Hypoxia Routes Tryptophan Homeostasis Towards Increased Tryptamine Production

The liver is the central hub for processing and maintaining homeostatic levels of dietary nutrients especially essential amino acids such as tryptophan (Trp). Trp is required not only to sustain protein synthesis but also as a precursor for the production of NAD, neurotransmitters and immunosuppressive metabolites. In light of these roles of Trp and its metabolic products, maintaining homeostatic levels of Trp is essential for health and well-being. The liver regulates global Trp supply by the immunosuppressive enzyme tryptophan-2,3-dioxygenase (TDO2), which degrades Trp down the kynurenine pathway (KP). In the current study, we show that isolated primary hepatocytes when exposed to hypoxic environments, extensively rewire their Trp metabolism by reducing constitutive Tdo2 expression and differentially regulating other Trp pathway enzymes and transporters. Mathematical modelling of Trp metabolism in liver cells under hypoxia predicted decreased flux through the KP while metabolic flux through the tryptamine branch significantly increased. In line, the model also revealed an increased accumulation of tryptamines under hypoxia, at the expense of kynurenines. Metabolic measurements in hypoxic hepatocytes confirmed the predicted reduction in KP metabolites as well as accumulation of tryptamine. Tdo2 expression in cultured primary hepatocytes was reduced upon hypoxia inducible factor (HIF) stabilisation by dimethyloxalylglycine (DMOG), demonstrating that HIFs are involved in the hypoxic downregulation of hepatic Tdo2. DMOG abrogated hepatic luciferase signals in Tdo2 reporter mice, indicating that HIF stability also recapitulates hypoxic rewiring of Trp metabolism in vivo. Also in WT mice HIF stabilization drove homeostatic Trp metabolism away from the KP towards enhanced tryptamine production, leading to enhanced levels of tryptamine in liver, serum and brain. As tryptamines are the most potent hallucinogens known, the observed upregulation of tryptamine in response to hypoxic exposure of hepatocytes may be involved in the generation of hallucinations occurring at high altitude. KP metabolites are known to activate the aryl hydrocarbon receptor (AHR). The AHR-activating properties of tryptamines may explain why immunosuppressive AHR activity is maintained under hypoxia despite downregulation of the KP. In summary our results identify hypoxia as an important factor controlling Trp metabolism in the liver with possible implications for immunosuppressive AHR activation and mental disturbances

Mouse mesenchymal stem cells suppress antigen-specific TH-cell immunity independent of indoleamine 2,3-dioxygenase 1 (IDO1)

Due to their immunosuppressive properties human mesenchymal stem cells (hMSC) represent a promising tool for cell-based therapies of autoimmune diseases such as multiple sclerosis (MS). Mouse MSC (mMSC) have been used extensively to characterize and optimize route of administration, motility, cellular targets and immunosuppressive mechanisms in mouse models of autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE). Tryptophan (trp) catabolism by indolamine-2,3-dioxygenase 1 (IDO1) is a chief endogenous metabolic pathway that tightly regulates unwanted immune responses through depletion of trp and generation of immunosuppressive kynurenines (kyn). IDO1 activity contributes to the immunosuppressive phenotype of hMSC. Here we demonstrate that although IDO1 is inducible in bone marrow-derived mMSC by proinflammatory stimuli such as interferon-gamma (IFN-gamma) and ligands of toll-like receptors (TLR), it does not lead to catabolism of trp in vitro. This failure to catabolize trp is not due to defective TLR signaling as demonstrated by induction of interleukin 6 (IL-6) by TLR activation. While mMSC suppressed the activation of antigen-specific myelin-oligodendrocyte glycoprotein (MOG)-reactive T cell receptor (TCR) transgenic T helper (TH) cells in coculture, neither pharmacologic inhibition nor genetic ablation of IDO1 reversed this suppressive effect. Finally, systemic administration of both, IDO1-proficient and phenotypically identical IDO1-deficient mMSC equally resulted in amelioration of EAE. mMSC, unlike hMSC, do not display IDO1-mediated suppression of antigen-specific T cell responses

Hepatocyte-intrinsic type I interferon signaling reprograms metabolism and reveals a novel compensatory mechanism of the tryptophan-kynurenine pathway in viral hepatitis.

The liver is a central regulator of metabolic homeostasis and serum metabolite levels. Hepatocytes are the functional units of the liver parenchyma and not only responsible for turnover of biomolecules but also act as central immune signaling platforms. Hepatotropic viruses infect liver tissue, resulting in inflammatory responses, tissue damage and hepatitis. Combining well-established in vitro and in vivo model systems with transcriptomic analyses, we show that type I interferon signaling initiates a robust antiviral immune response in hepatocytes. Strikingly, we also identify IFN-I as both, sufficient and necessary, to induce wide-spread metabolic reprogramming in hepatocytes. IFN-I specifically rewired tryptophan metabolism and induced hepatic tryptophan oxidation to kynurenine via Tdo2, correlating with altered concentrations of serum metabolites upon viral infection. Infected Tdo2-deficient animals displayed elevated serum levels of tryptophan and, unexpectedly, also vast increases in the downstream immune-suppressive metabolite kynurenine. Thus, Tdo2-deficiency did not result in altered serum homeostasis of the tryptophan to kynurenine ratio during infection, which seemed to be independent of hepatocyte-intrinsic compensation via the IDO-axis. These data highlight that inflammation-induced reprogramming of systemic tryptophan metabolism is tightly regulated in viral hepatitis

Autoantibodies against central nervous system antigens in a subset of B cell-dominant multiple sclerosis patients

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS), with characteristic inflammatory lesions and demyelination. The clinical benefit of cell-depleting therapies targeting CD20 has emphasized the role of B cells and autoantibodies in MS pathogenesis. We previously introduced an enzyme-linked immunospot spot (ELISpot)-based assay to measure CNS antigen-specific B cells in the blood of MS patients and demonstrated its usefulness as a predictive biomarker for disease activity in measuring the successful outcome of disease-modifying therapies (DMTs). Here we used a planar protein array to investigate CNS-reactive antibodies in the serum of MS patients as well as in B cell culture supernatants after polyclonal stimulation. AntiCNS antibody reactivity was evident in the sera of the MS cohort, and the antibodies bound a heterogeneous set of molecules, including myelin, axonal cytoskeleton, and ion channel antigens, in individual patients. Immunoglobulin reactivity in supernatants of stimulated B cells was directed against a broad range of CNS antigens. A group of MS patients with a highly active B cell component was identified by the ELISpot assay. Those antibody reactivities remained stable over time. These assays with protein arrays identify MS patients with a highly active B cell population with antibodies directed against a swathe of CNS proteins

Mouse Mesenchymal Stem Cells Suppress Antigen-Specific TH Cell Immunity Independent of Indoleamine 2,3-Dioxygenase 1 (IDO1)

Due to their immunosuppressive properties, human mesenchymal stem cells (hMSC) represent a promising tool for cell-based therapies of autoimmune diseases such as multiple sclerosis (MS). Mouse MSC (mMSC) have been used extensively to characterize and optimize route of administration, motility, cellular targets, and immunosuppressive mechanisms in mouse models of autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE). Tryptophan (trp) catabolism by indolamine-2,3-dioxygenase 1 (IDO1) is a chief endogenous metabolic pathway that tightly regulates unwanted immune responses through depletion of trp and generation of immunosuppressive kynurenines (kyn). IDO1 activity contributes to the immunosuppressive phenotype of hMSC. Here, we demonstrate that although IDO1 is inducible in bone marrow-derived mMSC by proinflammatory stimuli such as interferon-g (IFN-g) and ligands of toll-like receptors (TLR), it does not lead to catabolism of trp in vitro. This failure to catabolize trp is not due to defective TLR signaling as demonstrated by induction of interleukin 6 (IL-6) by TLR activation. While mMSC suppressed the activation of antigen-specific myelin oligodendrocyte glycoprotein (MOG)-reactive T-cell receptor (TCR) transgenic T-helper (TH) cells in co-culture, neither pharmacologic inhibition nor genetic ablation of IDO1 reversed this suppressive effect. Finally, systemic administration of both, IDO1-proficient and phenotypically identical IDO1-deficient mMSC, equally resulted in amelioration of EAE. mMSC, unlike hMSC, do not display IDO1-mediated suppression of antigen-specific T-cell responses

Angiotensin II sustains brain inflammation in mice via TGF-β

The renin-angiotensin-aldosterone system (RAAS) is a key hormonal system regulating blood pressure. However, expression of RAAS components has recently been detected in immune cells, and the RAAS has been implicated in several mouse models of autoimmune disease. Here, we have identified Ang II as a paracrine mediator, sustaining inflammation in the CNS in the EAE mouse model of MS via TGF-β. Ang II type 1 receptors (AT1Rs) were found to be primarily expressed in CNS-resident cells during EAE. In vitro, astrocytes and microglia responded to Ang II treatment by inducing TGF-β expression via a pathway involving the TGF-β–activating protease thrombospondin-1 (TSP-1). TGF-β upregulation in astrocytes and microglia during EAE was blocked with candesartan (CA), an inhibitor of AT1R. Treatment of EAE with CA ameliorated paralysis and blunted lymphocyte infiltration into the CNS, outcomes that were also seen with genetic ablation of AT1Ra and treatment with an inhibitor of TSP-1. These data suggest that AT1R antagonists, frequently prescribed as antihypertensives, may be useful to interrupt this proinflammatory, CNS-specific pathway in individuals with MS

Oral mucosal breaks trigger anti-citrullinated bacterial and human protein antibody responses in rheumatoid arthritis.

Periodontal disease is more common in individuals with rheumatoid arthritis (RA) who have detectable anti-citrullinated protein antibodies (ACPAs), implicating oral mucosal inflammation in RA pathogenesis. Here, we performed paired analysis of human and bacterial transcriptomics in longitudinal blood samples from RA patients. We found that patients with RA and periodontal disease experienced repeated oral bacteremias associated with transcriptional signatures of ISG15+HLADRhi and CD48highS100A2pos monocytes, recently identified in inflamed RA synovia and blood of those with RA flares. The oral bacteria observed transiently in blood were broadly citrullinated in the mouth, and their in situ citrullinated epitopes were targeted by extensively somatically hypermutated ACPAs encoded by RA blood plasmablasts. Together, these results suggest that (i) periodontal disease results in repeated breaches of the oral mucosa that release citrullinated oral bacteria into circulation, which (ii) activate inflammatory monocyte subsets that are observed in inflamed RA synovia and blood of RA patients with flares and (iii) activate ACPA B cells, thereby promoting affinity maturation and epitope spreading to citrullinated human antigens