Targeting Bacteria within Us to Diminish Infection and Autoimmunity.

Antibiotics improve clinical outcomes independent of their antibacterial effects. In this issue of Immunity, Almeida et al. and Colaço et al. demonstrate that antibiotic impairment of mitochondrial ribosomes modulates both T-cell-dependent inflammation and host tolerance to infection.

Hexokinase 1 cellular localization regulates the metabolic fate of glucose.

The product of hexokinase (HK) enzymes, glucose-6-phosphate, can be metabolized through glycolysis or directed to alternative metabolic routes, such as the pentose phosphate pathway (PPP) to generate anabolic intermediates. HK1 contains an N-terminal mitochondrial binding domain (MBD), but its physiologic significance remains unclear. To elucidate the effect of HK1 mitochondrial dissociation on cellular metabolism, we generated mice lacking the HK1 MBD (ΔE1HK1). These mice produced a hyper-inflammatory response when challenged with lipopolysaccharide. Additionally, there was decreased glucose flux below the level of GAPDH and increased upstream flux through the PPP. The glycolytic block below GAPDH is mediated by the binding of cytosolic HK1 with S100A8/A9, resulting in GAPDH nitrosylation through iNOS. Additionally, human and mouse macrophages from conditions of low-grade inflammation, such as aging and diabetes, displayed increased cytosolic HK1 and reduced GAPDH activity. Our data indicate that HK1 mitochondrial binding alters glucose metabolism through regulation of GAPDH.

HRD1-mediated METTL14 degradation regulates m6A mRNA modification to suppress ER proteotoxic liver disease.

Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen triggers an unfolded protein response (UPR) for stress adaptation, the failure of which induces cell apoptosis and tissue/organ damage. The molecular switches underlying how the UPR selects for stress adaptation over apoptosis remain unknown. Here, we discovered that accumulation of unfolded/misfolded proteins selectively induces N6-adenosine-methyltransferase-14 (METTL14) expression. METTL14 promotes C/EBP-homologous protein (CHOP) mRNA decay through its 3' UTR N6-methyladenosine (m6A) to inhibit its downstream pro-apoptotic target gene expression. UPR induces METTL14 expression by competing against the HRD1-ER-associated degradation (ERAD) machinery to block METTL14 ubiquitination and degradation. Therefore, mice with liver-specific METTL14 deletion are highly susceptible to both acute pharmacological and alpha-1 antitrypsin (AAT) deficiency-induced ER proteotoxic stress and liver injury. Further hepatic CHOP deletion protects METTL14 knockout mice from ER-stress-induced liver damage. Our study reveals a crosstalk between ER stress and mRNA m6A modification pathways, termed the ERm6A pathway, for ER stress adaptation to proteotoxicity.

Mitochondrial ubiquinol oxidation is necessary for tumour growth.

The mitochondrial electron transport chain (ETC) is necessary for tumour growth1-6 and its inhibition has demonstrated anti-tumour efficacy in combination with targeted therapies7-9. Furthermore, human brain and lung tumours display robust glucose oxidation by mitochondria10,11. However, it is unclear why a functional ETC is necessary for tumour growth in vivo. ETC function is coupled to the generation of ATP-that is, oxidative phosphorylation and the production of metabolites by the tricarboxylic acid (TCA) cycle. Mitochondrial complexes I and II donate electrons to ubiquinone, resulting in the generation of ubiquinol and the regeneration of the NAD+ and FAD cofactors, and complex III oxidizes ubiquinol back to ubiquinone, which also serves as an electron acceptor for dihydroorotate dehydrogenase (DHODH)-an enzyme necessary for de novo pyrimidine synthesis. Here we show impaired tumour growth in cancer cells that lack mitochondrial complex III. This phenotype was rescued by ectopic expression of Ciona intestinalis alternative oxidase (AOX)12, which also oxidizes ubiquinol to ubiquinone. Loss of mitochondrial complex I, II or DHODH diminished the tumour growth of AOX-expressing cancer cells deficient in mitochondrial complex III, which highlights the necessity of ubiquinone as an electron acceptor for tumour growth. Cancer cells that lack mitochondrial complex III but can regenerate NAD+ by expression of the NADH oxidase from Lactobacillus brevis (LbNOX)13 targeted to the mitochondria or cytosol were still unable to grow tumours. This suggests that regeneration of NAD+ is not sufficient to drive tumour growth in vivo. Collectively, our findings indicate that tumour growth requires the ETC to oxidize ubiquinol, which is essential to drive the oxidative TCA cycle and DHODH activity.

IFN-Induced Protein with Tetratricopeptide Repeats 2 Limits Autoimmune Inflammation by Regulating Myeloid Cell Activation and Metabolic Activity.

Besides antiviral functions, type I IFN expresses potent anti-inflammatory properties and is being widely used to treat certain autoimmune conditions, such as multiple sclerosis. In a murine model of multiple sclerosis, experimental autoimmune encephalomyelitis, administration of IFN-ß effectively attenuates the disease development. However, the precise mechanisms underlying IFN-ß-mediated treatment remain elusive. In this study, we report that IFN-induced protein with tetratricopeptide repeats 2 (Ifit2), a type I and type III IFN-stimulated gene, plays a previously unrecognized immune-regulatory role during autoimmune neuroinflammation. Mice deficient in Ifit2 displayed greater susceptibility to experimental autoimmune encephalomyelitis and escalated immune cell infiltration in the CNS. Ifit2 deficiency was also associated with microglial activation and increased myeloid cell infiltration. We also observed that myelin debris clearance and the subsequent remyelination were substantially impaired in Ifit2-/- CNS tissues. Clearing myelin debris is an important function of the reparative-type myeloid cell subset to promote remyelination. Indeed, we observed that bone marrow-derived macrophages, CNS-infiltrating myeloid cells, and microglia from Ifit2-/- mice express cytokine and metabolic genes associated with proinflammatory-type myeloid cell subsets. Taken together, our findings uncover a novel regulatory function of Ifit2 in autoimmune inflammation in part by modulating myeloid cell function and metabolic activity.

Mitochondrial complex III is essential for suppressive function of regulatory T cells.

Regulatory T cells (Treg cells), a distinct subset of CD4+ T cells, are necessary for the maintenance of immune self-tolerance and homeostasis1,2. Recent studies have demonstrated that Treg cells exhibit a unique metabolic profile, characterized by an increase in mitochondrial metabolism relative to other CD4+ effector subsets3,4. Furthermore, the Treg cell lineage-defining transcription factor, Foxp3, has been shown to promote respiration5,6; however, it remains unknown whether the mitochondrial respiratory chain is required for the T cell-suppression capacity, stability and survival of Treg cells. Here we report that Treg cell-specific ablation of mitochondrial respiratory chain complex III in mice results in the development of fatal inflammatory disease early in life, without affecting Treg cell number. Mice that lack mitochondrial complex III specifically in Treg cells displayed a loss of T cell-suppression capacity without altering Treg cell proliferation and survival. Treg cells deficient in complex III showed decreased expression of genes associated with Treg function, whereas Foxp3 expression remained stable. Loss of complex III in Treg cells increased DNA methylation as well as the metabolites 2-hydroxyglutarate (2-HG) and succinate that inhibit the ten-eleven translocation (TET) family of DNA demethylases7. Thus, Treg cells require mitochondrial complex III to maintain immune regulatory gene expression and suppressive function.

Mitochondria in the regulation of innate and adaptive immunity.

Mitochondria are well appreciated for their role as biosynthetic and bioenergetic organelles. In the past two decades, mitochondria have emerged as signaling organelles that contribute critical decisions about cell proliferation, death, and differentiation. Mitochondria not only sustain immune cell phenotypes but also are necessary for establishing immune cell phenotype and their function. Mitochondria can rapidly switch from primarily being catabolic organelles generating ATP to anabolic organelles that generate both ATP and building blocks for macromolecule synthesis. This enables them to fulfill appropriate metabolic demands of different immune cells. Mitochondria have multiple mechanisms that allow them to activate signaling pathways in the cytosol including altering in AMP/ATP ratio, the release of ROS and TCA cycle metabolites, as well as the localization of immune regulatory proteins on the outer mitochondrial membrane. In this Review, we discuss the evidence and mechanisms that mitochondrial dependent signaling controls innate and adaptive immune responses.

TCA Cycle and Mitochondrial Membrane Potential Are Necessary for Diverse Biological Functions.

Mitochondrial metabolism is necessary for the maintenance of oxidative TCA cycle function and mitochondrial membrane potential. Previous attempts to decipher whether mitochondria are necessary for biological outcomes have been hampered by genetic and pharmacologic methods that simultaneously disrupt multiple functions linked to mitochondrial metabolism. Here, we report that inducible depletion of mitochondrial DNA (ρ(ο) cells) diminished respiration, oxidative TCA cycle function, and the mitochondrial membrane potential, resulting in diminished cell proliferation, hypoxic activation of HIF-1, and specific histone acetylation marks. Genetic reconstitution only of the oxidative TCA cycle function specifically in these inducible ρ(ο) cells restored metabolites, resulting in re-establishment of histone acetylation. In contrast, genetic reconstitution of the mitochondrial membrane potential restored ROS, which were necessary for hypoxic activation of HIF-1 and cell proliferation. These results indicate that distinct mitochondrial functions associated with respiration are necessary for cell proliferation, epigenetics, and HIF-1 activation.

Futility sustains memory T cells.

Memory T cells display a distinct metabolic profile compared to effector T cells. In this issue of Immunity, O'Sullivan et al. (2014) report that memory T cells activate a "futile cycle" of de novo fatty-acid synthesis and concurrent fatty-acid oxidation to generate ATP for cell survival.

IL-17A mediates pyroptosis via the ERK pathway and contributes to steroid resistance in CRSwNP.

BACKGROUND: Pyroptosis is closely related to inflammation. However, the molecular mechanisms and pathologic contributions of pyroptotic epithelial cell are not yet fully understood. OBJECTIVE: This study aimed to explore the function and molecular mechanisms of IL-17A on human nasal epithelial cell (hNEC) pyroptosis. METHODS: The expression of pyroptosis-related biomarkers and IL-17A was assessed in sinonasal mucosa from control individuals, patients with chronic rhinosinusitis without nasal polyps, and patients with chronic rhinosinusitis with nasal polyps (CRSwNP) by using quantitative RT-PCR. Their localization was analyzed via immunohistochemistry and immunofluorescence. The ultrastructural characteristics of IL-17A-induced pyroptosis in hNECs were visualized by using electron microscopy. IL-17A functional assays were performed on hNECs and airway epithelial cell lines. Cytokine levels were quantified via ELISA. The signaling pathways involved in IL-17A-induced pyroptosis were studied via unbiased RNA sequencing and Western blotting. RESULTS: The expression of IL-17A and the pyroptotic biomarkers NOD-like receptor family, pyrin domain containing 3 (NLRP3), caspase-1, gasdermin D, and IL-1ß was increased in nasal mucosa from patients with CRSwNP compared with in those with chronic rhinosinusitis without nasal polyps and the control subjects. IL-17A was positively correlated and colocalized with the pyroptotic biomarkers. IL-17A treatment induced pyroptosis in the hNECs and cell lines analyzed, primarily through the extracellular signal-regulated kinase (ERK)-NLRP3/caspase-1 signaling pathway, and increased IL-1ß and IL-18 secretion in hNECs. Moreover, IL-17A-induced pyroptosis contributed to steroid resistance by affecting glucocorticoid receptor-α and glucocorticoid receptor-ß expression, and the inhibition of pyroptotic proteins partially abolished IL-17A-induced steroid resistance in hNECs. CONCLUSION: Elevated IL-17A level promotes pyroptosis in hNECs through the ERK-NLRP3/caspase-1 signaling pathway and contributes to glucocorticoid resistance by affecting glucocorticoid receptor homeostasis in patients with CRSwNP.

Overview of Inositol and Inositol Phosphates on Chemoprevention of Colitis-Induced Carcinogenesis.

Chronic inflammation is one of the most common and well-recognized risk factors for human cancer, including colon cancer. Inflammatory bowel disease (IBD) is defined as a longstanding idiopathic chronic active inflammatory process in the colon, including ulcerative colitis and Crohn's disease. Importantly, patients with IBD have a significantly increased risk for the development of colorectal carcinoma. Dietary inositol and its phosphates, as well as phospholipid derivatives, are well known to benefit human health in diverse pathologies including cancer prevention. Inositol phosphates including InsP3, InsP6, and other pyrophosphates, play important roles in cellular metabolic and signal transduction pathways involved in the control of cell proliferation, differentiation, RNA export, DNA repair, energy transduction, ATP regeneration, and numerous others. In the review, we highlight the biologic function and health effects of inositol and its phosphates including the nature and sources of these molecules, potential nutritional deficiencies, their biologic metabolism and function, and finally, their role in the prevention of colitis-induced carcinogenesis.

Metabolism and Skeletal Muscle Homeostasis in Lung Disease.

There is increased awareness that patients with lung diseases develop muscle dysfunction. Muscle dysfunction is a major contributor to a decreased quality of life in patients with chronic pulmonary diseases. Furthermore, muscle dysfunction exacerbates lung disease outcome, as a decrease in muscle mass and function are associated with increased morbidity, often long after critical illness or lung disease has been resolved. As we are learning more about the role of metabolism in health and disease, we are appreciating more the direct role of metabolism in skeletal muscle homeostasis. Altered metabolism is associated with numerous skeletal muscle pathologies and, conversely, skeletal muscle diseases are associated with significant changes in metabolic pathways. In this review, we highlight the role of metabolism in the regulation of skeletal muscle homeostasis. Understanding the metabolic pathways that underlie skeletal muscle wasting is of significant clinical interest for critically ill patients as well as patients with chronic lung disease, in which proper skeletal muscle function is essential to disease outcome.

Targeting mitochondria metabolism for cancer therapy.

Mitochondria have a well-recognized role in the production of ATP and the intermediates needed for macromolecule biosynthesis, such as nucleotides. Mitochondria also participate in the activation of signaling pathways. Overall, accumulating evidence now suggests that mitochondrial bioenergetics, biosynthesis and signaling are required for tumorigenesis. Thus, emerging studies have begun to demonstrate that mitochondrial metabolism is potentially a fruitful arena for cancer therapy. In this Perspective, we highlight recent developments in targeting mitochondrial metabolism for the treatment of cancer.

Acidity induces durable enhancement of Treg cell suppressive functions for tumor immune evasion.

The microenvironment within solid tumors often becomes acidic due to various factors associated with abnormal metabolism and cellular activities, including increased lactate production as a result of dysregulated tumor glycolysis. Recently, we have identified multiple tumor microenvironment (TME) factors that potentiate regulatory T (Treg) cell function in evading anti-tumor immunosurveillance. Despite the strong correlation between lactate and acidity, the potential roles of acidity in intratumoral Treg cell adaptation and underlying molecular mechanisms have gone largely unstudied. In this study, we demonstrate that acidity significantly enhances immunosuppressive functions of nTreg cells, but not iTreg cells, without altering the expression of either FoxP3 or the cell surface receptors CD25, CTLA4, or GITR in these cells. Surprisingly, the addition of lactate, often considered a major contributor to increased acidity of the TME, completely abolished the acidity-induced enhancement of nTreg suppressive functions. Consistently, metabolic flux analyses showed elevated basal mitochondrial respiratory capacity and ATP-coupled respiration in acidity-treated nTreg cells without altering glycolytic capacity. Genome-wide transcriptome and metabolomics analyses revealed alterations in multiple metabolic pathways, particularly the one-carbon folate metabolism pathway, with reduced SAM, folate, and glutathione, in nTreg cells exposed to low pH conditions. Addition of a one-carbon metabolic contributor, formate, diminished the acidity-induced enhancement in nTreg cell suppressive functions, but neither SAM nor glutathione could reverse the phenotype. Remarkably, in vitro transient treatment of nTreg cells resulted in sustained enhancement of their functions, as evidenced by more vigorous tumor growth observed in mice adoptively receiving acidity-treated nTreg cells. Further analysis of intratumoral infiltrated T cells confirmed a significant reduction in CD8+ T cell frequency and their granzyme B production. In summary, our study elucidates how acidity-mediated metabolic reprogramming leads to sustained Treg-mediated tumor immune evasion.

Characterization of hyperpolarization-activated cyclic nucleotide-gated channels in oligodendrocytes.

Mature oligodendrocytes (OLG) are the myelin-forming cells of the central nervous system. Recent work has shown a dynamic role for these cells in the plasticity of neural circuits, leading to a renewed interest in voltage-sensitive currents in OLG. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and their respective current (Ih) were recently identified in mature OLG and shown to play a role in regulating myelin length. Here we provide a biochemical and electrophysiological characterization of HCN channels in cells of the oligodendrocyte lineage. We observed that mice with a nonsense mutation in the Hcn2 gene (Hcn2ap/ap) have less white matter than their wild type counterparts with fewer OLG and fewer oligodendrocyte progenitor cells (OPCs). Hcn2ap/ap mice have severe motor impairments, although these deficits were not observed in mice with HCN2 conditionally eliminated only in oligodendrocytes (Cnpcre/+; Hcn2F/F). However, Cnpcre/+; Hcn2F/F mice develop motor impairments more rapidly in response to experimental autoimmune encephalomyelitis (EAE). We conclude that HCN2 channels in OLG may play a role in regulating metabolism.

Mitochondrial respiration in microglia is essential for response to demyelinating injury but not proliferation.

Microglia are necessary for central nervous system (CNS) function during development and play roles in ageing, Alzheimer's disease and the response to demyelinating injury1-5. The mitochondrial respiratory chain (RC) is necessary for conventional T cell proliferation6 and macrophage-dependent immune responses7-10. However, whether mitochondrial RC is essential for microglia proliferation or function is not known. We conditionally deleted the mitochondrial complex III subunit Uqcrfs1 (Rieske iron-sulfur polypeptide 1) in the microglia of adult mice to assess the requirement of microglial RC for survival, proliferation and adult CNS function in vivo. Notably, mitochondrial RC function was not required for survival or proliferation of microglia in vivo. RNA sequencing analysis showed that loss of RC function in microglia caused changes in gene expression distinct from aged or disease-associated microglia. Microglia-specific loss of mitochondrial RC function is not sufficient to induce cognitive decline. Amyloid-ß plaque coverage decreased and microglial interaction with amyloid-ß plaques increased in the hippocampus of 5xFAD mice with mitochondrial RC-deficient microglia. Microglia-specific loss of mitochondrial RC function did impair remyelination following an acute, reversible demyelinating event. Thus, mitochondrial respiration in microglia is dispensable for proliferation but is essential to maintain a proper response to CNS demyelinating injury.

Mediator complex subunit 1 architects a tumorigenic Treg cell program independent of inflammation.

While immunotherapy has revolutionized cancer treatment, its safety has been hampered by immunotherapy-related adverse events. Unexpectedly, we show that Mediator complex subunit 1 (MED1) is required for T regulatory (Treg) cell function specifically in the tumor microenvironment. Treg cell-specific MED1 deletion does not predispose mice to autoimmunity or excessive inflammation. In contrast, MED1 is required for Treg cell promotion of tumor growth because MED1 is required for the terminal differentiation of effector Treg cells in the tumor. Suppression of these terminally differentiated Treg cells is sufficient for eliciting antitumor immunity. Both human and murine Treg cells experience divergent paths of differentiation in tumors and matched tissues with non-malignant inflammation. Collectively, we identify a pathway promoting the differentiation of a Treg cell effector subset specific to tumors and demonstrate that suppression of a subset of Treg cells is sufficient for promoting antitumor immunity in the absence of autoimmune consequences.

AMP-activated protein kinase is necessary for Treg cell functional adaptation to microenvironmental stress.

CD4+FOXP3+ regulatory T (Treg) cells maintain self-tolerance, suppress the immune response to cancer, and protect against tissue injury in the lung and other organs. Treg cells require mitochondrial metabolism to exert their function, but how Treg cells adapt their metabolic programs to sustain and optimize their function during an immune response occurring in a metabolically stressed microenvironment remains unclear. Here, we tested whether Treg cells require the energy homeostasis-maintaining enzyme AMP-activated protein kinase (AMPK) to adapt to metabolically aberrant microenvironments caused by malignancy or lung injury, finding that AMPK is dispensable for Treg cell immune-homeostatic function but is necessary for full Treg cell function in B16 melanoma tumors and during acute lung injury caused by influenza virus pneumonia. AMPK-deficient Treg cells had lower mitochondrial mass and exhibited an impaired ability to maximize aerobic respiration. Mechanistically, we found that AMPK regulates DNA methyltransferase 1 to promote transcriptional programs associated with mitochondrial function in the tumor microenvironment. In the lung during viral pneumonia, we found that AMPK sustains metabolic homeostasis and mitochondrial activity. Induction of DNA hypomethylation was sufficient to rescue mitochondrial mass in AMPK-deficient Treg cells, linking DNA methylation with AMPK function and mitochondrial metabolism. These results define AMPK as a determinant of Treg cell adaptation to metabolic stress and offer potential therapeutic targets in cancer and tissue injury.

[WITHDRAWN] Hexokinase-1 mitochondrial dissociation and protein O-GlcNAcylation drive heart failure with preserved ejection fraction.

The authors have requested that this preprint be removed from Research Square.