A Germinal Center Checkpoint of AIRE in B Cells Limits Antibody Diversification.

In response to antigens, B cells undergo affinity maturation and class switching mediated by activation-induced cytidine deaminase (AID) in germinal centers (GCs) of secondary lymphoid organs, but uncontrolled AID activity can precipitate autoimmunity and cancer. The regulation of GC antibody diversification is of fundamental importance but not well understood. We found that autoimmune regulator (AIRE), the molecule essential for T cell tolerance, is expressed in GC B cells in a CD40-dependent manner, interacts with AID and negatively regulates antibody affinity maturation and class switching by inhibiting AID function. AIRE deficiency in B cells caused altered antibody repertoire, increased somatic hypermutations, elevated autoantibodies to T helper 17 effector cytokines and defective control of skin Candida albicans. These results define a GC B cell checkpoint of humoral immunity and illuminate new approaches of generating high-affinity neutralizing antibodies for immunotherapy.

TXNIP mediates high glucose-induced mitophagic flux and lysosome enlargement in human retinal pigment epithelial cells.

Thioredoxin-interacting protein (TXNIP) plays a critical role in oxidative stress, inflammation, apoptosis and the pathogenesis of diabetic retinopathy (DR). However, the role of TXNIP in high glucose-induced retinal pigment epithelium (RPE) dysfunction is still unknown. Here, we show that high glucose (HG; 25 mM,) significantly increases TXNIP expression at both the mRNA and protein levels when compared to low glucose (LG; 5.5 mM) in a human RPE cell line (ARPE-19) and primary human RPE (HRPE) cells. TXNIP upregulation is associated with mitochondrial membrane depolarization, fragmentation and mitophagic flux to lysosomes. We used confocal live-cell imaging of RPE cells expressing mt-Keima, a coral protein that emits green light in mitochondria (alkaline or neutral pH) and red light in the acidic lysosome, to measure mitophagic flux. We observed an elongated mitochondrial network of green mt-Keima under LG, which is fragmented in HG. Red mt-Keima accumulates in lysosomes as small punctate aggregations under LG in both ARPE-19 and HRPE cells, whereas they are significantly enlarged (two- to threefold) under HG. Lysosomal enlargement under HG is further illustrated by lysosomal membrane protein LAMP1-mCherry expression in both ARPE-19 and HRPE cells. Furthermore, HG causes lysosomal cathepsin L inactivation and pro-inflammatory caspase-1 activation in ARPE-19 cells. TXNIP knockdown by shRNA prevents mitochondrial fragmentation, mitophagic flux and lysosome enlargement under HG. In addition, antioxidant N-acetylcysteine (NAC) and Amlexanox (Amlx), an inhibitor of protein kinase TBK1 and of the mitophagic adaptors Optineurin (Optn) and Sequestosome 1 (p62/SQSTM1), prevent mitophagic flux and lysosome enlargement. These results suggest that TXNIP mediates several deleterious effects of high glucose on RPE, which may be implicated in the development of DR.

Interleukin-33-induced expression of PIBF1 by decidual B cells protects against preterm labor.

Preterm birth (PTB) is a leading cause of neonatal death worldwide. Intrauterine and systemic infection and inflammation cause 30-40% of spontaneous preterm labor (PTL), which precedes PTB. Although antibody production is a major immune defense mechanism against infection, and B cell dysfunction has been implicated in pregnancy complications associated with PTL, the functions of B cells in pregnancy are not well known. We found that choriodecidua of women undergoing spontaneous PTL harbored functionally altered B cell populations. B cell-deficient mice were markedly more susceptible than wild-type (WT) mice to PTL after inflammation, but B cells conferred interleukin (IL)-10-independent protection against PTL. B cell deficiency in mice resulted in a lower uterine level of active progesterone-induced blocking factor 1 (PIBF1), and therapeutic administration of PIBF1 mitigated PTL and uterine inflammation in B cell-deficient mice. B cells are a significant producer of PIBF1 in human choriodecidua and mouse uterus in late gestation. PIBF1 expression by B cells is induced by the mucosal alarmin IL-33 (ref. 9). Human PTL was associated with diminished expression of the α-chain of IL-33 receptor on choriodecidual B cells and a lower level of active PIBF1 in late gestation choriodecidua. These results define a vital regulatory cascade involving IL-33, decidual B cells and PIBF1 in safeguarding term pregnancy and suggest new therapeutic approaches based on IL-33 and PIBF1 to prevent human PTL.

Immunization of pregnant women: Future of early infant protection.

Children in early infancy do not mount effective antibody responses to many vaccines against commons infectious pathogens, which results in a window of increased susceptibility or severity infections. In addition, vaccine-preventable infections are among the leading causes of morbidity in pregnant women. Immunization during pregnancy can generate maternal immune protection as well as elicit the production and transfer of antibodies cross the placenta and via breastfeeding to provide early infant protection. Several successful vaccines are now recommended to all pregnant women worldwide. However, significant gaps exist in our understanding of the efficacy and safety of other vaccines and in women with conditions associated with increased susceptible to high-risk pregnancies. Public acceptance of maternal immunization remained to be improved. Broader success of maternal immunization will rely on the integration of advances in basic science in vaccine design and evaluation and carefully planned clinical trials that are inclusive to pregnant women.

Insertion of proteolipid protein into oligodendrocyte mitochondria regulates extracellular pH and adenosine triphosphate.

Proteolipid protein (PLP) and DM20, the most abundant myelin proteins, are coded by the human PLP1 and non-human Plp1 PLP gene. Mutations in the PLP1 gene cause Pelizaeus-Merzbacher disease (PMD) with duplications of the native PLP1 gene accounting for 70% of PLP1 mutations. Humans with PLP1 duplications and mice with extra Plp1 copies have extensive neuronal degeneration. The mechanism that causes neuronal degeneration is unknown. We show that native PLP traffics to mitochondria when the gene is duplicated in mice and in humans. This report is the first demonstration of a specific cellular defect in brains of PMD patients; it validates rodent models as ideal models to study PMD. Insertion of nuclear-encoded mitochondrial proteins requires specific import pathways; we show that specific cysteine motifs, part of the Mia40/Erv1 mitochondrial import pathway, are present in PLP and are required for its insertion into mitochondria. Insertion of native PLP into mitochondria of transfected cells acidifies media, partially due to increased lactate; it also increases adenosine triphosphate (ATP) in the media. The same abnormalities are found in the extracellular space of mouse brains with extra copies of Plp1. These physiological abnormalities are preventable by mutations in PLP cysteine motifs, a hallmark of the Mia40/Erv1 pathway. Increased extracellular ATP and acidosis lead to neuronal degeneration. Our findings may be the mechanism by which microglia are activated and proinflammatory molecules are upregulated in Plp1 transgenic mice (Tatar et al. (2010) ASN Neuro 2:art:e00043). Manipulation of this metabolic pathway may restore normal metabolism and provide therapy for PMD patients.

A Tet-on system for DRD1-expressing cells.

Cells expressing the dopamine D1 receptor (DRD1) have significant functional roles in diverse physiological processes including locomotion and drug addiction. The present work presents a novel in vivo DRD1-Bacterial Artificial Chromosome (BAC) Tet-on system allowing for the inducible activation of tet-operated transgenes specifically within DRD1-expressing cells of transgenic mice. It is shown that the DRD1-rtTA BAC-driven expression of a tet-operated reporter is under tight regulation by doxycycline and is restricted to DRD1-expressing brain regions. The model will be a useful research tool in studies of movement and reward and associated pathologies such as Parkinson's disease and addiction.

Lipasin, thermoregulated in brown fat, is a novel but atypical member of the angiopoietin-like protein family.

Hyperlipidemia is a major contributor to cardiovascular diseases. Members of the angiopoietin-like protein family (ANGPTLs) are important determinants of blood lipid levels. Lipasin, a newly identified gene that regulates serum triglycerides, is homologous to ANGPTL3's N-terminal domain, which is sufficient and necessary for blood lipid regulation. Brown fat is critical in mediating energy homeostasis. Thermogenesis is the primary function of brown fat, in which Lipasin and some ANGPTLs are abundant; it is unknown, however, whether these genes are thermoregulated. We therefore comprehensively examined the thermoregulation of Lipasin and ANGPTLs in brown fat. Here we show that Lipasin is a novel but atypical member of the ANGPTL family because it is within the same branch as ANGPTL3 and 4 by phylogenetic analysis. The mRNA levels of Lipasin are dramatically increased in the cold environment (4 °C for 4 h) whereas those of ANGPTL4 and ANGPTL2 are suppressed. Fasting dramatically suppresses Lipasin but increases ANGPTL4. High-fat diet treatment increases Lipasin, but reduces ANGPTL2. The distinct transcriptional regulations of Lipasin, ANGPTL2 and ANGPTL4 in brown fat in response to cold exposure and nutritional stimulation suggest distinct physiological roles for ANGPTL family members in mediating thermogenesis and energy homeostasis.

Nrac, a novel nutritionally-regulated adipose and cardiac-enriched gene.

Obesity increases the risk of multiple diseases, such as type 2 diabetes and coronary heart diseases, and therefore the current obesity epidemic poses a major public health issue. Therapeutic approaches are urgently needed to treat obesity as well as its complications. Plasma-membrane proteins with restricted tissue distributions are attractive drug targets, because of their accessibility to various drug delivery mechanisms and potentially alleviated side effects. To identify genes involved in metabolism, we performed RNA-Seq on fat in mice treated with a high-fat diet or fasting. Here we show that the gene A530016L24Rik (human ortholog C14orf180), named Nrac, is a novel nutritionally-regulated adipose and cardiac-enriched gene. Nrac is expressed specifically and abundantly in fat and the heart. Both fasting and obesity reduced Nrac expression in white adipose tissue, and fasting reduced its expression in brown fat. Nrac is localized to the plasma membrane, and highly induced during adipocyte differentiation. Nrac is therefore a novel adipocyte marker and has potential functions in metabolism.

Signal transducer and activator of transcription-3 is required in hypothalamic agouti-related protein/neuropeptide Y neurons for normal energy homeostasis.

Signal transducer and activator of transcription (Stat)-3 signals mediate many of the metabolic effects of the fat cell-derived hormone, leptin. In mice, brain-specific depletion of either the long form of the leptin receptor (Lepr) or Stat3 results in comparable obese phenotypes as does replacement of Lepr with an altered leptin receptor locus that codes for a Lepr unable to interact with Stat3. Among the multiple brain regions containing leptin-sensitive Stat3 sites, cells expressing feeding-related neuropeptides in the arcuate nucleus of the hypothalamus have received much of the focus. To determine the contribution to energy homeostasis of Stat3 expressed in agouti-related protein (Agrp)/neuropeptide Y (Npy) arcuate neurons, Stat3 was deleted specifically from these cells, and several metabolic indices were measured. It was found that deletion of Stat3 from Agrp/Npy neurons resulted in modest weight gain that was accounted for by increased adiposity. Agrp/Stat3-deficient mice also showed hyperleptinemia, and high-fat diet-induced hyperinsulinemia. Stat3 deletion in Agrp/Npy neurons also resulted in altered hypothalamic gene expression indicated by increased Npy mRNA and decreased induction of suppressor of cytokine signaling-3 in response to leptin. Agrp mRNA levels in the fed or fasted state were unaffected. Behaviorally, mice without Stat3 in Agrp/Npy neurons were mildly hyperphagic and hyporesponsive to leptin. We conclude that Stat3 in Agrp/Npy neurons is required for normal energy homeostasis, but Stat3 signaling in other brain areas also contributes to the regulation of energy homeostasis.

Signal transducer and activator of transcription (stat) binding sites but not stat3 are required for fasting-induced transcription of agouti-related protein messenger ribonucleic acid.

Energy homeostasis depends on the regulation of hypothalamic neurons by leptin, an adipocyte hormone whose circulating levels communicate body energy stores. Leptin activates the transcription factor signal transducer and activator of transcription 3 (Stat3) in hypothalamic neurons, including neuronal subtypes producing Agouti-related protein (Agrp), a neuropeptide that stimulates feeding. Previous studies have suggested a model in which high levels of Agrp transcription during fasting represent a default state that is actively repressed by phospho-Stat3 induced by leptin signaling in the fed state. We identify putative Stat3 binding elements in the Agrp promoter that have been highly conserved during vertebrate evolution. Using a reporter assay in transgenic mice that faithfully recapitulates normal regulation of Agrp, we show that these sites are required, but in a way opposite to that predicted by the existing model: mutation of the sites leads to a default state characterized by a low level of Agrp transcription and insensitivity to fasting. We also find that removing activatable Stat3 from Agrp neurons has no detectable effect on steady-state levels of Agrp mRNA in the fed or fasted state. These results suggest a new model for transcriptional regulation of orexigenic neuropeptides in which the default level of expression is low in the fed state, and transcriptional activation in response to fasting is mediated by factors other than Stat3.

Microarray analysis of fluoro-gold labeled rat dopamine neurons harvested by laser capture microdissection.

The cellular heterogeneity of brain tissue presents a challenge to gene expression profiling of specific neuronal cell types. The present study employed a fluorescent neural tracer to specifically label midbrain dopamine neurons and non-dopamine cortical neurons. The labeled cells were then used to visually guide harvesting of the cells by laser capture microdissection (LCM). RNA extracted from the two populations of harvested cells was then amplified, labeled and co-hybridized to high density cDNA microarrays for two-color differential expression profiling. Many of the genes most highly enriched in the dopamine neurons were found to be genes previously known to define the dopamine neuronal phenotype. However, results from the microarray were only partially validated by quantitative RT-PCR analysis. The results indicate that LCM harvesting of specific neuronal phenotypes can be effectively guided in a complex cellular environment by specific pre-labeling of the target cell populations and underlie the importance of independent validation of microarray results.

Mediation of the DCC apoptotic signal by DIP13 alpha.

DCC (deleted in colorectal cancer) is a candidate tumor suppressor gene. However the function of DCC remains elusive. Previously, we demonstrated that forced expression of DCC induces apoptosis or cell cycle arrest (Chen, Y. Q., Hsieh, J. T., Yao, F., Fang, B., Pong, R. C., Cipriano, S. C. & Krepulat, F. (1999) Oncogene 18, 2747-2754). To delineate the DCC-induced apoptotic pathway, we have identified a protein, DIP13 alpha, which interacts with DCC. The DIP13 alpha protein has a pleckstrin homology domain and a phosphotyrosine binding domain. It interacts with a region on the DCC cytoplasmic domain that is required for the induction of apoptosis. Although ectopic expression of DIP13 alpha alone causes only a slight increase in apoptosis, co-expression of DCC and DIP13 alpha results in an approximately 5-fold increase in apoptosis. Removal of the DCC-interacting domain on DIP13 alpha abolishes its ability to enhance DCC-induced apoptosis. Inhibition of endogenous DIP13 alpha expression by small interfering RNA blocks DCC-induced apoptosis. Our data suggest that DIP13 alpha is a mediator of the DCC apoptotic pathway.