Food poisoning is one of the leading causes of morbidity and mortality in the world. Citrobacter rodentium is an enteric pathogen which attaches itself to enterocytes and induces attachment and effacing (A/E) lesions. The ability of the bacterium to cause infection requires subversion of the host actin cytoskeleton. Rac-dependent actin polymerization is activated by a guanine nucleotide exchange factor known as Dedicator of cytokinesis 2 (DOCK2). However, the role of DOCK2 in infectious disease is largely unexplored. Here, we found that mice lacking DOCK2 were susceptible to C. rodentium infection. These mice harbored increased levels of C. rodentium bacteria, showed more pronounced weight loss and inflammation-associated pathology, and were prone to bacterial dissemination to the systemic organs compared with wild-type mice. We found that mice lacking DOCK2 were more susceptible to C. rodentium attachment to intestinal epithelial cells. Therefore, our results underscored an important role of DOCK2 for gastrointestinal immunity to C. rodentium infection.

The receptor tyrosine kinase Axl contributes to cell migration and invasion. Expression of Axl correlates with metastatic progression in cancer patients, yet the specific signaling events promoting invasion downstream of Axl are poorly defined. Herein, we report Elmo scaffolds to be direct substrates and binding partners of Axl. Elmo proteins are established to interact with Dock family guanine nucleotide exchange factors to control Rac-mediated cytoskeletal dynamics. Proteomics and mutagenesis studies reveal that Axl phosphorylates Elmo1/2 on a conserved carboxyl-terminal tyrosine residue. Upon Gas6-dependent activation of Axl, endogenous Elmo2 becomes phosphorylated on Tyr-713 and enters into a physical complex with Axl in breast cancer cells. Interfering with Elmo2 expression prevented Gas6-induced Rac1 activation in breast cancer cells. Similarly to blocking of Axl, Elmo2 knockdown or pharmacological inhibition of Dock1 abolishes breast cancer cell invasion. Interestingly, Axl or Elmo2 knockdown diminishes breast cancer cell proliferation. Rescue of Elmo2 knockdown cells with the wild-type protein but not with Elmo2 harboring Tyr-713-Phe mutations restores cell invasion and cell proliferation. These results define a new mechanism by which Axl promotes cell proliferation and invasion and identifies inhibition of the Elmo-Dock pathway as a potential therapeutic target to stop Axl-induced metastases.

White adipose tissue (eWAT) plays a crucial role in preventing metabolic syndrome. We aimed to investigate WAT distribution and gene expression and lipidomic profiles in epididymal WAT (eWAT) in diet-induced obese mice, reflecting a Western-style diet of humans to elucidate the bioactive properties of the dietary antioxidant curcumin in preventing lifestyle-related diseases. For 16 weeks, we fed C57BL/6J mice with a control diet, a high-fat, high-sucrose and high-cholesterol Western diet or Western diet supplemented with 0.1% (w/w) curcumin. Although the dietary intake of curcumin did not affect eWAT weight or plasma lipid levels, it reduced lipid peroxidation markers' levels in eWAT. Curcumin accumulated in eWAT and changed gene expressions related to eukaryotic translation initiation factor 2 (eIF2) signalling. Curcumin suppressed eIF2α phosphorylation, which is induced by endoplasmic reticulum (ER) stress, macrophage accumulation and nuclear factor-κB (NF-κB) p65 and leptin expression, whereas it's anti-inflammatory effect was inadequate to decrease TNF-α and IFN-γ levels. Lipidomic and gene expression analysis revealed that curcumin decreased some diacylglycerols (DAGs) and DAG-derived glycerophospholipids levels by suppressing the glycerol-3-phosphate acyltransferase 1 and adipose triglyceride lipase expression, which are associated with lipogenesis and lipolysis, respectively. Presumably, these intertwined effects contribute to metabolic syndrome prevention by dietary modification.

Isotope-labeling is a useful technique for understanding cellular metabolism. Recent advances in metabolomics have extended the capability of isotope-assisted studies to reveal global metabolism. For instance, isotope-assisted metabolomics technology has enabled the mapping of a global metabolic network, estimation of flux at branch points of metabolic pathways, and assignment of elemental formulas to unknown metabolites. Furthermore, some data processing tools have been developed to apply these techniques to a non-targeted approach, which plays an important role in revealing unknown or unexpected metabolism. However, data collection and integration strategies for non-targeted isotope-assisted metabolomics have not been established. Therefore, a systematic approach is proposed to elucidate metabolic dynamics without targeting pathways by means of time-resolved isotope tracking, i.e., "metabolic turnover analysis", as well as multivariate analysis. We applied this approach to study the metabolic dynamics in amino acid perturbation of Saccharomyces cerevisiae. In metabolic turnover analysis, 69 peaks including 35 unidentified peaks were investigated. Multivariate analysis of metabolic turnover successfully detected a pathway known to be inhibited by amino acid perturbation. In addition, our strategy enabled identification of unknown peaks putatively related to the perturbation.

The immunological synapse formed by a T lymphocyte on the surface of a target cell contains a peripheral ring of filamentous actin (F-actin) that promotes adhesion and facilitates the directional secretion of cytokines and cytolytic factors. We show that growth and maintenance of this F-actin ring is dictated by the annular accumulation of phosphatidylinositol trisphosphate (PIP3) in the synaptic membrane. PIP3 functions in this context by recruiting the exchange factor Dock2 to the periphery of the synapse, where it drives actin polymerization through the Rho-family GTPase Rac. We also show that synaptic PIP3 is generated by class IA phosphoinositide 3-kinases that associate with T cell receptor microclusters and are activated by the GTPase Ras. Perturbations that inhibit or promote PIP3-dependent F-actin remodeling dramatically affect T cell cytotoxicity, demonstrating the functional importance of this pathway. These results reveal how T cells use lipid-based signaling to control synaptic architecture and modulate effector responses.

Thymic epithelial cells (TECs) establish spatially distinct microenvironments in which developing T cells are selected to mature or die. A unique property of medullary TECs is their expression of thousands of tissue-restricted self-antigens that is largely under the control of the transcriptional regulator Aire. We previously showed that Jmjd6, a lysyl hydroxylase for splicing regulatory proteins, is important for Aire protein expression and that transplantation of Jmjd6-deficient thymic stroma into athymic nude mice resulted in multiorgan autoimmunity. Here we report that TEC-specific deletion of Jmjd6 exacerbates development of autoimmune diabetes in a mouse model, which express both ovalbumin (OVA) under the control of the rat insulin gene promoter and OT-I T cell receptor specific for OVA peptide bound to major histocompatibility complex class I Kb molecules. We found that Aire protein expression in mTECs was reduced in the absence of Jmjd6, with retention of intron 2 in Aire transcripts. Our results thus demonstrate the importance of Jmjd6 in establishment of immunological tolerance in a more physiological setting.

The sphingolipid ceramide elicits several stress responses, however, organisms survive despite increased ceramide but how they do so is poorly understood. We demonstrate here that the AKT/FOXO pathway regulates survival in increased ceramide environment by metabolic adaptation involving changes in glycolysis and lipolysis through novel downstream targets. We show that ceramide kinase mutants accumulate ceramide and this leads to reduction in energy levels due to compromised oxidative phosphorylation. Mutants show increased activation of Akt and a consequent decrease in FOXO levels. These changes lead to enhanced glycolysis by upregulating the activity of phosphoglyceromutase, enolase, pyruvate kinase, and lactate dehydrogenase to provide energy. A second major consequence of AKT/FOXO reprogramming in the mutants is the increased mobilization of lipid from the gut through novel lipase targets, CG8093 and CG6277 for energy contribution. Ubiquitous reduction of these targets by knockdown experiments results in semi or total lethality of the mutants, demonstrating the importance of activating them. The efficiency of these adaptive mechanisms decreases with age and leads to reduction in adult life span of the mutants. In particular, mutants develop cardiac dysfunction with age, likely reflecting the high energy requirement of a well-functioning heart. The lipases also regulate physiological triacylglycerol homeostasis and are important for energy metabolism since midgut specific reduction of them in wild type flies results in increased sensitivity to starvation and accumulation of triglycerides leading to cardiac defects. The central findings of increased AKT activation, decreased FOXO level and activation of phosphoglyceromutase and pyruvate kinase are also observed in mice heterozygous for ceramide transfer protein suggesting a conserved role of this pathway in mammals. These data reveal novel glycolytic and non-autonomous lipolytic pathways in response to increased ceramide for sustenance of high energy demanding organ functions like the heart.

Inflammation is a fundamental defensive response to harmful stimuli. However, it can cause damage if it does not subside. To avoid such damage, organisms have developed a mechanism called resolution of inflammation. Here we applied an untargeted metabolomics approach to a sterile and self-resolving animal model of acute inflammation, namely zymosan-induced peritonitis in mice, to examine the effect of inflammation and resolution on the metabolomic profiles. Significant and time-dependent changes in metabolite profiles after zymosan administration were observed in both peritoneal wash fluid (PWF) and plasma. These metabolomic changes correlated well with inflammatory chemokine or cytokine production. In PWF, most of metabolites that could detected increased in zymosan-treated mice, which is suggestive of inflammation, oxidative stress and increased energy demands. In plasma, most metabolites in the central metabolic pathway (glycolysis and TCA cycle) were significantly downregulated after zymosan administration. The concentration of the ketone body 3-hydroxybutyric acid (3-HB) in plasma and PWF increased in zymosan-injected animals indicating upregulation of fatty acid β-oxidation. Increased 3-HB level was observed in the cells that infiltrated into the peritoneal cavity and these infiltrated cells might contribute, at least in part, to the production of 3-HB in the peritoneal cavity.

Allograft rejection is induced by graft tissue infiltration of alloreactive T cells that are activated mainly in secondary lymphoid organs of the host. DOCK2 plays a critical role in lymphocyte homing and immunological synapse formation by regulating the actin cytoskeleton, yet its role in the in vivo immune response remains unknown. We show here that DOCK2 deficiency enables long-term survival of cardiac allografts across a complete mismatch of the major histocompatibility complex molecules. In DOCK2-deficient mice, alloreactivity and allocytotoxicity were suppressed significantly even after in vivo priming with alloantigens, which resulted in reduced intragraft expression of effector molecules, such as interferon-gamma, granzyme B, and perforin. This is mediated, at least in part, by preventing potentially alloreactive T cells from recruiting into secondary lymphoid organs. In addition, we found that DOCK2 is critical for CD28-mediated Rac activation and is required for the full activation of alloreactive T cells. Although DOCK2-deficient, alloreactive T cells were activated in vitro in the presence of exogenous interleukin-2, these T cells, when transferred adoptively, failed to infiltrate into the allografts that were transplanted into RAG1-deficient mice. Thus, DOCK2 deficiency attenuates allograft rejection by simultaneously suppressing multiple and key processes. We propose that DOCK2 could be a novel molecular target for controlling transplant rejection.

Chemokines guide lymphocytes from blood to secondary lymphoid organs by triggering integrin-dependent firm adhesion under vascular flow and directed migration of T and B lymphocytes within lymphoid tissue. Here, we analyze the roles of DOCK2, a mammalian homolog of Caenorhabditis elegans CED-5 and Drosophila melanogaster Myoblast City, and phosphoinositide-3-kinase (PI3K) during lymphocyte recirculation. DOCK2 mediated efficient lymphocyte migration in a largely PI3K-independent manner, although a minor, PI3K-dependent pathway for migration was observed in wild-type and DOCK2-deficient lymphocytes. In T cells, this residual migration depended mainly on PI3Kgamma, whereas other PI3K isoforms were implicated in B cells. In vitro adhesion assays and intravital microscopy of lymphoid organ vasculature uncovered an unexpected defect in integrin activation in DOCK2-/- B cells, whereas lack of DOCK2 did not affect chemokine-triggered integrin activation in T cells. DOCK2 and PI3Kgamma thus play distinct roles during T and B cell integrin activation and migration.

Dock2 has been shown to be indispensable for chemotaxis of mature lymphocytes as a critical Rac activator. However, the functional expression of Dock2 in immature hematopoietic cells is unclear. In this study, we demonstrate that Dock2 is broadly expressed in bone marrow (BM) hematopoietic compartment, including hematopoietic stem/progenitor cell (HSC/HPC) fraction. Response of Dock2-/- HPCs to CXCL12 in chemotaxis and actin polymerization in vitro was impaired, although alpha4 integrin activation by CXCL12 was not altered. Myelosuppressive stress on HSCs in vivo, such as consecutive 5-FU administration and serial bone marrow transplantation, did not show hematopoietic defect in Dock2-/- mice. Long-term engraftment of transplanted Dock2-/- BM cells was severely impaired in competitive reconstitution. However, this was not intrinsic to HSCs but originated from the defective competition of Dock2-/- lymphoid precursors. These results suggest that Dock2 plays a significant role in BM lymphopoiesis, but is dispensable for HSC engraftment and self-renewal.

Rac1 is a member of the Rho family of small GTPases that regulates cytoskeletal reorganization, membrane polarization, cell migration and proliferation. Recently, a self-activating mutation of Rac1, Rac1P29S, has been identified as a recurrent somatic mutation frequently found in sun-exposed melanomas, which possesses increased inherent GDP/GTP exchange activity and cell transforming ability. However, the role of cellular Rac1-interacting proteins in the transforming potential of Rac1P29S remains unclear. We found that the catalytic domain of DOCK1, a Rac-specific guanine nucleotide exchange factor (GEF) implicated in malignancy of a variety of cancers, can greatly accelerate the GDP/GTP exchange of Rac1P29S. Enforced expression of Rac1P29S induced matrix invasion and macropinocytosis in wild-type (WT) mouse embryonic fibroblasts (MEFs), but not in DOCK1-deficient MEFs. Consistently, a selective inhibitor of DOCK1 that blocks its GEF function suppressed the invasion and macropinocytosis in WT MEFs expressing Rac1P29S. Human melanoma IGR-1 and breast cancer MDA-MB-157 cells harbor Rac1P29S mutation and express DOCK1 endogenously. Genetic inactivation and pharmacological inhibition of DOCK1 suppressed their invasion and macropinocytosis. Taken together, these results indicate that DOCK1 is a critical regulator of the malignant phenotypes induced by Rac1P29S, and suggest that targeting DOCK1 might be an effective approach to treat cancers associated with Rac1P29S mutation.

Interleukin (IL)-17-producing T helper (Th17) cells are crucial for host defense against extracellular microbes and pathogenesis of autoimmune diseases. Here we show that the AP-1 transcription factor JunB is required for Th17 cell development. Junb-deficient CD4+ T cells are able to develop in vitro into various helper T subsets except Th17. The RNA-seq transcriptome analysis reveals that JunB is crucial for the Th17-specific gene expression program. Junb-deficient mice are completely resistant to experimental autoimmune encephalomyelitis, a Th17-mediated inflammatory disease, and naive T helper cells from such mice fail to differentiate into Th17 cells. JunB appears to activate Th17 signature genes by forming a heterodimer with BATF, another AP-1 factor essential for Th17 differentiation. The mechanism whereby JunB controls Th17 cell development likely involves activation of the genes for the Th17 lineage-specifying orphan receptors RORγt and RORα and reduced expression of Foxp3, a transcription factor known to antagonize RORγt function.

Glucose metabolism is remodeled in cancer, but the global pattern of cancer-specific metabolic changes remains unclear. Here we show, using the comprehensive measurement of metabolic enzymes by large-scale targeted proteomics, that the metabolism both carbon and nitrogen is altered during the malignant progression of cancer. The fate of glutamine nitrogen is shifted from the anaplerotic pathway into the TCA cycle to nucleotide biosynthesis, with this shift being controlled by glutaminase (GLS1) and phosphoribosyl pyrophosphate amidotransferase (PPAT). Interventions to reduce the PPAT/GLS1 ratio suppresses tumor growth of many types of cancer. A meta-analysis reveals that PPAT shows the strongest correlation with malignancy among all metabolic enzymes, in particular in neuroendocrine cancer including small cell lung cancer (SCLC). PPAT depletion suppresses the growth of SCLC lines. A shift in glutamine fate may thus be required for malignant progression of cancer, with modulation of nitrogen metabolism being a potential approach to SCLC treatment.

Gaucher disease (GD) is the most common lysosomal storage disease caused by recessive mutations in the degrading enzyme of β-glucosylceramide (β-GlcCer). However, it remains unclear how β-GlcCer causes severe neuronopathic symptoms, which are not fully treated by current therapies. We herein found that β-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.

One of the technical challenges in the field of metabolomics is the development of a single-run method to detect the full complement of polar metabolites in biological samples. However, an ideal method to meet this demand has not yet been developed. Herein, we proposed a simple methodology that enables the comprehensive and simultaneous analysis of polar metabolites using unified-hydrophilic-interaction/anion-exchange liquid chromatography mass spectrometry (unified-HILIC/AEX/MS) with a polymer-based mixed amines column composed of methacrylate-based polymer particles with primary, secondary, tertiary, and quaternary amines as functional groups. The optimized unified-HILIC/AEX/MS method is composed of two consecutive chromatographic separations, HILIC-dominant separation for cationic, uncharged, and zwitterionic polar metabolites [retention times (RTs) = 0-12.8 min] and AEX-dominant separation for polar anionic metabolites (RTs = 12.8-26.5 min), by varying the ratio of acetonitrile to 40 mM ammonium bicarbonate solution (pH 9.8). A total of 400 polar metabolites were analyzed simultaneously through a combination of highly efficient separation using unified-HILIC/AEX and remarkably sensitive detection using multiple reaction monitoring-based triple quadrupole mass spectrometry (unified-HILIC/AEX/MS/MS). A nontargeted metabolomic approach using unified-HILIC/AEX high-resolution mass spectrometry (unified-HILIC/AEX/HRMS) also provided more comprehensive information on polar metabolites (3242 metabolic features) in HeLa cell extracts than the conventional HILIC/HRMS method (2068 metabolic features). Our established unified-HILIC/AEX/MS/MS and unified-HILIC/AEX/HRMS methods have several advantages over conventional techniques, including polar metabolome coverage, throughput, and accurate quantitative performance, and represent potentially useful tools for in-depth studies on metabolism and biomarker discovery.

We have previously reported a novel homozygous 4-bp deletion in DDHD1 as the responsible variant for spastic paraplegia type 28 (SPG28; OMIM#609340). The variant causes a frameshift, resulting in a functionally null allele in the patient. DDHD1 encodes phospholipase A1 (PLA1) catalyzing phosphatidylinositol to lysophosphatidylinositol (LPI). To clarify the pathogenic mechanism of SPG28, we established Ddhd1 knockout mice (Ddhd1[-/-]) carrying a 5-bp deletion in Ddhd1, resulting in a premature termination of translation at a position similar to that of the patient. We observed a significant decrease in foot-base angle (FBA) in aged Ddhd1(-/-) (24 months of age) and a significant decrease in LPI 20:4 (sn-2) in Ddhd1(-/-) cerebra (26 months of age). These changes in FBA were not observed in 14 months of age. We also observed significant changes of expression levels of 22 genes in the Ddhd1(-/-) cerebra (26 months of age). Gene Ontology (GO) terms relating to the nervous system and cell-cell communications were significantly enriched. We conclude that the reduced signaling of LPI 20:4 (sn-2) by PLA1 dysfunction is responsible for the locomotive abnormality in SPG28, further suggesting that the reduction of downstream signaling such as GPR55 which is agonized by LPI is involved in the pathogenesis of SPG28.

Cancers disrupt host homeostasis in various manners but the identity of host factors underlying such disruption remains largely unknown. Here we show that nicotinamide-N-methyltransferase (NNMT) is a host factor that mediates metabolic dysfunction in the livers of cancer-bearing mice. Multiple solid cancers distantly increase expression of Nnmt and its product 1-methylnicotinamide (MNAM) in the liver. Multi-omics analyses reveal suppression of the urea cycle accompanied by accumulation of amino acids, and enhancement of uracil biogenesis in the livers of cancer-bearing mice. Importantly, genetic deletion of Nnmt leads to alleviation of these metabolic abnormalities, and buffers cancer-dependent weight loss and reduction of the voluntary wheel-running activity. Our data also demonstrate that MNAM is capable of affecting urea cycle metabolites in the liver. These results suggest that cancers up-regulate the hepatic NNMT pathway to rewire liver metabolism towards uracil biogenesis rather than nitrogen disposal via the urea cycle, thereby disrupting host homeostasis.

Metabolic profiling is an omics approach that can be used to observe phenotypic changes, making it particularly attractive for biomarker discovery. Although several candidate metabolites biomarkers for disease expression have been identified in recent clinical studies, the reference values of healthy subjects have not been established. In particular, the accuracy of concentrations measured by mass spectrometry (MS) is unclear. Therefore, comprehensive metabolic profiling in large-scale cohorts by MS to create a database with reference ranges is essential for evaluating the quality of the discovered biomarkers. In this study, we tested 8700 plasma samples by commercial kit-based metabolomics and separated them into two groups of 6159 and 2541 analyses based on the different ultra-high-performance tandem mass spectrometry (UHPLC-MS/MS) systems. We evaluated the quality of the quantified values of the detected metabolites from the reference materials in the group of 2541 compared with the quantified values from other platforms, such as nuclear magnetic resonance (NMR), supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS) and UHPLC-Fourier transform mass spectrometry (FTMS). The values of the amino acids were highly correlated with the NMR results, and lipid species such as phosphatidylcholines and ceramides showed good correlation, while the values of triglycerides and cholesterol esters correlated less to the lipidomics analyses performed using SFC-MS/MS and UHPLC-FTMS. The evaluation of the quantified values by MS-based techniques is essential for metabolic profiling in a large-scale cohort.

4-hydroxytamoxifen (OHT) is an anti-cancer drug that induces apoptosis in breast cancer cells. Although changes in lipid levels and mitochondrial respiration have been observed in OHT-treated cells, the overall mechanisms underlying these metabolic alterations are poorly understood. In this study, time-series metabolomics and lipidomics were used to analyze the changes in metabolic profiles induced by OHT treatment in the MCF-7 human breast cancer cell line. Lipidomic and metabolomic analyses revealed increases in ceramide, diacylglycerol and triacylglycerol, and decreases in citrate, respectively. Gene expression analyses revealed increased expression of ATP-dependent citrate lyase (ACLY) and subsequent fatty acid biosynthetic enzymes, suggesting that OHT-treated MCF-7 cells activate citrate-to-lipid metabolism. The significance of the observed metabolic changes was evaluated by co-treating MCF-7 cells with OHT and ACLY or a diacylglycerol O-acyltransferase 1 (DGAT1) inhibitor. Co-treatment ameliorated cell death and reduced mitochondrial membrane potential compared to that in OHT treatment alone. The inhibition of cell death by co-treatment with an ACLY inhibitor has been observed in other breast cancer cell lines. These results suggest that citrate-to-lipid metabolism is critical for OHT-induced cell death in breast cancer cell lines.