Background: Colorectal cancer (CRC) and the associated metastatic lesions are reported to be hypoxic. Hypoxia is a common feature in the tumor microenvironment and a potent stimulant of CRC. We have identified a regulatory role of Nur77 on Akt activation to enhance β-catenin signaling essential for CRC progression under hypoxic conditions.Methods: The functional role of Nur77 in hypoxia-induced EMT was examined by scattering assays to monitor the morphologies of CRC cell lines under 1% O2. Sphere formation assays were performed to investigate whether Nur77 induced cancer stem cell-like properties in hypoxic CRC cells. The expression of various epithelial-to-mesenchymal transition (EMT) and stemness markers was analyzed by qPCR and Western blotting. Finally, Nur77 function and signaling in vivo was ascertained in subcutaneous tumor xenograft or liver metastasis model in nude mice using CRC cells stably transfected with appropriate constructs.Results: Herein, we show, for the first time, that Nur77 is a novel regulator of microRNA biogenesis that may underlie its significant tumor-promoting activities in CRC cells under hypoxia. Mechanistically, Nur77 interacted with the tumor suppressor protein p63, leading to the inhibition of p63-dependent transcription of Dicer, an important miRNA processor and subsequent decrease in the biogenesis of let-7i-5p which targeted the 3'UTR of p110α mRNA and regulated its stability. Knockdown of Nur77 or overexpression of let-7i-5p inhibited the tumor metastasis in vivo.Conclusion: Our data uncovered a novel mechanistic link connecting Nur77, Akt, and invasive properties of CRC in the hypoxic microenvironment. Colorectal cancer (CRC) is the third most common cancer worldwide and accounts for ~10% of cancer-related mortality Hypoxia was shown to be a potent stimulant of Nur77 expression, which is transcriptionally activated by HIF-1α via inhibition of Dicer-let-7i-5p under hypoxia.In this study, we show, for the first time, that Nur77 plays a critical role in regulating microRNA (miRNA) biogenesis during hypoxia-induced epithelial-to-mesenchymal transition (EMT), a key metastatic process for cancer cells to acquire a more malignant phenotype for migration and invasion. Our results provide evidence that Nur77 promotes Akt activity through up-regulation of p110α expression 2. Media were supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin and 5% fetal bovine serum. For hypoxic conditions, the cells were grown at 1% O2 in a modulator.Human CRC cell line HCT116 was a gift from Dr. B. Vogelstein , and SW480 was obtained from American Type Culture Collection. HCT116 was grown in RPMI-1640 medium , and SW480 was grown in DMEM (Gibco) at 37°C with 5% COCells were transfected with 0.25 μg plasmid DNA per well in 24-well plates for 24 h using Lipofectamine 2000 , followed by hypoxia incubation. Constructs encoding Nur77 and Nur77ΔDBD were previously described Small interfering RNA (siRNA) targeting Nur77 (SMARTpool), Dicer (SMARTpool), p63 (SMARTpool), and an NS duplex oligo (5'-GGC TAC GTC CAG GAG CGC A-3') were purchased from Dharmacon . Let-7i-5p, non-specific miRNA mimic and let-7i-5p inhibitor were purchased from Ambion . Cells were transfected with 20 nM siRNA, miRNA mimic or miRNA inhibitor using siLectFect  following the manufacturer's instructions.To identify the miRNA that is regulated by p110α on the hypoxic colon cancer cells, three prediction softwares  were used. The analysis revealed 15 miRNAs  that have potential binding sites at the 3'UTR of p110α mRNA.-ΔΔCt method. Unspecific signals caused by primer dimers were excluded by non-template controls and dissociation curve analysis.Total RNA was extracted with Trizol Reagent (Invitrogen) and reverse transcribed into cDNAs using the M-MLV reverse transcriptase (Invitrogen). qPCR was done by AceQ qPCR SYBR Green Master Mix (High ROX Premixed) Kit  following the manufacturer's instructions. For precursor (pre-) and primary precursor (pri-) miRNA levels, immature miRNA was reversed-transcribed to cDNA by RevertAid First Strand cDNA Synthesis Kit (ThermoFisher) using random hexamer primer. The following specific primers were used: Nur77: forward: 5'-CCC TGA AGT TGT TCC CCT CAC-3' and reverse: 5'-GCC CTC AAG GTG TGG AGA AG-3'; E-cadherin: forward: 5'-ATT TTT CCC TCG ACA CCC GAT-3' and reverse: 5'-TCC CAG GCG TAG ACC AAG A-3'; N-cadherin: forward: 5'-AGC CAA CCT TAA CTG AGG AGT-3' and reverse: 5'-GGC AAG TTG ATT GGA GGG ATG-3'; Snail: forward: 5'-TCG GAA GCC TAA CTA CAG CGA-3' and reverse: 5'-AGA TGA GCA TTG GCA GCG AG-3'; Slug: forward: 5'-CGA ACT GGA CAC ACA TAC AGT G-3' and reverse: 5'-CTG AGG ATC TCT GGT TGT GGT-3'; CD133: forward: 5'-AGT CGG AAA CTG GCA GAT AGC-3' and reverse: 5'-GGT AGT GTT GTA CTG GGC CAA T-3'; CD44: forward: 5'-CTG CCG CTT TGC AGG TGT A-3' and reverse: 5'-CAT TGT GGG CAA GGT GCT ATT-3'; ALDH1: forward: 5'-CTG CTG GCG ACA ATG GAG T-3' and reverse: 5'-CGC AAT GTT TTG ATG CAG CCT-3'; EpCAM : forward: 5'-TGA TCC TGA CTG CGA TGA GAG-3' and reverse: 5'-CTT GTC TGT TCT TCT GAC CCC-3'; p110α: forward: 5'-CCA CGA CCA TCA TCA GGT GAA-3' and reverse: 5'-CCT CAC GGA GGC ATT CTA AAG T-3'; Dicer: forward: 5'-TGC TAT GTC GCC TTG AAT GTT-3' and reverse: 5'-AAT TTC TCG ATA GGG GTG GTC TA-3'; Drosha: forward: 5'-TGG CCA TGA CTC ATC CAA GT-3' and reverse: 5'-AGG GAG TTG GGT CAT CTT GG-3'; p63: forward: 5'-AAA GAC ATG CCC CAT CCA GA-3' and reverse:5'- CAT ACT GGG CAT GGC TGT TC-3'. Dicer promoter primer AB: forward: 5'-AAA TCA GAC AAA CCG GCC AG-3' and reverse: 5'-ATC TCG TCT CAC TGC AAC CT-3'; Dicer promoter primer C: forward: 5'- GGC GAC AAG AGT GAA ACT CT-3' and reverse: 5'- ATC TCA GTA TGG GGG TAG GC-3'; GAPDH: forward: 5'-GGA GCG AGA TCC CTC CAA AAT-3' and reverse: 5'- GGC TGT TGT CAT ACT TCT CAT GG-3'. Immature miRNA levels were amplified by qPCR using specific primers as follows: pre-let-7i: forward: 5'-TGT GCT GTT GGT CGG GT-3' and reverse: 5'- GCA GTA GCT TGC GCA GTT-3'; pri-let-7i: forward: 5'- CGA GGA AGG ACG GAG GA -3' and reverse: 5'-GCT GAG CAT CAC CAG CAC-3'; pre-U6: forward: 5'-CTC GCT TCG GCA GCA CA-3' and reverse: 5'-AAC GCT TCA CGA ATT TGC GT-3'. To detect miRNA levels, miRNA was first reversed-transcribed to cDNA by TaqMan miRNA reverse transcription kit  using specific stem-loop primers let-7i-5p: 5'-GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG AAA CAG C-3'; U6 small nuclear RNA : 5'-GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACA AAA AAT-3'. miRNA levels were then amplified by qPCR using specific primers as follows: let-7i-5p: forward: 5'- TGA GGT AGT AGT TTG TGC TGT T-3'; U6: Forward: 5'-TTC CTC CGC AAG GAT GAC ACG C-3'; Universal reverse: 5'-GTG CAG GGT CCG AGG T-3'. Relative quantification was achieved by normalization to the amount of GAPDH using 2To determine the half-life of p110α mRNA, cells transfected with NS or Nur77 siRNA were treated with actinomycin D (5 μg/mL) under hypoxia. Cells were harvested at various time points, and mRNA expression was quantified by qPCR as described above.Cells were lysed with SDS lysis buffer . Equal amounts of cell lysates were separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were electro-transferred to nitrocellulose membranes and incubated with primary antibodies against Snail , Slug , E-cadherin (CST), N-cadherin (CST), CD44 , CD133 , EpCAM (CST), ALDH1A1 , p110α (CST), Nur77 , p-mTOR , mTOR , p-p70S6K , p70S6K (CST), p-4E-BP1 , 4E-BP1 , p63 , p53 , Dicer1 , Drosha , phospho-Akt , total Akt  or β-actin (CST). For Western blotting, antibodies were used at the dilution of 1:1000. The membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Bio-Rad). The protein bands were visualized using enhanced chemiluminescence detection reagents . Densities of bands were measured using Image J. Relative quantification was achieved by normalization to the amount of β-actin.After incubation for 24 h under hypoxia or normoxia, HCT116 cells were lysed and sonicated in 500 μL IP lysis buffer containing 150 mM NaCl, 100 mM NaF, 50 mM Tris-HCl (pH 7.6), 0.5% NP-40 and 1 mM PMSF. The lysates were incubated with antibodies against Nur77  or p63  and purified with protein A/G beads  at 4 °C overnight. The eluted proteins were separated by 10% SDS-PAGE and detected with anti-Nur77, anti-p63, and anti-p53 antibodies.For 3'UTR luciferase assays, HCT116 and SW480 cells were co-transfected with let-7i-5p or NS mimic and p110α-3'UTR luciferase reporter constructs using Lipofectamine 2000, following the manufacturer's instructions. Renilla luciferase-expressing plasmid was also co-transfected for normalizing transfection efficiency. For the Dicer-promoter reporter assays, HEK293T cells were transfected with Dicer reporter construct and Nur77 and/or p63 expression plasmid, and Renilla luciferase plasmid. To further test for endogenous activity of Nur77, HCT116 and SW480 cells were co-transfected with Nur77 or non-specific siRNA, Dicer promoter construct and Renilla luciferase plasmid. After transfection for 48 h, cells were lysed with reporter lysis buffer . Luciferase activities were measured by VictorX4 multilabel plate reader  after the addition of luciferase substrates (Promega), according to the manufacturer's instructions.Cell scattering assays were performed to evaluate EMT morphologic changes. Briefly, 2000 cells were plated in six-well plates. When small colonies formed, cells were transfected with the indicated siRNA and incubated in the hypoxic chamber for 48 h. Scattered colonies were judged by a typical change in morphology characterized by cell-cell dissociation and the acquisition of a migratory fibroblast-like phenotype. Fifty colonies were randomly scored for each well under light microscope.Cells were transfected with NS or Nur77 siRNA for 48 h; empty vector pcDNA3.1 or Nur77 constructs for 24 h. Enrichment of cancer stem cells was performed in serum-free RPMI-1640 and DMEM supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin using non-tissue culture treated dished coated with 1% agarose. Cell were seeded at 5,000 cells/mL and cultured for 72 h under hypoxic condition till tumor spheres were formed. For serial spheroid formation, primary spheroids were dissociated and reseeded under the hypoxic condition to obtain secondary spheroids. Same procedures were repeated for the formation of tertiary spheroids.ChIP assay was performed with High-Sensitivity ChIP Kit  following manufacturer's instructions. Briefly, cells were lysed and the pellet containing chromatin was resuspended with 500 μl ChIP Buffer. The chromatin was then extracted by sonication shearing. The ChIP reaction mixtures were incubated at 4°C overnight, and the DNA was released and purified for the following PCR test.6 HCT116 cells alone or stably transfected with non-specific (NS) shRNA or Nur77 shRNA were subcutaneously injected into the right flank of BALB/c nude mice. Body weight and tumor volume were measured and recorded every day after injection. The length and width of tumors were measured with a caliper, and tumor volume was calculated as (length+width2)/2. After removing unwanted surrounding tissues, tumor samples were either fixed with formalin for paraffin embedding or cut into small pieces and homogenized for protein extraction, followed by co-immunoprecipitation or Western blot analysis. Total RNA was extracted from the paraffin blocks using the RNeasy FFPE kit  according to the manufacturer's instructions. For the liver metastasis model, HCT116 stably expressing non-specific or Nur77 shRNA were harvested from subconfluent cultures and washed once in PBS and resuspended in PBS. After the nude mice were anesthetized with pentobarbital sodium, a small left abdominal flank incision was made, and 5✕105 of cells in 50 μL PBS were injected into the spleen parenchyma . To avoid intrasplenic tumor growth, the spleen was removed after 10 minutes. After the mice were euthanized 6 weeks later, livers were removed, photographed and the numbers of metastatic nodules were counted.All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Hong Kong. 2✕10Paraffin sections from mice tumors were deparaffinized with xylene and rehydrated in graded ethanol. After blocking and heat-induced antigen retrieval using citrate buffer, the slides were separately incubated with antibodies against PCNA , Nur77  and IgG  overnight at 4 °C. After incubation with anti-rabbit or anti-mouse secondary antibodies (peroxidase-labeled) (1:200) for 2 h at room temperature, the slides were further detected with DAB Detection Kit .t test or two-way ANOVA were performed in GraphPad Prism  to determine the differences between the experimental groups. P < 0.05 is considered to be statistically significant.All experiments were repeated at least three times, and data were presented as mean ± S.D. Student's 2. Cells transfected with Nur77 siRNA adopted epithelial-like phenotypes capable of forming compact colonies, whereas the growth of control cells was scattered  could induce p110α mRNA and protein levels similar to wild-type Nur77 Figure A-B, suggNumerous factors are known to regulate mRNA stability, including miRNAs, small non-coding RNAs that complementarily bind to the 3' untranslated regions (UTRs) of target mRNAs to regulate their degradation or translation The down-regulation of Dicer by Nur77 prompted us to identify miRNAs that could potentially regulate p110α. To this end, we used three prediction tools, namely TargetScan, RNAhybrid, and miRBase (miRanda) to predict potential regulators , p110α mutant-transfected cells lost let-7i-5p binding ability compared to wild-type, confirming a role for let-7i-5p in regulating p110α mRNA at the predicted binding site.We transfected HCT116 and SW480 cells with let-7i-5p mimic to validate if it would regulate p110α/Akt. Let-7i-5p mimic remarkably inhibited p110α expression and Akt phosphorylation compared to NS miRNA mimic , we found three putative p63-binding sites  located between -1638 and -1380 bp in the Dicer promoter region   A. ChIP amoter C) B. The crmoter C) C. To expmoter C) D. Hypoximoter C) D. Interevia Dicer to prevent p110α degradation and casually induced the CSC phenotype.Given that hypoxia-induced Nur77 could enrich CSC properties Figure , we askein vivo, HCT116 cells were stably transfected with Nur77 shRNA. The depletion efficiency of Nur77 was shown  and CSC (CD44 and CD143) markers  dataset using SurvExpress, an online tool for survival analysis and risk assessment of cancer datasets Hypoxia is one of the main obstacles in treating solid tumors. Tumors growing under hypoxia exhibit an aggressive phenotype, resulting in increased metastasis and enhanced resistance to conventional radiotherapy and chemotherapy PI3K is a crucial heterodimeric lipid kinase in cancer development with three classes comprising different structures and biochemical features. Among them, Class IA is the most common type consisting of a regulatory (p85) and a catalytic subunit (p110) via Dicer, which cleaves pre-miRNA into mature miRNA. Low expression of Dicer, frequently observed in various malignancies, was reported to be associated with poor clinical outcome miRNAs are small non-coding regulatory RNAs that post-transcriptionally control gene expression and are vital regulators of many cellular processes and could serve as potential targets or biomarkers in the clinic In humans, the let-7 miRNA family consists of 10 mature members. As one of the earliest tumor suppressor miRNAs discovered, let-7 was shown to be under-expressed in many human malignancies We found that Nur77 could regulate Dicer expression independent of its transactivation ability. This is not unexpected since Nur77 has been shown to be a nucleocytoplasmic shuttling protein with various genomic and non-genomic roles during cancer progression To summarize, our data demonstrated a novel non-genomic mechanism of Nur77 to enhance cancer EMT and stemness properties. The inhibition of Dicer/let-7i and subsequent activation of the oncogenic p110α/Akt pathway suggest an attractive therapeutic potential for Nur77.Supplementary figures and tables.Click here for additional data file.


In vitro co-culture of human eosinophils and dermal fibroblasts upon AD-related IL-31 and IL-33 stimulation, and in vivo MC903-induced AD murine model were employed to investigate the anti-inflammatory mechanism of IL-1 family cytokine IL-37 in AD. Results showed that IL-37b could inhibit the in vitro induction of AD-related pro-inflammatory cytokines IL-6 and TNF-α, and chemokines CXCL8, CCL2 and CCL5, increase autophagosome biogenesis-related LC3B, and decrease autophagy-associated ubiquitinated protein p62 by regulating intracellular AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signaling pathway. In CRISPR/Cas9 human IL-37b knock-in mice, IL-37b could significantly alleviate MC903-stimulated ear tissue swelling, itching sensation and the level of circulating cytokine IL-6 and ear in situ expression of AD-related TNF-α, CCL5 and transforming growth factor-β. Moreover, IL-37b could significantly upregulate Foxp3+ regulatory T cells (Treg) in spleen and ear together with significantly increased serum Treg cytokine IL-10, and decrease eosinophil infiltration in ear lesion. IL-37b knock-in mice showed a distinct intestinal microbiota metabolic pattern upon MC903 stimulation. Furthermore, IL-37b restored the disordered gut microbiota diversity, through regulating the in vivo autophagy mechanism mediated by intestinal metabolite 3-methyladenine, adenosine monophosphate, 2-hydroxyglutarate, purine and melatonin. In summary, IL-37b could significantly ameliorate eosinophils-mediated allergic inflammation via the regulation of autophagy mechanism, intestinal bacterial diversity and their metabolites in AD. Results therefore suggest that IL-37 is a potential anti-inflammatory cytokine for AD treatment.Interaction between eosinophils and dermal fibroblasts is essential for provoking allergic inflammation in atopic dermatitis (AD).  Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disease, characterized by skin lesions, pruritic excoriations and susceptibility to cutaneous infections . There iIL-1 family cytokine IL-37/IL-1F7 can downregulate systemic and local inflammation and Th2 cytokines, by suppressing the production of pro-inflammatory mediators and cytokines in innate and adaptive immunity . ClinicaIL-37 has been reported to alleviate inflammation by regulating intracellular AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling mechanism, which is the key regulator of autophagy . Autophain vitro and in vivo experiments. Further non-targeted metabolomic analysis and GM profile of CRISPR/Cas9 human IL-37b knock-in and wild type mice with AD were employed to elucidate the anti-inflammatory mechanism of IL-37 in AD.In the present study, the mechanism of detailed immunoregulatory role of IL-37b in Th2-related eosinophils-mediated interaction with dermal fibroblasts in AD was explored using Inbred CRISPR/Cas9 human IL-37b knock-in mice  were purchased from Cyagen Biosciences (Guangzhou) Inc, China.The AD mouse model was established using MC903 stimulation for 16 days. A total of 2 nmol of MC903  was topically applied in 5 μL of ethanol to one ear of a mouse every other day for 16 days. Ear thickness was measured with a dial thickness gauge  and snatching time within 5 min were recorded to assess itching severity every other day for 17 days, followed by sacrificing the mice for post mortem analysis of AD skin lesions. Quick and continuous multiple scratching within a very short period was considered as one-time scratching. All animal experiments were approved by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong, Hong Kong.Sections (5 mm) were stained with H&E to assess the general morphology. Paraffin sections were immunostained with lineage-specific antibodies to identify eosinophils and dermal fibroblasts by immunohistofluorescence. Samples were incubated with rat anti-mouse major basic protein (MBP) antibody , rabbit anti-Vimentin antibody , AMPK alpha-1 monoclonal antibody, mTOR monoclonal antibody, and IL-37 polyclonal antibody , and LC3B antibody . Cy3-conjugated goat anti-rat immunoglobulin G (IgG) antibody  and Alexa Fluor 488-conjugated goat anti-rabbit and goat anti-mouse IgG antibody  were used as secondary antibodies. All images were acquired with a Leica DM6000B microscope  and processed using the Leica Application Suite software (Leica Microsystems GmbH).Single-cell suspensions were prepared from spleens of wild type and IL-37b Tg mice. Spleen was mechanically disrupted and homogenate collected by using a 70 μm cell strainer . Single splenic cells were washed with wash buffer (1 x PBS supplemented with 2 mM EDTA and 2% FBS), and analyzed using a FACSVia flow cytometer  with a mouse Treg cell staining kit (Invitrogen).Total RNA of mouse ear tissue (10 mg) was extracted using RNA extraction kit . cDNA was synthesized using SuperScript II Reverse Transcriptase (Invitrogen). Quantitative real-time RT-PCR analysis of the cDNA was performed using a StepOnePlus Real-Time PCR System  with SYBR Green Master Mix (Bio-Rad). The relative mRNA expression of each gene was determined using ddCt calculation method with GAPDH as internal housekeeping gene.Primer sequences used were: IL-4: Forward 5′-ACAGGA GAAGGGACGCCAT-3′, Reverse 5′-ACCTTGGAAGCCCTAC AGA-3′; CCL2: Forward 5′-GCATCTGCCCTAAGGTCTTCA-3′, Reverse 5′-GTGGAAAAGGTAGTGGATGCATT-3′; Foxp3: Forward 5′-CCCAGGAAAGACAGCAACCTT-3′, Reverse 5′-T TCTCACAACCAGGCCACTTG-3′; TNF-α: Forward 5′-CACA GAAAGCATGATCCGCGACGT-3′, Reverse 5′-CGGCAGA GAGGAGGTTGACTTTCT-3′; TGF-β: Forward 5′-CACAGAA AGCATGATCCGCGACGT-3′, Reverse 5′-CGGCAGAGAGGA GGTTGACTTTCT-3′; CCR3: Forward 5′-AAGCTTTGAGAC CACACCCTATG- 3′, Reverse 5′-GACCCCAGCTCTTTGATT CTGA-3′; CCL5: Forward 5′-CCCTCACCATCATCCTCACT-3′, Reverse 5′-TCCTTCGAGTGACAAACACG-3′; mGAPDH: Forward 5′-TGGTGAAGCAGGCATCTGAG-3′, Reverse 5′-TG TTGAAGTCGCAGGAGACAAC-3′.Human eosinophils were purified from fresh human buffy coats (Hong Kong Red Cross Blood Transfusion Service) using anti-CD16 magnetic beads and LS+ column within a magnetic field (Miltenyi Biotec). Purity of eosinophils (at least 96%) was assessed by Hemacolor rapid blood smear stain . The above protocol using human eosinophils was approved by Clinical Research Ethics Committee, The Chinese University of Hong Kong-New Territories East Cluster Hospitals according to the Declaration of Helsinki.5 cells) and confluent fibroblasts (1 × 105 cells) were co-cultured in RPMI 1640 medium containing 10% FBS with or without IL-37b (100 ng/mL) pre-treatment for 10 mins, followed by IL-31 and IL-33 (100 ng/mL) stimulation for 24 h prior to the determination of the expression of cytokines/chemokines and autophagy related proteins.Primary human dermal fibroblasts  were grown in Medium 106 supplemented with low serum growth supplement . Eosinophils , rapamycin (10 ng/mL), IL-37b (100 ng/mL), or chloroquine (20 μM) for 1, 2, 4, and 16 h. Cells were stained and autophagic vacuoles were detected at indicated time by using Autophagy Detection Kit . Fluorescent autophagic vacuoles were analyzed by a Leica DM6000B fluorescence microscope .Concentrations of human inflammatory cytokines and chemokines including TNF-α, IL-6, CCL2, CCL5, and CXCL8 in culture supernatant were measured using the cytometric bead array kit  on a BD FACSVia flow cytometer. Murine interferon IL-5, IL-13, IL-6, and IL-10 in serum were quantified with mouse cytokine Milliplex MAP assay kit  using the Bio-Plex 200 system .Cells from the co-culture and homogenized mouse ear tissue were lysed with RIPA lysis buffer (Thermo Fisher Scientific). Protein content from cell lysis and mouse ear tissue was determined with protease inhibitor and phosphatase inhibitor (Sigma-Aldrich) and Pierce BCA protein assay (Thermo Fisher Scientific). AMPK alpha-1 monoclonal antibody, mTOR monoclonal antibody and IL-37 polyclonal antibody were obtained from Thermo Fisher Scientific. SQSTM1/P62, β-actin rabbit mAb, HRP-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG were obtained from Cell Signaling Technology. Dorsomorphin (Compound C/CC AMPK inhibitor) and LC3B were obtained from Sigma-Aldrich Corp. Secondary antibodies were used at 1:2000 and other primary antibodies were diluted at 1:1000.1 and ChemSpider2 database.Stools (25 mg) obtained from mice at day 1, 15, and 17 were individually grounded with liquid nitrogen and the homogenate was resuspended with prechilled 80% methanol and 0.1% formic acid. The collected supernatant was diluted to a final concentration containing 60% methanol. LC-MS/MS analysis was performed using a Vanquish UPLC system (Thermo Fisher Scientific) coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific). Samples were injected onto a Hyperil Gold column  using a 16-min linear gradient at a flow rate of 0.2 mL/min. The eluents for positive polarity mode were eluent A (0.1% FA in Water) and eluent B (Methanol). The eluents for negative polarity mode were eluent A  and eluent B (methanol). The solvent gradient was set as follows: 2% B, 1.5 min; 2–100% B, 12.0 min; 100% B, 14.0 min; 100-2% B, 14.1 min; 2% B, 16 min. Q Exactive HF-X mass spectrometer was operated in positive/negative polarity mode with spray voltage of 3.2 kV, capillary temperature of 320°C, sheath gas flow rate of 35 arb and aux gas flow rate of 10 arb. Raw data files generated by UPLC-MS/MS were processed to perform peak alignment, peak picking and quantitation for each metabolite by using Compound Discoverer 3.0 . Peak intensities were normalized to predict the molecular formulas and metabolites based on additive ions, molecular ion peaks and fragment ions by matching with mzCloud® High-Fidelity PCR Master Mix (New England Biolabs). PCR products were purified with GeneJETTM Gel Extraction Kit (Thermo Fisher Scientific). Sequencing libraries were generated using Ion Plus Fragment Library Kit (Thermo Fisher Scientific) following the manufacturer’s recommendations, and quality control was conducted with the Qubit@ 2.0 Fluorometer (Thermo Fisher Scientific). 400/600bp single-end reads were generated on an Ion S5TM XL platform. 16S rRNA amplification and sequencing service were provided by Novogene Co., Ltd., Beijing, China.Total genome DNA from stool samples was extracted using CTAB/SDS method. DNA was diluted to 1 ng/μL using sterile water. 16S rRNA genes of distinct regions (16S V3-V4) were amplified using specific primer  with the barcode. All PCR reactions were carried out with PhusionPRJEB36050.Alpha diversity and beta diversity on both weighted and unweighted unifrac were calculated by QIIME software (Version1.7.0) and displayed with R software (Version 2.15.3) using Principal Component Analysis (PCA), Non-Metric Multi-Dimensional Scaling (NMDS) and Unweighted Pair-group Method with Arithmetic Means (UPGMA) analysis. Partial least-square discriminant analysis (PLS-DA) was performed to discriminate the microbial community profiles among groups. The metagenomic sequencing data (16S rRNA) has been deposited in European Nucleotide Archive (ENA) with accession number P < 0.05 was considered statistically significant.Data were presented as mean ± SEM, and analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple inter-group comparisons using GraphPad PRISM software version 6.01. P > 0.05) on both cell proliferation and viability , which is in concordance with previous study using a similar AD murine model (P < 0.05). QPCR results showed that the in situ Th2 cytokine IL-4 mRNA expression was decreased in IL-37b Tg AD mice  showed significantly less Treg cells in spleen compared with either sham mice (Treg/CD4+: 532 ± 115/8519 ± 1456) and IL-37b Tg (Treg/CD4+: 426 ± 35/7224 ± 659) AD mice (P < 0.05). Together with upregulating Treg cytokine IL-10 . Statistical indices of alpha diversity are summarized in As shown in Clusters of W1, T1, and T2  samples Large number of bacterial species were screened in related paired group . The detAs shown by the volcano map , differeThe identified differential metabolites are showed in a heat map  and moreIL-37b Tg AD mice showed higher cholic acid (CA) and melatonin levels than those in wild type AD mice , 6C. LowIncreased adenosine monophosphate (AMP) was reported to induce the cell autophagy process by activating AMPK . IL-37b in vivo. Significantly lower ratio of LC3II/LC3I and AMPK levels, and higher mTOR level were found in the ear tissue of wild type AD mice compared with IL-37b Tg AD mice  has been deposited in European Nucleotide Archive (ENA) with accession number The animal study was reviewed and approved by Animal Experimentation Ethics Committee, the Chinese University of Hong Kong, Hong Kong.TH and C-KW designed the experiments. C-KW obtained the funding. TH, XS, JZ, K-LH, PJ, IM-TC, and MS-MT performed the experiments and analyzed the data. TH, C-KW, HZ, and CW-KL drafted and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RIG-I plays important roles in pathogen sensing and activation of antiviral innate immune responses in response to RNA viruses. RIG-I-mediated signaling must be precisely controlled to maintain innate immune signaling homeostasis. Previous studies demonstrated that lysine 63 (K63)-linked polyubiquitination of RIG-I is vital for its activation, but the mechanisms through which RIG-I is deubiquitinated to control innate immune responses are not well understood. Here we identified USP27X as a negative regulator of antiviral signaling in response to RNA viruses through siRNA library screening. Further functional studies indicated that USP27X negatively modulated RIG-I-mediated antiviral signaling in a deubiquitinase-dependent manner. Mechanistically, we found that USP27X removed K63-linked polyubiquitin chains from RIG-I to negatively modulate type I interferon signaling. Collectively, these studies uncover a novel negative regulatory role of USP27X in targeting RIG-I to balance innate immune responses. Innate antiviral immunity provides the first line of the host defense system that rapidly detects invading virus through pattern recognition receptors, and subsequently inducing type I interferon production. Protein ubiquitination/deubiquitination play important roles in antiviral signaling, however how the dynamic regulation of ubiquitin modification controls innate immune signaling against RNA virus infection remains unclear. In this study, we screened E3 ligases and deubiquitinases (DUBs) using a siRNA library, and identified USP27X, a DUB, as a negative regulator of antiviral signaling in response to RNA virus infection. Moreover, we employed knockdown and knockout of USP27X various cell lines to perform functional analyses, and found that USP27X suppressed RIG-I-mediated antiviral signaling in a DUB activity-dependent manner. Mechanistically, we found that USP27X removed K63-linked polyubiquitin chains from RIG-I to negatively modulate type I IFN signaling. Collectively, our study reveals that in addition to removing K48-linked polyubiquitin chains, USP27X can target RIG-I for removing the K63-linked polyubiquitin chains, thereby balancing antiviral signaling to avoid deleterious effects on host cells. Viral infection can trigger innate immune responses, which serve as the first line of host defense in detecting and eliminating viruses. Innate immune responses are triggered by various pattern recognition receptors sensing different pathogen-associated molecular patterns , 2. HostRIG-I and MDA5 function as cytosolic viral RNA sensors. Their structures are very similar: both consist of two caspase activation and recruitment domains (CARDs), a central DexD/H helicase domain and a C-terminal regulatory domain (RD) , 7. ViraProtein ubiquitination plays a pivotal role in regulating RIG-I-mediated signaling. Previous studies found that K63-linked polyubiquitination of RIG-I was required for activation and downstream signaling transduction, while K48-linked polyubiquitination regulated RIG-I stability and inhibited type I IFN production . TRIM25 USP27X is a DUB and a member of the cysteine protease family. A recent study suggested that USP27X, together with USP22 and USP51, regulate monoubiquitination of histone H2B . MoreoveTo better understand the roles and molecular mechanisms of ubiquitination in RIG-I-mediated signaling, we screened E3 ligases and DUBs using a siRNA library, and identified USP27X as a negative regulator of antiviral signaling. We employed knockdown and knockout of USP27X various cell lines, performed further functional analyses, and demonstrated that USP27X inhibited RIG-I-mediated antiviral signaling in a DUB activity-dependent manner. Moreover, we found that USP27X removed K63-linked RIG-I polyubiquitin chains to negatively regulate type I IFN signaling. Our results demonstrate that in addition to removing K48-linked polyubiquitin chains, USP27X can remove K63-linked polyubiquitin chains from RIG-I, thus negatively modulating antiviral signaling to maintain innate immune response homeostasis.IFNB1, TNFα and IFIT1 following SeV infection  stable reporter cell line expressing firefly luciferase driven by a human IFNβ promoter for screening. From this screening, we identified a DUB, USP27X, which acted as a negative regulator of IFNβ expression induced by Sendai virus (SeV) infection . To furtnfection . Since dnfection . Moreovenfection . Taken tPrevious studies have shown that USP27X could employ another start site (CTG) upstream of the predicted ATG start site to code a long form (72-kDa) of USP27X (hereafter referred as USP27X-72) . CompareIfnb1, Ifit1 and Il6 mRNA following SeV infection compared with control cells. Consistent with the qRT-PCR results, levels of phosphorylated IRF3 and P65 were increased in Usp27x-knockdown cells following SeV infection , but not MAVS Figs . Moreovenfection . Of notenfection . Taken tTo further explore the molecular mechanisms of USP27X in regulation of RIG-I activity, we examined which domain of RIG-I was required for the interaction between RIG-I and USP27X. RIG-I contains two CARDs, a central DExD/H box RNA helicase domain, and a C-terminal RD domain. Co-IP showed that both RIG-I(N) and full-length RIG-I pulled down USP27X, but that the RIG-I helicase domain and RD domain failed to pull down USP27X. These data indicated that USP27X interacted with RIG-I through its CARDs Figs  and S11ANext, we mapped which region of USP27X was required for its interaction with RIG-I. Since structural analysis showed that USP27X contained a catalytic UCH domain (residues 78–421), we generated three deletion mutants of USP27X: USP27X-ΔN (deleting residues 1–77), USP27X-ΔUCH (deleting residues 78–421), and USP27X-ΔC (deleting residues 422–438). Co-IP experiments demonstrated that deletion of the UCH domain or the N-terminal region of USP27X significantly inhibited its interaction with RIG-I(N), but the C-terminal region was not required for this interaction . ReporteIFNB1 mRNA following SeV infection , and obtained results consistent with those of the in vivo assays , but failed to affect K48-linked ubiquitination , this product therefore contained an additional 198 aa fragment at its N-terminus , 30. AltA number of studies including ours indicated that K63-linked of RIG-I ubiquitination is regulated by several DUBs, including USP3, USP21, USP25, USP15 and CYLD , 34–37. Collectively, our studies uncovered a novel role of USP27X in regulating antiviral signaling. USP27X interacted with RIG-I and MDA5, and removed K63-linked polyubiquitin chains from RIG-I and MDA5 to suppress the excessive production of type I IFNs. Our data provide insights into the mechanisms through which USP27X functions as a negative regulator of antiviral signaling to maintain innate immune signaling homeostasis.HEK293T, HeLa, HepG2 and RAW264.7 cells were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences, and maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) containing 10% (v/v) fetal bovine serum (Invitrogen) and 1% streptomycin and penicillin. THP-1 cells were cultured in RPMI-1640 containing 10% fetal bovine serum, 1% streptomycin and penicillin and 10 μM β-mercaptoethanol.Rabbit anti-p-IRF3, anti-IRF3, and anti-p-P65 antibodies were from Cell Signaling Technology. Mouse anti-P65 and TRIM25 antibodies were from Santa Cruz. Rabbit anti-Flag antibody was from Sigma. Rabbit anti-Myc, anti-Sendai pAb and anti-HA antibodies were from MBL. Mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and mouse anti-HA antibodies were from Sungene Biotechnology.Flag-tagged RIG-I, RIG-I(N), RIG-I(Heli), RIG-I(RD), USP27X, USP27X-72, MAVS, TRAF3, TRAF6, NEMO, TBK1, IRF3, IKKβ and P65, Myc-tagged USP27X, USP27X-72, USP27X-72(N) and HA-tagged Ub, Ub-K63 and Ub-K48 were constructed using standard molecular biology methods. USP27X mutants were generated by PCR using Pfu DNA polymerase. The plasmids encoding IFNβ-Luc, NF-κB-Luc and ISRE-Luc have been described previously .HEK293T cells were seeded in 24-well plates and cultured overnight, then transfected with plasmids using polyethylenimine. A Renilla reporter plasmid and a firefly luciferase reporter plasmid encoding IFNβ-Luc, NF-κB-Luc or ISRE-Luc were transfected along with the indicated expression plasmids. In the same experiment, empty control plasmid was added to ensure that the same amount of total DNA was transfected. Twenty-four hours following transfection, cells were lysed to measure luciferase activity, which was normalized to Renilla activity. All reporter assays were repeated at least three times.Cells were lysed in lysis buffer . Clarified cell lysates were incubated with anti-Flag agarose beads (Sigma) for 4 h. The immunoprecipitated complexes were washed with lysis buffer containing 300 mM NaCl three times and subjected to immunoblotting analysis with the indicated antibodies. For semi-endogenous IP, cell lysates were incubated with RIG-I antibody overnight at 4°C, followed by further incubation with protein A/G beads (Pierce) for 2 h. Immunoblotting was carried out using standard procedures.HEK293T and HepG2 cells were grown on gelatin-coated glass coverslips, and transfected or infected by SeV as indicated. Cells were washed with phosphate-buffered saline, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 10 min, and then blocked with 5% (w/v) bovine serum albumin for 30 min. Cells were then incubated with primary and secondary antibodies. Imaging was conducted using a Zeiss LSM 710 META laser scanning confocal system.in vivo deubiquitination assays, HEK293T cells were transfected with the indicated plasmids. Cells were treated with MG132  before harvesting. Cells were lysed in lysis buffer A . The supernatants were denatured at 95°C for 5 min, diluted in buffer B  until the concentration of SDS was about 0.1%, then immunoprecipitated with anti-Flag beads for 4 h. After washing with wash buffer C  three times, the immunoprecipitated material was subjected to western blotting with the indicated antibodies.For in vitro deubiquitination assays, HEK293T cells were co-transfected with plasmids encoding Flag-RIG-I(N) and HA-Ub-K63. Cells were lysed with lysis buffer  and ubiquitinated RIG-I(N) was purified using anti-Flag beads. Ubiquitinated RIG-I(N) proteins were eluted with Flag peptide after extensive washing with lysis buffer . Flag-tagged USP27X proteins were purified using the same method. Ubiquitinated RIG-I(N) was incubated with USP27X in deubiquitination buffer  at 37°C for 2 h, then incubated at 16°C overnight, followed by immunoblotting assays.For USP27X+/+ and USP27X-/- cells were infected with VSVΔM51-GFP at a multiplicity of infection (MOI) of 0.01 for 9 h, and culture supernatants were collected and diluted to infect Vero cells. Thirty-six hours later, Vero cells were fixed with methanol for 30 min and stained with 1% crystal violet. Plaques were counted to quantitate viral titer and shown in plaque-forming units (p.f.u.)/mL.HEK293T A genome-wide siRNA screen using the Silencer Human Ubiquitin siRNA Library (Dharmacon) was conducted by transfection a HEK293T stable reporter cell line that expresses firefly luciferase driven by a human IFNβ promoter. Forty-eight hours later, cells were infected with SeV, followed by reporter assays.RAW264.7 cells were infected with lentiviral vectors targeting two different regions of mUsp27x  or empty vector for 48 h. The cells were either untreated or infected with SeV or VSVΔM51-GFP for the indicated times, followed by subsequent experiments. Knockdown efficiency was determined by qRT-PCR. The sequences for mouse Usp27x shRNAs were as follows (5’–3’): shUsp27x1: TCATGTGCCCTATAAGTTA; shUsp27x2: GCGCAAGATCACTACGTACAT. The sequence for human USP27X shRNA was (5’–3’): AAACAACCAAACCAGAATTA.-ΔΔCt method was used to calculate relative gene expression, and relative mRNA level for each gene was normalized to levels of GAPDH. Data shown are the relative abundance of mRNA as compared with control groups. All samples were assayed in triplicate. The gene-specific primers were as follows (5’–3’):Total RNA was extracted from cells using TRIZOL reagent (Invitrogen) and cDNA was synthesized using the SuperScript III First-Strand cDNA Synthesis kit (Invitrogen). qRT-PCRs were conducted using SYBR Green Master Mix (Thermo Fisher) and a Light Cycler 480 instrument (Roche). The 2hUSP27X-S CAACCTGGGAAACAACCAAACC;hUSP27X-AS GGCCTGGACAATGCAGTTCAT;hIFNB1-S AGGACAGGATGAACTTTGAC;hIFNB1-AS TGATAGACATTAGCCAGGAG;hIFIT1-S TACCTGGACAAGGTGGAGAA;hIFIT1-AS GTGAGGACATGTTGGCTAGA;hIL6-S TCCAGAACAGATTTGAGAGTAGTG;hIL6-AS GCATTTGTGGTTGGGTCAGG;hTNFα-S CTGCCCCAATCCCTTTATT;hTNFα-AS CCCAATTCTCTTTTTGAGCC;hGAPDH-S ATGACATCAAGAAGGTGGTG;hGAPDH-AS CATACCAGGAAATGAGCTTG;hUSP3-S GAGCTGCCTGTATGACCT;hUSP3-AS ACCACAGTCTCCTCGTCA;hUSP21-S ACCGAGCCAACCTAATGTG;hUSP21-AS GGCAGGGACAGGTCACAA;hCYLD-S CATAATAAACCAAAGGCTAC;hCYLD-AS GTGAAGAACGGTCAAAGT;hTRIM25-S AGCAGCTACAACAAGAATACACG;hTRIM25-AS GGCTCTGTTCAATCTCCTCCT;mGapdh-S AACTTTGGCATTGTGGAAGG;mGapdh-AS ACACATTGGGGGTAGGAACA;mIfnb1-S ATGGTGGTCCGAGCAGAGAT;mIfnb1-AS CCACCACTCATTCTGAGGCA;mIfit1-S CTGAGATGTCACTTCACATGGAA;mIfit1-AS GTGCATCCCCAATGGGTTCT;mIl6-S TAGTCCTTCCTACCCCAATTTCC;mIl6-AS TTGGTCCTTAGCCACTCCTTC;mUsp27x-S CCAAAGAAGAGCAAGGAG;mUsp27x-AS GTAAGCCGATGGTAAAGC.et al . The two-tailed Student’s t-test was used to analyze statistical significance. * P < 0.05; **P < 0.01; ***P < 0.001; n.s. not significant versus control groups.(A–C) HEK293T cells were co-transfected with the indicated expression plasmids along with luciferase reporter constructs driven by promoters of IFNβ (A), ISRE (B) or NF-κB (C) as well as Renilla as an internal control. Twenty-four hours after transfection, the cells were lysed for luciferase assays (upper panel) and immunoblotting assays (lower panels). (D–G) HEK293T (TIF)Click here for additional data file.S11 FigUSP27X+/+ and USP27X-/- cells were transfected with indicated expression plasmids. Twenty-four hours after transfection, the cells were infected with SeV for 9 h, followed by measurement of IFNB1 mRNA levels by qRT-PCR. The data shown in (B) are from one representative experiment of at least three independent experiments (mean ± SD of triplicate experiments). The two-tailed Student’s t-test was used to analyze statistical significance. *** P < 0.001 versus control groups.(A) HEK293T cells were transfected with the indicated expression plasmids. Cell lysates were immunoprecipitated with anti-Flag beads, followed by immunoblotting. (B) HEK293T (TIF)Click here for additional data file.S12 Fig(A–E) HEK293T cells were transfected with the indicated plasmids. Cell lysates were immunoprecipitated with anti-Flag or anti-HA beads, followed by immunoblotting with the indicated antibodies.(TIF)Click here for additional data file.S13 FigUSP27X+/+ and USP27X-/- cells were co-transfected with Myc-GFP and Flag-RIG-I(N) plasmids. Twenty-four hours after transfection, cell lysates were for immunoblotting analysis with the indicated antibodies.(A) HEK293T cells were transfected with the indicated plasmids. Twenty-four hours after transfection, the cells were lysed for immunoblotting with the indicated antibodies. (B) HEK293T (TIF)Click here for additional data file.S14 FigUSP27X+/+ and USP27X-/- cells were transfected with Flag-MDA5(N) plasmids. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-Flag beads, followed by immunoblotting analysis with the indicated antibodies. The expression levels of transfected proteins in whole cell lysates (WCL) are shown in the bottom panels.(A) HEK293T cells were transfected with the indicated expression plasmids. Cell lysates were immunoprecipitated with anti-Flag beads, followed by immunoblotting. (B–D) HEK293T cells were transfected with USP27X-72 and MDA5(N) together with HA-tagged wild-type Ub (HA-Ub) (B), HA-Ub-K63 (C), or HA-Ub-K48 (D) plasmids. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-Flag beads, followed by immunoblotting analysis with the indicated antibodies. The expression levels of transfected proteins in whole cell lysates (WCL) are shown in the bottom panels. (E) HepG2 (TIF)Click here for additional data file.S15 FigUSP27X and IFNB1 mRNA levels by qRT-PCR. The data shown in (A–B) are from one representative experiment of at least three independent experiments (mean ± SD of triplicate experiments). The two-tailed Student’s t-test was used to analyze statistical significance. ***P < 0.001, versus control groups.HEK293T (A) or HepG2 (B) were infected with SeV for the indicated times, then lysed for measurement of (TIF)Click here for additional data file.S16 FigUSP27X+/+ and USP27X-/- cells were transfected with Flag-RIG-I(N) or Flag-RIG-I(N)-K172R plasmids. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-Flag beads, followed by immunoblotting analysis with the indicated antibodies. The expression levels of transfected proteins in whole cell lysates (WCL) are shown in the bottom panels.HepG2 (TIF)Click here for additional data file.S17 FigUSP27X+/+ and USP27X-/- cells including HEK293T, HeLa and HepG2 cells were lysed for immunoblotting with the indicated antibodies. (B) Usp27x+/+ and Usp27x-/- cells including L929 and RAW264.7 cells were lysed for immunoblotting with the indicated antibodies.(A) (TIF)Click here for additional data file.S18 Fig(A–B) HEK293T cells were transfected with USP27X, USP27X-72(N) and RIG-I(N) (A) or MDA5(N) (B) together with HA-tagged Ub-K63 plasmids. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-Flag beads, followed by immunoblotting analysis with the indicated antibodies. The expression levels of transfected proteins in whole cell lysates (WCL) are shown in the bottom panels.(TIF)Click here for additional data file.S19 FigUSP3 (A), USP21 (B) and CYLD (C) mRNA in a number of cell lines. (D) HEK293T WT, USP27X-/-, USP3-/- and USP21-/- cells were infected with SeV for 9 h, then lysed for measurement of IFNB1 mRNA levels by qRT-PCR. (E) HEK293T WT and CYLD-/- cells were infected with SeV for the indicated times, then lysed for measurement of IFNB1 mRNA levels by qRT-PCR. The data shown in (A–E) are from one representative experiment of at least three independent experiments (mean ± SD of triplicate experiments). The two-tailed Student’s t-test was used to analyze statistical significance. * P < 0.05; **P < 0.01; ***P < 0.001, versus control groups.(A–C) qRT-PCR assays were performed to measure levels of (TIF)Click here for additional data file.S20 FigUSP27X-KO cells including HEK293T, HeLa, HepG2, L929 and RAW264.7 cells were generated by CRISPR/Cas9 gene editing system, and USP27X-KO cells were verified by DNA sequencing.(TIF)Click here for additional data file.


Immunotherapy with redirected T cells that express a chimeric antigen receptor (CAR) is a promising prospectin cancer treatment. Most CARs use murine-derived single-chain variable fragments (scFvs) as an antigen targeting moiety,which may lead to host immunogenic responses and engineered T cell disappearance. It seems that development of lessimmunogenic CARs, such as CARs composed of the camelid variable domain of heavy chain antibodies (VHHs) may likelyovercome this obstacle. Here, we improved the expression of the VHH-based anti-MUC1 CAR gene construct using a thirdgeneration lentiviral vector in primary human T cells and assessed its effect on antigen specific targeting, activation andcytotoxicity of redirected human T cells.+. Next, we constructed lentiviral vectors that contained thisCAR gene construct using an optimized transiently virus production method and transduced it into human T cells. Cellsurface expression of CAR, cytokine secretion and cytotoxic activity were assessed in the transduced CD3+T cells. In this experimental study, we established a second generation novel CAR (VHH-based anti-MUC1 CAR) that contained a camelid-derived anti-MUC1 VHH followed by an IgG3 hinge, a CD28 transmembranedomain and signalling endodomains of CD28 and CD3 The transduced T cells had high levels of surface expression of CAR. T cells that expressed anti-MUC1 CARshowed significantly increased secretion of Th1 cytokines, including IL-2, TNF alpha and IFN-γ, as well as cytotoxicactivity upon recognition of MUC1 on tumour cells after co-incubation with T47D or MCF-7 (MUC1-positive) comparedwith A431 (MUC1-negative) or untransduced T cells.Our results suggested that, given the unique properties of VHHs to prevent immunogenic responses andtonic signalling, our novel VHH-based anti-MUC1 CAR might be effective for clinical purposes in cancer immunotherapy. Adoptive cell-based immunotherapy is a promisingapproach in the treatment of cancers. In recentyears, chimeric antigen receptor (CAR)-T cell basedimmunotherapies have shown impressive successes inthe treatment of hematologic malignancies, especiallylymphoma. In 2017, the first two engineered cell-basedimmunotherapies (CD19-targeted CAR-T cells) wereapproved by the US Food and Drug Administration (FDA).Kymriah was introduced by Novartis for treatment ofB-cell acute lymphoblastic leukaemia (ALL) patients lessthan 25 years of age and about two months later, Yescartawas introduced by Kite Pharma (Gilead) for patients withrelapsed or refractory B-cell lymphoma , inhibitorycytokines (TGF-β and IL-10), and a hostile tumourmicroenvironment  in most solid tumours has presented problems forCAR-T cell therapy. Currently, several novel innovationssuch as Identification of neoantigens, designing inhibitoryCARs (iCARs), dual recognition of different antigens bytwo CARs (tandem CARs), co-expression of cytokinereceptor transgenes in CAR-T cells, and the combinationof checkpoint inhibitors with CAR-T cells have beendeveloped to overcome these barriers , 5.CAR transgene  gene locus, utilization of self-inactivating (SIN) lentiviralvectors instead of gamma-retroviruses, incorporation of 4-1BB in endodomain signalling ofCARs and substitution of single-domain antibodies instead of scFv  was purchased from BD Biosciences (USA) and FITC-conjugatedgoat anti-rabbit IgG secondary antibody was purchased from Abcam .In this experimental study, human breast cancer cell lines (T47D and MCF-7 cells), humanepidermoid squamous carcinoma cell line (A431), and Lenti-X 293T cells were purchased fromIranian Biological Resource Centre . T47D cells were cultured in RPMI 1640medium . MCF-7, A431 and Lenti-X 293T cells were culturedin Dulbecco’s Modified Eagle Medium . Both media weresupplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine . Furthermore, for packaging and production of lentiviral particles,Lenti-X 293T cells were maintained in DMEM  that consistedof 10% FBS and 2 mM L-glutamine. For flow cytometry assessment, APCconjugated anti-CD3 (BDPharmingen+ was made in our previous study(pLJM1-EGFP (Addgene plasmid #19319) transferplasmid (a gift from David Sabatini), pRSV-Rev), pMDLg/pRRE and pMD2.G plasmids were used to produce thethird generation lentiviral particles. Anti-MUC1 CARcassette that contained coding sequences for anti-MUCIVHH-IGg3-CD28-CD3F: 5ˊ-TATAGCTAGCGCCACCATGGCCGAGGTGGAG-3ˊ andR: 5ˊ-TATTACCGGTTTCGATCCTCCTCC-3ˊdesigned to introduce the NheI (3ˊ site) and AgeI (5ˊsite) restriction sites in the ends of the CAR construct.The modified anti-MUCI construct was subcloned to thepLJM1-EGFP plasmid using digestion and ligation of theNheI/AgeI fragment.6 Lenti-X 293Tcells were seeded onto a 24-well plate and incubated overnight in a 37˚C, 5%CO2 incubator. The next day, pMDLg/pRRE, pRSV-Rev, pMD2.G and pLJM1-EGFPplasmids were co-transfected to the Lenti-X 293T cells using branched polyethyleneimine  were co-transfected to theLenti-X 293T cells by the optimized PEI transfection method. Cell supernatants thatcontained the virus particles were harvested at 24, 48 and 72 hours after transfection andwere centrifuged at 500× g/4˚C. The supernatants that contained the virus particleswere filtered through 0.45 μm filters (Millipore) to remove cell debris, then transferredto sterile capped tubes and concentrated by ultra-centrifugation (20000 × g for 2 hoursat 4˚C). The probable virus pellet was resuspended in 100-200 μL of DMEM, from which weobtained aliquots that were stored at -80˚C until the subsequent transduction step.First, to determine the optimum transfection condition, 0.8×10ich, DE)  in PEI:DThe infective lentivirus titre was determined byquantitative PCR  for the pur-F: 5ˊ-GCAGCAACAGATGGAAGG-3ˊ andpur-R: 5ˊ-GAGGTCTCCAGGAAGGC-3ˊ primers.23)/(length(bp)×1×109×650). The titre of the recombinant anti-MUC1 CAR lentiviralvector, reported in transforming units (TU)/mL, was calculated to be 1.12×108TU/mL.Briefly, after transduction of Lenti-X293T cells, copy numbers of integrated lentiviralvectors were measured by the standard curve of real-time PCR (Rotor-Gene 6000 SeriesSoftware 1.7) and the following formula: (DNA amount (ng)×6.022×105 T cells were resuspended in 1 mL of complete media  to which we added the appropriate volume of concentrated virusparticles and soluble RetroNectin® (Clontech) for a final concentration of 8 μg/mL. Cellsuspensions were centrifuged at 800 × g for 90 minutes at 32˚C. T cells were thenresuspended in complete media supplemented with 100 IU/mL rIL-2 and plated in 24-wellplates for 72-96 hours .Peripheral blood samples were obtained from healthy males and non-pregnant femalesbetween the ages of 25-35 years in compliance with the Institutional Review Board.approvedresearch protocols of the Tehran University of Medical Sciences (IR.TUMS.VCR.REC.1395.538). All blood samples collected following donor informed consent.Peripheral blood mononuclear cells (PBMC) were isolated by the Ficollpaque density gradient separation method. Freshly isolated PBMCs were cultured in 2 mL 10% FBSRPMI1640 media supplemented with 100 IU/ mL rIL-2  and subsequentlymixed with Dynabeads Human T-Activator CD3/ CD28  at a1:1 (bead:cell) ratio. At 48 to 72 hours after activation, the T cells were transducedwith recombinant lentiviral particles at a multiplicity of infection (MOI) of >20 usingthe spinoculation protocol where the MOI was calculated by viral titre/number of cells.Briefly, 2×105transduced T cells. The purity of the activated T cells was verified by APC mouseanti-human . All samples were examined by BD FACSCanto IIequipment. FlowJo software (v10) was utilized for data analysis.In order to detect VHH-based CAR expression on T cells, FITC goat anti-rabbit IgGantibody, specific for VHH , was used to stain 2×10To examine antigen specific activation and cytotoxicityof the transduced T cells, confluent cells that highlyexpressed MUC1 (T47D and MCF-7) and also cellswith no or slight expression (A431) were co-culturedwith transduced T cells at a 10:1 (effector:target) ratio inRPMI 1640 (10% FBS) supplemented with rIL-2 (100IU/mL) (n=3). The untransduced T cells were co-culturedwith cancerous cells and a single culture of tumour cellswere used as the negative control in this assay. After72 hours of incubation, all supernatants were collectedand cytokine secretion was measured by the humanTNF alpha , human IL-2 and IFN-γ  ELISA assays according tomanufacturer’s instructions.2 incubator at 37˚C. Cellsupernatant media were aspirated after the incubation time and the formation of a formazanproduct (dissolved in DMSO) was detected at a 540 nm absorbance wavelength by an ELISAplate reader.Viable MUC1-positive or MUC1-negative cells were analysed after co-culture with eitheranti-MUCI CAR-T cells or untransduced T cells in a 10:1 ratio by the MTT assay (n=3). Forthis purpose, after 72 hours, the anti- MUC1 CAR-T cells or untransduced T cells wereremoved and MTT reagent was added directly into the cell media. Subsequently, the cellswere maintained for 3 hours in a humidified 5% COAnalysis of variance (ANOVA) was used to identifydifferences between groups. Data were analysed byANOVA and Tukey’s post hoc test using GraphPad Prismsoftware (version 7.05). The data are shown as mean ±SEM or mean ± SD. P<0.05 was considered statisticallysignificant.+. The anti-MUCI VHH-IgG3 hing-CD28-CD3+ construct was successfully cloned into the pLJM1-EGFP plasmid. Sub-cloning was verified by restrictionenzyme digestion and DNA sequencing according to theSanger sequencing method ,which were called anti-MUC1 CAR-T cells .Cytokine production is a functional hallmark ofCAR-T cells. Therefore, production of cytokines IL-2,TNF alpha and IFN-γ were measured using ELISA afterco-culturing the modified T cells with MUC1 positive(T47D and MCF-7) or MUC1 negative (A431) tumourcell lines. The results revealed that the MUC1-redirectedCAR-T cells could produce TNF alpha, IFN-γ and IL-2in response to recognition of MUC1 expressed on thetumour cells . The co-We sought to appraise the cytotoxicity of CAR-Tcells by assessing cell viability of the target cells aftera 72-hour co-culture with transduced or untransducedT cells . MTT assMUC1 is a heavily glycosylated glycoprotein withan extracellular domain that extends up to 200-500nm from the cell surface. MUC1 has been found to beoverexpressed on the cell surface in multiple epithelialadenocarcinomas, including thoseof the breast, ovary, and pancreas . In mali+ (transmembrane). Immediately after this region lies the signalling endodomains thattransmit activation and costimulatory signals upon antigen recognition by scFv. According tothese signalling endodomains, CARs are classified into three “generations”. Only CD3+ hasbeen used as a signalling domain in “first generation” CARs. They lack co-stimulatorymolecules and are unable to directing engineered T cell activation and expansion effectivelyupon antigen recognition. “Second generation” CARs are added to intracellular domain oftencomprising of CD28 or 4-1BB. "Third generation" CARs include CD3+ and two or morecostimulatory domains, such as CD28 and usually OX40 (CD134) or 4-1BB (CD137) - derived scFv, ii. Extracellularspacer (hinge) which is the connecting region between the ligand binding domain to thetransmembrane domain, and iii. Transmembrane and signalling domains. Between the hingeregion and signalling endodomains are sequences which are usually derived from CD8, CD28 orCD3 (CD137) .Although the use of third generation CARs has beenmore effective than second generation CARs based ontheir anti-tumour effects and persistence in animal studies, there wMost clinical CAR-T cell studies have utilizedmurine-derived scFvs that lead to the eliminationof the scFv-based CAR-expressing T cells by thehost anti-mouse or anti-IgE antibodies . TheseaCAR geneconstruct to human primary T cells. Moreover, we also optimized our T cell transductionmethod (data not shown). This high anti-MUC1 CAR expression was responsible for specificcytotoxicity and cytokine production observed upon co-cultivation of anti- MUC1 CARexpressing T cells with cancerous cells. Moreover, our results showed significantly reducedsurvival of MUC1-positive cells after co-culturing with transduced T cells (24.9% and22.73%), but not in co-culturing with untransduced T cells (85.01% and 86.9%), which showedthat specific recognition of the MUC1 antigen by anti-MUC1 CAR on the transduced T cellstriggered intracellular signal transduction and activation of redirected human T cells, andled to specific elimination of MUC1-positive cells by these anti-MUC1 CAR-T cells.Accordingly, recognition of MUC1 by our anti-MUC1 VHH antibody was in an antigen-specificmanner. In line with this result, we previously demonstrated specific and efficientrecognition of a tumor antigen (TAG-72) by CCRFCEM cells that expressed another VHH-basedCAR (anti-TAG-72), which led to proliferation and cytokine release  ratio and 6 hourincubation period. Although reagent cytotoxicity increased with increasing PEI:DNA ratiolevels, GFP expression did not increase in ratios higher than 1:1 PEI:DNA. Xie et al. have rep release .In several CAR constructs, tonic signalling has ledto prolonged expansion, constitutive cytokine releaseand exhaustion of T cells in the inexistence of targetligand and can be a cause of poor antitumor efficacy-38. Ligainvivo experiments must be performed to assess both efficacy and safety.Taken together, the present study successfully demonstrated transduction of a secondgeneration VHHbased CAR construct into human primary T cells. VHHbased anti-MUC1 redirectedT cells were activated upon recognition of MUC1 expressing cancer cells and showedsignificant cytokine production and cytotoxic activity. This study supported the idea thatVHH-based CARs might be a promising alternative strategy to efficiently target tumour cellsand potentially overcome immunogenic and target-independent signalling impediments observedin scFv-based CARs. Although this study was significant in targeting and cytolysis of MUC1positive cell lines due to VHH-based CAR-T cells specific for TAA-MUC1,


Cochliobolus sativus is one of the major constrains to barley production in warmer regions worldwide. The study was undertaken to identify and estimate effects of loci underlying quantitative resistance to SB at the seedling and adult plant stages. A panel of 261 high input (HI-AM) barley genotypes consisting of released cultivars, advanced breeding lines, and landraces, was screened for resistance to SB. The seedling resistance screening was conducted using two virulent isolates from Morocco (ICSB3 and SB54) while the adult plant stage resistance was evaluated at two hot spot locations, Faizabad and Varanasi, in India under artificial inoculation using a mixture of prevalent virulent isolates. The HI-AM panel was genotyped using DArT-Seq high-throughput genotyping platform. Genome wide association mapping (GWAM) was conducted using 13,182 PAV and 6,311 SNP markers, for seedling and adult plant resistance. Both GLM and MLM model were employed in TASSEL (v 5.0) using principal component analysis and Kinship Matrix as covariates. Final disease rating and Area Under Disease Progress Curve (AUDPC) were used for the evaluation of adult stage plant resistance. The GWAM analysis indicated 23 QTL at the seedling stage (14 for isolate ICSB3 and 9 for isolate SB54), while 15 QTL were detected at the adult plant stage resistance (6 at Faizabad and 9 at Varanasi) and 5 for AUDPC based resistance at Varanasi. Common QTL at seedling and adult plant stages were found across all barley chromosomes. Seedling stage QTL explained together 73.24% of the variance for seedling resistance to isolate ICSB3 and 49.26% for isolate SB54, whereas, QTL for adult plant stage resistance explained together 38.32%, 44.09% and 26.42% of the variance at Faizabad and Varanasi and AUDPC at Varanasi, respectively. Several QTL identified in this study were also reported in previous studies using bi-parental and association mapping populations, corroborating our results. The promising QTL detected at both stages, once validated, can be used for marker assisted selection (MAS) in SB resistance barley breeding program.Barley spot blotch (SB) caused by  Hordeum vulgare L.), also commonly referred as leaf blight, and is caused by Cochliobolus sativus [anamorph: Bipolaris sorokiniana (Sacc.) Shoem.]. It is one of the major concerns in South Asia including China, Nepal, Pakistan, Bangladesh and the humid north eastern regions of India (Spot blotch (SB) of barley  from 0 to 20% and disease severity from 20 to 29%. Barley lines carrying two QTL Rcs-qtl-1H-11_10764 and Rcs-qtl-3H-11_10565 reduced IR from 5 to 31% and disease severity from 52 to 56%. Furthermore, barley lines carrying three QTL Rcs-qtl-1H-11_10764, Rcs-qtl-3H-11_10565, Rcs-qtl-7H-11_20162 showed 47% lower IR and 83% lower disease severity when compared with lines lacking any of three QTL. Similar findings on additive effects of QTL for stripe rust of barley have been reported . The set is named as HI-AM (High Input Association Mapping) panel as most of barley genotypes were targeted toward optimum management  conditions. Out of the 261 genotypes (172 two-row and 89 six-row types), 124 were from ICARDA’s barley breeding program (50 two-row and 74 six-row type), 32 from Europe (28 two-row and 4 six-row type), 34 from North America (28 two-row and 6 six-row type), 67 from South America (62 two-row and 5 six-row type), and 4 from Australia . The full list of genotypes is available in C. sativus isolates under controlled conditions in the growth chamber at the International Center for Agricultural Research in the Dry Areas (ICARDA), Rabat, Morocco. These C. sativus isolates were collected from farmer’s field in Morocco during the disease survey of 2015 and were preserved as mono-conidial isolates in −80°C until further use (C. sativus isolates (ICSB3 and SB54) were classified into pathotypes by using three differential barley cultivars  as described by –1 supplemented with the surfactant (0.01% of Tween 20).The seedling resistance test (SRT) for HI-AM panel was done with two ther use . Two C. About 4–5 seeds of each barley genotype were sown in peat moss in a single cone of 3.8 cm diameter and 14 cm depth  supplemented with 14–14–14 NPK and the seedlings were raised in the growth chamber with photoperiod of 16 h light/8 h dark at 20°C. Each tray containing 96 test genotypes along with resistant (ND B112) and susceptible checks (Annoucer [a Moroccan variety highly susceptible to SB]), was inoculated with 100 ml of spore suspension with hand held sprayers (0.2 ml/seedling) till run off followed by incubation under 100% relative humidity for 24 h in the dark at 20°C. After 24 h, the seedlings were transferred to growth chamber under same conditions as described earlier . The expA disease rating scale of 0–9 , was use5 spores ml–1) of virulent SB isolates grown on sorghum grains at booting stage (GS 43–49) twice during evening hours by using knapsack sprayer  including two standard checks, Rihane-03 and VMorales, was sown in first week of December 2013 at the Agricultural Research Farms of the Banaras Hindu University (BHU), 25.2677°N, 82.9913°E, Varanasi, and at Narendra Dev University of Agriculture and Technology (NDUAT), 26.7732°N, 82.1442°E, Faizabad, both in Uttar Pradesh, India. These genotypes were sown in a 1-m row using augmented block design with a highly susceptible genotype, “RD2503” repeated at interval of 20 test genotypes. RD2503 was selected as a SB susceptible check because it showed highly susceptible reactions  at the seedling stage and 99 score (double-digit score) of SB severity at the adult stages in the field. Further, RD2503 was grown as long paired row perpendicular to the test plots as spreader rows on either side. The SB isolates  were multiplied on sterilized sorghum grains to get enough inoculum. Artificial inoculation was done with a spore mixture , extent of disease severity level  based on the maximum score on genotype as well as based on AUDPC values .adegenet package for R statistical software (The R Development Core Team). Finally, based on principal component analysis (PCA), genotypes were assigned to subgroups or considered admixed on the basis of 80% membership criterion. Linkage disequilibrium (LD) was calculated with TASSEL 5.2.32 . More information regarding genotyping population structure and LD analysis was reported by The 261 genotypes of the HI-AM panel were genotyped with DArT-Seq technology . The final marker sets  were obtained by removing heterozygous and monomorphic markers and markers with minor allele frequencies (MAF) < 5% and markers with missing data > 10%. Markers distribution across the seven barley chromosome is shown in L 5.2.32 . The ext2 values  using nl10p ≥ 3) was set for identifying significant marker-trait associations. Significant markers mapping within the interval of LD decay were considered as being linked to the same QTL and the marker with the highest p-value was chosen as representing the QTL. Considering the stringency of the model used for accounting population structure, in which most of the false positives were inherently controlled. The critical p-value for marker-trait association was firstly determined according to a liberal approach proposed by p = 0.0001 [−log(p) = 4] with the selected models (p) (p < 0.05).Genome wide association mapping was performed combining genotypic data and disease severity scores at the seedling and adult plant stages. Genome scans were performed using both General Linear Model (GLM) and Mixed Linear Model (MLM), the general equations for GLM and MLM were reported by d models . A furthd models , corresp1. The position of the marker representative of the QTL was compared with those of markers at QTL peaks reported in previous studies and considered adjacent on the base of LD value . Molecular markers sequences were aligned to the barley physical genome2. Putative candidate genes were then identified searching within the genes aligned and located within the LD interval at both sites of the markers at QTL peaks using PGSB database (Plant Genome System Biology3). The database provides access to the barley gene annotation described by the QTL detected for SB resistance were aligned with those previously reported in different barley germplasm by checking the position of markers at the QTL peak in the barley pseudomolecules Morex V.2.0 database. Markers sequences were mapped in the database using the IPK Barley Blast ServerIn the greenhouse, SB infection was uniform and reliable infection responses (IR) were recorded. The frequency distribution of IR HI-AM panel (261 genotypes) at the seedling stage has been presented in Interestingly, 9 genotypes (3.5%) were resistant (IR 1-3) to the isolate ICSB3, whereas, 18 genotypes (7%) were resistant to SB54. While 93 genotypes (36%) were moderately resistant (IR 4-5) to the isolate ICSB3, and 76 genotypes (29%) were moderately resistant to the isolate SB54 .The frequency distribution of SB severity of the HI-AM at BHU-14 (Varanasi) and NDUAT-14 (Faizabad) are presented in At BHU-14, higher disease severity of 74 ± 15 (mean ± SD) was recorded than at NDUAT-14 (55 ± 11). Furthermore, overall SB disease severity in NDUAT-14 was slightly higher in two-row barley (56.9 ± 9.9) than in six-row types (50.6 ± 10.7). But in BHU-14, the overall SB disease severity in six-row was slightly higher (75.5 ± 14.2) than two-row types (72.5 ± 15.6). Similarly, in BHU-15-AUDPC, the overall AUDPC value of 399 ± 176 was observed. The AUDPC for six-row was significantly higher (425.8 ± 175.4) than in two-row types (385.3 ± 175.6). Only three barley genotypes, HI-AM-3 , HI-AM-241 , and HI-AM-250  were found resistant and/or moderately resistant across three locations in two cropping seasons.R2 for isolate SB54 ranged from 4.53% to 6.82% and the total phenotypic variance explained by 9 QTL was 49.26%. The GWAM analyses at SRT for ICSB3 identified 14 QTL located on chromosomes 1H, 3H, 4H, 6H and 7H  overlaps with a QTL for APS located on the same chromosome at 12.75 cM (APS_Var_9).Performing GWAM for SRT to SB, using both PAVs and SNPs markers sets, the best fitting model for NDUAT-14 (Faizabad) and BHU-14 (Varanasi) was the MLM procedure using PCA + K for accounting for population structure and relatedness. When the data from BHU-15-AUDPC (Varanasi) was used for GWAM the best fitting models were MLM Q + K for the PAVs and MLM PCA + K for the SNPs marker sets, respectively. GWAM for APS showed a total of 15 QTL using disease severity data from two locations NDUAT-14 and BHU-14 and 5 QTL in BHU-15-AUDPC (Varanasi) by using AUDPC values. At BHU-14 (Varanasi) 9 QTL were located on chromosomes 2H, 3H, 4H, 5H, and 7H with marker variance . At NDUAvariance . QQ plotvariance . OverlapSRT-ICSB-12 overlaps with a QTL detected in the same panel for stripe rust and reported in SRT-SB54-6 was already reported in a previous studies (chromosome 4H 104 cM) using different isolates and genotypes with different genetic background  was already reported at both SRT using different germplasm and different isolates  detected in BHU-Varanasi in 2014 were already reported by APS-AUDPC-1 (4H 20 cM), APS-AUDPC-3 (5H 82 cM) and APS-AUDPC-4 (7H 127 cM) were earlier reported by Out of the 15 QTL for SB resistance at SRT for isolate ICSB3, four were coincident with prior reports and those QTL were identified at both SRT and APS stages using different germplasm and different isolates . Furtherckground , while Qme panel . For APSisolates . QTL APSAPS-Fai-3 detected at NDUAT-14, APS-Var14-1, APS-Var14-2 and APS-Var14-8 detected at BHU-14 and APS-AUDPC-2 at BHU-AUDPC-15  and pathotype 7 (ICSB3) to map loci conferring seedling resistance in a diverse barley germplasm from ICARDA, adapted specifically to the high input conditions. SB was not reported in Morocco until recently our group found this disease during disease survey in 2015 (unpublished data) Net Form of Net Blotch (NFNB) and Spot Form of Net Blotch (SFNB) have been prevalent with disease incidence up to 70% and disease severity from 40 to 90%, respectively . To our C. sativus isolates from Morocco  genotypes showed resistance reaction to isolate SB54 and 7 genotypes (2.7%) were resistant to isolate ICSB3 . However Morocco . This ca Morocco .Spot blotch is also a major constraint for barley production in South Asian countries like China, Nepal, India, Bangladesh, and Pakistan due to hot and humid climate prevailing during February to March . More spSRT-ICSB3-1) and 120 cM (SRT-ICSB3-2). SRT-ICSB3-1 was already reported in SRT by SRT-ICSB3-3 (2 cM), SRT-ICSB3-4 (67 cM), and SRT-ICSB3-5 (133 cM) explained a total of 18% phenotypic variation. All identified QTL remained to be novel.For pathotype 7 isolate (ICSB3), chromosome 1H harbors two SRT QTL on 45 cM (SRT-ICSB3-7 (17 cM), SRT-ICSB3-8 (25 cM), and SRT-ICSB3-9 (93 cM) explained an overall phenotypic variation of 15.76%. The QTL SRT-ICSB3-9 (93 cM) was already reported in seedling resistance to diverse SB isolates by SRT-ICSB3-10 (3 cM), SRT-ICSB3-11 (10 cM) and SRT-ICSB3-12 (116 cM) explained 15% phenotypic variation against ICSB3 isolate. Further, two SRT QTL SRT-ICSB3-11 (10 cM) and SRT-ICSB3-12 (116 cM) have been reported to be involved in seedling resistance to different SB isolates and in diverse barley germplasm in two different studies by SRT-ICSB3-12 also overlaps with a QTL for stripe rust resistance reported by On chromosome 6H, three SRT QTL SRT-SB54-3 (2 cM), and SRT-SB54-4 (133 cM) explained together the 12.75% of phenotypic variation. Furthermore on chromosome 4H, SRT-SB54-5 (68 cM), overlaps with a previous QTL mapped for stripe rust resistance by Visioni et al., 201, while SRT-SB54-6 (104 cM) had also shown to be involved in SRT (SRT-SB54-7 (17 cM) on chromosome 6H and on chromosome 7H SRT-SB54-8 (116 cM), to the best of our knowledge have never been reported. SRT-SB54-8 also overlaps with a previous QTL mapped by For pathotype 3 isolate (SB54), two novel QTL located on chromosome 3H two d in SRT . The sinSRT-ICSB3-7, SRT-SB54-7), all three SRT QTL on chromosome 3 and 7H showed negative effect, thus conditioning resistance response to both SB isolates tested.Common SRT QTL to pathotype 3 (SB54) and pathotype 7 (ICSB3) were identified in this study. Two SRT QTL on 3H (2 and 133 cM), one on 6H (17 cM) and one on 7H (116 cM) explained a total phenotypic variation of 24.64% to ICSB3 and 22.83% to SB54, respectively. Strikingly, except SRT QTL on chromosome 6H (APS-Fai-1) at 87 cM in NDUAT-14 showed the highest R2 explaining 9.85% of the variance. APS-Fai-1 was already reported to be involved in seedling resistance (APS-Fai-3 (146.72 cM), and two detected at BHU-14, APS-Vars-14-1 (40.08 cM) QTL APS-Vars-14-2 (94.72 cM) were already reported for stripe rust resistance in the same association mapping panel by In case of adult plant stage resistance, one QTL on 1H (sistance . In addiAPS-Var-14-3 (83.07 cM) and APS-Var-14-4 (128 cM) were found and both have been reported to be involved in SRT to SB (APS-Fai-4 (1.27 cM) at NDUAT-14, APS-Var-14-5 (79.76 cM) at BHU-14, and APS-AUDPC-1 (20 cM) and APS-AUDPC-2 (112 cM) at BHU-AUDPC-15. Only one QTL APS-AUDPC-1 (20 cM) has been reported to be involved in APR by APS-Var-14-6 (35.1 cM), APS-Var-14-7 (41.56 cM) and APS-Var-14-8 (137.22 cM). Moreover, BHU-AUDPC-15 contributed with one more QTL on 5H, APS-AUDPC-3 (82 cM) that was already reported by APS-Var-14-8 was already associated to stripe rust resistance and showed to be stable across years and environments (APS-Fai-5 (30 cM), and APS-Fai-6 (100.42 cM) were detected. Whereas, on chromosome 7H two QTL were found; one at BHU-14, APS-Var-14-9 (12.75 cM), the second QTL was detected at BHU-15 (APS-AUDPC-415-4 at 127 cM), the first remained to be novel while the second was already reported by Tamang et al. (2015)No QTL were found at NDUAT-14 on chromosome 3H while at BHU-14, two APR QTL RT to SB . Chromosronments . At NDUASRT-ICSB3-11) and APR QTL (APS-Var-14-9) are located on chromosome 7H at 10 and 12.75 cM, respectively, and hence could represent the same QTL.Only one common QTL could be detected on chromosome 3H to ICSB3 (133 cM) and SB54 (133 cM) at SRT, and at APR in BHU-14 (128 cM). Furthermore, both SRT , pathogenesis related proteins , and MYB transcription factors . NB-LRR disease resistance proteins have been implicated in effector triggered immunity to various pathogens and a similar role to SB resistance is envisaged here. Nucleotide-binding (NB)-LRR (leucine rich repeat) proteins (NLRs) have been associated with quantitative resistance to necrotrophs. A combination of transcriptomics and association mapping of pathogen or hosts will result in the identification of novel necrotrophic effectors (NEs) and corresponding QTL, respectively. A probable strategy would be to eliminate host plant susceptibility genes for both biotrophic and necrotrophic pathogens. Furthermore, minor R genes (APR) could be pyramided for durable control of diverse pathogens .SRT_ICSB3_6) on 60 cM (1H) and DArT9658 marker (APS_Var14-8) on 137.22 cM of chromosome 5H is a glucan endo-1,3-beta-glucosidase-4 like protein and it has more than 90% amino acid identity with orthologs from rice, maize, sorghum, wheat. The glucan endo-1,3-beta-glucosidase belongs to the family of PR proteins which are induced upon pathogen ingress. These beta-1,3-glucanases targets -beta-D-glucosidic linkages of glucans present in fungal cell wall and enhance fungal resistance in crop plants  and one APR QTL  are closely associated with MYB transcription factors. Further, MYB transcription factor (MYB15) was implicated as a regulator of defense-induced lignification and basal immunity to bacterial pathogen Pseudomonas syringae  could be a potential candidate for MAS.The CG associated with the peak marker DArT6694 QTL (p plants . Similarsyringae . In addisyringae . DependiAPS_Vars_14-1) on 2H at 40.08 cM encodes CsAtPR5 pathogenesis- related (PR) protein. PR proteins are conserved in many plant species and are induced upon biotic stresses conditioned by various pathogens  associated with the DArT2274 marker (athogens . The wheant line . Likewises teres .One two-row barley genotype HI-AM-32 (Issaria) was recorded as resistant and two six-row barley genotypes, HI-AM-241 (ZIGZIG/BLLU//PETUNIA 1) and HI-AM-250 (M104/TOCTE), were found as moderately resistant across two locations during two cropping seasons. The present study has further unlocked the genetic potential of HI-AM with the identification of 15 novel QTL for SRT and 14 novel QTL for APR. Furthermore, 11 previously mapped QTL were also identified (5 for SRT and 6 for APR). Markers at QTL peak will enrich the existing allelic diversity for SB resistance and once validated, could be used for MAS to pyramid multiple resistance alleles to curb losses induced by this economically important pathogen of barley. The three lines observed as resistant/moderately resistant across the three environments can be readily utilized in barley breeding program for incorporation of effective SB resistance targeted for South Asia and North Africa.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below:https://mel.cgiar.org/reporting/report/id/5949/del_id/20282https://hdl.handle.net/20.500.11766.1/FK2/R2AW6A orhttps://dx.doi.org/20.500.11766.1/FK2/R2AW6Ahttps://hdl.handle.net/20.500.11766.1/FK2/QRXB0W orhttps://dx.doi.org/20.500.11766.1/FK2/QRXB0WSR, AV, and RV conceived and coordinated the study. AV performed statistical and bioinformatics analysis. SR, SSV, SS, and RPSV collected the phenotypic data. SR, AV, RV, SG, and AA-A reviewed and contributed to the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The SGK1 inhibitor enhances the cytotoxic effects of bortezomib and ixazomib; however, FGFR1 and CCR2 inhibitors do not show such effect in KMS-20 cells. Moreover, SGK1 activation induces the phosphorylation of NF-κB p65, and an NF-κB inhibitor enhances the sensitivity of KMS-20 cells to bortezomib and ixazomib. Additionally, high levels of expression of SGK1 and NF-κB p65 is associated with a low sensitivity to bortezomib and a poor prognosis in MM patients. These results indicate that the activation of the SGK1/NF-κB pathway correlates with a low sensitivity to bortezomib and ixazomib, and a combination of bortezomib and ixazomib with an SGK1 or NF-κB inhibitor may be involved in the treatment of MM via activation of the SGK1/NF-κB pathway.Multiple myeloma (MM) is an incurable malignancy often associated with primary and acquired resistance to therapeutic agents, such as proteasome inhibitors. However, the mechanisms underlying the proteasome inhibitor resistance are poorly understood. Here, we elucidate the mechanism of primary resistance to bortezomib and ixazomib in the MM cell lines, KMS-20, KMS-26, and KMS-28BM. We find that low bortezomib and ixazomib concentrations induce cell death in KMS-26 and KMS-28BM cells. However, high bortezomib and ixazomib concentrations induce cell death only in KMS-20 cells. During Gene Expression Omnibus analysis, KMS-20 cells exhibit high levels of expression of various genes, including  Multiple myeloma (MM), the second most common hematological cancer, is defined as the monoclonal proliferation of plasma cells in bone marrow and the discharge of monoclonal immunoglobulins . The incBortezomib, the first proteasome inhibitor, binds to the caspase- and chymotrypsin-like active sites of the 20S proteasome, suppresses proteasome activity, and induces apoptosis associated with the endoplasmic reticulum (ER) stress response ,5. IxazoSerum and glucocorticoid regulated kinase 1 (SGK1) are family members of cyclic adenosine monophosphate (cAMP)-dependent, cyclic guanosine monophosphate (cGMP)-dependent, and protein kinase C (AGC) kinases, and regulates ion channels, transporters, transcription factors, and enzymes . SGK1 isHere, we investigate the mechanism underlying the primary resistance to bortezomib and ixazomib (low sensitivity to bortezomib and ixazomib) and aim to determine whether alternative small-molecule inhibitors can be used to enhance the cytotoxicity of bortezomib and ixazomib to MM cells.Bortezomib, ixazomib, GSK650394, and PD166866 were purchased from SelleckChem . A C-C chemokine receptor type 2 (CCR2) antagonist was obtained from Santa Cruz Biotechnology . Dimethyl fumarate (DMF) was purchased from FUJIFILM Wako . These reagents were dissolved in dimethyl sulfoxide (DMSO) and diluted in phosphate-buffered saline .2.KMS-20, KMS-26, and KMS-28BM cells were obtained from the Japanese Collection of Research Bioresources Cell Bank . These cells were maintained in RPMI 1640 medium  supplemented with 25 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid , 100 U/mL streptomycin , 100 μg/mL penicillin (Gibco), and 10% fetal bovine serum (Gibco) in an atmosphere of 5% COThe effect of bortezomib, ixazomib, GSK650394, PD166866, C-C chemokine receptor type 2 (CCR2) antagonist, and dimethyl fumarate (DMF) on cell viability was assessed by the trypan blue stain exclusion assay as previously described ,22,23,24Cellular cytoplasmic and nuclear fractions were extracted using the ProteoExtract Subcellular Proteome Extraction Kit . These fractions were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes . The membranes were incubated with the following primary antibodies: anti-proteasome 20S subunit β5 (PSMB5) antibodies , anti-phospho- serum and glucocorticoid regulated kinase (SGK)1 antibodies, anti-SGK1 antibodies, anti-phospho-fibroblast growth factor receptor 1 (FGFR1) antibodies, anti-FGFR1 antibodies, anti- chemokine receptor type (CCR)2 antibodies, anti-phospho-extracellular regulated protein kinase 1/2 (ERK1/2) (Thr202/Tyr204) antibodies, anti-ERK1/2 antibodies, anti-phospho-nuclear factor (NF)-κB antibodies (Ser536), anti-NF-κB antibodies, anti-phospho-Akt antibodies (Ser473), anti-Akt antibodies, anti-phospho-c-Jun N-terminal kinase (JNK) antibodies (Thr183/Tyr185), anti-JNK antibodies, anti-survivin antibodies, anti-X-linked inhibitor of apoptosis protein (XIAP) antibodies , anti-B-cell lymphoma-2 (Bcl-2)-associated X (Bax) antibodies, anti-Bcl-2-like protein 11 (Bim) antibodies, anti-Bcl-2 antibodies, anti-B-cell lymphoma-extra large (Bcl-xL) antibodies, anti-phorbol-12-myristate-13-acetate-induced protein 1 (Noxa) antibodies, anti-p53 upregulated modulator of apoptosis (Puma) antibodies, anti-lamin A/C antibodies (Santa Cruz Biotechnology), and anti-β-actin antibodies (Sigma). The membranes were then treated with horseradish peroxidase-coupled sheep anti-rabbit immunoglobulin G  and were visualized using Luminata Forte .Total RNA was extracted using RNAiso  and complementary deoxyribonucleic acid (cDNA) was synthesized from purified total RNA using the PrimeScript RT Master Mix . cDNA was subjected to quantitative real-time Polymerase Chain Reaction (PCR) using SYBR Premix Ex Taq  and the Thermal Cycler Dice Real-time System . The PCR conditions for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), anti-phospho-fibroblast growth factor receptor 1 (FGFR1), C-C chemokine receptor type 2 (CCR2), and anti-phospho- serum and glucocorticoid regulated kinase (SGK1) were: 94 °C for 2 min; followed by 40 cycles at 94 °C for 0.5 min, 50 °C for 0.5 min, and 72 °C for 0.5 min. The following primers were used: FGFR1, 5′- CTT CGT TTC TTG GTA TGC -3′ (5′-primer) and 5′- GGA CAG GAT GGA GTT TGG AC -3′ (3′-primer); CCR2, 5′- CTG TGT TTG CTT CTG TCC -3′ (5′-primer) and 5′- CCC TAT GCC TCT TCT C -3′ (3′-primer); SGK1, 5′- AGG ATG GGT CTG AAC GAC TTT -3′ (5′-primer) and 5′- GCC CTT TCC GAT CAC TTT CAA G -3′ (3′-primer); and GAPDH, 5′-ACT TTG TCA AGC TCA TTT-3′ (5′-primer) and 5′-TGC AGC GAA CTT TAT TG-3′ (3′-primer). The expression of FGFR1, CCR2, and SGK1 in KMS-26, KMS-28BM, and KMS-20 cells was analyzed as previously described ,26,27.Proteasome β5 subunit activity was assessed as described previously .Bortezomib- and ixazomib-induced autophagy was assessed using the Muse™ Cell Analyzer and Muse™ Autophagy LC3-antibody based Kit . The autophagy induction ratios were analyzed for bortezomib- and ixazomib-treated cells versus control cells (0.1% dimethyl sulfoxide (DMSO)-treated cells).TM (Merck Millipore), using the following antibodies: anti-phospho-mammalian target of rapamycin (mTOR) (Ser2448), anti-phospho-Akt (Ser473), anti-phospho-c-Jun N-terminal kinase (JNK) (Thr183/Tyr185), anti-phospho-extracellular regulated protein kinase 1/2 (ERK1/2) (Thr185/Tyr187), anti-phospho-signal transducer and activator of transcription 3 (STAT3) (Tyr705), anti-phospho-p38 mitogen-activated protein kinase (p38MAPK) (Thr180/Tyr182), and anti-phospho-nuclear factor (NF)-κB (Ser536) (Merck Millipore).Phosphorylated signaling protein expression was analyzed using a multiplex kit, Millipore Luminex 200http://www.ncbi.nlm.nih.gov/geo/). The expression of various genes in KMS-26, KMS-28BM, and KMS-20 cells was analyzed from GSE6205 and the expression of anti-phospho- serum and glucocorticoid regulated kinase (SGK)1 and nuclear factor (NF)-κB p65, and the overall survival rate of patients with multiple myeloma (MM) were analyzed from GSE9782.The gene expression profiles of the microarray datasets with the accession numbers GSE6205 and GSE9782 were obtained from the National Center of Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database  based on the method described by Chou and Talalay [All results are represented as the means and standard deviations (SDs) of several independent experiments. All analyses were conducted using SPSS version 21.0 software , and Shapiro-Wilk analysis and one-way analysis of variance (ANOVA) were performed. When no differences in the Shapiro-Wilk test and satisfactory differences in ANOVA were confirmed, the data from the control group and various drug-treated groups were compared and analyzed using Dunnett’s test. When our data did not show normal distribution, they were analyzed using the Kruskal-Wallis test followed by the Steel test. Survival rates were assessed using Kaplan-Meier curves and long-rank analysis.  Talalay . A CI vap < 0.05) and ixazomib  induced KMS-20 cell death, low concentrations of bortezomib  and ixazomib  significantly induced KMS-26 and KMS-28BM cell death (p < 0.05) (We investigated the cytotoxic effect of bortezomib (1–200 nM) and ixazomib (1–500 nM) on the KMS-20, KMS-26, and KMS-28BM cell lines. Although high concentrations of bortezomib  C. These p < 0.05)  A–C. Trea < 0.05) D,E.To reveal differences in gene expression between KMS-20 cells and KMS-26 or KMS-28BM cells, we used the publicly available gene expression profiling (GEP) dataset, GSE6205. These analyses revealed that the expression of several genes was elevated only in KMS-20 cells. Above all, we focused on three genes; anti-phospho-fibroblast growth factor receptor 1 (FGFR1), a member of the growth factor receptor tyrosine kinase family; C-C chemokine receptor type 2 (CCR2); and anti-phospho- serum and glucocorticoid regulated kinase (SGK1), a member of the serine/threonine kinase family . The FGFp < 0.05)  C–E. Addi < 0.05) . Moreove < 0.05) . These rp < 0.01)  F,G. Thusp < 0.01) (p < 0.01) (It has been indicated that activation of serum/glucocorticoid regulated kinase 1 (SGK1) promotes nuclear factor (NF)-κB and phospho-mammalian target of rapamycin (mTOR) activation and suppresses phospho- anti-phospho-c-Jun N-terminal kinase (JNK) activation ,39,40. I < 0.01) A,B. The  < 0.01) A. The ac < 0.01) A. Next,  < 0.01) C. Additi < 0.01) . Recentl < 0.01) . It has  < 0.01) D. Moreov < 0.01) . These rp < 0.01)  using the GSE9782 dataset. High expression of NF-κB p65 in patients with MM led to a low sensitivity to bortezomib and a shorter overall survival than low expression of NF-κB p65 in patients ( < 0.01) E,F. Thusp < 0.05) (Our results suggest that a low sensitivity to bortezomib and ixazomib is correlated with the activation of nuclear factor (NF)-κB by serum/glucocorticoid regulated kinase 1 (SGK1). We examined whether dimethyl fumarate (DMF), an NF-κB inhibitor, enhances the sensitivity of KMS-20 cells to bortezomib and ixazomib. The combination of DMF and bortezomib or ixazomib enhanced the sensitivity of KMS-20 cells to bortezomib and ixazomib ( < 0.05) . Additio < 0.05) .To validate these observations, we confirmed the sensitivity to bortezomib and ixazomib in other multiple myeloma (MM) cells. We found that L363 had a lower sensitivity to bortezomib and ixazomib, similar to that of KMS-20 cells, but ARH-77 and RPMI8226 cells showed a high sensitivity to bortezomib and ixazomib, which was similar to that of the KMS-26 and KMS-28BM cells . AdditioHere, KMS-20 cells presented a lower sensitivity to bortezomib and ixazomib than KMS-26 and KMS-28BM cells. Although proteasome β5 subunit overexpression and the induction of autophagy by a proteasome inhibitor have been associated with resistance in multiple myeloma (MM) cells ,46,47, oTo ascertain the mechanism underlying the low sensitivity to bortezomib and ixazomib, we analyzed the gene expression profiling (GEP) database to reveal the genes expressed in KMS-26 or KMS-28BM cells, compared to those in KMS-20 cells. Considering the possible resistance genes revealed in this study, fibroblast growth factor receptor 1 (FGFR1), C-C chemokine receptor type 2 (CCR2), and serum/glucocorticoid regulated kinase 1 (SGK1) were overexpressed in KMS-20 cells. FGFR1 contributes to tyrosine kinase inhibitor resistance and chemoresistance in lung, breast, and urothelial cancers ,49,50. TAlthough it is known that the activation of SGK1 accelerates the phosphorylation of N-myc downstream regulated gene 1 and activation of murine double minute 2, the effects of signaling crosstalk is unknown . The expIt has been demonstrated previously that SGK1 activation is involved in acquired bortezomib resistance in MM cells. Also, SGK1 inhibition by bortezomib or short hairpin RNA (shRNA) has been shown to enhance its cytotoxicity via activation of the JNK pathway and induction of endoplasmic reticulum (ER) stress . During It has previously been indicated that induction of SGK1 expression was regulated via activation of the JAK/STAT3, MEK/ERK, PI3K/Akt, or p38MAPK pathways ,41,42. HTo conclude, both the overexpression and activation of the serum/glucocorticoid regulated kinase 1 (SGK1)/Nuclear Factor (NF)-κB pathway are involved with the low sensitivity of multiple myeloma (MM) cells to bortezomib and ixazomib, and SGK1 or NF-κB inhibitors can be used to increase the cytotoxic effects of bortezomib and ixazomib. These results indicate that the combination of an SGK1 or NF-κB inhibitor and bortezomib or ixazomib may be a potential therapy for MM harboring the overexpression and activation of the SGK1/NF-κB pathway.


Scientific Reports 10.1038/s41598-020-57452-6, published online 17 January 2020Correction to: The Supplementary info2 file originally published with this Article was incorrectly provided as a LaTeX class file. The correct Supplementary info2 file now accompanies the Article.