Phagocytic Rate Research Articles

Effects of dietary dihydromyricetin on growth performance, antioxidant capacity, immune response and intestinal microbiota of shrimp (Litopenaeus vannamei)

A 56-day culture trial was conducted to evaluate the effects of dietary dihydromyricetin (DMY) on growth performance, antioxidant capacity, immune response and intestinal microbiota of shrimp (Litopenaeus vannamei). 840 healthy shrimp (1.60 ± 0.21 g) in total were fed with four different levels of DMY diets at 0 (Control), 100 (D1), 200 (D2), and 300 (D3) mg/kg, respectively. Samples were collected after the culture trial, and then, a 7-day challenge experiment against Vibrio parahaemolyticus was conducted. The results demonstrated that DMY significantly enhanced the activity of protease, amylase and lipase as well as the expression of lipid and protein transport-related genes (P < 0.05). The results of plasma lipid parameters indicated that DMY reduced lipid deposition, manifested by significantly (P < 0.05) decreased plasma total cholesterol (T-CHO), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C). The expression of genes involved in fatty acid β-oxidation and triglyceride catabolism was significantly up-regulated (P < 0.05), and genes involved in triglyceride synthesis were significantly down-regulated in DMY groups when compared to control group (P < 0.05). Moreover, dietary DMY also significantly (P < 0.05) increased the total antioxidant capacity (T-AOC), antioxidant enzymes activity and glutathione (GSH) content of shrimp, and a significant increase of total hemocytes count (THC), phagocytic rate (PR), antibacterial activity (AA) and bacteriolytic activity (BA) was observed in DMY groups (P < 0.05). The addition of DMY to the diet significantly augmented immune response by up-regulating the expression of genes related to toll-like receptors (Toll) signaling pathway, immune deficiency (IMD) signaling pathway and intestinal mucin. Furthermore, dietary DMY could modulate the composition and abundance of intestinal microbiota. In conclusion, DMY showed promising potential as a functional feed additive for shrimp to improve the growth performance and physiological health.

Functional characterization of Bombyx mori (Lepidoptera: Bombycidae) C-type lectin 5.

C-type lectins (CTLs) are an important family of pattern recognition receptors (PRRs) that regulate immune responses. The CTL5 gene of the silkworm Bombyx mori L. (Lepidoptera: Bombycidae) encodes a protein comprised of 223 amino acids, containing a signal peptide and a carbohydrate recognition domain (CRD). Our previous study showed that CTL5 can facilitate the clearance of bacteria from larval hemocoel but the underlying mechanisms are unclear. In this study, we found that CTL5 was mainly expressed in fourth-instar larvae, adult moths, and the larval epidermis. CTL5 expression showed differential responses to both pathogenic stimuli and the molting hormone 20-hydroxyecdysone. The full-length (FL) and truncated (ΔN/ΔC/ΔNC) CTL5 recombinant proteins can bind to hemocytes, polysaccharides, bacteria, and spores of the entomopathogenic fungus Beauveria bassiana. Yeast 2-hybrid assays showed that the recombinant proteins can interact with integrin β2-β5 subunits. Recombinant proteins increased the phagocytic rate of hemocytes. Injection of recombinant CTL5 stimulated the expression of many immune genes in hemocytes, mainly antimicrobial peptides and immune signaling molecules. Additionally, transcriptomic sequencing of CTL5-stimulated hemocytes revealed 265 upregulated and 580 downregulated genes. Functional enrichment and the gene set enrichment analyses showed that differentially expressed genes were mainly enriched in innate immune responses and signaling. Our study suggests that CTL5 may act as an opsonin to enhance the clearance of pathogens by regulating both humoral and cellular responses.

Phagocytosis of LM-Mel-45 Melanoma and Jurkat Cells by THP-1 Macrophages

Exposing macrophages to IFN-g or IL-4 polarizes them toward M1-like inflammatory or M2-like tissue repair phenotypes, respectively. It is uncertain whether the macrophage phenotypes are equally effective at phagocytosing melanoma and non-melanoma bait cells. We hypothesize that phenotypic polarization of macrophages will improve their phagocytic ability and that phagocytic vulnerability will depend on bait cell type. This study compares phenotypic polarization of macrophages on phagocytosis of LM-Mel-45 (LM45) melanoma cells and non-melanoma (Jurkat) cells at 2 h or 16 h. Macrophage differentiation to an M0 phenotype was induced by exposing THP-1 monocytes to 50 ng/ml PMA for two days, followed by 4 days rest in RPMI 1640, 10% FBS. M1-like and M2-like phenotypes were induced by 24 h exposure to 20 ng/ml IFN-γ and 20 ng/mL IL-4, respectively. Phenotypic state was measured by surface expression of CCR7, CD209, and CD172. Bait cells (LM45 or Jurkat) were treated with 0.5 mM staurosporine for 24 h to induce apoptosis and stained with CellTrace® Violet. Macrophages were stained with CellTracker® Orange CMRA. Bait cells were added to adherent macrophages to measure phagocytosis (2 or 16 h). To qualitatively monitor the phagocytic process, time-lapse imaging every 15 min for 16 h was done on a Zeiss Axiovert 200M microscope with a 10x objective. To quantify phagocytosis, adhered and loose cells were harvested and prepared for flow cytometry. Cells were analyzed for violet, orange, or both orange and violet staining. Positive phagocytosis was expressed as the percentage of orange macrophages with violet staining above the violet cutoff. M0 macrophages are positive for CD172. Exposure to IFN-γ increases levels of CD209 and CCR7, and CD172 remains present. Exposure to IL-4 increases levels of CD209 with little change in CCR7 and CD172 remains present. Time-lapse imaging shows phagocytosis of LM45 cells via a “nipping” behavior rather than engulfment. When Jurkat cells serve as bait, multiple bait cells will attach to a single macrophage. Blebbing in bait cells is seen by 8 h. Phagocytic rate toward LM45 cells over 2 h averaged 6.7 ± 1.8% for M0 macrophages and 5.6 ± 2.3% for IFN-γ treated macrophages. Phagocytic rate toward LM45 cells over 16 h averaged 13.9 ± 1.3% for M0 macrophages and 14.9 ± 1.8% for IFN-γ treated macrophages. The differences were not significant. Phagocytic rate toward Jurkat cells averaged 22.4 ± 3.5% for M0 macrophages and 26.9 ± 4.0% for IFN-γ treated macrophages. This difference was not significant. Phagocytosis was significantly greater for Jurkat cells than LM45 cells (P&lt;0.001). In summary, IFN-γ treatment does not increase macrophage phagocytic ability, refuting our hypothesis. However, macrophages phagocytose Jurkat cells more effectively than LM45 melanoma, suggesting that chemotherapy designed to increase phagocytic vulnerability may have potential in melanoma treatment. KCOM Graduate Program Grant #851-068 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lactobacillus plantarum isolated from kefir enhances immune responses and survival of white shrimp (Penaeus vannamei) challenged with Vibrio alginolyticus

Lactobacillus plantarum is known for its probiotics benefit to host, although the effects vary among strains. This study conducted a feeding experiment of three Lactobacillus strains, MRS8, MRS18 and MRS20, which were isolated from kefir and incorporated into the diets of shrimp to evaluate the effects of non-specific immunity, immune-related gene expression, and disease resistance of white shrimp (Penaeus vannamei) against Vibrio alginolyticus. To prepare the experimental feed groups, the basic feed was mixed with different concentrations of L. plantarum strains MRS8, MRS18, and MRS 20, which were incorporated at 0 CFU (control), 1 × 106 CFU (groups 8–6, 18–6, and 20–6), and 1 × 109 CFU (groups 8–9, 18–9, and 20–9) per gram of diet for an in vivo assay. During the rearing period for 28 days of feeding each group, immune responses, namely the total hemocyte count (THC), phagocytic rate (PR), phenoloxidase activity, and respiratory burst were examined on days 0, 1, 4, 7, 14, and 28. The results showed that groups 20–6, 18–9 and 20–9 improved THC, and groups 18–9 and 20–9 improved phenoloxidase activity and respiratory burst as well. The expression of immunity-related genes was also examined. Group 8–9 increased the expression of LGBP, penaeidin 2 (PEN2) and CP, group 18–9 increased the expression of proPO1, ALF, Lysozyme, penaeidin 3 (PEN3) and SOD, and group 20–9 increased the expression of LGBP, ALF, crustin, PEN2, PEN3, penaeidin 4 (PEN4) and CP (p < 0.05). Groups 18–6, 18–9, 2–6, and 20–9 were further used in the challenge test. After feeding for 7 days and 14 days, Vibrio alginolyticus was injected into white shrimp and observed the shrimp survival for 168 h. The results showed that compared to the control, all groups improved the survival rate. Especially, feeding group 18–9 for 14 days improved the survival rate of white shrimp (p < 0.05). After the challenge test for 14 days, the midgut DNA of survival white shrimps was extracted to analyze the colonization of L. plantarum. Among the groups, (6.61 ± 3.58) × 105 CFU/pre shrimp of L. plantarum in feeding group 18–9 and (5.86 ± 2.27) × 105 CFU/pre shrimp in group 20–9 were evaluated by qPCR. Taken together, group 18-9 had the best effects on the non-specific immunity, the immune-related gene expression, and the disease resistance, which might be due to the benefit of the probiotic colonization.

CD146+ Umbilical Cord Mesenchymal Stem Cells Exhibit High Immunomodulatory Activity and Therapeutic Efficacy in Septic Mice.

Several studies have shown that MSCs can significantly improve the survival of sepsis animals. CD146+ mesenchymal stem cells (MSCs) correlate with high therapeutic potency. However, their therapeutic effect on sepsis and detail mechanisms have not been explored. The effect of CD146±MSCs on differentiation of Treg, Th1, Th17 subsets was evaluated by flow cytometry. The effects of CD146±MSCs on RAW264.7 phagocytosis and LPS-stimulated polarization were studied using a co-culture protocol. Luminex bead array and RNA sequencing were employed to determine the mechanisms of MSCs on LPS-stimulated RAW264.7. The Arg1 protein was detected by Western blot. CD146±MSCs were injected into LPS-induced sepsis mice by tail vein. The therapeutic effect was assessed by organ HE staining, T-cell subsets, cytokine in plasma, peritoneal macrophages, infiltrating monocytes subpopulations. In vitro, CD146+MSCs could significantly increase the proportion of Treg cells. Co-culture with CD146+MSCs increase the phagocytic rate of RAW264.7. CD146+MSCs regulate M2-type macrophages production more rapidly. The transcript profile differences between the CD146+MSCs and CD146-MSCs groups were clustered in arginine metabolism pathways. CD146+MSCs decreased NO production and increased ARG1 expression. CD146+MSCs secreted higher level of IL15,IFNγ, VEGF and lower level of IL1β, IL8 under LPS stimuli. In vivo, The level of IL10 at 24h and CXCL1, IFNγ at 12h in CD146+MSCs group was the highest. CD146+MSCs treatment enhances the phagocytic capacity of peritoneal macrophages. CD146+MSCs also increases the ratios of CD11b+Ly6Clo reparative monocytes and CD11b+Ly6Chi inflammatory monocytes until 24h. Compared with CD146-MSCs, CD146+MSCs can accelerate the end of the inflammatory response and have robust anti-inflammatory effects, by increasing the Treg cells, promoting macrophage phagocytosis, enhancing the reparative macrophage, secreting more VEGF, etc.

A novel type II crustin in the innate immune response of the freshwater crab (Sinopotamon henanense) against infection and its expression changes by cadmium

Antibacterial peptide (AMP), an effector of the innate immune system, is an essential component of invertebrate innate immunity. Crustin is a family of antimicrobial peptides that are widely studied in crustaceans. Here we report a novel crustin (designated Shcrustin) from the freshwater crab Sinopotamon henanense. The results revealed that the full-length cDNA of Shcrustin was 691 bp with an open reading frame (ORF) of 510 bp. Phylogenetic analysis of the Shcrustin sequence showed that it clustered with type II crustin. Shcrustin exists in different tissues, among which the highest expression level is found in the gills. After the bacterial challenge, the expression of Shcrustin increased in hemocytes or gills. However, crustin expression was suppressed in the presence of cadmium (Cd). To elucidate the biological activity of Shcrustin, we constructed a recombinant Shcrustin protein. Purified rShcrustin could bind to a variety of bacteria and inhibit the growth of different bacteria indicating that Shcrustin has inhibitory activity against gram-positive and gram-negative bacteria. In addition, the phagocytic rate of hemocytes toward bacteria decreased after the interference of Shcrustin expression by RNA interference, suggesting that Shcrustin may be involved in such a process. Therefore, we conclude that Shcrustin may be involved in the innate immunity of S. henanense by binding to bacteria and promoting hemolymph phagocytosis to clear invading pathogens. It is an important immune effector against pathogen infection. In the presence of Cd, it may alter the expression of Shcrustin and suppress its immune function.

3-Hydroxybutyrate helps crayfish resistant to Vibrio parahaemolyticus infection in versatile ways

The bacterial storage compound poly-β-hydroxybutyrate (PHB) is a potential bio-control agent in aquaculture. It has been reported that PHB benefit to the survival and growth, and improve their immunity of aquatic animals. However, the cellular and molecular regulation mechanisms of PHB in immunity process remain unclear. This study investigated the immune mechanism of hemocytes regulated by Halomonas-PHB (PHB-HM) and PHB monomer 3-HB. Red claw crayfish Cherax quadricarinatus was used as the experimental animal in cytological study. Fluorescence microscopy and flow cytometry (FCM) analysis indicated that PHB-HM labeled with fluorescein isothiocyanate (FITC) could be engulfed by granulocytes (Gs) and semi-granulocytes (SGs) upon in vitro incubation. Transmission electron microscopy (TEM) further showed the ongoing degradation of PHB granules inside Gs and SGs after the injection of PHB-HM into crayfish sinus, but phagocytosis of PHB-HM by hyalinocyte (H) was not observed. Therefore, Gs and SGs are considered the main effector cells of cellular immunity induced by PHB-HM, and SGs likely played a particular important role in this process. To study the biosafety and molecular mechanism of PHB monomer 3-HB, hemocyte viability and expression of the related genes were determined after being exposed to 0–1 mg/mL of 3-HB, and Vibrio parahaemolyticus (VP) was used as the pathogenic bacterium. The results confirmed that 3-HB had no toxic effect on hemocytes by means of cell viability assay, and supplementation with 1 mg/mL of 3-HB suppressed the growth rate of VP in TSB medium. Moreover, injection of 3-HB into the blood sinus of crayfish remarkably improved the phagocytic rate of Gs and SGs on VP. Furthermore, transcriptome assay was designed to illuminate the molecular mechanism of 3-HB regulation using red swamp crayfish Procambarus clarkii as experimental animals. RNA-seq analysis and qRT-PCR verification revealed that the microtubule and cytoskeleton-related genes were high expressed 3 h after 3-HB injection, indicating both genes might involve in building up the innate immunity. In summary, bacterial storage PHB could be phagocytosed by main effector blood cells and likely to be degraded within the cells. 3-HB helped the crayfish resistant to pathogens through improving phagocytosis, suppressing the growth of pathogenic bacteria, and increasing the expression of microtubule-related genes. The findings in this work provide cytological and molecular evidence which will facilitate the application of PHB and 3-HB as immune-control agents in farming of aquatic animals.

Biofilm of Klebsiella pneumoniae minimize phagocytosis and cytokine expression by macrophage cell line

Infectious bacteria in biofilm mode are involved in many persistent infections. Owing to its importance in clinical settings, many in vitro and in vivo studies are being conducted to study the structural and functional properties of biofilms, their drug resistant mechanism and the s urvival mechanism of planktonic and biofilm cells. In this regard, there is not sufficient information on the interaction between Klebsiella biofilm and macrophages. In this study, we have attempted to unravel the interaction between Klebsiella biofilm and macrophages in terms of phagocytic response and cytokine expression. In vitro phagocytosis assays were performed for heat inactivated and live biofilms of K. pneumoniae, together with the expression analysis of TLR2, iNOS, inflammatory cytokines such as IL-β1, IFN-γ, IL-6, IL-12, IL-4, TNF-α and anti-inflammatory cytokine, IL-10. A phagocytic rate of an average of 15% was observed against both heat inactivated and live biofilms when LPS + IFN-γ activated macrophages were used. This was significantly higher than non-activated macrophages when tested against heat inactivated and live biofilms (average 8%). Heat-inactivated and live biofilms induced similar phagocytic responses and up-regulation of pro-inflammatory genes in macrophages, indirectly conveying that macrophage responses are to some extent dependent on the biofilm matrix.

The immunity-promoting activity of porcine placenta in mice as an immunomodulator for functional foods

This study was to explore the immunity-promoting activity of porcine placenta as a potential raw material for functional foods. Porcine placenta was subjected to the analysis for its bioactive substances, and their immunity-promoting activity was determined in mice supplemented with porcine placenta extract (PPE) and freeze-dried porcine placenta powder at high (PPH) and low (PPL) dosage. Results showed that porcine placenta contained placental peptides and 15 free amino acids, and the amounts of estrogen and progesterone in products developed from porcine placenta were within the limit of national standard. Mice model experiment revealed that compared with the control, the PPH treatment significantly improved the spleen index (P < 0.05) by increasing the phagocytic rate of macrophages from 20 % to 60 % and the conversion rate of T lymphocytes from 8 % to 60 %. The qPCR analysis disclosed that the porcine placenta powder enhanced mice immunity via promoting the expression of Th1 cytokines of interleukin-2 (IL-2) and IFN-γ, especially the former, by almost 8 times in the spleens of male mice, while inhibited Th2 cytokines of IL-4 and IL-10. This investigation has provided a reference for the development of porcine placenta as a raw material applied in functional foods to improve human immunity.

Effect of dietary supplementation with Moringa oleifera leaf extract and Lactobacillus acidophilus on growth performance, intestinal microbiota, immune response, and disease resistance in whiteleg shrimp (Penaeus vannamei)

This study investigated the effect of the moringa (Moringa oleifera) leaf extract and Lactobacillus acidophilus individually or combined on growth performance, enzyme activity, intestinal and hepatopancreatic histology, intestinal microbiota, immune response, and resistance against Vibrio alginolyticus and Vibrio parahaemolyticus in whiteleg shrimp (Penaeus vannamei). Six diets were formulated: three diets without L. acidophilus containining 0 (control, ME0), 2.5 (ME2.5), and 5.0 g/kg of moringa (ME5.0) and the same three diets containing L. acidophilus at 1 × 107 CFU/g of diet (ME0+P, ME2.5 + P, and ME5.0 + P, respectively). Growth performance was measured after 60 days of the rearing period. On the final day, the shrimp were sampled to assess enzyme activity, intestinal and hepatopancreatic histology, and gut microbiota. Shrimp hemocytes were examined on Days 0, 1, 2, 4, 7, 14, 21, and 28 to measure the immune response in terms of the total hemocyte count, phenoloxidase activity, phagocytosis, and superoxide anion production. Furthermore, the shrimp were challenged with V. alginolyticus and V. parahaemolyticus. The results revealed that ME2.5 + P significantly increased (P < 0.05) final weight, weight gain, specific growth rate, enzyme activities, and villi height compared with ME2.5 and control. Wall thickness was increased in the shrimp fed diet supplemented with moringa and L. acidophilus compared with the control shrimp. Hepatopancreatic histology revealed that R cells were more abundant in the shrimp fed diet containing moringa and L. acidophilus compared with those fed diet containing moringa alone (P < 0.05) at the same concentration. High-throughput sequencing analysis indicated that the dietary supplementation with moringa and L. acidophilus affected the gut microbiota composition. All gene functions, members of KEGG level 2, related to metabolism were increased in diet supplemented with moringa with or without L. acidophilus compared with the control group. The immune assay revealed that the total hemocyte count, phenoloxidase activity, phagocytic rate, superoxide anion production, and immune-related gene expression (including those of prophenoloxidase II, alpha-2-macroglobulin, penaeidin2, antilipopolysaccharide factor, crustin, lysozyme, glutathione peroxidase, and superoxide dismutase) were higher in the experimental groups than in the control group on several observed days; however, the increases were observed more often in the ME2.5 + P group than in the other treatment groups. Furthermore, the ME2.5 + P group exhibited a significantly higher survival rate (P < 0.05) in the challenge test against V. alginolyticus and V. parahaemolyticus. In conclusion, supplementation with dietary moringa and L. acidophilus at ME2.5 + P improved growth performance, immune system, and resistance against Vibrio in the shrimp.

Effects of dietary Lactobacillus reuteri and Pediococcus acidilactici on the cultured water qualities, the growth and non-specific immune responses of Penaeus vannamei

This study discussed the effects of two types of lactic acid bacteria, Lactobacillus reuteri (L. reuteri) and Pediococcus acidilactici (P. acidilactici), on the growth and nonspecific immunity of Penaeus vannamei (P. vannamei) and developed probiotic applications for shrimp cultivation.This study incorporated two types of lactic acid bacteria in shrimp feed through spraying. The shrimps were grouped according to the type and concentration of effective bacteria incorporated into their feed. This research was separated into 3 individual feeding treatment as control, L. reuteri (Lr groups) and P. acidilactici (Pa groups). The shrimp was feeding with 103, 105, and 107 cfu/feed (g) L. reuteri namely as Lr3, Lr5, and Lr7. The shrimp was feeding with 103, 105, and 107 cfu/feed (g) P. acidilactici were named Pa3, Pa5, and Pa7, respectively. Through 8 weeks of feeding, the results revealed that the use of shrimp feed incorporated with lactic acid bacteria did not cause negative effects on water quality. The testing items include ammonia-nitrogen concentration, nitrite-nitrogen concentration, and total vibrio count in the water. In addition, the lactic acid bacteria concentration in the water were in the range of 1.33 ± 0.58 × 101 to 9.77 ± 1.34 × 102 cfu/mL. Observations of the growth performance of the white shrimps after 8 weeks of feeding revealed that both bacteria were beneficial to shrimp growth. In particular, group Lr7 had the highest percentage weight gain (107.99 ± 3.92%), special growth rate (1.93 ± 0.07%), feed conversion ratio (3.34 ± 0.05), and survival rate (97.22 ± 4.81%).Furthermore, observations of the nonspecific immunity reactions of the white shrimps after 4 weeks of feeding indicated that on day 4, the total number of haemocyte in shrimps in groups Lr5, Lr7, Pa3, and Pa5 significantly increased. On days 1 and 4, the phenoloxidase activity and superoxide axion production rates of the Lr group and Ls group increased. This phenomenon was the most significant in group Lr7, and the effect continued until day 28. After day 7, the phagocytic rate of groups Lr5 and Lr7 significantly increased. In addition, Lr and Pa groups exhibited significant increases in the phagocytic index after days 4 and 14, respectively. This phenomenon was also the most significant in group Lr7.

Natural Feed Supplements Improve Growth, Non-Specific Immune Responses and Resistance against Vibrio alginolyticus in Lates calcarifer

This study aimed to investigate the effects of dietary natural feed supplement on the growth performance, non-specific responses, and disease resistance in Lates calcarifer. Three commercial products (A, B, and C) containing a basal probiotic mixture were tested. Product A contained a basal mixture of Paenibacillus sp., Bacillus subtilis, Bacillus amyloliquefaciens, and Lactobacillus rhamnosus (107 cfu/g for each probiotic); product B contained additional Lactobacillus plantarum (1010 cfu/g); and product C contained additional soybean peptides (500 g/kg) and garlic powder (1 g/kg). Each product was supplemented into subject diets at dosages of 1 or 2 g/kg (designated as the A1, A2, B1, B2, C1, and C2 groups, respectively). Following an eight-week trial, growth parameters (specific growth rate and feed conversion ratio), non-specific immune responses (O2− production, phagocytic rate, and phagocytic index), and the results of a challenge test against Vibrio alginolyticus were evaluated. The results show that all probiotic supplement groups exhibited an improvement in growth performance compared to the control group (non-probiotic diet). In terms of non-specific immunity parameters, a significant improvement in O2− production was found in the C2 group, whereas significant improvements in phagocytic activity were found in all the B and C groups. The C2 group displayed optimal O2− production, phagocytic rate, and phagocytic index results. For the challenge test, the C groups showed higher Vibrio resistance than the other experimental groups and the control group. These results suggest that product C, given at dosages of 2 g/kg, may serve as a growth-promoting and immunostimulatory additive for the cultivation of Asian seabass.

Exosomal miRNA-455 from Bone Marrow Stromal Cells (BMSCs) Promotes Macrophage Phagocytosis and Restrains Progression of Gastric Cancer (GC)

Multiple miRNAs are differentially expressed in gastric cancer (GC). Herein, this study aims to investigate miR-455’s role in GC and its mechanism. Exosomes (exo) separated from BMSCs after transfection were co-cultured with either phagocytes, GC cells (NCI-N87 cell), or macrophages combined with NCI-N87cells (mixed group) followed by analysis of the expression of PTEN, N-cadherin, E-cadherin, and PI3K, and AKT by RT-qPCR and Western blot. Increased miR-455 expression was observed in GC cells upon transfection. GC cells in the mixed group relative to NCI-N87 group exhibited a lower cell migration and invasion and impaired proliferative capacity (p &lt; 0.05), accompanied with higher expressions of N-cadherin, E-cadherin, PI3K, and AKT, and decreased level of PTEN (p &lt; 0.05). The combined treatment resulted in a higher phagocytic rate (12.38±0.21%) and phagocytic index (14.29±2.11%) compared to treatment with only phagocytes (p &lt; 0.05). In conclusion, BMSC-derived exosomal miR-455 inhibits the growth of GC cells and promotes the phagocytosis through inactivating PI3K/AKT signaling pathway.

Bioaccumulation of arsenic and immunotoxic effect in white shrimp (Penaeus vannamei) exposed to trivalent arsenic

Trivalent arsenic (As (III)) contamination in the marine environment can produce adverse effects in crustaceans. The present study investigated the chronic toxicity of As (III) in white shrimp (Penaeus vannamei) by analyzing the tissue bioaccumulation and non-specific immune responses. Shrimps were exposed to 0 (control), 50, 500, and 2500 μg/L of As (III) for 21 days. The results showed that the hepatopancreas was the main tissue of arsenic accumulation in white shrimp. The cumulative concentration of total arsenic and inorganic arsenic but not arsenobetaine was positively correlated with the exposure concentration. In vitro As (III) treatment (0–2500 μg/L) with haemocytes isolated from healthy shrimp did not cause the cytotoxicity, but this arsenic treatments inhibited the phagocytic rate and O2− production. Moreover, the decrease of total haemocyte count and the inhibition of phagocytic rate, phagocytic index, O2− production and phenoloxidase activity were observed in white shrimp under the exposure of As (III) over a period of 21 days. This study revealed that chronic As (III) stress could disturb arsenic metabolism and immune responses in P. vannamei.

Effect of Methylmercury Exposure on Bioaccumulation and Nonspecific Immune Respsonses in Hybrid Grouper Epinephelus fuscoguttatus × Epinephelus lanceolatus.

Simple SummaryThe head kidney was primary organ that accumulated methylmercury in hybrid grouper. Muscle tissue had lower methylmercury content than the head kidney and liver. Nonspecific immune responses and bioaccumulation of methylmercury were linked to hybrid grouper health.Mercury (Hg) is a dangerous heavy metal that can accumulate in fish and is harmful when consumed by humans. This study investigated the bioaccumulation of mercury in the form of methylmercury (MeHg) and evaluated nonspecific immune responses such as phagocytic activity and superoxide anion (O2−) production in hybrid grouper (Epinephelus fuscoguttatus × E. lanceolatus). The hybrid grouper leukocytes were incubated with methylmercury chloride (CH3HgCl) at concentrations of 10–10,000 µg/L to determine cell viability, phagocytic activity, and O2− production in vitro. Subsequently, the grouper were exposed daily to CH3HgCl mixed in the experimental diets at concentrations of 0, 1, 5, and 10 mg/kg for 28 days. The bioaccumulation of MeHg in the liver, head kidney, and muscle tissue was measured, and the phagocytic activity and O2− production were evaluated. In vitro results indicated that cell viability was significantly lower than that of the control group at concentrations > 500 µg/L. The phagocytic rate and O2− production at concentrations ˃ 500 and ˃ 200 µg/L, respectively, were significantly lower than those of the control group. The dietary exposure demonstrated that MeHg accumulated more substantially in the liver and head kidney compared with the muscle tissue in the treatment groups. Moreover, the cumulative concentration significantly increased with higher concentrations and more days of exposure. The phagocytic rate and O2− production in the treatment groups were significantly lower than those in the control group from days 2 and 1, respectively. In conclusion, hybrid grouper accumulated significant MeHg in the liver and head kidney compared with the muscle tissue, and higher concentrations and more exposure days resulted in decreased cell viability, phagocytic activity, and O2− production.

Hemolymph of triatomines presents fungistatic activity against Cryptococcus neoformans and improves macrophage function through MCP-I/TNF-α increase.

Background: Triatomines are blood-feeding arthropods belonging to the subfamily Triatominae (Hemiptera; Reduviidae), capable of producing immunomodulatory and water-soluble molecules in their hemolymph, such as antimicrobial peptides (AMPs). In this work, we evaluated the antifungal and immunomodulatory activity of the hemolymph of Meccus pallidipennis (MPH) and Rhodnius prolixus (RPH) against Cryptococcus neoformans. Methods:We assessed the activity of the hemolymph of both insects on fungal growth by a minimum inhibitory concentration (MIC) assay. Further, RAW 264.7 macrophages were cultivated with hemolymph and challenged with C. neoformans. Then, their phagocytic and killing activities were assessed. The cytokines MCP-1, IFN-γ, TNF-α, IL-10, IL-12, and IL-6 were measured in culture supernatants 4- and 48-hours post-infection. Results:Both hemolymph samples directly affected the growth rate of the fungus in a dose-dependent manner. Either MPH or RPH was capable of inhibiting fungal growth by at least 70%, using the lowest dilution (1:20). Treatment of RAW 264.7 macrophages with hemolymph of both insects was capable of increasing the production of MCP-I and TNF-α. In addition, when these cells were stimulated with hemolymph in the presence of C. neoformans, a 2- and a 4-fold increase in phagocytic rate was observed with MPH and RPH, respectively, when compared to untreated cells. For the macrophage killing activity, MPH decreased in approximately 30% the number of viable yeasts inside the cells compared to untreated control; however, treatment with RPH could not reduce the total number of viable yeasts. MPH was also capable of increasing MHC-II expression on macrophages. Regarding the cytokine production, MCP-I and TNF-α, were increased in the supernatant of macrophages treated with both hemolymphs, 4 and 48 hours after stimulation.Conclusion:These results suggested that hemolymph of triatomines may represent a source of molecules capable of presenting antifungal and immunomodulatory activity in macrophages during fungal infection.

Moringa oleifera Leaves' Extract Enhances Nonspecific Immune Responses, Resistance against Vibrio alginolyticus, and Growth in Whiteleg Shrimp (Penaeus vannamei).

Simple SummaryThis study found that moringa (Moringa oleifera) leaves’ water extract triggered phenoloxidase activity, phagocytic rate, and superoxide anion production in whiteleg shrimp (Penaeus vannamei) hemocytes by an in vitro assay. By an in vivo assay, a dietary moringa extract enhanced the total hemocyte count, phenoloxidase activity, phagocytic rate, immune-related gene expressions, and growth performance of the whiteleg shrimp. The administration of dietary moringa extract increased the survival rate after challenging the whiteleg shrimp with Vibrio alginolyticus.Moringa is widely known as a plant with high medicinal properties. Therefore, moringa has a high potential for use as an immunostimulant in shrimp. This study investigated the effect of a moringa water extract on the immune response, resistance against V. alginolyticus, and growth performance of whiteleg shrimp. To perform the in vitro assay, hemocytes were incubated with different concentrations of the moringa extract. Furthermore, the moringa extract was incorporated at 0 (control), 1.25 g (ME1.25), 2.5 g (ME2.5), and 5.0 g (ME5.0) per kg of diet for the in vivo assay. During the rearing period, immune responses, namely the total hemocyte count (THC), phenoloxidase (PO) activity, phagocytosis activity, superoxide anion production, and immune-related gene expression were examined on days 0, 1, 2, 4, 7, 14, 21, and 28. Growth performance was measured 60 days after the feeding period. Furthermore, the shrimp were challenged with V. alginolyticus after being fed for different feeding durations. The results of the in vitro assay revealed that 100–250 ppm of the moringa extract enhanced the PO activity, phagocytic rate (PR), and superoxide anion production. The findings of the in vivo assay demonstrated that the THC, PO activity, PR, and immune-related gene expression, including alpha-2-macroglobulin, prophenoloxidase II, penaeidin2, penaeidin3, anti-lipopolysaccharide factor, crustin, lysozyme, superoxide dismutase, and clotting protein, were higher in the group of ME.25 and ME5.0 than in the control and ME1.25 at several time points. Growth performance was significantly increased (p < 0.05) in the ME2.5 group compared to the control group. Furthermore, the dietary ME2.5 resulted in a higher survival rate compared to that of the control group after challenging with V. alginolyticus, especially at ME2.5 administered for 4 and 7 days. This study indicated that the incorporation of the moringa extract at 2.5 g per kg of diet enhanced the immune response, the growth performance of the whiteleg shrimp, and the resistance against V. alginolyticus infection.

CgRab1 regulates Cgcathepsin L1 expression and participates in the phagocytosis of haemocytes in oyster Crassostrea gigas

Rab protein plays an important role in a variety of cellular activities, especially the fusion process of the inner membrane during endocytosis. In the present study, a Rab1 protein (CgRab1) was identified from the Pacific oyster Crassostrea gigas. The full-length cDNA sequence of CgRab1 was of 2248 bp with an open reading frame of 618 bp, encoding a polypeptide of 205 amino acids containing a Rab domain. The deduced amino acid sequence of CgRab1 shared 94.2% and 89.3% identity with Rab1 from Pomacea canaliculata and Homo sapiens respectively. In the phylogenetic tree, CgRab1 was firstly clustered with the Rab1s from Aplysia californica and Pomacea canaliculata to form a sister group with Rab1 from invertebrates. The recombinant CgRab1 protein (rCgRab1) was able to bind GTP. The mRNA transcripts of CgRab1 were highly expressed in oyster haemocytes, and its expression level in oyster haemocytes was significantly up-regulated at 24 h after the stimulations with Vibro splendidus, which was 2.43-fold (p < 0.01) of that in the control group. After the expression of CgRab1 was knocked down (0.38-fold of that in EGFP-RNAi experimental group) by RNAi，the protein expression of Cgcathepsin L1 were reduced (0.63-fold, p < 0.01) compared with that in EGFP-RNAi experimental group. The phagocytic rate and phagocytic index of haemocytes in CgRab1-RNAi oysters decreased after V. splendidus stimulation, which was 0.50-fold (p < 0.01) and 0.58-fold (p < 0.01) of that in EGFP-RNAi experimental group. These results indicated that CgRab1 was involved in the process of haemocytes phagocytosis by regulating the expression of Cgcathepsin L1 in oyster C. gigas.

Immunotoxicity of polychlorinated biphenyls (PCBs) to the marine crustacean species, Scylla paramamosain

Polychlorinated biphenyls (PCBs) are persistent organic pollutants in environments, and they can negatively affect aquatic animal health. After 7 days of PCBs exposure, the activities of catalase, phenoloxidase, and superoxide dismutase and the total hemocyte count in the haemolymph were significantly decreased and the reactive oxygen species (ROS) content and phagocytic rate of hemocytes were significantly increased in mud crab Scylla paramamosain. Additionally, serum lysozyme, glutathione, glutathione-S-transferase, and glutathione peroxidase activities were significantly down-regulated in mud crab after PCBs exposure. The survival rate of crab hemocytes significantly declined as the PCBs concentration increased, indicating that PCBs had a cytotoxic effect on hemocytes. Exposure to increasing concentrations of PCBs also increased the degree of DNA damage in crab hemocytes. After PCBs exposure, the expression levels of P53 and caspase-3 in hemocytes were significantly up-regulated, which suggests that apoptosis was occurring. The apoptosis rate of hemocytes was up-regulated as the PCBs concentration increased, indicating that apoptosis was induced by the PCBs-activated caspase-3 pathway. These data suggest that exposure to PCBs hampered the immune response of mud crabs, most likely by (1) inducing ROS, causing DNA damage, and reducing the viability of hemocytes, (2) reducing the activities of antioxidant enzymes, and (3) inducing phagocytosis and apoptosis of hemocytes. And the final result of PCBs-induced immunotoxicity to mud crabs is the reduced bacterial disease resistance and survival rate of crabs under Vibrio alginolyticus challenge.

Melanin of Sporothrix globosa affects the function of THP-1 macrophages and modulates the expression of TLR2 and TLR4

BackgroundMelanin is an important virulence factor for Sporothrix globosa, the causative agent of sporotrichosis, a subcutaneous mycosis that occurs worldwide. Although previous research suggests that melanin is involved in the pathogenesis of sporotrichosis, little is known about its influence on the macrophages that represent the frontline components of innate immunity. ObjectivesTo evaluate the effects of melanin on phagocytic activity and the expression of Toll-like receptor (TLR)2 and TLR4 during S. globosa infection of macrophages in vitro. MethodsTo compare phagocytic activity and survival rates, THP-1 macrophages and primary mouse peritoneal macrophages were co-cultured with a wild-type S. globosa strain (Mel+), an albino mutant strain (Mel˗), a tricyclazole-treated Mel + strain (TCZ-Mel+), or melanin ghosts extracted from S. globosa conidia. Reactive oxygen species (ROS), nitric oxide (NO) generation, tumor necrosis factor (TNF)-α and interleukin (IL)-6 were assayed in THP-1 cells infected with S. globosa conidia. Quantitative PCR and western blotting were used to observe the effect of melanin on TLR2 and TLR4 expression. Knockdown of TLR2/4 expression with small interfering RNA was performed to further verify the role of these receptors during infection. ResultsMacrophages infected with Mel + conidia showed a lower phagocytosis index and a higher survival rate than TCZ-Mel+ and Mel˗ in vitro. After incubation with S. globosa, the release of ROS, NO, TNF-α and IL-6 by THP-1 were decreased in the presence of melanin. Increased mRNA and protein expression of TLR2 and TLR4 occurred upon S. globosa infection in THP-1, whereas the presence of melanin suppressed TLR2 and TLR4. Moreover, TLR2 or TLR4 knockdown showed a trend toward reducing the pernicious effect of S. globosa conidia on THP-1 cells in vitro. ConclusionsCollectively, our results indicated that melanin inhibits the phagocytosis of S. globosa and guards against macrophage attack by providing protection from oxygen- and nitrogen-derived radicals, as well as suppressing the host pro-inflammatory cytokine response (TNF-α and IL-6). Melanin was also involved in modulating TLR2 and TLR4 receptor expression, weakening the killing efficiency of S. globosa.