Rationale: The major cause of heart failure is myocardium death consequent to detrimental cardiac remodeling and fibrosis following myocardial infarction. The cardiac protective cytokine interleukin (IL)-33, which signals by ST2 receptor binding, is associated with group 2 innate lymphoid cell (ILC2) activation and regulates tissue homeostasis and repair following tissue injury in various tissues. However, the distribution and role of IL-33-responsive ILC2s in cardiac fibrosis remain unclear. In this study, we elucidated the roles of IL-33-responsive cardiac-resident ILC2s and IL-33-mediated immunomodulatory functions in cardiac fibrosis.Methods: We examined the distribution of cardiac ILC2s by using flow cytometry. The roles of IL-33-mediated ILC2 expansion in cardiac fibrosis was evaluated in the mouse model of catecholamine-induced cardiac fibrosis. ILC-deficient ‒/‒IL2Rγc‒/‒Rag2 mice were implemented to determine the contribution of endogenous ILC in the progression of cardiac fibrosis. Histopathological assessments, speckle tracking echocardiography, and transcriptome profile analysis were performed to determine the effects of IL-33-mediated cardiac protective functions.Results: We identified the resident cardiac ILC2s, which share similar cell surface marker and transcriptional factor expression characteristics as peripheral blood and lung tissue ILC2s. IL-33 treatment induced ILC2 expansion via ST2. In vivo, ILC-deficient ‒/‒IL2Rγc‒/‒Rag2 mice developed exacerbated cardiac fibrosis following catecholamine-induced stress cardiac injury. IL-33 treatment expanded cardiac ILC2s and revealed protective effects against cardiac tissue damage with reduced cardiomyocyte death, immune cell infiltration, tissue fibrosis, and improved myocardial function. Transcriptome analysis revealed that IL-33 attenuated extracellular matrix synthesis- and fibroblast activation-associated gene expressions. IL13-knockout or epidermal growth factor receptor (EGFR) inhibition abolished IL-33-mediated cardiac protective function, confirming IL-13 and EGFR signaling as crucial for IL-33-mediated cardioprotective responses. Moreover, ILC2-produced BMP-7 served as a novel anti-fibrotic factor to inhibit TGF-β1-induced cardiac fibroblast activation.Conclusion: Our findings indicate the presence of IL-33-responsive ILC2s in cardiac tissue and that IL-33-mediated ILC2 expansion affords optimal cardioprotective function via ILC2-derived factors. IL-33-mediated immunomodulation is thus a promising strategy to promote tissue repair and alleviate cardiac fibrosis following acute cardiac injury. Cardiac fibrosis, a condition attributed to the excessive deposition of extracellular matrix (ECM) components produced by activated fibroblasts and/or myofibroblasts, constitutes a common feature associated with nearly all forms of heart disease IL1RL1 gene Interleukin (IL)-33, an IL-1 family cytokine that functions as a tissue-derived nuclear alarmin We and others found that IL-33 exerts cardioprotective functions in animal models of cardiac pressure overload induced by transverse aortic constriction surgery In this study, we identified and characterized the IL-33-responsive cardiac ILC2 population resident in the mouse heart. IL-33 treatment expanded the cardiac ILC2 population via the ST2 receptor and elicited protective effects against catecholamine-induced cardiac fibrosis with reduced cardiomyocyte death, immune cell infiltration, cardiac fibroblast (CF) activation, fibrotic gene expressions, and improved myocardial function. Transcriptome analysis revealed that IL-33 attenuated ECM synthesis- and fibroblast activation-associated gene expression. We demonstrated that ILC2-derived factors including IL-13, Areg, and BMP-7 concurrently contribute to IL-33-mediated anti-fibrotic responses following acute cardiac injury. Overall, our results revealed that IL-33-responsive ILC2s contribute to IL-33-mediated cardiac protective function in alleviation of cardiac fibrosis.ad libitum access to regular food and water. All experimental animal protocols were approved by the KCGMH IACUC committee (2015122507 and 2018122122). Both male and female mice aged 6-14 weeks were used for all experiments. Wild-type C57BL/6JNarl and BALB/cByJNarl mice were purchased from National Laboratory of Animal Center (NLAC), Taiwan. B6.ST2‒/‒, B6.‒/‒ IL33‒/‒ST2, and B6.‒/‒IL33 mice were generated as described in our previous study  Il1rl1-/- (ST2 knockout) Il13-/-‒/‒Rag2 , and BALB/c.‒/‒IL2Rγc‒/‒Rag2  mice were maintained on a BALB/c background.Mice were maintained in the AAALAC-certified specific pathogen-free animal facility of Kaohsiung Chang Gung Memorial Hospital (KCGMH) with controlled temperature and light cycles (24 ºC and 12/12 h light/dark cycle) with Murine blood was harvested through the retro-orbital route and transferred into heparin-containing canonical tubes. Blood leukocytes were purified by using Histopaque 1077 and 1119 (Sigma) following the manufacturer's instructions. For isolation of cells from the lung and heart, mice were anesthetized with Isoflurane (3%) and the heart and lung tissues were dissected and then flushed by phosphate-buffered saline (PBS) through the ventricles to remove excessive blood content. Lung tissues were minced by razor and then digested in DMEM (3 mL) containing collagenase IV (1.6 mg/mL) and DNase I (0.1 mg/mL) , using an 18-gauge needle to shear tissue during digestion. Homogenates were filtered using 75-μm mesh and then centrifuged . Pellets were incubated in RBC lysis buffer (Gibco or Biolegend) . The isolated cells were neutralized with PBS. Similar procedure was performed for isolation of cardiac cells, except the digestion buffer comprised DMEM (2 mL) containing collagenase B (1.0 mg/mL) and DNase I (0.1 mg/mL) .Table S1.After homogenization, the cells were treated with 2.4G2 conditioned medium or TruStain FcX (Biolegend 101320)  to block Fc gamma receptors on leukocytes. Fixable Viability Dye (Thermo Fisher) and fluorescence-conjugated antibodies were then applied . To stain transcription factors, cells were initially permeabilized using the Foxp3 Transcription Factor Staining Buffer Set (Thermo Fisher). Then, the 2% FCS/PBS-washed cell suspensions were analyzed using the fluorescence-activated cell sorter (FACS) LSRII (BD) or by Gallios (Beckman Coulter). Flow diagrams were generated using FlowJo v.10.1 (TreeStar). Details of antibodies used for flow cytometry are listed in 109-Ile266) containing a 6X Histidine tags was synthesized with E. Coli favorable codons (Genewise Inc.). The synthesized cDNA was then cloned into pET15b expression vector . The protein was expressed mainly in soluble fraction. Recombinant mouse IL-33 protein was purified by using Ni-NTA affinity chromatography  using BioLogic LP System (Bio-Rad). Following the protein purification, the protein solution was applied through the Endotoxin Removal Spin Columns  to remove endotoxin. The endotoxin levels in the purified protein was analyzed and confirmed with <0.01 EU per 1 μg of the protein by the LAL method . The purity of the recombinant proteins was evaluated by Coomassie blue staining analysis and Western blot, which was performed with anti-His antibodies.Mouse IL-33 cDNA (Ser-1 d-1) to induce acute cardiac injury as previously described ‒/‒IL13 mice .For acute isoproterenol-induced cardiac injury, male or female wild-type BALB/cByJ mice (age between 8-12 week-old) were randomly subjected into groups and were subcutaneously injected with normal saline as a control or ISO  for three consecutive days , and BALB/c.‒/‒IL2Rγc‒/‒Rag2 mice, the mice  were randomly subjected to subcutaneous injection with Saline or ISO (30mg/kg per day for 3 days). The hearts were isolated for histological assessments on day 4 post last ISO treatment. For evaluation of the effect of IL-33 on cardiac fibrosis in BALB/c.‒/‒IL2Rγc‒/‒Rag2, the mice  were randomly subjected to subcutaneous injection with Saline or ISO (30mg/kg per day for 3 days). Recombinant mouse IL-33  was intraperitoneally administrated on day 3, 5, and 7 following the last ISO injection and the hearts and sera were collected on day 10 for histology, mRNA, and serum cytokine analyses.For ISO-induced cardiac fibrosis in BALB/c.Table S1. Alexa Fluor-conjugated secondary antibodies were purchased from ThermoFisher. Slides were incubated with 1% Sudan black (Sigma-Aldrich) in 75% ethanol at room temperature for 20 min to reduce tissue auto-fluorescence before mounting. Confocal images were captured using an Olympus FLUOVIEW FV10i confocal microscope. For fibrosis area quantification, paraffin sections were stained with hematoxylin and eosin or Picrosirius red stain kit  per manufacturer instruction. Bright-field images were scanned using 3D Histech Pannoramic MIDI (3DHISTECH) under 20 X magnification. Eight random fields of the left ventricular regions were captured using the Pannoramic viewer (3DHISTECH) and the red-stained area quantified using ImageJ software (NIH). Red-stained fibrotic areas and non-stained myocyte areas from each section were determined using color-based thresholding. Total fibrosis area percentage was calculated as the summed red-stained areas divided by the total cardiac area. All histological assessments were performed by two technicians blinded to treatment groups.Mouse heart tissues were fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained using standard immunohistochemistry and fluorescence microscopy methods as previously described ‒/‒Rag2 mice were intranasally treated with IL-33 (1 μg) and sacrificed five days later. Lung ILC2 cells (Lin-ST2+ subset) were sorted using FACSAria III and cultivated in RPMI-1640 supplemented with IL-2, IL-7, and IL-33 . Cells were maintained for 72 h in RPMI-1640 containing only IL-2 and IL-7 (10 ng/mL) prior to each experiment. To collect IL-33-activated ILC2 conditioned medium (ILC2-CM), the cells were treated with IL-2, IL-7, and IL-33 (10 ng/mL) for 48 h. CMs were collected for ELISA and CF activation experiments. For the adoptive transfer experiments, ‒/‒IL2Rγc‒/‒Rag2mice were challenged with ISO for three doses (30mg/kg/d). Then, ILC2 cells (5 × 106 cells) were injected via tail vein following last dose of ISO. The hearts were harvested for histological assessments on day 4 post last ISO injection.https://string-db.org).The mouse heart or cardiac fibroblast RNA samples were subjected to gene expression profiling using Affymetrix MTA 1.0 microarray chips. RNA samples were prepared using the WT PLUS Reagent kit (Affymetrix) followed by microarray hybridization. The raw data were first subjected to quality control examination per manufacturer guidelines. Chips passing the quality control criteria were then analyzed using Partek Genomics Suite . CEL files were further analyzed using the Affymetrix Transcriptome Analysis Console. Differentially expressed gene clustering, functional annotation, and gene ontology analyses were performed by using STRING . The average of three consecutive cardiac cycles was used. Left ventricular fractional shortening was calculated as a percentage as follows: (EDD - ESD)/EDD × 100, where EDD is end-diastolic dimension and ESD is end-systolic dimension. The observer was blind to the treatments. Cardiac functions including heart rate, left ventricular end-diastolic diameter, left ventricular end-systolic diameter, posterior wall thickness in diastole, interventricular septum thickness in diastole, fractional shorting, and ejection fraction were assessed. To minimize the confounding influence of different heart rates on aortic pressure gradient and left ventricular function, the flow of isoflurane  was adjusted to anesthetize the mice while maintaining their heart rates at 450-550 beats per minute. During speckle tracking echocardiography analysis, the left ventricle was automatically divided into six segments. Strain curves were made available for each analyzed segment. Data for the peak longitudinal and radial segmental strain and strain rate, and the time to peak strain  were analyzed according to instrumental instruction. Echocardiography was performed by an experienced cardiologist blinded to the treatment group. M-mode standard two-dimensional (2D) long axis echocardiographic ex-amination was conducted, and the fractional shortening (FS), ejection fraction (EF) were calculated by Vevo 2100 software. For measuring the pulsed wave Doppler spectral waveforms, we measured the peak early‐ and late‐diastolic transmitral velocities (E and A waves) to obtain the E/A ratio.2 for 4 h to allow cell attachment. Non-attached cells were removed, and the dishes were washed 2 to 3 times using 1X PBS. The cells were cultured in CF culture medium and the medium was changed every 2 days and cultured for 7 days before further subculture.Primary CFs were isolated from 2-day old neonatal C57BL/6JNarl mice (NLAC). Briefly, neonatal mouse hearts were isolated and quickly placed in a cell culture dish containing 75% ethanol solution, then transferred to ice-cold PBS, minced into small pieces using curved scissors, and transferred into 50 mL of Trypsin-EDTA solution  with 100 U/mL collagenase II . The tissues were incubated , gently agitating 2 to 3 times. The tube was then centrifuged  and the supernatant was removed. The cells were re-suspended by adding CF culture medium  (10 mL) containing L-ascorbic acid  (100 μM), 10% fetal bovine serum (Gibco #10437-028), and 1 X penicillin/streptomycin solutions ), gently mixed by repeatedly pipetting up and down 20-30 times, plated into 6 well cell culture dishes  pre-coated with gelatin (1% in PBS) for 1 day prior to the experiment, and incubated at 37 ºC in a cell culture incubator containing 5% CO5 cells) were treated with Saline control, mIL-33 , ILC2-CM (1:100), hTGF-β1 , TGF-β1+IL-33, or TGF-β1+ILC2-CM for 48 h. The cells were harvested, and the mRNA of the samples was isolated for qRT-PCR analysis of the gene expression fibroblast activation markers (Postn and Tnc).To evaluate IL-33 or ILC2-CM effects on CF activation, primary CFs (1 × 10TM Kit (ABI (4387406). qRT-PCR was performed using an ABI 7300 Real-Time PCR system (Applied Biosystems) with the Quantinova SYBR Green PCR Kit . The primer sequences used are listed in  The relative quantity of the target transcript was normalized to endogenous Hprt transcript levels using the 2-∆∆Ct method. Each sample was run in triplicate and data are presented as the means ± SD.Total RNA from the heart tissues or cells was isolated using TRIzol reagent (Invitrogen). cDNA was synthesized from 1 µg of total RNA and random hexamers using the High-Capacity RNA-to-cDNATable S3. Serum cytokine concentrations  were measured using an antibody-coated microsphere-based multiplex cytokine immunoassay with 25 µL of serum samples .Supernatants of sorted ILC2s were harvested after 48 h of culture with or without IL-33. The cytokine levels in the supernatants  and mouse serum  were analyzed by ELISA according to manufacturer instruction. ELISA kits used are listed in P<0.05.Statistical significance between two groups was analyzed using the nonparametric Mann-Whitney test. The significance of differences among the groups was evaluated using one-way analysis of variance, followed by the Bonferroni multiple comparison post-hoc test. Statistical analysis was performed using Prism 6 statistical software (GraphPad). All data are reported as the means ± SD. Statistical significance was set at All data supporting this study can be found within the paper and its +Lin-Thy1.2+), which is consistent with the characteristics of ILC2s found in peripheral blood and lung tissues Figure A-B+Lin-Thy1.2+) was relatively lower than that in lung ILC2s. However, IL-33 treatment increased the marker expression and ILC2 number, indicating cardiac ILC2 population expansion and activation by IL-33 treatment Figure A-B. Moreover, IL-33 treatment increased transcription factor GATA3 expression in the total cardiac ILCs (CD45+Lin-Thy1.2+), suggesting the increased ILC2-committed population upon IL-33 treatment Figure B, D.To examine whether ILC2s are localized in mouse cardiac tissue, we performed flow cytometry analysis of isolated suspension cells from mouse hearts. This identified a resident cardiac ILC population expressing surface markers  in heterozygote littermates and ‒/‒IL33 mice, but not in ‒/‒ST2 mice Figure G-H. ILC2-derived cytokine  gene expression was also increased by exogenous IL-33 in heterozygote littermates and ‒/‒IL33 but not ‒/‒ST2 mice Figure I, indicating that IL-33-mediated ILC2 expansion is dependent on ST2 signaling and that -/+ST2 heterozygosity is sufficient for IL-33-mediated ILC2 expansion in vivo.We next examined whether IL-33 expands ILC2s via its receptor ST2. The hearts from Saline or IL-33-treated Rag2‒/‒ mice (which lack T and B lymphocytes with preserved ILCs) and ‒/‒IL2Rγc‒/‒Rag2 mice  and compared the tissue fibrosis levels to delineate the contribution of ILC2s in the tissue responses following cardiac injury. Histopathological assessment by Picrosirius red staining revealed that ILC-deficient ‒/‒IL2Rγc‒/‒Rag2 mice were more susceptible to ISO-induced cardiac fibrosis when compared with wild-type BALB/cByJ and ILC-sufficient Rag2‒/‒ mice Figure A. We did not observe any difference in the fibrosis area between wild-type and Rag2‒/‒ mice. The average percentage of cardiac fibrosis area was greater in ‒/‒IL2Rγc‒/‒Rag2 mice than in Rag2‒/‒ and BALB/cByJ mice Figure B.As tissue damage, inflammation, and repair responses occur following cardiac injury, the ILC2 population might be altered in the injured heart and contribute to inflammation and tissue reparative responses. To test this hypothesis, we implemented the isoproterenol (ISO)-induced cardiac injury model for catecholamine-induced stress cardiomyopathy ‒/‒IL2Rγc‒/‒Rag2 mice. Strikingly, reconstitution of FACS-sorted ILC2s via tail vein injection ameliorated ISO-induced cardiac fibrosis in‒/‒IL2Rγc‒/‒ Rag2 mice, confirming the anti-fibrotic role of ILC2s Figure C-D.To specifically address the role of ILC2s in ISO-induced cardiac fibrosis, we further examined whether adoptive transfer of FACS-sorted ILC2 into + endothelial cells . IL-33+ cell number increased and peaked on day 1, being sustained until day 12 following ISO-induced cardiac injury A-B.We next determined whether IL-33-mediated ILC2 expansion protects against ISO-induced cardiac fibrosis. We employed BALB/cByJ mice for the ISO-induced fibrosis model as these are more susceptible to ISO-induced cardiac fibrosis A. The hearts were harvested on day 4 after the last ISO injection for flow cytometry analysis for ILC2 populations and pathological assessments. Notably, flow cytometry analysis revealed no difference in ILC2 frequency in the heart following ISO treatment compared with that for Saline control, suggesting that the increased endogenous IL-33 expression is likely not sufficient to trigger ILC2 expansion B-C. Conversely, exogenous IL-33 treatment increased ILC2 frequency and number in the heart compared to that in the ISO+IL-33 group B-C. Picrosirius red staining for fibrosis area showed that IL-33 co-treatment significantly reduced cardiac fibrosis lesion areas at day 4 following ISO challenge D. This result indicated that IL-33 treatment increased cardiac ILC2 accumulation in the cardiac tissue following ISO-induced fibrosis. In addition, we also showed that prophylactic pre-treatment of IL-33 protects against ISO-induced fibrosis E-F.To further examine the effect of IL-33 in ISO-induced cardiac fibrosis, the wild-type BALB/cByJ mice were treated with Saline, ISO, or ISO+IL-33 Figure A. The frequency and cell number of ILC2 in the heart tissue were increased by IL-33 at day 10 following ISO challenge in the post-treatment experiment Figure B-C. Histopathological assessment also showed that IL-33 treatment reduced ISO-induced cardiac fibrosis Figure D. Together, these results suggested an anti-fibrotic effect of IL-33 in ISO-induced cardiac fibrosis.Next, we examined whether IL-33 post-treatment could reduce ISO-induced cardiac injury to determine the therapeutic potential of IL-33 treatment for established myocardium fibrosis. Mice were administered three doses of ISO challenges followed by three doses of IL-33 or saline on days 3, 5, and 7 post-ISO Figure E-F. ILC2 occupied only a small proportion but showed the highest expansion among ST2+ leukocytes after IL-33 was administrated Figure F-G. ILC2 also showed the highest ST2 expression level on the cell surface Figure F-H. These indicate that ILC2 is a major recipient in the heart after IL-33 treatment. Additionally, the dominance of ILC2 in ST2+ leukocytes can be ascertained in the post-treatment protocol with or without ISO stimuli Figure E-H.Furthermore, we analyzed the expression level of ST2 on the cell surface of major leukocytes in the heart using flow cytometry Figure A. Analysis of the heart weight to tibial ratio did not show significant cardiac hypertrophy in this model Figure B. Cardiac function analysis by M-mode echocardiography analysis did not show functional impairments on ejection fraction and fraction shortening between groups Figure C. Pulse wave Doppler analysis of the ratio of peak velocity of early to late filling of mitral inflow (E/A) showed a decreased E/A ratio in the ISO group compared with the saline control, whereas this was reversed by IL-33 treatment Figure D. Using speckle-tracking echocardiography, the segmental strains in the groups at days 0 (baseline), 3 , and 10 (endpoint) were assessed. Intriguingly, we did not observe any considerable significant difference in the segmental strain or strain rate at baseline and day 3 post-ISO Figure F-I, suggesting that segmental strain is not altered on day 3 despite myocardial injury. Quantification results of the segmental cardiac contractility revealed that ISO treatment markedly decreases the global longitudinal strain, whereas this was reversed by IL-33 treatment on day 10 Figure F, H. The global radial peak strain remained unchanged Figure G, I. Further quantification of the segmental longitudinal strain revealed that ISO-induced functional impairment is restricted at the apical segments, including posterior apices (AP) and anterior apices (AA), which was consistent with the pathological findings of endocardium fibrosis on day 10 Figure D. Conversely, IL-33 treatment attenuated the ISO-induced apical segment functional impairment.Traditional M-mode echocardiography and the speckle-tracking echocardiography, which allows more precise analysis of cardiac tissue segmental impairments in vivo, we analyzed the changes of the transcriptome profiles in the hearts from Saline-, IL-33-, ISO-, and ISO+IL-33-treated groups by microarray analysis Figure . Principal component analysis revealed that the ISO-induced transcriptome profile alteration was reversed following IL-33 treatment Figure A. Among the 477 differentially expressed genes , the up- or down-regulation of 221 DEGs (46.3%) by ISO treatment was reversed by IL-33 Figure B. Functional annotation of the IL-33-reversed DEGs revealed clusters enriched for ECM organization, collagen fibril organization, and immune responses Figure C. The expression of IL-33-regulated DEGs associated with ECM, ECM remodeling, inflammatory cytokines and chemokines, and cardiac injury markers was altered upon ISO challenge albeit reversed by IL-33 treatment Figure D.To further investigate the mechanisms underlying IL-33-mediated anti-fibrotic responses Figure ). Consistent with the transcriptome analysis results, IL-33 treatment reduced ISO-induced gene expression associated with fibroblast activation (Postn and Tnc) Col1a2 and Col3a1) LOX) Timp1). This suggested that exogenous IL-33 treatment attenuated stress-induced cardiac fibrosis and reversed the transcriptional profile related to cardiac remodeling and fibrosis . In addition to the reduced collagen deposition Figure D, immunohistochemical staining further confirmed that IL-33 treatment reduced ISO-upregulated cardiac periostin protein levels, which is associated with cardiac fibroblast activation Figure F-G. These results together demonstrated that IL-33 post-treatment alleviated adverse cardiac fibrotic responses by reducing ECM and ECM remodeling-associated gene expressions.To validate the microarray data, we performed qRT-PCR to analyze ECM and ECM remodeling-associated gene expressions  immunofluorescence staining, a marker of cell death/or apoptosis. ISO-induced cardiac injury increased the number of TUNELFigure A. Additionally, we also found that IL-33 increased the BMP-7, IL-10, and G-CSF levels in ILC2s Figure A.We next tested the hypothesis that ILC2-derived factors contribute to IL-33-mediated effector functions in the injured myocardium. We collected and analyzed cytokine levels in the supernatants from FACS-sorted ILC2s cultured for 48 h with control (IL-2+IL-7) or activation medium (IL-2+IL-7+IL-33). IL-5, IL-13, and Areg levels were higher in the supernatants following IL-33 stimulation Arg1 and Ym1) in ISO+IL-33 hearts compared with those in the ISO group Figure A. However, IL13-deficiency abolished IL-33-mediated M2 marker activation and IL-33-mediated anti-fibrotic effects Figure B-D. In addition, we directly assayed RELMα, an M2ϕ marker Figure ) and validated that cardiac M2ϕ conversion was abolished in IL13-deficient mice after ISO+IL-33 treatment Figure F-G. These results indicated that IL-13 is a crucial downstream mediator for M2ϕ polarization for optimal anti-fibrotic response following IL-33 treatment.As IL-13 is a crucial downstream effector of ILC2, we evaluated whether IL-13 is required for IL-33-mediated anti-fibrotic responses. In wild-type BALB/cByJ mice, IL-33 treatment increased M2ϕ-associated markers  transcript levels compared with those of Saline control Figure C. CF co-culture with ILC2-CM, but not with IL-33, reduced TGF-β1-induced Postn and Tnc gene expression Figure C. Blocking of BMP-7 by neutralizing antibody abolished the inhibitory effect of ILC2-CM on TGF-β1-induced Postn and TncFigure D, suggesting an essential role of BMP-7 on anti-fibrotic function in ILC2-CM. Together, our results demonstrated that IL-33-responsive ILC2-derived factors concurrently contribute to IL-33-mediated anti-fibrotic responses for alleviation of adverse myocardial fibrosis.qRT-PCR analysis showed that TGF-β1, but not IL-33 or ILC2-CM alone, stimulated mouse CF activation as shown by increased fibroblast marker (-Thy1.2+KLRG1+ST2+) and transcriptional factor (GATA3+) expression with other tissue resident ILC2s ‒/‒IL2Rγc‒/‒Rag2 mice were more susceptible to ISO-induced cardiac fibrosis compared with ILC-sufficient ‒/‒Rag2 mice, implying that impaired ILC function might attribute to detrimental cardiac remodeling and tissue fibrosis. The protective role of ILC2s was further confirmed by adoptive transfer ‒/‒IL2Rγc‒/‒Rag2 mice following ISO challenge. Transcriptome analysis revealed that IL-33 attenuated ECM, ECM remodeling, and CF activation-associated gene expression. IL-33-activated ILC2s constitute sources of type 2 cytokines, EGF ligands, and the anti-fibrotic factor BMP-7. We further revealed that IL-33-responsive ILC2s contribute to IL-33-mediated tissue reparative functions involving EGFR signaling, IL-13-mediated M2 macrophage activation, and BMP-7-mediated anti-fibrotic responses. These factors concurrently facilitate IL-33-mediated cardiac protective effects in alleviating cardiac fibrosis.In the present study, we demonstrated the evidence of presence of IL-33-responsive ILC2 cells in cardiac tissue. We identified the presence of a resident cardiac ILC2 population that shares similar characteristics of cell surface marker (LinIL33- or ST2-deficiency did not affect cardiac-resident ILC2 abundance, suggesting that IL-33/ST2 signaling primarily mediates ILC2 expansion and activation rather than maintaining homeostasis. Recently, IL-33 was shown to promote egress of bone marrow ILC2s Notably, although endogenous IL-33 was upregulated in the injured hearts, ILC2 frequency was not altered; indicating that the local IL-33 elevation was not sufficient to trigger ILC2 expansion. Additionally,  in vivo. The EGFR ligand Areg exerts cardioprotective functions in a mouse model of myocardial infarction ST2 signaling underlies IL-33-mediated cardiac protective function in pressure overload in vivo, which may imply that the IL-33/ILC2/IL-13 axis contributes to tissue repair via M2ϕ activation. Although IL-13 activation exerts both beneficial and detrimental functions in tissue repair and fibrosis, our results demonstrated that IL-13-mediated M2ϕ activation is required for optimal IL-33 cardiac protection. Further study to dissect the optimal time window for IL-33-based therapy for treating tissue fibrosis might be crucial as excessive ILC2 or IL-13 signaling activation in the context of allergic conditions might lead to adverse tissue remodeling and detrimental side effects Figure G, whereas we did not observe clinical or histological phenotypes of myocarditis according to echocardiography and histological assessments. This discrepancy might due to the difference in the dose of IL-33 administered. In addition to their pro-inflammatory role, eosinophils have been reported to be beneficial in the repairing process of the injured myocardium IL-13, a crucial mediator to promote M2ϕ activation, may mediate the effector function of ILC2-associated type 2 immune responses ‒/‒ Rag2and ILC-deficient ‒/‒IL2Rγc‒/‒Rag2 mice did not respond to IL-33 treatment , suggesting that cells other than ILC2s contributed to the elevation of circulating BMP-7 levels. The local ILC2-derived BMP-7 protein elevated by IL-33 might function as an anti-fibrotic paracrine factor within the myocardium. A recent study showed that circulating ILC2 are not able infiltrate into the heart tissues Previous studies using deep RNA sequencing have identified several markers including BMP-7 uniquely enriched in ILC2 compared to other innate lymphoid cells Postn and Tnc gene expressions in CFs in vitro, suggesting that IL-33-mediated paracrine factors from ILC2s contribute to the reduced CF activation toward myofibroblast phenotype.CF activation and excessive ECM production contribute to the process of tissue fibrosis. In our present study, transcriptome analysis revealed that IL-33 treatment reduced ISO-induced ECM and ECM remodeling-associated gene expression. However, IL-33 alone did not alter TGF-β1-induced Figure E. Harnessing IL-33-mediated immunomodulatory function thus constitutes a promising strategy to promote tissue repair and alleviate cardiac fibrosis following acute cardiac injury.Here, our results demonstrated that ILC2s are present in the cardiac tissue and that IL-33 mediates its cardiac protective function through expansion of ILC2s in the heart. Using a mouse model of cardiac fibrosis, we showed that IL-33-mediated ILC2 expansion is responsible for optimal anti-fibrotic functions via ILC2-derived factors Supplementary figures and tables.Click here for additional data file.


Th17-mediated immune responses are notably important as they promote ongoing inflammation. The purpose of this study is to determine the impact of Fli-1 on expression of interleukin-17A (IL-17A) and the infiltration of immune cells into the kidney. IL-17A concentrations were measured by ELISA in sera collected from MRL/lpr Fli-1-heterozygotes (+/−Fli-1) and MRL/lpr +/+Fli-1 control littermates. Expression of IL-17A and related proinflammatory mediators was measured by real-time polymerase chain reaction (RT-PCR). Immunofluorescence staining was performed on renal tissue from MRL/lpr +/−Fli-1 and control littermates using anti-CD3, anti-CD4, and anti-IL-17A antibodies to detect Th17 cells and anti-CCL20 and anti-CD11b antibodies to identify CCL20+ monocytes. The expression of IL-17A in renal tissue was significantly reduced; this was accompanied by decreases in expression of IL-6, signal transducer and activator of transcription 3 (STAT3), and IL-1β. Likewise, we detected fewer CD3+IL-17+ and CD4+IL-17+ cells in renal tissue of MLR/lpr +/−Fli-1 mice and significantly fewer CCL20+CD11b+ monocytes. In conclusion, partial deletion of Fli-1 has a profound impact on IL-17A expression and on renal histopathology in the MRL/lpr mouse. The transcription factor Friend leukemia integration 1 (Fli-1) regulates the expression of numerous cytokines and chemokines and alters the progression of lupus nephritis in humans and in the MRL/MpJ- Infiltrmorrhage . Nonetheus model ,8. Our pus model ,13,14,15controls ; elevatecontrols . The cytcontrols ,19. Prevcontrols ,21,22,23patients . In this+/+Fli-1 mice and MRL/lpr +/−Fli-1 mice were generated as previously reported [+/−Fli-1 mice were generated by back-crossing with +/−Fli-1 C57BL/6 strain for more than 12 generations, as described in our previous report [Wild-type (WT) MRL/lpr reported . MRL/lprMice were genotyped using PCR to differentiate between the wild-type and mutant Fli-1 alleles as reported previously . Briefly+, CD4+, and IL-17A+ cells were detected in tissue with FITC-conjugated rat anti-mouse CD3 or anti-mouse CD4 antibodies (Biolegend) and rabbit polyclonal anti-IL-17A (Abcam) with secondary anti-rabbit IgG conjugated to Alexa Fluor® 647 (Abcam). Similarly, FITC-conjugated anti-mouse CD11b (Biolegend) and anti-CCL20 (Abcam) were used to detect immunoreactive CD11b cells and CCL20 (a representative chemoattractant for Th17 cells), respectively [+, CD4+, and CD11b+ cells within the kidney infiltrates  was as described previously [+IL-17A+, CD4+IL-17A+, and CD11b+ CCL20+ cells per high-power field (magnification 400×); cells were photographed with a microscope equipped with digital camera .Kidneys were removed from adult mice, fixed in formalin, and embedded in paraffin. The deparaffinized section was stained with hematoxylin and eosin (H&E). Kidney sections were evaluated by light microscopy and the grade of kidney inflammation was evaluated using the pathology scoring system that was previously described . Brieflyectively . Detectieviously . Briefly+/+Fli-1) group [Transcripts encoding cytokines, chemokines, and signaling molecules were evaluated in total RNA extracted from renal tissue from adult mice with TRIzol reagent (Invitrogen). SuperScript First-Strand Synthesis System (Invitrogen) was used to generate cDNA. Real-time PCR (RT-PCR) was performed in duplicate using the SYBR Green PCR Master Mix  according to the manufacturer’s instructions, using two independent RNA preparations. The primers for detection of transcripts encoding IL-6, Janus kinase 2 (JAK2), and signal transducer and activator of transcription 3 (STAT3) were purchased from SABiosciences (QIAGEN) and primers for all other transcripts are listed in +) group .Immunoreactive IL-6 and IL-17A in sera from adult mice (4 months or older) were evaluated quantitatively by enzyme-linked immunosorbent assay (ELISA)  as per the manufacturer’s instructions.Serum double-strand (ds) DNA antibody levels in sera from adult mice were measured by ELISA as per the manufacturer’s instructions . Urinary protein levels in adult mice (4 to 6 months old) were evaluated by dipstick (Wako).p-value <0.05 was considered significant. Statistical analyses were conducted using Excel add-ins and Statcel 4 Software .Results are presented as means ± standard deviation (SD). Quantitative data were analyzed using Student’s t-test with Welch’s correction or Mann–Whitney test as appropriate. A +/−Fli-1 mice and compared to those from WT MRL/lpr +/+Fli-1 littermate controls. As shown in +/−Fli-1 mice were somewhat lower than those detected in littermate controls, although the difference did not reach statistical significance (p = 0.16). By contrast, and consistent with our earlier reports, levels of IL-6 in sera from MRL/lpr +/−Fli-1 mice were significantly lower than those detected in littermate MRL/lpr +/+Fli-1 controls (p < 0.05). Similarly, serum anti-ds DNA antibody levels and urine protein levels were significantly decreased in MRL/lpr +/−Fli-1 mice . By contrast, expression of transcripts encoding Janus kinase 2 (JAK2) and suppressor of cytokine signaling 3 (SOCS3) was somewhat diminished, although the results did not achieve statistical significance.Messenger RNA was isolated from renal tissue from MRL/lpr +/−Fli-1 mice compared to their MRL/lpr +/+Fli-1 counterparts. By contrast, we observed no differences in leukocyte infiltration to inflamed tubulointerstitial lesions and in part, perivascular lesions into the kidney  as well as CD4+IL-17+ cells (Th17 cells) were detected in renal tissue from MRL/lpr mice +/−Fli-1 compared to littermate controls. By contrast, the renal tissues received similar scores , which is predominantly expressed in Th17 cells, CCL20+ monocytes/macrophages induce migration of CCR6+ Th17 cells into the inflammatory cites [+ monocytes affect local CCL20 expression and, in part, result in more decreased CCR6+ Th17 cells into the kidney of nephritic mice. Further investigation is needed to clarify the association of Fli-1 in CCL20 expression and infiltration of CCR6+ Th17 cells in lupus nephritis. In contrast to the findings presented here, several groups reported that IL-17A and the Th17 immune response have a relatively minor impact on the pathogenesis of lupus nephritis. For example, Schmidt et al. found that the degree of leukocyte infiltration in the kidneys of IL-17A-deficient MRL/lpr mice could not be distinguished from that detected in their IL17A-sufficient counterparts [We are also interested in understanding why fewer IL-17A+/− mice ; this mae kidney . CXCR3 pe kidney ,31. Indery cites ,32,33. Ary cites . These oterparts . Likewisterparts . Interesterparts ,22. Futu


IGF-1 and IGF receptor. Trans-10-hydroxy-2-decenoic acid (10H2DA) and 10-hydroxydecanoic acid (10HDAA), two major fatty acids contained in RJ. After ingestion, 10H2DA and 10HDAA are metabolized into 2-decenedioic acid (2DA) and sebacic acid (SA) respectively. We found that 10H2DA, 10HDAA, 2DA, and SA all regulated myogenesis of C2C12 cells, murine myoblast cells. These novel findings may be useful for potential preventative and therapeutic applications for muscle atrophy disease included in Sarcopenia, an age-related decline in skeletal muscle mass and strength.Honeybees produce royal jelly (RJ) from their cephalic glands. Royal jelly is a source of nutrition for the queen honey bee throughout its lifespan and is also involved in fertility and longevity. Royal jelly has long been considered beneficial to human health. We recently observed that RJ delayed impairment of motor function during aging, affecting muscle fiber size. However, how RJ affects skeletal muscle metabolism and the functional component of RJ is as of yet unidentified. We demonstrate that feeding mice with RJ daily prevents a decrease in myofiber size following denervation without affecting total muscle weight. RJ did not affect atrophy-related genes but stimulated the expression of myogenesis-related genes, including  Sarcopenia is an age-related decline in skeletal muscle mass and strength . Loss ofSatellite cells are skeletal muscle stem cells that reside beneath the basal lamina. Satellite cells play a central role in postnatal muscle growth, repair, and regeneration in adults. Upon activation, satellite cells proliferate extensively and upregulate expression of MyoD, followed by increasing myogenic differentiation marker genes such as myogenin, muscle creatine kinase (Mck), and myosin heavy chain (Myhc) ,10,11. UApis mellifera) produce royal jelly (RJ) from their cephalic glands. Royal jelly is a source of nutrition for the queen honey bee throughout its lifespan and is also involved in fertility and longevity. RJ has long been considered beneficial to health [Honeybees , proteins (9–18%), sugars 7.5–23%), lipids (3–8%), and other trace compounds. The two major fatty acids in RJ are trans-10-hydroxy-2-decenoic acid (10H2DA) and 10-hydroxydecanoic acid (10HDAA), which comprise 60–80% of RJ lipids . 10H2DA %, lipidsRoyal jelly also appears to have a function in skeletal muscle metabolism. In mice, feeding of RJ increases the serum IGF1 levels and stimulates regeneration of injured muscle via the IGF1-Akt pathway in satellite cells . AdminisHere, we demonstrate that daily feeding of pRJ in mice cancels the in of muscle fiber size induced by denervation. In addition, treatment of C2C12 myoblasts with pRJ and pRJ-related fatty acids stimulated differentiation and proliferation. C57BL/6J mice were purchased from CLEA Japan Inc. . Seven-week-old mice were anesthetized, after which a 5 mm section of the sciatic nerve on the right leg was cut and excised. A sham operation was performed on the left leg as a control . Six dayn = 7) or 1% pRJ diet (n = 7) for 3 weeks pre-operation and for 6 days post-operation. Chow was refreshed every 2 days. Lyophilized protease-treated RJ  was prepared at Yamada Bee Company, Inc. . Protease-treated RJ contained a standardized amount of specific fatty acids (3.5% 10H2DA and 0.6% 10HDAA). Experimental diets were prepared by thoroughly mixing pRJ with MF powder diet  at a concentration of 1% (v/v) . Four-weTibialis anterior (TA) muscle was isolated after sacrifice and immediately frozen in chilled isopentane and liquid nitrogen and stored at −80 °C . SectionC2C12 cells and C3H10T1/2 cells were purchased from American Type Culture Collection . C2C12 cells and C3H10T1/2 cells were maintained as previously described  and cultSkeletal muscle differentiation in C2C12 cells was initiated by replacing growth medium  with differentiation medium (medium supplemented with 2% horse serum) in sub-confluent cultures . TATA box binding protein (Tbp) using the 2-ΔΔCt method [murine atrogin-1 , murine murf1 , murine foxo1 , murine myogenin , murine myoD , murine mck , murinecyclin A2 , murine cyclin D1 , murine cyclin E1 , murine Myhc1 , murine Myhc7 , murine Myhc2 , murine Myhc4 , murine IGF1 , murine IGF1R , and murine tbp .Total RNA was isolated from cells using a FastGeneTM RNA Basic Kit  and then reverse-transcribed into cDNA using High Capacity cDNA Reverse Transcription Kit (Applied biosystems). SYBR green-based qPCR was performed in 96-well plates using PowerUp SYBR Green Master Mix  and a QuantStudio 3 Real-Time PCR System (ThermoFisher Scientific). Expression levels were normalized to t method . The folC2C12 cells were incubated with primary antibody for 1 hour at room temperature after blocking and permeabilization with phosphate-buffered saline containing 0.3% Triton X100 and 5% goat serum for 30 minutes at room temperature. Anti-Myhc mouse monoclonal antibody  or anti-Ki-67 rabbit polyclonal antibody  were used for immunocytochemistry. Target proteins were visualized using Alexa 488-conjugated secondary antibody  and imaged with an ABZ-9000  microscope.Antibodies used for Western blot analysis were anti-Myogenin mouse monoclonal antibody , anti-Myhc mouse monoclonal antibody , anti-CyclinD1 mouse monoclonal antibody , anti-Cyclin A2 rabbit polyclonal antibody , Phosopho-anti-AMPKα (Thr172) Rabbit monoclonal antibody , anti-AMPKα Rabbit monoclonal antibody , anti-Fbx32 (Atrogin-1) Rabbit monoclonal antibody , and HRP-conjugated anti-Gapdh mouse monoclonal antibody . Target proteins were detected using anti-mouse or anti-rabbit IgG antibody conjugated with horseradish peroxidase  and ImmunoStar LD .Proliferation of C2C12 cells was assessed using a Cell Counting kit-8  according to the manufacturer’s protocol . p < 0.01 and *, p < 0.05.Comparisons were made using an unpaired analysis of variance (ANOVA) with Tukey–Kramer post-hoc test and Wilcoxon’s signed rank test. The results are shown as the mean ± S.D. The statistical significance is indicated as follows: **, Atrogin-1 (Muscle ring finger protein 1 (MuRF1) (Forkhead box O-1 (Foxo-1) (Cyclin E1 (Cyclin A2 (Myogenin (Mych1 (Myhc2 (Myhc4 (Myhc7 (IGF-1 (IGF receptor (IGFR)  B, or For(Foxo-1) C. Howeveyclin E1 D, Cyclinyclin A2 E, or MyoMyogenin G. Protean  J and phor (IGFR) K. Next, we examined the effect of pRJ on proliferation using an in vitro cell culture system. C2C12 cells are from a murine myoblast cell line isolated from satellite cells  commonlyProtease-treated royal jelly treatment for 48 hours increased the number of cells A,B as weFinally, we examined the effect of pRJ and RJ-related fatty acid products on myoblast differentiation. C2C12 cells were induced to differentiate in the presence or absence of pRJ. Myosin heavy-chain immunostaining showed that pRJ treatment led to an increase in myotube formation compared to control treatment cells A,B. FurtIn this study, we demonstrate that feeding mice daily with pRJ prevents a decrease in myofiber size following denervation. In a previous study, we showed that pRJ affects muscle fiber size in elderly mice without changing total muscle weight . Our cur10-hydroxy-2-decenoic acid and 10-hydroxydecanoic acid, two major fatty acids contained in RJ, are associated with health benefits such as anti-tumor activity , anti-hyWe observed that RJ treatment stimulated both proliferation and differentiation. RJ stimulates cell proliferation and increases the size of Myhc positive fibers in primary satellite cells isolated from aged mice via upregulation of IGF-1 and IGFR . In thisRoyal jelly has been known to regulate global epigenetic changes . EpigeneIn conclusion, daily oral administration of pRJ prevented a decrease in myofiber size following denervation. pRJ also increased the expression of regeneration-related genes in vivo. Although we could not determine the proliferative cell population responding to pRJ in vivo, pRJ and RJ related fatty acids strongly stimulated proliferation and differentiation of C2C12 myoblasts in vitro.


The human ortholog to him-4, HMCN 1 (hemicentin-1), is implicated in age related macular degeneration 1 . HIM-4/HMCN1 is a highly conserved extracellular matrix structural component and contains six RGD (Arg-Gly-Asp) cell-binding motifs within its 48 immunoglobin (Ig)-like repeats, while HMCN1 contains only one RGD motif . Two novel mutant alleles of C. eleganshim-4 were generated using CRISPR-Cas9 gene editing in this study. In one mutant allele, three of the aforementioned six RGD motifs were mutated to RGE (Arg-Gly-Glu) . Four of the six RGD motifs were altered to RGE in the second mutant line. Neither of these mutant lines exhibited outstanding abnormal phenotypes when screened for behavioral or morphological abnormalities. We examined the mutants for Him  phenotype, kq8207  and kq8297 . However, the examined animals failed to show an outstanding number of males compared to N2 . Mutants were also examined for behavioral abnormalities in a thrashing assay. A Mann-Whitney U Test revealed possible differences between kq8207 mutants and N2 worms (p=0.037), and revealed no significant difference between kq8297 mutants and N2 worms (p=0.447). 53 triple RGD mutants (average 19.85 thrashes), 51 RGD quadruple mutants (average 21 thrashes), and 51 N2 worms (average 22.16 thrashes) were used in this assay.Morrissey et al. demonstrated that PAT-3/INA-1integrin is essential for assembly of HIM-4/hemicentin puncta during anchor cell invasion . However, integrin and HIM-4 failed to colocalize in other tissues . Although anchor cell invasion was not examined in this study, these him-4 RGD mutants hold potential for further research pertaining to the functions of cell to extracellular matrix binding domains in C. elegans.The him-4, CRISPR target sites were identified within the gene using the CRISPR guide RNA Selection Tool (http://genome.sfu.ca/crispr/). The triple mutation line, BU8207 (him-4(kq8207)), contains RGE mutations at amino acid numbers 2618, 3352, and 4527 , in which the mutations were added sequentially into each position. The quadruple mutation line, BU8297 (him-4(kq8297)), contained these three loci as well as amino acid number 3519 as described here. Briefly, the mixture of custom template DNA , custom crRNA (HIM4RGD3519), tracrRNA (cat. #1072532), and Alt-R Cas9 nuclease (cat. #1081058) was annealed at room temperature  and micro-injected into the syncytial gonad arms of N2 wild-type worms (P0) . The mixture also included dpy-10 crRNA as a co-CRISPR marker . The F1 generation was then screened for the intended mutation by identifying animals exhibiting the Dpy phenotype and by PCR genotyping using mutation specific primers (HIM4RGE3519R and HIM4RGE3519SEQF). PCR was then conducted on F2 offspring using both wild-type him-4 and mutant-specific primers (HIM4RGD3519WTR and HIM4RGE3519SEQF) in order to isolate homozygous mutants. Once homozygotes were isolated, PCR products were sequenced in order to confirm that the mutation was successful . The repair DNA  for these loci was designed and produced at IDT Inc., Coralville, IA. Both lines of mutant animals were backcrossed (1x) to N2 then studied for phenotype characterization. For Him phenotypes, N2, kq8207, and kq8297 worms were self-fertilized, and males were randomly identified from NGM agar plates. The thrashing assay was performed by counting the number of body bends for 15 seconds in a 10 μl drop of M9 buffer . A Mann-Whitney U Test was then run to confirm statistical significance of results.In order to induce specific RGD to RGE mutations in crRNA sequenceHIM4RGD3519 AAUACACUGGCUUGTCUCCA(dpy-10) ZQDP10A GCUACCAUAGGCACCACGAGPCR PrimersHIM4RGE3519SEQF TACGCCGCAGAAGTGATTGGHIM4RGE3519SEQR TCCTGCTTCGTTGGATGCACHIM4RGD3519WTR ATACACTGGCTTGTCTCCACGGHIM4RGE3519R ACAAGTAGACCGGTTTCTCACCTCTARepair oligo, crRNA, and primer sequences for screening are readily available upon request.him-4(kq8207) and BU8297 him-4(kq8297) are available upon request.BU8207


Mechanistically, vIRF1 interacts with transcription factor Lef1 to promote SPAG9 transcription. vIRF1-induced SPAG9 promotes the interaction of mitogen-activated protein kinase kinase 4 (MKK4) with JNK1/2 to increase their phosphorylation, resulting in enhanced VEGFA expression, angiogenesis, cell proliferation and migration. Finally, genetic deletion of ORF-K9 from KSHV genome abolishes KSHV-induced cellular transformation and impairs angiogenesis. Our results reveal that vIRF1 transcriptionally activates SPAG9 expression to promote angiogenesis and tumorigenesis via activating JNK/VEGFA signaling. These novel findings define the mechanism of KSHV induction of the SPAG9/JNK/VEGFA pathway and establish the scientific basis for targeting this pathway for treating KSHV-associated cancers.Kaposi’s sarcoma (KS), caused by Kaposi’s sarcoma-associated herpesvirus (KSHV), is a highly angioproliferative disseminated tumor of endothelial cells commonly found in AIDS patients. We have recently shown that KSHV-encoded viral interferon regulatory factor 1 (vIRF1) mediates KSHV-induced cell motility :e1007578). However, the role of vIRF1 in KSHV-induced cellular transformation and angiogenesis remains unknown. Here, we show that vIRF1 promotes angiogenesis by upregulating sperm associated antigen 9 (SPAG9) using two  Kaposi’s sarcoma-associated herpesvirus (KSHV)-encoded viral interferon regulatory factor 1 (vIRF1) is essential for KSHV-induced angiogenesis and tumorigenesis, but the underlying mechanism has not been elucidated. Sperm associated antigen 9 (SPAG9) is a well-characterized oncoprotein, highly expressed in a variety of human cancer. However, whether SPAG9 could act as a promoter of KS development has not been investigated. Here, we reveal that vIRF1 enhances SPAG9 transcription by forming a transcription factor complex with Lef1. vIRF1-induced SPAG9 upregulation promotes the interaction of mitogen-activated protein kinase kinase 4 (MKK4) with JNK together leading to enhanced JNK phosphorylation. Activation of the JNK pathway induces VEGFA expression, contributing to KSHV-induced cellular transformation and angiogenesis. In summary, this work discovers a novel mechanism of vIRF1 induction of angiogenesis and tumorigenesis, and reveals SPAG9/JNK/VEGFA pathway as a potential target for KS therapy. Kaposi’s sarcoma (KS), a highly angiogenic and disseminated tumor of endothelial cells commonly found in AIDS patients, is caused by infection of a human oncogenic virus Kaposi’s sarcoma-associated herpesvirus (KSHV). KSHV is a DNA tumor virus of the gammaherpesvirus subfamily, which was identified in an AIDS-associated Kaposi's sarcoma (AIDS-KS) lesion in 1994 .KSHV encodes over 90 open reading frames (ORFs) and at least 25 microRNAs . SimilarKSHV vIRFs (vIRF1 to vIRF4) are encoded by a cluster of ORFs , which are resided between ORF57 and ORF58 of the viral genome and transcribed in the opposite orientation . vIRFs aSPAG9 gene has four isoforms, the longest one is 1,321 amino acids. SPAG9, also called c-Jun-terminal kinase (JNK)-associated leucine zipper protein (JLP) or c-Jun-amino-terminal kinase-interacting protein 4 (JIP4), is a member of the cancer/testis (CT) antigen family. SPAG9 could function as a scaffold protein mediating the interactions of mitogen-activated protein kinase kinases (MKKs) and their targets to activate specific signaling pathways [Sperm associated antigen 9 (SPAG9) encoded by pathways , 21. SPApathways –26. SPAGpathways , 23, 27.In this work, we demonstrated that vIRF1-Lef1 transcription factor complex was able to bind to SPAG9 promoter to enhance its transcription and expression. High level of SPAG9 was responsible for vIRF1-induced cell proliferation, migration, angiogenesis, and cellular transformation. vIRF1-induced SPAG9 promotes the interaction of mitogen-activated protein kinase kinase 4 (MKK4) with JNK, resulting in an increase of JNK phosphorylation and activation of VEGFA signaling. Moreover, treatment with the JNK inhibitor, SP600125, abolished vIRF1-induced cell oncogenic behaviors. Our novel findings suggest that the SPAG9/JNK/VEGFA pathway induced by vIRF1 is a potential therapeutic target for KSHV-associated malignancies.SPAG9 is a well-characterized oncoprotein , but itshttp://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3)  . To subs=TF_8.3) . Notably=TF_8.3) . Using a=TF_8.3) . As expe=TF_8.3) . The red=TF_8.3) . These rin vivo model of angiogenesis, following silencing of SPAG9 expression in vIRF1-transduced endothelial cell line with lentivirus-mediated shRNAs , which is an d shRNAs . We found shRNAs . To furt in vivo . Convers in vivo . We inve in vivo .In addition, we transduced KSHV-infected HUVECs with lentivirus-mediated shRNAs targeting SPAG9 and examined the functional consequence on KSHV-induced cell behaviors. We found that knockdown of SPAG9 in KSHV-infected HUVECs decreased cell proliferation and migration .SPAG9, as a scaffolding protein, could tether mitogen-activated protein kinase kinases (MKKs) and their targets together to activate JNK signaling pathway . Given tNext, we determined whether JNK1/2 signaling mediated vIRF1-induced cell proliferation, migration and angiogenesis. As expected, treatment with SP600125, an inhibitor of JNK pathway, reduced JNK phosphorylation , as wellTaken together, these results suggested that vIRF1 transcriptional activation of SPAG9 induced JNK1/2 signaling, which promoted angiogenesis, cell proliferation and migration by recruiting MKK4.It is well known that activation of JNK pathway could enhance the binding of phosphorylated c-Jun to the VEGFA promoter to induce VEGFA production . We assuTogether these data suggested that by hijacking Lef1, vIRF1 transcriptionally activated SPAG9 to promote angiogenesis, cell proliferation and migration via activation of the JNK/VEGFA pathway.To determine the role of vIRF1 in cellular transformation and angiogenesis, we examined cellular transformation of a KSHV mutant virus with deleted ORF-K9  in rat pTo examine the role of the JNK1/2 pathway in KSHV-induced cellular transformation, we treated KSHV-infected cells with SP600125, and found that inhibition of JNK activation not only reduced KSHV-induced efficiency of colony formation  and cellO-tetradecanoyl-phorbol-13-acetate (TPA) and sodium butyrate (NaB) in KSHV-positive primary effusion lymphoma (PEL) cells [de novo infection, virus-encoded cytokines and host-induced cytokines contribute to KS pathogenesis through autocrine and paracrine mechanisms [KSHV vIRF1 was characterized as a lytic protein and its expression can be induced by 12-L) cells , 35. HowL) cells –38. HencL) cells –42. The chanisms . Importachanisms . In thisAs the first identified oncogenic protein encoded by KSHV, vIRF1 has been reported to transform mouse embryonic fibroblasts (NIH3T3) cells ; howeverSPAG9, resulting in enhancement of SPAG9 transcription and expression. Silencing of SPAG9 inhibited vIRF1-induced angiogenesis, cell proliferation, and migration, indicating the importance of SPAG9 in the development of KS. Interestingly, a higher antibody response against SPAG9 was observed in cancer patients [Human tumor antigens are potential targets for tumor immunotherapy. Cancer/testis (CT) antigens, a unique group of antigens, could induce spontaneous immune responses in cancer patients and have been proposed to be potential cancer vaccine targets . Sperm apatients , suggestJNK activation leads to phosphorylation of downstream molecules, which is associated with a wide spectrum of cellular processes, including cell death/survival, inflammation, cell differentiation, and cell proliferation . JNK actVEGFA is a well-known angiogenic factor important for vascular permeability, and survival of newly-formed vasculature . VEGFA, In conclusion, we have found that KSHV vIRF1 upregulates SPAG9, which is essential for vIRF1-induced cellular transformation, angiogenesis and cell invasion. vIRF1 enhances SPAG9 transcription by interacting with Lef1 to promote the transcriptional activity of Lef1. Increased SPAG9 expression enhances the activation of JNK1/2 pathway and VEGFA transcription, contributing to KSHV-induced angiogenesis and tumorigenesis . Our finThe Institutional Ethics Committee of the First Affiliated Hospital of Nanjing Medical University reviewed and ethically approved the clinical section of the research . Written informed consent was obtained from all participants, and all samples were anonymized. All participants were adults. Embryonated chicken eggs were purchased from Qian Yuan Hao Biological Co., LTD. Nanjing Biological Pharmaceutical Factory , and were housed under SPF conditions. Male athymic BALB/c nu/nu mice of four-week-old were from Nanjing Biomedical Research Institute of Nanjing University  and were maintained under pathogen-free conditions. All animal care and use protocols were performed in accordance with Laboratory Animal Management Regulations approved by the State Council of People’s Republic of China. The Institutional Animal Care and Use Committee of Nanjing Medical University approved the animal experiments .The iSLK-RGB-BAC16 and iSLK-RGB-K9 mutant cells were established and cultivated in DMEM supplemented with 10% fetal bovine serum (FBS), 1 μg/ml puromycin, 250 μg/ml G418, and 1.2 mg/ml hygromycin B . Rat priFlag-vIRF1 was cloned by inserting the coding sequences into plasmid pHAGE-CMV-MCSIzsGreen as previously described , 72. A 2Transfection of HEK293T cells were performed with the Lipofectamine 2000 Reagent . Transfection of HUVECs was performed with the Effectence transfection reagent . SP600125 were purchased from Selleck Chemicals . Antibodies used were anti-SPAG9 rabbit antibody from Abcam , anti-p-JNK rabbit antibody and anti-JNK rabbit antibody from Beyotime Institute of Biotechnology , anti-Flag rabbit antibody from Cell Signaling Technologies , anti-α-Tubulin mouse antibody, anti-GAPDH mouse antibody, anti-Actin mouse antibody, and anti-VEGFA rabbit antibody from Santa Cruz Biotechnology . The polyclonal rabbit anti-vIRF1 antibodies were kindly provided by Dr. Gary Hayward from Viral Oncology Program, The Johns Hopkins School of Medicine.5) were seeded into chambers, and the chambers were harvested, fixed and stained after 6 h or 12 h incubation. The migrated cells were photographed and calculated by counting stained cells in a double-blinded manner by two observers.Cell Count Kit-8  was used for Cell proliferation assay as previously described . Transwe4) were suspended in 0.4% top agar (BD Biosciences) and then plated onto 0.8% base agar in six well-plates. The plates were incubated at 37°C with 5% CO2 for 2 weeks. Random fields were chosen and photographed. Colony areas were counted by NIH Image J software. The colony sizes of 20 μm or larger were scored for calculation of the percentage of soft agar colony.Soft agar assay was performed as previously described . BrieflyPromega dual-luciferase reporter assay system were adopted for luciferase reporter assay according to the manufacturer’s protocol as previously described . The renTotal RNA was extracted using RNA Isolator Total RNA Extraction Reagent  according to the manufacturer’s instructions. Reverse transcription was performed by HiScript Q RT SuperMix . Quantitative PCR (qPCR) analysis was performed by AceQ qPCR SYBR Green Master Mix . The sequences of the primers for PCR are as follows: 5’-GAAGGTGAAGGTCGGAGTC-3’ (forward) and 5’-GAAGATGGTGATGGGATTTCC-3’ (reverse) for GAPDH; 5’-CAAGGCGGATCTAAAGCTACC-3’ (forward) and 5’- TTGGCGCATCTGTAACCTTCA -3’ (reverse) for SPAG9.CAMs were performed with 9-day-old embryos as previously described . At leasnu/nu nude mice were used. The plug assay was performed as previously described [6 cells with the high concentration Matrigel , and then injected into the right flanks of nude mice. 10 days later, harvest and photograph the plugs using stereomicroscope. Drabkin’s reagent kit (Sigma-Aldrich) was used for the measurement of the hemoglobin content. Nude mice were randomly divided and no statistical method was used to predetermine sample sizes.For Matrigel Plug Assay, four-week-old male escribed . All pro7) cross-linked by 1% formaldehyde were harvested to suffer sonication. Precipitate obtained DNA fragments with indicated antibodies. Specific primers were used for RT-qPCR. The sequences of SPAG9 promoter primers are as follows: 5’- TTTCCCGTTGTAGCTGCGT -3’ and 5’- GTGACGAGTTAACTTAGCTGG’ for site (1); 5’- TCCGGGGCCGTGATGTCGGAG -3’ and 5’- AGCTCCAGCTCCACCTGGT’ for site (2).EZ-Magna ChIP A/G Chromatin Immunoprecipitation Kit  was used for ChIP analysis as previously described . Cells . Magnification, ×200.Immunohistochemical staining of isotype control immunoglobulin G (IgG) in normal skin, and skin KS of patient #1 ((TIF)Click here for additional data file.S2 Fig(A). RT-qPCR analysis of mRNA level of vIRF1 in HUVECs infected by KSHV wild-type virus (KSHV) (MOI of 3). Samples were collected at 0 h, 1 h, 6 h, 12 h, 24 h, 48 h, and 72 h after KSHV infection. Undet., undetermined. (B). RT-qPCR analysis of mRNA level of SPAG9 in HUVECs infected by KSHV wild-type virus (KSHV) (MOI of 3). Samples were collected at 0 h, 1 h, 6 h, 12 h, 24 h, 48 h, and 72 h after KSHV infection. (C). Lack of vIRF1 reduces SPGA9 mRNA transcript induced by KSHV. RT-qPCR analysis of mRNA level of SPAG9 in HUVECs treated with PBS (PBS) or infected with wild-type KSHV (KSHV_WT) (MOI of 3) or vIRF1 mutant virus (K9_mut) (MOI of 3) for 30 h. Data were shown as mean ± SD. *** P < 0.001, Student's t-test. n.s, not significant.(TIF)Click here for additional data file.S3 Fig(A). Western-blotting analysis of vIRF1 (Anti-Flag) in HUVECs transduced with different MOIs  of lentiviral-vIRF1 or its control lentiviral-pHAGE. (B). Luciferase reporter assay of the activity of SPAG9 promoter in HUVECs transduced with different MOIs  of lentiviral-vIRF1 or its control lentiviral-pHAGE. Data were shown as mean ± SD. * P < 0.05 and *** P < 0.001, Student's t-test.(TIF)Click here for additional data file.S4 FigPBS) or infected with wild-type KSHV (KSHV_WT) (MOI of 3) or vIRF1 mutant virus (K9_mut) (MOI of 3) for 30 h. Data were shown as mean ± SD. ** P < 0.01 and *** P < 0.001, Student's t-test.Luciferase reporter assay of the activity of SPAG9 promoter in HUVECs treated with PBS ((TIF)Click here for additional data file.S5 Fig(A). RT-qPCR analysis of mRNA level of SPAG9 expression in KSHV-infected HUVECs transduced with a mixture of lentivirus-mediated shRNAs targeting Lef1 (shLef1). Data were shown as mean ± SD. ** P < 0.01 and *** P < 0.001, Student's t-test. (B). Western-blotting analysis of SPAG9 expression in KSHV-infected HUVECs transduced with a mixture of lentivirus-mediated shRNAs targeting Lef1 (shLef1).(TIF)Click here for additional data file.S6 FigP < 0.05 and ** P < 0.01, Student's t-test.Lentiviral vIRF1- or its control pHAGE-infected endothelial cell line were transduced with lentivirus-SPAG9 (SPAG9), lentivirus-Lef1 (Lef1) or its control pHAGE (pHAGE), respectively, and then were subjected to chicken chorioallantoic membranes (CAMs) assay. Quantification of CAMs assay was showed. Data were shown as mean ± SD. * (TIF)Click here for additional data file.S7 FigWestern-blotting analysis of VEGFA expression in iSLK-RGB cells and iSLK-RGB-K9 mutant cells treated with doxycycline (Doxy) (1 μg/ml) for 48 h.(TIF)Click here for additional data file.S8 Fig(A). Western-blotting analysis of the expressions of SPAG9, p-JNK1/2, and VEGFA in KSHV-infected HUVECs transduced with a mixture of lentivirus-mediated shRNAs targeting Lef1 (shLef1). (B). CCK-8 assay of HUVECs treated as in (A). (C). Transwell migration analysis of HUVECs treated as in (A). The migrated HUVECs were counted at 6 h and 12 h post seeding. (D). PBS-treated or KSHV-infected endothelial cell line were transduced with a mixture of lentivirus-mediated shRNAs targeting Lef1 (shLef1) for 48 h, and then were subjected to chicken chorioallantoic membranes (CAMs) assay. Data were shown as mean ± SD. * P < 0.05, ** P < 0.01 and *** P < 0.001, Student's t-test.(TIF)Click here for additional data file.S9 Fig(A). Western-blotting analysis of Lef1, SPAG9, p-JNK1/2 and VEGFA expression in MM and KMM cells transduced with a mixture of lentivirus-mediated shRNAs targeting Lef1 (shLef1). (B). CCK-8 assay of cells treated as in (A). (C). Western-blotting analysis of SPAG9, p-JNK1/2 and VEGFA expression in MM and KMM cells transduced with a mixture of lentivirus-mediated shRNAs targeting SPAG9 (shSPAG9). (D). CCK-8 assay of cells treated as in (C). Data were shown as mean ± SD. ** P < 0.01 and *** P < 0.001, Student's t-test.(TIF)Click here for additional data file.


For BNCT to be successful, a high (500 mg/kg) dose of 10BPA must be administered over a few hours. Here, we report BNCT efficacy after rapid, ultralow-dose administration of either tumor vasculature-specific annexin A1-targeting IFLLWQR (IF7)-conjugated 10BPA or borocaptate sodium (10BSH).10B drugs intravenously injected into MBT2 bladder tumor-bearing mice and biodistribution of 10B in tumors and normal organs analyzed by prompt gamma-ray analysis. (2) Therapeutic effect of IF7-10B drug-mediated BNCT was assessed by either MBT2 bladder tumor bearing C3H/He mice and YTS-1 tumor bearing nude mice.(1) IF7 conjugates of either 10B drugs into MBT2 bladder tumor-bearing mice promoted rapid 10B accumulation in tumor and suppressed tumor growth. Moreover, multiple treatments at ultralow (10–20 mg/kg) doses of IF7-10B drug-mediated BNCT significantly suppressed tumor growth in a mouse model of human YTS-1 bladder cancer, with increased Anxa1 expression in tumors and infiltration by CD8-positive lymphocytes.Intravenous injection of IF7C conjugates of either 10B delivery vehicle by targeting tumor tissues via the tumor vasculature and could serve as a relevant vehicle for BNCT drugs.We conclude that IF7 serves as an efficient The online version contains supplementary material available at 10.1186/s12885-020-07760-x. Mice subjected to neutron irradiation RR- or IF7K(10BSH)RR-mediated BNCT , although body weight was comparable between groups RR- and IF7K(10BSH)RR-mediated BNCT groups  at sacrifice were significantly less than those seen in untreated control mice (0.790 g [0.113–1.130]), the IF7C(10BPA)RR-treated group (0.800 g [0.263–1.293]), the IF7K(10BSH)RR-treated group (0.955 g [0.295–1.155]), and the neutron-irradiated control group (0.455 g [0.195–1.273]) RR- and IF7K(10BSH)RR-mediated BNCT groups compared with those in the other four groups RR-treated mice, IF7K(10BSH)RR-treated mice, and neutron-irradiated control did not differ significantly, both the IF7C(10BPA)RR- and IF7K(10BSH)RR-mediated BNCT groups RR- and IF7K(10BSH)RR-mediated BNCT groups  RR-treated mice, IF7K(10BSH)RR-treated mice . CD31-positive blood vessels were evident in tumor tissue of all groups RR-mediated- or IF7K(10BSH)RR-mediated BNCT RR-treated mice, IF7K(10BSH)RR-treated mice RR-mediated- or IF7K(10BSH)RR-mediated BNCT  RR-treated mice, IF7K(10BSH)RR-treated mice, or irradiated controls . Anxa 1 expression in the IF7C(10BPA)RR-mediated or IF7K(10BSH)RR-mediated BNCT groups RR-treated mice, IF7K(10BSH)RR-treated mice  and requires a few hours to accumulate at tumor sites. Many researchers have tried to induce more effective intratumoral 10B accumulation using clinically effective doses of 10B drugs, but achieving this goal has been challenging. Here, we devised a novel delivery approach using the short 7-mer IF7 peptide, which is easily synthesized and can be readily modified. IF7 is, however, degraded by proteases in plasma and thus would not be antigenic, minimizing concerns regarding immune reactions. In our previous study using IF7-based chemotherapy, we were able to significantly reduce the dose of a conjugated anticancer drug, and the intravenously-injected IF7 construct reached tumor tissue within a minute ICF01012 induces immunogenic tumor cell death, marked by a significant increase in cell surface Anxa1 and calreticulin [10B accumulation in tumor tissues might occur following the second injection of IF7-10B drugs. Thus, multiple ultralow doses of IF7-10B drug-mediated BNCT likely boost BNCT therapeutic potential. Future studies should further examine mechanisms underlying Anxa1 upregulation in tumor tissue after BNCT. Although we did not observe a significant difference in the number of CD31-positive blood vessels per tumor area between groups, IF7-10B drug-mediated BNCT may destroy Anxa1-positive tumor vasculature as a way of boosting BNCT therapeutic potential (Fig. Previously, we demonstrated that IF7 binds to the Anxa1 N-terminus . Annexinwth Fig. . Here, tors Fig. . Our imment Fig. . In addites Fig. g. It is ups Fig. a–f. Anxaeticulin . This fiial Fig. .Fig. 6S10B drug-mediated BNCT. Combining extremely efficient tumor vasculature targeting activity by IF7 with local radiation therapy such as BNCT could be an excellent dual targeting strategy. Further preclinical studies and Phase I clinical trials are needed to evaluate the clinical efficacy of 10B drugs conjugated to Anxa1-binding peptides in patients.In summary, here we have assessed the therapeutic potential of Anxa1-binding IF7-Additional file 1.


Drosophila ortholog Kuduk consists of a transmembrane and an intramembrane domain and localizes to the outer nuclear membrane, where it regulates myonuclear positioning and morphology . Kuduk is the first characterized cytoplasmic regulator of the LINC (linker of nucleoskeleton and cytoskeleton) complex. LINC complexes are conserved throughout eukaryotes and consist of SUN (Sad-1 and UNC-84) proteins, which localize to the inner nuclear membrane and bind to lamin, and KASH proteins  that span the outer nuclear membrane and interact with cytoskeletal elements. KASH and SUN bind in the perinuclear space to bridge the nucleus and cytoskeleton and transfer force across the nuclear envelope . Drosophila Kuduk has been shown to help anchor the nucleus in place via the LINC complex, but the molecular mechanisms it uses to carry out nuclear migration remain unknown.Human TMEM258 is involved in stress pathways and has been implicated in inflammatory intestinal disorders . The tmem-258  is required for nuclear positioning and migration in C. elegans. We used CRISPR-Cas9 to introduce non-specific mutations in tmem-258 and generated two frameshift mutants, yc63 and yc66, which created early stop codons . We then assayed nuclear migration in two different tissues. First, nuclei migrate across the dorsal midline in hypodermal (hyp7) precursors during normal C. elegans embryogenesis . If hyp7 nuclear migration fails, ectopic nuclei can be seen in the dorsal cord. For example, in an unc-84(n369) null line, an average of 15.0 nuclei were abnormally in the dorsal cord . Neither tmem-258(yc63) nor tmem-258(yc66) animals exhibited mislocalized nuclei, suggesting hyp7 nuclear migration was successful . Second, we quantified P-cell nuclear migration in L1 larvae. P-cell nuclei migrate from the lateral to the ventral side of the worm where they divide and differentiate to form the vulva and GABA neurons. At 25˚C, unc-84(n369) mutants typically are missing 6.5±0.60 GABA neurons . To determine whether tmem-258 mutants enhance the P-cell nuclear migration defect of unc-84, we crossed tmem-258(yc63) worms to unc-84(n369) and quantified P-cell nuclear migration failure at 15°C by counting GABA neurons with UNC-47::GFP. The tmem-258(yc63); unc-84(n369) double mutant were missing an average of 0.70±0.3  GABA neurons, equal to the 0.60±0.4 GABA neurons of the unc-84(n369) single mutant at 15˚C. Finally, we tested whether tmem-258 is required for nuclear anchorage in C. elegans by counting the number of syncytial hyp7 nuclei clustering together in adults. The tmem-258 mutants had no significant nuclear anchorage defects . We therefore conclude that tmem-258 is not required for nuclear positioning in C. elegans hyp7 cells.We hypothesized that tmem-258 mutations were generated using the guide sequence 5’-TCGCGAAGTTTACTGGAG-3’ injected into UD398: him-8 (e1489) ycIs10  animals containing a hyp7 GFP nuclear marker. Injections were done with dpy-10 co-CRISPR according to previous methods . tmem-258(yc63) corresponds to UD600 and tmem-258(yc66) is UD609. tmem-258(yc63) was crossed to UD87: unc-84(n369); oxIs12; ycEx60[odr-1::rfp] to create UD623: tmem-258(yc63), him-8(e1489) ycIs10; unc-84 (n369) oxIs12[unc-47::gfp] ycEx60[odr-1::rfp unc-84(+)] which was used to assay P-cell nuclear migration.The


Cytochrome P450 CYP2E1 is an enzyme engaged in the breakdown of various compounds  as well as endogenous compounds (steroids and fatty acids), resulting in both detoxification and elimination of these agents. Often, it can also convert these agents, i.e., Acetaminophen, into more toxic products. Elevated liver CYP2E1 content is implicated in various metabolic diseases including alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH), diabetes and obesity. While liver CYP2E1 elevation is considered essential to the pathogenesis of these liver diseases, our findings of elevated liver CYP2E1 content in two genetic mouse models with impaired degradation and thus a disrupted disposal of CYP2E1 when fed a regular lab chow diet, argue that it is not sufficient for triggering NAFLD/NASH. Thus, despite comparable hepatic CYP2E1 elevation and functional stabilization in these two models, NAFLD/NASH was only observed in the mouse livers that exhibited concurrently enhanced liver fat production. These findings reinforce the notion that in addition to elevated liver CYP2E1 content, CYP2E1-mediated NAFLD/NASH requires liver fat accumulation derived from either enhanced liver fat-production or ingestion of a high fat/high carbohydrate diet.Hepatic cytochrome P450 CYP2E1 is an enzyme engaged in the metabolic biotransformation of various xenobiotics and endobiotics, resulting in both detoxification and/or metabolic activation of its substrates to more therapeutic or toxic products. Elevated hepatic CYP2E1 content is implicated in various metabolic diseases including alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH), diabetes and obesity. While hepatic CYP2E1 elevation is considered essential to the pathogenesis of these liver diseases, our findings in two mouse models of E3 ubiquitin ligase genetic ablation fed a regular lab chow diet, argue that it is not sufficient for triggering NAFLD/NASH. Thus, albeit comparable hepatic CYP2E1 elevation and functional stabilization in these two models upon E3 ubiquitin ligase genetic ablation and consequent disruption of its ubiquitin-dependent proteasomal degradation, NAFLD/NASH was only observed in the mouse livers that exhibited concurrent SREBP1c-transcriptional upregulation of hepatic lipogenesis. These findings reinforce the critical complicity of an associated prolipogenic scenario induced by either an inherently upregulated hepatic lipogenesis or a high fat/high carbohydrate diet in CYP2E1-mediated NAFLD/NASH. Human liver CYP2E1 is an endoplasmic reticulum (ER)-anchored cytochrome P450 involved in the biotransformation of clinically relevant drugs , ethanol and other xenobiotics, carcinogens (nitrosamines), endogenous acetone and fatty acids (arachidonic acid) to toxic/reactive intermediates ,4. DespiSimilar observations have also been documented in CYP2E1-overexpressing hepatocyte cell lines  apparently cultured in regular cell culture media, not particularly enriched in fatty acids or other lipogenic sources ,12,27. I−/−) resulted in a hepatic ≈180–200% stabilization of CYP2E1 ER-content in these mice [−/−-mice was associated not only with a correspondingly increased functional activity, but also with increased lipid peroxidation with concomitantly increased 4-hydroxynonenal tissue conjugates and 15-F2T-isoprostane levels, marked activation of the hepatic JNK-cascade and microvesicular fat accumulation quite early at 2 months, which within 8–9 months of age progressed to macrovesicular fat accumulation, hepatocyte ballooning and injury, typical of clinical NASH [−/−-mice had been fed a regular, non-fat/carbohydrate-enriched chow-diet, suggesting that overexpression of hepatic CYP2E1 content and consequent oxidative stress were sufficient for NASH-development [Intriguingly, we encountered a similar pathogenic scenario in mice upon genetic ablation of the E3 ubiquitin (Ub)-ligase C-terminus of Hsc70-interacting protein (CHIP) , an esseese mice . This CYcal NASH . Surpriselopment . This fielopment  that hep−/−-mice [−/−-mice relative to wild type (WT) mice [−/−-mouse hepatocytes, gp78−/−-mouse hepatocytes, albeit from comparably age-matched mice similarly fed dietary chow, exhibited no comparable elevation of hepatic lipid peroxidation, fatty acids and/or triglyceride content over corresponding age-matched WT-controls [−/−-mice versus gp78−/−-mice suggested that factors additional to elevated hepatic CYP2E1 content most likely contributed to the early microvesicular hepatic fat accumulation observed in CHIP−/−-mice.Puzzled by these findings, we examined whether a comparable hepatic CYP2E1 stabilization observed upon liver-specific genetic ablation of gp78/AMFR (autocrine motility factor receptor), an ER-polytopic E3 Ub-ligase also known to participate in CYP2E1-UPD ,33, woul−/−-mice . Indeed,controls . These dIn search of clues, we focused on the hepatic Insig 1/2-SREBP1-SCAP ER-complex involved in lipogenic regulation ,36,37,38−/−-mice, we found that hepatic Insig-1 and Insig-2 mRNA expression albeit comparable at 2 months of age, markedly decreased at 9 months of age relative to corresponding WT [fas1 and scd-1 mRNA expression at 2 months with further increases at 9 months of age [−/−-mouse hepatocytes revealed not only a decrease in hepatic Insig-1 (36% of WT levels) and Insig-2 (42% of WT levels) proteins, but also a corresponding increase in precursor (208% of WT levels) and nuclear (151% of WT levels) SREBP-1 protein species  The transcriptional upregulation of hepatic adiponectin receptors R1/R2 and adipose adipoQ gene, along with the concomitant stabilization of AMP-Kinase AMPKα1 , its ROP-target ; and (2)P-target  and of SP-target , both of of NASH .


Microbial conversion of dietary or drug substrates into small bioactive molecules represents a regulatory mechanism by which the gut microbiota alters intestinal physiology. Here, we show that a wide variety of gut bacteria can metabolize the dietary supplement and antidepressant 5-hydroxytryptophan (5-HTP) to 5-hydroxyindole (5-HI) via the tryptophanase (TnaA) enzyme. Oral administration of 5-HTP results in detection of 5-HI in fecal samples of healthy volunteers with interindividual variation. The production of 5-HI is inhibited upon pH reduction in in vitro studies. When administered orally in rats, 5-HI significantly accelerates the total gut transit time (TGTT). Deciphering the underlying mechanisms of action reveals that 5-HI accelerates gut contractility via activation of L-type calcium channels located on the colonic smooth muscle cells. Moreover, 5-HI stimulation of a cell line model of intestinal enterochromaffin cells results in significant increase in serotonin production. Together, our findings support a role for bacterial metabolism in altering gut motility and lay the foundation for microbiota-targeted interventions. The gastrointestinal (GI) tract is home to trillions of microbes. The gut microbiota produces a wide range of small bioactive molecules derived from various substrates, including dietary precursors and medications ,2. Such 2+ entry and intracellular Ca2+ release + m/z = 134,0602, which corresponds to exact molecular weight of 5-HI in positive mode. (B) No basal levels of 5-HTP were detected in the control samples (gray line) in the fecal samples from healthy individuals at time point 0 h. (C) Chromatogram shows that tryptophan was not converted to indole in the fecal samples of the Non-Converters group. The raw data of B and C can be found in (TIF)Click here for additional data file.S2 Fig(A) Overnight culture of E. coli BW25113WT (black line) and E. coli BW25113ΔtnaA (red line) incubated at 37 °C with agitation with Trp present in the culture medium for 24 h. Curves represent 1 example of 3 biological replicates. The raw data can be found in B) High levels of Trp do not prevent the production of 5-HI in the fecal samples after 24 h of anaerobic incubation. (C) Cell lysates of B. thetaiotamicron VPI-5482 incubated at 37 °C converted 5-HTP to 5-HI after 48 h. The raw data of B and C can be found in (TIF)Click here for additional data file.S3 FigE. coli, F. nucleatum, and B. thetaiotamicron. Levels of the conserved amino acids are highlighted in the shades of blue . The level of conservation in the multiple active sites of TnaA is highlighted in red. MSA, multiple sequence alignment; TnaA, tryptophanase.An MSA of the 15 closest orthologs of (TIF)Click here for additional data file.S4 Fig(A) Pie charts represent the difference in relative percentage of Bifidobacterium genus annotated in light blue (not exploded). Exploded slices represent the difference in tnaA-encoding bacterial phyla among High, Intermediate, and Non-Converters. Slices annotated in pink (not exploded) represent sum of the Bacteroides species which do not contain TnaA enzyme. Parts annotated in the shades of gray represent the rest of bacterial genera detected in the samples. (B) PCA score plot indicates clear separation of High Converters from Intermediate and Non-Converters group. Two PCs explained 43.1% and 14.9% of total variances in 16S rRNA sequencing data. Only the first 5 top most contributing species are shown. (C) RDA plot indicates significant influence of 5-HI levels, being the explanatory variable . Only the first 5 top most contributing species are shown. (D) Graph shows OD600 measurements of F. nucleatum subsp. animalis cultured anaerobically at 37 °C in Enriched Beef Broth adjusted to different pH. (E) Graph shows the relative 5-HI concentration produced in the F. nucleatum subsp. animalis lysates after 48 h of incubation with 100 μM 5-HTP at different pH levels. (F) Graph shows the relative 5-HI concentration produced in the B. thetaiotamicron lysates after 48 h of incubation with 100 μM 5-HTP at different pH levels. The raw data used for quantification of D, E, and F can be found in (TIF)Click here for additional data file.S5 Fig(A) Bar graph represents remaining percentage of 5-HI after incubation for 90 min and 24 h in rat colonic tissue extracts (n = 3). Error bars demonstrate SEM. (B–E) Bar graphs and illustrative recordings representing the inhibitory effect of either (B) 1 μM ondansetron, (C) 1 μM SB-207266, (D) 1 μM TTX, or (E) 1 μM hexamethonium on the 5-HT-induced response. (F) Bar graph represents no inhibitory effect of 1 μM hexamethonium on 5-HI-induced response. (G–I) Bar graphs represent an inhibitory effect on ACh-induced response by the addition of either (G) 1 μM AQ-RA 741, (H) 100 nM DAU 5884 hydrochloride, or (I) 1 μM ML 204 and SAR 7334. (J) Bar graph and its representative recording of colonic contractions showing no inhibitory effect on 5-HI-induced response when 1 μM ML 204 together with 1 μM SAR 7334 were added. (K) Bar graph represents contractile agent (ATP) acting independently of L-type Ca2+ channels. Data represent 3–5 biological replicates. Data were analyzed using the Wilcoxon matched-pairs (before/after) signed rank test . Error bars represent SEM. Quantitative analysis of the organ bath data is described in Materials and method section. The raw data used for quantification of A–K can be found in (TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S4 Data(XLSX)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.S2 Raw images(PDF)Click here for additional data file.S3 Raw images(PDF)Click here for additional data file.S1 Fig Data(TIF)Click here for additional data file.S2 Fig Data(TIF)Click here for additional data file.S1 TableContains Table A , Table B , Table C , Table D (Pearson R correlations between 5-HI levels and tnaA-encoding bacteria), Table E (Constituents of Liquid Amies + 20% Glycerol medium), Table F (Constituents of Enriched Beef Broth medium), Table G , Table H (Primers), Table I (Plasmids), and Table J .(XLSX)Click here for additional data file.


We then sought to identify and clone blocking mAbs from the memory B cell repertoire of recently recovered COVID-19 patients to prevent the entry of COVID-19 virus to the host cells.Currently, there are no approved prophylactic vaccines or therapeutic drugs that are specific to COVID-19. Blocking monoclonal antibodies (mAbs), due to their extraordinary antigen specificity, are one of the best candidates for neutralizing virus infection.15 The S protein of SARS-CoV-2 consists of two subunits, S1 and S2. To engage host cell receptor human angiotensin-converting enzyme 2 (hACE2), shared by both SARS-CoV and SARS-CoV-2, S protein undergoes dramatic conformational changes to expose the RBD and key residues for receptor binding. S protein is metastable, and binding of RBD to hACE2 receptor likely leads to the shedding of S1 protein from S2 protein, thus promoting S2-mediated virus-host membrane fusion and virus entry.18 Given the critical role of the RBD in initiating invasion of SARS-CoV-2 into host cells, it becomes a vulnerable target for neutralizing antibodies. Thus far, the human mAbs specifically target the SARS-CoV-2 RBD-hACE2 interaction have not been reported, and a monoclonal antibody targeting S1 made from immunized transgenic mice expressing human Ig variable heavy and light chains has been recently shown to neutralize both SARS-CoV-2 and SARS-CoV infection, but by an unknown mechanism that is independent of the blockade of RBD-hACE2 interaction.19Similar to SARS-CoV, SARS-CoV-2 also utilizes highly glycosylated homotrimeric spike (S) protein for receptor binding and virus entry.20Prior to cloning SARS-CoV-2 RBD-specific human mAbs, we first examined whether patients recently recovered from COVID-19 had mounted anti-SARS-CoV-2 S1 protein IgG antibodies in sera. Among 26 recovered COVID-19 patients, we found that the majority of these recruited patients were able to produce high titers of SARS-CoV-2 S1-specific IgG antibodies and only three patients mounted relatively lower anti-S1 IgG responses, by enzyme-linked immunosorbent assay (ELISA) Fig. . Consist21 We found that SARS-CoV-2 RBD-specific, IgG-positive memory B cells only enriched in COVID-19 recovered patients, but not in healthy controls  and light chain (VL) from each single memory B cell. After cloning both VH and VL, we inserted both sequences into expression plasmids that encoding constant regions of human IgG1 heavy chain and light chain Fig. .21 We fools Fig. , suggest50 = 0.0332, and 0.0450 μg/ml, respectively), while 311mab–31B9 clone failed to inhibit such an interaction . As expected, 311mab-31B9 clone failed to show any neutralization activities (Fig. Finally, we expressed these paired plasmids encoding IgG VH and VL sequences and named these three mAbs as 311mab–31B5, 311mab–32D4 and 311mab–31B9, respectively. We first examined whether these human mAbs were able to bind to SARS-CoV-2 RBD protein by ELISA. The results showed that all three mAbs strongly and specifically bind to the RBD protein Fig. . Next, wion Fig. . The 31Bion Fig. . Furtheries Fig. .In conclusion, we have successfully cloned two human blocking mAbs using SARS-CoV-2 RBD-specific memory B cells isolated from recovered COVID-19 patients. These two mAbs can specifically bind to SARS-CoV-2 RBD, block the interaction between SARS-CoV-2 RBD and hACE2 receptor, and lead to efficient neutralization of SARS-CoV-2 S protein pseudotyped virus infection. Such human anti-SARS-CoV-2 RBD-hACE2 blocking mAbs are first reported, and hold great promise to be exploited as specific prophylactic and therapeutic agents against ongoing SARS-CoV-2 pandemic.Materials and Methods