DC-SIGN of Largemouth Bass (Micropterus salmoides) Mediates Immune Functions against Aeromonas hydrophila through Collaboration with the TLR Signaling Pathway.

C-type lectins in organisms play an important role in the process of innate immunity. In this study, a C-type lectin belonging to the DC-SIGN class of Micropterus salmoides was identified. MsDC-SIGN is classified as a type II transmembrane protein. The extracellular segment of MsDC-SIGN possesses a coiled-coil region and a carbohydrate recognition domain (CRD). The key amino acid motifs of the extracellular CRD of MsDC-SIGN in Ca2+-binding site 2 were EPN (Glu-Pro-Asn) and WYD (Trp-Tyr-Asp). MsDC-SIGN-CRD can bind to four pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), glucan, peptidoglycan (PGN), and mannan. Moreover, it can also bind to Gram-positive, Gram-negative bacteria, and fungi. Its CRD can agglutinate microbes and displays D-mannose and D-galactose binding specificity. MsDC-SIGN was distributed in seven tissues of the largemouth bass, among which the highest expression was observed in the liver, followed by the spleen and intestine. Additionally, MsDC-SIGN was present on the membrane of M. salmoides leukocytes, thereby augmenting the phagocytic activity against bacteria. In a subsequent investigation, the expression patterns of the MsDC-SIGN gene and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) exhibited an up-regulated expression response to the stimulation of Aeromonas hydrophila. Furthermore, through RNA interference of MsDC-SIGN, the expression level of the DC-SIGN signaling pathway-related gene (RAF1) and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) was decreased. Therefore, MsDC-SIGN plays a pivotal role in the immune defense against A. hydrophila by modulating the TLR signaling pathway.

Acanthopanax senticosus cultures fermented by Lactobacillus rhamnosus enhanced immune response through improvement of antioxidant activity and inflammation in crucian carp (Carassius auratus).

Lactic acid bacteria (LAB) have been recognized as safe microorganism that improve micro-flora disturbances and enhance immune response. A well-know traditional herbal medicine, Acanthopanax senticosus (As) was extensively utilized in aquaculture to improve growth performance and disease resistance. Particularly, the septicemia, skin wound and gastroenteritis caused by Aeromonas hydrophila threaten the health of aquatic animals and human. However, the effects of probiotic fermented with A. senticosus product on the immune regulation and pathogen prevention in fish remain unclear. Here, the aim of the present study was to elucidate whether the A. senticosus fermentation by Lactobacillus rhamnosus improve immune barrier function. The crucian carp were fed with basal diet supplemented with L. rhamnosus fermented A. senticosus cultures at 2 %, 4 %, 6 % and 8 % bacterial inoculum for 8 weeks. After trials, the weight gain rate (WGR), specific growth rate (SGR) were significantly increased, especially in LGG-6 group. The results confirmed that the level of the CAT, GSH-PX, SOD, lysozyme, and MDA was enhanced in fish received with probiotic fermented product. Moreover, the L. rhamnosus fermented A. senticosus cultures could trigger innate and adaptive immunity, including the up-regulation of the C3, C4, and IgM concentration. The results of qRT-PCR revealed that stronger mRNA transcription of IL-1ß, IL-10, IFN-γ, TNF-α, and MyD88 genes in the liver, spleen, kidney, intestine and gills tissues of fish treated with probiotic fermented with A. senticosus product. After infected with A. hydrophila, the survival rate of the LGG-2 (40 %), LGG-4 (50 %), LGG-6 (60 %), LGG-8 (50 %) groups was higher than the control group. Meanwhile, the pathological damage of the liver, spleen, head-kidney, and intestine tissues of probiotic fermentation-fed fish could be alleviated after pathogen infection. Therefore, the present work indicated that L. rhamnosus fermented A. senticosus could be regard as a potential intestine-target therapy strategy to protecting fish from pathogenic bacteria infection.

Climate change can impair bacterial pathogen defences in sablefish via hypoxia-mediated effects on adaptive immunity.

Low-oxygen levels (hypoxia) in aquatic habitats are becoming more common because of global warming and eutrophication. However, the effects on the health/disease status of fishes, the world's largest group of vertebrates, are unclear. Therefore, we assessed how long-term hypoxia affected the immune function of sablefish, an ecologically and economically important North Pacific species, including the response to a formalin-killed Aeromonas salmonicida bacterin. Sablefish were held at normoxia or hypoxia (100% or 40% air saturated seawater, respectively) for 6-16 weeks, while we measured a diverse array of immunological traits. Given that the sablefish is a non-model organism, this involved the development of a species-specific methodological toolbox comprised of qPCR primers for 16 key immune genes, assays for blood antibacterial defences, the assessment of blood immunoglobulin (IgM) levels with ELISA, and flow cytometry and confocal microscopy techniques. We show that innate immune parameters were typically elevated in response to the bacterial antigens, but were not substantially affected by hypoxia. In contrast, hypoxia completely prevented the ∼1.5-fold increase in blood IgM level that was observed under normoxic conditions following bacterin exposure, implying a serious impairment of adaptive immunity. Since the sablefish is naturally hypoxia tolerant, our results demonstrate that climate change-related deoxygenation may be a serious threat to the immune competency of fishes.

Transcriptome profiling of lumpfish (Cyclopterus lumpus) head kidney to Renibacterium salmoninarum at early and chronic infection stages.

Renibacterium salmoninarum causes Bacterial Kidney Disease (BKD) in several fish species. Atlantic lumpfish, a cleaner fish, is susceptible to R. salmoninarum. To profile the transcriptome response of lumpfish to R. salmoninarum at early and chronic infection stages, fish were intraperitoneally injected with either a high dose of R. salmoninarum (1 × 109 cells dose-1) or PBS (control). Head kidney tissue samples were collected at 28- and 98-days post-infection (dpi) for RNA sequencing. Transcriptomic profiling identified 1971 and 139 differentially expressed genes (DEGs) in infected compared with control samples at 28 and 98 dpi, respectively. At 28 dpi, R. salmoninarum-induced genes (n = 434) mainly involved in innate and adaptive immune response-related pathways, whereas R. salmoninarum-suppressed genes (n = 1537) were largely connected to amino acid metabolism and cellular processes. Cell-mediated immunity-related genes showed dysregulation at 98 dpi. Several immune-signalling pathways were dysregulated in response to R. salmoninarum, including apoptosis, alternative complement, JAK-STAT signalling, and MHC-I dependent pathways. In summary, R. salmoninarum causes immune suppression at early infection, whereas lumpfish induce a cell-mediated immune response at chronic infection. This study provides a complete depiction of diverse immune mechanisms dysregulated by R. salmoninarum in lumpfish and opens new avenues to develop immune prophylactic tools to prevent BKD.

Haemolysin Ahh1 secreted from Aeromonas dhakensis activates the NLRP3 inflammasome in macrophages and mediates severe soft tissue infection.

Severe soft tissue infections caused by Aeromonas dhakensis, such as necrotizing fasciitis or cellulitis, are prevalent in southern Taiwan and around the world. However, the mechanism by which A. dhakensis causes tissue damage remains unclear. Here, we found that the haemolysin Ahh1, which is the major virulence factor of A. dhakensis, causes cellular damage and activates the NLR family pyrin domain containing 3 (NLRP3) inflammasome signalling pathway. Deletion of ahh1 significantly downregulated caspase-1, the proinflammatory cytokine interleukin 1ß (IL-1ß) and gasdermin D (GSDMD) and further decreased the damage caused by A. dhakensis in THP-1 cells. In addition, we found that knockdown of the NLRP3 inflammasome confers resistance to A. dhakensis infection in both THP-1 NLRP3-/- cells and C57BL/6 NLRP3-/- mice. In addition, we demonstrated that severe soft-tissue infections treated with antibiotics combined with a neutralizing antibody targeting IL-1ß significantly increased the survival rate and alleviated the degree of tissue damage in model mice compared control mice. These findings show that antibiotics combined with therapies targeting IL-1ß are potential strategies to treat severe tissue infections caused by toxin-producing bacteria.

Expression and functional characterization of three ß-defensins in grass carp (Ctenopharyngodon idella).

ß-defensins (BDs) are a group of cysteine-rich cationic antimicrobial peptides and play important roles in the first line of defense against infection. In this study, the expression and antibacterial activities of three grass carp (Ctenopharyngodon idella) (Ci) ß-defensin (BD) peptides were comparatively investigated. Expression analysis reveals that CiBD1-3 were constitutively expressed in tissues, with the highest expression detected in the skin. The CiBD-1 transcripts were more abundant than CiBD-2 and CiBD-3. In the primary head kidney leukocytes, CiBDs were induced by PHA, LPS, poly(I:C) and cytokines such as IL-1ß and IFN-γ. In vivo challenge of fish with Aeromonas hydrophila resulted in the up-regulation of CiBDs in the head kidney and hindgut. To determine the biological activities, recombinant CiBD proteins were produced in the HEK293-F cells and purified for the minimum inhibitory concentration assay. It was found that all three recombinant CiBD proteins were effective to inhibit the growth of Gram-negative fish bacterial pathogens including Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium columnare and Klebsiella pneumoniae and Gram-positive Staphylococcus aureus. CiBD-2 and CiBD-3 were more effective than CiBD-1. Our results demonstrate that all the three CiBDs have broad antibacterial activity against fish bacterial pathogens.

Transcriptional Signatures of Immune, Neural, and Endocrine Functions in the Brain and Kidney of Rainbow Trout (Oncorhynchus mykiss) in Response to Aeromonas salmonicida Infection.

Rainbow trout (Oncorhynchus mykiss) serves as one of the most important commercial fish with an annual production of around 800,000 tonnes. However, infectious diseases, such as furunculosis caused by Aeromonas salmonicida infection, results in great economic loss in trout culture. The brain and kidney are two important organs associated with "sickness behaviors" and immunomodulation in response to disease. Therefore, we worked with 60 trout and investigated transcriptional responses and enrichment pathways between healthy and infected trout. We observed that furunculosis resulted in the activation of toll-like receptors with neuroinflammation and neural dysfunction in the brain, which might cause the "sickness behaviors" of infected trout including anorexia and lethargy. We also showed the salmonid-specific whole genome duplication contributed to duplicated colony stimulating factor 1 (csf-1) paralogs, which play an important role in modulating brain immunomodulation. Enrichment analyses of kidneys showed up-regulated immunomodulation and down-regulated neural functions, suggesting an immune-neural interaction between the brain and kidney. Moreover, the kidney endocrine network was activated in response to A. salmonicida infection, further convincing the communications between endocrine and immune systems in regulating internal homeostasis. Our study provided a foundation for pathophysiological responses of the brain and kidney in response to furunculosis and potentially offered a reference for generating disease-resistant trout strains.

Bactericidal efficacy of non-thermal plasma activation against Aeromonas hydrophila and immunological responses of koi (Cyprinus carpio haematopterus).

In the aquaculture industry, an efficient and safe water purification system is important to prevent mass mortality by virulent pathogens. As extensive use of traditional methods (e.g.: povidone-iodine, ozone, ultraviolet irradiation, formalin, and chlorine dioxide) have adverse effects on cultured fish, an appropriate and alternative water purification method is vital for the sustainability of the industry. Non-thermal plasma technology has been successfully used for various biomedical purposes (e.g: food sterilization, medical device disinfection, wound healing, cancer therapy, etc.) and has great potential to be used as a sterilizing system. However, few studies have been conducted on its usefulness in the aquaculture industry. In this study, we investigated the bactericidal efficacy of plasma-activated water induced by non-thermal plasma and its histopathological as well as immunological adverse effects on koi. A highly virulent Aeromonas hydrophila SNU HS7, which caused massive mortality of koi, was used for this study. Non-thermal plasma was applied for 10 min to the fish tanks with 1.2 × 109 CFU/mL SNU HS7 using PLMB-20 system to confirm the sterilization efficacy and to observe the survival and immunological reaction of koi for 14 days. As a result, gross pathological, histopathological, and immunological investigations did not reveal any significant adverse effects in fish as compared to the control groups. To the best of our knowledge, this is the first study showing that non-thermal plasma can be used for sterilization of rearing water without giving significant physiological damage to the fish, even under the assumption of extreme situations. As plasma can effectively sterilize not only bacteria but also other unknown pathogens, the results of this study are showing a promising future in purifying water in aquaculture practice.

Evolution and expression analysis of STAT family members in blunt snout bream (Megalobrama amblycephala).

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is involved in regulating the body's immunity, cell proliferation, differentiation, and apoptosis. Members of the STAT family have been extensively studied in different mammalian species. However, there are few studies on the STAT family genes in farmed economic fish. In this study, eight STAT genes including STAT1a, STAT1b, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6, in blunt snout bream (Megalobrama amblycephala), an economically important fish in China, were identified and characterized. Analyses of gene location, phylogeny and conserved synteny were conducted to infer the evolutionary origin of these STAT family genes. Furthermore, the evolutionary origin model of STATs was constructed based on the 2R hypothesis and teleost genome duplication (TGD) hypothesis, which clarified the evolutionary origin of the eight STATs in blunt snout bream. Besides, expression of the eight STATs was detected in 10 tissues of healthy blunt snout bream, which showed different expression patterns, and all had the highest level in the blood. In addition, expression of the STATs was significantly induced in the spleen, liver, and kidney after infection of Aeromonas hydrophila, suggesting that they play an important role in protecting the host from pathogens. In general, the evolution of cytokine-related genes parallels that of the immune system, which has likely been a main evolutionary driver. Therefore, the evolutionary model of STAT genes, constructed in the non-model organism pioneeringly, may provide some enlightenment for the evolution of the fish STAT family genes and their involvement in the immune function.

Genome-wide identification of endosialin family of C-type lectins in common carp (Cyprinus carpio) and their response following Aeromonas hydrophila infection.

The endosialin family is the group XIV of C-type lectin, regulating several processes involved in innate immunity and inflammation. Endosialin family genes have been extensively studied in human and mammals, however, rarely reported in teleost. In the present study, a set of 8 endosialin family genes was identified across the entire common carp genome. Functional domain and motif prediction and phylogenetic analysis supported their annotation and orthologies. Through examining gene copy number across several vertebrates, endosialin family genes were found have undergone gene duplication. Most of the endosialin family genes were ubiquitously expressed during common carp early developmental stages, and presented tissue-specific expression patterns in various healthy tissues, with relatively high expression in intestine, liver, gill, spleen and kidney, indicating their likely essential roles in maintaining homeostasis and host immune response. After Aeromonas hydrophila infection, gene thbd-1, thbd-2 and cd93-2 were significantly up-regulated at one or more timepoints in spleen and kidney, while gene cd248a-1, cd248a-2, cd248b-1, cd248b-2, and cd93-1 were significantly down-regulated. Taken together, all these results suggested that endosialin family genes were involved in host immune response to A. hydrophila infection in common carp, and provided fundamental genomic resources for better understanding the critical roles of endosialin family on the primary innate immune processes in teleost.

Grass carp MAP3K4 participates in the intestinal immune response to bacterial challenge.

Mitogen-activated protein kinase kinase kinase 4 (MAP3K4) is a multifunctional mediator of the conserved MAPK signaling pathway that plays essential roles in the regulation of immune responses in mammals. However, the function of teleost MAP3K4s in innate immunity, especially in the intestinal immune system, is still poorly understood. In the current study, we identified a fish MAP3K4 homolog (CiMAP3K4) in Ctenopharyngodon idella as well as its immune function in intestine following bacterial infection in vivo and in vitro. The open reading frame (ORF) of CiMAP3K4 encodes putative peptide of 1544 amino acids containing a predicted serine/threonine protein kinase (S_TKc) domain with high identity with other fish MAP3K4s. Phylogenetic analysis revealed the CiMAP3K4 belonged to the fish cluster and showed the closest relationship to Pimephales promelas. Quantitative real-time PCR (qRT-PCR) analysis revealed that CiMAP3K4 transcripts were widely distributed in all tested tissues, especially with high expression in the muscle and intestine of healthy grass carp. In vitro, CiMAP3K4 gene expression was upregulated by bacterial PAMPs (lipolysaccharide (LPS), peptidoglycan (PGN), L-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) and muramyl dipeptide (MDP)) and pathogens (Aeromonas hydrophila and Aeromonas veronii) in primary intestinal cells. In vivo, the mRNA expression levels of CiMAP3K4 in the intestine were significantly induced by bacterial MDP challenge in a time-dependent manner; however, this effect could be inhibited by the bioactive dipeptides ß-alanyl-l-histidine (carnosine) and alanyl-glutamine (Ala-Gln). Moreover, CiMAP3K4 was located primarily in the cytoplasm, and its overexpression increased the transcriptional activity of AP-1 in HEK293T cells. Collectively, these results suggested that CiMAP3K4 might play an important role in the intestinal immune response to bacterial infections, which paves the way for a better understanding of the intestinal immune system of grass carp.

Blood cell characterization and transcriptome analysis reveal distinct immune response and host resistance of different ploidy cyprinid fish following Aeromonas hydrophila infection.

Aeromonas hydrophila can pose a great threat to survival of freshwater fish. In this study, A. hydrophila infection could decrease blood cell numbers, promote blood cell damage as well as alter the levels of alkaline phosphatase (ALP), lysozyme (LZM), aspartate aminotransferase (AST), total antioxidant capacity (T-AOC), total superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) in immune-related tissues of red crucian carp (RCC, 2 N = 100) and triploid cyprinid fish (3 N fish, 3 N = 150). In addition, the significant alternation of antioxidant status was observed in PBMCs isolated from RCC and 3 N following LPS stimulation. The core differential expression genes (DEGs) involved in apoptosis, immunity, inflammation and cellular signals were co-expressed differentially in RCC and 3 N following A. hydrophila challenge. NOD-like receptor (NLR) signals appeared to play a critical role in A. hydrophila-infected fish. DEGs of NLR signals in RCCah vs RCCctl were enriched in caspase-1-dependent Interleukin-1ß (IL-1ß) secretion, interferon (IFN) signals as well as cytokine activation, while DEGs of NLR signals in 3Nah vs 3Nctl were enriched in caspase-1-dependent IL-1ß secretion and antibacterial autophagy. These results highlighted the differential signal regulation of different ploidy cyprinid fish to cope with bacterial infection.

Remarkable positive effects of figwort (Scrophularia striata) on improving growth performance, and immunohematological parameters of fish.

This study was conducted to investigate the effect of figwort on the growth and immunohematological parameters of common carp (14.20 ± 0.53 g). Four experimental diets were developed to feed fish for eight weeks: control, Figw10 (10 g/kg figwort), Figw20 (20 g/kg figwort), and Figw30 (30 g/kg figwort). The results showed that fish fed dietary Figw10 gained more weight (38.25 g) than control (P < 0.05). Regarding immunohematological parameters, fish fed dietary Figw30 had a higher level of white blood cells (31.2 103/mm3), hematocrit (35.82%), blood performance (14.63), total protein (1.96 g/dL), albumin (0.79 g/dL), globulin (1.17 g/dL), lymphocyte (70.53%), monocyte (3.03%), alternative hemolytic complement activity (ACH50) (147.76 u/mL), lysozyme (62.19 u/mL), and bactericidal activities (135.24) than the control group (P < 0.05). After 14 days of the challenge with Aeromonas hydrophila, the Figw30 treatment had the highest survival ratio (61.76%) compared to the control with 26.46%. Further, after the challenge, fish fed dietary Figw30 had a higher value of immunoglobulin M (42.00 µg/mL), antibody titer (19.23), complement component 3 (296.39 µg/mL), and complement component 4 (97.91 µg/mL) when compared with those fed control diet (P < 0.05). In conclusion, the optimum dosage for providing the best immune response was 30 g/kg in diet.

SCFAs improve disease resistance via modulate gut microbiota, enhance immune response and increase antioxidative capacity in the host.

To evaluate the effects of dietary short chain fatty acids (SCFAs) on the intestinal health and innate immunity in crucian carp, a six-week feeding trial was carried out with following treatments: basal diet (BD), basal diet supplementation with 1% sodium acetate (BDSA), basal diet supplementation with 1% sodium propionate (BDSP) and basal diet supplementation with 1% sodium butyrate (BDSB). The results showed dietary BDSA, BDSP and BDSB could protect the host against oxidative stress by improving the activity of certain antioxidative enzymes (T-SOD, GSH-Px and CAT). Additionally, dietary SCFAs could enhance mucosal and humoral immune responses by improving certain innate immune parameters in serum and skin mucus productions (IgM, ACH50 and T-SOD). Furthermore, dietary BDSA and BDSP could up-regulate the expression of immune related genes (TNF-α, TGF-ß and IL-8) and tight junction protein genes (occludin and ZO-1). Dietary BDSB could also elevate the expression of IL-8, TGF-ß, ZO-1 and Occludin in the midgut. Although dietary differences of SCFAs didn't alter the α-diversity of the intestinal flora, they altered the core microbiota. Finally, the challenge trial showed that dietary basal diet supplementation with SCFAs could protect zebrafish against Aeromonas hydrophila. These results suggest that dietary SCFAs could improve innate immunity, modulate gut microbiota and increase disease resistance in the host, which indicated the potential of SCFAs as immunostimulants in aquaculture.

Pediococcus pentosaceus Enhances Host Resistance Against Pathogen by Increasing IL-1ß Production: Understanding Probiotic Effectiveness and Administration Duration.

Probiotic administration is a potential strategy against enteric pathogen infection in either clinical treatment or animal nutrition industry, but the administration duration of probiotics varied and the underlying mechanisms remain unclear. A strain (YC) affiliated to Pediococcus pentosaceus, a commonly used probiotic, was isolated from fish gut and the potential role of YC against Aeromonas hydrophila was detected in zebrafish. We found that 3- or 4-week YC administration (YC3W or YC4W) increased the resistance against A. hydrophila while 1- or 2-week treatment (YC1W or YC2W) did not. To determine the possible reason, intestinal microbiota analysis and RNAseq were conducted. The results showed that compared with CON and YC1W, YC4W significantly increased the abundance of short-chain fatty acids (SCFAs) producing bacteria and elevated the gene expression of nlrp3. Higher butyrate content and enhanced expression of IL1ß were subsequently found in YC4W. To identify the causal relationship between butyrate and the higher pathogen resistance, different concentrations of sodium butyrate (SB) were supplemented. The results suggested that 10 mmol/kg SB addition mirrored the protective effect of YC4W by increasing the production of IL-1ß. Furthermore, the increased IL-1ß raised the percentage of intestinal neutrophils, which endued the zebrafish with A. hydrophila resistance. In vivo knockdown of intestinal il1b eliminated the anti-infection effect. Collectively, our data suggested that the molecular mechanism of probiotics determined the administration duration, which is vital for the efficiency of probiotics. Promoting host inflammation by probiotic pretreatment is one potential way for probiotics to provide their protective effects against pathogens.

JAK2-STAT Epigenetically Regulates Tolerized Genes in Monocytes in the First Encounter With Gram-Negative Bacterial Endotoxins in Sepsis.

Microbial challenges, such as widespread bacterial infection in sepsis, induce endotoxin tolerance, a state of hyporesponsiveness to subsequent infections. The participation of DNA methylation in this process is poorly known. In this study, we perform integrated analysis of DNA methylation and transcriptional changes following in vitro exposure to gram-negative bacterial lipopolysaccharide, together with analysis of ex vivo monocytes from septic patients. We identify TET2-mediated demethylation and transcriptional activation of inflammation-related genes that is specific to toll-like receptor stimulation. Changes also involve phosphorylation of STAT1, STAT3 and STAT5, elements of the JAK2 pathway. JAK2 pathway inhibition impairs the activation of tolerized genes on the first encounter with lipopolysaccharide. We then confirm the implication of the JAK2-STAT pathway in the aberrant DNA methylome of patients with sepsis caused by gram-negative bacteria. Finally, JAK2 inhibition in monocytes partially recapitulates the expression changes produced in the immunosuppressive cellular state acquired by monocytes from gram-negative sepsis, as described by single cell-RNA-sequencing. Our study evidences both the crucial role the JAK2-STAT pathway in epigenetic regulation and initial response of the tolerized genes to gram-negative bacterial endotoxins and provides a pharmacological target to prevent exacerbated responses.

Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity.

Non-canonical inflammasome activation by mouse caspase-11 (or human CASPASE-4/5) is crucial for the clearance of certain gram-negative bacterial infections, but can lead to severe inflammatory damage. Factors that promote non-canonical inflammasome activation are well recognized, but less is known about the mechanisms underlying its negative regulation. Herein, we identify that the caspase-11 inflammasome in mouse and human macrophages (Mϕ) is negatively controlled by the zinc (Zn2+) regulating protein, metallothionein 3 (MT3). Upon challenge with intracellular lipopolysaccharide (iLPS), Mϕ increased MT3 expression that curtailed the activation of caspase-11 and its downstream targets caspase-1 and interleukin (IL)-1ß. Mechanistically, MT3 increased intramacrophage Zn2+ to downmodulate the TRIF-IRF3-STAT1 axis that is prerequisite for caspase-11 effector function. In vivo, MT3 suppressed activation of the caspase-11 inflammasome, while caspase-11 and MT3 synergized in impairing antibacterial immunity. The present study identifies an important yin-yang relationship between the non-canonical inflammasome and MT3 in controlling inflammation and immunity to gram-negative bacteria.

Mannan Oligosaccharides Application: Multipath Restriction From Aeromonas hydrophila Infection in the Skin Barrier of Grass Carp (Ctenopharyngodon idella).

The objective of this study was to evaluate the efficacy of dietary Mannan oligosaccharides (MOS) supplementation on skin barrier function and the mechanism of on-growing grass carp (Ctenopharyngodon idella). Five hundred forty grass carp were fed for 60 days from the growing stage with six different levels of MOS diets (0, 200, 400, 600, 800, and 1,000 mg kg-1). At the end of the growth trial, the 14-day Aeromonas hydrophila challenge experiment has proceeded. The obtained data indicate that MOS could (1) decline skin lesion morbidity after being challenged by the pathogenic bacteria; (2) maintain physical barrier function via improving antioxidant ability, inhibiting excessive apoptosis, and strengthening the tight junction between the epithelial cell and the related signaling pathway (Nrf2/Keap1, p38MAPK, and MLCK); and (3) regulate immune barrier function by modulating the production of antimicrobial compound and expression of involved cytokines and the related signaling pathway (TOR and NFκB). Finally, we concluded that MOS supplementation reinforced the disease resistance and protected the fish skin barrier function from Aeromonas hydrophila infection.

Identification of Polyvalent Vaccine Candidates From Extracellular Secretory Proteins in Vibrio alginolyticus.

Bacterial infections cause huge losses in aquaculture and a wide range of health issues in humans. A vaccine is the most economical, efficient, and environment-friendly agent for protecting hosts against bacterial infections. This study aimed to identify broad, cross-protective antigens from the extracellular secretory proteome of the marine bacterium Vibrio alginolyticus. Of the 69 predicted extracellular secretory proteins in its genome, 16 were randomly selected for gene cloning to construct DNA vaccines, which were used to immunize zebrafish (Danio rerio). The innate immune response genes were also investigated. Among the 16 DNA vaccines, 3 (AT730_21605, AT730_22220, and AT730_22910) were protective against V. alginolyticus infection with 47-66.7% increased survival compared to the control, while other vaccines had lower or no protective effects. Furthermore, AT730_22220, AT730_22910, and AT730_21605 also exhibited cross-immune protective effects against Pseudomonas fluorescens and/or Aeromonas hydrophila infection. Mechanisms for cross-protective ability was explored based on conserved epitopes, innate immune responses, and antibody neutralizing ability. These results indicate that AT730_21605, AT730_22220, and AT730_22910 are potential polyvalent vaccine candidates against bacterial infections. Additionally, our results suggest that the extracellular secretory proteome is an antigen pool that can be used for the identification of cross-protective immunogens.

The Era of Antimicrobial Peptides: Use of Hepcidins to Prevent or Treat Bacterial Infections and Iron Disorders.

The current treatments applied in aquaculture to limit disease dissemination are mostly based on the use of antibiotics, either as prophylactic or therapeutic agents, with vaccines being available for a limited number of fish species and pathogens. Antimicrobial peptides are considered as promising novel substances to be used in aquaculture, due to their antimicrobial and immunomodulatory activities. Hepcidin, the major iron metabolism regulator, is found as a single gene in most mammals, but in certain fish species, including the European sea bass (Dicentrarchus labrax), two different hepcidin types are found, with specialized roles: the single type 1 hepcidin is involved in iron homeostasis trough the regulation of ferroportin, the only known iron exporter; and the various type 2 hepcidins present antimicrobial activity against a number of different pathogens. In this study, we tested the administration of sea bass derived hepcidins in models of infection and iron overload. Administration with hamp2 substantially reduced fish mortalities and bacterial loads, presenting itself as a viable alternative to the use of antibiotics. On the other hand, hamp1 seems to attenuate the effects of iron overload. Further studies are necessary to test the potential protective effects of hamp2 against other pathogens, as well as to understand how hamp2 stimulate the inflammatory responses, leading to an increased fish survival upon infection.