL9, LTB, CCL5, IL-8, GZMA, PSMB9, CD38, and VCAM1) were significantly overexpressed in the CS group; whereas expressions of 2 genes (CD24 and CD164) were increased in the CR group. Results of qPCR were consistent with those of the NanoString nCounter® analysis. Stromal TIL levels were significantly associated with adjuvant chemotherapeutic response (p = 0.001).ConclusionsSignificant differences between the CS and CR groups were observed in the expression levels of immune-related genes. Immune-related gene expressions were significantly higher in the CS group, which also had higher levels of TILs. We, therefore, suggest that, in patients with HGSC, immune-related gene expressions and TIL levels may be associated with chemotherapeutic sensitivity.

Construction of immune-related risk model for prognosis of hepatocellular carcinoma.

e16683 Background: Hepatocellular carcinoma is the most common malignant tumor. Although the treatment of HCC has significantly improved, the 5-year survival rate is still only 18%. There is increasing evidence that tumor immune microenvironment (TIM) plays critical roles during cancer initiation and progression. Based on the comprehensive exploration of the immunogenomic, an immune-related risk model was constructed to predict hepatocellular carcinoma prognosis. Methods: Transcriptomic data of HCC patients were downloaded from the TCGA database, and the differentially expressed immune-related genes (IRGs) (FDR &lt; 0.01, |log2fold change| &gt; 2) were identified. Functional enrichment analysis was performed to explore potential molecular mechanisms of the differentially expressed IRGs. By univariate and multivariate Cox regression analysis, we identified eight prognosis-related IRGs. Based on the expression levels of IRGs, we constructed the immune-related risk model. The Kaplan‐Meier (K‐M) survival curves, ROC curves, univariate and multivariate analysis were used to evaluate the immune-related risk model. According to the risk score, HCC patients were stratified into low and high-risk groups. CIBERSORT was applied to analyze the profiling difference of infiltrating immune cells between the two groups. Results: A total of 113 differentially expressed IRGs were identified, of which nine IRGs were correlated with the prognosis of HCC patients. Functional enrichment analysis showed that these genes were involved in immune response and immune signal pathway. The immune-related risk model consisted of eight IRGs (FABP6, RBP2, MAPT, BIRC5, PLXNA3, CSPG5, IL17D and STC2). The immune risk score was an independent prognostic factor (HR, 2.63 [1.93−3.58]; P = 8.16E−10) and the patients with a high-risk score tended to have a shorter OS than those with a low-risk score. In the TCGA cohort, high-risk patients tended to have an advanced stage. Moreover, we found that the patients in the high-risk group had higher fractions of T follicular cells helper and macrophages M0. The patients with low-risk scores had higher fractions of CD8+ T cells and CD4+ T cells. Conclusions: We have identified the immune-related risk model of hepatocellular carcinoma based on the expression profiles of eight immune-related genes. This model could predict prognosis and reflect the tumor immune microenvironment of HCC patients, which can provide new insights in the individualized treatment of HCC and potential novel targets for immunotherapy.

Bioinformatics profiling integrating a three immune-related long non-coding RNA signature as a prognostic model for clear cell renal cell carcinoma

BackgroundRenal cell carcinoma (RCC) is one of the most common aggressive malignant tumors in urogenital system, and the clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal carcinoma. Immune related long non-coding RNAs (IRlncRs) plentiful in immune cells and immune microenvironment (IME) are potential in evaluating prognosis and assessing the effects of immunotherapy. A completed and meaningful IRlncRs analysis based on abundant ccRCC gene samples from The Cancer Genome Atlas (TCGA) will provide insight in this field.MethodsBased on the TCGA dataset, we integrated the expression profiles of IRlncRs and overall survival (OS) in the 611 ccRCC patients. The immune score of each sample was calculated based on the expression level of immune-related genes and used to identify the most meaningful IRlncRs. Survival-related IRlncRs (sIRlncRs) was estimated by calculating the algorithm of difference and COX regression analysis in ccRCC patients. Based on the median immune-related risk score (IRRS) developed from the screened sIRlncRs, the high-risk and low-risk components were distinguished. Functional annotation was detected by gene set enrichment analysis (GSEA) and principal component analysis (PCA), and the immune composition and purity of the tumor was evaluated by microenvironment cell population records. The expression levels of three sIRlncRs were verified in various tissues and cell lines.ResultsA total of 39 IRlncRs were collected by Pearson correlation analyses among immune score and the lncRNA expression. A total of 7 sIRlncRs were significantly associated with the clinical outcomes of ccRCC patients. Three sIRlncRs (ATP1A1-AS1, IL10RB-DT and MELTF-AS1) with the most significant prognostic values were enrolled to build the IRRS model in which the OS of in the high-risk group was shorter than that in the low-risk group. The IRRS was identified as an independent prognosis factor and correlated with the OS. The high-risk group and low-risk group illustrated different distributions in PCA and different immune status in GSEA. Besides, we found the more significant expression in certain ccRCC cell lines and tumor tissues of ccRCC patients compared with the HK-2 and adjacent tissues respectively. Additionally, the expression levels of lncR-MELTF-AS1 and IL10RB-DT were remarkably enhanced along the more advanced T-stages, but the lncR-ATP1A1-AS1 showed the inverse gradient.ConclusionOur results demonstrate some sIRlncRs with remark clinical relevance show the latent monitoring and prognosis values for ccRCC patients and may provide new insight in immunological researches and treatment strategies of ccRCC patients.

Interleukin 34 Serves as a Novel Molecular Adjuvant against Nocardia Seriolae Infection in Largemouth Bass (Micropterus Salmoides).

DNA vaccines have been widely employed in controlling viral and bacterial infections in mammals and teleost fish. Co-injection of molecular adjuvants, including chemokines, cytokines, and immune co-stimulatory molecules, is one of the potential strategies used to improve DNA vaccine efficacy. In mammals and teleost fish, interleukin-34 (IL-34) had been described as a multifunctional cytokine and its immunological role had been confirmed; however, the adjuvant capacity of IL-34 remains to be elucidated. In this study, IL-34 was identified in largemouth bass. A recombinant plasmid of IL-34 (pcIL-34) was constructed and co-administered with a DNA vaccine encoding hypoxic response protein 1 (Hrp1; pcHrp1) to evaluate the adjuvant capacity of pcIL-34 against Nocardia seriolae infection. Our results indicated that pcIL-34 co-injected with pcHrp1 not only triggered innate immunity and a specific antibody response, but also enhanced the mRNA expression level of immune-related genes encoding for cytokines, chemokines, and humoral and cell-mediated immunity. Moreover, pcIL-34 enhanced the protection of pcHrp1 against N. seriolae challenge and conferred the relative percent survival of 82.14%. Collectively, IL-34 is a promising adjuvant in a DNA vaccine against nocardiosis in fish.

Single or combined effects of dietary arabinoxylan-oligosaccharide and inulin on growth performance, gut microbiota, and immune response in Pacific white shrimp Litopenaeus vannamei

The single and synergistic effects of dietary arabinoxylan-oligosaccharide (AXOS) and inulin at different doses (2, 4, and 8 mg/g diet) on survival, growth performance, gut microbiota, and immune response in Pacific white shrimp Litopenaeus vannamei were assessed. Singular application of either inulin or AXOS at doses of 4 mg/g diet showed the most stimulatory effects on the growth rate and gene expression levels of chitinase, cathepsin L, chymotrypsin, and extracellular signal-regulated kinase (ERK) in each single prebiotic feeding trial. Compared with single prebiotic treatments, simultaneous application of AXOS and inulin at 4 mg/g diet enhanced remarkably the growth parameters of shrimp and the expression of related genes (chitinase, cathepsin L, chymotrypsin, ERK, myeloid differentiation factor 88, and phenoloxidase) after 8-week feeding (P < 0.05). Additionally, gut microbiota analysis indicated the dietary supplementation with combined prebiotics increased significantly the bacterial community richness and relative abundance of Bacillus, Pseudomonas, Bacteriovorax, and Lactobacillus, and reduced the abundance of Vibrio, Rhodococcus, and Photobacterium in the digestive tract of L. vannamei. Compared with the single prebiotic treatment and the control, combined prebiotics supplementation boosted notably the survival rate and expression levels of immune-related genes in shrimp infected with Vibrio alginolyticus or white spot syndrome virus. Therefore, simultaneous application of AXOS and inulin shall have a great potential of dietary supplement in the culture of L. vannamei.

Priming TLR3 and TLR4 in human adipose- and olfactory mucosa-derived mesenchymal stromal cells and comparison of their cytokine secretions.

Mesenchymal stromal cells (MSCs) have potent immunomodulatory abilities to regulate most of the immune cells. Not only the tissue origin of MSCs can affect their functions, but also their microenvironment can strongly influence their biology, particularly through toll-like receptors (TLR)/ligands interaction. In the present study, we compared MSCs derived from two different sources, i.e. human olfactory ecto-mesenchymal stem cells (OE-MSCs) and adipose tissue (AT-MSCs), in terms of their immunosuppressive effects before and after TLR3 and TLR4 stimulation through low-level and short-term TLR-priming protocol. After isolation and characterization of OE-MSCs and AT-MSCs, flow cytometry analyses were used to assess the expression of TLR3, TLR4 by MSCs. Secretion and expression levels of immune-related genes were analyzed using ELISA and RT-qPCR techniques. Based on the results, the proliferation potential of OE-MSCs was significantly higher than that of AT-MSCs. The gene expression and also protein levels of both TLR3 and TLR4 were significantly higher in OE-MSCs, compared to AT-MSCs. Among the examined cytokines and chemokines, OE-MSCs exhibited significantly higher levels of CCL5, IL-8, and TGF-β production, in comparison with AT-MSCs. However, IL-6 secretion by AT-MSCs was considerably more than that by OE-MSCs. OE-MSCs were only affected by the TLR4 ligand, and IL-8 and IL-6 production levels increased after LPS treatment. However, only IL-8 significantly increased after adding LPS or Poly (I:C) to the AT-MSC media. According to the obtained data, OE-MSCs exhibited a higher proliferative potential and greater expression levels of TLR3 and TLR4 genes, compared to AT-MSCs. However, unlike AT-MSCs, the expression of TLR3 by OE-MSCs was nonfunctional. Finally, based on our findings, OE-MSCs have a stronger secretion of immunosuppressive cytokines both before and after LPS or PIC treatment, compared to AT-MSCs.

Effects of dietary cottonseed meal protein hydrolysate on growth, antioxidants and immunity of Chinese mitten crab Eriocheir Sinensis

One hundred and sixty crabs (average initial weight: 51.32±0.08 g) were fed with four experimental diets containing cottonseed meal protein hydrolysate (CPH) at 0% (CPH0, control), 0.3% (CPH0.3), 0.6% (CPH0.6), and 1.2% CPH (CPH1.2). The experiment results show that no difference was observed in specific growth rate and survival rate of crabs fed with CPH diet. Moisture content of crabs fed with CPH0.6 diet was significantly reduced than that of the CPH0 group. Superoxide dismutase and catalase activities of crabs fed with CPH0.6 diet were significantly increased and the difference was not significant between the CPH0.3 and CPH0.6 groups. Malondialdehyde content of CPH0.3 group was significantly lower than that of the CPH0 group. Lysozyme, alkaline phosphatase, and acid phosphatase activities of CPH0.3 diet crabs were significantly higher than that of the CPH0 group. Glutamic-pyruvic transaminase activity of crabs fed with CPH0.3 diet was significantly decreased compared to the CPH0 group. The relative expression levels of Toll1, Toll2, MyD88, LITAF, and ILF-2 of crabs fed with CPH0.3 diet were significantly higher than that of the CPH0 group. The expression level of SOCS2 showed an opposite pattern. After CPH perfusion, the expression levels of SOCS2 and Toll1 in intestine at time 3 h and SOCS2 in hepatopancreas at time 18 h increased significantly to the highest value. The expression level of Toll2, MyD88, LITAF decreased at times 6 h, 6 h, 12 h, respectively, then increased gradually. Therefore, supplementation of dietary CPH could improve antioxidant capacity and immune function; the appropriate supplement dosage of CPH for crab could be 0.3%–0.6%. Furthermore, the short-term CPH stimulation could significantly increase or decline the expression levels of immune-related genes at different times after CPH perfusion.

Cloning and characterization of the virulence factor Hop from Vibrio splendidus

BackgroundVibrio splendidus is an aquaculture pathogen that can cause skin ulcer syndrome (SUS) in Apostichopus japonicus. HopPmaJ is a type III system effector (T3SE) that has been reported to be an important virulence factor. In this study, a gene named hop, which encodes HopPmaJ in V. splendidus was cloned and its cytotoxicity to coelomocytes and its effects on the expression of immune-related genes in A. japonicus were characterized. MethodsReal time reverse transcription PCR (RT-PCR) was used to determine the expression of the hop gene under various conditions. To obtain the purified Hop, hop gene was conditionally expressed in Escherichia coli BL21(DE3) and was purified by GST tag. The cytotoxicity of Hop to coelomocyte was determined using MTT method, and the effect of Hop on the expression of immune-related genes was determined using real time RT-PCR. ResultsThe deduced amino acid sequence of Hop from V. splendidus shared 84%–96% homology with those of Hops from other Vibrio spp. The expression of hop gene was induced not only by host-pathogen contact but also by high cell density. Purified recombinant Hop (rHop) showed cytotoxicity to the coelomocyte of A. japonicus. The cell viability decreased to approximately 42%, 26%, 32%, 30% and 20%, when 30, 50, 60, 80 and 100 μL of purified rHop was added, respectively. After being injected with rHop, the expression levels of immune-related genes that encode complement component (C1q) and caspase were significantly increased, and the production of reactive oxygen species were also increased in A. japonicus. ConclusionOur results indicated that Hop not only contributed to the cytotoxicity to coelomocyte, but also caused immune response in A. japonicus.

Deletion of the Infectious spleen and kidney necrosis virus ORF069L reduces virulence to mandarin fish Siniperca chuatsi

Mandarin fish (Siniperca chuatsi) is a significant cultured species with high added value in China. With the expansion of farming, diseases of mandarin fish such as Infectious spleen and kidney necrosis virus (ISKNV) diseases are becoming more and more serious. Human endogenous retrovirus subfamily H long terminal repeat associating protein 2 (HHLA2) is a type 1 transmembrane molecule with three extracellular Ig domains (IgV-IgC-IgV) and plays important roles in the T cell proliferation and tumorigenesis. The HHLA2-homologues have not been found in virus. In this study, a viral HHLA2 protein encoded by ISKNV ORF069L was identified and the virulence of the deleted ORF069L reconstruction ISKNV strain (ΔORF069L) was investigated. ISKNV ORF069L gene was predicted to encode a 222-amino acids peptide. The bioinformation analysis revealed that ISKNV ORF069L contained an Ig HHLA2 domain and was homologous to vertebrate B7-CD28 family proteins. The recombinant virus strain of ΔORF069L was constructed by homologous recombination technology. The virus titer and growth curves between ISKNV wild type (WT) and ΔORF069L on cellular level showed no significant differences indicating that the ORF069L did not influence the ISKNV replication. The expression levels of immune-related genes (Mx1, IL-1β, IL-8, TNF-a and IgM) were increased in fish infected with ΔORF069L, compared to those in fish infected with ISKNV WT. Furthermore, the lethality caused by ΔORF069L declined by 40% compared with ISKNV WT, indicating that ORF069L was a virulence gene of ISKNV. Most importantly, the protection rate was nearly 100% for fish immunized with ΔORF069L strain. Those results suggested that ΔORF069L could be developed as a potential attenuated vaccine against ISKNV. Our work will be beneficial to promote the development of gene deletion attenuated vaccines for ISKNV disease.

A New Isolate of Pediococcus pentosaceus (SL001) With Antibacterial Activity Against Fish Pathogens and Potency in Facilitating the Immunity and Growth Performance of Grass Carps.

Probiotic-feeding continues to be a promising strategy to control the bacterial pathogens in aquaculture. A new Pediococcus pentosaceus strain (SL001) was isolated from 1000s of soil samples, which exhibited wide antimicrobial spectrum of against fish pathogens, involving Aeromonas hydrophila, Aeromonas veronii, Aeromonas sobria, Edwardsiella tarda, Lactococcus garvieae, and Plesiomonas shigelloide. The challenge test against A. hydrophila showed that the survival rate of SL001-supplemented group was significantly higher than that of control group (P < 0.05). Moreover, SL001 could stably colonize in gut of grass carp and increased mucus-secreting goblet cells and extended intestinal villi could be observed in SL001-supplemented group (P < 0.05). Feeding with SL001 supplemented diet could significantly enhance the growth rate (P < 0.05) and markedly affect gut microbiota structure of grass carps, resulting in reduced potential pathogens and increased potential probiotics. Furthermore, feeding grass carps with SL001 caused the up-regulated expression of insulin-like growth factor (IGF-1 and IGF-2) and down-regulated expression of myostatin (MSTN-1 and MSTN-2) (P < 0.05), which probably also account for the increased growth rate of SL001-fed group. Meanwhile, relative mRNA expression levels of immune-related genes in liver, spleen, and head kidney were analyzed in grass carps after feeding for 30 days with SL001 supplemented diets. In all three immune organs, the expression levels of immunoglobulin M (IgM) and complement 3 (C3) were significantly increased (P < 0.05), whereas the interleukin-8 (IL-8) was down-regulated (P < 0.05). Besides, whole genome sequencing revealed several probiotics properties of SL001, including organic acid synthesis, bacteriocin synthesis (coagulin), superoxide dismutase, and digestive enzymes. In conclusion, P. pentosaceus SL001 which could enhance immunity and promoter growth rate of grass carps, is prospective to be used as a dietary probiotic in freshwater fish aquaculture.

Comparative evaluation of the immune responses and disease resistance of Nile tilapia (Oreochromis niloticus) induced by yeast β-glucan and crude glucan derived from mycelium in the spent mushroom substrate of Schizophyllum commune

β-glucan has been applied as an immunostimulant that enhances the fish immunity to contend with bacterial diseases, which have caused devastating losses in diverse economic cultured fish worldwide. The Schizophyllum commune or split gill mushroom is widely cultured in southern Thailand. A large number of undesirable waste, including spent mushroom substrate (SMS), increases along with its production. This work was designed to investigate the immunostimulatory effect of glucan extracted from S. commune cultivation waste in Nile tilapia (Oreochromis niloticus) to discover an alternative source of low-cost β-glucan. Crude glucan, Cr-glucan, which is obtained through pressurized hot water extraction, comprises mainly β-glucan and residues of α-glucan, protein and phenolic compounds. The immunostimulatory potential of Cr-glucan was determined compared with that of commercial β-glucan from Saccharomyces cerevisiae (Yb-glucan) by intraperitoneally (i.p.) injection (10 μg per fish). Immune parameters in blood and skin mucus and the transcriptional expression level of immune-related genes were examined at 6, 12 and 24 h post injection (hpi). The results showed that Cr-glucan could induce these immune parameters similar to Yb-glucan which was significantly higher (P < 0.05) than those of the control fish. In addition, both glucans upregulated all studied genes. Cr-glucan could extend the expression of immune-related genes over those induced by Yb-glucan. The disease resistance against a pathogenic bacterium Aeromonas veronii, a disease-causing agent of motile Aeromonas septicemia (MAS), was also established. The survival proportion in Cr-glucan treatment was higher than that of Yb-glucan and control PBS. The results together suggested that Cr-glucan enhanced the immunity contributing to protect Nile tilapia against A. veronii. These findings provide the first evidence to promote the feasibility of crude glucan from valueless waste as a potent immunostimulant that might be used in tilapia culture.

Changes in the intestine microbial, digestion and immunity of Litopenaeus vannamei in response to dietary resistant starch

Resistant starch (RS) is a constituent of dietary fibre that has beneficial effects on the intestine physiological function of animals. However, the roles of RS on shrimp intestine health is unknown. In this study, we investigated the the effects of dietary RS on the microbial composition, and digestive and immune-related indices in the intestine of Litopenaeus vannamei. The shrimp were fed with diets containing different levels of RS: 0 g/kg (Control), 10 g/kg (RS1), 30 g/kg (RS2) and 50 g/kg (RS3) for 56 days. The results showed that dietary RS improved the morphology of the intestine mucosa. RS also increased the activity of digestive enzymes (AMS, LPS, Tryp, and Pep) and immune enzymes (PO, T-AOC, T-NOS, and NO), and the expression levels of immune-related genes (proPO, ALF, Lys, HSP70, Trx, Muc-1, Muc-2, Muc-5AC, Muc-5B, and Muc-19). A microbiome analysis indicated that dietary RS increased the short-chain fatty acids (SCFAs) contents and altered the composition of the intestine microbial. Specifically, RS increased the abundances of Proteobacteria and decreased the abundance of Bacteroidetes. At the genus level, the beneficial bacteria (Lutimonas, Ruegeria, Shimia, Mesoflavibacter, and Mameliella) were enriched, which might be involved in degrading toxins and producing beneficial metabolites; while potential pathogens (Formosa and Pseudoalteromonas) were decreased in response to dietary RS. Our results revealed that dietary RS could improve the intestine health of L. vannamei, probably via modulating the intestine microbial composition and SCFAs contents, and enhancing the digestion and immunity of the shrimp.