Microbial metabolites, including B complex vitamins contribute to diverse aspects of human health. Folate, or vitamin B9, refers to a broad category of biomolecules that include pterin, para-aminobenzoic acid (pABA), and glutamate subunits. Folates are required for DNA synthesis and epigenetic regulation. In addition to dietary nutrients, the gut microbiota has been recognized as a source of B complex vitamins, including folate. This study evaluated the predicted folate synthesis capabilities in the genomes of human commensal microbes identified in the Human Microbiome Project and folate production by representative strains of six human intestinal bacterial phyla. Bacterial folate synthesis genes were ubiquitous across 512 gastrointestinal reference genomes with 13% of the genomes containing all genes required for complete de novo folate synthesis. An additional 39% of the genomes had the genetic capacity to synthesize folates in the presence of pABA, an upstream intermediate that can be obtained through diet or from other intestinal microbes. Bacterial folate synthesis was assessed during exponential and stationary phase growth through the evaluation of expression of select folate synthesis genes, quantification of total folate production, and analysis of folate polyglutamylation. Increased expression of key folate synthesis genes was apparent in exponential phase, and increased folate polyglutamylation occurred during late stationary phase. Of the folate producers, we focused on the commensal Lactobacillus reuteri to examine host–microbe interactions in relation to folate and examined folate receptors in the physiologically relevant human enteroid model. RNAseq data revealed segment-specific folate receptor distribution. Treatment of human colonoid monolayers with conditioned media (CM) from wild-type L. reuteri did not influence the expression of key folate transporters proton-coupled folate transporter (PCFT) or reduced folate carrier (RFC). However, CM from L. reuteri containing a site-specific inactivation of the folC gene, which prevents the bacteria from synthesizing a polyglutamate tail on folate, significantly upregulated RFC expression. No effects were observed using L. reuteri with a site inactivation of folC2, which results in no folate production. This work sheds light on the contributions of microbial folate to overall folate status and mammalian host metabolism.
Mitochondrial reactive oxygen species (ROS) are indispensible for T cell activation-induced expression of interleukin 2 (IL-2) and CD95 ligand (CD95L, FasL/Apo-1L) genes, and in turn, for CD95L-mediated activation-induced cell death (AICD). Here, we show that manganese superoxide dismutase (MnSOD/SOD2), a major mitochondrial antioxidative enzyme, constitutes an important control switch in the process of activation-induced oxidative signal generation in T cells. Analysis of the kinetics of T cell receptor (TCR)-triggered ROS production revealed a temporal association between higher MnSOD abundance/activity and a shut-down phase of oxidative signal generation. Transient or inducible MnSOD overexpression abrogated T cell activation-triggered mitochondrial ROS production as well as NF-κB- and AP-1-mediated transcription. Consequently, lowered expression of IL-2 and CD95L genes resulted in decreased IL-2 secretion and CD95L-dependent AICD. Moreover, upregulation of the mitochondrial MnSOD level is dependent on oxidation-sensitive transcription and not on the increase of mitochondrial mass. Thus, MnSOD-mediated negative feedback regulation of activation-induced mitochondrial ROS generation exemplifies a process of retrograde mitochondria-to-nucleus communication. Our finding underlines the critical role for MnSOD and mitochondria in the regulation of human T cell activation.
Early scientific reports limited the cell biological role of reactive oxygen species (ROS)
Edited by Quan Chen Keywords:T cell Interleukin Reactive oxygen species (ROS) Mitochondrial dynamics Dynamin related protein 1 (Drp1) Apoptosis Activation induced T cell death (AICD) CD95L/FASL a b s t r a c tIn T cells mitochondria-derived reactive oxygen species (ROS) are indispensible for activation of the transcription factor NF-jB, expression of cytokines and the CD95 ligand (CD95L/FasL). Here we show that activation-induced ROS generation is dependent on mitochondrial fission. Inhibition of dynamin related protein 1 (Drp1) results in reduced ROS levels and transcriptional activity of NF-jB leading to diminished proliferation and CD95L-dependent activation-induced cell death (AICD). Upon stimulation Drp1 is S-nitrosylated, which is required for oxidative signalling, AICD and cytokine production. In conclusion, we describe a novel signalling pathway that links TCR-induced nitric oxide release to mitochondrial fission and oxidative signalling.
The “free radical theory of aging” suggests that reactive oxygen species (ROS) are responsible for age‐related loss of cellular functions and, therefore, represent the main cause of aging. Redox regulation by thioredoxin‐1 (TRX) plays a crucial role in responses to oxidative stress. We show that thioredoxin‐interacting protein (TXNIP), a negative regulator of TRX, plays a major role in maintaining the redox status and, thereby, influences aging processes. This role of TXNIP is conserved from flies to humans. Age‐dependent upregulation of TXNIP results in decreased stress resistance to oxidative challenge in primary human cells and in Drosophila. Experimental overexpression of TXNIP in flies shortens lifespan due to elevated oxidative DNA damage, whereas downregulation of TXNIP enhances oxidative stress resistance and extends lifespan.
Colonization of the gut by certain probiotic Lactobacillus reuteri strains has been associated with reduced risk of inflammatory diseases and colorectal cancer. Previous studies pointed to a functional link between immunomodulation, histamine production, and folate metabolism, the central 1‐carbon pathway for the transfer of methyl groups. Using mass spectrometry and NMR spectroscopy, we analyzed folate metabolites of L. reuteri strain 6475 and discovered that the bacterium produces a 2‐carbon‐transporting folate in the form of 5, 10‐ethenyl‐tetrahydrofolyl polyglutamate. Isotopic labeling permitted us to trace the source of the 2‐carbon unit back to acetate of the culture medium. We show that the 2C folate cycle of L. reuteri is capable of transferring 2 carbon atoms to homocysteine to generate the unconventional amino acid ethionine, a known immunomodulator. When we treated monocytic THP‐1 cells with ethionine, their transcription of TNF‐α was inhibited and cell proliferation reduced. Mass spectrometry of THP‐1 histones revealed incorporation of ethionine instead of methionine into proteins, a reduction of histone‐methylation, and ethylation of histone lysine residues. Our findings suggest that the microbiome can expose the host to ethionine through a novel 2‐carbon transporting variant of the folate cycle and modify human chromatin via ethylation.—Roth, D., Chiang, A. J., Hu, W., Gugiu, G. B., Morra, C. N., Versalovic, J., Kalkum, M. The two‐carbon folate cycle of commensal Lactobacillus reuteri 6475 gives rise to immunomodulatory ethionine, a source for histone ethylation. FASEB J. 33,3536‐3548 (2019). http://www.fasebj.org
Constitutively active NFκB promotes survival of many cancers, especially T-cell lymphomas and leukemias by upregulating antiapoptotic proteins such as inhibitors of apoptosis (IAPs) and FLICE-like inhibitory proteins (cFLIPs).Nuclear factor-κ B (NFκ B) is a central transcription factor orchestrating innate and adaptive immune responses. In acute inflammation, NFκ B activity is tightly regulated. However, aberrantly activated NFκ B is associated with chronic inflammatory diseases and a variety of human cancers including both solid and hematopoietic malignancies. Cancers such as T-cell acute lymphoblastic leukemia (T-ALL), cutaneous T-cell lymphoma (CTCL), and its leukemic variant, Sézary Syndrome, revealed constitutive NFκ B activity [1][2][3][4] .The NFκ B family consists of five Rel related proteins: RelA (p65), RelB, cRel, p50 and p52, which can form both homo-and heterodimers. The typical NFκ B complex is a p65/p50 heterodimer critical for NFκ B mediated anti-apoptotic effects 5 . In its inactive form, NFκ B is sequestered in the cytoplasm by Iκ Bα . Phosphorylation and proteasomal degradation of Iκ Bα releases NFκ B. Subsequent nuclear translocation and full activation of NFκ B is redox-dependent and mediated by phosphorylation 6 . The redox regulator Thioredoxin-1 (Trx1) promotes DNA binding activity of NFκ B by reduction of a cysteine residue within its DNA binding domain 7,8 . During oncogenesis, NFκ B promotes cell survival and proliferation by inducing expression of molecules associated with suppression of programmed cell death (PCD), such as cFLIPs 9 , IAP proteins 6,10 , and members of the Bcl-2 family 11 . PCD is a mechanism of tumor suppression and manifests itself in, e.g. apoptosis and necroptosis. Necroptosis is a form of regulated necrosis, which has been implicated to trigger strong immune responses by release of damage-associated molecular patterns (DAMPs) 12 . Moreover, necroptosis is critical for T-cell homeostasis as backup to eliminate an excess of activated T-cells after clonal expansion preventing autoimmunity 13 .The ripoptosome is a signaling platform triggering cell death in an apoptotic or necroptotic manner [14][15][16] . The core components of the ripoptosome include caspase-8, FADD (Fas-associated death domain) and RIPK1 (Receptor-interacting kinase 1). Formation and activation of the ripoptosome are negatively regulated by IAPs (cIAP1, cIAP2 and XIAP) and cFLIPs (cFLIP L and cFLIP S ), respectively. IAPs are regulated by Smac (Second
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