Cholinergic regulation of vascular endothelial function by human ChAT+ T cells.

Endothelial dysfunction and impaired vasodilation are linked with adverse cardiovascular events. T lymphocytes expressing choline acetyltransferase (ChAT), the enzyme catalyzing biosynthesis of the vasorelaxant acetylcholine (ACh), regulate vasodilation and are integral to the cholinergic antiinflammatory pathway in an inflammatory reflex in mice. Here, we found that human T cell ChAT mRNA expression was induced by T cell activation involving the PI3K signaling cascade. Mechanistically, we identified that ChAT mRNA expression was induced following the attenuation of RE-1 Silencing Transcription factor REST-mediated methylation of the ChAT promoter, and that ChAT mRNA expression levels were up-regulated by GATA3 in human T cells. In functional experiments, T cell-derived ACh increased endothelial nitric oxide-synthase activity, promoted vasorelaxation, and reduced vascular endothelial activation and promoted barrier integrity by a cholinergic mechanism. Further, we observed that survival in a cohort of patients with severe circulatory failure correlated with their relative frequency of ChAT +CD4+ T cells in blood. These findings on ChAT+ human T cells provide a mechanism for cholinergic immune regulation of vascular endothelial function in human inflammation.

PGC-1ß maintains mitochondrial metabolism and restrains inflammatory gene expression.

Metabolic programming of the innate immune cells known as dendritic cells (DCs) changes in response to different stimuli, influencing their function. While the mechanisms behind increased glycolytic metabolism in response to inflammatory stimuli are well-studied, less is known about the programming of mitochondrial metabolism in DCs. We used lipopolysaccharide (LPS) and interferon-ß (IFN-ß), which differentially stimulate the use of glycolysis and oxidative phosphorylation (OXPHOS), respectively, to identify factors important for mitochondrial metabolism. We found that the expression of peroxisome proliferator-activated receptor gamma co-activator 1ß (PGC-1ß), a transcriptional co-activator and known regulator of mitochondrial metabolism, decreases when DCs are activated with LPS, when OXPHOS is diminished, but not with IFN-ß, when OXPHOS is maintained. We examined the role of PGC-1ß in bioenergetic metabolism of DCs and found that PGC-1ß deficiency indeed impairs their mitochondrial respiration. PGC-1ß-deficient DCs are more glycolytic compared to controls, likely to compensate for reduced OXPHOS. PGC-1ß deficiency also causes decreased capacity for ATP production at steady state and in response to IFN-ß treatment. Loss of PGC-1ß in DCs leads to increased expression of genes in inflammatory pathways, and reduced expression of genes encoding proteins important for mitochondrial metabolism and function. Collectively, these results demonstrate that PGC-1ß is a key regulator of mitochondrial metabolism and negative regulator of inflammatory gene expression in DCs.

Epigenomic analysis of Parkinson's disease neurons identifies Tet2 loss as neuroprotective.

Parkinson's disease (PD) pathogenesis may involve the epigenetic control of enhancers that modify neuronal functions. Here, we comprehensively examine DNA methylation at enhancers, genome-wide, in neurons of patients with PD and of control individuals. We find a widespread increase in cytosine modifications at enhancers in PD neurons, which is partly explained by elevated hydroxymethylation levels. In particular, patients with PD exhibit an epigenetic and transcriptional upregulation of TET2, a master-regulator of cytosine modification status. TET2 depletion in a neuronal cell model results in cytosine modification changes that are reciprocal to those observed in PD neurons. Moreover, Tet2 inactivation in mice fully prevents nigral dopaminergic neuronal loss induced by previous inflammation. Tet2 loss also attenuates transcriptional immune responses to an inflammatory trigger. Thus, widespread epigenetic dysregulation of enhancers in PD neurons may, in part, be mediated by increased TET2 expression. Decreased Tet2 activity is neuroprotective, in vivo, and may be a new therapeutic target for PD.

Isolation of Cardiomyocytes Undergoing Mitosis With Complete Cytokinesis.

RATIONALE: Adult human cardiomyocytes do not complete cytokinesis despite passing through the S-phase of the cell cycle. As a result, polyploidization and multinucleation occur. To get a deeper understanding of the mechanisms surrounding division of cardiomyocytes, there is a crucial need for a technique to isolate cardiomyocytes that complete cell division/cytokinesis. OBJECTIVE: Markers of cell cycle progression based on DNA content cannot distinguish between mitotic cardiomyocytes that fail to complete cytokinesis from those cells that undergo true cell division. With the use of molecular beacons (MBs) targeting specific mRNAs, we aimed to identify truly proliferative cardiomyocytes derived from human induced pluripotent stem cells. METHODS AND RESULTS: Fluorescence-activated cell sorting combined with MBs was performed to sort cardiomyocyte populations enriched for mitotic cells. Expressions of cell cycle specific genes were confirmed by means of reverse transcription-quantitative polymerase chain reaction and single-cell RNA sequencing (scRNA-seq) combined with gene signatures of cell cycle progression. We characterized the sorted groups by proliferation assays and time-lapse microscopy which confirmed the proliferative advantage of MB-positive cell populations relative to MB-negative and G2/M populations. Gene expression analysis revealed that the MB-positive cardiomyocyte subpopulation exhibited patterns consistent with the processes of nuclear division, chromosome segregation, and transition from M to G1 phase. The use of dual-MBs targeting CDC20 and SPG20 mRNAs enabled the enrichment of cytokinetic events (CDC20highSPG20high). Interestingly, cells that did not complete cytokinesis and remained binucleated were found to be CDC20lowSPG20high while polyploid cardiomyocytes that replicated DNA but failed to complete karyokinesis were found to be CDC20lowSPG20low. CONCLUSIONS: This study demonstrates a novel alternative to existing DNA content-based approaches for sorting cardiomyocytes with true mitotic potential that can be used to study the unique dynamics of cardiomyocyte nuclei during mitosis. Our technique for sorting live cardiomyocytes undergoing cytokinesis would provide a basis for future studies to uncover mechanisms underlying the development and regeneration of heart tissue.

Inhibition of pro-inflammatory myeloid cell responses by short-term S100A9 blockade improves cardiac function after myocardial infarction.

AIMS: Neutrophils have both detrimental and beneficial effects in myocardial infarction (MI), but little is known about the underlying pathways. S100A8/A9 is a pro-inflammatory alarmin abundantly expressed in neutrophils that is rapidly released in the myocardium and circulation after myocardial ischaemia. We investigated the role of S100A8/A9 in the innate immune response to MI. METHODS AND RESULTS: In 524 patients with acute coronary syndrome (ACS), we found that high plasma S100A8/A9 at the time of the acute event was associated with lower left ventricular ejection fraction (EF) at 1-year and increased hospitalization for heart failure (HF) during follow-up. In wild-type C57BL/6 mice with MI induced by permanent coronary artery ligation, treatment with the S100A9 blocker ABR-238901 during the inflammatory phase of the immune response inhibited haematopoietic stem cell proliferation and myeloid cell egression from the bone marrow. The treatment reduced the numbers of neutrophils and monocytes/macrophages in the myocardium, promoted an anti-inflammatory environment, and significantly improved cardiac function compared with MI controls. To mimic the clinical scenario, we further confirmed the effects of the treatment in a mouse model of ischaemia/reperfusion. Compared with untreated mice, 3-day ABR-238901 treatment significantly improved left ventricular EF (48% vs. 35%, P = 0.002) and cardiac output (15.7 vs. 11.1 mL/min, P = 0.002) by Day 21 post-MI. CONCLUSION: Short-term S100A9 blockade inhibits inflammation and improves cardiac function in murine models of MI. As an excessive S100A8/A9 release is linked to incident HF, S100A9 blockade might represent a feasible strategy to improve prognosis in ACS patients.

TIM-4 is differentially expressed in the distinct subsets of dendritic cells in skin and skin-draining lymph nodes and controls skin Langerhans cell homeostasis.

T cell immunoglobulin and mucin-4 (TIM-4), mainly expressed on dendritic cells (DC) and macrophages, plays an essential role in regulating immune responses. Langerhans cells (LC), which are the sole DC subpopulation residing at the epidermis, are potent mediators of immune surveillance and tolerance. However, the significance of TIM-4 on epidermal LCs, along with other cutaneous DCs, remains totally unexplored. For the first time, we discovered that epidermal LCs expressed TIM-4 and displayed an increased level of TIM-4 expression upon migration. We also found that dermal CD207+ DCs and lymph node (LN) resident CD207-CD4+ DCs highly expressed TIM-4, while dermal CD207- DCs and LN CD207-CD4- DCs had limited TIM-4 expressions. Using TIM-4-deficient mice, we further demonstrated that loss of TIM-4 significantly upregulated the frequencies of epidermal LCs and LN resident CD207-CD4+ DCs. In spite of this, the epidermal LCs of TIM-4-deficient mice displayed normal phagocytic and migratory abilities, comparable maturation status upon the stimulation as well as normal repopulation under the inflamed state. Moreover, lack of TIM-4 did not affect dinitrofluorobenzene-induced contact hypersensitivity response. In conclusion, our results indicated that TIM-4 was differentially expressed in the distinct subsets of DCs in skin and skin-draining LNs, and specifically regulated epidermal LC and LN CD207-CD4+ DC homeostasis.

Emerging biomarkers in psoriatic arthritis.

Psoriasis is an immune-mediated skin disease which affects 2-4% of the worldwide population. Approximately 20-30% of patients with psoriasis develop psoriatic arthritis (PsA), a frequently destructive and disabling condition. As skin manifestations precede joint symptoms in nearly all patients with PsA, identification of biomarkers for early prediction of joint damage is an important clinical need. Because not all patients with PsA respond to treatment in the same fashion, identification of biomarkers capable of predicting therapeutic response is also imperative. Here, we review existing literature and discuss current investigations to identify potential biomarkers for PsA disease activity, with particular emphasis on microRNAs as novel markers of interest. Serum (soluble) biomarkers, peripheral osteoclast precursor as cellular biomarkers, and genetic loci associated with skin and joint disease are also reviewed.

Clearance of genital warts in pregnant women by mild local hyperthermia: a pilot report.

Genital warts acquired during pregnancy tend to grow fast, and management is challenging. We treated two cases of primipara with extensive genital warts by local hyperthermia at 44°C for 30 minutes a day for 3 consecutive days plus 2 additional days 1 week later, then once a week till there showed signs of clinical regression. The warty lesions in the patients resolved in 5 and 7 weeks, respectively. There was no sign of recurrence during a 6-month follow-up. This suggests that local hyperthermia seems to be a promising method for treating genital warts in pregnant women.

Serum miRNA expression profiles change in autoimmune vitiligo in mice.

It is widely believed that non-segmental vitiligo results from the autoimmune destruction of melanocytes. MicroRNAs (miRNAs), a class of small non-coding RNAs that negatively regulate gene expression, are involved in the immune cell development and function and regulate the development of autoimmune diseases. Recent studies demonstrate that functional miRNAs can be detected in the serum and serve as biomarkers of various diseases. In the present study, we used a mouse autoimmune vitiligo model, in which melanocyte autoreactive CD4+ T cells were adoptively transferred into Rag1(-/-) host mice. Serum miRNA expression was profiled in vitiligo developed mice and control mice using TaqMan RT-PCR arrays. We have found that the expressions of 20 serum miRNAs were changed in vitiligo mice compared to control mice. Three increased miRNAs, miR-146a, miR-191, and miR-342-3p, were further confirmed by a single TaqMan RT-PCR. Our findings suggest that miRNAs may be involved in vitiligo development and serum miRNAs could serve as serum biomarkers for vitiligo in mice.

Small RNAs have a large impact: circulating microRNAs as biomarkers for human diseases.

The highly conserved RNAs, microRNAs (miRNAs), are a class of small single stranded noncoding RNAs that function through translational repression of specific target mRNAs. miRNAs exhibit a wide range of involvement regulating gene expressions. miRNA expression dysregulated in cancer cells and damaged tissues from different diseases implicates a functional role of miRNAs in the disease development. More recently miRNAs have been detected in cell-free serum, and these circulating miRNAs can distinguish diseased individuals from healthy controls. The noninvasive nature of circulating miRNA collection and their sensitivity and specificity in diseases has encouraged a pursuit of miRNA biomarker research. As a result, approximately 100 circulating miRNAs have been identified as biomarkers for different diseases, and the number is growing. Here we review recently reported circulating miRNA biomarkers and discuss their values and challenges for the disease biomarkers.

Identification of mouse serum miRNA endogenous references by global gene expression profiles.

MicroRNAs (miRNAs) are recently discovered small non-coding RNAs and can serve as serum biomarkers for disease diagnosis and prognoses. Lack of reliable serum miRNA endogenous references for normalization in miRNA gene expression makes single miRNA assays inaccurate. Using TaqMan® real-time PCR miRNA arrays with a global gene expression normalization strategy, we have analyzed serum miRNA expression profiles of 20 female mice of NOD/ShiLtJ (n = 8), NOR/LtJ (n = 6), and C57BL/6J (n = 6) at different ages and disease conditions. We identified five miRNAs, miR-146a, miR-16, miR-195, miR-30e and miR-744, to be stably expressed in all strains, which could serve as mouse serum miRNA endogenous references for single assay experiments.

BRAF exon 15 T1799A mutation is common in melanocytic nevi, but less prevalent in cutaneous malignant melanoma, in Chinese Han.

Frequent somatic mutations of BRAF (v-raf murine sarcoma viral oncogene homolog B) exon T1799A, which are implicated in the initial events of promutagenic cellular proliferation, are detected in both malignant melanomas (MM) and melanocytic nevi (MN). Most of the data regarding BRAF exon T1799A mutation have been from Caucasian cohorts, and a comprehensive screening of a homogeneous population is lacking. A total of 379 cases of MN and 195 cases of MM were collected from Chinese Han living in three geographical regions in China, i.e., northeast, southwest, and northwest China. BRAF exon T1799A mutation was detected by PCR and sequencing from microdissected tumors. In all, 59.8% cases of MN harbored BRAF exon T1799A mutation. Samples from regions with high UV exposure had higher detection rates than regions with lower UV exposure (73.5, 67.0, and 38.9%, respectively; χ(2) = 31.674, P = 1.59E-7). There were no differences in mutation rates between congenital and acquired MN; however, acquired MN with advanced age of onset had a higher mutation rate than those with younger age of onset (χ(2) = 13.23, P = 0.02). In all, 15.0% cases of MM harbored the BRAF mutation. The mutation rate in MM was not affected by region, histological type, gender, pattern of UV exposure, and age. The study suggests that the mutation is not necessarily associated with malignant transformation.

Invariant NKT cell development and function in microRNA-223 knockout mice.

Invariant natural killer T (iNKT) cells, potent regulators of diverse immune responses, have been implicated in a number of diseases. The detailed mechanisms that drive iNKT cell development and maturation are still not completely understood. MicroRNAs (miRNAs) are small noncoding RNAs that regulate vast networks of genes that share miRNA target sequences. Our previous studies indicate that Dicer-dependent miRNAs play important roles in iNKT cell development, maturation, and function, but the roles of specific single miRNAs in this context are still lacking. Accumulated studies indicated that the miRNA miR-223 is a myeloid-specific miRNA. Here we report that miR-223 is highly expressed in thymic immature and activated splenic iNKT cells. To identify the role of miR-223 in iNKT cell development and function, miRNA-223-deficient mice were used. We have found that miR-223 deletion does not significantly interrupt iNKT cell development in the thymus, and miR-223-deficient mice have a normal frequency and number of iNKT cells in the thymus and peripheral immune organs. Furthermore, cytokine production of iNKT cells activated in vivo and in vitro shows no significant differences between miR-223 deficient mice and wild-type control. Thus, our data suggest that miR-223 may not be required for iNKT cell development and function.