TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7-9.

Toll-like receptors (TLRs) have a crucial role in the recognition of pathogens and initiation of immune responses1-3. Here we show that a previously uncharacterized protein encoded by CXorf21-a gene that is associated with systemic lupus erythematosus4,5-interacts with the endolysosomal transporter SLC15A4, an essential but poorly understood component of the endolysosomal TLR machinery also linked to autoimmune disease4,6-9. Loss of this type-I-interferon-inducible protein, which we refer to as 'TLR adaptor interacting with SLC15A4 on the lysosome' (TASL), abrogated responses to endolysosomal TLR agonists in both primary and transformed human immune cells. Deletion of SLC15A4 or TASL specifically impaired the activation of the IRF pathway without affecting NF-κB and MAPK signalling, which indicates that ligand recognition and TLR engagement in the endolysosome occurred normally. Extensive mutagenesis of TASL demonstrated that its localization and function relies on the interaction with SLC15A4. TASL contains a conserved pLxIS motif (in which p denotes a hydrophilic residue and x denotes any residue) that mediates the recruitment and activation of IRF5. This finding shows that TASL is an innate immune adaptor for TLR7, TLR8 and TLR9 signalling, revealing a clear mechanistic analogy with the IRF3 adaptors STING, MAVS and TRIF10,11. The identification of TASL as the component that links endolysosomal TLRs to the IRF5 transcription factor via SLC15A4 provides a mechanistic explanation for the involvement of these proteins in systemic lupus erythematosus12-14.

Antifungal mechanisms investigation of lactic acid bacteria against Aspergillus flavus: through combining microbial metabolomics and co-culture system.

AIMS: To develop antifungal lactic acid bacteria (LAB) and investigate their antifungal mechanisms against Aspergillus flavus in aflatoxin (AF) production. METHODS AND RESULTS: We isolated 179 LABs from cereal-based fermentation starters and investigated their antifungal mechanism against A. flavus through liquid chromatography-mass spectrometry and co-culture analysis techniques. Of the 179 isolates, antifungal activity was identified in Pediococcus pentosaceus, Lactobacillus crustorum, and Weissella paramesenteroides. These LABs reduced AF concentration by (i) inhibiting mycelial growth, (ii) binding AF to the cell wall, and (iii) producing antifungal compounds. Species-specific activities were also observed, with P. pentosaceus inhibiting AF production and W. paramesenteroides showing AF B1 binding activity. In addition, crucial extracellular metabolites for selecting antifungal LAB were involved in the 2',3'-cAMP-adenosine and nucleoside pathways. CONCLUSIONS: This study demonstrates that P. pentosaceus, L. crustorum, and W. paramesenteroides are key LAB strains with distinct antifungal mechanisms against A. flavus, suggesting their potential as biological agents to reduce AF in food materials.

Analytical factors for eight short-chain fatty acid analyses in mouse feces through headspace solid-phase microextraction-triple quadrupole gas chromatography tandem mass spectrometry.

This study developed a method for quantifying eight short-chain fatty acids (SCFAs) in mouse fecal samples using solid-phase microextraction (SPME) coupled with triple quadrupole gas chromatography tandem mass spectrometry. Furthermore, significant factors affecting SCFA analysis, including SPME fiber selection, pH, salting-out agent, and sample collection time, were investigated. Contrary to previous studies, we found that the CAR/PDMS fiber had the highest extraction efficiency for all SCFAs. The optimal extraction efficiency was observed at pH 2.0, particularly for low-molecular-weight SCFAs. NaH2PO4 showed a more effective extraction efficiency than NaCl, owing to its pH stability and less interference with the solvent matrix. Additionally, our results showed that the SCFA concentration increased over collection time. The composition ratio of the eight SCFAs was maintained for up to 24 h; thus, we concluded that samples should be collected within four hours to obtain reliable results. Our findings may improve laboratory methods for SCFA extraction and mouse fecal sample analysis.

Determination of the SLAMF1 self-association affinity constant with sedimentation velocity ultracentrifugation.

Signaling lymphocytic activating molecule family member 1 (SLAMF1 or CD150) is a cell surface glycoprotein expressed on various immune populations, regulating cell-cell interactions, activation, differentiation, and inflammatory responses and has been suggested as a potential target for inflammatory diseases. Signaling is believed to be mediated by high-affinity homophilic interactions; the recombinant soluble form of SLAMF1 has optimal activity in the range of 20 µg/mL. This contradicts with a rather weak homo-dimerization binding constant (KD) value reported previously; however, the analytical approach and data analysis suffered from various technical limitations at the time and therefore warrants re-examination. To address this apparent discrepancy, we determined the KD of soluble SLAMF1 using sedimentation velocity analytical ultracentrifuge (SV-AUC). A globally fitted monomer-dimer model properly explains the data from a wide concentration range obtained with both UV and fluorescence detection systems. The analysis suggests the dimerization KD value for human SLAMF1 is 0.48 µM. Additionally, our data show that SLAMF1 self-association is not driven by non-specific binding to glycans supporting the view of specific protein-protein interaction. We anticipate antibody biotherapeutics capable of modulating the biological consequences of SLAMF1 interactions will be readily identified.

Tumor necrosis factor receptor- associated factor 6 (TRAF6) regulation of development, function, and homeostasis of the immune system.

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is an adapter protein that mediates a wide array of protein-protein interactions via its TRAF domain and a RING finger domain that possesses non-conventional E3 ubiquitin ligase activity. First identified nearly two decades ago as a mediator of interleukin-1 receptor (IL-1R)-mediated activation of NFκB, TRAF6 has since been identified as an actor downstream of multiple receptor families with immunoregulatory functions, including members of the TNFR superfamily, the Toll-like receptor (TLR) family, tumor growth factor-ß receptors (TGFßR), and T-cell receptor (TCR). In addition to NFκB, TRAF6 may also direct activation of mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and interferon regulatory factor pathways. In the context of the immune system, TRAF6-mediated signals have proven critical for the development, homeostasis, and/or activation of B cells, T cells, and myeloid cells, including macrophages, dendritic cells, and osteoclasts, as well as for organogenesis of thymic and secondary lymphoid tissues. In multiple cellular contexts, TRAF6 function is essential not only for proper activation of the immune system but also for maintaining immune tolerance, and more recent work has begun to identify mechanisms of contextual specificity for TRAF6, involving both regulatory protein interactions, and messenger RNA regulation by microRNAs.

Regulator of fatty acid metabolism, acetyl coenzyme a carboxylase 1, controls T cell immunity.

Fatty acids (FAs) are essential constituents of cell membranes, signaling molecules, and bioenergetic substrates. Because CD8(+) T cells undergo both functional and metabolic changes during activation and differentiation, dynamic changes in FA metabolism also occur. However, the contributions of de novo lipogenesis to acquisition and maintenance of CD8(+) T cell function are unclear. In this article, we demonstrate the role of FA synthesis in CD8(+) T cell immunity. T cell-specific deletion of acetyl coenzyme A carboxylase 1 (ACC1), an enzyme that catalyzes conversion of acetyl coenzyme A to malonyl coenzyme A, a carbon donor for long-chain FA synthesis, resulted in impaired peripheral persistence and homeostatic proliferation of CD8(+) T cells in naive mice. Loss of ACC1 did not compromise effector CD8(+) T cell differentiation upon listeria infection but did result in a severe defect in Ag-specific CD8(+) T cell accumulation because of increased death of proliferating cells. Furthermore, in vitro mitogenic stimulation demonstrated that defective blasting and survival of ACC1-deficient CD8(+) T cells could be rescued by provision of exogenous FA. These results suggest an essential role for ACC1-mediated de novo lipogenesis as a regulator of CD8(+) T cell expansion, and may provide insights for therapeutic targets for interventions in autoimmune diseases, cancer, and chronic infections.

IL-18 synergizes with IL-7 to drive slow proliferation of naive CD8 T cells by costimulating self-peptide-mediated TCR signals.

Naive T cell populations are maintained in the periphery at relatively constant levels via mechanisms that control expansion and contraction and are associated with competition for homeostatic cytokines. It has been shown that in a lymphopenic environment naive T cells undergo expansion due, at least in part, to additional availability of IL-7. We have previously found that T cell-intrinsic deletion of TNFR-associated factor (TRAF) 6 (TRAF6ΔT) in mice results in diminished peripheral CD8 T cell numbers. In this study, we report that whereas naive TRAF6ΔT CD8 T cells exhibit normal survival when transferred into a normal T cell pool, proliferation of naive TRAF6ΔT CD8 T cells under lymphopenic conditions is defective. We identified IL-18 as a TRAF6-activating factor capable of enhancing lymphopenia-induced proliferation (LIP) in vivo, and that IL-18 synergizes with high-dose IL-7 in a TRAF6-dependent manner to induce slow, LIP/homeostatic-like proliferation of naive CD8 T cells in vitro. IL-7 and IL-18 act synergistically to upregulate expression of IL-18R genes, thereby enhancing IL-18 activity. In this context, IL-18R signaling increases PI3K activation and was found to sensitize naive CD8 T cells to a model noncognate self-peptide ligand in a way that conventional costimulation via CD28 could not. We propose that synergistic sensitization by IL-7 and IL-18 to self-peptide ligand may represent a novel costimulatory pathway for LIP.

Gellan gum prevents non-alcoholic fatty liver disease by modulating the gut microbiota and metabolites.

Gellan gum (GG) is an anionic polysaccharide used as an additive in the food industry. However, the effect of GG on gut microbiota regulation and nonalcoholic fatty liver disease (NAFLD) has not yet been investigated. In vitro fermentation experiments have demonstrated that GG promoted the growth of probiotic strains such as Lactiplantibacillus rhamnosus and Bifidobacterium bifidum, producing metabolites beneficial to gut health. In mice, GG reduced hepatic triglyceride content, serum biomarkers, and body fat mass and weight gain induced by a high fat diet. Additionally, GG regulated the gut microbiota including Desulfovibrionales, Deferribacterales, Bacteroidales, and Lactobacillales at the order level and also promoted short-chain fatty acid production. Moreover, GG improved the expression of proteins related to hepatic inflammation and lipid metabolism. Taken together, GG ameliorated NAFLD, possibly by acting on the gut-liver axis via improving the gut health, indicating its potential as a food supplement and/or prebiotic against NAFLD.

Innate-adaptive crosstalk: how dendritic cells shape immune responses in the CNS.

Dendritic cells (DCs) are a heterogeneous group of professional antigen presenting cells that lie in a nexus between innate and adaptive immunity because they recognize and respond to danger signals and subsequently initiate and regulate effector T-cell responses. Initially thought to be absent from the CNS, both plasmacytoid and conventional DCs as well as DC precursors have recently been detected in several CNS compartments where they are seemingly poised for responding to injury and pathogens. Additionally, monocyte-derived DCs rapidly accumulate in the inflamed CNS where they, along with other DC subsets, may function to locally regulate effector T-cells and/or carry antigens to CNS-draining cervical lymph nodes. In this review we highlight recent research showing that (a) distinct inflammatory stimuli differentially recruit DC subsets to the CNS; (b) DC recruitment across the blood-brain barrier (BBB) is regulated by adhesion molecules, growth factors, and chemokines; and (c) DCs positively or negatively regulate immune responses in the CNS.

Untargeted Metabolomics reveals Doenjang metabolites affected by manufacturing process and microorganisms.

Doenjang is a traditional Korean fermented soybean-based food that is now produced industrially as traditional Doenjang and modernized Doenjang, depending on the conditions of the manufacturing process. In this study, the effect of the production process on Doenjang metabolites was analyzed using untargeted capillary electrophoresis time-of-flight mass spectrometry (CE-TOF/MS). Of the 247 metabolites detected in 19 traditional and three modernized Doenjang samples, the level of 55 metabolites were significantly different between the two production groups. The S-plot of the orthogonal projection to latent structures-discriminant analysis (OPLS-DA) revealed that nine compounds (tryptamine, 2-phenylethylamine, citrulline, gamma-aminobutyric acid, putrescine, tyramine, 2-aminoisobutyric acid, stachydrine, and N5-ethylglutamine) were highly distributed in the traditional group, and six compounds (arginine, citric acid, choline, cytidine, hypoxanthine, and glucaric acid) were considered distinguishable metabolites of the modernized group. Microbial community analysis indicated that the levels of these metabolites were significantly altered by the presence of Bacillus spp., Enterococcus faecium, Tetragenococcus halophilus, Aspergillus oryzae, Penicillium spp., and Saccharomyces cerevisiae. These findings will give better understanding of the influence of the manufacturing process on Doenjang production in terms of both microbial activity and metabolite profiles.

Importance of lactic acid bacteria in Asian fermented foods.

Lactic acid bacteria play important roles in various fermented foods in Asia. Besides being the main component in kimchi and other fermented foods, they are used to preserve edible food materials through fermentation of other raw-materials such as rice wine/beer, rice cakes, and fish by producing organic acids to control putrefactive microorganisms and pathogens. These bacteria also provide a selective environment favoring fermentative microorganisms and produce desirable flavors in various fermented foods. This paper discusses the role of lactic acid bacteria in various non-dairy fermented food products in Asia and their nutritional and physiological functions in the Asian diet.

Characterization, prebiotic and immune-enhancing activities of rhamnogalacturonan-I-rich polysaccharide fraction from molokhia leaves.

Plant-derived polysaccharides possess potential health benefits that improve intestinal health and the immune system. Molokhia leaves have a large amount of mucilage polysaccharide; in the present study, crude polysaccharide extract was prepared from molokhia leaves. The molecular weight of molokhia leaf polysaccharide fraction (MPF) was estimated to be 51.2 × 103 Da. Polysaccharide was methylated and the structure of MPF was mainly composed of rhamnogalacturonan-I structure with side chains, such as galactans and linear glucan (starch), as shown by GC-MS analysis. To study the biofunctional effects of MPF, its prebiotic and intestinal immune-enhancing activities were assayed in vitro. MPF exhibited good prebiotic activity, as shown by its high prebiotic scores, and increased contents of total short-chain fatty acids on five probiotic strains. In addition, MPF showed immune-enhancing activity on Peyer's patches, as revealed by the high bone marrow cell proliferating activity and production of immunoglobulin A and cytokines. These results demonstrate that MPF may be a potential beneficial prebiotic and intestinal immune-enhancer, which may have wide implications in the food industry.

Intracerebral dendritic cells critically modulate encephalitogenic versus regulatory immune responses in the CNS.

Dendritic cells (DCs) appear in higher numbers within the CNS as a consequence of inflammation associated with autoimmune disorders, such as multiple sclerosis, but the contribution of these cells to the outcome of disease is not yet clear. Here, we show that stimulatory or tolerogenic functional states of intracerebral DCs regulate the systemic activation of neuroantigen-specific T cells, the recruitment of these cells into the CNS and the onset and progression of experimental autoimmune encephalomyelitis (EAE). Intracerebral microinjection of stimulatory DCs exacerbated the onset and clinical course of EAE, accompanied with an early T-cell infiltration and a decreased proportion of regulatory FoxP3-expressing cells in the brain. In contrast, the intracerebral microinjection of DCs modified by tumor necrosis factor alpha induced their tolerogenic functional state and delayed or prevented EAE onset. This triggered the generation of interleukin 10 (IL-10)-producing neuroantigen-specific lymphocytes in the periphery and restricted IL-17 production in the CNS. Our findings suggest that DCs are a rate-limiting factor for neuroinflammation.

Mycobacterium bovis bacille Calmette-Guérin infection in the CNS suppresses experimental autoimmune encephalomyelitis and Th17 responses in an IFN-gamma-independent manner.

Multiple sclerosis and an animal model resembling multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), are inflammatory demyelinating diseases of the CNS that are suppressed by systemic mycobacterial infection in mice and BCG vaccination in humans. Host defense responses against Mycobacterium in mice are influenced by T lymphocytes and their cytokine products, particularly IFN-gamma, which plays a protective regulatory role in EAE. To analyze the counter-regulatory role of mycobacterial infection-induced IFN-gamma in the CNS on the function of the pathological Th17 cells and the clinical outcome of EAE, we induced EAE in mice that were intracerebrally infected with Mycobacterium bovis bacille Calmette-Guerin (BCG). In this study, we demonstrate that intracerebral (i.c.) BCG infection prevented inflammatory cell recruitment to the spinal cord and suppressed the development of EAE. Concomitantly, there was a significant decrease in the frequency of myelin oligodendrocyte glycoprotein-specific IFN-gamma-producing CD4(+) T cells in the CNS. IL-17(+)CD4(+) T cell responses were significantly suppressed in i.c. BCG-infected mice following EAE induction regardless of T cell specificity. The frequency of Foxp3(+)CD4(+) T cells in these mice was equivalent to that of control mice. Intracerebral BCG infection-induced protection of EAE and suppression of myelin oligodendrocyte glycoprotein-specific IL-17(+)CD4(+) T cell responses were similar in both wild-type and IFN-gamma-deficient mice. These data show that live BCG infection in the brain suppresses CNS autoimmunity. These findings also reveal that the regulation of Th17-mediated autoimmunity in the CNS can be independent of IFN-gamma-mediated mechanisms.

Influence of aflatoxin in Nuruk on the safety of starch-based alcoholic beverage.

Nuruk is a fermentation agent used to manufacture alcoholic beverages, which contains a variety of microorganisms. Most microorganisms in Nuruk are useful for the production of alcoholic beverages; however, Nuruk can be infected with Aspergillus flavus, which produces aflatoxin (AF). Therefore, this study analyzed total AF concentrations in Nuruk, the transfer of AF from Nuruk to alcoholic beverages, and AF-producing microorganisms to determine the safety of alcoholic beverages with respect to this toxin. ELISA showed that total AF levels in 14 of 61 Nuruk samples exceeded 15 ppb, the Korean permissible level in cereal products. In alcoholic beverages produced with Nuruk containing AF levels >15 ppb, only AF G1 was detected, at a level of 0.3 ppb, and the transfer ratio of AF G1 was approximately 1.2% to 1.3%. The dominant genera in Nuruk were Aspergillus and Rhizopus. Among 30 strains belonging to Aspergillus, 10 produced only AF B1 at levels of 0.1 to 2.4 ppb after incubation at 25 °C for 8 days on potato dextrose agar plates. Although AF in Nuruk was rarely transferred to alcoholic beverages and the aflatoxigenic strains were found to possess poor AF-producing capacity, further efforts to reduce AF in Nuruk are needed to ensure the safety of alcoholic beverages.

Alteration of gut microbiota composition by short-term low-dose alcohol intake is restored by fermented rice liquor in mice.

Excessive alcohol consumption can increase gut permeability and alter the gut bacterial communities, influencing the pathogenesis of several disorders including alcoholic liver disease, endotoxemia, and systemic inflammation. Although recent studies have highlighted a pivotal role for gut microbiota in alcohol metabolism and alcoholic liver injury, the results have primarily been obtained from binge models treated with physiologically acute dose of alcohol. We sought to investigate the effect of low-dose (0.8 g/kg/day) and short-term (one week) consumption of alcohol on changes in gut microbiota composition in mice. The low-dose consumption of alcohol immediately altered fecal microbiota composition in mice after 1 and 7 days of treatment. We also evaluated the influence of alcoholic beverages containing various microbes using fermented rice liquors (FRLs, called Makgeolli in Korea) on gut microbiota and systemic inflammation. One week of FRLs consumption restored fecal microbiota compositions altered by alcohol administration in mice, with the abundance of Bacteroidetes and Firmicutes phyla recovered to levels of the control group. In addition, mice receiving FRLs exhibited increased fecal production of short-chain fatty acids (SCFA) such as butyric acid and propionic acid within 7 days, and reduced inflammatory responses induced by alcohol administration in the serum and colon. Taken together, these results suggest that short-term and low-dose alcohol intake induces alterations in fecal microbiota composition, and FRLs administration can restore microbial composition and suppress intestinal inflammation, highlighting potential benefits of FRLs as fermented foods.

Quantification of myelin and axon pathology during relapsing progressive experimental autoimmune encephalomyelitis in the Biozzi ABH mouse.

Multiple sclerosis is an immune-mediated demyelinating disease, with axonal loss underlying long-term progressive disability. In this study, we have analyzed axonal and myelin pathology in a chronic relapsing-remitting experimental autoimmune encephalomyelitis model in Biozzi ABH mice induced by immunization with a syngeneic spinal cord homogenate. The animals were followed for3 months; inflammation, T-cell infiltration, demyelination, and axonal loss were examined at various time points throughout the disease course. We found that macrophage infiltration and microglia activation preceded detectable T-cell infiltration. Axonal loss was first evident at the acute phase of disease before demyelination was detected. Demyelination and axonal loss occurred after each relapse and correlated with increasing residual motor deficits in remission. The resulting lesions displayed evidence of demyelination, remyelination, axonal degeneration, and axon loss. After a series of 3 relapses, animals entered a chronic progressive phase with permanent paralysis and a relative absence of inflammation. Axonal loss continued in this phase, although demyelinated axons persisted. These findings indicate that experimental autoimmune encephalomyelitis in Biozzi ABH mice has important similarities to multiple sclerosis with a relapsing-remitting disease course followed by a secondary progressive phase; it is thus a suitable model in which to explore remyelination and neuroprotective therapies for multiple sclerosis.

Surface Film Formation in Static-Fermented Rice Vinegar: A Case Study.

In the present study, we aimed to determine the cause of surface film formation in three rice vinegars fermented using the traditional static fermentation method. The pH and total acidity of vinegar were 3.0-3.3 and 3.0-8.7%, respectively, and acetic acid was the predominant organic acid present. Colonies showing a clear halo on GYC medium were isolated from the surface film of all vinegars. Via 16S rDNA sequencing, all of the isolates were identified as Acetobacter pasteurianus. Furthermore, field-emission scanning electron microscopy analysis showed that the bacterial cells had a rough surface, were rod-shaped, and were ∼1 × 2 µm in size. Interestingly, cells of the isolate from one of the vinegars were surrounded with an extremely fine threadlike structure. Thus, our results suggest that formation of the surface film in rice vinegar was attributable not to external contamination, to the production of bacterial cellulose by A. pasteurianus to withstand the high concentrations of acetic acid generated during fermentation. However, because of the formation of a surface film in vinegar is undesirable from an industrial perspective, further studies should focus on devising a modified fermentation process to prevent surface film formation and consequent quality degradation.

Short-term sPECAM-Fc treatment ameliorates EAE while chronic use hastens onset of symptoms.

The homophilic cell adhesion molecule PECAM-1 is a major participant in the migration of leukocytes across endothelium. We examined the ability of a chimeric soluble PECAM-1 fused to human IgG-Fc to impair leukocyte entry through the blood-brain barrier and reduce CNS autoimmunity. sPECAM-Fc impaired migration of lymphocytes across brain endothelial monolayers and diminished the severity of EAE, an experimental model of MS, when administered at the onset of symptoms. However, in mice transgenic for sPECAM-Fc, the chronically elevated levels of sPECAM-Fc hastened onset of EAE disease without significantly changing clinical score severity. Our data suggest that short-term treatment of diseases like MS with sPECAM-Fc has therapeutic potential.

Restoration of Traditional Korean Nuruk and Analysis of the Brewing Characteristics.

In this study, a total of 58 different kinds of nuruk (a traditional Korean fermentation starter) were prepared, including 46 kinds of restored nuruk from ancient documents. Each nuruk was evaluated by analysis of its saccharification power, and the enzyme activities of glucoamylase, α-amylase, ß-amylase, protease, and ß-glucanase. The range of saccharification power (sp) of the restored nuruk ranged between 85 and 565 sp. The diastatic enzymes, α-amylase, ß-amylase, and glucoamylase, were significantly correlated to the saccharification power value; conversely, ß-glucanase and protease did not have a correlative relationship with saccarification power. In addition, their brewing properties on chemical and organoleptic aspects of traditional alcoholic beverage production were compared. Each raw and supplementary material contained in nuruk showed its own unique characteristics on Korean alcoholic beverage brewing. For the first time, in this study, the traditional Korean nuruk types mentioned in ancient documents were restored using modernized production methods, and also characterized based on their brewing properties. Our results could be utilized as a basis for further study of traditional alcoholic beverages and their valuable microorganisms.