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Luo, W.

Publications and source records attributed to Luo, W..

At least 91 records · Page 5Linked to original sources

Ets1 and IL17RA cooperate to regulate autoimmune responses as well as skin immunity to Staphylococcus aureus

Ets1 is a lymphoid-enriched transcription factor that regulates B and T cell functions in development and disease. Mice that lack Ets1 (Ets1 KO) develop spontaneous autoimmune disease with high levels of autoantibodies. Naive CD4+ T cells isolated from Ets1 KO mice differentiate more readily to Th17 cells that secrete IL-17, a cytokine implicated in autoimmune disease pathogenesis. To determine if increased IL-17 production contributes to the development of autoimmunity in Ets1 KO mice, we crossed Ets1 KO mice to mice lacking the IL-17 receptor A subunit (IL17RA KO) to generate double knock out (DKO) mice. We found that the absence of IL17RA signaling did not prevent or ameliorate the autoimmune phenotype of Ets1 KO mice, but rather that DKO animals exhibited worse symptoms with striking increases in activated B cells and secreted autoantibodies. This was correlated with a prominent increase in the numbers of T follicular helper (Tfh) cells. In addition to the autoimmune phenotype, DKO mice also showed signs of immunodeficiency and developed spontaneous skin lesions colonized by Staphylococcus xylosus. When DKO mice were experimentally infected with S. aureus they were unable to clear the bacteria, suggesting a general immunodeficiency to Staphylococcal species. {gamma}{delta} T cells are important for control of skin Staphylococcal infections. We found that mice lacking Ets1 have a complete deficiency of the {gamma}{delta} T cell subset dendritic epidermal T cells (DETC), which are involved in skin wound healing responses. To determine if loss of DETC might promote susceptibility to Staph infection, we depleted DETC from IL17RA KO mice and found that the combined loss of DETC and IL-17 signaling leads to a failure to clear the infection. Our studies suggest that defects in wound healing, such as that caused by loss of DETC, can cooperate with impaired IL-17 responses to lead to increased susceptibility to skin Staph infections.

immunology↗

A dual hit of α-synuclein internalization and immune challenge leads to formation and maintenance of Lewy body-like inclusions in human dopaminergic neurons

Lewy bodies (LBs), rich in -synuclein, are a hallmark of Parkinsons disease (PD). Understanding their biogenesis is likely to provide insight into the pathophysiology of PD, yet a cellular model for LB formation remains elusive. The realization that the immune challenge is a trigger for neurodegenerative diseases has been a breakthrough in the understanding of PD. Here, iPSC-derived human dopaminergic (DA) neurons from multiple healthy donors were found to form LB-like inclusions following treatment with - synuclein preformed fibrils, but only when coupled to an immune challenge (interferon-gamma or interleukin-1 beta) or when co-cultured with activated microglia. Human cortical neurons derived from the same iPSC lines did not form LB-like inclusions. Exposure to interferon-gamma impairs autophagy in a lysosomal-specific manner in vitro, similar to the disruption of proteostasis pathways that contribute to PD. We find that lysosomal membrane proteins LAMP1 and LAMP2 and transcription factors regulating lysosomal biogenesis and function are downregulated in DA but not cortical neurons. Finally, due to the excellent sample preservation afforded by cells compared to post-mortem PD brain tissue, we conclude that the LB-like inclusions in DA neurons are membrane-bound, suggesting they are not limited to the cytoplasmic compartment. In Brief O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC="FIGDIR/small/542776v3_figabs.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1f41855org.highwire.dtl.DTLVardef@69798dorg.highwire.dtl.DTLVardef@ab55bdorg.highwire.dtl.DTLVardef@8a16b8_HPS_FORMAT_FIGEXP M_FIG C_FIG Bayati et al. identify that iPSC-derived dopaminergic neurons undergoing a dual hit treatment of exogenous -synuclein fibrils and proinflammatory cytokines form Lewy body-like inclusions. The dual hit treatment also led to the downregulation of lysosomal proteins. Characterization of inclusions revealed that inclusions were membrane-bound and LC3B-positive, suggesting they are dysfunctional autophagosomes. HighlightsO_LI-synuclein preformed fibril administration coupled with Interferon-gamma exposure leads dopaminergic neurons to form Lewy body-like inclusions C_LIO_LIInclusions are filamentous, membranous, and filled with aberrant organelles C_LIO_LIImpaired autophagic flux and downregulation of TFEB, NRF2, LAMP1, and LAMP2 correlated with inclusion formation C_LIO_LIActivation of NRF2 through the treatment of neurons with the antioxidant perillaldehyde, prevents inclusion formation C_LI

neuroscience↗

Distinct Local and Global Functions of Aβ Low-Threshold Mechanoreceptors in Mechanical Pain Transmission

The roles of A{beta} low-threshold mechanoreceptors (LTMRs) in transmitting mechanical hyperalgesia and in alleviating chronic pain have been of great interest but remain contentious. Here we utilized intersectional genetic tools, optogenetics, and high-speed imaging to specifically examine functions of SplitCre labeled A{beta}-LTMRs in this regard. Genetic ablation of SplitCre-A{beta}-LTMRs increased mechanical pain but not thermosensation in both acute and chronic inflammatory pain conditions, indicating their modality-specific role in gating mechanical pain transmission. Local optogenetic activation of SplitCre-A{beta}-LTMRs triggered nociception after tissue inflammation, whereas their broad activation at the dorsal column still alleviated mechanical hypersensitivity of chronic inflammation. Taking all data into consideration, we propose a new model, in which A{beta}-LTMRs play distinctive local and global roles in transmitting and alleviating mechanical hyperalgesia of chronic pain, respectively. Our model suggests a new strategy of global activation plus local inhibition of A{beta}-LTMRs for treating mechanical hyperalgesia.

neuroscience↗

iFlpMosaics: A method for the ratiometric induction and high-throughput comparative analysis of mutant and wildtype cells

To understand gene function, it is necessary to compare cells carrying the mutated target gene with normal cells. In most biomedical studies, the cells being compared are in different mutant and control animals and therefore do not experience the same epigenetic changes and tissue microenvironment. The experimental induction of genetic mosaics is essential to determine a gene cell-autonomous function and to model the etiology of diseases caused by somatic mutations. Current technologies used to induce genetic mosaics in mice lack either accuracy, throughput or barcoding diversity. Here, we present a large set of new genetic tools and mouse lines that enable Flp recombinase-dependent ratiometric induction and single-cell clonal tracking of multiple fluorescently labeled wildtype and Cre-mutant cells within the same time window and tissue microenvironment. The labeled cells can be profiled by multispectral imaging or by FACS and scRNA-seq. This technology facilitates the induction and analysis of genetic mosaics in any cell type and for any given single or combination of floxed genes. iFlpMosaics enables a more accurate understanding of how induced genetic mutations affect the biology of single cells during tissue development, homeostasis, and disease.

cell biology↗

Single-Soma Deep RNA sequencing of Human DRG Neurons Reveals Novel Molecular and Cellular Mechanisms Underlying Somatosensation

The versatility of somatosensation arises from heterogeneous dorsal root ganglion (DRG) neurons. However, soma transcriptomes of individual human DRG (hDRG) neurons - critical information to decipher their functions - are lacking due to technical difficulties. Here, we developed a novel approach to isolate individual hDRG neuron somas for deep RNA sequencing (RNA-seq). On average, >9,000 unique genes per neuron were detected, and 16 neuronal types were identified. Cross-species analyses revealed remarkable divergence among pain-sensing neurons and the existence of human-specific nociceptor types. Our deep RNA-seq dataset was especially powerful for providing insight into the molecular mechanisms underlying human somatosensation and identifying high potential novel drug targets. Our dataset also guided the selection of molecular markers to visualize different types of human afferents and the discovery of novel functional properties using single-cell in vivo electrophysiological recordings. In summary, by employing a novel soma sequencing method, we generated an unprecedented hDRG neuron atlas, providing new insights into human somatosensation, establishing a critical foundation for translational work, and clarifying human species-specific properties.

neuroscience↗

MerTK mediates the immunologically silent uptake of alpha-synuclein fibrils by human microglia

MerTK is a receptor tyrosine kinase that mediates the immunologically silent phagocytic uptake of diverse types of cellular debris. Highly expressed on the surface of microglial cell, MerTK is of importance in brain development, homeostasis, plasticity, and disease. Yet, involvement of this receptor in the clearance of protein aggregates that accumulate with aging and in neurodegenerative diseases has yet to be defined. The current study explored the function of MerTK in the microglial uptake of alpha-synuclein fibrils which play a causative role in the pathobiology of synucleinopathies. Using human primary and induced pluripotent stem cell-derived microglia, the MerTK- dependence of alpha-synuclein fibril internalization was investigated in vitro. Relevance of this pathway to synucleinopathies was assessed by analyzing MerTK expression in patient-derived cells and tissues. Pharmacological inhibition of MerTK and siRNA-mediated MERTK knockdown both caused a decreased rate of alpha-synuclein fibril internalization by human microglia. Consistent with the immunologically silent nature of MerTK-mediated phagocytosis, alpha-synuclein fibril internalization did not induce secretion of pro-inflammatory cytokines from microglia. In addition, burden analysis in two independent patient cohorts revealed a significant association between rare functionally deleterious MERTK variants and Parkinsons disease in one of the cohorts (p = 0.002). Accordingly, MERTK expression was significantly upregulated in nigral microglia from Parkinsons disease/Lewy body dementia patients compared to those from non-neurological control donors in a single-nuclei RNA-sequencing dataset (p = 5.08x10-21), and MerTK protein expression positively correlated with alpha-synuclein level in human cortex lysates (p = 0.0029). Taken together, our findings define a novel role for MerTK in mediating the uptake of alpha-synuclein aggregates by human microglia, with possible involvement in limiting alpha-synuclein spread in synucleinopathies such as Parkinsons disease.

cell biology↗

CXCR2 expression during melanoma tumorigenesis controls transcriptional programs that facilitate tumor growth

BackgroundThough the CXCR2 chemokine receptor is known to play a key role in cancer growth and response to therapy, a direct link between expression of CXCR2 in tumor progenitor cells during induction of tumorigenesis has not been established. MethodsTo characterize the role of CXCR2 during melanoma tumorigenesis, we generated tamoxifen-inducible tyrosinase-promoter driven BrafV600E/Pten-/-/Cxcr2-/- and NRasQ61R/INK4a-/-/Cxcr2-/- melanoma models. In addition, the effects of a CXCR1/CXCR2 antagonist, SX-682, on melanoma tumorigenesis were evaluated in BrafV600E/Pten-/- and NRasQ61R/INK4a-/- mice and in melanoma cell lines. Potential mechanisms by which Cxcr2 affects melanoma tumorigenesis in these murine models were explored using RNAseq, mMCP-counter, ChIPseq, and qRT-PCR; flow cytometry, and reverse phosphoprotein analysis (RPPA). ResultsGenetic loss of Cxcr2 or pharmacological inhibition of CXCR1/CXCR2 during melanoma tumor induction resulted in key changes in gene expression that reduced tumor incidence/growth and increased anti-tumor immunity. Interestingly, after Cxcr2 ablation, Tfcp2l1, a key tumor suppressive transcription factor, was the only gene significantly induced with a log2 fold-change greater than 2 in these three different melanoma models. ConclusionsHere, we provide novel mechanistic insight revealing how loss of Cxcr2 expression/activity in melanoma tumor progenitor cells results in reduced tumor burden and creation of an anti-tumor immune microenvironment. This mechanism entails an increase in expression of the tumor suppressive transcription factor, Tfcp2l1, along with alteration in the expression of genes involved in growth regulation, tumor suppression, stemness, differentiation, and immune modulation. These gene expression changes are coincident with reduction in the activation of key growth regulatory pathways, including AKT and mTOR.

cancer biology↗

α-synuclein preformed fibrils bind to β-neurexins and impair β-neurexin-mediated presynaptic organization

Synucleinopathies form a group of neurodegenerative diseases defined by misfolding and aggregation of alpha-synuclein (-syn). Abnormal accumulation and spreading of -syn aggregates lead to synapse dysfunction and neuronal cell death. Yet, little is known about synaptic mechanisms underlying -syn pathology. Here we identified {beta}-isoforms of neurexins ({beta}-NRXs) as presynaptic organizing proteins that interact with -syn preformed fibrils (-syn PFFs), toxic -syn aggregates, but not -syn monomers. Our cell surface protein binding assays and surface plasmon resonance assays reveal that -syn PFFs bind directly to {beta}-NRX through their N-terminal histidine-rich domain (HRD) at nanomolar range (Kd: ~500 nM monomer equivalent). Furthermore, our artificial synapse formation assays show that -syn PFFs diminish excitatory and inhibitory presynaptic organization induced by a specific isoform of neuroligin 1 that binds only {beta}-NRXs, but not -isoforms of neurexins. Thus, our data suggest that -syn PFFs interact with {beta}-NRXs to inhibit {beta}-NRX-mediated presynaptic organization, providing novel molecular insight into how -syn PFFs induce synaptic pathology in synucleinopathies such as Parkinsons disease and dementia with Lewy bodies.

neuroscience↗

Spike substitution T813S increases Sarbecovirus fusogenicity by enhancing the usage of TMPRSS2.

SARS-CoV Spike (S) protein shares considerable homology with SARS-CoV-2 S, especially in the conserved S2 subunit (S2). S protein mediates coronavirus receptor binding and membrane fusion, and the latter activity can greatly influence coronavirus infection. We observed that SARS-CoV S is less effective in inducing membrane fusion compared with SARS-CoV-2 S. We identify that S813T mutation is sufficient in S2 interfering with the cleavage of SARS-CoV-2 S by TMPRSS2, reducing spike fusogenicity and pseudoparticle entry. Conversely, the mutation of T813S in SARS-CoV S increased fusion ability and viral replication. Our data suggested that residue 813 in the S was critical for the proteolytic activation, and the change from threonine to Serine at 813 position might be an evolutionary feature adopted by SARS-2-related viruses. This finding deepened the understanding of Spike fusogenicity and could provide a new perspective for exploring Sarbecovirus evolution. Author SummaryThe Spike strain of SARS-CoV-2 has accumulated many mutations during its time in circulation, most of which have occurred in the S1 region, and more specifically in the RBD, in an effort to either improve the viruss affinity for the receptor ACE2 or to enhance its ability to evade the immune system. Mutations in the Spike S2 region have more far-reaching effects than those in the S1 region because it is more conserved across sarbecoviruses. By comparing SARS and SARS2, we found that an important substitution at amino acid position 813 in the S2 region (T813S) disrupts the utilization of TMPRSS2 and can significantly influence viral entry into cells. This discovery deepens our knowledge of S proteins and provides new prospects for tracing the evolution of Sarbecoviruses.

microbiology↗

Genetic inactivation of the USP19 deubiquitinase regulates a-synuclein ubiquitination and inhibits accumulation of Lewy body like aggregates in mice

The USP19 deubiquitinase is found in a locus associated with Parkinsons Disease (PD), interacts with heat shock proteins and promotes secretion of a-synuclein (a-syn) through the misfolding associated protein secretion (MAPS) pathway. Since these processes might modulate the processing of a-syn aggregates during the progression of PD, we tested the effect of USP19 knockout (KO) in mice expressing the A53T mutation of a-syn and in whom a-syn preformed fibrils (PFF) had been injected in the striatum. Compared to WT, KO brains showed decreased accumulation of phospho-synuclein (pSyn) positive aggregates. The improved pathology was associated with less activation of microglia, higher levels of synaptic marker proteins and improved performance in a tail suspension test. Exposure of primary neurons from WT and KO mice to PFF in vitro also led to decreased accumulation of pSyn aggregates. KO did not affect uptake of PFF in the cultured neurons. It also did not affect the propagation of aggregates as assessed by exposing WT or KO neurons to PFF and measuring pSyn positive aggregates in non-exposed adjacent neurons separated using a microfluidics device. We conclude that USP19 instead modulates intracellular dynamics of aggregates. Indeed, at the early time following PFF injection when the number of pSyn positive neurons were similar in WT and KO brains, the KO neurons contained less aggregates. KO brain aggregates stained more intensely with anti-ubiquitin antibodies. Immunoprecipitation of soluble proteins from primary neurons exposed to PFF with antibodies to ubiquitin or pSyn showed higher levels of ubiquitinated a-syn oligomeric species in the KO neurons. We propose that the improved pathology in USP19 KO brains may arise from decreased formation or enhanced clearance of the more ubiquitinated aggregates and/or enhanced disassembly towards more soluble oligomeric species. USP19 inhibition may represent a novel therapeutic approach that targets the intracellular dynamics of a-syn complexes.

neuroscience↗

Single-cell multi-omics reveals dynamics of purifying selection of pathogenic mitochondrial DNA across human immune cells

Cells experience intrinsic and extrinsic pressures that affect their proclivity to expand and persist in vivo. In congenital disorders caused by loss-of-function mutations in mitochondrial DNA (mtDNA), metabolic vulnerabilities may result in cell-type specific phenotypes and depletion of pathogenic alleles, contributing to purifying selection. However, the impact of pathogenic mtDNA mutations on the cellular hematopoietic landscape is not well understood. Here, we establish a multi-omics approach to quantify deletions in mtDNA alongside cell state features in single cells derived from Pearson syndrome patients. We resolve the interdependence between pathogenic mtDNA and lineage, including purifying selection against deletions in effector/memory CD8 T-cell populations and recent thymic emigrants and dynamics in other hematopoietic populations. Our mapping of lineage-specific purifying selection dynamics in primary cells from patients carrying pathogenic heteroplasmy provides a new perspective on recurrent clinical phenotypes in mitochondrial disorders, including cancer and infection, with potential broader relevance to age-related immune dysfunction.

genetics↗

Regulation of adventitial interstitial fluid circulation by heartbeat and respiration

Converging studies showed interstitial fluid (ISF) adjacent to blood vessels flows along adventitia of vasculature into heart and lungs. We aim to reveal circulatory pathways and regulatory mechanism of such adventitial ISF flow in rat model. By MRI, real-time fluorescent imaging, micro-CT and histological analysis, ISF was found to flow in adventitial matrix surrounded by fascia and along systemic vessels into heart, then flow into lungs via pulmonary arteries and back to heart via pulmonary veins, which was neither perivascular tissues nor blood or lymphatic vessels. Under physiological conditions, speckle-like adventitial ISF flow rate was positively correlated with heart rate, increased when holding breath, became pulsative during heavy breathing. During cardiac or respiratory cycle, each dilation or contraction of heart or lungs can generate to-and-fro adventitial ISF flow along femoral veins. Discovered regulatory mechanisms of adventitial ISF flow along vasculature by heart and lungs will revolutionize understanding of cardiovascular system. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/512678v3_ufig1.gif" ALT="Figure 1"> View larger version (68K): org.highwire.dtl.DTLVardef@1d29c16org.highwire.dtl.DTLVardef@583f5org.highwire.dtl.DTLVardef@1a551eorg.highwire.dtl.DTLVardef@ca4780_HPS_FORMAT_FIGEXP M_FIG Graphic abstract of adventitial interstitial fluid cycle by heartbeat and respiration.The heart and lungs can work together to regulate the ISF (green arrow) adjacent to blood vessels to flow centripetally in an adventitial pathway along the major arteries and veins of the systemic vasculature to the epicardium and myocardium, then flow into the lungs via the pulmonary arteries and back to the heart via the pulmonary veins, forming an adventitial ISF circulatory network. Under physiological conditions, the speckle-like adventitial ISF flow rate was positively correlated with heart rate, increased when holding breath, and became pulsative during heavy breathing. During respiratory cycle, each expansion of the lungs during inflation generates a centrifugal flow of adventitial ISF along the systemic veins, while each contraction of the lungs during deflation generates a centripetal adventitial ISF flow. During cardiac cycle, each dilation of atria or ventricles generates a centripetal flow of adventitial ISF along the systemic veins, while each contraction of atria or ventricles generates a centrifugal adventitial ISF flow. C_FIG

physiology↗

Presymptomatic neuroanatomical and cognitive biomarkers of alpha-synuclein propagation in a mouse model of synucleinopathy

There is significant evidence suggesting aggregated misfolded alpha-synuclein, a major component of Lewy bodies, propagates in a prion-like manner contributing to disease progression in Parkinsons disease (PD) and other synucleinopathies. Animal models are essential for understanding and developing treatments for these diseases. However, despite modelling human pathology, most endpoints studied in mice do not translate to humans. Furthermore, the progression by which alpha-synuclein misfolding affects human-relevant measures such as brain volume and underlying subtle, high-level cognitive deficits is poorly understood. Here we used a mouse model of synucleinopathy; hemizygous M83 human A53T alpha-synuclein transgenic mice inoculated with recombinant human alpha-synuclein preformed fibrils (PFF) injected in the right striatum to initiate alpha-synuclein misfolding and aggregation. We examined alpha-synuclein-induced atrophy at 90 days post-injection using ex vivo magnetic resonance imaging as well as high-level cognition and motor function, as biomarkers of alpha-synuclein toxicity. We observed widespread atrophy in bilateral regions that project to or receive input from the injection site, highlighting a network of regions that are consistent with structural changes observed in humans with PD. Moreover, we detected early deficits in reversal learning with touchscreen testing in PFF-injected mice prior to motor dysfunction, consistent with the pathology observed in cortical-striatal and thalamic loops. We show, using translational approaches in mice, that progression of prion-like spreading of alpha-synuclein causes selective atrophy via connected brain regions leading to high-level cognitive deficits. We propose that precise imaging and cognitive biomarkers can provide a more direct and human-relevant measurement of alpha-synuclein-induced toxicity in pre-clinical testing. Significance StatementThe work described in this manuscript showcases the utility of state-of-the-art methodologies (magnetic resonance imaging and touchscreen behavioural tasks) to examine endophenotypes, both in terms of symptomatology and neuroanatomy, of alpha-synuclein propagation in a mouse model of synucleinopathy. Our work further validates the M83-Hu-PFF mouse model of synucleinopathy-associated pathogenesis of neurodegenerative diseases while highlighting precise imaging and cognitive biomarkers of protein misfolding toxicity. Specifically, we identified rapid and translational biomarkers that can serve as a proxy for the direct examination of cellular levels for pathology. We anticipate that these biomarkers can measure progression of toxicity, specifically in the early phases, and may be more reliable than end stage pathology and more useful as endpoints in the examination of novel therapeutics.

neuroscience↗

Scratch-AID: A Deep-learning Based System for Automatic Detection of Mouse Scratching Behavior with High Accuracy

Mice are the most commonly used model animals for itch research and for development of antiitch drugs. Most labs manually quantify mouse scratching behavior to assess itch intensity. This process is labor-intensive and limits large-scale genetic or drug screenings. In this study, we developed a new system, Scratch-AID (Automatic Itch Detection), which could automatically identify and quantify mouse scratching behavior with high accuracy. Our system included a custom-designed videotaping box to ensure high-quality and replicable mouse behavior recording and a convolutional recurrent neural network (CRNN) trained with frame-labeled mouse scratching behavior videos, induced by nape injection of chloroquine (CQ). The best trained network achieved 97.6% recall and 96.9% precision on previously unseen test videos. Remarkably, Scratch-AID could reliably identify scratching behavior in other major mouse itch models, including the acute cheek model, the histaminergic model, and a chronic itch model. Moreover, our system detected significant differences in scratching behavior between control and mice treated with an anti-itch drug. Taken together, we have established a novel deep learning-based system that is ready to replace manual quantification for mouse scratching behavior in different itch models and for drug screening.

neuroscience↗

Transposase N-terminal phosphorylation and asymmetric transposon ends inhibit piggyBac transposition in mammalian cells

Mechanistic regulation of DNA transposon systems in mammalian cells remains poorly understood. Using modeling, biochemical, and cell-based assays, we sought to extend the recent cryoEM structural insight into the piggyBac transpososome to evaluate the previously unexplained role of the transposase N-terminus, the need for asymmetric transposon ends, and the complexity of transposase tetramer formation for transposition in mammalian cells. We found that N-terminal phosphorylation by casein kinase II inhibits transposase-DNA interaction and designed deletion of this phosphorylated domain releases inhibition thereby enhancing activity. We also found that the N-terminal domain promotes transposase dimerization in the absence of transposon DNA. N-terminal deletion enables transposition of symmetric transposon ends that was previously not achievable with piggyBac. The complex transposase tetramer needed for transposition of asymmetric transposon ends can be overcome via appending a second transposase C-terminal domain in combination with symmetric transposon ends overcoming the negative regulation by asymmetric ends. Our results demonstrate that N-terminal transposase phosphorylation and the requirement for asymmetric transposon ends both negatively regulate piggyBac transposons in mammalian cells. These novel insights into mechanism and structure of the piggyBac transposase expand its potential use for genomic applications.

molecular biology↗

The role and mechanism of NRF2 in endothelial cells and smooth muscle cells of pulmonary artery

Pulmonary hypertension (PH) is a kind of fatal disease. There are no existing drugs that could reverse the diseas. NF-E2-related factor 2 (NRF2) is one of the most important moleculars in the range of cell protection. We examined the expression of NRF2 in PH models and explored the role of NRF2 in regulating abnormal phenotypes in pulmonary artery cells. We determined the expression level of NRF2 in lung tissues of PH model decreased significantly. We found that NRF2 was reduced in rat pulmonary artery endothelial cells (rPAEC) under hypoxia, while it was overexpressed in rat pulmonary artery smooth muscle cells (rPASMC) under hypoxia. Next, the results showed that knockdown NRF2 in rPAEC promoted endothelial-mesenchymal transformation and upregulated reactive oxygen species level. After the rPASMC was treated with siRNA or activator, we found that NRF2 could promote cell proliferation by regulating PDGFR/ERK1/2 and mTOR/P70S6K pathways, and accelerate cell migration by affecting MMP2/3/7. Therefore, the study suggests that the combination of NRF2 activators should be considered to eliminate the promoting effect of NRF2 activators on proliferation and migration of rPASMC. Summary statementHypoxia regulates NRF2 in PAECs and PASMCs differently. We found that NRF2 could promote cell proliferation by regulating PDGFR/ERK1/2 and mTOR/P70S6K pathways, and accelerate cell migration by affecting MMP2/3/7.

cell biology↗

Blocking necroptosis reduces inflammation and tumor incidence in a mouse model of diet-induced hepatocellular carcinoma

Background & AimsNonalcoholic fatty liver disease (NAFLD) is one of the etiologies that contribute to hepatocellular carcinoma (HCC), and chronic inflammation is one of the proposed mediators of HCC. As necroptosis is a cell death pathway that induces inflammation, we tested whether necroptosis- induced inflammation contributes to the progression of NAFLD to HCC in a mouse model of diet- induced HCC. MethodsMale and female wild-type (WT) mice or mouse models where necroptosis is blocked (Ripk3-/- or Mlkl-/- mice) were fed a control diet or choline-deficient low fat diet (CD-LFD) or CD-high fat diet (CD-HFD) for 6 months. Changes in inflammation, immune cell infiltration, activation of oncogenic pathways, and tumor incidence were assessed by gene expression analysis, western blotting, and flow cytometry. RNA sequencing (RNA-seq) was performed to assess the changes in liver transcriptome. ResultsBlocking necroptosis by deleting either Ripk3 or Mlkl reduced markers of inflammation [proinflammatory cytokines (TNF, IL-6, and IL-1{beta}), F4/80+ve macrophages, CCR2+ve infiltrating monocytes], inflammation associated oncogenic pathways (JNK, PD-L1/PD-1, {beta}-catenin), and HCC in male mice. In female mice, blocking necroptosis reduced HCC independent of inflammation. Blocking necroptosis reduced cell senescence markers in males and females, suggesting a novel cross-talk between necroptosis and cell senescence. ConclusionsOur data show that hepatic necroptosis promotes recruitment and activation of liver macrophages leading to chronic inflammation, which in turn trigger oncogenic pathways leading to the progression of NAFLD to HCC in male mice. In female mice necroptosis contributes to HCC independent of inflammation. Thus, our study suggests that necroptosis is a valid target for NAFLD-mediated HCC. SynopsisNecroptosis is a cell death pathway that mediate inflammation. Blocking necroptosis attenuated chronic inflammation by reducing recruitment and activation of liver macrophages, which in turn reduced activation of oncogenic pathways and progression of NAFLD to HCC in mice. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/502666v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1bf82d7org.highwire.dtl.DTLVardef@26fc68org.highwire.dtl.DTLVardef@18538f4org.highwire.dtl.DTLVardef@15e338b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

Type 17 Follicular Helper T (Tfh17) Cells are Superior for Memory Maintenance

A defining feature of successful vaccination is the ability to induce long-lived antigen- specific memory cells. Follicular helper T (Tfh) cells specialize in providing help to B cells in mounting protective humoral immunity in infection and after vaccination. Memory Tfh cells that retain the CXCR5 expression can confer protection through enhancing humoral response upon antigen re-exposure but how they are maintained is poorly understood. CXCR5+ memory Tfh cells in human blood are divided into Tfh1, Tfh2 and Tfh17 cells by the expression of chemokine receptors CXCR3 and CCR6 associated with Th1 and Th17 respectively. Here, we developed a new method to induce Tfh1, Tfh2 and Tfh17-like (iTfh1, iTfh2 and iTfh17) cells in vitro. Although all three iTfh subsets efficiently support antibody responses in recipient mice with immediate immunization, iTfh17 cells are superior to iTfh1 and iTfh2 cells in supporting antibody response to a later immunization after extended resting in vivo to mimic memory maintenance. Notably, the counterpart human Tfh17 cells are selectively enriched in CCR7+ central memory Tfh (TfhCM) with survival and proliferative advantages. Furthermore, the analysis of multiple human cohorts that received different vaccines for HBV, influenza virus, tetanus toxin or measles revealed that vaccine-specific Tfh17 cells outcompete Tfh1 or Tfh2 cells for the persistence in memory phase. Therefore, the complementary mouse and human results showing the advantage of Tfh17 cells in maintenance and memory function supports the notion that Tfh17-induced immunization might be preferable in vaccine development to confer long-term protection.

immunology↗