Cre-LoxP and tamoxifen-induced deletion of ovarian quiescin sulfhydryl oxidase 2 showed disruption of ovulatory activity in mice.

BACKGROUND: Quiescin sulfhydryl oxidase 2 (QSOX2) is a flavin adenine dinucleotide-dependent sulfhydryl oxidase that is known to be involved in protein folding, cell growth regulation, and redox state modification through oxidative activities. Earlier studies demonstrated the tissue and cellular localization of QSOX2 in the male reproductive tract, as well as the highly-regulated mechanism of QSOX2 protein synthesis and expression through the coordinated action of testosterone and epididymal-enriched amino acid, glutamate. However, the presence and the functions of QSOX2 in female reproduction are unknown. In this study, we applied the Cre-loxP gene manipulation system to generate the heterozygous and homozygous Qsox2 knockout mice and examined its effects on ovarian function. RESULTS: We demonstrated that QSOX2 was detected in the follicle-supporting cells (granulosa and cumulus cells) of ovarian follicles of all stages but was absent in the corpus luteum, suggesting its supportive role in folliculogenesis. In comparison with reproductive organogenesis in wild-type mice, there was no difference in testicular and epididymal structure in male Qsox2 knockout; however, Qsox2 knockout disrupted the regular ovulation process in female mice as a drastic decrease in the formation of the corpus luteum was detected, and no pregnancy was achieved when mating males with homozygous Qsox2 knockout females. RNAseq analyses further revealed that Qsox2 knockout altered critical signaling pathways and genes that are responsible for maintaining ovarian functions. CONCLUSION: Our data demonstrated for the first time that Qsox2 is critical for ovarian function in mice.

Rational evolution for altering the ligand preference of estrogen receptor alpha.

Estrogen receptor α is commonly used in synthetic biology to control the activity of genome editing tools. The activating ligands, estrogens, however, interfere with various cellular processes, thereby limiting the applicability of this receptor. Altering its ligand preference to chemicals of choice solves this hurdle but requires adaptation of unspecified ligand-interacting residues. Here, we provide a solution by combining rational protein design with multi-site-directed mutagenesis and directed evolution of stably integrated variants in Saccharomyces cerevisiae. This method yielded an estrogen receptor variant, named TERRA, that lost its estrogen responsiveness and became activated by tamoxifen, an anti-estrogenic drug used for breast cancer treatment. This tamoxifen preference of TERRA was maintained in mammalian cells and mice, even when fused to Cre recombinase, expanding the mammalian synthetic biology toolbox. Not only is our platform transferable to engineer ligand preference of any steroid receptor, it can also profile drug-resistance landscapes for steroid receptor-targeted therapies.

AAV-Mediated Delivery of Plakophilin-2a Arrests Progression of Arrhythmogenic Right Ventricular Cardiomyopathy in Murine Hearts: Preclinical Evidence Supporting Gene Therapy in Humans.

BACKGROUND: Pathogenic variants in PKP2 (plakophilin-2) cause arrhythmogenic right ventricular cardiomyopathy, a disease characterized by life-threatening arrhythmias and progressive cardiomyopathy leading to heart failure. No effective medical therapy is available to prevent or arrest the disease. We tested the hypothesis that adeno-associated virus vector-mediated delivery of the human PKP2 gene to an adult mammalian heart deficient in PKP2 can arrest disease progression and significantly prolong survival. METHODS: Experiments were performed using a PKP2-cKO (cardiac-specific, tamoxifen-activated PKP2 knockout murine model). The potential therapeutic, adeno-associated virus vector of serotype rh.74 (AAVrh.74)-PKP2a (PKP2 variant A; RP-A601) is a recombinant AAVrh.74 gene therapy viral vector encoding the human PKP2 variant A. AAVrh.74-PKP2a was delivered to adult mice by a single tail vein injection either before or after tamoxifen-activated PKP2-cKO. PKP2 expression was confirmed by molecular and histopathologic analyses. Cardiac function and disease progression were monitored by survival analyses, echocardiography, and electrocardiography. RESULTS: Consistent with prior findings, loss of PKP2 expression caused 100% mortality within 50 days after tamoxifen injection. In contrast, AAVrh.74-PKP2a-mediated PKP2a expression resulted in 100% survival for >5 months (at study termination). Echocardiographic analysis revealed that AAVrh.74-PKP2a prevented right ventricle dilation, arrested left ventricle functional decline, and mitigated arrhythmia burden. Molecular and histological analyses showed AAVrh.74-PKP2a-mediated transgene mRNA and protein expression and appropriate PKP2 localization at the cardiomyocyte intercalated disc. Importantly, the therapeutic benefit was shown in mice receiving AAVrh.74-PKP2a after disease onset. CONCLUSIONS: These preclinical data demonstrate the potential for AAVrh.74-PKP2a (RP-A601) as a therapeutic for PKP2-related arrhythmogenic right ventricular cardiomyopathy in both early and more advanced stages of the disease.

Tamoxifen for the treatment of myeloproliferative neoplasms: A Phase II clinical trial and exploratory analysis.

Current therapies for myeloproliferative neoplasms (MPNs) improve symptoms but have limited effect on tumor size. In preclinical studies, tamoxifen restored normal apoptosis in mutated hematopoietic stem/progenitor cells (HSPCs). TAMARIN Phase-II, multicenter, single-arm clinical trial assessed tamoxifen's safety and activity in patients with stable MPNs, no prior thrombotic events and mutated JAK2V617F, CALRins5 or CALRdel52 peripheral blood allele burden ≥20% (EudraCT 2015-005497-38). 38 patients were recruited over 112w and 32 completed 24w-treatment. The study's A'herns success criteria were met as the primary outcome ( ≥ 50% reduction in mutant allele burden at 24w) was observed in 3/38 patients. Secondary outcomes included ≥25% reduction at 24w (5/38), ≥50% reduction at 12w (0/38), thrombotic events (2/38), toxicities, hematological response, proportion of patients in each IWG-MRT response category and ELN response criteria. As exploratory outcomes, baseline analysis of HSPC transcriptome segregates responders and non-responders, suggesting a predictive signature. In responder HSPCs, longitudinal analysis shows high baseline expression of JAK-STAT signaling and oxidative phosphorylation genes, which are downregulated by tamoxifen. We further demonstrate in preclinical studies that in JAK2V617F+ cells, 4-hydroxytamoxifen inhibits mitochondrial complex-I, activates integrated stress response and decreases pathogenic JAK2-signaling. These results warrant further investigation of tamoxifen in MPN, with careful consideration of thrombotic risk.

Genetic lineage tracing in skin reveals predominant expression of HEY2 in dermal papilla during telogen and that HEY2+ cells contribute to the regeneration of dermal cells during wound healing.

Dermal papilla (DP) cells are specialized mesenchymal cells that play a crucial role in regulating hair morphology, colour and growth through the secretion of specific factors. It is still unclear what the source of progenitor cells is for dermal cell regeneration during wound healing, and whether DP cells are involved in this process. We analyzed the gene expression profile of various skin cell populations using existing datasets and found that the Hey2 gene was predominantly expressed in DP cells. We introduced Hey2-CreERT2 knockin mice and crossed them with Rosa26-ZsGreen reporter mice. After induction in the double transgenic mice by administration of tamoxifen, the reporter ZsGreen was found to be predominantly expressed in DP cells both at anagen and telogen phases, and broadly expressed in some other dermal cells at anagen. We also created a wound after tamoxifen induction, and found there were abundant ZsGreen+ cells in the regenerated dermis. We conclude that the HEY2+ DP cells and dermal cells exhibit some stemness properties and can contribute to the dermal cell regeneration during wound healing.

Characterization of Breast Cancer Aggressiveness by Cell Mechanics.

In healthy tissues, cells are in mechanical homeostasis. During cancer progression, this equilibrium is disrupted. Cancer cells alter their mechanical phenotype to a softer and more fluid-like one than that of healthy cells. This is connected to cytoskeletal remodeling, changed adhesion properties, faster cell proliferation and increased cell motility. In this work, we investigated the mechanical properties of breast cancer cells representative of different breast cancer subtypes, using MCF-7, tamoxifen-resistant MCF-7, MCF10A and MDA-MB-231 cells. We derived viscoelastic properties from atomic force microscopy force spectroscopy measurements and showed that the mechanical properties of the cells are associated with cancer cell malignancy. MCF10A are the stiffest and least fluid-like cells, while tamoxifen-resistant MCF-7 cells are the softest ones. MCF-7 and MDA-MB-231 show an intermediate mechanical phenotype. Confocal fluorescence microscopy on cytoskeletal elements shows differences in actin network organization, as well as changes in focal adhesion localization. These findings provide further evidence of distinct changes in the mechanical properties of cancer cells compared to healthy cells and add to the present understanding of the complex alterations involved in tumorigenesis.

Exploring the Anticancer Activity of Tamoxifen-Based Metal Complexes Targeting Mitochondria.

Two new 'hybrid' metallodrugs of Au(III) (AuTAML) and Cu(II) (CuTAML) were designed featuring a tamoxifen-derived pharmacophore to ideally synergize the anticancer activity of both the metal center and the organic ligand. The compounds have antiproliferative effects against human MCF-7 and MDA-MB 231 breast cancer cells. Molecular dynamics studies suggest that the compounds retain the binding activity to estrogen receptor (ERα). In vitro and in silico studies showed that the Au(III) derivative is an inhibitor of the seleno-enzyme thioredoxin reductase, while the Cu(II) complex may act as an oxidant of different intracellular thiols. In breast cancer cells treated with the compounds, a redox imbalance characterized by a decrease in total thiols and increased reactive oxygen species production was detected. Despite their different reactivities and cytotoxic potencies, a great capacity of the metal complexes to induce mitochondrial damage was observed as shown by their effects on mitochondrial respiration, membrane potential, and morphology.

Inducible and Conditional Activation of Adult Neurogenesis Rescues Cadmium-Induced Hippocampus-Dependent Memory Deficits in ApoE4-KI Mice.

The apolipoprotein E (ApoE) gene is a genetic risk factor for late-onset Alzheimer's disease, in which ε4 allele carriers have increased risk compared to the common ε3 carriers. Cadmium (Cd) is a toxic heavy metal and a potential neurotoxicant. We previously reported a gene-environment interaction (GxE) effect between ApoE4 and Cd that accelerates or increases the severity of the cognitive decline in ApoE4-knockin (ApoE4-KI) mice exposed to 0.6 mg/L CdCl2 through drinking water compared to control ApoE3-KI mice. However, the mechanisms underlying this GxE effect are not yet defined. Because Cd impairs adult neurogenesis, we investigated whether genetic and conditional stimulation of adult neurogenesis can functionally rescue Cd-induced cognitive impairment in ApoE4-KI mice. We crossed either ApoE4-KI or ApoE3-KI to an inducible Cre mouse strain, Nestin-CreERTM:caMEK5-eGFPloxP/loxP (designated as caMEK5), to generate ApoE4-KI:caMEK5 and ApoE3-KI:caMEK5. Tamoxifen administration in these mice genetically and conditionally induces the expression of caMEK5 in adult neural stem/progenitor cells, enabling the stimulation of adult neurogenesis in the brain. Male ApoE4-KI:caMEK5 and ApoE3-KI:caMEK5 mice were exposed to 0.6 mg/L CdCl2 throughout the experiment, and tamoxifen was administered once Cd-induced impairment in spatial working memory was consistently observed. Cd exposure impaired spatial working memory earlier in ApoE4-KI:caMEK5 than in ApoE3-KI:caMEK5 mice. In both strains, these deficits were rescued after tamoxifen treatment. Consistent with these behavioral findings, tamoxifen treatment enhanced adult neurogenesis by increasing the morphological complexity of adult-born immature neurons. These results provide evidence for a direct link between impaired spatial memory and adult neurogenesis in this GxE model.

Evaluation of a polymeric topical formulation of Endoxifen in an estrogen receptor positive breast cancer murine model.

Breast cancer (BC) has surpassed lung cancer as the most diagnosed cancer and, in terms of mortality, is the fifth leading cause with 684,996 new deaths (6.7% of all cancer-related deaths) and the highest mortality amongst all cancers (15.5%) in women. Selective estrogen-receptor modulators (SERMs) have been used for the last thirty years for estrogen receptor-positive (ER+) BC prevention and treatment. Tamoxifen (TAM), the most widely used SERM, is orally administered and its long-term oral administration has been associated to toxicity and adverse side effects. Endoxifen (EDX) is one of the known active metabolites of TAM, with an affinity to ERα 100 times higher than TAM. Furthermore, EDX has shown antiproliferative activity against the ER+ BC cell line MCF-7. Alternative administration routes that avoid the metabolic processing of TAM seem an appealing alternative to its oral administration. With this aim, we have prepared a polymeric gel-like solution of Pluronic® F127 as vehicle for topical administration of EDX. In order to shed light on the potential clinical use of this formulation, we have compared it with the standard pharmaceutical form, i.e. orally administered TAM. The biodistribution, antitumor efficacy and toxic effects of topical EDX and oral TAM were evaluated in ER+ tumor xenograft athymic nu/nu mouse models. The results showed a statistically significant antitumor effect and reduced toxicity of topical EDX as compared to oral TAM or empty F127 gel. This novel administration route of SERMs could also have a strong impact in the prevention of BC at early development stages and could help to ameliorate the mortality and morbidity related to this disease.

Maternal tamoxifen exposure leads to abnormal primordial follicle assembly.

Tamoxifen (TAM) is an accredited drug used for treatment and prevention of breast cancer. Due to the long-term taking and the trend for women to delay childbearing, inadvertent conception occasionally occurs during TAM treatment. To explore the effects of TAM on a fetus, pregnant mice at gestation day 16.5 were orally administrated with different concentrations of TAM. Molecular biology techniques were used to analyze the effects of TAM on primordial follicle assembly of female offspring and the mechanism. It was found that maternal TAM exposure affected primordial follicle assembly and damaged the ovarian reserve in 3 dpp offspring. Up to 21 dpp, the follicular development had not recovered, with significantly decreased antral follicles and decreased total follicle number after maternal TAM exposure. Cell proliferation was significantly inhibited; however, the cell apoptosis was induced by maternal TAM exposure. Epigenetic regulation was also involved in the process of TAM induced abnormal primordial follicle assembly. The changed levels of H3K4me3, H3K9me3, and H3K27me3 presented the function of histone methylation in the regulation of the effects of maternal TAM exposure on the reproduction of female offspring. Moreover, the changed level of RNA m6A modification and the changed expression of genes related to transmethylation and demethylation proved the role of m6A in the process. Maternal TAM exposure led to abnormal primordial follicle assembly and follicular development by affecting cell proliferation, cell apoptosis, and epigenetics.

Genetically engineered multicistronic allele of Pmel yielding highly specific CreERT2-mediated recombination in the melanocyte lineage.

Genetic approaches that allow lineage tracing are essential to our future understanding of melanocytes and melanoma. To date, the approaches used to label melanocytes in mice have relied on random integration of transgenes driven by the promoters of the Tyrosinase and Dopachrome tautomerase genes, knock-in to the Dopachrome tautomerase locus or knock-in to the Mlana locus in a bacterial artificial chromosome. These strategies result in expression in other tissues such as telencephalon and other cell types such as nerves. Here we used homologous recombination in mouse embryonic stem cells to generate a targeted multicistronic allele of the Pmel locus that drives melanocyte-specific expression of CreERT2, nuclear localised H2B-Cerulean and membrane localised marcks-mKate2 allowing live imaging of melanocytes and activation of other conditional alleles. We combined this allele with R26R-EYFP mice allowing induction of EYFP expression on administration of tamoxifen or its metabolite 4-OHT. The fluorescent proteins H2B-Cerulean and marcks-mKate2 label the cell nucleus and plasma membrane respectively allowing live imaging and FACS isolation of melanoblasts and melanocytes as well as serving to provide an internal control allowing estimation of recombination efficiency after administration of tamoxifen. We demonstrate the utility of the transgene in embryonic and adult tissues.

Process development for the total synthesis of the novel drug metabolite Carboxy toremifene as a standard reference material along with characterization and purity assessment for the Antidoping quality Control Purposes.

Tamoxifen and toremifene are two selective estrogen receptor modulators (SERMs) commonly used to treat breast cancer in women. Toremifene is well-known as a triphenylethylene derivative. Carboxy toremifene is a common metabolite of toremifene and tamoxifen. Since 2005, the World Anti-Doping Agency (WADA) has banned the SERMs category during in and out of competition. These substances are in the S4 category in the WADA prohibited list as "agents with anti-oestrogenic activity." However, there is no commercially accessible carboxy toremifene reference material in the market. This research highlights the novel synthetic procedure, the development of a carboxy toremifene HPLC method, and validation, along with detailed characterization using advanced analytical techniques using 1 H NMR, HRMS, FT-IR-ATR and UV-visible spectroscopy. RP-HPLC-DAD method was developed and validated to assess the purity of carboxy toremifene. Developed reference material has shown 100% purity. Therefore, we recommend that this synthesized carboxy toremifene may be used as reference material to strengthen the WADA-accredited lab to maintain a clean sports mission during sports competitions.

Nestin is a marker of unipotent embryonic and adult progenitors differentiating into an epithelial cell lineage of the hair follicles.

Nestin is an intermediate filament protein transiently expressed in neural stem/progenitor cells. We previously demonstrated that outer root sheath (ORS) keratinocytes of adult hair follicles (HFs) in mice descend from nestin-expressing cells, despite being an epithelial cell lineage. This study determined the exact stage when nestin-expressing ORS stem/precursor cells or their descendants appear during HF morphogenesis, and whether they are present in adult HFs. Using Nes-Cre/CAG-CAT-EGFP mice, in which enhanced green fluorescent protein (EGFP) is expressed following Cre-based recombination driven by the nestin promoter, we found that EGFP+ cells appeared in the epithelial layer of embryonic HFs as early as the peg stage. EGFP+ cells in hair pegs were positive for keratin 14 (K14) and K5, but not vimentin, SOX2, SOX10, or S100 alpha 6. Tracing of tamoxifen-induced EGFP+ cells in postnatal Nes-CreERT2/CAG-CAT-EGFP mice revealed labeling of some isthmus HF epithelial cells in the first anagen stage. EGFP+ cells in adult HFs were not immunolabeled for K15, an HF multipotent stem cell marker. However, when hairs were depilated in Nes-CreERT2/CAG-CAT-EGFP mice to induce the anagen stage after tamoxifen injection, the majority of ORS keratinocytes in depilation-induced anagen HFs were labeled for EGFP. Our findings indicate that nestin-expressing unipotent progenitor cells capable of differentiating into ORS keratinocytes are present in HF primordia and adult HFs.

Cardiomyocyte p38 MAPKα suppresses a heart-adipose tissue-neutrophil crosstalk in heart failure development.

Although p38 MAP Kinase α (p38 MAPKα) is generally accepted to play a central role in the cardiac stress response, to date its function in maladaptive cardiac hypertrophy is still not unambiguously defined. To induce a pathological type of cardiac hypertrophy we infused angiotensin II (AngII) for 2 days via osmotic mini pumps in control and tamoxifen-inducible, cardiomyocyte (CM)-specific p38 MAPKα KO mice (iCMp38αKO) and assessed cardiac function by echocardiography, complemented by transcriptomic, histological, and immune cell analysis. AngII treatment after inactivation of p38 MAPKα in CM results in left ventricular (LV) dilatation within 48 h (EDV: BL: 83.8 ± 22.5 µl, 48 h AngII: 109.7 ± 14.6 µl) and an ectopic lipid deposition in cardiomyocytes, reflecting a metabolic dysfunction in pressure overload (PO). This was accompanied by a concerted downregulation of transcripts for oxidative phosphorylation, TCA cycle, and fatty acid metabolism. Cardiac inflammation involving neutrophils, macrophages, B- and T-cells was significantly enhanced. Inhibition of adipose tissue lipolysis by the small molecule inhibitor of adipocytetriglyceride lipase (ATGL) Atglistatin reduced cardiac lipid accumulation by 70% and neutrophil infiltration by 30% and went along with an improved cardiac function. Direct targeting of neutrophils by means of anti Ly6G-antibody administration in vivo led to a reduced LV dilation in iCMp38αKO mice and an improved systolic function (EF: 39.27 ± 14%). Thus, adipose tissue lipolysis and CM lipid accumulation augmented cardiac inflammation in iCMp38αKO mice. Neutrophils, in particular, triggered the rapid left ventricular dilatation. We provide the first evidence that p38 MAPKα acts as an essential switch in cardiac adaptation to PO by mitigating metabolic dysfunction and inflammation. Moreover, we identified a heart-adipose tissue-immune cell crosstalk, which might serve as new therapeutic target in cardiac pathologies.

Synthesis and SAR Analysis of Novel 4-Hydroxytamoxifen Analogues Based on Their Cytotoxic Activity and Electron-Donor Character.

Utilizing McMurry reactions of 4,4'-dihydroxybenzophenone with appropriate carbonyl compounds, a series of 4-Hydroxytamoxifen analogues were synthesized. Their cytotoxic activity was evaluated in vitro on four human malignant cell lines (MCF-7, MDA-MB 231, A2058, HT-29). It was found that some of these novel Tamoxifen analogues show marked cytotoxicity in a dose-dependent manner. The relative ROS-generating capability of the synthetized analogues was evaluated by cyclic voltammetry (CV) and DFT modeling studies. The results of cell-viability assays, CV measurements and DFT calculations suggest that the cytotoxicity of the majority of the novel compounds is mainly elicited by their interactions with cellular targets including estrogen receptors rather than triggered by redox processes. However, three novel compounds could be involved in ROS-production and subsequent formation of quinone-methide preventing proliferation and disrupting the redox balance of the treated cells. Among the cell lines studied, HT-29 proved to be the most susceptible to the treatment with compounds having ROS-generating potency.

Sensory Neuron-Specific Deletion of Tropomyosin Receptor Kinase A (TrkA) in Mice Abolishes Osteoarthritis (OA) Pain via NGF/TrkA Intervention of Peripheral Sensitization.

Tropomyosin receptor kinase A (TrkA/NTRK1) is a high-affinity receptor for nerve growth factor (NGF), a potent pain mediator. NGF/TrkA signaling elevates synovial sensory neuronal distributions in the joints and causes osteoarthritis (OA) pain. We investigated the mechanisms of pain transmission as to whether peripheral sensory neurons are linked to the cellular plasticity in the dorsal root ganglia (DRG) and are critical for OA hyperalgesia. Sensory neuron-specific deletion of TrkA was achieved by tamoxifen injection in 4-week-old TrkAfl/fl;NaV1.8CreERT2 (Ntrk1 fl/fl;Scn10aCreERT2) mice. OA was induced by partial medial meniscectomy (PMM) in 12-week-old mice, and OA-pain-related behavior was analyzed for 12 weeks followed by comprehensive histopathological examinations. OA-associated joint pain was markedly improved without cartilage protection in sensory-neuron-specific conditional TrkA knock-out (cKO) mice. Alleviated hyperalgesia was associated with suppression of the NGF/TrkA pathway and reduced angiogenesis in fibroblast-like synovial cells. Elevated pain transmitters in the DRG of OA-induced mice were significantly diminished in sensory-neuron-specific TrkA cKO and global TrkA cKO mice. Spinal glial activity and brain-derived neurotropic factor (BDNF) were significantly increased in OA-induced mice but were substantially eliminated by sensory-neuron-specific deletion. Our results suggest that augmentation of NGF/TrkA signaling in the joint synovium and the peripheral sensory neurons facilitate pro-nociception and centralized pain sensitization.

Functional architecture of pancreatic islets identifies a population of first responder cells that drive the first-phase calcium response.

Insulin-secreting ß-cells are functionally heterogeneous. Whether there exist cells driving the first-phase calcium response in individual islets, has not been examined. We examine "first responder" cells, defined by the earliest [Ca2+] response during first-phase [Ca2+] elevation, distinct from previously identified "hub" and "leader" cells. We used islets isolated from Mip-CreER; Rosa-Stop-Lox-Stop-GCamP6s mice (ß-GCamP6s) that show ß-cell-specific GCamP6s expression following tamoxifen-induced CreER-mediated recombination. First responder cells showed characteristics of high membrane excitability and lower electrical coupling to their neighbors. The first-phase response time of ß-cells in the islet was spatially organized, dependent on the cell's distance to the first responder cell, and consistent over time up to approximately 24 h. When first responder cells were laser ablated, the first-phase [Ca2+] was slowed down, diminished, and discoordinated compared to random cell ablation. Cells that were next earliest to respond often took over the role of the first responder upon ablation. In summary, we discover and characterize a distinct first responder ß-cell state, critical for the islet first-phase response to glucose.

Essential phospholipids decrease apoptosis and increase membrane transport in human hepatocyte cell lines.

BACKGROUND: Essential phospholipids (EPL) have hepatoprotective effects across many liver diseases/conditions. The impact of EPL on hepatocyte function in vitro was investigated. METHODS: Effects of noncytotoxic concentrations of EPL (0.1 and 0.25 mg/ml), and its constituents, polyenylphosphatidylcholine (PPC) and phosphatidylinositol (PI) (both at 0.1 and 1 mg/ml), on membrane fluidity, apoptosis and extracellular transport versus controls were investigated in human hepatocyte cell lines (HepG2, HepaRG, steatotic HepaRG).  RESULTS: Significantly increased membrane fluidity occurred with all 3 phospholipids (PLs) in HepG2 cultures, and with PI (1 mg/ml) in steatotic HepaRG cells. Significantly decreased tamoxifen-induced apoptosis was observed in HepG2 cells with EPL, PPC and PI. Breast cancer resistance protein (BCRP) activity was significantly increased by EPL and PI in HepG2 cells. Multidrug resistance-associated protein 2 (MRP-2) activity was unaffected by any PL in HepG2 cells, and significantly increased by EPL, PI and PPC (1 mg/ml) in HepaRG cells, and by PI (1 mg/ml) in steatotic HepaRG cells. Bile salt export protein (BSEP) activity in HepG2 cells and steatotic HepaRG cells was significantly increased by EPL (0.25 mg/ml), and PPC (both concentrations), but not by PI. The PLs had no effects on HepaRG cell BSEP activity. P-glycoprotein (P-GP) activity was significantly increased by all compounds in HepG2 cells. PI (1 mg/ml) significantly increased P-GP activity in HepaRG and steatotic HepaRG cells. CONCLUSIONS: EPL, PPC, and PI increased hepatocyte membrane fluidity, decreased apoptosis and increased hepatocellular export, all of which may improve liver function. These in-vitro investigations provide valuable insights into the mechanism of action of EPL.

Inflammatory perturbations in early life long-lastingly shape the transcriptome and TCR repertoire of the first wave of regulatory T cells.

The first wave of Foxp3+ regulatory T cells (Tregs) generated in neonates is critical for the life-long prevention of autoimmunity. Although it is widely accepted that neonates are highly susceptible to infections, the impact of neonatal infections on this first wave of Tregs is completely unknown. Here, we challenged newborn Treg fate-mapping mice (Foxp3eGFPCreERT2xROSA26STOP-eYFP) with the Toll-like receptor (TLR) agonists LPS and poly I:C to mimic inflammatory perturbations upon neonatal bacterial or viral infections, respectively, and subsequently administrated tamoxifen during the first 8 days of life to selectively label the first wave of Tregs. Neonatally-tagged Tregs preferentially accumulated in non-lymphoid tissues (NLTs) when compared to secondary lymphoid organs (SLOs) irrespective of the treatment. One week post challenge, no differences in the frequency and phenotypes of neonatally-tagged Tregs were observed between challenged mice and untreated controls. However, upon aging, a decreased frequency of neonatally-tagged Tregs in both NLTs and SLOs was detected in challenged mice when compared to untreated controls. This decrease became significant 12 weeks post challenge, with no signs of altered Foxp3 stability. Remarkably, this late decrease in the frequency of neonatally-tagged Tregs only occurred when newborns were challenged, as treating 8-days-old mice with TLR agonists did not result in long-lasting alterations of the first wave of Tregs. Combined single-cell T cell receptor (TCR)-seq and RNA-seq revealed that neonatal inflammatory perturbations drastically diminished TCR diversity and long-lastingly altered the transcriptome of neonatally-tagged Tregs, exemplified by lower expression of Tigit, Foxp3, and Il2ra. Together, our data demonstrate that a single, transient encounter with a pathogen in early life can have long-lasting consequences for the first wave of Tregs, which might affect immunological tolerance, prevention of autoimmunity, and other non-canonical functions of tissue-resident Tregs in adulthood.

B cell intrinsic and extrinsic factors impacting memory recall responses to SRBC challenge.

MBCs (MBCs) generated in T-dependent immune responses can persist for a lifetime and rapidly react upon secondary antigen exposure to differentiate into plasma cells (PCs) and/or to improve the affinity of their BCR through new rounds of hypermutation in germinal centers (GCs). The fate of a MBC in secondary immune reactions appears to depend upon multiple parameters, whose understanding is mandatory for the design of efficient vaccine strategies. We followed the behavior of MBCs in recall responses to SRBCs using an inducible AID fate mapping mouse model in which B cells engaged in a germinal center (GC) response are irreversibly labeled upon simultaneous tamoxifen ingestion and immunization. We used different schemes of mouse immunization and tamoxifen feeding in adoptive-transfer experiments of total splenic B cells into congenic mice that have been pre-immunized or not, to assess the contribution of the different effector subsets in a physiological competitive context. We were able to show that naive B cells can differentiate into GC B cells with kinetics similar to MBCs in the presence of previously activated T follicular helper (TFH) cells and a primed microenvironment. We also showed that MBCs are recruited into secondary GCs, together with naive B cells. In contrast, PC differentiation, which dominated secondary MBC responses, was not dependent upon a previous TFH activation. We observed that the presence of persisting germinal centers and circulating antibody levels are key factors determining the germinal center versus plasma cell fate in a recall response. Notably, disruption of persistent germinal center structures by a lymphotoxin beta-receptor fusion protein or a longer timing between the prime and the boost, which correlated with reduced antigen-specific immunoglobulin levels in serum, were two conditions with an opposite impact, respectively inhibiting or promoting a GC fate for MBCs. Altogether, these studies highlight the complexity of recall responses, whose outcome varies according to immunization contexts.