Adaptive immune regulation in the gut: T cell-dependent and T cell-independent IgA synthesis.

In mammals, the gastrointestinal tract harbors an extraordinarily dense and complex community of microorganisms. The gut microbiota provide strong selective pressure to the host to evolve adaptive immune responses required for the maintenance of local and systemic homeostasis. The continuous antigenic presence in the gut imposes a dynamic remodeling of gut-associated lymphoid tissues (GALT) and the selection of multiple layered strategies for immunoglobulin (Ig) A production. The composite and dynamic gut environment also necessitates heterogeneous, versatile, and convertible T cells, capable of inhibiting (Foxp3(+) T cells) or helping (T(FH) cells) local immune responses. In this review, we describe recent advances in our understanding of dynamic pathways that lead to IgA synthesis, in gut follicular structures and in extrafollicular sites, by T cell-dependent and T cell-independent mechanisms. We discuss the finely tuned regulatory mechanisms for IgA production and emphasize the role of mucosal IgA in the selection and maintenance of the appropriate microbial composition that is necessary for immune homeostasis.

Interspecies Chimerism with Mammalian Pluripotent Stem Cells.

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

Selective elimination of mitochondrial mutations in the germline by genome editing.

Mitochondrial diseases include a group of maternally inherited genetic disorders caused by mutations in mtDNA. In most of these patients, mutated mtDNA coexists with wild-type mtDNA, a situation known as mtDNA heteroplasmy. Here, we report on a strategy toward preventing germline transmission of mitochondrial diseases by inducing mtDNA heteroplasmy shift through the selective elimination of mutated mtDNA. As a proof of concept, we took advantage of NZB/BALB heteroplasmic mice, which contain two mtDNA haplotypes, BALB and NZB, and selectively prevented their germline transmission using either mitochondria-targeted restriction endonucleases or TALENs. In addition, we successfully reduced human mutated mtDNA levels responsible for Leber's hereditary optic neuropathy (LHOND), and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in mammalian oocytes using mitochondria-targeted TALEN (mito-TALENs). Our approaches represent a potential therapeutic avenue for preventing the transgenerational transmission of human mitochondrial diseases caused by mutations in mtDNA. PAPERCLIP.

Metabolic shift induced by systemic activation of T cells in PD-1-deficient mice perturbs brain monoamines and emotional behavior.

T cells reorganize their metabolic profiles after being activated, but the systemic metabolic effect of sustained activation of the immune system has remained unexplored. Here we report that augmented T cell responses in Pdcd1-/- mice, which lack the inhibitory receptor PD-1, induced a metabolic serum signature characterized by depletion of amino acids. We found that the depletion of amino acids in serum was due to the accumulation of amino acids in activated Pdcd1-/- T cells in the lymph nodes. A systemic decrease in tryptophan and tyrosine led to substantial deficiency in the neurotransmitters serotonin and dopamine in the brain, which resulted in behavioral changes dominated by anxiety-like behavior and exacerbated fear responses. Together these data indicate that excessive activation of T cells causes a systemic metabolomic shift with consequences that extend beyond the immune system.

B cell-derived GABA elicits IL-10+ macrophages to limit anti-tumour immunity.

Small, soluble metabolites not only are essential intermediates in intracellular biochemical processes, but can also influence neighbouring cells when released into the extracellular milieu1-3. Here we identify the metabolite and neurotransmitter GABA as a candidate signalling molecule synthesized and secreted by activated B cells and plasma cells. We show that B cell-derived GABA promotes monocyte differentiation into anti-inflammatory macrophages that secrete interleukin-10 and inhibit CD8+ T cell killer function. In mice, B cell deficiency or B cell-specific inactivation of the GABA-generating enzyme GAD67 enhances anti-tumour responses. Our study reveals that, in addition to cytokines and membrane proteins, small metabolites derived from B-lineage cells have immunoregulatory functions, which may be pharmaceutical targets allowing fine-tuning of immune responses.

Endonuclease Q as a robust enhancer for nucleic acid amplification.

Isothermal nucleic acid amplification techniques are attracting increasing attention in molecular diagnosis and biotechnology. However, most existing techniques are complicated by the need for intricate primer design and numerous enzymes and primers. Here, we have developed a simple method, termed NAQ, that employs adding both endonuclease Q (EndoQ) and dUTP/dITP to conventional rolling circle amplification reactions to increase DNA amplification. NAQ does not require intricate primer design or DNA sequence-specific enzymes, and existing isothermal amplification techniques could be readily adapted to include both EndoQ and dUTP/dITP.

In vivo reprogramming of wound-resident cells generates skin epithelial tissue.

Large cutaneous ulcers are, in severe cases, life threatening1,2. As the global population ages, non-healing ulcers are becoming increasingly common1,2. Treatment currently requires the transplantation of pre-existing epithelial components, such as skin grafts, or therapy using cultured cells2. Here we develop alternative supplies of epidermal coverage for the treatment of these kinds of wounds. We generated expandable epithelial tissues using in vivo reprogramming of wound-resident mesenchymal cells. Transduction of four transcription factors that specify the skin-cell lineage enabled efficient and rapid de novo epithelialization from the surface of cutaneous ulcers in mice. Our findings may provide a new therapeutic avenue for treating skin wounds and could be extended to other disease situations in which tissue homeostasis and repair are impaired.

Modeling the marmoset brain using embryonic stem cell-derived cerebral assembloids.

Studying the non-human primate (NHP) brain is required for the translation of rodent research to humans, but remains a challenge for molecular, cellular, and circuit-level analyses in the NHP brain due to the lack of in vitro NHP brain system. Here, we report an in vitro NHP cerebral model using marmoset (Callithrix jacchus) embryonic stem cell-derived cerebral assembloids (CAs) that recapitulate inhibitory neuron migration and cortical network activity. Cortical organoids (COs) and ganglionic eminence organoids (GEOs) were induced from cjESCs and fused to generate CAs. GEO cells expressing the inhibitory neuron marker LHX6 migrated toward the cortical side of CAs. COs developed their spontaneous neural activity from a synchronized pattern to an unsynchronized pattern as COs matured. CAs containing excitatory and inhibitory neurons showed mature neural activity with an unsynchronized pattern. The CAs represent a powerful in vitro model for studying excitatory and inhibitory neuron interactions, cortical dynamics, and their dysfunction. The marmoset assembloid system will provide an in vitro platform for the NHP neurobiology and facilitate translation into humans in neuroscience research, regenerative medicine, and drug discovery.

Correction of a pathogenic gene mutation in human embryos.

Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR-Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains to be considered before clinical applications, including the reproducibility of the technique with other heterozygous mutations.

Altered Pharmacological Efficacy of Phenobarbital with the Treatment of 7,8-Dihydroxyflavone, an Agonist of Tropomyosin Receptor Kinase B, in Rats.

From our previous observation that the anesthetic effects of phenobarbital potentiate in rats with a decreased cerebral protein expression of the potassium chloride cotransporter KCC2 (SLC12A5), an in vivo study was conducted to clarify whether the pharmacological effect of phenobarbital alters by stimulating the cerebral tropomyosin receptor kinase B (TrkB) that is known to down-regulate the KCC2 protein expression. The stimulation was performed in rats with repetitious intraperitoneal administration of a TrkB agonist, namely 7,8-dihydroxyflavone (DHF). After that, the rats underwent an intraventricular infusion of phenobarbital using a dwelled cannula, and the onset time of the phenobarbital-induced general anesthesia was determined. In addition, their brain tissues were excised and cerebral cortices were collected. Then, subcellular fractions were prepared and the cerebral expression of various proteins involving the anesthetic effects of phenobarbital was examined. It was demonstrated that phenobarbital induced general anesthesia about 2 times faster in rats receiving the DHF treatment than in control rats, and that the phenobarbital amount in the brain tissue at the onset time of anesthesia was lower in rats with the treatment. Western blotting showed that the cerebral protein expression of KCC2 decreases, and the phosphorylation of the TrkB protein increases with the DHF treatment. These observations indicate that the anesthetic effects of phenobarbital potentiate with the TrkB stimulation and the resultant decrease in the cerebral KCC2 protein expression. The results also suggest that the TrkB protein and its phosphorylation status may be a key modulator of the pharmacological efficacy of phenobarbital.

B cell-specific and stimulation-responsive enhancers derepress Aicda by overcoming the effects of silencers.

Activation-induced cytidine deaminase (AID) is essential for the generation of antibody memory but also targets oncogenes, among other genes. We investigated the transcriptional regulation of Aicda (which encodes AID) in class switch-inducible CH12F3-2 cells and found that Aicda regulation involved derepression by several layers of positive regulatory elements in addition to the 5' promoter region. The 5' upstream region contained functional motifs for the response to signaling by cytokines, the ligand for the costimulatory molecule CD40 or stimuli that activated the transcription factor NF-kappaB. The first intron contained functional binding elements for the ubiquitous silencers c-Myb and E2f and for the B cell-specific activator Pax5 and E-box-binding proteins. Our results show that Aicda is regulated by the balance between B cell-specific and stimulation-responsive elements and ubiquitous silencers.

Usefulness of rapid MR angiography using two-point Dixon for evaluating carotid and aortic plaques.

PURPOSE: Recently, various magnetic resonance imaging (MRI) modalities have been developed to easily detect carotid and aortic plaques, but these techniques are time-consuming and vulnerable to motion artifacts. We investigated the utility of a gradient echo MRI technique known as liver acquisition with volume acceleration flexible (LAVA-Flex) to detect carotid and aortic atherosclerotic plaques. METHODS: Ten patients who underwent carotid endarterectomy (CEA) were assessed regarding the correspondence between LAVA-Flex findings and the histopathology of excised carotid plaques. In addition, 47 patients with cryptogenic ischemic stroke underwent LAVA-Flex and transesophageal echocardiography (TEE) for detection of embolic sources in the thoracic aorta. We analyzed the relationship between the thickness of the aortic plaque measured by TEE and the presence of high-intensity lesions on LAVA-Flex. RESULTS: Nine of 10 patients (90.0%) who underwent CEA showed a high-intensity carotid lesion on LAVA-Flex, which corresponded pathologically to plaques containing large lipid cores and hemorrhage. Twenty-four (51.1%) of 47 cryptogenic stroke patients showed a high-intensity lesion in the thoracic aorta on LAVA-Flex; of these, 21 (87.5%) also demonstrated a large plaque (thickness ≥4 mm) on TEE. Twenty-two (95.7%) of 23 patients without a high-intensity lesion on LAVA-Flex demonstrated no large plaque on TEE. LAVA-Flex had a sensitivity of 95.5% and a specificity of 88.0% in patients with large plaques. CONCLUSION: This study showed that LAVA-Flex successfully detected carotid and aortic plaques. This imaging technique may be useful to rapidly diagnose and evaluate carotid and aortic plaques, which are critical risk factors for aortogenic stroke.

In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration.

Targeted genome editing via engineered nucleases is an exciting area of biomedical research and holds potential for clinical applications. Despite rapid advances in the field, in vivo targeted transgene integration is still infeasible because current tools are inefficient, especially for non-dividing cells, which compose most adult tissues. This poses a barrier for uncovering fundamental biological principles and developing treatments for a broad range of genetic disorders. Based on clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, we demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.

ChREBP deficiency prevents high sucrose diet-induced obesity through reducing sucrase expression.

Obesity appears to be a major contributing factor for many health problems. Effective treatments for reducing weight gain, other than caloric restriction and exercise, are limited. The consumption of sugars is a major factor in the development of obesity in part by stimulating the transcription factor, carbohydrate response element binding protein (ChREBP), a process that is driven by de novo lipogenesis. Therefore, we hypothesized that inhibiting the action of ChREBP would be a promising strategy for alleviating these diseases. Using ChREBP deficient mice, the effect of a high intake of sucrose on body weight and blood glucose levels were investigated. Unlike wild type mice, ChREBP deficient mice did not gain much weight and their blood glucose and cholesterol levels remained relatively constant. In tracing it's cause, we found that the levels of expression of sucrase, an enzyme that digests sucrose, and both Glut2 and Glut5, a transporter of glucose and fructose, were not induced by feeding a high sucrose diet in the small intestine of ChREBP deficient mice. Our findings suggest that the inhibition of ChREBP could suppress weight gain even on a high sucrose diet.

A lack of ChREBP inhibits mitochondrial cristae formation in brown adipose tissue.

The carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor that increases the transcription of multiple genes. ChREBP is highly localized in the liver, where it upregulates the expression of genes that code for glycolytic and lipogenic enzymes, resulting in the conversion of excess carbohydrate into storage fat. ChREBP knockout (KO) mice display an anti-obese phenotype. However, at this time, role of ChREBP in adipose tissue remains unclear. Therefore, the energy metabolism and morphology of mitochondrial brown adipose tissue (BAT) in ChREBP KO mice was examined. We found increased expression levels of electron transport system proteins including the mitochondrial uncoupling protein (UCP1), and mitochondrial structural alterations such as dysplasia of the cristae and the presence of small mitochondria in BAT of ChREBP KO mice. Mass spectrometry analyses revealed that fatty acid synthase was absent in the BAT of ChREBP KO mice, which probably led to a reduction in fatty acids and cardiolipin, a regulator of various mitochondrial events. Our study clarified the new role of ChREBP in adipose tissue and its involvement in mitochondrial function. A clearer understanding of ChREBP in mitochondria could pave the way for improvements in obesity management.

An alternative pluripotent state confers interspecies chimaeric competency.

Pluripotency, the ability to generate any cell type of the body, is an evanescent attribute of embryonic cells. Transitory pluripotent cells can be captured at different time points during embryogenesis and maintained as embryonic stem cells or epiblast stem cells in culture. Since ontogenesis is a dynamic process in both space and time, it seems counterintuitive that these two temporal states represent the full spectrum of organismal pluripotency. Here we show that by modulating culture parameters, a stem-cell type with unique spatial characteristics and distinct molecular and functional features, designated as region-selective pluripotent stem cells (rsPSCs), can be efficiently obtained from mouse embryos and primate pluripotent stem cells, including humans. The ease of culturing and editing the genome of human rsPSCs offers advantages for regenerative medicine applications. The unique ability of human rsPSCs to generate post-implantation interspecies chimaeric embryos may facilitate our understanding of early human development and evolution.

Acute Peripheral Inflammation Increases Plasma Concentration of Hypoglycemic Agent Nateglinide with Decreased Hepatic Drug-Metabolizing Activity in Rats.

The effects of inflammation on hypoglycemic agents were evaluated in male rats with acute peripheral inflammation (API). Nateglinide (NTG) was utilized as a model compound, since it is a hepatically-metabolized compound and its metabolism is mainly mediated by CYP 2C11 enzyme. In the experiments, rats were subjected to carrageenan injection into their hind paws for API induction, and the plasma concentration profiles of NTG were then examined. In addition, pooled liver microsomes were prepared from control and API rats, and the hepatic drug-metabolizing activity toward NTG and the hepatic expression of CYP2C11 protein were evaluated. It was shown that the plasma concentration of NTG following its intravenous administration decreases at a slower rate in API rats than that in control rats. It was also indicated in the incubation study with the liver microsomes that the hepatic drug-metabolizing activity toward NTG decreases in API rats. Additionally, it was revealed in Western immunoblotting that the hepatic expression of CYP2C11 protein decreases in API rats. These findings suggest that inflammation occurring in peripheral tissues brings about a decrease in hepatic NTG metabolism by suppressing the hepatic expression of CYP2C11 protein, causing an alteration of the plasma concentration profile of NTG with its impaired elimination.

Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts.

Embryonic stem cells (ESCs) are derived from the inner cell mass of preimplantation blastocysts. From agricultural and biomedical perspectives, the derivation of stable ESCs from domestic ungulates is important for genomic testing and selection, genome engineering, and modeling human diseases. Cattle are one of the most important domestic ungulates that are commonly used for food and bioreactors. To date, however, it remains a challenge to produce stable pluripotent bovine ESC lines. Employing a culture system containing fibroblast growth factor 2 and an inhibitor of the canonical Wnt-signaling pathway, we derived pluripotent bovine ESCs (bESCs) with stable morphology, transcriptome, karyotype, population-doubling time, pluripotency marker gene expression, and epigenetic features. Under this condition bESC lines were efficiently derived (100% in optimal conditions), were established quickly (3-4 wk), and were simple to propagate (by trypsin treatment). When used as donors for nuclear transfer, bESCs produced normal blastocyst rates, thereby opening the possibility for genomic selection, genome editing, and production of cattle with high genetic value.

Diversified IgA-Bacteria Interaction in Gut Homeostasis.

Immunoglobulin A (IgA) is the major immunoglobulin isotype produced by the gut immune system, and many studies revealed key roles of IgA in establishing host-bacteria mutualism. This chapter will review current understandings for the function of gut IgA in regulating commensal microbiota. IgA specifically recognizes bacterial species that strongly stimulate host's immune responses, and suppresses their overgrowth or reduces the expressions of bacterial pro-inflammatory genes. On the other hand, IgA coatings on symbiotic bacteria enhance bacteria-mucus and bacteria-bacteria interactions, which induce production of metabolites enforcing mucosal barrier functions. Such diversified effects suggest that multiple factors may be involved in the mechanisms of IgA-bacteria interactions, including IgA specificity to microbial epitopes, mode of cellular responses of IgA synthesis (T-dependent and T-independent) and post-translational modifications of IgA proteins, such as glycosylation.

Fostering of advanced mutualism with gut microbiota by Immunoglobulin A.

Immunoglobulin A (IgA), the most abundantly secreted antibody isotype in mammals, not only provides direct immune protection to neonates via maternal milk but also helps program the infant immune system by regulating the microbiota. IgA continues to maintain dynamic interactions with the gut microbiota throughout life and this influences immune system homeostasis as well as other physiological processes. The secretory IgA produced independently of T-cell selection are commonly referred to as natural or innate antibodies. Our studies have shown that innate-IgA, while effective at excluding microorganisms from the gut, does not promote mutualism with the microbiota in the same way as adaptive-IgA that is selected in T cell-dependent germinal center reactions. Adaptive-IgA fosters more advanced mutualism with the microbiota than innate-IgA by selecting and diversifying beneficial microbial communities. In this review, we suggest that the diversified microbiota resulting from adaptive-IgA pressure was pivotal in promoting ecological adaptability and speciation potential of mammals.