SUMMARY Tight regulation of NF-κB signaling is essential for innate and adaptive immune responses, yet the molecular mechanisms responsible for its negative regulation are not completely understood. Here we report that NLRX1, a NOD-like receptor family member, negatively regulates Toll-like receptor-mediated NF-κB activation. NLRX1 interacts with TRAF6 or IκB kinase (IKK) in an activation signal-dependent fashion. Upon LPS stimulation, NLRX1 is rapidly ubiquitinated, disassociates from TRAF6 and then binds to the IKK complex, resulting in inhibition of IKKα/β phosphorylation and NF-κB activation. Knockdown of NLRX1 in various cell types markedly enhances IKK phosphorylation and the production of NF-κB-responsive cytokines after LPS stimulation. We further provide in vivo evidence that NLRX1 knockdown in mice markedly enhances susceptibility to LPS-induced septic shock and plasma IL-6 level. Our study identifies a previously unrecognized role for NLRX1 in the negative regulation of TLR-induced NF-κB activation by dynamically interacting with TRAF6 and the IKK complex.

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Background Lactobacillus reuteri strains are widely used as probiotics to prevent and treat inflammatory bowel disease by modulating the host’s immune system. However, the underlying mechanisms by which they communicate with the host have not been clearly understood. Bacterial extracellular vesicles (EVs) have been considered as important mediators of host-pathogen interactions, but their potential role in commensals-host crosstalk has not been widely studied. Here, we investigated the regulatory actions of EVs produced by L. reuteri BBC3, a gut-associated commensal bacterium of Black-Bone chicken, in the development of lipopolysaccharide (LPS)-induced intestinal inflammation in a chicken model using both in vivo and in vitro experiments. Results L. reuteri BBC3 produced nano-scale membrane vesicles with the size range of 60–250 nm. Biochemical and proteomic analyses showed that L. reuteri BBC3-derived EVs (LrEVs) carried DNA, RNA and several bioactive proteins previously described as mediators of other probiotics’ beneficial effects such as glucosyltransferase, serine protease and elongation factor Tu. In vivo broiler experiments showed that administration of LrEVs exerted similar effects as L. reuteri BBC3 in attenuating LPS-induced inflammation by improving growth performance, reducing mortality and decreasing intestinal injury. LrEVs suppressed the LPS-induced expression of pro-inflammatory genes (TNF-α, IL-1β, IL-6, IL-17 and IL-8), and improved the expression of anti-inflammatory genes (IL-10 and TGF-β) in the jejunum. LrEVs could be internalized by chicken macrophages. In vitro pretreatment with LrEVs reduced the gene expression of TNF-α, IL-1β and IL-6 by suppressing the NF-κB activity, and enhanced the gene expression of IL-10 and TGF-β in LPS-activated chicken macrophages. Additionally, LrEVs could inhibit Th1- and Th17-mediated inflammatory responses and enhance the immunoregulatory cells-mediated immunosuppression in splenic lymphocytes of LPS-challenged chickens through the activation of macrophages. Finally, we revealed that the reduced content of both vesicular proteins and nucleic acids attenuated the suppression of LrEVs on LPS-induced inflammatory responses in ex vivo experiments, suggesting that they are essential for the LrEVs-mediated immunoregulation. Conclusions We revealed that LrEVs participated in maintaining intestinal immune homeostasis against LPS-induced inflammatory responses in a chicken model. Our findings provide mechanistic insight into how commensal and probiotic Lactobacillus species modulate the host’s immune system in pathogens-induced inflammation.

This experiment was conducted to investigate the effects of 1alpha-hydroxycholecalciferol (1alpha-OH D3) on the growth performance, tibia and plasma parameters, nutrient utilization, meat quality of the breast and thigh, and type IIb sodium phosphate cotranspoter gene expression of broilers. A total of 96 males of 1-d-old Arbor Acres broilers were randomly assigned to 8 cages of 12 birds each. Two dietary treatments were applied to 4 cages each. Diet 1 was prepared as the basal diet (nonphytate phosphorus, 0.21%), whereas diet 2 was the basal diet supplemented with 5 microg/kg of 1alpha-OH D3. Results showed that supplementation of the basal diet with 1alpha-OH D3 increased growth performance, tibia ash and strength, plasma inorganic phosphate concentration, utilization of total phosphorus and nonphytate phosphorus, lightness and yellowness of the breast and thigh meat, and intestinal type IIb sodium phosphate cotranspoter mRNA expression, whereas it decreased the shear force and water-holding capacity of the thigh meat. These data suggest that the addition of 1alpha-OH D3 might improve growth performance, tibia development, and meat quality in 1- to 21-d-old broilers by increasing the absorption and retention of phosphorus.

BackgroundLimited research has focused on the effect of Lactobacillus on the intestinal toxicity of deoxynivalenol (DON). The present study was conducted to investigate the role of Lactobacillus plantarum (L. plantarum) JM113 in protecting against the intestinal toxicity caused by DON.MethodsA total of 144 one-day-old healthy Arbor Acres broilers were randomly distributed into 3 treatments, including the CON (basal diet), the DON (extra 10 mg/kg deoxynivalenol), and the DL (extra 1 × 109 CFU/ kg L. plantarum JM113 based on DON group) treatments. The growth performance, organ indexes, intestinal morphology, pancreatic digestive enzymes, intestinal secreted immunoglobulin A (sIgA), jejunal transcriptome, and intestinal microbiota were evaluated.ResultsCompared with the CON and DL groups, the DON supplementation altered intestinal morphology, especially in duodenum and jejunum, where villi were shorter and crypts were deeper (P < 0.05). Meanwhile, the significantly decreased mRNA expression of jejunal claudin-1 and occludin (P < 0.05), ileal rBAT and jejunal GLUT1 of 21-day-old broilers (P < 0.05), as well as duodenal PepT1 and ileal rBAT of 42-day-old broilers were identified in the DON group. Moreover, supplementation with L. plantarum JM113 could increase duodenal expression of IL-10 and IL-12 of 21-day-old broilers, ileal sIgA of 42-day-old broilers, and the bursa of Fabricius index of 21-day-old broilers. Further jejunal transcriptome proved that the genes related to the intestinal absorption and metabolism were significantly reduced in the DON group but a significant increase when supplemented with extra L. plantarum JM113. Furthermore, the bacteria related to nutrient utilization, including the Proteobacteria, Escherichia, Cc-115 (P < 0.05), Lactobacillus and Prevotella (P < 0.1) were all decreased in the DON group. By contrast, supplementation with L. plantarum JM113 increased the relative abundance of beneficial bacterium, including the Bacteroidetes, Roseburia, Anaerofustis, Anaerostipe, and Ruminococcus bromii (P < 0.05). Specifically, the increased abundance of bacteria in the DL group could be proved by the significantly increased caecal content of propionic acid, n-Butyric acid, and total short-chain fatty acid.ConclusionsL. plantarum JM113 enhanced the digestion, absorption, and metabolic functions of the gut when challenged with DON by reducing the injury to intestinal barriers and by increasing the abundance of beneficial bacterium.Electronic supplementary materialThe online version of this article (10.1186/s40104-018-0286-5) contains supplementary material, which is available to authorized users.

The aim of this experiment was to study the antioxidant capacity of JM113 isolated from healthy intestinal contents of Tibetan chicken and its protective effect on broiler chickens challenged with deoxynivalenol (DON). Compared with PZ01 and M23, JM113 demonstrated maximum reducing ( < 0.05) activity and resistance in the presence of 1.2 mmol/L hydrogen peroxide, and great scavenging ability ( < 0.05) against hydroxyl, superoxide anion, and 1,1-diphenyl-2-picrylhydrazyl radicals in vitro. For each strain, the antioxidant activities of live bacterial strains were greater ( < 0.05) than of cell free extracts and dead bacterial strains. To examine the antioxidant capacity of JM113 in vivo, 192 1-d-old Arbor Acres chicks were randomly divided into 4 treatments groups consisting of 6 replicates with 8 birds per replicate. The dietary treatments were 1) control; 2) control diet supplemented with JM113 at 1 × 10 cfu/kg; 3) control diet contaminated with DON at 10 mg/kg; 4) control diet contaminated with DON at 10 mg/kg and supplemented with JM113 at 1 × 10 cfu/kg. Dietary supplementation with DON decreased ( < 0.05) superoxide dismutase activity in serum and increased ( < 0.05) malondialdehyde in the jejunal mucosa of broilers, compared to the control. However, supplementation with JM113 to both the DON-contaminated diet and the control diet, caused a significant reduction ( < 0.05) in malondialdehyde activity in the jejunal mucosa. A reduction ( < 0.05) in expression of nuclear factor erythroid 2-related factor 2 was observed in the jejunal mucosa of broilers fed dietary supplementation with DON, whereas the mRNA levels of and its corresponding downstream gene increased ( < 0.05) with JM113 treatment. Addition of JM113 resulted in longer villi ( < 0.05), even in combination with DON compared to the DON group. JM113 treatment, especially in the DON plus JM113 group, up-regulated ( < 0.05) the expression of mRNA. In conclusion, the present study demonstrates that the JM113 strain has great antioxidant activity and supplementation in feed protected the integrity of the intestinal barrier in broilers challenged with DON, suggesting its use for alleviation of negative effects of DON in poultry.

In our previous study, several iron-related outer membrane proteins in Aeromonas hydrophila , a serious pathogen of farmed fish, conferred high immunoprotectivity to fish, and were proposed as potential vaccine candidates. However, the protective efficacy of these extracellular proteins against A. hydrophila remains largely unknown. Here, we identified secreted proteins that were differentially expressed in A. hydrophila LP-2 in response to iron starvation using an iTRAQ-based quantitative proteomics method. We identified 341 proteins, of which 9 were upregulated in response to iron starvation and 24 were downregulated. Many of the differently expressed proteins were associated with protease activity. We confirmed our proteomics results with Western blotting and qPCR. We constructed three mutants by knocking out three genes encoding differentially expressed proteins (Δ orf01830 , Δ orf01609 , and Δ orf03641 ). The physiological characteristics of these mutants were investigated. In all these mutant strains, protease activity decreased, and Δ orf01609 , and Δ orf01830 were less virulent in zebrafish. This indicated that the proteins encoded by these genes may play important roles in bacterial infection. We next evaluated the immune response provoked by the six iron-related recombinant proteins (ORF01609, ORF01830, ORF01839, ORF02943, ORF03355, and ORF03641) in zebrafish as well as the immunization efficacy of these proteins. Immunization with these proteins significantly increased the zebrafish immune response. In addition, the relative percent survival (RPS) of the immunized zebrafish was 50–80% when challenged with three virulent A. hydrophila strains, respectively. Thus, these extracellular secreted proteins might be effective vaccine candidates against A. hydrophila infection in fish.

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