Chronic HIV-1 infection preferentially depleted CD34+CD38- early HPCs in the BM and reduced their proliferation potential in vivo in both HIV-1-infected patients and humanized mice, while CD34+CD38+ intermediate HSCs were relatively unaffected. Strikingly, depletion of plasmacytoid dendritic cells (pDCs) prevented human CD34+CD38- early HPCs from HIV-1 infection-induced depletion and functional impairment and restored the gene expression profile of purified CD34+ HPCs in humanized mice. These findings suggest that pDCs contribute to the early hematopoietic suppression induced by chronic HIV-1 infection and provide a novel therapeutic target for the hematopoiesis suppression in HIV-1 patients.Chronic human immunodeficiency virus-1 (HIV-1) infection in patients leads to multi-lineage hematopoietic abnormalities or pancytopenia. The deficiency in hematopoietic progenitor cells (HPCs) induced by HIV-1 infection has been proposed, but the relevant mechanisms are poorly understood. We report here that both human CD34 in vivo. Our study clarifies a novel mechanism underlying hemato-suppression induced by chronic HIV-1 infection and provides a novel strategy to halt HIV-1 disease.Multi-lineage hematopoietic abnormalities generally occur during chronic infection which results in a disorder of human leukocyte development and differentiation, contributing to human immunodeficiency virus-1 (HIV-1)-infection induced immune-pathogenesis in AIDS patients. Although successful antiretroviral therapy can reduce plasma viral loads to undetectable levels and ameliorate HIV-1-associated hemato-suppression, immune cell development is only partially restored. The mechanism for the abnormal hematopoiesis occurring during chronic HIV-1 infection remains unclear. HIV-1 infection may directly or indirectly functionally impair hematopoietic progenitors by either viral products or induction of persistent inflammatory responses, leading to hematopoiesis obstacles. Here, we show that HIV-1 infection significantly depleted and functionally impaired human hematopoietic progenitors in the bone marrow of both HIV-1-infected patients and humanized mice through a plasmacytoid dendritic cell (pDC)-dependent mechanism, as depletion of pDCs significantly recovered cell numbers and functions and gene expression profiles of hematopoietic progenitor cells in humanized mice  Tissues were digested with liver digest medium . The suspension was filtered through a 70-μm cell strainer  and centrifuged at 150 × g for 5 minutes to isolate mononuclear cells by Ficoll. After selection with the CD34+ magnetic-activated cell sorting (MACS) kit, CD34+ HPCs (0.5 × 106) were injected into the liver of each 2- to 6-day-old DKO or NRG mice, which had been previously irradiated at 300 rad. More than 95% of the humanized mice were stably reconstituted with human leukocytes in the blood (60–90% at 12–14 weeks). Each cohort had similar levels of engraftment. All mice were housed at the University of North Carolina at Chapel Hill.We constructed humanized Balb/c reported . BrieflyAn R5-tropic strain of HIV-1, JR-CSF, was used for chronic HIV-1 infection. All viruses were generated by transfection of 293 T cells  with pYK-JRCSF . Humanized mice with stable human leukocyte reconstitution were infected with JR-CSF at a dose of 10 ng p24/mouse, through intravenous (i.v.) injection. Humanized mice infected with 293 T mock supernatant were used in control groups . Viral gA monoclonal antibody specific to blood dendritic cell antigen-2 (BDCA2), 15B, was used to treat humanized mice through intra-peritoneal (i.p.) injection (4 mg/kg) as previously reported . BrieflyFor chronic JR-CSF infection, mice were terminated at 12 week post-infection. On termination, total leukocytes were isolated from mouse lymphoid organs as previously described ,34–36. L-mCD45-hCD45+) were analyzed for HPC subsets or phenotypic expression by using the CyAn FACS instrument (Dako). Live/dead fixable violet dead cell dye (LD7) was purchased from Molecular Probes . For intracellular p24 staining, freshly isolated cells were collected for surface staining, followed by cell permeabilization using a Cytofix/Cytoperm kit (BD Bioscience) and intracellular staining and washing. The data were analyzed using Summit Software.Surface and intracellular fluorochrome-conjugated antibodies or reagents from Biolegend, BD Bioscience, eBioscience and R&D Systems were used in this study. For humanized mice, live human leukocytes  was first dissolved in water at a concentration of 10 mg/mL for stock in -20°C. The BrdU stock was then diluted in 200 μL PBS and injected i.p. at 100 mg/kg body weight. Four hours later, the mice were terminated, and BM cells were collected. BrdU staining was performed according to the manufacturer’s instructions. In brief, cells were first stained for surface markers and then incubated with a working solution of the BrdU staining buffer for 15 minutes, followed by incubation with DNase I  for 1 hour at 37°C in the dark. Thereafter, the cells were stained with the FITC-conjugated anti-BrdU antibody for 30 minutes at room temperature in the dark and subsequently washed. The data were analyzed using Summit Software.+ selection kit  was used to isolate CD34+ cells from frozen BM cells. The purity of CD34+ cells was greater than 90%. The CD34+ cells were then counted and seeded in complete methylcellulose  at a concentration of 500 cells/mL and plated in 35-mm grid plates, 1 mL/plate, in triplicate per mouse according to the manufacturer’s instructions. Colonies were counted 2 weeks later in a blinded fashion using a QImaging Micropublisher 3.3 CCD digital camera and QCapture software version 3.0 .The EasySep human CD34+ cells sorting. Cells were stained with human CD45, mouse CD45 and 7-Aminoactinomycin D (7-AAD). For human CD34+ HSC sorting, anti-lineage  and anti-CD34 antibodies were added to the antibody mix. Cell sorting was performed by the UNC Flow Cytometry Core.BM cells were pooled by mouse groups for human CD45+ cells were isolated from human fetal liver tissues. Then the cells were cultured in StemSpan SFEM medium (Stem Cell Technologies) with heparin , recombinant human SCF , thrombopoietin  and CHIR99021  for 48 hours in the presence of IFN-α or IFN-β at the dose of 20 IU/ml and 200 IU/ml, respectively. The cells were counted and collected for the detection of CD38 expression on HPCs.CD34RNA purification was carried out using the RNeasy Plus Mini Kit  according to the manufacturer’s instructions. DNase (QIAGEN) treatment was added to the column to eliminate any potential DNA contamination during RNA preparations. Total RNA was checked for quantity, purity and integrity by capillary electrophoresis. RNA was amplified with Cy3- and Cy5-labeled CTP in separate reactions to produce differentially labeled samples and reference cDNAs. Total RNA (200 ng to 400 ng) was used as the starting material to prepare cDNA. Both samples were hybridized to the same microarray (UNC Genomic and Bioinformatics Core) using SurePrint G3 Human Gene Expression 8 ◊ 60K Microarray Kit (Agilent). Agilent Feature Extraction v18 software was used to analyze all images. Gene expression values were quantified by the log2 ratio of the red channel intensity (mean) vs. green channel intensity (mean), followed by LOWESS normalization to remove the intensity-dependent dye bias.P values < 0.05.Data were analyzed using GraphPad Prism software version 5.0 . Data from different cohorts of mice were compared using a 2-tailed unpaired T test. All results were considered significant for S1 Table(DOCX)Click here for additional data file.S1 Fig+ cells, the lineage-CD34+ cells that remained were identified as total HPCs. Based on CD38 expression, HPCs were further divided into CD38- early and CD38+ intermediate HPC subpopulations. The lineage markers included CD3, CD14, CD16, CD19, CD20 and CD56.After gating on lymphocytes (FSC-SSC), singlets and live human CD45(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigCFU-GM, colony-forming unit-granulocyte, macrophage. CFU-E, colony-forming unit-erythroid. CFU-GEMM, colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte.(TIF)Click here for additional data file.S4 Fig+CD38- early HPC proportion (C), CD34+CD38+ intermediate HPC proportion (D), BM pDC percentage (E), plasma IFN-α level (F) and plasma HIV-1 load (G) between rescued (n = 7) and non-rescued (n = 6) groups in humanized mice with HIV-1 infection after pDC depletion. Mice with less than the median percentage of CD34+CD38- HPCs were defined as the non-rescued group (n = 6), while others were defined as the rescued group (n = 7). (H) Correlation analysis between the percentage of CD34+CD38- HPCs and the percentage of pDCs among CD45+ cells in BM of HIV-1 infected humanized mice with pDC depletion (Spearman correlation test). r, correlation coefficient; P values are shown.(A) Plasma HIV-1 load at the termination of humanized mice with HIV-1 infection with or without pDC depletion. (B) Depletion of pDCs did not influence the CD38 expression and BrdU expression on HPCs in the BM from humanized mice  in the absence of HIV-1 infection. (C-G) Differences in CD34(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig+Lin-CD34+ cells from HIV-1-infected mice treated with IgG and 15B relative to mock samples . (B) Table showing relative expression levels of ISGs in HIV-1-infected mice treated with IgG and 15B relative to mock mice.(A) Heat map showing the relative expression of ISGs indicated by the color bars in huCD45(TIF)Click here for additional data file.S7 Fig+Lin-CD34+ cells from HIV-1-infected mice over mock samples . (B) Table showing numbers of significantly up-regulated or down-regulated genes in the HPC pathway in HIV-1-infected mice treated with IgG and 15B relative to mock mice. Δ change, D-value between HIV-1+IgG and HIV-1+15B. (C) Fold changes in expression of 88 genes from hematopoietic cell lineage in HIV-1-infected mice with or without pDC depletion in relation to mock mice.(A) Heat map showing relative expression of 88 genes in the hematopoietic cell lineage pathway indicated by the color bars in huCD45(TIF)Click here for additional data file.S8 Fig+ cells from the spleen of HIV-1-infected humanized mice over mock samples . (B) Table showing the number of genes significantly up-regulated and down-regulated in splenic CD45+ cells from HIV-1-infected humanized mice with or without pDC depletion. (C) Table showing the KEGG pathway analysis of the top 8 most altered pathways in splenic CD45+ cells from HIV-1-infected humanized mice relative to mock samples. (D) Heat map showing the relative gene expression of the SLE pathway indicated by the colored bars in splenic huCD45+ cells of HIV-1-infected humanized mice over mock samples . (E) Table showing numbers of significantly up-regulated or down-regulated genes in SLE pathway in HIV-1-infected mice with IgG and 15B relative to mock mice. Δ change, D-value between HIV-1+IgG minus HIV-1+15B. (F) Fold changes of 88 genes from SLE pathway in HIV-1-infected mice with or without pDC depletion in relation to mock mice.(A) Heat map showing the relative gene expression indicated by the colored bars in huCD45(TIF)Click here for additional data file.S9 Fig+ fetal liver-derived HPCs in vitro of 24- and 48-hours in the presence of IFN-α and IFN-β at 20 IU/ml and 200 IU/ml doses. The numbers in (A) indicated that the CD38 percentages on HPCs. * p < 0.05. (C) Pool data indicated the cell counts of CD34+ HPCs in vitro after 24- and 48-hour culture in the presence of IFN-I (n = 3).(A and B) Representative dot plots (A) and pool data  indicated CD38 expression on purified CD34(TIF)Click here for additional data file.S10 Fig+CD38- HPCs and dysregulates gene expression profile in HPCs leading to their depletion and functional impairment. This effect on early HSCs subsequently contributes to HIV-1-induced pathogenesis such as loss of all human leukocyte cells (pancytopenia). Suppression of HIV-1 replication by ① cART, ② depletion of pDC or ③ blocking reagents against pDC-derived cytokines will restore HSC number and function.HIV-1 infection activates pDCs, possibly through type I IFNs, IL-6 and TNF-α, suppresses self-renewal proliferation of early CD34(TIF)Click here for additional data file.


For TLR4+896A/G variant allele carriers with severe sepsis, high plasma endotoxin/IL-10 inhibits HLA-DR expression and impaired phagocytosis were noted in their classical monocyte. In the same group, increased non-classical monocyte subset, enhanced LPS-stimulated TLR4 expression and TNFα/nitrite production, and systemic inflammation [high plasma soluble CD14 (sCD14) and total nitric oxide (NOx) levels] were noted. For CD14-159C/T variant allele carriers with severe sepsis, persist endotoxemia inhibited mCD14/HLA-DR expression and impaired phagocytosis of their classical monocyte. In the same group, increased non-classical monocyte subset up-regulated TLR4-NFκB-iNOS and p38MAPK pathway, stimulated TNFα/nitrite production and elicited systemic inflammation. In febrile acute de-compensated cirrhotic patients, TLR4+896A/G and CD14-159C/T polymorphisms-related non-classical and classical monocytes dysfunction resulted in increased severe sepsis risk. Malnutrition, high plasma endotoxin and sCD14 levels, single TLR4+896A/G or CD14-159C/T variant allele carriers and double variant allele carriers are significant predictive factors for the development of severe sepsis among them.Genetic variants and dysfunctional monocyte had been reported to be associated with infection susceptibility in advanced cirrhotic patients. This study aims to explore genetic predictive markers and relevant immune dysfunction that contributed to severe sepsis in febrile acute de-compensated cirrhotic patents. Polymorphism analysis of candidate genes was undergone in 108 febrile acute de-compensated cirrhotic patients and 121 healthy volunteers. Various plasma inflammatory/regulatory cytokines, proportion of classical (CD 16 Acute de-compensated cirrhotic patients are suffered from compromise hepatic immune surveillance, impaired phagocytosis and high severe sepsis-related mortality . Poor reTLR4 genetic polymorphism and infection susceptibility has been reported in patients with and without cirrhosis  were enrolled consecutively . The diaExclusion criteria were: human immunodeficiency virus infection, previous transplantation or any other type of immunodeficiency, steroid treatment, pituitary or adrenal disease, hepatocellular carcinoma, severe chronic heart (New York Heart Association function class III or IV) or pulmonary disease , chronic dialysis, acute respiratory distress syndrome, and refusal of patient to participate. Patients gave written informed consent to participate in the study which was approved by the Institutional Review Board Taipei Veterans General Hospital, Taiwan, R.O.C. .Demographic and the baseline clinical evaluation  were completed within 48 hours of hospitalization. Subjective global nutritional assessment (SGNA) score of >1 (2 to 4) was defined as malnourish . All cliRetrospectively, we determined the time of the first de-compensation of cirrhosis  and the period from this time until the first day of the present hospitalization/time of entering current study (pre-study period). Previous incidence and total episode of infections during the pre-study period were recorded.Additionally, age and sex-matched unrelated 121 healthy controls and 51 afebrile compensated cirrhotic patients with available blood sample for genetic analysis were included as comparison group. Healthy controls were those whose visit our hospital for health check-up without clinical or biochemical evidence of liver, renal and cardiovascular disease. The afebrile compensated cirrhotic patients were identified from ongoing studies on cirrhosis in outpatient department of our hospital.TLR4 +896A/G, Asp299Gly substitution mutation, rs4986790; TLR4 3′UTR, G/C, rs11536889) &] and inflammatory response [ &  & ] genes that had available polymorphic information for a Han population by using Applied Biosystems SNP browser software version 3.0  groups. The HLA-DR expression on CD16-  and CD16+  monocytes were measured by staining with anti-HLA-DR-APC, anti-CD14-FITC  and anti-CD16-PE antibodies.The isolated CD16-  monocytes between groups. It is well-established that classical monocyte are phagocytic with no inflammatory attributes ×100%, where MFI stands for mean fluorescence intensity , duration (months) from first de-compensation to entering study, serum bilirubin, prothrombin time prolongation,8plasma CRP level (3.6±0.8 vs. 2.9±1.7mg/dL) and plasma TNFα/IL-6/IL-1β levels were not different between febrile de-compensated cirrhotic patients with and without severe sepsis  monocytes were not different between variant and wild-type allele carriers of TLR4+896A/G or CD14-159C/T as well as between non-severe sepsis and severe sepsis cases (data not shown).The baseline and LPS-stimulated TLR4 expression on the CD16+ monocyte subsets were observed in TLR4+896A/G or CD14-159C/T variant allele carriers and severe sepsis cases  monocyte subset–related mechanisms for increased severe sepsis risk  monocytes also resulted in increased severe sepsis risk among TLR4+896A/G and CD14-159C/T variant allele carriers  monocytes contributed to increased severe sepsis risk in CD14-159C/T variant allele carriers  of double TLR+896A/G and CD14-159C/T variant allele carriers were complicated with severe sepsis compared to either single TLR+896A/G or CD14-159C/T variant alleles carriers (59% or43%). In multivariate analysis, malnutrition (SGNA score>1), high plasma sCD14 level (>3.7 μg/mL), high endotoxin level (>2.3 EU/mL), single or double TLR4 +896A/G or CD14 -159C/T variant alleles carriers were associated with increased severe sepsis risk in febrile de-compensated cirrhotic patients  variants and the SBP-related mortality in de-compensated cirrhotic patients [Guarner-Argente carriers . Appenropatients . HoweverTLR4, mCD14, NFκBp65 and iNOS expression and corresponding intracellular and extracellular IL-6, TNFα, nitrite levels on cultured classical and non-classical monocytes) activities were surveyed in our current study. Notably, current study revealed that variant alleles of TLR4+896A/G and CD14-159C/T modulate immunologic protein abundance  and function  in our febrile acute de-compensated cirrhotic patients with severe sepsis.Thoughtfully, the correlation among the plasma sCD14/endotoxin/NOx levels and various immune regulatory effectors (LPS-stimulated TLR4 +896A/G in our febrile acute de-compensated cirrhotic patients did show increased LPS-stimulated IL-10 production and complicated with higher frequency (59%) of severe sepsis compared to wild-type allele carriers (32%).It was suggested that chronic endotoxemia is associated with increased plasma IL-10 levels in cirrhotic patients ,34. In dTLR4+896A/G and CD14-159C/T variant alleles carriers with severe sepsis, increased TLR4 expression was accompanied by the up-regulation of in vitro LPS-stimulated NFκB, TNF-α, and iNOS signals on their cultured non-classical  monocytes.For host protection from pathogen, the LPS-stimulated NFκB/MAP kinases and TNF-α release depend on the up-regulation of TLR4 expression on activated monocytes ,9. SequeCD14-159C/T variant allele carriers.Membrane bound CD14 (mCD14) are pattern recognition receptors generate early innate immune response against bacterial pathogens. Decreased mCD14 expression on activated classical (phagocytic) monocyte may decrease the pathogen elimination ability of individuals. Previous study had revealed elevated levels of sCD14 in patients with sepsis . AdditioTLR4+896A/G variant allele carriers with severe sepsis, a negative correlation was noted between plasma IL-10 and LPS-stimulated CD16-  monocyte HLA-DR expression. Due to persistent endotoxemia, acute de-compensated cirrhotic patients were suffered from the down-regulation of monocyte HLA-DR expression and immune paralysis [Reduced monocyte HLA-DR expression has been reported in patients with septic de-compensation of acute-on-chronic liver failure . In advaaralysis .-  monocyte HLA-DR expression in our TLR4+896A/G or CD14-159C/T variant allele carriers with severe sepsis. It had been reported that iNOS-derived NO can down-regulate HLA-DR expression and suppress systemic monocyte activation [TLR4+896A/G or CD14-159C/T variant allele carriers with severe sepsis, the significantly higher LPS-stimulated iNOS expression and nitrite production on their cultured non-classical (inflammatory) monocytes and significantly higher plasma NOx levels compared to their control groups were observed.Accordingly, it was reasonable to observe a significant negative correlation between plasma endotoxin levels and LPS-stimulated CD16tivation . In our TLR4+896A/G or CD14-159C/T variant and wild-type allele carriers as well as between severe sepsis and non-severe sepsis cases. Actually, recent meta-analysis reported a lack of association between IL-6 -174G/C polymorphism and sepsis risk [TNFα-308G/A, TNFα-238G/A, IL-6-174G/C, IL-6-597G/A, IL-1β-31T/C and +3954C/T polymorphisms and severe sepsis risk suggested that these inflammatory cytokines might influence sepsis progression via mechanisms other than regulations by these polymorphisms. Probably, the phenotypic heterogeneity of the sepsis syndrome including the infection location, and the amount of time passed since the onset of infection, as well as other individual parameters including background and environmental factors, contributed to some dis-concordance with previous reports.Our study revealed that LPS-stimulated IL-1β/IL-6 production in cultured supernatant of non-classical monocyte and plasma IL-1β/IL-6 levels were not significantly different between our sis risk . In our Serum CRP level, MELD score and APACH score, were well known as a predictor of severity of inflammation/disease. However, the parameters revealed non-significance to predict severe sepsis in current study. In fact, the study about the predictor for severe sepsis in febrile de-compensated cirrhotic patients is limited. It had been suggested that the concentration of lipopolysaccharide binding protein (LBP) is associated inversely with disease severity scores and outcomes in critically ill cirrhotic patients with severe sepsis . AnotherTLR4+896A/G and CD14-159C/T polymorphism-related immune dysfunction including increased non-classical (inflammatory) monocyte proportion-related LPS hyper-inflammatory response and decreased classical (phagocytic) monocyte proportion-related impaired phagocytosis in febrile acute de-compensated cirrhotic patients complicated with severe sepsis. In addition to regular predictive factors such as malnutrition, high plasma endotoxin and sCD14 levels for development of severe sepsis in high risk groups, the novel predictive factors were either single or double TLR4+896A/G and CD14-159C/T variant alleles carriers observed in current study. Accordingly, treatment strategies should change from uniform management to rapid stratifications and sub-categorization, with subsequent aggressive targeted therapeutic intervention in those most at risk.Using multi-modalities comprehensive approaches, our data showed the contribution on the S1 Fig(A-E) Schematic representative SNPs that explored within various cytokines genes in current study; the comparison of (F) previous episode of infection during the pre-study period and (G) duration of first de-compensation to entering study (pre-study period) between cases with and without severe sepsis. *P < 0.05 or **P < 0.001 vs. TLR4/CD14 variant alleles carriers/severe sepsis cases. Pre-study period: first de-compensation of cirrhosis and the period from this time until the first day of the present hospitalization/time of entering current study.(TIF)Click here for additional data file.S2 Fig(A-C). Plasma TNFα, IL-6 and IL-1β levels of all cases; (D). LPS-stimulated IL-1β production; . LPS-stimulated p38MAPβ and ERK1 mRNA expression on CD16+  monocytes of all cases; #p<0.05 vs. un-stimulated group.(TIF)Click here for additional data file.S3 FigA). surface HLA-DR expression on CD16+ monocyte; (B).correlation between LPS-stimulated surface HLA-DR expression on CD16- monocyte and levels of TNFα/nitrite in the LPS-stimulated on the culture supernatant in CD14-159C/T variant allele carrier with severe sepsis. (C). correlation between LPS-stimulated mCD14+ density (Abc) on CD16-  monocyte and LPS-stimulated phagocytic index TLR4+896A/G variant allele carriers with severe sepsis. *P < 0.05 vs. TLR4/CD14 variant alleles carriers/severe sepsis cases; #p<0.05 vs. un-stimulated group.((TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


C. elegans alongside human cell culture models, we establish a new WDR23-DDB1-CUL4 regulatory axis for NRF2 activity that operates independently of the canonical KEAP1-CUL3 system. WDR23 binds the DIDLID sequence within the Neh2 domain of NRF2 to regulate its stability; this regulation is not dependent on the KEAP1-binding DLG or ETGE motifs. The C-terminal domain of WDR23 is highly conserved and involved in regulation of NRF2 by the DDB1-CUL4 complex. The addition of WDR23 increases cellular sensitivity to cytotoxic chemotherapeutic drugs and suppresses NRF2 in KEAP1-negative cancer cell lines. Together, our results identify WDR23 as an alternative regulator of NRF2 proteostasis and uncover a cellular pathway that regulates NRF2 activity and capacity for cytoprotection independently of KEAP1.Cellular adaptation to stress is essential to ensure organismal survival. NRF2/NFE2L2 is a key determinant of xenobiotic stress responses, and loss of negative regulation by the KEAP1-CUL3 proteasome system is implicated in several chemo- and radiation-resistant cancers. Advantageously using  C. elegans WDR-23 has been shown to regulate the worm cytoprotective transcription factor SKN-1. We have leveraged C. elegans genetic approaches to identify conserved regulatory mechanisms of mammalian cytoprotection by NRF2. Collectively, our studies suggest control of NRF2 homeostasis is much more sophisticated than previously appreciated.Chronic exposure to environmental stressors throughout life (“the exposome”) has been tied to several cancers in humans. Cellular adaptation to stress is essential to ensure organismal survival, and NRF2 is an exceptionally well-studied and key determinant of cellular stress responses that plays complex roles in cancer biology and responses to xenobiotics, including chemotherapies. Our studies have established a functional and evolutionarily conserved role for WDR23 as a substrate receptor for the Cullin4(CUL4)-DDB1 E3-ubiquitin ligase, which regulates NRF2 protein levels and activity, and which operates independently of the canonical KEAP1-CUL3 pathway. KEAP1 has been the most highly studied regulator of NRF2, as mutations in KEAP1, which result in uncontrolled activation of NRF2 and chemo-resistance, are found in many aggressive cancers. Importantly, increased expression of WDR23 in KEAP1(-/-) cancer cells restores aberrant NRF2 regulation. In the absence of a KEAP1-like system,  In response to environmental and cellular stress, organisms must activate specific pathways to defend and protect against damage–3. Such The regulation of NRF2 is of particular importance to the progression of human diseases where oxidative stress plays a mechanistic role, including: cancer, inflammC. elegans, a mechanistically similar pathway negatively regulates the abundance of SKN-1, the worm equivalent of NRF2, but via the action of WDR-23. Alt. AltCeWDin worms . The locCeWDR-23 did not impact ARE-luciferase expression in unstressed cells or in KEAP1 siRNA treated cells. Thus, although WDR23 is an ancient regulator of cytoprotection, its functionality in the SKN-1 and NRF2 pathways is species specific  activates NRF2-dependent transcription of cytoprotection genes, SPC296[wdr-23], SPC318[wdr-23], SPC302[wdr-23], SPC306[wdr-23], SPC299[wdr-23], SPC315[wdr-23], SPC303[wdr-23], and SPC317[wdr-23]. Double mutants were generated by standard genetic techniques. For RNAi experiments, NGM plates containing 5 mM IPTG and 100 μg ml-1 carbencillin were seeded with overnight cultures of double-stranded RNAi-expressing HT115 bacteria. Plates were allowed to induce overnight followed by transfer of age-synchronous populations of C. elegans. For arsenite survival, L4 worms of indicated genotype were transferred to plates containing 5mM arsenite (J.T.Baker) and counted for survival after 24 hours.echniques. The folC. elegans strain harboring the SKN-1 transcriptional reporter gst-4p::gfp was mutagenized with EMS, and F1 worms with high GFP expression (indicating SKN-1 activation) were selected. A complementation group of eight recessive alleles were isolated and mapped to chromosome I. The wdr-23 gene was sequenced in each mutant isolated to determine the specific mutation in each strain.Ethyl methanesulfonate (EMS) mutagenesis was performed as previously described. BrieflyCells were grown on coverslips coated with poly-D-lysine (Corning) and transiently transfected with indicated plasmids. Twenty-four hours post-transfection, coverslips were mounted on cover slides and imaged with a Zeiss Axio Imager.M2m microscope, Axio Cam MRm camera, and Zen Blue software.snb-1 and B2M were used to normalize samples in worms and human cells, respectively. Primer sequences are listed in Supplemental Quantitative PCR was performed as previously described. BrieflyHEK-293T cells were transiently transfected with the indicated plasmids and/or siRNA and Cignal antioxidant response luciferase reporter (Qiagen). Forty-eight hours post-transfection, cells were assayed using the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer’s protocol. Firefly luciferase activity was normalized to renilla luciferase activity .HEK-293T cells were transiently transfected with indicated plasmids. Twenty-four hours post-transfection and without treatment of the proteasome inhibitor MG-132, cells were lysed in 0.5% CHAPS buffer  containing Halt Protease Inhibitor (Thermo Fisher). Immunoprecipitation of GFP:WDR23 was performed according to the manufacturer’s protocol (ChromoTek). Briefly, cell lysates were precleared with blocked magnetic agarose GFP Trap beads for 1 hour at 4°C, followed by incubation with magnetic agarose GFP Trap beads for 1 hour at 4°C. After three washes  post-immunoprecipitation, immunoprecipitated protein complexes were eluted in 2X sample buffer  by boiling for 10 minutes at 95°C. Samples were analyzed by Western blot.For detection of protein expression in total cell lysates, cells were lysed in RIPA buffer ). Protein concentrations were measured with Bradford (Amaresco), then prepared with 5X sample buffer , electrophoresed through Bolt 4–12% bis-tris polyacrylamide gels in MOPS running buffer (Thermo Fisher), transferred to nitrocellulose membranes, and subjected to immunoblot analysis. Antibodies used include: GFP GF28R (Thermo Fisher), FLAG M2 (Sigma), NRF2 H-300 (Santa Cruz), NRF2 C-20 (Santa Cruz), DDB1 A300-462 (Bethyl), CUL4A 113876 (GeneTex), KEAP1 ab66620 (Abcam), Actin A5441 (Sigma), Tubulin 21485 (CST), Ubiquitin 1859660 (Thermo Fisher).A549 cells were grown on coverslips coated with poly-D-lysine (Corning) and transiently transfected with indicated plasmids. Forty-eight hours post-transfection, cells were fixed in 100% methanol in -20°C for 5 minutes, blocked in 10% normal goat serum/PBS for 20 minutes, incubated in primary antibody for 1 hour each, incubated in secondary Alexa Fluor antibody (Abcam) for 1 hour each, and mounted with Vectashield with DAPI (Vector Labs). Images were taken with a Zeiss Axio Imager.M2m microscope, Axio Cam MRm camera, and Zen Blue software .HEK-293T cells were transiently transfected with indicated plasmids. Forty-eight hours post-transfection, cells were treated with the indicated chemical: Etoposide (Cayman), Doxorubicin (Cayman), or Cisplatin (Cayman). For viability assays, after forty-eight hours of treatment, cells were assayed using the Vybrant MTT Cell Proliferation Assay Kit (Thermo Scientific), performed according to the manufacturer’s protocol. Viability was calculated relative to the vehicle treatment for each transfection condition. For DNA damage analysis, after twenty-four hours of treatment, cells were assayed using the Muse Multi-Color DNA Damage Kit (EMD Millipore) and Muse Cell Analyzer (EMD Millipore), performed according to the manufacturer’s protocol. For apoptotic cell analysis, cells were assayed using the Muse Annexin V and Dead Cell Assay Kit (EMD Millipore) and Muse Cell Analyzer (EMD Millipore), performed according to the manufacturer’s protocol.In Vitro Protein Synthesis Kit (NEB). Briefly, Flag tagged NRF2 was purified from HEK-293T cells with Flag-M2 affinity resin (Sigma) for Reactions were performed as described in Broderson M. M. L. et al. (2016), with the exception of NRF2 eluate from a PURExpress Statistical analyses were performed with GraphPad Prism 6 software. Data are presented as mean ± s.e.m. Data were analyzed by using unpaired Student’s t-test, one-way ANOVA, and Fisher’s exact test, where indicated. P<0.05 was considered as significant.S1 FigA) Homology table of selected WDR23 proteins with BLAST e-values among invertebrates and vertebrates. (B-E) Subcellular localization of human WDR23 isoform 1 in the cytoplasm and nucleus (B) and WDR23 isoform 2 primarily in the nucleus (C) in HepG2 cells overexpressing GFP-tagged WDR23 is similar to the overexpression of these same constructs observed in HEK-293T cells (D) and WDR-23B (E), respectively. (F-G) The subcellular localization of overexpressed GFP:WDR23 isoform 1 (F) and GFP:WDR23 isoform 2 (H) in untreated cells is not measurably altered in cells treated with tBHQ and overexpressing GFP:WDR23 isoform 1 (G) or GFP:WDR23 isoform 2 (I).(3T cells  and of w(PDF)Click here for additional data file.S2 FigA,B) Overexpression of C. elegans WDR-23A or WDR23B is unable to alter NRF2 transcriptional responses in normal cells  (A) or in cells with reduced KEAP1 expression following KEAP1 siRNA treatment  (B). (C-E) Overexpression of WDR23 isoform 1 or isoform 2 does not significantly reduce the expression of the NRF2 targets GCLM  (C), ABCC1  (D) or CYP4A11  (E) in the absence of stress. (F-H) The increased expression of NRF2 targets following exposure to tBHQ does not occur for PRDX1  (F) when WDR23 is ectopically expressed while expression of NQO1  (G) and HO-1  (H) is still increased. (I) The compensatory increased expression of KEAP1 following stress is attenuated when WDR23 isoform 1 or isoform 2 are overexpressed.  (J) Wdr23 knockout (KO) MEF cells display increased expression of Keap1 .  Slbp siRNA treatment does not significantly alter transcript levels of (M) NRF2 or the NRF2 transcriptional targets (N) Nqo1, (O) Ho-1, (P) Gclc, or (Q) Acadl in HEK293T cells. .((PDF)Click here for additional data file.S3 FigA) Overexpression of GFP and subsequent IP of the GFP protein does not pull down NRF2, DDB1, CUL4A, or KEAP1. (B) Schematic NRF2 protein and localization of the binding site of a NRF2 siRNA and domains used as antigens H-300 and C-20 for the production of NRF2 specific antibodies.  Specificity of the NRF2 protein detected in co-IPs when detected by H-300 (C) and C-20 (D).((PDF)Click here for additional data file.S4 FigA-C) The decreased level of NRF2 protein when WDR23 is expressed is dependent on WDR23 and reversed by WDR23 siRNA treatment (A), which efficiently reduces WDR23 mRNA levels (B), and also reduces endogenous WDR23 protein (C). (D) NRF2 protein levels are reduced when WDR23 is expressed; however a significant change in turnover rate is not detected when cells are treated with cyclohexamide (CHX). (E) Time dependent polyubiquitination of NRF2 by the CUL4-DDB1-WDR23 complex. *, non-specific cross reacting band.((PDF)Click here for additional data file.S5 FigA) The interaction of WDR23 isoform 1 or WDR23 isoform 2 with NRF2 occurs even in the presence of oxidative stress.((PDF)Click here for additional data file.S6 FigA) Schematic of EMS mutagenesis screen to identify wdr-23 mutations, which lead to activation of the SKN-1 reporter gst-4::gfp. (B) Location of conserved mutations in the crystal structure of DCAF provided by the Protein Model Portal[C) Strains harboring mutant versions of WDR-23 display compensatory increased expression of wdr-23 itself. . (D-S) The increased expression of the SKN-1/NRF2 transcriptional reporter gst-4::gfp in wdr-23 mutants  is dependent on skn-1 . Str(PDF)Click here for additional data file.S7 FigA-D) The H306Y  and W335Stop  mutations do not measurably change the subcellular localization of WDR23 isoform 1 while the same mutations in WDR23 isoform 2 leads to more cytoplasmic protein. (E) The W335Stop mutation in WDR23 isoform 1 reduces the interaction with NRF2 and DDB1-CUL4 complexes. (F) The H306Y mutation in WDR23 isoform 2 modestly reduces the interaction with NRF2 and DDB1-CUL4.((PDF)Click here for additional data file.S8 FigA) WDR23 isoform 2 does not interact with NRF2ΔNeh2. (B-I) WDR23 isoform 1  or WDR23 isoform 2  still interact with NRF2ΔNeh4,5 , NRF2ΔNeh6 , NRF2ΔNeh1 , and NRF2ΔNeh3 .((PDF)Click here for additional data file.S9 FigA) Although overexpression of WDR23 can suppress some NRF2 transcriptional targets when KEAP1 is reduced, NQO1 expression remains high. . (B-C) Deletion of the first half of the Neh2 domain (ΔNeh2A) abolishes binding to WDR23 (B), while deletion of the second half of the Neh2 domain (ΔNeh2B) can still interact (C). (D) Single blot assessment of differential binding of WDR23 to full length NRF2, ΔNeh2A, and ΔDIDLID.  Mutation of the DLG (E) or ETGE (F) motifs in the Neh2 domain of NRF2 does not abolish binding by WDR23.((PDF)Click here for additional data file.S10 FigA) Viability of cells with treatment of the vehicle DMSO.  (B-D) Cells overexpressing GFP:WDR23 isoform 1 (pink) or GFP:WDR23 isoform 2 (orange) are more sensitive to etoposide  (B), doxorubicin  (C), and cisplatin  (D). (E-J) Cells overexpressing GFP:WDR23 isoform 1 (F) or GFP:WDR23 isoform 2 (G) have increased overall levels of DNA double-stranded breaks compared to cells overexpressing GFP alone (E), and upon treatment with etoposide, cells overexpressing GFP:WDR23 isoform 1 (I) or GFP:WDR23 isoform 2 (J) results in enhanced DNA double-stranded breaks compared to cells overexpressing GFP alone (H). (K-M) Cells overexpressing GFP:WDR23 isoform 1 (L) or GFP:WDR23 isoform 2 (M) have increased number of apoptotic cells compared to cells overexpressing GFP alone (K). Data are mean ± s.e.m.; one-tailed t-test relative to control GFP overexpression for each treatment condition.((PDF)Click here for additional data file.S11 FigA) Location of WDR23 mutations sequence-confirmed from somatic tumors in human patients. Pink lines are in regions identified in C. elegans and human in this study that result in SKN-1/NRF2 activation. (B) Somatic tumors isolated from lung cancer patients with confirmed KEAP1 mutations have increased expression of WDR23. (C) Samples from stomach cancers with confirmed WDR23 mutations have increased expression of KEAP1.  Total NRF2 protein is reduced in A549 cells transiently transfected with WDR23 isoform 1 or isoform 2; quantified in (E). (F) Increased expression of GFP:WDR23 isoform 1 or isoform 2 can deplete activated endogenous NRF2  from the nucleus  in H460 cells. Scale bar, 20um. (G) Quantification of nuclear or diffuse localization of endogenous NRF2 in H460 cells overexpressing GFP:WDR23 isoform 1 (n = 188), GFP:WDR23 isoform 2 (n = 130), or GFP (n = 225) from the same experiment shown. Each bar represents an independent immunostaining experiment. Fisher’s exact test; **P<0.01, ***P<0.001.((PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S8 Table(PDF)Click here for additional data file.S9 Table(PDF)Click here for additional data file.


Myocarditis is a heterogeneous disease with a large spectrum of possible clinical courses ranging from complete healing to end stage heart failure. However, there is a lack of reliable prognostic factors to identify patients with an adverse outcome. T1 and T2 mapping are promising novel diagnostic cardiovascular magnetic resonance (CMR) tools for a quantitative assessment of the dynamic tissue changes such as edema, necrosis and fibrosis during the course of myocarditis. Thus, we evaluated T1 and T2 mapping CMR for the evaluation of disease activity in patients with myocarditis.This study included 26 patients with myocarditis, who underwent CMR at 1.5 Tesla acute at baseline (BL) and at 3 months follow-up (FU) after the initial presentation with myocarditis, respectively. T1 and T2 mapping CMR were performed in addition to a conventional CMR protocol in myocarditis. T1 quantification was performed on three short axis using the modified Look-Locker inversion-recovery (MOLLI) sequence before and 15 minutes after administration of 0.075 mmol/kg Gadolinium-BOPTA. T2 quantification was performed using a free-breathing, navigator-gated multi-echo sequence. Maps were generated using a dedicated plug-in for the OsiriX software to calculate global myocardial T2, native/post-contrast T1 and extracellular-volume-fraction (ECV).Left ventricular ejection fraction improved from 49 (29-62) % at BL to 54 (42-61) % at FU (p < 0.001). Global myocardial T2 significantly decreased from 60 (58-63) ms at BL to 58 (55-61) ms at FU (p < 0.05). Similarly, global myocardial native T1 decreased from 1119 (1097-1149) ms to 1075 (1037-1113) ms (p < 0.01). Furthermore, global myocardial ECV was significantly lower at FU with 26 (25-29) % compared to 28 (26-31) % at BL (p < 0.05). There were no significant differences between global myocardial T2, native T1 and ECV values of patients with myocarditis at FU and healthy controls.T1 and T2 mapping CMR techniques offer novel insights into the course of myocarditis. Our findings suggest that mapping techniques are useful for the assessment of disease activity and may serve as prognostic factors for patients with myocarditis.


C. elegans to identify novel genetic modulators in SV cycling. One hundred seventy six genes that up-regulating SpH fluorescence and 96 genes that down-regulating SpH fluorescence were identified after multi-round screen. Among these genes, B0035.1 (bugz-1) encodes ortholog of mammalian C2H2 zinc-finger protein BuGZ/ZNF207, which is a spindle assembly checkpoint protein essential for mitosis in human cells. Combining electrophysiology, imaging and behavioral assays, we reveal that depletion of BuGZ-1 results in defects in locomotion. We further demonstrate that BuGZ-1 promotes SV recycling by regulating the expression levels of endocytosis-related genes such as rab11.1. Therefore, we have identified a bunch of potential genetic modulators in SV cycling, and revealed an unexpected role of BuGZ-1 in regulating synaptic transmission.Synaptic vesicles (SV) store various neurotransmitters that are released at the synapse. The molecular mechanisms of biogenesis, exocytosis, and endocytosis for SV, however, remain largely elusive. In this study, using Complex Object Parametric Analysis and Sorter (COPAS) to monitor the fluorescence of synapto-pHluorin (SpH), we performed a whole-genome RNAi screen in  These steps include biogenesis of SVs, transport to release sites, docking with plasma membrane, priming, and calcium-triggered fusion  store neurotransmitters, concentrate in the presynaptic nerve terminals, and undergo Ca2+ via synaptotagmin and complexin, and is additionally regulated by a presynaptic active zone proteins that includes Munc13 and RIM as central components  protein, synaptobrevin (on vesicle membrane), and two target membrane SNARE (t-SNARE) proteins, syntaxin-1, and SNAP-25 (on the plasma membrane) , a pH-sensitive variant of GFP (pHluorin) fused to the luminal domain of synaptobrevin, is widely used to quantitatively measure the exocytosis and endocytosis of SVs  of corresponding genes , which provides a method for high-throughput, reproducible quantitative analysis  expression in the nerve system for optical measurements of presynaptic activity. SpH is a PH-sensitive variant of GFP fused to the luminal domain of synaptobrevin. Previous studies have confirmed that the fluorescence of SpH is quenched in the acidic environment of the SV lumen but increased dramatically when the SVs fused to the plasma membrane ; lin-15b(n744)  cycling, we performed an automatic whole-genome RNAi screen in m Figure . For higm Figure . We deveunc-11/AP180, which has been reported to play an important role in recycling synaptobrevin from plasma membrane . Knocking down pHluorin by feeding worms with gfp RNAi bacteria decreased the fluorescent intensity of SpH to <0.5-fold in worms. The fluorescent intensities of worms cultured in 96-well plates in liquid which detected by COPAS were consistent with the fluorescent intensities of worms cultured on RNAi plates which detected by the confocal microscope. These results indicated that positive genes can be knocked down by feeding worms with corresponding RNAi bacteria in liquid culture, and the changes of fluorescent intensity can be detected efficiently by the COPAS.We chose n Figure . We usedunc-11 RNAi clones, down-regulated control of gfp RNAi clones, and empty vector control of L4440 clones into the empty wells of each 96-well plate. We rearranged a few clones in 96-well plates to insure each experimental plate contains two empty vector controls, one up-regulated control and one down-regulated control. Synchronized L1 worms were fed with each RNAi clone in each well of 96-well plates, and SpH fluorescence was detected in their progeny via the COPAS. For those RNAi clones that caused embryonic lethal or sterile, SpH fluorescence was detected in young adults of the same generation ;lin-15b(hd126);rhIs13[unc-119p::GFP + dpy-20(+)]  were excluded. Finally, 176 genes with up-regulated fluorescent intensity of SpH and 96 genes with down-regulated fluorescent intensity of SpH were identified after multi-round screen  secretion can be indirectly detected by measuring their resistance to the acetylcholine esterase inhibitor aldicarb  as a positive control in aldicarb resistance assay  is a specific mutant that is important for locomotion and SV morphology in motoneurons , Y71G12B.11(ok1648), Y25C1A.7(tm2889), F41D9.3(ok695), F45E4.3(ok2285), Y76A2A.2(gk107), T23F11.1(tm3480), F59E12.11(tm3828), C18B12.2(tm1690)  encodes an ortholog of mammalian C2H2 zinc-finger protein BuGZ/ZNF207. Very interestingly, recent studies have been implicated that mammalian BuGZ binds to and stabilizes spindle check point protein Bub3 during interphase and facilitates mitosis  and BuGZ-1L (B0035.1b), both containing a nuclear localization sequence (NLS) and two C2H2 zinc finger domains at the N-terminal of the protein  generated by the National Bioresource Project . Five out of six exons are deleted in bugz-1(tm578) mutant worms. The deletion starts from the promoter region of bugz-1, which makes bugz-1(tm578) a null allele  animals displayed more hesitating and uncoordinating movement with decreased trajectories and locomotion speed , is expressed specifically in cholinergic motor neurons  and evoked EPSCs at NMJs in  Figures . The evo Figures . The def Figures . Furthers Figure . These rBuGZ-1 was knocked down by RNAi treatment (1.43-fold change compared with L4440 empty vector). Significant increases of the SpH fluorescence were also observed in the nerve ring, the ventral nerve cord, as well as the axonal SpH puncta and inter-puncta of the dorsal nerve cord in bugz-1(tm578) mutations  background. Given the neurotransmitter release was decreased at NMJs in bugz-1 mutant animals  from wild type and bugz-1(tm578) worms  worms  worms ;unc-11(e47) double mutant worms, more significant increases of the SpH fluorescence were observed both in the nerve ring and the ventral nerve cord compared to either bugz-1(tm578) or unc-11(e47) single mutant worms. These strongly enhanced phenotypes in double mutants suggest that BuGZ-1 and UNC-11 mainly function in parallel pathways, though loss of BuGZ-1 may result in down-regulation of UNC-11  shows similar phenotypes with increased SpH fluorescence with bugz-1(RNAi) worms, no synthetic effect was observed in bugz-1(RNAi) combined with rab-11.1(RNAi) depletion, suggesting that BuGZ-1 and RAB-11.1 may function sequentially in the recycling pathway ;lin-15b(n744) ;lin-15b(n744);kanIs8[snb-1::pHluorin] for RNAi screening.All Brenner, . The plabugz-1(tm578), Y25C1A.7(tm2889), T23F11.1(tm3480), F59E12.11(tm3828), C18B12.2(tm1690), RB1445 Y71G12B.11(ok1648), RB861 F41D9.3(ok695), RB1777 F45E4.3(ok2285), VC194 Y76A2A.2(gk107), CB189 unc-32(e189), VH624 rhIs13[Punc-119::GFP + dpy-20(+)]; nre-1(hd20); lin-15b(hd126); zxIs6 (a gift from Dr. Mei Zhen). Single copy worm strain xtl1186 kanIs54 II; unc-119(ed9) IIIkanEx378[bugz-1p::BUGZ-1::gfp+ rab-3p::mCherry]kanEx379[bugz-1p::gfp+ rab-3p::mCherry]bugz-1(tm578); kanEx380[lin-44p::gfp+ rab-3p:: BUGZ-1::mCherry]bugz-1(tm578); kanEx392[lin-44p::gfp+ myo-3p:: BUGZ-1::mCherry]bugz-1(tm578);kanEx380[lin-44p::gfp+ rab-3p:: BUGZ-1::mCherry]; zxIs6bugz-1(tm578);kanEx392[lin-44p::gfp+ myo-3p:: BUGZ-1::mCherry]; zxIs6.The following strains were used in this study:gfp RNAi down-regulated control and one unc-11 RNAi up-regulated control.Bacterial glycerol stocks of RNAi library was replicated onto LB-agar square plates containing 25 μg/ml carbenicillin and 15 μg/ml tetracycline using a 96-pin replicator. After overnight culture at 37°C, bacterial clones were replicated from square plates into 1.2 mL 96-well plates containing 400 μl LB liquid medium with 25 μg/ml carbenicillin in each well. The RNAi bacterial clones in 1.2 mL 96-well plates were cultured overnight at 37°C with shaking. Added IPTG to bacterial cultures to a final 1 mM concentration to induce transcription of double-stranded RNA and incubated at 37°C for 1 h with shaking. Bacterial clones were spun down and resuspended with 200 μl S-Basal buffer containing 50 μg/ml carbenicillin and 1 mM IPTG. Forty microliters concentrated bacterial suspension was added into each well of standard 96-well plates. RNAi bacteria from original 96-well RNAi library were rearranged to make each experimental 96-well plate contained two L4440 controls, one Adult hermaphrodite worms were bleached using standard method. Synchronized L1s were washed and resuspended in S-Basal containing 1 mM IPTG, 50 μg/ml carbenicillin, and 0.01% Tween-20. 5–8 L1s/10 μl S-Basal for F1 generation screen or 120 L1s/10 μl for P0 generation screen. Ten microliters worm solution was added into each well of 96-well plates which pre-added 40 μl concentrated RNAi bacterial suspension. Experimental 96-well plates containing bacterial clones and L1s were incubated in humid chambers at 20°C. To increase RNAi efficiency, we used two-generation RNAi treatment for whole-genome screen: synchronized L1 worms were cultured for 6 days, and fluorescent intensity of their progeny were detected by COPAS  to represent synapto-pHluorin (SpH) signal of each worm. The green fluorescent signals  divided by the EXT signals  would result in the normalized SpH signals. LogRobust Z-score (rZ) was used to normalize screen data in all the plates in the whole-genome screen. Robust Z-score was similar to Z-score except that the sample median and sample median absolute deviation (MAD) were used instead of sample mean and sample standard deviation, and thus was not sensitive to outliers , one tailed t-test was used to identify the experimental RNAi bacteria that were significantly different from L4440 empty control.In the secondary and cytoplasmic GFP screen, student's p < 0.05 in secondary screen and >0.05 in the cytoplasmic GFP screen were considered as positive hits.Those RNAi bacteria with Aldicarb-sensitivity experiments were performed on NGM plates containing 1 mM aldicarb as previously described  repair template, 50 ng/μl Mos1 transposase pJL43.1(Pglh-2::transposase), 5 ng/μl myo-3p::mCherry which as a negative marker. Injected animals were transferred to NGM plates, one worm per plate. Individual injected worms were allowed to exhaust the food source of each plate. Once starved, L1 progeny were screened for insertion events with GFP fluorescence and wild-type movement but lack of co-injection marker expression.Transgenic worms were generated by injection constructs into EG4322  animals  on agarose pads. For quantitative analysis, images were acquired in young adult worms and maximum fluorescent intensity of Z-series was stacked. Background fluorescence was subtracted before analysis. P-values were calculated by student t-tests. Images were quantified and analyzed using FV10-ASW Viewer and Image J. For fluorescent analysis of SpH signal in the nerve ring and ventral nerve cord, average of stacked maximum fluorescent intensity were calculated to represent florescent intensity. For fluorescent analysis in the dorsal nerve cord, florescent intensity of puncta, and inter-puncta were measured using Igor Pro software.All the images were obtained using a FV 1000 laser scanning confocal microscope (Olympus). Confocal images were captured using a 60X objective with NA 1.4 at 1x or 2x digital zoom. Worms were immobilized with 30 mM NaNE. Coli OP50 8–12 h before. Fifteen young adult animals (12–14 h after L4 stage) were transferred to each 60 mm NGM plate. Ten min after transfer, a 1 min movie of animal moving was recorded using a digital camera installed on a Zeiss dissecting microscope. Movies of locomotion behaviors were analyzed by the “Imaging the Behavior of Nematodes (iBeN)” system developed in our lab. Software of iBeN system is developed based on the computer vision library, OpenCV (Open Source Computer Vision). Locomotion rates and movement status were achieved automatically by iBeN system.Locomotion experiments were performed by capturing movies of animals free moving on NGM plates with fresh seeded C. elegans as previously described -YFP was expressed in cholinergic neurons, was used for recording evoked EPSCs  with a GFP filter controlled by the Patchmaster software.Electrophysiology assays were performed at the neuromuscular junctions of dissected RNA was extracted from young adult animals using Trizol reagent (Invitrogen). Total RNA was reversed to cDNA using Reverse Transcription System A3500 (Promega). For quantitative RT-PCR, Bio-Rad CFX96 real-time PCR Amplifier was used to run the cycles. qPCR was performed in triplicate for three independent biological experiments. Relative gene expression levels were calculated by ΔCt method.C. elegans WS235 was used to align reads. Significant binding peaks were called with SPP and IDR algorithms .ChIP assays were performed as previously described (Kudron et al., t-test was used for data comparison and P < 0.05 were considered to be statistically significant.Data analysis was performed using Excel or Igor 5. All data were presented in mean ± SEM. Unpaired two-tailed MH, YY, LK, and TX conceived and designed the experiments. MH, WZ, HC, YY, HZ, SL, HKC, GW, and YC performed molecular genetics, optogenetics, behavioral, and electrophysiological experiments. MH, VR, LK, and TX analyzed and interpreted results. MH, YY, and LK wrote the manuscript and modified by all the other authors.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.


Functional inhibition of Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) can cause the death of EBV infected cells. In this study, a bioinformatics tool predicted the existence of putative extracellular signal-regulated kinase (ERK) docking and substrate consensus sites on EBNA1, suggesting that ERK2 could bind to and phosphorylate EBNA1. In accordance, ERK2 was found to phosphorylate EBNA1 serine 383 in a reaction suppressed by H20 , U0126 (an inhibitor of MEK kinase), and mutations at substrate (S383A) or putative ERK docking sites. Wild-type (S383) and phosphomimetic (S383D) EBNA1 demonstrated comparable transactivation function, which was suppressed by H20 or U0126. In contrast, non-phosphorylated EBNA1 mutants displayed significantly impaired transactivation activity. ERK2 knock-down by siRNA, or treatment with U0126 or H20 repressed EBNA1-dependent transactivation. Collectively, these data indicate that blocking ERK2-directed phosphorylation can suppress EBNA1-transactivation function in latent EBV-infected cells, validating ERK2 as a drug target for EBV-associated disorders. Epstein-Barr virus (EBV) usually infects and replicates in oropharyngeal epithelial cells -3. In lain silico analyses suggested that H20 docked in pocket 2 of EBNA1, surrounded by K514, Y518, R521, and P535. Interestingly, this pocket overlapped with the predicted ERK docking motif and the structure of H20 was similar to that of the ERK docking inhibitor (ERKi), which prevented ERK association with the substrate ). The eukaryotic linear motif (http://elm.eu.org) and Netphos 2.0 (http://www.cbs.dtu.dk/services/NetPhos/) tool predicted the existence of a putative motif for ERK2-directed proline-dependent phosphorylation at a.a. 381-384 (PRSP), and a potential ERK docking site at a.a. 521-528RRGTALAI) on the surface of EBNA1 . EB. EB23]. EBV-negative human Burkitt's lymphoma cells (BJAB), BJAB cells that stably expressed FE1 (BJ-FE1), BJ-FE1 cells with EBNA1-dependent episomal OriPCp-FL (or POLP2), and BJ-FE1 with integrated EBNA1-independent SV40p-RL (BJ-FE1-OF) have been described previously . Where nCytoplasmic and nuclear proteins from BJ-FE1 S383 WT, and S383A MT cells treated with DMSO and U0126 for 3 days were washed in 1 mL of ice cold PBS and then fractionated. Cytoplasmic extracts were obtained using 5× hypotonic buffer ; nuclear extracts were obtained using 1× hypertonic buffer . Extracts were stored at −80°C until assayed. Subcellular localization of EGFP-EBNA1 S383 WT and S383A MT in BJAB cells was assayed by live confocal microscopy as described previously  ATP in the presence or absence of compounds, as indicated previously [Different EBNA1 constructs  as a source of cellular kinases , subsequently pulled-down by His-bind magnetic agarose beads , washed and subjected to western blotting using anti-His-tag, anti-ERKs, and anti-pyruvate kinase muscle type isoform 2 (PKM2) antibodies.Cell extracts from stable BJAB cells expressing vector control or FE1 were prepared inlysis buffer . Cell extracts were precleared with Protein G/A agarose beads (10% v/v of 50% bead slurry), washed 3× with 1% lysis buffer, and immunoprecipitated using anti-FLAG, anti-ERK2, or IgG isotype control antibodies as described previously . Immune


Chlamydia muridarum (Cm) genital infection. In this report, we investigated immune cell type-specific miRs, i.e. miR-155 and -182, and the role in Ag-specific immunity. We observed significant up-regulation of miR-155 in C57BL/6 bone marrow derived dendritic cells (BMDC), and miR-182 in splenic Ag-specific CD4+ T-cells. Using mimics and inhibitors, we determined that miR-155 contributed to BMDC activation following Cm infection. Co-cultures of miR-155 over-expressed in BMDC and miR-182 over-expressed in Ag-specific CD4+ T-cells, or miR-155−/− BMDC with miR-182 inhibitor treated Ag-specific CD4+ T-cells, resulted in IFN-γ production comparable to Ag-specific CD4+ T-cells isolated from Cm infected mice. Additionally, miR-182 was significantly up-regulated in intranasally vaccinated mice protected against Cm infection. In vivo depletion of miR-182 resulted in reduction in Ag-specific IFN-γ and genital pathology in Cm infected mice. To the best of our knowledge, this is the first study to report an interaction of miR-155 (in Cm infected DC) and miR-182 (in CD4+ T-cell) resulting in Ag specific immune responses against genital Cm.Anti-chlamydial immunity involves efficient presentation of antigens (Ag) to effector cells resulting in Ag-specific immune responses. There is limited information on inherent underlying mechanisms regulating these events. Previous studies from our laboratory have established that select microRNAs (miRs) function as molecular regulators of immunity in  Chlamydia trachomatis (Ct) immune response involves neutrophils, macrophages, and dendritic cells followed by antigen (Ag)-specific and non-specific T-cells homing to the infected genital tract . T. TC. murted mice , and theted mice .in vivo results from a complex of various immune cell types including Ag-presenting cells (APC) and CD4+ T-cells [+ T-cells  involved in anti-Cm immunity [+ T-cells isolated at day 12 post infection, respectively. Activation of bone marrow derived DC (BMDC) as assessed by major histocompatibility complex II expression was regulated by miR-155. Co-culture of miR-155 treated BMDC (transfected with a miR-155 inhibitor) or miR-155−/− BMDC with Ag-specific CD4+ T-cells resulted in significant up-regulation of IFN-γ production. Ag-specific IFN-γ production was abrogated in total splenocytes from miR-182 inhibitor treated mice compared to scramble treated or mock treated Cm infected mice. Moreover, following Cm infection, miR-182 inhibitor treated mice displayed significant reduction in development of upper genital pathology compared to scramble or mock treated mice. Importantly, IFN-γ production in miR-155 mimic treated BMDC co-cultured with miR-182 mimic treated Ag-specific CD4+ T-cells was comparable to untransfected co-cultures. Untransfected co-cultures surrogately demonstrated/ mimicked the specific role of miR-155 and -182 in IFN-γ production during an in vivo Cm infection. These findings were further corroborated by comparable IFN-γ production in miR-155−/− BMDC cocultured with CD4+ T-cells from miR-182 inhibitor treated mice compared to WT BMDC co-cultured with CD4+ T-cells from Cm infected mice. Taken together, these results strongly demonstrate the combined effect of 2 miRs  in contributing to anti-Cm immune responses and IFN-γ production reported previously to be critical for protection against a genital Cm infection [Anti-Ct immunity immunity , 24, 25.post Cm infection [i.e., miR-214 regulates ICAM-1) contributes to upper genital pathology in Cm infected mice [post infection  in Cm infected BMDC compared to mock infection was observed . Collectively, these results demonstrate that miR-155 contributes to MHC-II expression in CD11c+ BMDC following Cm infection.DCs are professional Ag-presenting cells (APC) that upon activation, initiate adaptive immune responses . MiR-155d Figure . In ordes Figure . In conts Figure . Transfed Figure . In conts Figure . We obse−/− or WT BMDC with splenic CD4+ T cells isolated at day 12 from mock or Cm infected mice and measured IFN-γ production by ELISA   might ps Figure . MiR-182s Figure . Additios Figure , and in s Figure  compareds Figure . Express) Figure , and res) Figure  from all) Figure  in Cm va+ T cells from Cm infected and vaccinated mice, we determined the in vivo relevance of miR-182 in genital Cm infection. We used a depletion regimen for up to day 80 post infection , the primary source of Ag-specific IFN-γ production [+ T cells are the most important effector cell population that have the potential to provide protective responses against a subsequent infection [post infection in a well characterized 27-33 day murine genital infection model [Tbx21 indicating clonal expansion of Th1 cells (data not shown). Interestingly, regulation of MHC-II by miR-155 inhibitor  by miR-155 via pathways involving molecules such as c-Fos, SHIP1, SOCS-1 and cytokines, IL-12 and IL-6, are warranted for a better understanding of Ag-specific IFN-γ production in Ct infections [Identification of miRs in cultured DC infected with Cm revealed only 12/88 miRs probed being regulated compared to mock infected controls Figure . Of thesent mice . Additiofections , 46. Impn Figure . We co-coduction ; (2), CDnfection  and; 3)on model . Increason model fections , 47, 48.FOXO-1, and up-regulation of its controlling microRNA, i.e., miR-182 [+ T cells isolated at day 12 post infection from Cm infected mice compared to mock infected animals  and may also be involved in vaccine mediated protection (isolated at day 6 post infection in vaccinated mice). We have previously reported that miRs 182 and -183 are significantly modulated in the Cm infected genital tract, and are up-regulated in the lower genital tract of Cm infected CD4−/− mice compared to the wild type [in vivo delivery [+ T cells , our findings , both miRs-155 and -182 were up-regulated following Cm infection  respectively, lead to reduction in Ag-specific IFN-γ; (3), miR-182 was observed to be up-regulated in vaccinated mice protected against a subsequent Cm infection where protection may be mediated by production of Ag-specific IFN-γ , Ct is the leading cause of STIs globally , (2) a kSeed stocks of Cm were propagated in HeLa 229 cells. At 24 h post infection, HeLa cells were mechanically disrupted, and following high speed centrifugation, bacterial pellets were purified on Renografin gradient as previously described .−/− were purchased from Jackson Laboratory.All procedures were carried out in compliance with Institutional Animal Care and Use Committee (IACUC) guidelines. Female, 4-6 week old wild type C57BL/6 (WT) mice and miR-1554 IFU [post infection, genital tracts were removed from all groups of mice, fixed in 10% neutral formalin, and embedded into paraffin blocks. Serial horizontal sections (5 μm) were prepared and every tenth section (∼8-10 sections per tissue) was stained using hematoxylin and eosin (H&E). Stained sections were visualized using a Zeiss Axioskop 2 Plus research microscope and images were acquired using an Axiocam digital camera . Representative sections stained with H&E were scored in a blinded fashion by a trained pathologist using a scoring scheme as described previously [n = 10). From a subset of mice (n = 3)/ treatment group genital tract  and splenic CD4+ T cells (day 12) were collected and stored in −80° C till further use. Genital tissue was ground using mortar and pestle and the crushed tissue was divided into 2 portions for isolating RNA (for miR PCR) and DNA (16s encoding gene PCR) respectively. Expression of miR-182 and -183 using miR-specific RT-PCR was performed as described below and bacterial burdens  were determined using primers and PCR parameters as described by Wooters et al [In order to render the mice anestrous and more receptive to the genital infection, a single subcutaneous injection containing 2.5 mg Depo progesterone  was administered five days prior to infection. Mice were intravaginally infected with purified Cm diluted in 15 μL sucrose/phosphate/glutamate (SPG) buffer at a final inoculating dose of 5 × 104 IFU . For miR4 IFU . At day eviously . Resultsrs et al .4 IFU Cm, mice were swabbed at days 2, 4, and 6 for cervical-vaginal bacterial shedding. At day 6 post Cm infection, mice were euthanized for isolating splenocytes.For vaccination studies, mice were intranasally immunized with 500 IFU live Cm and rested for a month. Following intravaginal infection with 5 × 10n = 3-5/ experiment) were euthanized and bone marrow was collected from the tibia and femur under aseptic conditions. BMDC were prepared as described previously with slight modifications [Female C57BL/6 mice (ications . At day + T-cells were purified (purity > 85-90% by flow cytometry) using a CD4 enrichment protocol per manufacturer's recommendation (StemCell). CD4+ T-cells were manipulated with miR-182 mimic prior to co-culture with treated BMDC.At day 12 post infection, mock or Cm infected mice were euthanized and spleens isolated. Single cells were prepared as described previously . CD4+ T-post Cm infection in treated BMDC, CD4+ T-cells were over-layed at a BMDC: CD4 ratio of 1:2.5 (for miR-155 manipulation experiments), and 1:2 (for miR-155 and -182 treatment experiments). Co-cultured cells were incubated at 37°C for 72 h. After 72 h, cells were centrifuged at 1200 rpm at 4°C for 10 min, and the supernatant was collected and stored at −80 °C for ELISA.At 24 h For miR-inhibitor delivery, polymeric nanoparticles based on polyethylenimine  were employed. More specifically, PEI F25-LMW / inhibitor complexes was prepared as previously described for siRNA . In brie+ T-cells were seeded at a density of 2 × 105 or 5 × 105 respectively, per well. MiR-155 or -182 agomiR , and antagomiR (MiScript miRNA Inhibitor) were transfected using Attractene as transfection reagent at a concentration of 20 μM by fast forward transfection per manufacturer's recommendation (Qiagen). Transfection was carried out for 24 h (BMDC), and 18 h (CD4+ T-cells) followed by Cm infection (BMDC) or co-culture with infected BMDC (CD4+ T-cells). Prior to infection or co-culture, efficiency of transfection or in vivo depletion and cell viability was determined by Real Time PCR and MTS assay , respectively, (data not shown).BMDC and CD4+ T-cells at 24 h post infection or 12 days post infection, respectively. For vaccination studies, splenic CD4+ T-cells were isolated 6 days post infection. RNA was obtained using a miRNeasy RNA extraction Kit (Qiagen) according to the manufacturer's instructions. Total RNA was assessed using a Nanodrop Spectrophotometer . RNA samples exhibiting A260/280 2.0 and A260/230 1.8 were converted to cDNA using a RT2 First Strand cDNA Kit (Qiagen) according to the manufacturer's instructions. RNA (1 μg) was used in all miR PCR amplifications, and were performed using custom designed miScript miR-155, -182, and -183 primers (Qiagen) according to the manufacturer's instructions. For FOXO-1, Tbx21, FoxP3, GATA3, and RoRγ expression, RNA (1 μg) was converted to cDNA using an iScript™ cDNA Synthesis Kit followed by real time PCR using the SsoAdvanced™ Universal SYBR®Green Supermix  with gene specific primer pairs  on a CFX 96 instrument (Bio-Rad). All miR expression analyses were normalized to housekeeping miRs RNU6-2_1 and SNORD68 expression values (for BMDC experiments) and SNORD68 and 85 (for CD4+ T-cell experiments). All gene expression analyses were normalized to housekeeping genes GAPDH. Data are represented as fold-change relative to mock control as indicated in the Results and Figure legends using the comparative cycle threshold method [Total RNA was extracted from BMDC and splenic CD4d method .Cells were stained with fluorochrome-conjugated CD-11c, and MHC-II labeled antibodies , and FACS analysis was performed using a LSR II instrument (BD Biosciences) .Myelin Oligodendrocyte Glycoprotein MOG35-55).Supernatants from ‘co-culture’ wells were analyzed for IFN-γ production using BD OptELISA kits  as previously described . Cells wpost Cm infection, splenocytes were isolated from all groups of mice in vaccination studies, and single cell suspensions made as previously described [per specifications [At day 6 escribed . IFN-γ Eications .2 Profiler PCR Array Data Analysis . All experimental results were calculated as the mean ± SE of 2-5 independent experiments (n = 3-5) indicated in respective figure legends. Student's t-test was used for comparison between two groups. ANOVA with post-hoc multiple comparison test was used for multiple groups. Kruskall-Wallis test was used for comparing bacterial shedding profiles. Differences were considered statistically significant if P values were < 0.05. All statistical analyses were conducted using the GraphPad Prism 5 software package .MiR analysis was performed using RT


Accordingly, we developed a lymphoma progression model comprising cell lines established at indolent (MAC-1) and aggressive (MAC-2A) CTCL stages. We discovered activating JAK3 (V722I) mutations present at indolent disease, reinforced in aggressive disease by novel compound heterozygous SOCS1 (G78R/D105N) JAK-binding domain inactivating mutations. Though isogenic, indolent and aggressive-stage cell lines had diverged phenotypically, the latter expressing multiple Th17 related cytokines, the former a narrower profile. Importantly, indolent stage cells remained poised for Th17 cytokine expression, readily inducible by treatment with IL-2 - a cytokine which mitigates Th17 differentiation in mice. In indolent stage cells JAK3 expression was boosted by IL-2 treatment. Th17 conversion of MAC-1 cells by IL-2 was blocked by pharmacological inhibition of JAK3 or STAT5, implicating IL2RG - JAK3 – STAT5 signaling in plasticity responses. Like IL-2 treatment, SOCS1 knockdown drove indolent stage cells to mimic key aggressive stage properties, notably IL17F upregulation. Co-immunoprecipitation experiments showed that SOCS1 mutations abolished JAK3 binding, revealing a key role for SOCS1 in regulating JAK3/STAT5 signaling. Collectively, our results show how JAK/STAT pathway mutations contribute to disease progression in CTCL cells by potentiating inflammatory cytokine signaling, widening the potential therapeutic target range for this intractable entity. MAC-1/2A cells also provide a candidate human Th17 laboratory model for identifying potentally actionable CTCL markers or targets and testing their druggability  Cutaneous T-cell lymphomas (CTCL) form a diverse group of skin neoplasms, including  lymphomatoid papulosis (LyP), malignant anaplastic large cell lymphoma and mycosis fungoides, together with the systemic, aggressive Sézary syndrome . The molLike those observed in non-malignant T-helper cells, cytokine profiles of malignant cells in CTCL depend on stimuli they receive from their environment, and phenotypic changes acquired during disease progression invite comparisons with physiological T-cell development . While eJAK3 is a tyrosine kinase associated with common gamma-chain receptors of cytokine signaling, thereby preferentially phosphorylating and activating STAT5 . JAK3 muSOCS proteins constitute a group of negative feedback regulators of JAK/STAT signaling . The funin vitro platforms for investigating both the molecular pathology and the druggability of therapeutic targets identified therein or clinically. MAC-1 and MAC-2A cell lines were respectively established at indolent and aggressive disease phases from a cutaneous lymphoma patient initially presenting with LyP [Human CTCL derived cell lines provide infinitely verifiable with LyP , 19. To http://genetics.bwh.harvard.edu/pph2/), Sift (http://sift.jcvi.org/) and Condel (http://omictools.com/consensus-deleteriousness-score-of-missense-snvs-tool) annotation tools. Alterations are summarized in Figure Despite sharing identical TCR sequences  and primMAC-1 and MAC-2A carry heterozygous JAK3 mutations at chr19: 17,945,696 (C- > T) with allelic depths of 0.530 and 0.500, respectively, but absent from other ALCL/CTCL cell lines . The identical mutation was also present in MAC-2B established alongside MAC-2A, from a separate skin lesion with an allelic depth of 0.524 (not shown). This gain-of-function mutation JAK3 (V722I) replaces valine with isoleucine at position 722 inside the pseudokinase domain leading to constitutive JAK3 activation, as described in megakaryoblastic, prolymphocytic and in T-cell acute lymphoblastic leukemias .While the JAK3 mutation was common to all three MAC cell lines, the SOCS1 gene carried two mutations in aggressive stage MAC-2A cells: at chr16: 11,349,104 , and 11,349,023  Figure . Again tin vivo [T-helper subsets, notably Th17 cells, display phenotypic plasticity –23. Morein vivo , 24. MicWe next tested the effects of IL-2 alone, or in combination with IL-6 and/or TGFβ. Unaided, IL-6 and TGFβ - classic agents for inducing Th17 differentiation, elicited minimal responses, limited to upregulation of IL9 by TGFβ Fig . In markSince IL-2 reportedly activates Treg signature genes , we alsoIL-2 acts via the heterotrimeric IL-2 receptor, belonging to the common gamma-chain receptors and comprising the subunits IL2RA, IL2RB and IL2RG. Importantly, the common gamma-chain receptors are associated with JAK3, which upon stimulation initiate JAK-STAT signaling through STAT phosphorylation (STAT5A/B - Y694/Y699) . This prTo confirm transcriptional signaling by IL-2 or IL-15 conducted via their cognate receptors, we respectively quantified expression of inducible Th17 program genes in MAC-1 cells after IL-2 or IL-15 receptor blockade, respectively using IL-2RA or IL-15RA blocking antibodies. Antibodies against IL-2RA or IL-15RA both reduced expression of key genes, while the inductive effects of IL-2/IL-15 on, e.g. IFN-gamma or IL17F were partially abrogated after blockade of either receptor . CollectGiven the central importance of JAK3 signaling in T-cell biology , we now These conclusions were further supported by the proliferation-resistance of JAK3wt HH cells to tofacitinib Figure  which coSince the effects of tofacitinib and IL-2 are antagonistic and coupled via the common gamma-chain receptors, we investigated transcriptional differences after combined IL-2 and tofacitinib treatments. Significantly, the IL-2 driven induction of gene expression in MAC-1 was abrogated by tofacitinib Figure . ConversBecause the IL-2 - common γ-chain receptor - JAK3 cascade also promotes STAT5 phosphorylation [SOCS1 binds to JAK via the SH2 domain, forming a complex which inhibits IL-2 induced STAT5 phosphorylation , 32. SinTo further test this model, we compared the JAK3 binding efficiencies of SOCS1 G78R/D105N (in MAC-2A) and wt SOCS1 (in MAC-1) to JAK3 in co-immunoprecipitation (IP) experiments. This exercise confirmed interaction between wt SOCS1 and JAK3 in MAC-1 cells, as shown by the presence of JAK3 in SOCS1 precipitated fractions Figure . This inin vitro system for modelling lymphomatous progression and inflammatory cytokine differentiation driven by JAK/STAT pathway mutations which may be recapitulated using IL-2 and which highlights this cytokine in driving IL-17F expression, at least in the cell contexts described herein.Here we characterize a dual lymphoma progression/candidate Th17 cell line model. Our model is two-stage - “before-and after”, comprising cell lines respectively established at indolent and aggressive disease stages providing controlled inducible cytokine expression which vividly recalls the inflammatory cytokine profiles seen at advanced CTCL. Using this model we reveal a two-step mutational process reflecting disease progression, whereby an activating JAK3 mutation known to drive lymphoma formation is joined in progressed disease by novel inactivating mutations of SOCS1, thereby weakening the latter's inhibitory potency. We further show that expression of a core inflammatory element - one reportedly associated with progressed CTCL, namely IL17F, can be induced by SOCS1 knockdown performed in indolent disease (MAC-1) CTCL cells bearing wild type SOCS1. Co-IP experiments showed that the SOCS1 mutations in question impaired binding to JAK3 upon which their inhibitory interactions are known to be contingent, thus providing a cogent explanation for exuberant JAK3 downstream cytokine signaling in MAC-2A cells bearing both SOCS1 mutations. As a corollary, the Th17-like cytokine profile seen in aggressive disease MAC-2A cells was partially abolished by pharmacological inhibition of JAK3 or STAT5 implicating this signaling pathway in inflammatory disease progression. It should be noted, however, that doubts have been raised over the reported selectivity of pimozide, implying the need to verify conclusions based on the use of this inhibitor alone. Moreover, the wide Th17 profile seen in aggressive stage cells was readily inducible in plastic, indolent stage MAC-1 cells by IL-2, a cytokine reportedly inimical to Th17 differentiation - while “classical” IL-6/TGFβ treatments proved ineffectual. And although MAC-2A cells secrete copious amounts of IL-6, antibody blockade of IL6R (tocilizumab) effected only moderate (circa twofold) inhibition of IL17F expression . Thus collectively, MAC-1/2A cells comprise a dynamic Isogenic systems like the T-cell neoplasia progression model described here - the first reported hitherto - help distinguish indictable mutations from neutral polymorphisms. Key phenotypes studied here were the plasticity of indolent phase cells in response to IL-2, traded against the exuberant cytokine expression of aggressive phase cells, matching that described clinically , 33, 34.in vitro artifact be excluded. Accordingly, the JAK3 (V722I) mutation occurred in three karyotypically distinguishable cell lines studied at early passage: MAC-1/2A as described here plus MAC-2B established along with MAC-2A from a separate skin lesion (data not shown). These observations imply earlier formation in the putative neoplastic stem cell compartment, while rendering implausible an artifactual origin in vitro. Moreover, the identical mutation has been described in a variety of hematopoietic neoplasms, including T-cell lymphoma [in vivo origin is also shown, again in an evolving stem cell. In short, given their multiple occurrence an in vivo origin for both sets of mutations is warranted, plausibly in a neoplastic stem cell.Both JAK3 and SOCS1 mutations involve C > T transitions occurring at CpG sites. C > T enrichment of mutational signatures, for which our sequencing data provide some support, has been attributed to spontaneous deamination of 5-methylcytosines or DNA mismatch repair . The prihe JAK3 V22I mutatAssembling these findings into a unified picture Figure , we propAlthough IL2RG – JAK3 – STAT5 signaling may involve JAK1, the last is usually activated via the IL-2 beta chain receptor . MoreoveEschewal of T-regulatory in favor of Th17 differentiation in the CTCL system described here may be partly attributable to co-induction of IL-21 which is known to promote IL-17 expression at the expense of FOXP3 . CollectSOCS1 inactivating mutations or deletions have been previously shown to abolish control of phospho-JAK2 or phospho-STAT5 signaling in primary mediastinal B-cell and Hodgkin lymphomas , 17, recKeeping step with IL17F and IFN-gamma, TNFRSF9  was conspicuously upregulated by IL-2 treatment. Induction of TNFRSF9 by ionomycin or phorbol 12-myristate 13-acetate (PMA) has already been described . TNFRSF9While this study has focused on STAT5, STAT3 is also implicated in IL-2 signaling via JAK3 in driving IL-17 expression in CTCL . We receOur picture of T-cell plasticity derives from nonmalignant cells, primarily in mouse, as do the key features of main T-helper classes . However, the applicability of these defining characteristics to humans is uncertain. Moreover, the original clear picture of signature cytokines distinguishing four key Th-subsets, Th1, Th2, Th17 and Tregs, has become hazier and multidimensional with the recognition of additional overlapping categories and transgressive plasticity. The most notable discrepancy reported here is the Th17 promotional role of IL-2 and ineffectuality of IL-6 which contradicts that already described in mice. Whether such discrepancies are restricted to CTCL or occur more generally among human Th cells requires additional investigation.This study extends the emerging paradigm of JAK3 activating mutations , 7, 41 tAuthenticated leukemia/lymphoma cell lines established from patients with ALK-positive ALCL (L-82); ALK-negative ALCL (FE-PD), and CTCL  - are detailed in Drexler  and drawhttp://www.dsmz.de). Treatment of cell lines with effector molecules/cytokines/chemicals was performed with indicated factors for various incubation times. For transfer of nucleic acids 80 pmol siRNA construct directed against SOCS1 was tranfected into 1 × 106 cells using the EPI-2500 impulse generator  at 350 V for 10 ms [Cells were cultured as recommended . cDNA was subsequently synthesized from 3 μg RNA by random priming using Superscript II (Invitrogen). RQ-PCR was performed by the 7500 Fast Real-time PCR System  using SsoFast EvaGreen Real Mastermix  and custom designed oligonucleotides (MWG Eurofins (Martinsried/Germany - sequences available upon request). For normalization of expression levels we used TATA box binding protein (TBP). Quantitative analyses were performed in triplicate, repeated on two or more occasions. The analysis of relative quantitative expression was performed using the Applied Biosystems software, followed by statistical analysis and data visualization using the R-based ggplot2 package .http://www.bioconductor.org). Minimum Information about a Microarray Experiment (MIAME) is available upon request for all microarray experiments. Gene ontology and pathway analyses were performed using the Broad Institute Gene Set Enrichment Analysis (http://software.broadinstitute.org/gsea/index.jsp) and DAVID [To parse gene expression, we generated a cDNA expression array using the Affymetrix HGU133plus2 Chip (Santa Clara/CA). For statistical analyses of primary expression data (CEL-files) and subsequent generation of heat maps we used the R-based Bioconductor Linear Models for Microarray Data (LIMMA) package . For thend DAVID  platformWestern blot analysis was performed by the semi-dry method. Proteins obtained from cell lysates were transferred onto nitrocellulose membranes  and blocked with 5% bovine serum albumin (BSA) or milk powder dissolved in phosphate-buffered-saline buffer (PBS). GAPDH served as loading control .8 cells were lysed in  containing protease inhibitors (Sigma). Lysates were precipitated using an anti-SOCS1 antibody  bound to protein-G-sepharose beads (Sigma). Eluted fractions were analyzed by Western Blotting using anti-JAK3 antibody . Image documentation employed the digital ChemoStar Imager system .For co-IP experiments 1.5 × 10Cytokine secretion was measured by enzyme-linked immunosorbent assay (ELISA). For analysis of IFN-gamma, IL-1A, IL-2, IL-4, IL-6, IL-12, IL-13, IL-17A, TARC, TGFβ1, TNFα, a customer designed Mix-N-Match ELISArray Kit (Qiagen) was used, analysis of IL-17F and IL-22 was performed with Quantikine ELISA Kits  following the manufacturer's protocol. Briefly, 50 μl of each sample, control or standard were added to 96-well plates pre-coated with the relevant capture antibodies. After 2 h incubation, plates were washed thrice and 100 μl conjugated detection antibody added to each well, followed by 1 h incubation. Plates were washed and avidin-HRP added and incubated for 30 min, followed by four washes. Development solution was added and plates incubated for 15 min in the dark. Finally, stop solution was added and optical density measured by ELISA reader .Whole exome sequencing was performed by Oxford Gene Technologies (Oxford/UK), using the Illumina HiSeq platform. Enrichment and library preparation samples were prepared according to Agilent's SureSelect Protocol Version 1.2. DNA concentration of each library was determined using Agilent's QPCR NGS Library Quantification Kit (G4880A) and samples were adjusted to a final concentration of 10 nM. Mapping and alignment was performed using 89.9% of the on-target regions covered to a depth of at least 20 ×. A total 16.59 Gbp of sequence data were read and aligned at high quality and 268,493 variations from the reference genome were identified. Reads were mapped according to build hg19/b37 using the Burrows-Wheeler Aligner package, version 0.6.2. Local realignment was carried out with the Genome Analysis Tool Kit (Broad Institute GATK version 1.6). Base quality  scores were recalibrated using GATK's covariance recalibration. SNP and indel variants were called using the GATK Unified Genotyper for each sample. Genotyping includes dbSNP release 135 to improve quality of calls. SNP novelty was determined against dbSNP release 132.For Sanger sequencing, genomic DNA was extracted using the Gentra Puregene DNA-Isolation Kit (Qiagen). PCR primers were obtained from MWG Eurofins (Ebersberg/Germany). Genomic PCR was performed using the C1000 Thermo Cycler . PCR products were separated with 2% agarose gel and purified with the QIAquick Gel Extraction Kit (Qiagen). Purified PCR products were directly sequenced by MWG. PCR products for SOCS1 were cloned into pGEMT vector  for sequencing (MWG).For a conspectus of gene rearrangements the Circos program was used . Sequenc