Direct activation of microglia by ß-glucosylceramide causes phagocytosis of neurons that exacerbates Gaucher disease.

Gaucher disease (GD) is the most common lysosomal storage disease caused by recessive mutations in the degrading enzyme of ß-glucosylceramide (ß-GlcCer). However, it remains unclear how ß-GlcCer causes severe neuronopathic symptoms, which are not fully treated by current therapies. We herein found that ß-GlcCer accumulating in GD activated microglia through macrophage-inducible C-type lectin (Mincle) to induce phagocytosis of living neurons, which exacerbated Gaucher symptoms. This process was augmented by tumor necrosis factor (TNF) secreted from activated microglia that sensitized neurons for phagocytosis. This characteristic pathology was also observed in human neuronopathic GD. Blockade of these pathways in mice with a combination of FDA-approved drugs, minocycline (microglia activation inhibitor) and etanercept (TNF blocker), effectively protected neurons and ameliorated neuronopathic symptoms. In this study, we propose that limiting unrestrained microglia activation using drug repurposing provides a quickly applicable therapeutic option for fatal neuronopathic GD.

Efficient lipidomic approach for the discovery of lipid ligands for immune receptors by combining LC-HRMS/MS analysis with fractionation and reporter cell assay.

C-type lectin receptors (CLRs), which are pattern recognition receptors responsible for triggering innate immune responses, recognize damaged self-components and immunostimulatory lipids from pathogenic bacteria; however, several of their ligands remain unknown. Here, we propose a new analytical platform combining liquid chromatography-high-resolution tandem mass spectrometry with microfractionation capability (LC-FRC-HRMS/MS) and a reporter cell assay for sensitive activity measurements to develop an efficient methodology for searching for lipid ligands of CLR from microbial trace samples (crude cell extracts of approximately 5 mg dry cell/mL). We also developed an in-house lipidomic library containing accurate mass and fragmentation patterns of more than 10,000 lipid molecules predicted in silico for 90 lipid subclasses and 35 acyl side chain fatty acids. Using the developed LC-FRC-HRMS/MS system, the lipid extracts of Helicobacter pylori were separated and fractionated, and HRMS and HRMS/MS spectra were obtained simultaneously. The fractionated lipid extract samples in 96-well plates were thereafter subjected to reporter cell assays using nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing mouse or human macrophage-inducible C-type lectin (Mincle). A total of 102 lipid molecules from all fractions were annotated using an in-house lipidomic library. Furthermore, a fraction that exhibited significant activity in the NFAT-GFP reporter cell assay contained α-cholesteryl glucoside, a type of glycolipid, which was successfully identified as a lipid ligand molecule for Mincle. Our analytical platform has the potential to be a useful tool for efficient discovery of lipid ligands for immunoreceptors.

Mycobacterial mycolic acids trigger inhibitory receptor Clec12A to suppress host immune responses.

Mycobacteria often cause chronic infection. To establish persistence in the host, mycobacteria need to evade host immune responses. However, the molecular mechanisms underlying the evasion strategy are not fully understood. Here, we demonstrate that mycobacterial cell wall lipids trigger an inhibitory receptor to suppress host immune responses. Mycolic acids are major cell wall components and are essential for survival of mycobacteria. By screening inhibitory receptors that react with mycobacterial lipids, we found that mycolic acids from various mycobacterial species bind to mouse Clec12A, and more potently to human Clec12A. Clec12A is a conserved inhibitory C-type lectin receptor containing immunoreceptor tyrosine-based inhibitory motif (ITIM). Innate immune responses, such as MCP-1 production, and PPD-specific recall T cell responses were augmented in Clec12A-deficient mice after infection. In contrast, human Clec12A transgenic mice were susceptible to infection with M. tuberculosis. These results suggest that mycobacteria dampen host immune responses by hijacking an inhibitory host receptor through their specific and essential lipids, mycolic acids. The blockade of this interaction might provide a therapeutic option for the treatment or prevention of mycobacterial infection.

Quantitative metabolomics for dynamic metabolic engineering using stable isotope labeled internal standards mixture (SILIS).

The production of chemicals and fuels from renewable resources using engineered microbes is an attractive alternative for current fossil-dependent industries. Metabolic engineering has contributed to pathway engineering for the production of chemicals and fuels by various microorganisms. Recently, dynamic metabolic engineering harnessing synthetic biological tools has become a next-generation strategy in this field. The dynamic regulation of metabolic flux during fermentation optimizes metabolic states according to each fermentation stage such as cell growth phase and compound production phase. However, it is necessary to repeat the evaluation and redesign of the dynamic regulation system to achieve the practical use of engineered microbes. In this study, we performed quantitative metabolome analysis to investigate the effects of dynamic metabolic flux regulation on engineered Escherichia coli for γ-amino butyrate (GABA) fermentation. We prepared a stable isotope-labeled internal standard mixture (SILIS) for the stable isotope dilution method (SIDM), a mass spectrometry-based quantitative metabolome analysis method. We found multiple candidate bottlenecks for GABA production. Some metabolic reactions in the GABA production pathway should be engineered for further improvement in the direct GABA fermentation with dynamic metabolic engineering strategy.