ILC2s and Adipose Tissue Homeostasis: Progress to Date and the Road Ahead.

Group 2 innate lymphoid cells (ILC2s) were initially identified as a new type of lymphocytes that produce vigorous amounts of type 2 cytokines in adipose tissue. Subsequent studies revealed that ILC2s are present not only in adipose tissue but also in various other tissues such as lung and skin. ILC2s are generally recognized as tissue-resident immune cells that regulate tissue homeostasis. ILC2s express receptors for various humoral factors and thus can change their functions or distribution depending on the environment and circumstances. In this review, we will outline our recent understanding of ILC2 biology and discuss future directions for ILC2 research, particularly in adipose tissue and metabolic homeostasis.

Nanaomycin E inhibits NLRP3 inflammasome activation by preventing mitochondrial dysfunction.

Nod-like receptor family pyrin domain-containing 3 (NLRP3) is a cytosolic innate immune receptor that senses organelle dysfunction induced by various stimuli, such as infectious, environmental, metabolic and drug stresses. Upon activation, NLRP3 forms an inflammasome with its adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1, to trigger the release of inflammatory cytokines. The development of effective anti-inflammatory drugs targeting the NLRP3 inflammasome is in high demand as its aberrant activation often causes inflammatory diseases. Here, we found that nanaomycin A (NNM-A), a quinone-based antibiotic isolated from Streptomyces, effectively inhibited NLRP3 inflammasome-mediated inflammatory responses induced by imidazoquinolines, including imiquimod. Interestingly, its epoxy derivative nanaomycin E (NNM-E) showed a comparable inhibitory effect against the NLRP3 inflammasome-induced release of interleukin (IL)-1ß and IL-18 from macrophages, with a much lower toxicity than NNM-A. NNM-E inhibited ASC oligomerization and caspase-1 cleavage, both of which are hallmarks of NLRP3 inflammasome activation. NNM-E reduced mitochondrial damage and the production of reactive oxygen species, thereby preventing the activation of the NLRP3 inflammasome. NNM-E treatment markedly alleviated psoriasis-like skin inflammation induced by imiquimod. Collectively, NNM-E inhibits NLRP3 inflammasome activation by preventing mitochondrial dysfunction with little toxicity and showed an anti-inflammatory effect in vivo. Thus, NNM-E could be a potential lead compound for developing effective and safe anti-inflammatory agents for the treatment of NLRP3 inflammasome-mediated inflammatory diseases.

Thousands of induced germline mutations affecting immune cells identified by automated meiotic mapping coupled with machine learning.

Forward genetic studies use meiotic mapping to adduce evidence that a particular mutation, normally induced by a germline mutagen, is causative of a particular phenotype. Particularly in small pedigrees, cosegregation of multiple mutations, occasional unawareness of mutations, and paucity of homozygotes may lead to erroneous declarations of cause and effect. We sought to improve the identification of mutations causing immune phenotypes in mice by creating Candidate Explorer (CE), a machine-learning software program that integrates 67 features of genetic mapping data into a single numeric score, mathematically convertible to the probability of verification of any putative mutation-phenotype association. At this time, CE has evaluated putative mutation-phenotype associations arising from screening damaging mutations in ∼55% of mouse genes for effects on flow cytometry measurements of immune cells in the blood. CE has therefore identified more than half of genes within which mutations can be causative of flow cytometric phenovariation in Mus musculus The majority of these genes were not previously known to support immune function or homeostasis. Mouse geneticists will find CE data informative in identifying causative mutations within quantitative trait loci, while clinical geneticists may use CE to help connect causative variants with rare heritable diseases of immunity, even in the absence of linkage information. CE displays integrated mutation, phenotype, and linkage data, and is freely available for query online.

SLFN2 protection of tRNAs from stress-induced cleavage is essential for T cell-mediated immunity.

Reactive oxygen species (ROS) increase in activated T cells because of metabolic activity induced to support T cell proliferation and differentiation. We show that these ROS trigger an oxidative stress response that leads to translation repression. This response is countered by Schlafen 2 (SLFN2), which directly binds transfer RNAs (tRNAs) to protect them from cleavage by the ribonuclease angiogenin. T cell-specific SLFN2 deficiency results in the accumulation of tRNA fragments, which inhibit translation and promote stress-granule formation. Interleukin-2 receptor ß (IL-2Rß) and IL-2Rγ fail to be translationally up-regulated after T cell receptor stimulation, rendering SLFN2-deficient T cells insensitive to interleukin-2's mitogenic effects. SLFN2 confers resistance against the ROS-mediated translation-inhibitory effects of oxidative stress normally induced by T cell activation, permitting the robust protein synthesis necessary for T cell expansion and immunity.

Essential cell-extrinsic requirement for PDIA6 in lymphoid and myeloid development.

In a forward genetic screen of N-ethyl-N-nitrosourea (ENU)-induced mutant mice for aberrant immune function, we identified mice with a syndromic disorder marked by growth retardation, diabetes, premature death, and severe lymphoid and myeloid hypoplasia together with diminished T cell-independent (TI) antibody responses. The causative mutation was in Pdia6, an essential gene encoding protein disulfide isomerase A6 (PDIA6), an oxidoreductase that functions in nascent protein folding in the endoplasmic reticulum. The immune deficiency caused by the Pdia6 mutation was, with the exception of a residual T cell developmental defect, completely rescued in irradiated wild-type recipients of PDIA6-deficient bone marrow cells, both in the absence or presence of competition. The viable hypomorphic allele uncovered in these studies reveals an essential role for PDIA6 in hematopoiesis, but one extrinsic to cells of the hematopoietic lineage. We show evidence that this role is in the proper folding of Wnt3a, BAFF, IL-7, and perhaps other factors produced by the extra-hematopoietic compartment that contribute to the development and lineage commitment of hematopoietic cells.

Mutual inhibition between Prkd2 and Bcl6 controls T follicular helper cell differentiation.

T follicular helper cells (TFH) participate in germinal center (GC) development and are necessary for B cell production of high-affinity, isotype-switched antibodies. In a forward genetic screen, we identified a missense mutation in Prkd2, encoding the serine/threonine kinase protein kinase D2, which caused elevated titers of immunoglobulin E (IgE) in the serum. Subsequent analysis of serum antibodies in mice with a targeted null mutation of Prkd2 demonstrated polyclonal hypergammaglobulinemia of IgE, IgG1, and IgA isotypes, which was exacerbated by the T cell-dependent humoral response to immunization. GC formation and GC B cells were increased in Prkd2-/- spleens. These effects were the result of excessive cell-autonomous TFH development caused by unrestricted Bcl6 nuclear translocation in Prkd2-/- CD4+ T cells. Prkd2 directly binds to Bcl6, and Prkd2-dependent phosphorylation of Bcl6 is necessary to constrain Bcl6 to the cytoplasm, thereby limiting TFH development. In response to immunization, Bcl6 repressed Prkd2 expression in CD4+ T cells, thereby committing them to TFH development. Thus, Prkd2 and Bcl6 form a mutually inhibitory positive feedback loop that controls the stable transition from naïve CD4+ T cells to TFH during the adaptive immune response.

Enhanced susceptibility to chemically induced colitis caused by excessive endosomal TLR signaling in LRBA-deficient mice.

LPS-responsive beige-like anchor (LRBA) protein deficiency in humans causes immune dysregulation resulting in autoimmunity, inflammatory bowel disease (IBD), hypogammaglobulinemia, regulatory T (Treg) cell defects, and B cell functional defects, but the cellular and molecular mechanisms responsible are incompletely understood. In an ongoing forward genetic screen for N-ethyl-N-nitrosourea (ENU)-induced mutations that increase susceptibility to dextran sodium sulfate (DSS)-induced colitis in mice, we identified two nonsense mutations in Lrba Although Treg cells have been a main focus in LRBA research to date, we found that dendritic cells (DCs) contribute significantly to DSS-induced intestinal inflammation in LRBA-deficient mice. Lrba-/- DCs exhibited excessive IRF3/7- and PI3K/mTORC1-dependent signaling and type I IFN production in response to the stimulation of the Toll-like receptors (TLRs) 3, TLR7, and TLR9. Substantial reductions in cytokine expression and sensitivity to DSS in LRBA-deficient mice were caused by knockout of Unc93b1, a chaperone necessary for trafficking of TLR3, TLR7, and TLR9 to endosomes. Our data support a function for LRBA in limiting endosomal TLR signaling and consequent intestinal inflammation.

LMBR1L regulates lymphopoiesis through Wnt/ß-catenin signaling.

Precise control of Wnt signaling is necessary for immune system development. In this study, we detected severely impaired development of all lymphoid lineages in mice, resulting from an N-ethyl-N-nitrosourea-induced mutation in the limb region 1-like gene (Lmbr1l), which encodes a membrane-spanning protein with no previously described function in immunity. The interaction of LMBR1L with glycoprotein 78 (GP78) and ubiquitin-associated domain-containing protein 2 (UBAC2) attenuated Wnt signaling in lymphocytes by preventing the maturation of FZD6 and LRP6 through ubiquitination within the endoplasmic reticulum and by stabilizing "destruction complex" proteins. LMBR1L-deficient T cells exhibited hallmarks of Wnt/ß-catenin activation and underwent apoptotic cell death in response to proliferative stimuli. LMBR1L has an essential function during lymphopoiesis and lymphoid activation, acting as a negative regulator of the Wnt/ß-catenin pathway.

The RAB2B-GARIL5 Complex Promotes Cytosolic DNA-Induced Innate Immune Responses.

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that induces the IFN antiviral response. However, the regulatory mechanisms that mediate cGAS-triggered signaling have not been fully explored. Here, we show the involvement of a small GTPase, RAB2B, and its effector protein, Golgi-associated RAB2B interactor-like 5 (GARIL5), in the cGAS-mediated IFN response. RAB2B-deficiency affects the IFN response induced by cytosolic DNA. Consistent with this, RAB2B deficiency enhances replication of vaccinia virus, a DNA virus. After DNA stimulation, RAB2B colocalizes with stimulator of interferon genes (STING), the downstream signal mediator of cGAS, on the Golgi apparatus. The GTP-binding activity of RAB2B is required for its localization on the Golgi apparatus and for recruitment of GARIL5. GARIL5 deficiency also affects the IFN response induced by cytosolic DNA and enhances replication of vaccinia virus. These findings indicate that the RAB2B-GARIL5 complex promotes IFN responses against DNA viruses by regulating the cGAS-STING signaling axis.

Mitochondria-Endoplasmic Reticulum Contact Sites Mediate Innate Immune Responses.

Mitochondria and the endoplasmic reticulum (ER) are fundamental organelles that coordinate high-order cell functions. Mitochondria are centers of energy production, whereas the ER is responsible for folding, transport, and degradation of proteins. In addition to their specific functions, mitochondria and ER actively communicate with each other to promote a variety of cellular events, such as material transfer and signal transduction. Recent studies have shown the critical involvement of these organelles in regulation of the innate immune system, which functions in host defense. The innate immune system utilizes a wide range of germ-line-encoded pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) and induces inflammatory and antiviral responses. Contact sites between mitochondria and the ER function in assembly of the NLR family pyrin domain containing 3 (NLRP3)-inflammasome to promote the inflammatory response. The NLRP3-inflammasome is a protein complex composed of the receptor NLRP3 on the ER side and the adaptor apoptosis-associated speck-like protein containing a CARD on the mitochondrial side; it induces caspase-1-dependent maturation of proinflammatory cytokines such as interleukin (IL)-1ß and IL-18. Furthermore, ER-mitochondria contact sites function in initiation and mediation of signal transduction pathways downstream of intracellular PRRs, such as retinoic acid-inducible gene I-like receptor and cyclic GMP-AMP synthase, to promote the antiviral response. Therefore, ER-mitochondria contact sites, also known as mitochondria-associated membranes, play key roles in regulation of innate immune responses.

Mitochondrial damage elicits a TCDD-inducible poly(ADP-ribose) polymerase-mediated antiviral response.

The innate immune system senses RNA viruses by pattern recognition receptors (PRRs) and protects the host from virus infection. PRRs mediate the production of immune modulatory factors and direct the elimination of RNA viruses. Here, we show a unique PRR that mediates antiviral response. Tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP ribose) polymerase (TIPARP), a Cysteine3 Histidine (CCCH)-type zinc finger-containing protein, binds to Sindbis virus (SINV) RNA via its zinc finger domain and recruits an exosome to induce viral RNA degradation. TIPARP typically localizes in the nucleus, but it accumulates in the cytoplasm after SINV infection, allowing targeting of cytoplasmic SINV RNA. Redistribution of TIPARP is induced by reactive oxygen species (ROS)-dependent oxidization of the nuclear pore that affects cytoplasmic-nuclear transport. BCL2-associated X protein (BAX) and BCL2 antagonist/killer 1 (BAK1), B-cell leukemia/lymphoma 2 (BCL2) family members, mediate mitochondrial damage to generate ROS after SINV infection. Thus, TIPARP is a viral RNA-sensing PRR that mediates antiviral responses triggered by BAX- and BAK1-dependent mitochondrial damage.

IgD class switching is initiated by microbiota and limited to mucosa-associated lymphoid tissue in mice.

Class-switch recombination (CSR) alters the Ig isotype to diversify antibody effector functions. IgD CSR is a rare event, and its regulation is poorly understood. We report that deficiency of 53BP1, a DNA damage-response protein, caused age-dependent overproduction of secreted IgD resulting from increased IgD CSR exclusively within B cells of mucosa-associated lymphoid tissues. IgD overproduction was dependent on activation-induced cytidine deaminase, hematopoietic MyD88 expression, and an intact microbiome, against which circulating IgD, but not IgM, was reactive. IgD CSR occurred via both alternative nonhomologous end-joining and homologous recombination pathways. Microbiota-dependent IgD CSR also was detected in nasal-associated lymphoid tissue of WT mice. These results identify a pathway, present in WT mice and hyperactivated in 53BP1-deficient mice, by which microbiota signal via Toll-like receptors to elicit IgD CSR.

Bone-protective Functions of Netrin 1 Protein.

Netrin 1 was initially identified as an axon guidance factor, and recent studies indicate that it inhibits chemokine-directed monocyte migration. Despite its importance as a neuroimmune guidance cue, the role of netrin 1 in osteoclasts is largely unknown. Here we detected high netrin 1 levels in the synovial fluid of rheumatoid arthritis patients. Netrin 1 is potently expressed in osteoblasts and synovial fibroblasts, and IL-17 robustly enhances netrin 1 expression in these cells. The binding of netrin 1 to its receptor UNC5b on osteoclasts resulted in activation of SHP1, which inhibited VAV3 phosphorylation and RAC1 activation. This significantly impaired the actin polymerization and fusion, but not the differentiation of osteoclast. Strikingly, netrin 1 treatment prevented bone erosion in an autoimmune arthritis model and age-related bone destruction. Therefore, the netrin 1-UNC5b axis is a novel therapeutic target for bone-destructive diseases.

Application of Two-Dimensional Nuclear Magnetic Resonance for Signal Enhancement by Spectral Integration Using a Large Data Set of Metabolic Mixtures.

Nuclear magnetic resonance (NMR) spectroscopy has tremendous advantages of minimal sample preparation and interconvertibility of data among different institutions; thus, large data sets are frequently acquired in metabolomics studies. Previously, we used a novel analytical strategy, named signal enhancement by spectral integration (SENSI), to overcome the low signal-to-noise ratio (S/N ratio) problem in (13)C NMR by integration of hundreds of spectra without additional measurements. In this letter, the development of a SENSI 2D method and application to >1000 2D JRES NMR spectra are described. Remarkably, the obtained SENSI 2D spectrum had an approximate 14-fold increase in the S/N ratio and 80-250 additional peaks without any additional measurements. These results suggest that SENSI 2D is a useful method for assigning weak signals and that the use of coefficient of variation values can support the assignment information and extraction of features from the population characteristics among large data sets.

Application of Market Basket Analysis for the Visualization of Transaction Data Based on Human Lifestyle and Spectroscopic Measurements.

With the innovation of high-throughput metabolic profiling methods such as nuclear magnetic resonance (NMR), data mining techniques that can reveal valuable information from substantial data sets are constantly desired in this field. In particular, for the analytical assessment of various human lifestyles, advanced computational methods are ultimately needed. In this study, we applied market basket analysis, which is generally applied in social sciences such as marketing, and used transaction data derived from dietary intake information and urinary chemical data generated using NMR and inductively coupled plasma optical emission spectrometry measurements. The analysis revealed several relationships, such as fish diets with high trimethylamine N-oxide excretion and N-methylnicotinamide excreted at higher levels in the morning and produced from a protein that was consumed one day prior. Therefore, market basket analysis can be applied to metabolic profiling to effectively understand the relationships between metabolites and lifestyle.

SENSI: signal enhancement by spectral integration for the analysis of metabolic mixtures.

The method provided here can overcome the low S/N problem in (13)C NMR, by the integration of plural spectra to take advantage of high-resolution potential based on non-bucketing analysis without additional measurements. In addition, a new metabolite annotation approach using advanced STOCSY and quantum chemistry calculations was introduced in this study.

Resveratrol inhibits the acetylated α-tubulin-mediated assembly of the NLRP3-inflammasome.

With its adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), Nod-like receptor family, pyrin domain containing 3 (NLRP3) forms the inflammasome and mediates inflammatory innate immune responses. Development of an anti-inflammatory drug targeting the NLRP3-inflammasome is urgently required because its aberrant activation often causes inflammatory diseases, including gout. We show that resveratrol, a natural polyphenol in grapes and wine, is a safe and effective phytochemical that inhibits NLRP3-inflammasome activation. Resveratrol inhibits the accumulation of acetylated α-tubulin caused by mitochondrial damage in macrophages stimulated with inducers of the NLRP3-inflammasome. Consequently, resveratrol inhibits the acetylated-α-tubulin-mediated spatial arrangement of mitochondria and their subsequent contact with the endoplasmic reticulum (ER), causing insufficient assembly of ASC on the mitochondria and NLRP3 on the ER. These findings indicate that resveratrol targets the generation of an optimal site for the assembly of NLRP3 and ASC, thus inhibiting NLRP3-inflammasome activation. Therefore, resveratrol could be an effective medication for the treatment of NLRP3-related inflammatory diseases.

Role of zinc-finger anti-viral protein in host defense against Sindbis virus.

Accumulating evidence indicates that type I interferon (IFN) mediates the host protective response to RNA viruses. However, the anti-viral effector molecules involved in this response have not been fully identified. Here, we show that zinc-finger anti-viral protein (ZAP), an IFN-inducible gene, plays a critical role in the elimination of Sindbis virus (SINV) in vitro and in vivo. The loss of ZAP greatly enhances the replication of SINV but does not inhibit type I IFN production in primary mouse embryonic fibroblasts (MEFs). ZAP binds and destabilizes SINV RNA, thereby suppressing the replication of SINV. Type I IFN fails to suppress SINV replication in ZAP-deficient MEFs, whereas the ectopic expression of ZAP is sufficient to suppress the replication of SINV in MEFs lacking the expression of type I IFN and the IFN-inducible genes. ZAP-deficient mice are highly susceptible to SINV infection, although they produce sufficient amounts of type I IFN. Therefore, ZAP is an RNA-sensing anti-viral effector molecule that mediates the type-I-IFN-dependent host defense against SINV.

Human metabolic, mineral, and microbiota fluctuations across daily nutritional intake visualized by a data-driven approach.

Daily intake information is important for an understanding of the metabolic fluctuation of humans exposed to environmental stimuli. However, little investigation has been performed on the variations in dietary intake as an input and the relationship with human fecal, urinary, and salivary metabolic fluctuations as output information triggered by daily dietary intake. In the present study, we describe a data-driven approach for visualizing the daily intake information on a nutritional scale and for evaluating input-output responses under uncontrolled diets in a human study. For the input evaluation of nutritional intake, we collected information about daily dietary intake and converted this information to numeric data of nutritional elements. Furthermore, for the evaluation of output metabolic, mineral, and microbiota responses, we characterized the metabolic, mineral, and microbiota variations of noninvasive human samples of feces, urine, and saliva. The data-driven approach captured significant differences in the fluctuation of intestinal microbiota and some metabolites caused by a high-protein and a high-fat diet in daily life. This approach should contribute to the metabolic assessment of humans affected by environmental and nutritional factors under unlimited and uncontrolled diets.

Zinc-finger antiviral protein mediates retinoic acid inducible gene I-like receptor-independent antiviral response to murine leukemia virus.

When host cells are infected by an RNA virus, pattern-recognition receptors (PRRs) recognize the viral RNA and induce the antiviral innate immunity. Toll-like receptor 7 (TLR7) detects the genomic RNA of incoming murine leukemia virus (MLV) in endosomes and mediates the antiviral response. However, the RNA-sensing PRR that recognizes the MLV in the cytosol is not fully understood. Here, we definitively demonstrate that zinc-finger antiviral protein (ZAP) acts as a cytosolic RNA sensor, inducing the degradation of the MLV transcripts by the exosome, an RNA degradation system, on RNA granules. Although the retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 detect various RNA viruses in the cytosol and induce the type I IFN-dependent antiviral response, RLR loss does not alter the replication efficiency of MLV. In sharp contrast, the loss of ZAP greatly enhances the replication efficiency of MLV. ZAP localizes to RNA granules, where the processing-body and stress-granule proteins assemble. ZAP induces the recruitment of the MLV transcripts and exosome components to the RNA granules. The CCCH-type zinc-finger domains of ZAP, which are RNA-binding motifs, mediate its localization to RNA granules and MLV transcripts degradation by the exosome. Although ZAP was known as a regulator of RIG-I signaling in a human cell line, ZAP deficiency does not affect the RIG-I-dependent production of type I IFN in mouse cells. Thus, ZAP is a unique member of the cytosolic RNA-sensing PRR family that targets and eliminates intracellular RNA viruses independently of TLR and RLR family members.