D-2-hydroxyglutarate supports a tolerogenic phenotype with lowered major histocompatibility class II expression in non-malignant dendritic cells and acute myeloid leukemia cells.

D-2-hydroxyglutarate (D-2-HG) accumulates in primary acute myeloid leukemia (AML) patients with mutated isocitrate dehydrogenase (IDH) and other malignancies. D-2-HG suppresses antitumor T cell immunity but little is known about potential effects on non-malignant myeloid cells. Here we show that D-2-HG impairs human but not murine dendritic cell (DC) differentiation, resulting in a tolerogenic phenotype with low major histocompatibility (MHC) class II expression. In line, IDH-mutated AML blasts exhibited lower expression of HLA-DP and were less susceptible to lysis by HLA-DP-specific T cells. Interestingly, D-2-HG reprogrammed metabolism towards increased lactate production in DCs and AML besides its expected impact on DNA demethylation. Vitamin C accelerated DNA demethylation, but only the combination of vitamin C and glycolytic inhibition lowered lactate levels and supported MHC class II expression. Our results indicate an unexpected link between the immunosuppressive metabolites 2-HG and lactic acid and suggest a potentially novel therapeutic strategy with combinations of anti-glycolytic drugs and epigenetic modulators (hypomethylating agents) or other therapeutics for the treatment of AML.

MCT4 blockade increases the efficacy of immune checkpoint blockade.

BACKGROUND & AIMS: Intratumoral lactate accumulation and acidosis impair T-cell function and antitumor immunity. Interestingly, expression of the lactate transporter monocarboxylate transporter (MCT) 4, but not MCT1, turned out to be prognostic for the survival of patients with rectal cancer, indicating that single MCT4 blockade might be a promising strategy to overcome glycolysis-related therapy resistance. METHODS: To determine whether blockade of MCT4 alone is sufficient to improve the efficacy of immune checkpoint blockade (ICB) therapy, we examined the effects of the selective MCT1 inhibitor AZD3965 and a novel MCT4 inhibitor in a colorectal carcinoma (CRC) tumor spheroid model co-cultured with blood leukocytes in vitro and the MC38 murine CRC model in vivo in combination with an antibody against programmed cell death ligand-1(PD-L1). RESULTS: Inhibition of MCT4 was sufficient to reduce lactate efflux in three-dimensional (3D) CRC spheroids but not in two-dimensional cell-cultures. Co-administration of the MCT4 inhibitor and ICB augmented immune cell infiltration, T-cell function and decreased CRC spheroid viability in a 3D co-culture model of human CRC spheroids with blood leukocytes. Accordingly, combination of MCT4 and ICB increased intratumoral pH, improved leukocyte infiltration and T-cell activation, delayed tumor growth, and prolonged survival in vivo. MCT1 inhibition exerted no further beneficial impact. CONCLUSIONS: These findings demonstrate that single MCT4 inhibition represents a novel therapeutic approach to reverse lactic-acid driven immunosuppression and might be suitable to improve ICB efficacy.

Anomaly detection in mixed high-dimensional molecular data.

MOTIVATION: Mixed molecular data combines continuous and categorical features of the same samples, such as OMICS profiles with genotypes, diagnoses, or patient sex. Like all high-dimensional molecular data, it is prone to incorrect values that can stem from various sources for example the technical limitations of the measurement devices, errors in the sample preparation, or contamination. Most anomaly detection algorithms identify complete samples as outliers or anomalies. However, in most cases, not all measurements of those samples are erroneous but only a few one-dimensional features within the samples are incorrect. These one-dimensional data errors are continuous measurements that are either located outside or inside the normal ranges of their features but in both cases show atypical values given all other continuous and categorical features in the sample. Additionally, categorical anomalies can occur for example when the genotype or diagnosis was submitted wrongly. RESULTS: We introduce ADMIRE (Anomaly Detection using MIxed gRaphical modEls), a novel approach for the detection and correction of anomalies in mixed high-dimensional data. Hereby, we focus on the detection of single (one-dimensional) data errors in the categorical and continuous features of a sample. For that the joint distribution of continuous and categorical features is learned by mixed graphical models, anomalies are detected by the difference between measured and model-based estimations and are corrected using imputation. We evaluated ADMIRE in simulation and by screening for anomalies in one of our own metabolic datasets. In simulation experiments, ADMIRE outperformed the state-of-the-art methods of Local Outlier Factor, stray, and Isolation Forest. AVAILABILITY AND IMPLEMENTATION: All data and code is available at https://github.com/spang-lab/adadmire. ADMIRE is implemented in a Python package called adadmire which can be found at https://pypi.org/project/adadmire.

IDH3γ functions as a redox switch regulating mitochondrial energy metabolism and contractility in the heart.

Redox signaling and cardiac function are tightly linked. However, it is largely unknown which protein targets are affected by hydrogen peroxide (H2O2) in cardiomyocytes that underly impaired inotropic effects during oxidative stress. Here, we combine a chemogenetic mouse model (HyPer-DAO mice) and a redox-proteomics approach to identify redox sensitive proteins. Using the HyPer-DAO mice, we demonstrate that increased endogenous production of H2O2 in cardiomyocytes leads to a reversible impairment of cardiac contractility in vivo. Notably, we identify the γ-subunit of the TCA cycle enzyme isocitrate dehydrogenase (IDH)3 as a redox switch, linking its modification to altered mitochondrial metabolism. Using microsecond molecular dynamics simulations and experiments using cysteine-gene-edited cells reveal that IDH3γ Cys148 and 284 are critically involved in the H2O2-dependent regulation of IDH3 activity. Our findings provide an unexpected mechanism by which mitochondrial metabolism can be modulated through redox signaling processes.

Glutamine synthetase expression rescues human dendritic cell survival in a glutamine-deprived environment.

Introduction: Glutamine deficiency is a well-known feature of the tumor environment. Here we analyzed the impact of glutamine deprivation on human myeloid cell survival and function. Methods: Different types of myeloid cells were cultured in the absence or presence of glutamine and/or with L-methionine-S-sulfoximine (MSO), an irreversible glutamine synthetase (GS) inhibitor. GS expression was analyzed on mRNA and protein level. GS activity and the conversion of glutamate to glutamine by myeloid cells was followed by 13C tracing analyses. Results: The absence of extracellular glutamine only slightly affected postmitotic human monocyte to dendritic cell (DC) differentiation, function and survival. Similar results were obtained for monocyte-derived macrophages. In contrast, proliferation of the monocytic leukemia cell line THP-1 was significantly suppressed. While macrophages exhibited high constitutive GS expression, glutamine deprivation induced GS in DC and THP-1. Accordingly, proliferation of THP-1 was rescued by addition of the GS substrate glutamate and 13C tracing analyses revealed conversion of glutamate to glutamine. Supplementation with the GS inhibitor MSO reduced the survival of DC and macrophages and counteracted the proliferation rescue of THP-1 by glutamate. Discussion: Our results show that GS supports myeloid cell survival in a glutamine poor environment. Notably, in addition to suppressing proliferation and survival of tumor cells, the blockade of GS also targets immune cells such as DCs and macrophages.

Bovine blood derived macrophages are unable to control Coxiella burnetii replication under hypoxic conditions.

Background: Coxiella burnetii is a zoonotic pathogen, infecting humans, livestock, pets, birds and ticks. Domestic ruminants such as cattle, sheep, and goats are the main reservoir and major cause of human infection. Infected ruminants are usually asymptomatic, while in humans infection can cause significant disease. Human and bovine macrophages differ in their permissiveness for C. burnetii strains from different host species and of various genotypes and their subsequent host cell response, but the underlying mechanism(s) at the cellular level are unknown. Methods: C. burnetii infected primary human and bovine macrophages under normoxic and hypoxic conditions were analyzed for (i) bacterial replication by CFU counts and immunofluorescence; (ii) immune regulators by westernblot and qRT-PCR; cytokines by ELISA; and metabolites by gas chromatography-mass spectrometry (GC-MS). Results: Here, we confirmed that peripheral blood-derived human macrophages prevent C. burnetii replication under oxygen-limiting conditions. In contrast, oxygen content had no influence on C. burnetii replication in bovine peripheral blood-derived macrophages. In hypoxic infected bovine macrophages, STAT3 is activated, even though HIF1α is stabilized, which otherwise prevents STAT3 activation in human macrophages. In addition, the TNFα mRNA level is higher in hypoxic than normoxic human macrophages, which correlates with increased secretion of TNFα and control of C. burnetii replication. In contrast, oxygen limitation does not impact TNFα mRNA levels in C. burnetii-infected bovine macrophages and secretion of TNFα is blocked. As TNFα is also involved in the control of C. burnetii replication in bovine macrophages, this cytokine is important for cell autonomous control and its absence is partially responsible for the ability of C. burnetii to replicate in hypoxic bovine macrophages. Further unveiling the molecular basis of macrophage-mediated control of C. burnetii replication might be the first step towards the development of host directed intervention measures to mitigate the health burden of this zoonotic agent.

Heterogeneity of Amino Acid Profiles of Proneural and Mesenchymal Brain-Tumor Initiating Cells.

Glioblastomas are highly malignant brain tumors that derive from brain-tumor-initiating cells (BTICs) and can be subdivided into several molecular subtypes. Metformin is an antidiabetic drug currently under investigation as a potential antineoplastic agent. The effects of metformin on glucose metabolism have been extensively studied, but there are only few data on amino acid metabolism. We investigated the basic amino acid profiles of proneural and mesenchymal BTICs to explore a potential distinct utilization and biosynthesis in these subgroups. We further measured extracellular amino acid concentrations of different BTICs at baseline and after treatment with metformin. Effects of metformin on apoptosis and autophagy were determined using Western Blot, annexin V/7-AAD FACS-analyses and a vector containing the human LC3B gene fused to green fluorescent protein. The effects of metformin on BTICs were challenged in an orthotopic BTIC model. The investigated proneural BTICs showed increased activity of the serine and glycine pathway, whereas mesenchymal BTICs in our study preferably metabolized aspartate and glutamate. Metformin treatment led to increased autophagy and strong inhibition of carbon flux from glucose to amino acids in all subtypes. However, oral treatment with metformin at tolerable doses did not significantly inhibit tumor growth in vivo. In conclusion, we found distinct amino acid profiles of proneural and mesenchymal BTICs, and inhibitory effects of metformin on BTICs in vitro. However, further studies are warranted to better understand potential resistance mechanisms against metformin in vivo.

Macrophages inhibit Coxiella burnetii by the ACOD1-itaconate pathway for containment of Q fever.

Infection with the intracellular bacterium Coxiella (C.) burnetii can cause chronic Q fever with severe complications and limited treatment options. Here, we identify the enzyme cis-aconitate decarboxylase 1 (ACOD1 or IRG1) and its product itaconate as protective host immune pathway in Q fever. Infection of mice with C. burnetii induced expression of several anti-microbial candidate genes, including Acod1. In macrophages, Acod1 was essential for restricting C. burnetii replication, while other antimicrobial pathways were dispensable. Intratracheal or intraperitoneal infection of Acod1-/- mice caused increased C. burnetii burden, weight loss and stronger inflammatory gene expression. Exogenously added itaconate restored pathogen control in Acod1-/- mouse macrophages and blocked replication in human macrophages. In axenic cultures, itaconate directly inhibited growth of C. burnetii. Finally, treatment of infected Acod1-/- mice with itaconate efficiently reduced the tissue pathogen load. Thus, ACOD1-derived itaconate is a key factor in the macrophage-mediated defense against C. burnetii and may be exploited for novel therapeutic approaches in chronic Q fever.

Metabolic Heterogeneity of Brain Tumor Cells of Proneural and Mesenchymal Origin.

Brain-tumor-initiating cells (BTICs) of proneural and mesenchymal origin contribute to the highly malignant phenotype of glioblastoma (GB) and resistance to current therapies. BTICs of different subtypes were challenged with oxidative phosphorylation (OXPHOS) inhibition with metformin to assess the differential effects of metabolic intervention on key resistance features. Whereas mesenchymal BTICs varied according to their invasiveness, they were in general more glycolytic and less responsive to metformin. Proneural BTICs were less invasive, catabolized glucose more via the pentose phosphate pathway, and responded better to metformin. Targeting glycolysis may be a promising approach to inhibit tumor cells of mesenchymal origin, whereas proneural cells are more responsive to OXPHOS inhibition. Future clinical trials exploring metabolic interventions should account for metabolic heterogeneity of brain tumors.

Isotope Ratio Outlier Analysis (IROA) for HPLC-TOFMS-Based Metabolomics of Human Urine.

Metabolic fingerprinting by mass spectrometry aims at the comprehensive, semiquantitative analysis of metabolites. Isotope dilution, if successfully implemented, may provide a more reliable, relative quantification. Therefore, the 13C labeled yeast extract of the IROA TruQuant kit was added as an internal standard (IS) to human urine samples measured in full-scan mode on a high-performance liquid chromatography-time-of-flight mass spectrometer (HPLC-TOFMS) system. The isotope ratio approach enabled the analysis of 112 metabolites. The correlation with reference data did not improve significantly using 12C/13C ratios compared to absolute 12C peak areas. Moreover, using an intricate 13C-labeled standard increased the complexity of the mass spectra, which made correct signal annotation more challenging. On the positive side, the ratio approach helps to reduce batch effects, but it does not perform better than computational methods such as the "removebatcheffect" function in the R package Limma.

LDHB Overexpression Can Partially Overcome T Cell Inhibition by Lactic Acid.

Accelerated glycolysis leads to secretion and accumulation of lactate and protons in the tumor environment and determines the efficacy of adoptive T cell and checkpoint inhibition therapy. Here, we analyzed effects of lactic acid on different human CD4 T cell subsets and aimed to increase CD4 T cell resistance towards lactic acid. In all CD4 T cell subsets analyzed, lactic acid inhibited metabolic activity (glycolysis and respiration), cytokine secretion, and cell proliferation. Overexpression of the lactate-metabolizing isoenzyme LDHB increased cell respiration and mitigated lactic acid effects on intracellular cytokine production. Strikingly, LDHB-overexpressing cells preferentially migrated into HCT116 tumor spheroids and displayed higher expression of cytotoxic effector molecules. We conclude, that LDHB overexpression might be a promising strategy to increase the efficacy of adoptive T cell transfer therapy.

Acidic Microenvironments Found in Cutaneous Leishmania Lesions Curtail NO-Dependent Antiparasitic Macrophage Activity.

Local tissue acidosis affects anti-tumor immunity. In contrast, data on tissue pH levels in infected tissues and their impact on antimicrobial activity is sparse. In this study, we assessed the pH levels in cutaneous Leishmania lesions. Leishmania major-infected skin tissue displayed pH levels of 6.7 indicating that lesional pH is acidic. Next, we tested the effect of low extracellular pH on the ability of macrophages to produce leishmanicidal NO and to fight the protozoan parasite Leishmania major. Extracellular acidification led to a marked decrease in both NO production and leishmanicidal activity of lipopolysaccharide (LPS) and interferon γ (IFN-γ)-coactivated macrophages. This was not directly caused by a disruption of NOS2 expression, a shortage of reducing equivalents (NAPDH) or substrate (L-arginine), but by a direct, pH-mediated inhibition of NOS2 enzyme activity. Normalization of intracellular pH significantly increased NO production and antiparasitic activity of macrophages even in an acidic microenvironment. Overall, these findings indicate that low local tissue pH can curtail NO production and leishmanicidal activity of macrophages.

Prolonged Suppression of Butyrate-Producing Bacteria Is Associated With Acute Gastrointestinal Graft-vs-Host Disease and Transplantation-Related Mortality After Allogeneic Stem Cell Transplantation.

BACKGROUND: Butyrogenic bacteria play an important role in gut microbiome homeostasis and intestinal epithelial integrity. Previous studies have demonstrated an association between administration of short-chain fatty acids like butyrate and protection from acute graft-vs-host disease (GvHD) after allogeneic stem cell transplantation (ASCT). METHODS: In the current study, we examined the abundance and butyrogenic capacity of butyrate-producing bacteria in 28 healthy donors and 201 patients after ASCT. We prospectively collected serial stool samples and performed polymerase chain reaction analysis of the butyrate-producing bacterial enzyme butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) in fecal nucleic acid extracts. RESULTS: Our data demonstrate a strong and prolonged suppression of butyrogenic bacteria early in the course of ASCT. In a multivariable analysis, early use of broad-spectrum antibiotics before day 0 (day of transplantation) was identified as an independent factor associated with low BCoAT copy numbers (odds ratio, 0.370 [95% confidence interval, .175-.783]; P = .009). Diminished butyrogens correlated with other biomarkers of microbial diversity, such as low 3-indoxylsulfate levels, reduced abundance of Clostridiales and low inverse Simpson and effective Shannon indices (all P < .001). Low BCoAT copy numbers at GvHD-onset were correlated with GI-GvHD severity (P = .002) and associated with a significantly higher GvHD-associated mortality rate (P = .04). Furthermore, low BCoAT copy numbers at day 30 were associated with a significantly higher transplantation-related mortality rate (P = .02). CONCLUSIONS: Our results are consistent with the hypothesis that alterations in the microbiome play an important role in GvHD pathogenesis and that microbial parameters such as BCoAT might serve as biomarkers to identify patients at high risk of lethal GI-GvHD.

Kynurenine induces T cell fat catabolism and has limited suppressive effects in vivo.

BACKGROUND: L-kynurenine is a tryptophan-derived immunosuppressive metabolite and precursor to neurotoxic anthranilate and quinolinate. We evaluated the stereoisomer D-kynurenine as an immunosuppressive therapeutic which is hypothesized to produce less neurotoxic metabolites than L-kynurenine. METHODS: L-/D-kynurenine effects on human and murine T cell function were examined in vitro and in vivo (homeostatic proliferation, colitis, cardiac transplant). Kynurenine effects on T cell metabolism were interrogated using [13C] glucose, glutamine and palmitate tracing. Kynurenine was measured in tissues from human and murine tumours and kynurenine-fed mice. FINDINGS: We observed that 1 mM D-kynurenine inhibits T cell proliferation through apoptosis similar to L-kynurenine. Mechanistically, [13C]-tracing revealed that co-stimulated CD4+ T cells exposed to L-/D-kynurenine undergo increased ß-oxidation depleting fatty acids. Replenishing oleate/palmitate restored effector T cell viability. We administered dietary D-kynurenine reaching tissue kynurenine concentrations of 19 µM, which is close to human kidney (6 µM) and head and neck cancer (14 µM) but well below the 1 mM required for apoptosis. D-kynurenine protected Rag1-/- mice from autoimmune colitis in an aryl-hydrocarbon receptor dependent manner but did not attenuate more stringent immunological challenges such as antigen mismatched cardiac allograft rejection. INTERPRETATION: Our dietary kynurenine model achieved tissue concentrations at or above human cancer kynurenine and exhibited only limited immunosuppression. Sub-suppressive kynurenine concentrations in human cancers may limit the responsiveness to indoleamine 2,3-dioxygenase inhibition evaluated in clinical trials. FUNDING: The study was supported by the NIH, the Else Kröner-Fresenius-Foundation, Laffey McHugh foundation, and American Society of Nephrology.

Balancing of mitochondrial translation through METTL8-mediated m3C modification of mitochondrial tRNAs.

Mitochondria contain a specific translation machinery for the synthesis of mitochondria-encoded respiratory chain components. Mitochondrial tRNAs (mt-tRNAs) are also generated from the mitochondrial DNA and, similar to their cytoplasmic counterparts, are post-transcriptionally modified. Here, we find that the RNA methyltransferase METTL8 is a mitochondrial protein that facilitates 3-methyl-cytidine (m3C) methylation at position C32 of the mt-tRNASer(UCN) and mt-tRNAThr. METTL8 knockout cells show a reduction in respiratory chain activity, whereas overexpression increases activity. In pancreatic cancer, METTL8 levels are high, which correlates with lower patient survival and an enhanced respiratory chain activity. Mitochondrial ribosome profiling uncovered mitoribosome stalling on mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons. Further analysis of the respiratory chain complexes using mass spectrometry revealed reduced incorporation of the mitochondrially encoded proteins ND6 and ND1 into complex I. The well-balanced translation of mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons through METTL8-mediated m3C32 methylation might, therefore, facilitate the optimal composition and function of the mitochondrial respiratory chain.

Cold Atmospheric Plasma Changes the Amino Acid Composition of Solutions and Influences the Anti-Tumor Effect on Melanoma Cells.

Cold Atmospheric Plasma (CAP) is an ionized gas near room temperature. Its anti-tumor effect can be transmitted either by direct treatment or mediated by a plasma-treated solution (PTS), such as treated standard cell culture medium, which contains different amino acids, inorganic salts, vitamins and other substances. Despite extensive research, the active components in PTS and its molecular or cellular mechanisms are not yet fully understood. The purpose of this study was the measurement of the reactive species in PTS and their effect on tumor cells using different plasma modes and treatment durations. The PTS analysis yielded mode- and dose-dependent differences in the production of reactive oxygen and nitrogen species (RONS), and in the decomposition and modification of the amino acids Tyrosine (Tyr) and Tryptophan (Trp). The Trp metabolites Formylkynurenine (FKyn) and Kynurenine (Kyn) were produced in PTS with the 4 kHz (oxygen) mode, inducing apoptosis in Mel Im melanoma cells. Nitrated derivatives of Trp and Tyr were formed in the 8 kHz (nitrogen) mode, elevating the p16 mRNA expression and senescence-associated ß-Galactosidase staining. In conclusion, the plasma mode has a strong impact on the composition of the active components in PTS and affects its anti-tumor mechanism. These findings are of decisive importance for the development of plasma devices and the effectiveness of tumor treatment.

Lactonization of the Oncometabolite D-2-Hydroxyglutarate Produces a Novel Endogenous Metabolite.

In recent years, onco-metabolites like D-2-hydroxyglutarate, which is produced in isocitrate dehydrogenase-mutated tumors, have gained increasing interest. Here, we report a metabolite in human specimens that is closely related to 2-hydroxyglutarate: the intramolecular ester of 2-hydroxyglutarate, 2-hydroxyglutarate-γ-lactone. Using 13C5-L-glutamine tracer analysis, we showed that 2-hydroxyglutarate is the endogenous precursor of 2-hydroxyglutarate-lactone and that there is a high exchange between these two metabolites. Lactone formation does not depend on mutated isocitrate dehydrogenase, but its formation is most probably linked to transport processes across the cell membrane and favored at low environmental pH. Furthermore, human macrophages showed not only striking differences in uptake of 2-hydroxyglutarate and its lactone but also in the enantiospecific hydrolysis of the latter. Consequently, 2-hydroxyglutarate-lactone may play a critical role in the modulation of the tumor microenvironment.

Cytokine-specific autoantibodies shape the gut microbiome in autoimmune polyendocrine syndrome type 1.

BACKGROUND: Gastrointestinal dysfunction is a frequent and disabling manifestation of autoimmune polyendocrine syndrome type 1 (APS-1), a rare monogenic multiorgan autoimmune disease caused by the loss of central AIRE-controlled immune tolerance. OBJECTIVES: This study aimed to understand the role of the gut microbiome in APS-1 symptoms and potentially alleviate common gastrointestinal symptoms by probiotic intervention. METHODS: This study characterized the fecal microbiomes of 28 patients with APS-1 and searched for associations with gastrointestinal symptoms, circulating anti-cytokine autoantibodies, and tryptophan-related metabolites. Additionally, daily doses of the probiotic Lactobacillus rhamnosus GG were administered for 3 months. RESULTS: Of 581 metagenomic operational taxonomic units (mOTUs) characterized in total, 14 were significantly associated with patients with APS-1 compared with healthy controls, with 6 mOTUs depleted and 8 enriched in patients with APS-1. Four overabundant mOTUs were significantly associated with severity of constipation. Phylogenetically conserved microbial associations with autoantibodies against cytokines were observed. After the 3-month intervention with the probiotic L rhamnosus GG, a subset of gastrointestinal symptoms were alleviated. L rhamnosus GG abundance was increased postintervention and corresponded with decreased abundances of Alistipes onderdonkii and Collinsella aerofaciens, 2 species positively associated with severity of diarrhea in patients with APS-1. CONCLUSIONS: The APS-1 microbiome correlates with several APS-1 symptoms, some of which are alleviated after a 3-month L rhamnosus GG intervention. Autoantibodies against cytokines appear to shape the gut microbiome by positively correlating with a taxonomically consistent group of bacteria.

Mitochondrial arginase-2 is essential for IL-10 metabolic reprogramming of inflammatory macrophages.

Mitochondria are important regulators of macrophage polarisation. Here, we show that arginase-2 (Arg2) is a microRNA-155 (miR-155) and interleukin-10 (IL-10) regulated protein localized at the mitochondria in inflammatory macrophages, and is critical for IL-10-induced modulation of mitochondrial dynamics and oxidative respiration. Mechanistically, the catalytic activity and presence of Arg2 at the mitochondria is crucial for oxidative phosphorylation. We further show that Arg2 mediates this process by increasing the activity of complex II (succinate dehydrogenase). Moreover, Arg2 is essential for IL-10-mediated downregulation of the inflammatory mediators succinate, hypoxia inducible factor 1α (HIF-1α) and IL-1ß in vitro. Accordingly, HIF-1α and IL-1ß are highly expressed in an LPS-induced in vivo model of acute inflammation using Arg2-/- mice. These findings shed light on a new arm of IL-10-mediated metabolic regulation, working to resolve the inflammatory status of the cell.

Associations between urinary 3-indoxyl sulfate, a gut microbiome-derived biomarker, and patient outcomes after intensive care unit admission.

PURPOSE: 3-indoxyl sulfate (3-IS) is an indole metabolism byproduct produced by commensal gut bacteria and excreted in the urine; low urinary 3-IS has been associated with increased mortality in bone marrow transplant recipients. This study investigated urinary 3-IS and patient outcomes in the ICU. MATERIALS AND METHODS: Prospective study that collected urine samples, rectal swabs, and clinical data on 78 adult ICU patients at admission and again 72 h later. Urine was analyzed for 3-IS by mass spectrometry. RESULTS: Median urinary 3-IS levels were 17.1 µmol/mmol creatinine (IQR 9.5 to 26.2) at admission and 15.6 (IQR 4.2 to 30.7) 72 h later. 22% of patients had low 3-IS (≤6.9 µmol/mmol) on ICU admission and 28% after 72 h. Low 3-IS at 72 h was associated with fewer ICU-free days (22.5 low versus 26 high, p = 0.03) and with death during one year of follow-up (36% low versus 9% high 3-IS, p < 0.01); there was no detectable difference in 30-day mortality (18% low versus 5% high, p = 0.07). CONCLUSIONS: Low urinary 3-IS level 72 h after ICU admission was associated with fewer ICU-free days and with increased one-year but not 30-day mortality. Further studies should investigate urinary 3-IS as an ICU biomarker.