The TNF Family of Ligands and Receptors: Communication Modules in the Immune System and Beyond.

The tumor necrosis factor (TNF) and TNF receptor (TNFR) superfamilies (TNFSF/TNFRSF) include 19 ligands and 29 receptors that play important roles in the modulation of cellular functions. The communication pathways mediated by TNFSF/TNFRSF are essential for numerous developmental, homeostatic, and stimulus-responsive processes in vivo. TNFSF/TNFRSF members regulate cellular differentiation, survival, and programmed death, but their most critical functions pertain to the immune system. Both innate and adaptive immune cells are controlled by TNFSF/TNFRSF members in a manner that is crucial for the coordination of various mechanisms driving either co-stimulation or co-inhibition of the immune response. Dysregulation of these same signaling pathways has been implicated in inflammatory and autoimmune diseases, highlighting the importance of their tight regulation. Investigation of the control of TNFSF/TNFRSF activities has led to the development of therapeutics with the potential to reduce chronic inflammation or promote anti-tumor immunity. The study of TNFSF/TNFRSF proteins has exploded over the last 30 yr, but there remains a need to better understand the fundamental mechanisms underlying the molecular pathways they mediate to design more effective anti-inflammatory and anti-cancer therapies.

Metabolism and epigenetics at the heart of T cell function.

T cell subsets adapt and rewire their metabolism according to their functions and surrounding microenvironment. Whereas naive T cells rely on mitochondrial metabolic pathways characterized by low nutrient requirements, effector T cells induce kinetically faster pathways to generate the biomass and energy needed for proliferation and cytokine production. Recent findings support the concept that alterations in metabolism also affect the epigenetics of T cells. In this review we discuss the connections between T cell metabolism and epigenetic changes such as histone post-translational modifications (PTMs) and DNA methylation, as well as the 'extra-metabolic' roles of metabolic enzymes and molecules. These findings collectively point to a new group of potential therapeutic targets for the treatment of T cell-dependent autoimmune diseases and cancers.

Glutathione Primes T Cell Metabolism for Inflammation.

Activated T cells produce reactive oxygen species (ROS), which trigger the antioxidative glutathione (GSH) response necessary to buffer rising ROS and prevent cellular damage. We report that GSH is essential for T cell effector functions through its regulation of metabolic activity. Conditional gene targeting of the catalytic subunit of glutamate cysteine ligase (Gclc) blocked GSH production specifically in murine T cells. Gclc-deficient T cells initially underwent normal activation but could not meet their increased energy and biosynthetic requirements. GSH deficiency compromised the activation of mammalian target of rapamycin-1 (mTOR) and expression of NFAT and Myc transcription factors, abrogating the energy utilization and Myc-dependent metabolic reprogramming that allows activated T cells to switch to glycolysis and glutaminolysis. In vivo, T-cell-specific ablation of murine Gclc prevented autoimmune disease but blocked antiviral defense. The antioxidative GSH pathway thus plays an unexpected role in metabolic integration and reprogramming during inflammatory T cell responses.

MondoA drives malignancy in B-ALL through enhanced adaptation to metabolic stress.

Cancer cells are in most instances characterized by rapid proliferation and uncontrolled cell division. Hence, they must adapt to proliferation-induced metabolic stress through intrinsic or acquired antimetabolic stress responses to maintain homeostasis and survival. One mechanism to achieve this is reprogramming gene expression in a metabolism-dependent manner. MondoA (also known as Myc-associated factor X-like protein X-interacting protein [MLXIP]), a member of the MYC interactome, has been described as an example of such a metabolic sensor. However, the role of MondoA in malignancy is not fully understood and the underlying mechanism in metabolic responses remains elusive. By assessing patient data sets, we found that MondoA overexpression is associated with worse survival in pediatric common acute lymphoblastic leukemia (ALL; B-precursor ALL [B-ALL]). Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and RNA-interference approaches, we observed that MondoA depletion reduces the transformational capacity of B-ALL cells in vitro and dramatically inhibits malignant potential in an in vivo mouse model. Interestingly, reduced expression of MondoA in patient data sets correlated with enrichment in metabolic pathways. The loss of MondoA correlated with increased tricarboxylic acid cycle activity. Mechanistically, MondoA senses metabolic stress in B-ALL cells by restricting oxidative phosphorylation through reduced pyruvate dehydrogenase activity. Glutamine starvation conditions greatly enhance this effect and highlight the inability to mitigate metabolic stress upon loss of MondoA in B-ALL. Our findings give novel insight into the function of MondoA in pediatric B-ALL and support the notion that MondoA inhibition in this entity offers a therapeutic opportunity and should be further explored.

DJ-1 depletion prevents immunoaging in T-cell compartments.

Decline in immune function during aging increases susceptibility to different aging-related diseases. However, the underlying molecular mechanisms, especially the genetic factors contributing to imbalance of naïve/memory T-cell subpopulations, still remain largely elusive. Here, we show that loss of DJ-1 encoded by PARK7/DJ-1, causing early-onset familial Parkinson's disease (PD), unexpectedly diminished signs of immunoaging in T-cell compartments of both human and mice. Compared with two gender-matched unaffected siblings of similar ages, the index PD patient with DJ-1 deficiency showed a decline in many critical immunoaging features, including almost doubled non-senescent T cells. The observation was further consolidated by the results in 45-week-old DJ-1 knockout mice. Our data demonstrated that DJ-1 regulates several immunoaging features via hematopoietic-intrinsic and naïve-CD8-intrinsic mechanisms. Mechanistically, DJ-1 depletion reduced oxidative phosphorylation (OXPHOS) and impaired TCR sensitivity in naïve CD8 T cells at a young age, accumulatively leading to a reduced aging process in T-cell compartments in older mice. Our finding suggests an unrecognized critical role of DJ-1 in regulating immunoaging, discovering a potent target to interfere with immunoaging- and aging-associated diseases.

Regulatory T cell metabolism at the intersection between autoimmune diseases and cancer.

Regulatory T cells (Tregs) are critical for peripheral immune tolerance and homeostasis, and altered Treg behavior is involved in many pathologies, including autoimmunity and cancer. The expression of the transcription factor FoxP3 in Tregs is fundamental to maintaining their stability and immunosuppressive function. Recent studies have highlighted the crucial role that metabolic reprogramming plays in controlling Treg plasticity, stability, and function. In this review, we summarize how the availability and use of various nutrients and metabolites influence Treg metabolic pathways and activity. We also discuss how Treg-intrinsic metabolic programs define and shape their differentiation, FoxP3 expression, and suppressive capacity. Lastly, we explore how manipulating the regulation of Treg metabolism might be exploited in different disease settings to achieve novel immunotherapies.

Reactive Oxygen Species: Involvement in T Cell Signaling and Metabolism.

T cells are a central component of defenses against pathogens and tumors. Their effector functions are sustained by specific metabolic changes that occur upon activation, and these have been the focus of renewed interest. Energy production inevitably generates unwanted products, namely reactive oxygen species (ROS), which have long been known to trigger cell death. However, there is now evidence that ROS also act as intracellular signaling molecules both in steady-state and upon antigen recognition. The levels and localization of ROS contribute to the redox modeling of effector proteins and transcription factors, influencing the outcome of the T cell response. We discuss here how ROS can directly fine-tune metabolism and effector functions of T cells.

Fragile X mental retardation protein protects against tumour necrosis factor-mediated cell death and liver injury.

OBJECTIVE: The Fragile X mental retardation (FMR) syndrome is a frequently inherited intellectual disability caused by decreased or absent expression of the FMR protein (FMRP). Lack of FMRP is associated with neuronal degradation and cognitive dysfunction but its role outside the central nervous system is insufficiently studied. Here, we identify a role of FMRP in liver disease. DESIGN: Mice lacking Fmr1 gene expression were used to study the role of FMRP during tumour necrosis factor (TNF)-induced liver damage in disease model systems. Liver damage and mechanistic studies were performed using real-time PCR, Western Blot, staining of tissue sections and clinical chemistry. RESULTS: Fmr1null mice exhibited increased liver damage during virus-mediated hepatitis following infection with the lymphocytic choriomeningitis virus. Exposure to TNF resulted in severe liver damage due to increased hepatocyte cell death. Consistently, we found increased caspase-8 and caspase-3 activation following TNF stimulation. Furthermore, we demonstrate FMRP to be critically important for regulating key molecules in TNF receptor 1 (TNFR1)-dependent apoptosis and necroptosis including CYLD, c-FLIPS and JNK, which contribute to prolonged RIPK1 expression. Accordingly, the RIPK1 inhibitor Necrostatin-1s could reduce liver cell death and alleviate liver damage in Fmr1null mice following TNF exposure. Consistently, FMRP-deficient mice developed increased pathology during acute cholestasis following bile duct ligation, which coincided with increased hepatic expression of RIPK1, RIPK3 and phosphorylation of MLKL. CONCLUSIONS: We show that FMRP plays a central role in the inhibition of TNF-mediated cell death during infection and liver disease.

Protection from EAE in DOCK8 mutant mice occurs despite increased Th17 cell frequencies in the periphery.

Mutation of Dedicator of cytokinesis 8 (DOCK8) has previously been reported to provide resistance to the Th17 cell dependent EAE in mice. Contrary to expectation, we observed an elevation of Th17 cells in two different DOCK8 mutant mouse strains in the steady state. This was specific for Th17 cells with no change in Th1 or Th2 cell populations. In vitro Th cell differentiation assays revealed that the elevated Th17 cell population was not due to a T cell intrinsic differentiation bias. Challenging these mutant mice in the EAE model, we confirmed a resistance to this autoimmune disease with Th17 cells remaining elevated systemically while cellular infiltration in the CNS was reduced. Infiltrating T cells lost the bias toward Th17 cells indicating a relative reduction of Th17 cells in the CNS and a Th17 cell specific migration disadvantage. Adoptive transfers of Th1 and Th17 cells in EAE-affected mice further supported the Th17 cell-specific migration defect, however, DOCK8-deficient Th17 cells expressed normal Th17 cell-specific CCR6 levels and migrated toward chemokine gradients in transwell assays. This study shows that resistance to EAE in DOCK8 mutant mice is achieved despite a systemic Th17 bias.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

Tumor Necrosis Factor-Mediated Survival of CD169+ Cells Promotes Immune Activation during Vesicular Stomatitis Virus Infection.

Innate immune activation is essential to mount an effective antiviral response and to prime adaptive immunity. Although a crucial role of CD169+ cells during vesicular stomatitis virus (VSV) infections is increasingly recognized, factors regulating CD169+ cells during viral infections remain unclear. Here, we show that tumor necrosis factor is produced by CD11b+ Ly6C+ Ly6G+ cells following infection with VSV. The absence of TNF or TNF receptor 1 (TNFR1) resulted in reduced numbers of CD169+ cells and in reduced type I interferon (IFN-I) production during VSV infection, with a severe disease outcome. Specifically, TNF triggered RelA translocation into the nuclei of CD169+ cells; this translocation was inhibited when the paracaspase MALT-1 was absent. Consequently, MALT1 deficiency resulted in reduced VSV replication, defective innate immune activation, and development of severe disease. These findings indicate that TNF mediates the maintenance of CD169+ cells and innate and adaptive immune activation during VSV infection.IMPORTANCE Over the last decade, strategically placed CD169+ metallophilic macrophages in the marginal zone of the murine spleen and lymph nodes (LN) have been shown to play a very important role in host defense against viral pathogens. CD169+ macrophages have been shown to activate innate and adaptive immunity via "enforced virus replication," a controlled amplification of virus particles. However, the factors regulating the CD169+ macrophages remain to be studied. In this paper, we show that after vesicular stomatitis virus infection, phagocytes produce tumor necrosis factor (TNF), which signals via TNFR1, and promote enforced virus replication in CD169+ macrophages. Consequently, lack of TNF or TNFR1 resulted in defective immune activation and VSV clearance.

IDH1(R132H) mutation increases murine haematopoietic progenitors and alters epigenetics.

Mutations in the IDH1 and IDH2 genes encoding isocitrate dehydrogenases are frequently found in human glioblastomas and cytogenetically normal acute myeloid leukaemias (AML). These alterations are gain-of-function mutations in that they drive the synthesis of the 'oncometabolite' R-2-hydroxyglutarate (2HG). It remains unclear how IDH1 and IDH2 mutations modify myeloid cell development and promote leukaemogenesis. Here we report the characterization of conditional knock-in (KI) mice in which the most common IDH1 mutation, IDH1(R132H), is inserted into the endogenous murine Idh1 locus and is expressed in all haematopoietic cells (Vav-KI mice) or specifically in cells of the myeloid lineage (LysM-KI mice). These mutants show increased numbers of early haematopoietic progenitors and develop splenomegaly and anaemia with extramedullary haematopoiesis, suggesting a dysfunctional bone marrow niche. Furthermore, LysM-KI cells have hypermethylated histones and changes to DNA methylation similar to those observed in human IDH1- or IDH2-mutant AML. To our knowledge, our study is the first to describe the generation and characterization of conditional IDH1(R132H)-KI mice, and also the first report to demonstrate the induction of a leukaemic DNA methylation signature in a mouse model. Our report thus sheds light on the mechanistic links between IDH1 mutation and human AML.

Autophagy-independent functions of UVRAG are essential for peripheral naive T-cell homeostasis.

UV radiation resistance-associated gene (UVRAG) encodes a tumor suppressor with putative roles in autophagy, endocytic trafficking, and DNA damage repair but its in vivo role in T cells is unknown. Because conditional homozygous deletion of Uvrag in mice results in early embryonic lethality, we generated T-cell-specific UVRAG-deficient mice that lacked UVRAG expression specifically in T cells. This loss of UVRAG led to defects in peripheral homeostasis that could not be explained by the increased sensitivity to cell death and impaired proliferation observed for other autophagy-related gene knockout mice. Instead, UVRAG-deficient T-cells exhibited normal mitochondrial clearance and activation-induced autophagy, suggesting that UVRAG has an autophagy-independent role that is critical for peripheral naive T-cell homeostatic proliferation. In vivo, T-cell-specific loss of UVRAG dampened CD8(+) T-cell responses to LCMV infection in mice, delayed viral clearance, and impaired memory T-cell generation. Our data provide novel insights into the control of autophagy in T cells and identify UVRAG as a new regulator of naïve peripheral T-cell homeostasis.

Deficiency of MALT1 paracaspase activity results in unbalanced regulatory and effector T and B cell responses leading to multiorgan inflammation.

The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold, recruiting downstream signaling proteins, as well as by proteolytic cleavage of multiple substrates. However, the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation, we generated MALT1 protease-deficient mice (Malt1(PD/PD)) and compared their phenotype with that of MALT1 knockout animals (Malt1(-/-)). Malt1(PD/PD) mice displayed defects in multiple cell types including marginal zone B cells, B1 B cells, IL-10-producing B cells, regulatory T cells, and mature T and B cells. In general, immune defects were more pronounced in Malt1(-/-) animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro, inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation, impaired IL-2 and TNF-α production, as well as defective Th17 differentiation. Consequently, Malt1(PD/PD) mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly, Malt1(PD/PD) animals developed a multiorgan inflammatory pathology, characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels, which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1(PD/PD) animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease.

Toso controls encephalitogenic immune responses by dendritic cells and regulatory T cells.

The ability to mount a strong immune response against pathogens is crucial for mammalian survival. However, excessive and uncontrolled immune reactions can lead to autoimmunity. Unraveling how the reactive versus tolerogenic state is controlled might point toward novel therapeutic strategies to treat autoimmune diseases. The surface receptor Toso/Faim3 has been linked to apoptosis, IgM binding, and innate immune responses. In this study, we used Toso-deficient mice to investigate the importance of Toso in tolerance and autoimmunity. We found that Toso(-/-) mice do not develop severe experimental autoimmune encephalomyelitis (EAE), a mouse model for the human disease multiple sclerosis. Toso(-/-) dendritic cells were less sensitive to Toll-like receptor stimulation and induced significantly lower levels of disease-associated inflammatory T-cell responses. Consistent with this observation, the transfer of Toso(-/-) dendritic cells did not induce autoimmune diabetes, indicating their tolerogenic potential. In Toso(-/-) mice subjected to EAE induction, we found increased numbers of regulatory T cells and decreased encephalitogenic cellular infiltrates in the brain. Finally, inhibition of Toso activity in vivo at either an early or late stage of EAE induction prevented further disease progression. Taken together, our data identify Toso as a unique regulator of inflammatory autoimmune responses and an attractive target for therapeutic intervention.

RAIDD Mediates TLR3 and IRF7 Driven Type I Interferon Production.

BACKGROUND/AIMS: Viral infections represent a global health problem with the need for new viral therapies and better understanding of the immune response during infection. The most immediate and potent anti-viral defense mechanism is the production of type I interferon (IFN-I) which are activated rapidly following recognition of viral infection by host pathogen recognition receptors (PRR). The mechanisms of innate cellular signaling downstream of PRR activation remain to be fully understood. In the present study, we demonstrate that CASP2 and RIPK1 domain-containing adaptor with death domain (CRADD/RAIDD) is a critical component in type I IFN production. METHODS: The role of RAIDD during IFN-I production was investigated using western blot, shRNA mediated lentiviral knockdown, immunoprecipitation and IFN-I driven dual luciferase assay. RESULTS: Immunoprecipitation analysis revealed the molecular interaction of RAIDD with interferon regulatory factor 7 (IRF7) and its phosphorylating kinase IKKε. Using an IFN-4α driven dual luciferase analysis in RAIDD deficient cells, type I IFN activation by IKKε and IRF7 was dramatically reduced. Furthermore, deletion of either the caspase recruitment domain (CARD) or death domain (DD) of RAIDD inhibited IKKε and IRF7 mediated interferon-4α activation. CONCLUSION: We have identified that the adaptor molecule RAIDD coordinates IKKε and IRF7 interaction to ensure efficient expression of type I interferon.

Lymphocyte-derived ACh regulates local innate but not adaptive immunity.

Appropriate control of immune responses is a critical determinant of health. Here, we show that choline acetyltransferase (ChAT) is expressed and ACh is produced by B cells and other immune cells that have an impact on innate immunity. ChAT expression occurs in mucosal-associated lymph tissue, subsequent to microbial colonization, and is reduced by antibiotic treatment. MyD88-dependent Toll-like receptor up-regulates ChAT in a transient manner. Unlike the previously described CD4(+) T-cell population that is stimulated by norepinephrine to release ACh, ChAT(+) B cells release ACh after stimulation with sulfated cholecystokinin but not norepinephrine. ACh-producing B-cells reduce peritoneal neutrophil recruitment during sterile endotoxemia independent of the vagus nerve, without affecting innate immune cell activation. Endothelial cells treated with ACh in vitro reduced endothelial cell adhesion molecule expression in a muscarinic receptor-dependent manner. Despite this ability, ChAT(+) B cells were unable to suppress effector T-cell function in vivo. Therefore, ACh produced by lymphocytes has specific functions, with ChAT(+) B cells controlling the local recruitment of neutrophils.

2-Methoxyestradiol inhibits experimental autoimmune encephalomyelitis through suppression of immune cell activation.

The endogenous metabolite of estradiol, 2-Methoxyestradiol (2ME2), is an antimitotic and antiangiogenic cancer drug candidate that also exhibits disease-modifying activity in animal models of rheumatoid arthritis (RA). We found that 2ME2 dramatically suppresses development of mouse experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis (MS). 2ME2 inhibits in vitro lymphocyte activation, cytokine production, and proliferation in a dose-dependent fashion. 2ME2 treatment of lymphocytes specifically reduced the nuclear translocation and transcriptional activity of nuclear factor of activated T-cells (NFAT) c1, whereas NF-κB and activator protein 1 (AP-1) activation were not adversely affected. We therefore propose that 2ME2 attenuates EAE through disruption of the NFAT pathway and subsequent lymphocyte activation. By extension, our findings provide a molecular rationale for the use of 2ME2 as a tolerable oral immunomodulatory agent for the treatment of autoimmune disorders such as MS in humans.