Single-cell transcriptome landscape of circulating CD4+ T cell populations in autoimmune diseases.

CD4+ T cells are key mediators of various autoimmune diseases; however, their role in disease progression remains unclear due to cellular heterogeneity. Here, we evaluated CD4+ T cell subpopulations using decomposition-based transcriptome characterization and canonical clustering strategies. This approach identified 12 independent gene programs governing whole CD4+ T cell heterogeneity, which can explain the ambiguity of canonical clustering. In addition, we performed a meta-analysis using public single-cell datasets of over 1.8 million peripheral CD4+ T cells from 953 individuals by projecting cells onto the reference and cataloging cell frequency and qualitative alterations of the populations in 20 diseases. The analyses revealed that the 12 transcriptional programs were useful in characterizing each autoimmune disease and predicting its clinical status. Moreover, genetic variants associated with autoimmune diseases showed disease-specific enrichment within the 12 gene programs. The results collectively provide a landscape of single-cell transcriptomes of CD4+ T cell subpopulations involved in autoimmune disease.

A genetic method specifically delineates Th1-type Treg cells and their roles in tumor immunity.

Regulatory T (Treg) cells expressing the transcription factor (TF) Foxp3 also express other TFs shared by T helper (Th) subsets under certain conditions. Here, to determine the roles of T-bet-expressing Treg cells, we generate a mouse strain, called VeDTR, in which T-bet/Foxp3 double-positive cells are engineered to be specifically labeled and depleted by a combination of Cre- and Flp-recombinase-dependent gene expression control. Characterization of T-bet+Foxp3+ cells using VeDTR mice reveals high resistance under oxidative stress, which is involved in accumulation of T-bet+Foxp3+ cells in tumor tissues. Moreover, short-term depletion of T-bet+Foxp3+ cells leads to anti-tumor immunity but not autoimmunity, whereas that of whole Treg cells does both. Although ablation of T-bet+Foxp3+ cells during Toxoplasma infection slightly enhances Th1 immune responses, it does not affect the course of the infection. Collectively, the intersectional genetic method reveals the specific roles of T-bet+Foxp3+ cells in suppressing tumor immunity.

Protocol to evaluate cell lineage stability of mouse natural and induced regulatory T cells using bisulfite sequencing.

The establishment of regulatory T cells (Treg)-specific demethylation regions (TSDRs) is essential for the Treg-lineage stability. Here, we present a protocol using bisulfite sequencing to assess Treg-lineage stability. The protocol describes the isolation of lymphocytes and DNA extraction, followed by bisulfite conversion in unmethylated CpG DNA, bisulfite PCR and cloning, and sequencing to define the TSDR methylation. This protocol uses lymph nodes and spleen tissues and can be adapted to assess the methylation status of Tregs in other tissue types.

Myasthenia gravis-specific aberrant neuromuscular gene expression by medullary thymic epithelial cells in thymoma.

Myasthenia gravis (MG) is a neurological disease caused by autoantibodies against neuromuscular-associated proteins. While MG frequently develops in thymoma patients, the etiologic factors for MG are not well understood. Here, by constructing a comprehensive atlas of thymoma using bulk and single-cell RNA-sequencing, we identify ectopic expression of neuromuscular molecules in MG-type thymoma. These molecules are found within a distinct subpopulation of medullary thymic epithelial cells (mTECs), which we name neuromuscular mTECs (nmTECs). MG-thymoma also exhibits microenvironments dedicated to autoantibody production, including ectopic germinal center formation, T follicular helper cell accumulation, and type 2 conventional dendritic cell migration. Cell-cell interaction analysis also predicts the interaction between nmTECs and T/B cells via CXCL12-CXCR4. The enrichment of nmTECs presenting neuromuscular molecules within MG-thymoma is further confirmed immunohistochemically and by cellular composition estimation from the MG-thymoma transcriptome. Altogether, this study suggests that nmTECs have a significant function in MG pathogenesis via ectopic expression of neuromuscular molecules.

Distinct Foxp3 enhancer elements coordinate development, maintenance, and function of regulatory T cells.

The transcription factor Foxp3 plays crucial roles for Treg cell development and function. Conserved non-coding sequences (CNSs) at the Foxp3 locus control Foxp3 transcription, but how they developmentally contribute to Treg cell lineage specification remains obscure. Here, we show that among Foxp3 CNSs, the promoter-upstream CNS0 and the intergenic CNS3, which bind distinct transcription factors, were activated at early stages of thymocyte differentiation prior to Foxp3 promoter activation, with sequential genomic looping bridging these regions and the promoter. While deletion of either CNS0 or CNS3 partially compromised thymic Treg cell generation, deletion of both completely abrogated the generation and impaired the stability of Foxp3 expression in residual Treg cells. As a result, CNS0 and CNS3 double-deleted mice succumbed to lethal systemic autoimmunity and inflammation. Thus, hierarchical and coordinated activation of Foxp3 CNS0 and CNS3 initiates and stabilizes Foxp3 gene expression, thereby crucially controlling Treg cell development, maintenance, and consequently immunological self-tolerance.

Morphological and phenotypical diversity of eosinophils in the rat ileum.

Eosinophils are abundantly present in intestinal mucosa. However, the morphological characteristics of their cellular population are still largely unknown. In this study, we examine their characteristics in the rat ileal mucosa using histological and ultrastructural methods. The results indicated that ileal eosinophils could be distinguished into two main groups based on their nuclear shapes and distribution: eosinophils with spheric or reniform nuclei mainly localized in the villous region and eosinophils with annular or bacilliform nuclei as the major population around crypts. Immunohistochemical analysis revealed that all eosinophils in the lamina propria (LP) were immunopositive for CD11b, whereas eosinophils in LP of the intestinal villus but not those in LP around the crypt, were immunopositive for CD11c. Three-dimensional ultrastructural analysis using serial block-face scanning electron microscopy showed that the eosinophils with spheric or reniform nuclei were abundant in the upper portions of the intestinal villus, whereas those with annular nuclei were abundant in the lower portions of the intestinal villus and around crypts. The eosinophils with spheric or reniform nuclei possessed broader cellular bodies with greater abundance of surface projections compared with those with annular nuclei. Eosinophils in the upper portions of intestinal villus frequently extended their cellular bodies into the intraepithelial space. The number of total and eosinophil-specific granules was positively correlated with the minor axis of the nuclear holes in the annular nuclei. These data suggest that ileal eosinophils exhibit not homogenous but rather diverse characteristics, possible due to the mixture of eosinophils at different maturation and/or activation stages.

Effects of the expansion of bacterial colonies into the intervillous spaces on the localization of several lymphocyte lineages in the rat ileum.

The effect of bacterial colonies expanded into the intervillous spaces on the localization of several lymphocyte lineages was immunohistochemically investigated in two types of mucosa: ordinary mucosa of rat ileum, which consists of mucosa without any mucosal lymphatic tissue; and follicle-associated mucosa (FAM), which accompanies the parafollicular area under the muscularis mucosae in the rat ileal Peyer's patch. The results showed that bacterial colonies in the intervillous spaces induced increased populations of CD8+ cells in the epithelium of the intestinal villus in ordinary mucosa (IV) and intestinal villus in FAM (IV-FAM). Bacterial colonies in the intervillous spaces were also associated with increased numbers of IgA+ cells, which were mainly localized in the lamina propria of basal portions of IV and IV-FAM, and with expanded localization of IgA+ cells into the villous apex in both IV and IV-FAM. Moreover, IgA+ cells around the intestinal crypts adjacent to IV or IV-FAM were also increased in response to bacterial colonies. In the IV-FAM, but not IV, L-selectin+ cells, which were found to be immunopositive for TCRαß or CD19, were drastically increased in the lamina propria from the crypt to middle portion of IV-FAM and in the lumen of central lymph vessel of IV-FAM in response to the bacterial colonies in the intervillous spaces. These findings revealed that the expansion of bacterial colonies into the intervillous spaces accompanies the change of histological localization of the lymphocyte lineage in both the ordinary mucosa and FAM.

Immunohistochemical study on the distribution of ß-defensin 1 and ß-defensin 2 throughout the respiratory tract of healthy rats.

The distributions of ß-defensin 1 and 2 in secretory host defense system throughout respiratory tract of healthy rats were immunohistochemically investigated. In the nasal epithelium, a large number of non-ciliated and non-microvillous cells (NCs) were immunopositive for both ß-defensin 1 and 2, whereas a small number of goblet cells (GCs) were immunopositive only for ß-defensin 1. Beta-defensin 2-immunopositive GCs were few. In the nasal glands, a small number of acinar cells and a large number of ductal epithelial cells were immunopositive for both ß-defensins. In the laryngeal and tracheal epithelia, a very few NCs and GCs were immunopositive for both ß-defensins. In laryngeal and tracheal glands, a very few acinar cells and a large number of ductal epithelial cells were immunopositive for both ß-defensins. In the extra-pulmonary bronchus, a small number of NCs were immunopositive for both ß-defensins. A small number of GCs were immunopositive for ß-defensin 1, whereas few GCs were immunopositive for ß-defensin 2. From the intra-pulmonary bronchus to alveoli, a very few or no epithelial cells were immunopositive for both ß-defensins. In the mucus and periciliary layers, ß-defensin 1 was detected from the nose to the extra-pulmonary bronchus, whereas ß-defensin 2 was weakly detected only in the nose and the larynx. These findings suggest that the secretory sources of ß-defensin 1 and 2 are mainly distributed in the nasal mucosa and gradually decrease toward the caudal airway in healthy rats.

Immunohistochemical study on the secretory host defense system with lysozyme and secretory phospholipase A2 throughout rat respiratory tract.

The host defense system with lysozyme and secretory phospholipase A2 (sPLA2) was immunohistochemically investigated in rat respiratory tract under healthy conditions. In the nasal epithelium, a large number of non-ciliated and non-microvillous cells (NC) and a small number of goblet cells (GC) were immunopositive for lysozyme and sPLA2. A few acinar cells and almost all epithelial cells of intercalated ducts were immunopositive for both bactericidal substances in the nasal glands. In the laryngeal and tracheal epithelia, few NC and GC were immunopositive for both bactericidal substances. In the laryngeal and tracheal glands, a few acinar cells and most ductal epithelial cells were immunopositive for both bactericidal substances. In extra-pulmonary bronchus, small numbers of NC and GC were immunopositive for lysozyme and sPLA2, whereas few NC and no GC were immunopositive in the intra-pulmonary bronchus. No secretory source of either bactericidal substance was located in the bronchioles. In the alveolus, many glandular epithelial cells and alveolar macrophages were immunopositive for lysozyme but immunonegative for sPLA2. Moreover, lysozyme and sPLA2 were detected in the mucus layer and in the periciliary layer from the nose to the extra-pulmonary bronchus. These findings suggest that secretory sources of lysozyme and sPLA2 are distributed in almost all the respiratory tract. Their secretory products are probably transported to the pharynx and contribute to form the first line of defense against inhaled bacteria throughout the respiratory tract.

Mechanism of M-cell differentiation accelerated by proliferation of indigenous bacteria in rat Peyer's patches.

The mechanism by which indigenous bacteria on the follicle-associated epithelium (FAE) of lymphatic follicles (LFs) accelerate the differentiation of microvillous columnar epithelial cells (MV) into M-cells was immunohistochemically investigated in rat Peyer's patches. The results showed that the number of Toll-like receptor (TLR) -4+ M-cells was greater in the FAE with expansion of bacterial colonies (LFs with bacterial colonies on the FAE: b-LF) than the FAE without expansion of bacterial colonies (nb-LF). TLR-4 was also expressed in the striated borders of MV upstream next to M-cells in the FAE of the b-LF. TLR-4+ vesicles were frequently detected in the cytoplasms of MV with TLR-4+ striated borders upstream next to TLR-4+ M-cells in the FAE of b-LF. These findings suggest that TLR-4+ MV take up TLR-4 ligands and differentiate into M-cells in the b-LF. Neither the distribution of RANK nor that of RANKL was coincident with that of M-cells in the b-LF. Moreover, RANK, but not RANKL, was expressed in intestinal villi, whereas cleaved caspase-3 was immunonegative in the MV and M-cells of the FAE, unlike in villous epithelial cells. Therefore, RANK/RANKL signaling in the LF might contribute to the down-regulation of epithelial apoptosis to facilitate the differentiation of MV into M-cells in rat Peyer's patches.

Neutralized nanoparticle composed of SS-cleavable and pH-activated lipid-like material as a long-lasting and liver-specific gene delivery system.

Charge-neutralized lipid envelope-type nanoparticles formed with SS-cleavable and pH-activated lipid-like materials (ssPalm) accumulate rapidly in the liver without forming aggregates in the blood circulation, and result in a liver-specific gene expression for a long duration (>2 weeks) with neither immunological responses nor hepatotoxicity after intraveneous administration, when it carries pDNA free from CpG-motifs.

Neutral biodegradable lipid-envelope-type nanoparticle using vitamin A-Scaffold for nuclear targeting of plasmid DNA.

Biomembranes and cytoplasm, a diffusion-limited region for nanoparticles are critical barriers to be overcome for the successful gene delivery. We herein report on a neutral, and intracellularly degradable lipid nanoparticle (LNP), containing encapsulated plasmid DNA (pDNA) that can be effectively delivered to the nucleus. A key material component in this particle is a vitamin A-scaffold SS-cleavable Proton-Activated Lipid-like Material ((SS)PalmA), which contains tertiary amine groups as proton sponge units that can respond to the acidic pH in endosomes, disulfide bonding for programmed collapse in the cytoplasm, and retinoic acid (RA) as a hydrophobic unit for assembly into LNP. LNP prepared using (SS)PalmA (LNP(PalmA)) exhibited a 15-fold higher gene expression activity compared to particles prepared with a simple acyl chain (myristoyl group)-scaffold one (LNPPalmM). Intracellular imaging studies revealed that LNP(PalmA) unexpectedly showed excessive endosome-disruptive characteristics. Furthermore, the decapsulation of pDNA slowly, but successively occurred in parallel with peri-nuclear accumulation. Nuclear targeting was blocked in the presence of native RA. Collectively, LNP(PalmA) is an intelligent particle that passes through the cytoplasm in particle form with the aid of the intrinsic nuclear transport system of RA, and thereafter releases its encapsulated pDNA for effective gene expression.

The application of pH-sensitive polymer-lipids to antigen delivery for cancer immunotherapy.

For production of pH-sensitive liposomes, we developed pH-sensitive polymer-lipids that consists of pH-sensitive fusogenic polymer moieties such as 3-methyl glutarylated poly(glycidol) and 2-carboxycyclohexane-1-carboxylated poly(glycidol), connected to a phosphatidylethanolamine head group. Incorporation of these pH-sensitive polymer-lipids into egg yolk phosphatidylcholine liposomes produced highly pH-sensitive liposomes that were stable at neutral pH but which destabilized markedly in response to very small pH change in weakly acidic pH region. These liposomes delivered their contents (pyranine) into cytosol of dendritic cell-derived DC2.4 cells. When these polymer-lipid-incorporated liposomes loaded with antigenic protein ovalbumin (OVA) were administered subcutaneously to mice, the antigen-specific cellular immunity was induced efficiently in the mice. Furthermore, immunization of mice with these OVA-loaded pH-sensitive polymer-lipid-incorporated liposomes induced strong OVA-specific immunity, which achieved complete rejection of OVA-expressing E.G7-OVA cells and marked regression of E.G7-OVA tumors.

A neutral envelope-type nanoparticle containing pH-responsive and SS-cleavable lipid-like material as a carrier for plasmid DNA.

SS-cleavable proton-activated lipid-like material (ssPalm) functions as a key element in a lipid nanoparticle in which pDNA is encapsulated. The ssPalm contains dual sensing motifs that can respond to the intracellular environment; a proton-sponge unit (tertiary amines) that functions in response to an acidic environment (endosome/lysosome), and disulfide bonding that can be cleaved in a reducing environment (cytosol).

Vitamin E inhibits oxidative stress-induced denaturation of nerve terminal proteins involved in neurotransmission.

One characteristic of age-related neurodegeneration is thought to be cognitive deficits caused by oxidative stress. Neurons in the brain are considered to be particularly vulnerable to oxidative stress, leading to neuronal oxidative damage and neurodegenerative disorders such as Alzheimer's disease (AD) and senile dementia. The process of fusing synaptic plasma membranes and synaptic vesicles involves particular proteins, such as the soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) proteins for docking both membranes, and is integral to neurotransmission. To elucidate whether oxidative stress induces denaturation of SNARE proteins, and whether vitamin E can counteract this process, changes in the expression of synaptobrevin, synaptotagmin, SNAP-25, and syntaxin-1 in rat brain nerve terminals were analyzed using an immunoblotting method. The results showed that oxidative stress induced significant reductions in the levels synaptobrevin and synaptotagmin in synaptic vesicles. Similarly, marked decreases in the levels of SNAP-25 and syntaxin-1 in pre-synaptic plasma membranes were also observed. In the absence of oxidative stress, vitamin E-deficient rats exhibited similar decreases in these proteins. In contrast, it was found that decreases in SNARE proteins, except for SNAP-25, were not observed in vitamin E-supplemented rats, even when the rats were subjected to oxidative stress. These results suggest that reactive oxygen species generated by oxidative stress are detrimental to neurons, resulting in the oxidation of SNARE proteins, thereby disrupting neurotransmission. Additionally, vitamin E is capable of protecting against such neurodegeneration.

Dysfunction of the fusion of pre-synaptic plasma membranes and synaptic vesicles caused by oxidative stress, and its prevention by vitamin E.

To define whether hyperoxia induces the dysfunction of membrane fusion between synaptic vesicles with pre-synaptic plasma membranes in the nerve terminals, and whether vitamin E prevents this abnormal event, we investigated the influence of hyperoxia on the fusion ability of isolated synaptic vesicles and the inside-out type pre-synaptic plasma membrane vesicles from rat brain using the fluorescence tracing method. The membrane fusion ability of both membranes from rats subjected to hyperoxia was markedly decreased compared with the membranes from a normal rat. Rats subjected to hyperoxia in the form of oxidative stress showed significant increases in the levels of thiobarbituric acid reactive substances (TBARS), conjugated dienes, and protein carbonyl moieties in both synaptic vesicles and pre-synaptic plasma membranes. When rats were supplemented with vitamin E, these abnormalities were inhibited even when rats were subjected to hyperoxia.

Influence of oxidative stress on fusion of pre-synaptic plasma membranes of the rat brain with phosphatidyl choline liposomes, and protective effect of vitamin E.

Influence of oxidative stress on fusion of pre-synaptic plasma membranes with phosphatidylcholine (PC) liposomes as a model of synaptic vesicle was investigated. The inhibitory effect of vitamin E on the decline in the fusion caused by oxidative stress was also assessed. Rats subjected to hyperoxia as oxidative stress showed significant increases in the levels of lipid hydroperoxides and protein carbonyl moieties in pre-synaptic plasma membranes in the brain. The zeta potential of pre-synaptic membrane surface was decreased markedly. When synaptosomes were incubated with PC liposomes labeled by either rhodamine B or calcein as a fluorescence probe, or 12-doxyl stearic acid as an ESR spin trapping agent, translocation of each probe into oxidatively damaged pre-synaptic membranes was decreased significantly. Fatty acid composition analysis in pre-synaptic membranes obtained from normal rats revealed a marked increase in linoleic acid and a moderate decrease in docosahexaenoic content after the incubation with liposomes. However, rats subjected to hyperoxia did not show marked changes in these fatty acid contents in their pre-synaptic membranes after the incubation. Such changes caused by hyperoxia were inhibited by vitamin E treatment of rats. These results suggest that oxidative damage of pre-synaptic membranes caused by oxidative stress lowers the lipid-mixing for the membrane fusion. The results of this study imply that vitamin E prevents the deficit in neurotransmission at nerve terminals due to the decline in fusion between pre-synaptic membrane and synaptic vesicles caused by oxidative membrane damage.

Synthesis and isolation of cobalt hexacyanoferrate/chromate metal coordination nanopolymers stabilized by alkylamino ligand with metal elemental control.

This Communication describes the novel isolation with metal elemental control of cobalt hexacyanoferrate/chromate metal coordination polymers, stabilized by stearylamine (Co-Fe/Cr-SA) as a protecting coordination ligand in a reverse micelle technique. Each Co-Fe/Cr-SA can be isolated with high uniformity of particle size and elemental composition, and the ratio of the metal component depends on the fundamental characteristics of Co-Fe/Cr-SA: the nanopolymer's shape, color, and magnetism.