Effects of hypernatremia on the microglia.

Signs and symptoms of hypernatremia largely indicate central nervous system dysfunction. Acute hypernatremia can cause demyelinating lesions similar to that observed in osmotic demyelination syndrome (ODS). We have previously demonstrated that microglia accumulate in ODS lesions and minocycline protects against ODS by inhibiting microglial activation. However, the direct effect of rapid rise in the sodium concentrations on microglia is largely unknown. In addition, the effect of chronic hypernatremia on microglia also remains elusive. Here, we investigated the effects of acute (6 or 24 h) and chronic (the extracellular sodium concentration was increased gradually for at least 7 days) high sodium concentrations on microglia using the microglial cell line, BV-2. We found that both acute and chronic high sodium concentrations increase NOS2 expression and nitric oxide (NO) production. We also demonstrated that the expression of nuclear factor of activated T-cells-5 (NFAT5) is increased by high sodium concentrations. Furthermore, NFAT5 knockdown suppressed NOS2 expression and NO production. We also demonstrated that high sodium concentrations decreased intracellular Ca2+ concentration and an inhibitor of Na+/Ca2+ exchanger, NCX, suppressed a decrease in intracellular Ca2+ concentrations and NOS2 expression and NO production induced by high sodium concentrations. Furthermore, minocycline inhibited NOS2 expression and NO production induced by high sodium concentrations. These in vitro data suggest that microglial activity in response to high sodium concentrations is regulated by NFAT5 and Ca2+ efflux through NCX and is suppressed by minocycline.

Molecular Pathways and Animal Models of Tricuspid Atresia and Univentricular Heart.

The process of valve formation is a complex process that involves intricate interplay between various pathways at precise times. Although we have not completely elucidated the molecular pathways that lead to normal valve formation, we have identified a few major players in this process. We are now able to implicate TGF-ß, BMP, and NOTCH as suspects in tricuspid atresia (TA), as well as their downstream targets: NKX2-5, TBX5, NFATC1, GATA4, and SOX9. We know that the TGF-ß and the BMP pathways converge on the SMAD4 molecule, and we believe that this molecule plays a very important role to tie both pathways to TA. Similarly, we look at the NOTCH pathway and identify the HEY2 as a potential link between this pathway and TA. Another transcription factor that has been implicated in TA is NFATC1. While several mouse models exist that include part of the TA abnormality as their phenotype, no true mouse model can be said to represent TA. Bridging this gap will surely shed light on this complex molecular pathway and allow for better understanding of the disease process.

Association between skin lymphangiogenesis parameters and arterial hypertension status in patients: An observational study.

BACKGROUND: Recent studies have indicated that the skin lymphatic system and interstitium may play a role in the pathophysiology of arterial hypertension (AH). OBJECTIVES: We aimed to determine whether the set of pathway parameters described previously in rodents would allow for the distinction between hypertensive and normotensive patients. MATERIAL AND METHODS: Molecular and histopathological parameters from the skin and blood of patients with AH (AH group, n = 53), resistant AH (RAH group, n = 32) and control (C group, n = 45) were used, and a statistical multivariate bootstrap methodology combining partial least squares-discriminant analysis (PLS-DA) and selectivity ratio (SR) were applied. RESULTS: The C vs RAH model presented the best prediction performance (AUC test = 0.90) and had a sensitivity and specificity of 73.68% and 83.33%, respectively. However, the parameters selected for the C vs AH group model were the most important for the pathway described in the rodent model, i.e., greater density of the skin lymphatic vessels (D2-40 expression) and greater number of macrophages (CD68 expression), higher expression of the messenger ribonucleic acid (mRNA) of nuclear factor of activated T cells 5 (NFAT5), vascular endothelial growth factor C (VEGFC) and podoplanin (PDPN) in the skin, greater concentration of hyaluronic acid (HA) in the skin, and lower serum concentration of VEGF-C. CONCLUSIONS: Our study suggests that the NFAT5/VEGF-C/lymphangiogenesis pathway, previously described in rodent studies, may also be present in human HA. Further experiments are needed to confirm our findings.

Anti­osteoclastogenic effect of fermented mealworm extract by inhibiting RANKL­induced NFATc1 action.

Augmented osteoclast activity and differentiation can lead to destructive bone diseases, such as arthritis and osteoporosis. Therefore, modulating osteoclastogenesis and differentiation may serve to be a possible strategy for treating such diseases. Tenebrio molitor larvae, also known as mealworms, are considered a good source of protein with nutritional value, digestibility, flavor and functional properties, such as antioxidant, anti-diabetic and anti-obesity effects. However, the role of mealworms in osteoclastogenesis remains poorly understood. The present study therefore investigated the effects of fermented mealworm extract (FME) on receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in bone marrow-derived macrophages (BMMs) whilst also attempting to understand the underlying mechanism, if any. The cells treated with RANKL were used as the negative control. To prepare FME, defatted mealworm powder was fermented with a Saccharomyces cerevisiae strain, and then extracted with fermented alcohol. Cell viability of BMMs isolated from 5-week-old Institute of Cancer Research mice was measured using Cell Counting Kit-8 assay. Subsequently, the effects of FME on osteoclast differentiation were measured using tartrate-resistant acid phosphatase (TRAP) staining. In addition, expression of markers associated with osteoclast differentiation was assessed by reverse transcription-quantitative PCR. Expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) was assessed by western blotting. TRAP staining revealed that FME inhibited osteoclast differentiation in a dose-dependent manner (10-100 µg/ml) without causing cytotoxicity. Specifically, the formation of osteoclasts appear to have been suppressed by FME as indicated by the reduction in the number of TRAP-positive multinucleated cells observed. Furthermore, FME treatment significantly decreased the mRNA expression of c-Fos, whilst also significantly decreasing the expression of NFATc1 on both protein and mRNA levels. c-Fos and NFATc1 are transcription factors that can regulate osteoclast differentiation. FME treatment also reduced the expression of genes associated with osteoclast differentiation and function, including dendritic cell-specific transmembrane protein, osteoclast associated Ig-like receptor, Cathepsin K and TRAP, compared with that in the control group. Subsequently, FME was found to effectively suppress RANKL-induced osteoclast differentiation compared with that by the non-fermented mealworm extract. These findings suggest that FME may confer anti-osteoclastogenic effects, providing insights into its potential application in treatment of osteoporosis.

The mechanism of the NFAT transcription factor family involved in oxidative stress response.

As a transcriptional activator widely expressed in various tissues, nuclear factor of activated T cells (NFAT) is involved in the regulation of the immune system, the development of the heart and brain systems, and classically mediating pathological processes such as cardiac hypertrophy. Oxidative stress is an imbalance of intracellular redox status, characterized by excessive generation of reactive oxygen species, accompanied by mitochondrial dysfunction, calcium overload, and subsequent lipid peroxidation, inflammation, and apoptosis. Oxidative stress occurs during various pathological processes, such as chronic hypoxia, vascular smooth muscle cell phenotype switching, ischemia-reperfusion, and cardiac remodeling. Calcium overload leads to an increase in intracellular calcium concentration, while NFAT can be activated through calcium-calcineurin, which is also the main regulatory mode of NFAT factors. This review focuses on the effects of NFAT transcription factors on reactive oxygen species production, calcium overload, mitochondrial dysfunction, redox reactions, lipid peroxidation, inflammation, and apoptosis in response to oxidative stress. We hope to provide a reference for the functions and characteristics of NFAT involved in various stages of oxidative stress as well as related potential targets.

Characterization of gene expression in the kidney of renal tubular cell-specific NFAT5 knockout mice.

Nuclear factor of activated T cells 5 (NFAT5; also called TonEBP/OREBP) is a transcription factor that is activated by hypertonicity and induces osmoprotective genes to protect cells against hypertonic conditions. In the kidney, renal tubular NFAT5 is known to be involved in the urine concentration mechanism. Previous studies have suggested that NFAT5 modulates the immune system and exerts various effects on organ damage, depending on organ and disease states. Pathophysiological roles of NFAT5 in renal tubular cells, however, still remain obscure. We conducted comprehensive analysis by performing transcription start site (TSS) sequencing on the kidney of inducible and renal tubular cell-specific NFAT5 knockout (KO) mice. Mice were subjected to unilateral ureteral obstruction to examine the relevance of renal tubular NFAT5 in renal fibrosis. TSS sequencing analysis identified 722 downregulated TSSs and 1,360 upregulated TSSs, which were differentially regulated ≤-1.0 and ≥1.0 in log2 fold, respectively. Those TSSs were annotated to 532 downregulated genes and 944 upregulated genes, respectively. Motif analysis showed that sequences that possibly bind to NFAT5 were enriched in TSSs of downregulated genes. Gene Ontology analysis with the upregulated genes suggested disorder of innate and adaptive immune systems in the kidney. Unilateral ureteral obstruction significantly exacerbated renal fibrosis in the renal medulla in KO mice compared with wild-type mice, accompanied by enhanced activation of immune responses. In conclusion, NFAT5 in renal tubules could have pathophysiological roles in renal fibrosis through modulating innate and adaptive immune systems in the kidney.NEW & NOTEWORTHY TSS-Seq analysis of the kidney from renal tubular cell-specific NFAT5 KO mice uncovered novel genes that are possibly regulated by NFAT5 in the kidney under physiological conditions. The study further implied disorders of innate and adaptive immune systems in NFAT5 KO mice, thereby exacerbating renal fibrosis at pathological states. Our results may implicate the involvement of renal tubular NFAT5 in the progression of renal fibrosis. Further studies would be worthwhile for the development of novel therapy to treat chronic kidney disease.

Effect of Osteoking on Inflammatory Injury and NF-κB/NFATc1 Signaling Pathway in Model Mice with Postmenopausal Osteoporosis / 中国实验方剂学杂志

ObjectiveTo explore the possible mechanism of Osteoking （OK） on postmenopausal osteoporosis （PMOP）. MethodForty adult female mice were randomly divided into a sham operation （Sham） group， osteoporosis model （OVX） group， estradiol intervention （E2） group， and OK group， with 10 mice in each group. The modeling was completed by conventional back double incision ovariectomy， and the corresponding drugs were given one week later. After 12 weeks， the body mass and uterine index of mice were measured， and the pathological changes of bone tissue and the number of osteoclasts （OCs） were determined by hematoxylin-eosin （HE） and tartrate-resistant acid phosphatase （TRAP） staining， respectively. Bone mineral density （BMD）， trabecular number （Tb.N）， trabecular separation （Tb.Sp）， and bone volume fraction （BV/TV） were measured by microcomputed tomography （Micro-CT）. The maximum load of the femur was detected by a three-point bending test. The contents of tumor necrosis factor-α （TNF-α） and bone resorption marker C-terminal telopeptide of type Ⅰ collagen （CTX-1） were measured by enzyme linked immunosorbent assay （ELISA）. The protein expression levels of nuclear factor-kappa B p65 （NF-κB p65）， phosphorylated nuclear factor-kappa B p65 （p-NF-κB p65）， nuclear factor kappa B inhibitor alpha （IκBα）， phosphorylated nuclear factor kappa B alpha （p-IκBα）， nuclear factor of activated T cells 1 （NFATc1）， and proto-oncogene （c-Fos） were detected by Western blot. The mRNA expressions of OCs-related specific genes matrix metalloproteinase-9 （MMP-9）， NFATc1， TRAP， cathepsin K （CTSK）， and c-Fos were detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the Sham group， the uterine index decreased significantly in the OVX group， and the body mass （BMI） increased significantly. The structure of bone trabeculae was completely damaged， and the number of OCs increased. BMD， Tb.N， BV/TV， and maximum load decreased， while Tb.Sp was up-regulated. The levels of TNF-α and CTX-1 in serum were up-regulated. The protein expressions of c-Fos， p-NF-κB p65/NF-κB p65， NFATc1， and p-IκBα/IκBα were increased. The mRNA expressions of NFATc1， c-Fos， CTSK， TRAP， and MMP-9 were up-regulated （P<0.05， P<0.01）. Compared with the OVX group， the body mass of the OK and E2 groups decreased， and the uterine index increased. The bone trabeculae increased， and the number of OCs decreased. BMD， Tb.N， BV/TV， and maximum load increased， while Tb.Sp decreased. The levels of TNF-α and CTX-1 in serum were decreased. The protein expressions of c-Fos， p-NF-κB p65/NF-κB p65， NFATc1， and p-IκBα/IκBα were decreased， and the mRNA expressions of NFATc1， c-Fos， CTSK， TRAP， and MMP-9 were decreased （P<0.05， P<0.01）. ConclusionOK can inhibit the NF-κB/NFATc1 signaling pathway and reduce bone mass loss by reducing the level of inflammatory injury factors in PMOP mice， which is one of the mechanisms for treating PMOP.

Britanin inhibits titanium wear particle­induced osteolysis and osteoclastogenesis.

Wear particle­induced osteolysis is a serious complication that occurs in individuals with titanium (Ti)­based implants following long­term usage due to loosening of the implants. The control of excessive osteoclast differentiation and inflammation is essential for protecting against wear particle­induced osteolysis. The present study evaluated the effect of britanin, a pseudoguaianolide sesquiterpene isolated from Inula japonica, on osteoclastogenesis in vitro and Ti particle­induced osteolysis in vivo. The effect of britanin was examined in the osteoclastogenesis of mouse bone marrow­derived macrophages (BMMs) using TRAP staining, RT­PCR, western blotting and immunocytochemistry. The protective effect of britanin was examined in a mouse calvarial osteolysis model and evaluated using micro­CT and histomorphometry. Britanin inhibited osteoclast differentiation and F­actin ring formation in the presence of macrophage colony­stimulating factor and receptor activator of nuclear factor kB ligand in BMMs. The expression of osteoclast­specific marker genes, including tartrate­resistant acid phosphatase, cathepsin K, dendritic cell­specific transmembrane protein, matrix metallopeptidase 9 and nuclear factor of activated T­cells cytoplasmic 1, in the BMMs was significantly reduced by britanin. In addition, britanin reduced the expression of B lymphocyte­induced maturation protein­1, which is a transcriptional repressor of negative osteoclastogenesis regulators, including interferon regulatory factor­8 and B­cell lymphoma 6. Conversely, britanin increased the expression levels of anti­oxidative stress genes, namely nuclear factor erythroid­2­related factor 2, NAD(P)H quinone oxidoreductase 1 and heme oxygenase 1 in the BMMs. Furthermore, the administration of britanin significantly reduced osteolysis in a Ti particle­induced calvarial osteolysis mouse model. Based on these findings, it is suggested that britanin may be a potential therapeutic agent for wear particle­induced osteolysis and osteoclast­associated disease.

Macrophage Activation in Follicular Conjunctivitis during the COVID-19 Pandemic.

Among the symptoms of SARS-CoV-2, follicular conjunctivitis has become relevant. The conjunctiva acts as an open lymph node, reacting to the viral antigen that binds the epithelial cells, forming follicles of B cells with activated T cells and NK cells on its surface, which, in turn, talk to monocyte-derived inflammatory infected macrophages. Here, the NLRP3 inflammasome is a major driver in releasing pro-inflammatory factors such as IL-6 and caspase-1, leading to follicular conjunctivitis and bulbar congestion, even as isolated signs in the 'asymptomatic' patient.

Gleditsiae fructus regulates osteoclastogenesis by inhibiting the c­Fos/NFATc1 pathway and alleviating bone loss in an ovariectomy model.

Medical and economic developments have allowed the human lifespan to extend and, as a result, the elderly population has increased worldwide. Osteoporosis is a common geriatric disease that has no symptoms and even a small impact can cause fractures in patients, leading to a serious deterioration in the quality of life. Osteoporosis treatment typically involves bisphosphonates and selective estrogen receptor modulators. However, these treatments are known to cause severe side effects, such as mandibular osteonecrosis and breast cancer, if used for an extended period of time. Therefore, it is essential to develop therapeutic agents from natural products that have fewer side effects. Gleditsiae fructus (GF) is a dried or immature fruit of Gleditsia sinensis Lam. and is composed of various triterpenoid saponins. The anti­inflammatory effect of GF has been confirmed in various diseases, and since the anti­inflammatory effect plays a major role in inhibiting osteoclast differentiation, GF was expected to be effective in osteoclast differentiation and menopausal osteoporosis; however, to the best of our knowledge, it has not yet been studied. Therefore, the present study was designed to examine the effect of GF on osteoclastogenesis and to investigate the mechanism underlying inhibition of osteoclast differentiation. The effects of GF on osteoclastogenesis were determined in vitro by tartrate­resistant acid phosphatase (TRAP) staining, pit formation assays, filamentous actin (F­actin) ring formation assays, western blotting and reverse transcription­quantitative PCR analyses. Furthermore, the administration of GF to an animal model exhibiting menopausal osteoporosis allowed for the analysis of alterations in the bone microstructure of the femur using micro­CT. Additionally, assessments of femoral tissue and serum were conducted. The present study revealed that the administration of GF resulted in a reduction in osteoclast levels, F­actin rings, TRAP activity and pit area. Furthermore, GF showed a dose­dependent suppression of nuclear factor of activated T­cells cytoplasmic, c­Fos and other osteoclastogenesis­related markers.

Genetic association of nuclear factor of activated T cells' 3'UTR and structural polymorphisms with susceptibility to generalized vitiligo in Gujarat population.

Generalized vitiligo (GV) is an autoimmune skin depigmenting disease characterized by loss of functional melanocytes. Nuclear factor of activated T cells (NFATs) play a key role in regulatory T cells' (Tregs) activation and function. Our previous studies have highlighted the role of reduced NFATs expression and activity in impaired Tregs suppressive capacity, leading to GV pathogenesis. 3'UTR region and structural single nucleotide polymorphisms(SNPs) could lead to reduced NFAT expression and activity. Therefore, we studied the association of NFATs 3'UTR [NFATC2 rs4811198 (T > G) & NFATC4 rs11848279 (A > G)] and structural [NFATC1 rs754093 (T > G) & NFATC2 rs12479626 (T > C)] SNPs in 427 GV patients and 415 controls from Gujarat population by Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP). Additionally, we carried out genotype-phenotype correlation and in silico analysis to assess the effect of NFATs SNPs on NFATs expression and structure. NFATC2 rs4811198 (T > G) 3' UTR & NFATC2 rs12479626 (T > C) structural SNPs were significantly associated with GV (p < 0.0001). Interestingly, for NFATC2 rs4811198 (T > G) SNP, there was a significant difference in the TT vs GG genotypes' frequencies (p = 0.0034; Table 2), and for NFATC2 rs12479626 (T > C) SNP there was a significant difference between TT vs TC and CC genotypes' frequencies (p < 0.0001 & p = 0.0002) between GV patients and controls. Furthermore, Odds ratio suggested that the susceptible alleles for NFATC2 rs4811198 (T > G) & NFATC2 rs12479626 (T > C) SNPs increased the risk of GV by 1.38 & 3.04 fold. However, the NFAT 3' UTR [NFATC2 rs4811198 (T > G)] and structural [NFATC1 rs754093 (T > G)] SNPs were not significantly associated with GV. Interestingly, the genotype-phenotype correlation suggested that the susceptible 'G' allele of NFATC2 rs4811198 (T > G) & NFATC4 rs11848279 (A > G) 3' UTR SNPs lead to reduced NFATC2 and NFATC4 expression (p < 0.0001). Furthermore, in silico analysis suggested that hsa-miR-3183 & hsa-miR-6720-3p miRNAs specifically bound to 'G' allele of NFATC2 rs4811198 SNP and has-miR-4652-3p miRNA specifically bound to 'G' allele of NFATC4 rs11848279 SNP. Overall, our study suggests that NFATC2 rs4811198 (T > G) 3' UTR & NFATC2 rs12479626 (T > C) structural SNPs may be associated with GV susceptibility in Gujarat population. Moreover, the susceptible alleles for the 3' UTR SNPs could lead to reduced NFATs levels, which may further possibly, affect the Treg suppressive function leading to GV.

Cytotoxic NK cells phenotype and activated lymphocytes are the main characteristics of patients with alcohol-associated liver disease.

T cells, natural killer (NK) and NKT cells have opposing actions in the development of alcohol-associated liver fibrosis. We aimed to evaluate the phenotype of NK cells, NKT cells and activated T cells in patients with alcohol use disorder (AUD) according to the presence of advanced liver fibrosis (ALF). Totally, 79 patients (51-years, 71% males) were admitted to treatment of AUD. ALF was defined as FIB4-score > 2.67. Immunophenotyping of NK cells (CD3-CD56+CD16+, CD3-CD56+CD16-, CD3-CD56-CD16+), NKT-like (CD3+CD56+), and the activation status of CD4+, CD8+ and regulatory T cells (Tregs) were evaluated according to the HLA-DR expression. Patients had an AUD duration of 18 ± 11 years with a daily alcohol consumption of 155 ± 77 gr/day prior to hospital admission. The values of absolute cells were 2 ± 0.9 cells/L for total lymphocytes, 1054 ± 501 cells/µL for CD4+, 540 ± 335 cells/µL for CD8+, 49.3 ± 24.8 cells/µL for Tregs, 150.3 ± 97.5 cells/µL for NK cells and 69.8 ± 78.3 cells/µL for NKT-like. The percentage of total NK cells (11.3 ± 5.5% vs. 7 ± 4.3%, p < 0.01), CD3-CD56+CD16+ regarding total lymphocytes (9.7 ± 5.1% vs. 5.8 ± 3.9%, p < 0.01), activated CD4+ cells (5.2 ± 3.2% vs. 3.9 ± 3%, p = 0.04) and activated CD8+ cells (15.7 ± 9.1% vs. 12.2 ± 9%, p = 0.05) were significantly higher in patients with ALF. The percentage of CD3-CD56+CD16- regarding NK cells (5.1 ± 3.4% vs. 7.6 ± 6.2%, p = 0.03) was significantly lower in patients with ALF. Activated Tregs (39.9 ± 11.5 vs. 32.4 ± 9.2, p = 0.06) showed a tendency to be higher in patients with ALF. The proportion of activated CD4+ cells (r = 0.40, p < 0.01) and activated CD8+ cells (r = 0.51, p < 0.01) was correlated with the proportion of NKT-like in patients without ALF. Patients with ALF presented an increased NK cytotoxic phenotype and activated T cells concomitant with a decreased NK cytokine-secreting phenotype.

Oncogenic Role of the NFATC2/NEDD4/FBP1 Axis in Cholangiocarcinoma.

Nuclear factor of activated T cells 2 (NFATC2) is reported to contribute to the initiation and progression of various cancers; however, its expression and function in cholangiocarcinoma (CCA) tissues remain elusive. Herein, we investigated the expression pattern, clinicopathologic characteristics, cell biological functions, and potential mechanisms of NFATC2 in CCA tissues. Real-time reverse-transcription PCR (RT-qPCR) and immunohistochemistry were performed to analyze the expression of NFATC2 in human CCA tissues. Cell counting kit 8, colony formation, flow cytometry, Western blotting, and Transwell assays, and in vivo xenograft and pulmonary metastasis models, were used to explore the effect of NFATC2 on the proliferation and metastasis of CCA. A dual-luciferase reporter system, oligonucleotide pull-down, chromatin immunoprecipitation, immunofluorescence, and coimmunoprecipitation were performed to reveal the potential mechanisms. We found that NFATC2 was upregulated in CCA tissues and cells, and its aberrantly high levels were associated with a poorer differentiation pattern. Functionally, NFATC2 overexpression promoted CCA cell proliferation and metastasis, whereas knockdown of NFATC2 led to opposite result. Mechanistically, NFATC2 could be enriched in the promoter region of neural precursor cell-expressed developmentally downregulated protein 4 (NEDD4) to facilitate its expression. Furthermore, NEDD4 targeted fructose-1, 6-bisphosphatase 1 (FBP1) and inhibited FBP1 expression via ubiquitination. In addition, silencing NEDD4 rescued the effects of NFATC2 overexpression on CCA cells. NEDD4 was upregulated in human CCA tissues, and its expression levels were positively correlated with those of NFATC2. We thus conclude that NFATC2 promotes the progression of CCA via the NEDD4/FBP1 axis, emphasizing the oncogenic role of NFATC2 in CCA progression.

Rubiginosin B selectively inhibits Treg cell differentiation and enhances anti-tumor immune responses by targeting calcineurin-NFAT signaling pathway.

BACKGROUND: The accumulation of CD4+Foxp3+ regulatory T cells (Tregs) in the tumor microenvironment (TME) dampens anti-tumor immune responses and promotes tumor progression. Therefore, the elimination of Tregs has become a strategy to enhance the efficacy of tumor immunotherapy, although it is still a daunting challenge. Rhododendron brachypodum (R. brachypodum) is a perennial shrub mainly distributed in Southwestern China, whereas the chemical constituents in this plant remain elusive. PURPOSE: To identify small-molecule inhibitors of Tregs from R. brachypodum. METHODS: Meroterpenoids in R. brachypodum were isolated by column chromatography under the guidance of LCMS analyses. The structures of isolates were identified by spectroscopic data and quantum calculations. The activities of compounds were first evaluated on CD4+ T cell differentiation by flow cytometry in Th1, Th2, Th17, and Treg polarizing conditions, and then on CT26 and MC38 murine colorectal carcinoma cells-allografted mice models. The mechanism of action was first investigated by determining Foxp3 degradation in Jurkat T cells transfected with pLVX-TetOne-Puro-Foxp3-tGFP, and then through analyses of Foxp3 expression on several pre-transcriptional signaling molecules. RESULTS: Two new prenylated phenolic acids (1 and 2) and a chromane meroterpenoid, rubiginosin B (RGB, 3) were obtained from R. brachypodum. The structure of S-anthopogochromene C (1) was rectified according to the electronic circular dichroism (ECD) experiment, and rhodobrachypodic acid (2) was proposed as the precursor of RGB by photochemical transformation. In this investigation, we first found that RGB (3) selectively suppressed the de novo differentiation of TGFß-induced CD4+Foxp3+ regulatory T cells (iTregs), overcome the immunosuppressive TME, and consequently inhibited the growth of tumor in mouse models. The mechanistic study revealed that RGB could target calcineurin, inhibited the nuclear factor of activated T cells (NFAT) dephosphorylation, and down-regulated Foxp3 expression. The hypothetical binding modes of RGB with calcineurin were predicted by molecular docking, and the interactions were mainly hydrophobic effects and hydrogen bonds. CONCLUSION: These results suggest that RGB enhances anti-tumor immune responses by inhibiting Treg cell differentiation through calcineurin-NFAT signaling pathway, and therefore RGB or its analogs may be used as adjuvant agents meriting further investigation.

Interruption of TRPC6-NFATC1 signaling inhibits NADPH oxidase 4 and VSMCs phenotypic switch in intracranial aneurysm.

Intracranial aneurysm (IA) is a frequent cerebrovascular disorder with unclear pathogenesis. The vascular smooth muscle cells (VSMCs) phenotypic switch is essential for IA formation. It has been reported that Ca2+ overload and excessive reactive oxygen species (ROS) are involved in VSMCs phenotypic switch. The transient receptor potential canonical 6 (TRPC6) and NADPH oxidase 4 (NOX4) are the main pathway to participate in Ca2+ overload and ROS production in VSMCs. Ca2+ overload can activate calcineurin (CN), leading to nuclear factor of activated T cell (NFAT) dephosphorylation to regulate the target gene's transcription. We hypothesized that activation of TRPC6-NFATC1 signaling may upregulate NOX4 and involve in VSMCs phenotypic switch contributing to the progression of IA. Our results showed that the expressions of NOX4, p22phox, p47phox, TRPC6, CN and NFATC1 were significantly increased, and VSMCs underwent a significant phenotypic switch in IA tissue and cellular specimens. The VIVIT (NFATC1 inhibitor) and BI-749327 (TRPC6 inhibitor) treatment reduced the expressions of NOX4, p22phox and p47phox and the production of ROS, and significantly improved VSMCs phenotypic switch in IA rats and cells. Consistent results were obtained from IA Trpc6 knockout (Trpc6-/-) mice. Furthermore, the results also revealed that NFATC1 could regulate NOX4 transcription by binding to its promoter. Our findings reveal that interrupting the TRPC6-NFATC1 signaling inhibits NOX4 and improves VSMCs phenotypic switch in IA, and regulating Ca2+ homeostasis may be an important therapeutic strategy for IA.

[The Distribution and Significance of Activated T Cells and Lymphocyte Subsets in Myelodysplastic Syndrome].

OBJECTIVE: To investigate the distribution of bone marrow lymphocyte subsets in patients with myelodysplastic syndrome(MDS),the proportion of activated T cells with immunophenotype CD3+HLA-DR+ in the lymphocytes and its clinical significance, and to understand the effects of different types of MDS, different immunophenotypes, and different expression levels of WT1 on the proportion of lymphocyte subsets and activated T cells. METHODS: The immunophenotypes of 96 MDS patients, the subsets of bone marrow lymphocytes and activated T cells were detected by flow cytometry. The relative expression of WT1 was detected by real-time fluorescent quantitative PCR, and the first induced remission rate (CR1) was calculated, the differences of lymphocyte subsets and activated T cells in MDS patients with different immunophenotype, different WT1 expression, and different course of disease were analyzed. RESULTS: The percentage of CD4+T lymphocyte in MDS-EB-2, IPSS high-risk, CD34+ cells >10%, and patients with CD34+CD7+ cell population and WT1 gene overexpression at intial diagnosis decreased significantly (P<0.05), and the percentage of NK cells and activated T cells increased significantly (P<0.05), but there was no significant difference in the ratio of B lymphocytes. Compared with the normal control group, the percentage of NK cells and activated T cells in IPSS-intermediate-2 group was significantly higher(P<0.05), but there was no significant difference in the percentage of CD3+T, CD4+T lymphocytes. The percentage of CD4+T cells in patients with complete remission after the first chemotherapy was significantly higher than in patients with incomplete remission(P<0.05), and the percentage of NK cells and activated T cells was significantly lower than that in patients with incomplete remission (P<0.05). CONCLUSION: In MDS patients, the proportion of CD3+T and CD4+T lymphocytes decreased, and the proportion of activated T cells increased, indicating that the differentiation type of MDS is more primitive and the prognosis is worse.

Venetoclax resistance induced by activated T cells can be counteracted by sphingosine kinase inhibitors in chronic lymphocytic leukemia.

The treatment of chronic lymphocytic leukemia (CLL) patients with venetoclax-based regimens has demonstrated efficacy and a safety profile, but the emergence of resistant cells and disease progression is a current complication. Therapeutic target of sphingosine kinases (SPHK) 1 and 2 has opened new opportunities in the treatment combinations of cancer patients. We previously reported that the dual SPHK1/2 inhibitor, SKI-II enhanced the in vitro cell death triggered by fludarabine, bendamustine or ibrutinib and reduced the activation and proliferation of chronic lymphocytic leukemia (CLL) cells. Since we previously showed that autologous activated T cells from CLL patients favor the activation of CLL cells and the generation of venetoclax resistance due to the upregulation of BCL-XL and MCL-1, we here aim to determine whether SPHK inhibitors affect this process. To this aim we employed the dual SPHK1/2 inhibitor SKI-II and opaganib, a SPHK2 inhibitor that is being studied in clinical trials. We found that SPHK inhibitors reduce the activation of CLL cells and the generation of venetoclax resistance induced by activated T cells mainly due to a reduced upregulation of BCL-XL. We also found that SPHK2 expression was enhanced in CLL cells by activated T cells of the same patient and the presence of venetoclax selects resistant cells with high levels of SPHK2. Of note, SPHK inhibitors were able to re-sensitize already resistant CLL cells to a second venetoclax treatment. Our results highlight the therapeutic potential of SPHK inhibitors in combination with venetoclax as a promising treatment option for the patients.

Inhibiting 5-hydroxytryptamine receptor 3 alleviates pathological changes of a mouse model of Alzheimer's disease.

Extracellular amyloid beta (Aß) plaques are main pathological feature of Alzheimer's disease. However, the specific type of neurons that produce Aß peptides in the initial stage of Alzheimer's disease are unknown. In this study, we found that 5-hydroxytryptamin receptor 3A subunit (HTR3A) was highly expressed in the brain tissue of transgenic amyloid precursor protein and presenilin-1 mice (an Alzheimer's disease model) and patients with Alzheimer's disease. To investigate whether HTR3A-positive interneurons are associated with the production of Aß plaques, we performed double immunostaining and found that HTR3A-positive interneurons were clustered around Aß plaques in the mouse model. Some amyloid precursor protein-positive or ß-site amyloid precursor protein cleaving enzyme-1-positive neurites near Aß plaques were co-localized with HTR3A interneurons. These results suggest that HTR3A -positive interneurons may partially contribute to the generation of Aß peptides. We treated 5.0-5.5-month-old model mice with tropisetron, a HTR3 antagonist, for 8 consecutive weeks. We found that the cognitive deficit of mice was partially reversed, Aß plaques and neuroinflammation were remarkably reduced, the expression of HTR3 was remarkably decreased and the calcineurin/nuclear factor of activated T-cell 4 signaling pathway was inhibited in treated model mice. These findings suggest that HTR3A interneurons partly contribute to generation of Aß peptide at the initial stage of Alzheimer's disease and inhibiting HTR3 partly reverses the pathological changes of Alzheimer's disease.

OMIP-089: Cattle T-cell phenotyping by an 8-color panel.

This multiplex staining panel was developed to differentiate cattle T cells into conventional (CD4 and CD8) and unconventional (γδ-TCR) subsets as well as their stage of differentiation and activation. The combination of CD45RO and CD62L allows the identification of naïve (TNaïve ), central memory (TCM ), effector memory (TEM ) and terminal effector (TTE ) T cells. Activated cattle T cells (TAV ) can be identified by the cell surface expression of CD25. This panel was developed using cryopreserved cattle peripheral blood mononuclear cells (PBMCs) and tested on fresh as well as stimulated PBMCs. Therefore, this 8-color, 10-parameter flow cytometry panel simultaneously identifies cattle TNaïve , TAV , TCM , TEM , TTE and γδ-TCR cells. This panel will improve our ability to examine T-cell response to pathogens and vaccines in cattle including the potential to identify previously undescribed subpopulations. Furthermore, this panel can be readily optimized for other bovid species as many of these reagents are likely to cross react.

NOD2 Agonism Counter-Regulates Human Type 2 T Cell Functions in Peripheral Blood Mononuclear Cell Cultures: Implications for Atopic Dermatitis.

Atopic dermatitis (AD) is known as a skin disease; however, T cell immunopathology found in blood is associated with its severity. Skin Staphylococcus aureus (S. aureus) and associated host-pathogen dynamics are important to chronic T helper 2 (Th2)-dominated inflammation in AD, yet they remain poorly understood. This study sought to investigate the effects of S. aureus-derived molecules and skin alarmins on human peripheral blood mononuclear cells, specifically testing Th2-type cells, cytokines, and chemokines known to be associated with AD. We first show that six significantly elevated Th2-related chemokine biomarkers distinguish blood from adult AD patients compared to healthy controls ex vivo; in addition, TARC/CCL17, LDH, and PDGF-AA/AB correlated significantly with disease severity. We then demonstrate that these robust AD-associated biomarkers, as well as associated type 2 T cell functions, are readily reproduced from healthy blood mononuclear cells exposed to the alarmin TSLP and the S. aureus superantigen SEB in a human in vitro model, including IL-13, IL-5, and TARC secretion as well as OX-40-expressing activated memory T cells. We further show that the agonism of nucleotide-binding oligomerization domain-containing protein (NOD)2 inhibits this IL-13 secretion and memory Th2 and Tc2 cell functional activation while inducing significantly increased pSTAT3 and IL-6, both critical for Th17 cell responses. These findings identify NOD2 as a potential regulator of type 2 immune responses in humans and highlight its role as an endogenous inhibitor of pathogenic IL-13 that may open avenues for its therapeutic targeting in AD.