A Bioactive Extract Rich in Triterpenic Acid and Polyphenols from Olea europaea Promotes Systemic Immunity and Protects Atlantic Salmon Smolts Against Furunculosis

In the present study, the modulation of the transcriptional immune response (microarray analysis) in the head kidney (HK) of the anadromous fish Atlantic salmon (Salmo salar) fed a diet supplemented with an olive fruit extract (AQUOLIVE®) was evaluated. At the end of the trial (133 days), in order to investigate the immunomodulatory properties of the phytogenic tested against a bacterial infection, an in vivo challenge with Aeromonas salmonicida was performed. A total number of 1,027 differentially expressed genes (DEGs) (805 up- and 222 downregulated) were found when comparing the transcriptomic profiling of the HK from fish fed the control and AQUOLIVE® diets. The HK transcripteractome revealed an expression profile that mainly favored biological processes related to immunity. Particularly, the signaling of i-kappa B kinase/NF-kappa and the activation of leukocytes, such as granulocytes and neutrophils degranulation, were suggested to be the primary actors of the innate immune response promoted by the tested functional feed additive in the HK. Moreover, the bacterial challenge with A. salmonicida that lasted 12 days showed that the cumulative survival was higher in fish fed the AQUOLIVE® diet (96.9 ± 6.4%) than the control group (60.7 ± 13.5%). These results indicate that the dietary supplementation of AQUOLIVE® at the level of 0.15% enhanced the systemic immune response and reduced the A. salmonicida cumulative mortality in Atlantic salmon smolts.

TBK1 and IKKε act like an OFF switch to limit NLRP3 inflammasome pathway activation

NACHT, LRR, and PYD domains–containing protein 3 (NLRP3) inflammasome activation is beneficial during infection and vaccination but, when uncontrolled, is detrimental and contributes to inflammation-driven pathologies. Hence, discovering endogenous mechanisms that regulate NLRP3 activation is important for disease interventions. Activation of NLRP3 is regulated at the transcriptional level and by posttranslational modifications. Here, we describe a posttranslational phospho-switch that licenses NLRP3 activation in macrophages. The ON switch is controlled by the protein phosphatase 2A (PP2A) downstream of a variety of NLRP3 activators in vitro and in lipopolysaccharide-induced peritonitis in vivo. The OFF switch is regulated by two closely related kinases, TANK-binding kinase 1 (TBK1) and I-kappa-B kinase epsilon (IKKε). Pharmacological inhibition of TBK1 and IKKε, as well as simultaneous deletion of TBK1 and IKKε, but not of either kinase alone, increases NLRP3 activation. In addition, TBK1/IKKε inhibitors counteract the effects of PP2A inhibition on inflammasome activity. We find that, mechanistically, TBK1 interacts with NLRP3 and controls the pathway activity at a site distinct from NLRP3-serine 3, previously reported to be under PP2A control. Mutagenesis of NLRP3 confirms serine 3 as an important phospho-switch site but, surprisingly, reveals that this is not the sole site regulated by either TBK1/IKKε or PP2A, because all retain the control over the NLRP3 pathway even when serine 3 is mutated. Altogether, a model emerges whereby TLR-activated TBK1 and IKKε act like a “parking brake” for NLRP3 activation at the time of priming, while PP2A helps remove this parking brake in the presence of NLRP3 activating signals, such as bacterial pore-forming toxins or endogenous danger signals.

Akt+ IKKα/β+ Rab5+ Signalosome Mediate the Endosomal Recruitment of Sec61 and Contribute to Cross-Presentation in Bone Marrow Precursor Cells

Cross-presentation in dendritic cells (DC) requires the endosomal relocations of internalized antigens and the endoplasmic reticulum protein Sec61. Despite the fact that endotoxin-containing pathogen and endotoxin-free antigen have different effects on protein kinase B (Akt) and I-kappa B Kinase α/β (IKKα/β) activation, the exact roles of Akt phosphorylation, IKKα or IKKβ activation in endotoxin-containing pathogen-derived cross-presentation are poorly understood. In this study, endotoxin-free ovalbumin supplemented with endotoxin was used as a model pathogen. We investigated the effects of endotoxin-containing pathogen and endotoxin-free antigen on Akt phosphorylation, IKKα/β activation, and explored the mechanisms that the endotoxin-containing pathogen orchestrating the endosomal recruitment of Sec61 of the cross-presentation in bone marrow precursor cells (BMPC). We demonstrated that endotoxin-containing pathogen and endotoxin-free antigen efficiently induced the phosphorylation of Akt-IKKα/β and Akt-IKKα, respectively. Endotoxin-containing pathogen derived Akt+ IKKα/β+ Rab5+ signalosome, together with augmented the recruitment of Sec61 toward endosome, lead to the increased cross-presentation in BMPC. Importantly, the endosomal recruitment of Sec61 was partly mediated by the formation of Akt+ IKKα/β+ signalosome. Thus, these data suggest that Akt+ IKKα/β+ Rab5+ signalosome contribute to endotoxin-containing pathogen-induced the endosomal recruitment of Sec61 and the superior efficacy of cross-presentation in BMPC.

NF-κB Signaling in Astrocytes Modulates Brain Inflammation and Neuronal Injury Following Sequential Exposure to Manganese and MPTP During Development and Aging

Chronic exposure to manganese (Mn) is associated with neuroinflammation and extrapyramidal motor deficits resembling features of Parkinson’s disease. Activation of astrocytes and microglia is implicated in neuronal injury from Mn but it is not known whether early life exposure to Mn may predispose glia to more severe inflammatory responses during aging. We therefore examined astrocyte nuclear factor kappa B (NF-κB) signaling in mediating innate immune inflammatory responses during multiple neurotoxic exposures spanning juvenile development into adulthood. MnCl2 was given in drinking water for 30-day postweaning to both wildtype mice and astrocyte-specific knockout (KO) mice lacking I kappa B kinase 2, the central upstream activator of NF-κB. Following juvenile exposure to Mn, mice were subsequently administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at 4 months of age. Animals were evaluated for behavioral alterations and brain tissue was analyzed for catecholamine neurotransmitters. Stereological analysis of neuronal and glial cell counts from multiple brain regions indicated that juvenile exposure to Mn amplified glial activation and neuronal loss from MPTP exposure in the caudate-putamen and globus pallidus, as well as increased the severity of neurobehavioral deficits in open field activity assays. These alterations were prevented in astrocyte-specific I kappa B kinase 2 KO mice. Juvenile exposure to Mn increased the number of neurotoxic A1 astrocytes expressing C3 as well as the number of activated microglia in adult mice following MPTP challenge, both of which were inhibited in KO mice. These results demonstrate that exposure to Mn during juvenile development heightens the innate immune inflammatory response in glia during a subsequent neurotoxic challenge through NF-κB signaling in astrocytes.

TBK1, a central kinase in innate immune sensing of nucleic acids and beyond

Sensing of intracellular and extracellular environments is one of the fundamental processes of cell. Surveillance of aberrant nucleic acids, derived either from invading pathogens or damaged organelle, is conducted by pattern recognition receptors (PRRs) including RIG-I-like receptors, cyclic GMP-AMP synthase, absent in melanoma 2, and a few members of toll-like receptors. TANK-binding kinase 1 (TBK1), along with its close analogue I-kappa-B kinase epsilon, is a central kinase in innate adaptor complexes linking activation of PRRs to mobilization of transcriptional factors that transcribe proinflammatory cytokines, type I interferon (IFN-α/β), and myriads interferon stimulated genes. However, it still remains elusive for the precise mechanisms of activation and execution of TBK1 in signaling platforms formed by innate adaptors mitochondrial antiviral signaling protein (MAVS), stimulator of interferon genes protein (STING), and TIR-domain-containing adapter-inducing interferon-β (TRIF), as well as its complex regulations. An atlas of TBK1 substrates is in constant expanding, setting TBK1 as a key node of signaling network and a dominant player in contexts of cell biology, animal models, and human diseases. Here, we review recent advancements of activation, regulations, and functions of TBK1 under these physiological and pathological contexts.

The Dynamic Changes of Gut Microbiota in Muc2 Deficient Mice

Gut dysbiosis is associated with colitis-associated colorectal carcinogenesis, and the genetic deficiency of the Muc2 gene causes spontaneous development of colitis and colorectal cancer. Whether there are changes of gut microbiota and a linkage between the changes of microbiota and intestinal pathology in Muc2−/− mice are unclear. Muc2−/− and Muc2+/+ mice were generated by backcrossing from Muc2+/− mice, and the fecal samples were collected at different dates (48th, 98th, 118th, 138th, and 178th day). Gut microbiota were analyzed by high-throughput sequencing with the universal 16S rRNA primers (V3–V5 region). All mice were sacrificed at day 178 to collect colonic tissue and epithelial cells for the analysis of histopathology and inflammatory cytokines. On the 178th day, Muc2−/− mice developed colorectal chronic colitis, hyperplasia, adenomas and adenocarcinomas, and inflammatory cytokines (e.g., cyclooxygenase 2 (COX-2), interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin 1 β (IL-1β), i-kappa-B-kinase β (IKKβ)) were significantly increased in colonic epithelial cells of Muc2−/− mice. In general, structural segregation of gut microbiota was observed throughout the experimental time points between the Muc2−/− and Muc2+/+ mice. Impressively, in Muc2−/− mice, Alpha diversities reflected by Shannon and Chao indexes were higher, the phylum of Firmicutes was enriched and Bacteroidetes was decreased, and Desulfovibrio, Escherichia, Akkermansia, Turicibacter, and Erysipelotrichaceae were significantly increased, but Lactobacilli and Lachnospiraceae were significantly decreased. Moreover, the abundance of Ruminococcaceae and butyrate-producing bacteria was significantly higher in the Muc2−/− mice. There were significant differences of gut microbiota between Muc2−/− and Muc2+/+ mice. The dynamic changes of microbiota might contribute to the development of colitis and colitis-associated colorectal carcinogenesis. Therefore, this study revealed specific functional bacteria in the development of colitis and colitis-associated colorectal carcinogenesis, which will benefit the development of preventive and therapeutic strategies for chronic inflammation and its malignant transformation.