Interestingly, treatment of patient PBMCs with the Nuclear Factor-Erythroid-2-Related Factor 2 (NRF2) agonist, CDDO-Me(bardoxolone methyl), reduced MCP-1 production but not IL-6 or Interleukin-10 (IL-10) secretion. In enriched monocytes from healthy donors, CDDO-Me(bardoxolone methyl) also reduced LPS-induced MCP1/CCL2 production but did not alter IL-6 or IL-10 secretion. Similar immunomodulatory effects were observed with Compound 7, which activates the NRF2 pathway through a different and non-covalent Mechanism Of Action (MOA). Hence, our findings with CDDO-Me(bardoxolone methyl) and Compound 7 are likely to reflect a generalizable aspect of NRF2 activation. These observed effects were not specific to LPS-induced immune responses, as NRF2 activation also reduced MCP-1/CCL2 production after stimulation with IL-6. Pharmacological NRF2 activation reduced Mcp-1/Ccl2 transcript accumulation without inhibiting either Il-6 or Il-10 transcript levels. Hence, we describe a novel aspect of NRF2 activation that may contribute to the beneficial effects of NRF2 agonists during disease. Our work also demonstrates that the NRF2 pathway is retained and can be modulated to regulate important immunomodulatory functions in burn patient immune cells.Burn patients suffer from immunological dysfunction for which there are currently no successful interventions. Similar to previous observations, we find that burn shock patients  injury) have elevated levels of the innate immune cytokines Interleukin-6 (IL-6) and Monocyte Chemoattractant Protein-1 (MCP-1)/CC-motif Chemokine Ligand 2(CCL2) early after hospital admission . Functional immune assays with patient Peripheral Blood Mononuclear Cells (PBMCs) revealed that burn shock patients (≥15% TBSA) produced elevated levels of MCP-1/CCL2 after innate immune stimulation  Thermal injuries cause approximately 300,000 human deaths per year worldwide and are among the most expensive traumatic injuries due to long-term hospitalization and wound treatment , 2. Two Nrf2-/- mice have shown that the loss of Nrf2 alters inflammatory cytokine production such as Tumor Necrosis Factor-Alpha (TNF-α) and Interleukin 1-beta (IL-1β) in murine fibroblasts and macrophages, respectively [in vitro findings correlate with changes in inflammatory mediator production in vivo during models of sepsis, peritonitis, and burn injury [Nrf2-/- deficient peritoneal macrophages produce elevated amounts of Monocyte Chemoattractant Protein-1 (MCP-1) [In the context of disease, an important regulator of host defense against stress and inflammation is Nuclear Factor-Erythroid-2-Related Factor (NRF2). NRF2 is a transcription factor that regulates over 250 genes . During ectively , 9. Thesn injury –10. Addi (MCP-1) .MCP-1 was the first discovered chemokine and functions by recruiting monocytes and T cells to sites of inflammation . The maiAn agonist of the NRF2 pathway is Bardoxolone methyl (CDDO-Me) . CDDO-Me2/FiO2 ratio) have elevated levels of IL-6 and MCP-1 early(0–48 hours) after hospital admission. LPS-stimulated Peripheral Blood Mononuclear Cells (PBMCs) from moderate/severe burn patients(≥15% TBSA) produced elevated amounts of MCP-1 ex vivo indicating an altered innate immune profile. Treatment of patient PBMCs with CDDO-Me(bardoxolone methyl) effectively reduced MCP-1 production and induced the NRF2-target gene NQO1. Using enriched monocyte populations from healthy donors, we find similar immunomodulatory effects occur with multiple small molecule activators of the NRF2 pathway. NRF2 activation also reduced MCP-1 production in response to the host factor IL-6. Collectively, we demonstrate a connection between selectively activating the NRF2 pathway and inhibiting MCP-1 production that is both novel and may have important clinical implications.In this study, we demonstrate that patients with moderate/severe(≥15% TBSA) or pulmonary dysfunction . Cells were then subjected to Ammonium Chloride Potassium (ACK)  lysis, filtered through a 70μM cell strainer , and washed 2X with HBSS . Cells were counted with a disposable hemocytometer .16 cc of blood was collected from burn patients or ~50 cc of blood was collected from healthy donors . Blood was overlaid onto Histopaque 1077  and centrifuged at 450 x 5 PBMCs were plated in 200 μL of RPMI 1640  with 2% Human Serum , Beta-mercaptoethanol, Sodium Pyruvate, Non-essential Amino Acids  and antibiotics  in 96-well plates . Monocyte purification was performed with CD14 microbeads according to the manufacturer’s directions. Purified monocytes were re-suspended in RPMI media(See above) and plated the same as PBMCs. PBMCs and monocytes were treated with CDDO-Me(bardoxolone methyl), Vehicle (0.00005% DMSO), Compound 7 [g for 5 minutes and supernatants were collected. Cell pellets were subjected to protein or RNA analysis (See below).5 x 10olGibco, 985–027, Protein lysates were generated using RIPA buffer  with 1% SDS, 5mM DTT, Protease Inhibitor , and Phosphatase inhibitors . Lysates were resolved on Novex 4–12% gels  and wet-transfer blotting was performed onto PVDF filter paper . Membranes were blocked with 5% milk in a 1X TBST buffer  for 1 hour at room temperature. Blots were washed 3X with 1X TBST and subjected to over-night incubation at 4’C with anti-NQO1  or anti-GAPDH-HRP antibodies. Membranes were then washed 3X with 1X TBST and incubated for 1 hour at room temperature with an Perioxidase-conjugate AffiniPure Anti-Rabbit IgG antibody . Blots were washed 3X with 1X TBST and developed with Super Signal West Femto . Chemiluminescence was measured with a Biorad Chemi-dock system. ImageJ software was used to quantify the intensity of the NQO1 bands relative to the intensity of the loading control (GAPDH). For each lane, a ratio of NQO1/GAPDH was generated and relative values were determined based on setting the NQO1/GAPDH ratio for the unstimulated, vehicle control-treated cells to 1.Mcp-1 , Il-6 , Il-10 , Nqo-1 , Hmox-1 , and Gapdh  using TaqMan Gene Expression Master Mix . Relative target transcript expression to Gapdh was determined with the 2-ΔΔCT method.RNA was isolated using RNeasy Mini Kit and cDNA was synthesized using SuperScript VILO Master Mix Kit. Real time PCR was performed for 6 PBMCs or enriched monocytes(CD14+) were washed 2X in FACS buffer) before 1 hour incubation on ice with Human TruStain FcX  according to the manufacturer’s directions. Cells were pelleted and washed 3X with FACS buffer and stained with CD11b-FITC and HLA-DR-APC, according the manufacture’s protocol. Stained cells were acquired using a LSRII Fortessa flow cytometer and analyzed with FlowJo software.~2 x 10Plasma and cellular supernatants were measured using ELISA kits for MCP-1 , IL-6 , TNFα , IL-8 , MCP-2 (CCL8) , and IL-10 . 1-Step Slow TMB ELISA substrate  and Stop Solution  were used for colorimetric detection.Analysis of Variation (ANOVA), T-tests, Chi Square (with Fisher’s Exact Test) analysis and interpolation of cytokine values were performed with GraphPad Prism 6 Software.P < .01).There were a total of 50 patients admitted that met criteria to be included in our analysis. Clinical characteristics of the burn patient cohort are shown . The patP < .0001 and P < .001, respectively). An important resource that is used to quantified the level of a critical ill patients’ severity of illness is the Sequential Organ Failure Assessment (SOFA) score [2/FiO2 ratio of <357 represents dysfunction of the pulmonary compartment [2/FiO2 ratio) at 0–24 HPA also had significantly heightened levels of IL-6 and MCP-1 (P < .05). Longitudinal analysis of systemic IL-6 and MCP-1 levels revealed that differences between the mild (<15% TBSA) and moderate/large (≥15% TBSA) burn injury groups were most consistent early post-hospital admission  analysis in plasma fractions for IL-6 and MCP-1. Within the first 48 Hours Post-hospital Admittance (HPA), the moderate/severe burn injury group (≥15% TBSA) had significantly elevated levels of IL-6 and MCP-1 (A) score . Within partment . Indepennd MCP-1 , P < .05dmission .Nrf2-dependent in vivo [Since in many cases the cellular sources of IL-6 and MCP-1 are innate immune cells, we next evaluated how burn injury influenced the production of these cytokines after innate immune stimulation. Patient PBMCs were stimulated with LPS and supernatants were assayed for the production of IL-6 and MCP-1. Interestingly, the moderate/severe burn injury patient group had no difference in LPS-induced IL-6 secretion but produced more MCP-1 after innate immune stimulation . We next in vivo  and has  in vivo . Our resThe analysis was expanded across the burn patient cohort and we found that treatment of PBMCs with CDDO-Me(bardoxolone methyl) significantly reduced LPS-induced MCP-1 production but had no distinct effects on either IL-6 or IL-10 secretion . MeasureSince PBMCs are a heterogenous population of mononuclear immune cells , we assessed if activating the NRF2 pathway could effectively modulate MCP1 production within a homogenous monocyte population. Monocytes were enriched from healthy donor PBMC populations  and stimWe next evaluated a second, independently synthesized, NRF2 agonist to determine if MCP-1 reduction was a general aspect of pharmacological NRF2 activation. Compound 7 is a new and selective small molecule activator of the NRF2 pathway that functions through blocking the KEAP1-NRF2 interface . Hence, In vitro studies have demonstrated that IL-6 can induce MCP-1 production in human PBMCs and monocytic cell lines [Systemic IL-6 is elevated in burn shock patients and correlates with an increased risk of mortality . In vitrll lines , 26. We Mcp-1 transcript. Real time PCR analysis revealed that NRF2 activation with CDDO-Me(bardoxolone methyl) reduced Mcp-1 transcript levels but did not inhibit LPS-induced transcript accumulation of either Il-6 or Il-10  in stroke patients [Mcp-1 through pharmacological NRF2 activation may provide unique benefit.MCP-1 is a multifaceted component of the immune system that has numerous genetic and therapeutic connections to human disease. Genetic polymorphisms in patients . Elevatepatients , 30. Addpatients . From a patients . In humapatients . Therapepatients . Importapatients . Hence, Nrf2-/- mice have shown that the NRF2 pathway has a beneficial function in models of Acute Kidney Injury (AKI), Atherosclerosis, and Sepsis [Nrf2-/- mice suffered higher levels of mortality [Nrf2-/- mice correlated with 1) pulmonary immune cell infiltration, 2) elevated systemic levels of pro-inflammatory cytokines, and 3) intestinal permeability [in vivo activation of the NRF2 pathway led to improved physiological outcomes in an animal model of pulmonary hypertension [Keap1 and Nrf2 are associated with tumorigenesis and clinical outcomes in cancer patients [Nrf2 are also associated with blood pressure and cardiovascular mortality in hemodialysis patients [Nrf2 are associated with an increased risk for the development of Acute Lung Injury (ALI) [Both genetic and therapeutic studies have demonstrated a protective role for the NRF2 pathway during disease. Characterization of d Sepsis , 35, 36.ortality . The eleeability . Additiopatients . Geneticpatients . In the ry (ALI) . Additiory (ALI) .Nrf2-/-, Keap1-/-), and the usage of different small molecule activators of the NRF2 pathway , Compound 7, Diethylmaleate (DEM), etc.). It is imprudent to assume that pharmacological activation of the NRF2 pathway will have the opposite biological effect than Nrf2 genetic deficiency. Using Nrf2-/- mice, studies have shown that peritoneal macrophages and Mouse Embryonic Fibroblasts (MEFs) have enhanced NF-κB signaling which coincided with heightened TNFα production [Keap1-/- deficient mice, which have elevated NRF2 activation, have reduced transcript accumulation of Il-6 and Il-1β but not Tnfα in murine Bone Marrow-derived Macrophages (BMMs) [Il-6 and Il-1β transcript accumulation in both murine BMMs and immortalized human monocyte cells (THP-1) [κB p65 recruitment to the Il-6 and Il-1β promoters [Nrf2-/- mice produce reduced amounts of IL-1β [κB signaling is required for Nlrp3 expression and therefore subsequent NLRP3 inflammasome function [κB pathway. It is possible that different levels of NRF2 activation may induce unique immunomodulatory effects regarding innate immune cytokine production and the NF-κB pathway. This possibility is consistent with the findings that pharmacological NRF2 activation can induce either apoptosis or cytoprotection based on compound dosage.The role of the NRF2 pathway during innate immune signaling is complex due to variable host genetic systems  . Additio (THP-1) . This woromoters . Alternaof IL-1β , indicatfunction . Hence, Il-6 transcript accumulation although there was target engagement as measured by elevated expression of Nqo-1 [In PBMCs and monocytes from sepsis patients, CDDO-Me(bardoxolone methyl) treatment did not reduce LPS-induced of Nqo-1 . Using pof Nqo-1 , 21. Henof Nqo-1 .Small molecule agonists of the NRF2 pathway are in various stages of clinical development. Dimethyl Fumarate(Tecfidera) is an effective treatment for Multiple Sclerosis patients and has been shown to induce NRF2 activity in patients . SulforaWe demonstrate that induction of the NRF2 pathway has immunological properties that could be beneficial in numerous contexts. It is worth noting that a phase III trial with CDDO-Me(bardoxolone methyl) for Chronic Kidney Disease (CKD) was terminated due to adverse cardiovascular events in a patient population with severe kidney disease  althoughS1 Fig(A) Scatter plot analysis of systemic IL-6 levels between mild  and moderate/severe  patients at 72–144 HPA. (B) Scatter plot analysis of systemic MCP-1 levels between mild  and moderate/severe  patients at 72–144 HPA. Error bars represent ± SEM.(TIF)Click here for additional data file.S2 Fig(A) Relative intensity of NQO1 expression ratio among the indicated treatment groups. Values are relative to unstimulated, vehicle-treated PBMCs for each patient. In the figure legend, values represent nM concentrations of CDDO-Me(bardoxolone methyl). Results represent the combination of the 4 patient samples shown in (TIF)Click here for additional data file.S3 Fig(A) Top. Dot plot analysis depicting Forward and Side Scatter Events (K) after CD14 microbead-mediated enrichment. Bottom. Dot plot analysis depicted expression of the monocyte markers HLA-DR and CD11b in cell populations after CD14 microbead-mediated enrichment. Results are representative of 2 independent donors.(PNG)Click here for additional data file.S4 Fig(A) ELISA analysis for TNFα in healthy donor monocytes after treatment with CDDO-Me(bardoxolone methyl) (50nM) at indicated Hours Post-LPS Stimulation (HPS). Results are representative of 5 independent donors. (B) Bar graph depicting the percentage of CDDO-Me(bardoxolone methyl)-altered TNFα production at 4, 8, 16 HPS. Relative values represent the combination of 5 independent sets of healthy donor monocytes. Error bars represent SEM.(TIF)Click here for additional data file.S5 Figp < .05.(A) ELISA analysis for IL-8 in healthy donor monocytes after treatment with CDDO-Me(bardoxolone methyl) (50nM) at indicated Hours Post-LPS Stimulation (HPS). Results are representative of 3 independent donors. (B) Bar graph depicting the percentage of CDDO-Me(bardoxolone methyl)-altered IL-8 production at 4, 8, 16, 24 HPS. Relative values represent the combination of 3–4 independent sets of healthy donor monocytes. (C) ELISA analysis for MCP-2 in healthy donor monocytes after treatment with CDDO-Me(bardoxolone methyl) (50nM) at indicated Hours Post-LPS Stimulation (HPS). Results are representative of 7 independent donors. (D) Bar graph depicting the percentage of CDDO-Me(bardoxolone methyl)-altered MCP-2 production at 4, 8, 16, 24 HPS. Relative values represent the combination of 7 independent sets of healthy donor monocytes. ns = not significant. Error bars represent SEM. * = (TIF)Click here for additional data file.S6 FigHo-1) in healthy donor monocytes treated with CDDO-Me(bardoxolone methyl) (50nM). Values are relative to unstimulated, vehicle treated cells at 16 Hours Post-LPS Stimulation (HPS). Results are representative of 2 independent donors. Error bars represent SEM. * = p < .05.(A) Real time PCR analysis for Heme Oxygenase-1((TIF)Click here for additional data file.S7 FigRaw data values for all graphs.(XLSX)Click here for additional data file.


In vitro colony-forming unit cell (CFU-C) assays are usually-used to detect the quantitative and qualitative features of haematopoietic stem cells (HSCs). We studies CFU-C assays in bone marrow samples from 365 consecutive subjects with newly-diagnosed myelodysplastic syndrome (MDS). Data were interrogated for associations with prognosis.CFU-C assays were performed according to the protocol of MethoCultTM H4435 Enriched. 365 consecutive newly-diagnosed, untreated subjects with MDS diagnosed from July, 2007 to April, 2014 were studied. All subjects were reclassified according to the 2008 WHO criteria. Subjects were observed for survival until July 31, 2015. Follow-up data were available for 289 (80%) subjects. Median follow-up of survivors was 22 months  months. Erythroid and myeloid colonies were isolated from each subject with one cytogenetic abnormality such as del(5/5q), +8, del(7/7q) or del(20q). Cytogenetic abnormalities of each colony were analyzed by fluorescence in situ hybridization (FISH). SPSS 17.0 software was used to make statistical analysis.P = 0.005) in very high-risk cohort.The numbers of burst-forming units-erythroid (BFU-E), colony forming unit-erythroid (CFU-E) and colony forming unit-granulocytes/macrophages (CFU-G/M) were significantly lower than normals. A high ratio of cluster- to CFU-G/M was associated with poor-risk cytogenetics. In multivariable analyses a cluster- to CFU-G/M ratio >0.6 was an independent risk-factor for OS after adjusting for IPSS-R (HR 3.339, [95%CI 1.434-7.778]; in vitro parallel the ineffective hematopoiesis typical of MDS and may be useful in predicting outcomes of persons with higher-risk MDS.These data suggest abnormalities of proliferation and differentiation of erythroid and myeloid precursor cells  Myelodysplastic syndrome (MDS) is a heterogeneous neoplasm characterized by ineffective hematopoiesis with resultant cytopenias and a substantial risk of progression to acute myeloid leukemia (AML) , 2. ThisIn vitro colony-forming unit cell (CFU-C) assays are usually-used to detect the quantitative and qualitative features of haematopoietic stem cells (HSCs) from normals and persons with diverse haematologic abnormalities including MDS [in vitro parallel the ineffective hematopoiesis typical of MDS and a cluster- to CFU-G/M ratio >0.6 was an independent risk-factor for overall survival after adjusting for conventional prognostic scoring systems [ding MDS . Lower tding MDS -13. We a systems .Numbers of BFU-E and CFU-E in subjects with different forms of MDS were significantly lower than normals, and the ratio of cluster-G/M was higher than normals Table . The numn situ hybridization (FISH) analyses of interphase nuclei to determine whether CFU-C from subjects with MDS were from the MDS clone or from residual normal progenitor cells . Numbers of CFU-G/M were significantly associated with neutrophil level (P = 0.032). Subjects with hemoglobin concentrations < 100g/L (P = 0.001) or platelets <30×10E+9/L (P = 0.006) had significantly fewer BFU-E. Ratio of cluster- to CFU-G/M was not significantly-associated with percent bone marrow blasts. Subjects with higher-risk IPSS-R (P = 0.04) or more complex chromosome abnormalities (P = 0.039) were more likely to have a higher ratio of cluster- to CFU-G/M than those in the lower risk cohort or those with non-complex chromosome abnormalities  in very high-risk cohort.Subjects with a >0.6 ratio of cluster- to CFU-G/M were more likely to have briefer OS . 240 (66%) were male. Cases were classified according to the 2008 WHO criteria . 5 subjeSubjects were observed for survival until July 31, 2015. Follow-up data were available for 289 (80%)subjects. Median follow-up of survivors was 22 months  months. 142 (39%) subjects received immune suppressive drugs including cyclosporine and thalidomide. 46 subjects (13%) received anti-cancer therapy(ies) including aclacinomycin or homoharringtonine combined with cytarabine and granulocyte-colony stimulating factor , idarubicin or daunorubicin combined with cytarabine (IA or DA) or melphalan. 75 (21%) subjects received erythropoietin with or without G-CSF, RBC and/or platelet transfusions and/or iron chelation with desferrioxamine. 11 (3%) subjects received decitabine, 19 (5%) an allotransplant and 72 (20%), traditional Chinese medicines. Subjects receiving an allotransplants were censored in survival analyses.5-10 mL of bone marrow was collected in sterile, preservative-free heparin tubes and mononuclear cells were separated by Ficoll-Hypaque gradient density centrifugation. CFU-C assays were performed using the MethoCultTM H4435 Enriched . Briefly, cells were plated at 1×10E+5 cells/mL into 2 wells (1 ml/well) of 24-wells plates and placed into an incubator maintained at 37°C with 5% CO2 and >85% humidity. A colony was defined as an aggregate of >40 cells. Clusters consisted of 4 to 40 cells. Colony forming unit-erythroid (CFU-E) Figure  and 3B wCells were isolated from individual BFU-E and CFU-G/M after 14 days of culture and centrifuged onto microscopic slides. ≥5 erythroid and myeloid colonies were isolated from each subject with one cytogenetic abnormality such as del(5/5q), +8, del(7/7q), or del (20q). ≥8 colonies were analyzed by FISH using DNA probes to detect del(5/5q), +8, del(7/7q) and del(20q): 5p (D5S721), 5q (EGR1), CEP8, CEP7, D7S486 and D20S108 . Probe sensitivities were D5S721, 3%, EGR1, 3%, CEP8, 2.5%, CEP7, 2.5%, D7S486 3%, D20S108, 2%. At least 100 interphase nuclei per colony were analyzed. Hybridization and analysis were performed by standard techniques.The Mann-Whitney U-test or Kruskal-Wallis analysis of variance were used for continuous variables. Survival distributions were estimated by the Kaplan-Meier method. Cox proportional hazards regression model was used to assess the correlation between variables and survival. Overall survival (OS) was defined as time from diagnosis to date of death or last seen. Two-tailed P-values ≤0.05 were considered significant.


Scientific Reports 10.1038/s41598-017-17777-1, published online 11 Decemeber 2017Correction to: This Article contains typographical errors in the Acknowledgements section.“Authors gratefully acknowledge the financial support from Russian National Foundation under the grant 15-11-00033.”should read:“Authors gratefully acknowledge the financial support from Russian Science Foundation under the grant 15-11-00033.”


Theobroma cacao production, which can be intensified by global climate changes. In this study, we aimed to investigate the phenotypic correlation among morphological characteristics of cacao progenies submitted to irrigation and drought conditions and their partitions into direct and indirect effects. Path analysis with phenotypic plasticity index was used as criteria for estimation of basic and explanatory variables. The experiment was conducted in a greenhouse at the Cacao Research Center (CEPEC), Ilhéus, Bahia, Brazil, in a randomized block 21 x 2 factorial arrangement [Drought is worldwide considered one of the most limiting factors of  Theobroma cacao L.) productivity  and 6 replications. The average of each progeny in different water regimes were compared with the overall average of each growth variable using Student’s t test (p < 0.05 and p < 0.01).The data were analyzed as a randomized block design considering 42 treatments  .Regarding plant height (H) and stem diameter (SD), under drought conditions, only progenies originated from MOC-01 x TSH-1188 (11) and PUCALA x Catongo (14) crosses showed significant reductions of their averages, with values below the overall average. Under the same conditions, progenies of SCA-6 x IMC-67 (5) and RB-40 x MOC-01 (16) showed values above the expected average.Reduction in the leaf number (LN) and of total leaf area (TLA) was also observed in progenies subjected to soil water limitation. Progenies of RB-40 x IMC-67 (2), SCA-6 x IMC-67 (5) and Catongo x TSH-1188 (9) showed values above the overall average. In contrast, TSH-1188 x SCA-6 (10), PUCALA x Catongo (14) and PUCALA x IMC-67 (20) showed significant reductions in leaf area when subjected to soil water deficit.The decrease of leaf area (TLA) had a direct impact in the leaf biomass (LB) of cacao progenies submitted to soil water deficit, reducing the overall average by almost 20% when compared to the control . HoweverProgenies of RB-40 x SCA-6 (1), SCA-6 X IMC-67 (5), Catongo X IMC-67 (12) and RB-40 x MOC-01 (16) showed increment in stem biomass (SB) above the overall average under limiting soil water conditions. In contrast, progenies of MOC-01 x SCA-6 (7), PUCALA x Catongo (14), PUCALA x SCA-6 (19) and PUCALA x IMC-67 (20) showed averages below the overall average.Under water limitation, the increase in root biomass was higher than the control, with effects in RB, R/S, RA and RV . The proRegarding fine-roots length RD[<1mm], comparing the overall mean of the variable with the average of each progeny it was found that SCA-6 x IMC-67 (5), PUCALA x MOC-01 (8) and PUCALA x Catongo (14) progenies showed a significant increase of fine root values under drought stress . Also, cWL between –0.1 and –0.5 MPa; and drought: ΨWL between –2.0 and –2.5 MPa), demonstrating different schemes of phenotypic plasticity in response to drought. In this study, RV showed the highest IPF value (0.86), which points out the high plasticity in response to drought  was positively correlated with RB (0.66**), SB (0.74**), LB (0.77**) and TB (0.82**) under control condition . Under dThe root volume (RV) was positively correlated with TB (0.45*) under drought conditions . The incA breakdown of phenotypic correlations in direct and indirect effects, through path analysis, indicated that TLA (0.11), LB (0.27), RD[<1mm]  and RD[1-2mm]  showed the largest direct effects on the development of the root system (RV) of the cacao progenies under control conditions . On the The variables SD, RL and RD showed a positive and significant correlation with root volume (RV) under drought conditions. Despite the direct effect of RD[1-2mm] on RV in the drought conditions, the correlation, although positive, was not significant. This may also have been due to the growth of coarse roots (RD[>2mm] (–0.28).Water stress is one of the factors limiting agricultural production at the global scale , 36. TheCoffea canephora also detected differences between drought tolerant and sensitive clones  and RD[1-2mm] above the overall average across the sample . This caThe root volume (RV) was the growth variable most influenced by the water regimes among the analyzed cacao progenies . These rUnder drought conditions, stem diameter (SD), leaf dry biomass (LB), average root diameter (RD) and fine roots (RD[<1mm] were the growth variables exerting the greatest direct influence on the progenies root volume increase . These rThere was a direct positive effect of stem diameter (SD) in root volume (RV) increase under conditions of low soil water availability . The eff2 assimilation rate to produce carbohydrates. The need to solve this conflict takes the plant to develop morphophysiological mechanisms that lead to conserve water for use in later periods  = fine roots length (cm cm-3); RD[1-2mm] = medium roots length (cm cm-3); RD[>2mm] = coarse roots length (cm cm-3).Mean values for growth characters of cacao progenies, obtained from complete diallel crosses, under control (predawn Ψ(XLSX)Click here for additional data file.


The switch between latency and the lytic cycle of Kaposi’s sarcoma-associated herpesvirus (KSHV) is controlled by the expression of virally encoded ORF50 protein. Thus far, the regulatory mechanism underlying the protein stability of ORF50 is unknown. Our earlier studies have demonstrated that a protein abundance regulatory signal (PARS) at the ORF50 C-terminal region modulates its protein abundance. The PARS region consists of PARS-I (aa 490–535) and PARS-II (aa 590–650), and mutations in either component result in abundant expression of ORF50. Here, we show that ORF50 protein is polyubiquitinated and its abundance is controlled through the proteasomal degradation pathway. The PARS-I motif mainly functions as a nuclear localization signal in the control of ORF50 abundance, whereas the PARS-II motif is required for the binding of ubiquitin enzymes in the nucleus. We find that human oncoprotein MDM2, an ubiquitin E3 ligase, is capable of interacting with ORF50 and promoting ORF50 degradation in cells. The interaction domains between both proteins are mapped to the PARS region of ORF50 and the N-terminal 220-aa region of MDM2. Additionally, we identify lysine residues at positions 152 and 154 in the N-terminal domain of ORF50 critically involved in MDM2-mediated downregulation of ORF50 levels. Within KSHV-infected cells, the levels of MDM2 were greatly reduced during viral lytic cycle and genetic knockdown of MDM2 in these cells favored the enhancement of ORF50 expression, supporting that MDM2 is a negative regulator of ORF50 expression. Collectively, the study elucidates the regulatory mechanism of ORF50 stability and implicates that MDM2 may have a significant role in the maintenance of viral latency by lowering basal level of ORF50. The switch of Kaposi’s sarcoma-associated herpesvirus (KSHV) from latency to lytic replication is an essential process for controlling viral dissemination and the pathogenesis of KSHV-associated diseases. The virally encoded ORF50 protein is known as the master controller of viral reactivation. We previously reported that a two-component PARS region, which contains PARS-I and PARS-II, in ORF50 influences its protein abundance. In this study, we elucidate the regulatory mechanism of the ORF50 protein stability. We find that degradation of ORF50 involves at least two critical steps, including nuclear translocation and the subsequent degradation in the nucleus. The PARS-I motif is responsible for the nuclear translocation of ORF50, whereas the PARS-II motif is required for ORF50 degradation in the nucleus. Furthermore, we show that the PARS region is the primary docking site for cellular oncoprotein MDM2, an E3 ubiquitin ligase. Direct interaction of MDM2 with ORF50 promotes ORF50 degradation. The putative ubiquitin acceptor sites targeted by MDM2 are mapped to K152 and K154 in the N-terminal region of ORF50. In the context of KSHV-infected cells, we also provide evidence that MDM2 is involved in the regulation of the ORF50 expression. Kaposi’s sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus-8 (HHV-8), is the etiologic agent of Kaposi’s sarcoma (KS), primary effusion lymphomas (PELs) and multicentric Castleman’s disease –3. Numerreplication and transcription activator), is a multifunctional protein, which transcriptionally activates many KSHV or cellular genes and is involved in the assembly of replication complexes required for lytic DNA synthesis [protein abundance regulatory signal) that controls the protein abundance of ORF50. The PARS is composed of two sub-components: PARS-I and PARS-II. PARS-I region partially overlaps DBIS (aa 490–535), whereas the PARS-II region is located between aa 590 and 650  large T antigen to the C-terminus of GFP-ORF50 and GFP-ORF50(KK/EE), two green fluorescent protein (GFP) tagging constructs . As expeSince OFR50 oligomerizes , this stWe created C-terminal deletions in F-ORF50 and F-ORF50(KK/EE), from amino acid 650, 590, 564, and 537 , to study the action of PARS-II . ConfocaBesides wild-type GFP-ORF50, we tested whether different PARS-II deletion effectors, including GFP-ORF50(1–590) and GFP-ORF50(1–564), could rescue the subcellular localization and protein abundance of F-ORF50(KK/EE) . Both GFWe further investigated whether the abundance of ORF50 is attributed to the control by the ubiquitin-proteasome pathway. We transfected 293T cells with pCMV-FLAG-ORF50 to express F-ORF50, and then treated the cells with a 26S proteasome inhibitor, MG132, at 16 h after transfection. In an immunoblot study, we found that the amount of F-ORF50 in the cells that were treated with MG132 was higher than the protein from the cells untreated with MG132 . ImmunobTo further map the critical regions in ORF50 that are responsible for ubiquitination, we first tested the PARS-II deletion mutant F-590 . 293T ceTo identify the E3 ubiquitin ligase of ORF50, the possible roles of cullin-RING E3 ligases, the largest family of ubiquitin ligases , in the in vivo. Accordingly, a lysate was prepared from 293T cells that had been transfected with a plasmid expressing GFP-ORF50. Immunoblotting revealed that anti-MDM2 antibody not only immunoprecipitated MDM2 but also coimmunoprecipitated GFP-ORF50  and His-tagged ORF50 (His-ORF50). The integrity of both GST-MDM2 and His-ORF50 proteins was verified by immunoblotting analysis using at least two different antibodies that recognize either the N-terminal or C-terminal part of the target proteins . Additio E. coli . Only MD E. coli . The mut E. coli , lane 6, E. coli . Our res E. coli , lane 8.in vitro ubiquitination assay using purified components. The reconstitution reactions contained purified His-ORF50, GST-MDM2, E1, E2 (UbcH5B), Mg-ATP and ubiquitin. We did not detect ubiquitination of His-ORF50 by GST-MDM2 when Mg-ATP was omitted in the reaction , but not the PARS-II region only (from aa 590 to 691), is sufficient to interact with MDM2 . These rin vitro ubiquitination assay, we showed that purified GST-MDM2 could promote ubiquitination of His-ORF50 protein expressed in E. coli . The QuickChange site-directed mutagenesis kit (Stratagene) was used to introduce point mutations at the indicated sites of the orf50 gene. Plasmids encoding GFP-ORF50 and its mutant derivatives were constructed by inserting appropriate orf50 gene fragments into pEGFP-C2 (Clontech). To construct the plasmids expressing GFP-ORF50+NLS and GFP-ORF50(KK/EE)+NLS, double-stranded oligonucleotides corresponding to the coding sequence of the SV40 NLS were first inserted into pEGFP-C2, and then specific DNA fragments of the orf50 gene were cloned upstream of the SV40 NLS coding sequence. The coding sequence of the SV40 NLS is 5’-ATGGGCCCTAAAAAGAAGCGTAAAGTC, which encodes a peptide MGPKKKRKV. The plasmid pCMV6-XL-MDM2, an MDM2 expression vector, was purchased from OriGene . Plasmids that expressed GST-MDM2, GST-MDM2(1–220), GST-MDM2(100–291), and GST-MDM2(221–491) were generated by inserting corresponding MDM2 DNA fragments into pGEXT-4T1 . For expression of the recombinant His-tagged ORF50 or its deletion mutants in bacteria, DNA fragments encoding specific ORF50 domains were inserted into either pET-32a or pET-22b (Novagen).Several plasmids that express wild-type FLAG-tagged ORF50 (pCMV-FLAG-ORF50) and its mutants used in the study have been described previously . For gen5) were grown on coverslips in 6-well tissue culture plates and transiently transfected with plasmids for 24 hr. After transfection, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C for 30 min, and then permeabilized with 0.1% Triton X-100 in PBS for 7 min. To detect the FLAG-tagged proteins, cells were initially incubated with blocking solution (1% BSA) at room temperature for 1 hr, and then treated with polyclonal rabbit anti-FLAG antibody  for 2 hr. After three washing steps, Alexa Fluor 594-conjugated goat anti-rabbit IgG polyclonal antibody  was added to treat the cell samples. Staining with 4′-6-diamidino-2-phenylindole (DAPI) was performed at room temperature for 15 min. Cells were mounted in Citifluor (Agar Scientific) and observed under a confocal laser-scanning microscope  [293T cells  .Cell extracts were prepared and mixed with 3x sodium dodecyl sulfate (SDS) gel loading buffer. After the mixtures were boiled for 5 min, proteins were resolved on an 8% to 12% polyacrylamide gel. The proteins on the gels were then transferred onto a PVDF membrane (Bio-Rad) and were probed with specific antibodies. The anti-ORF50 polyclonal antibody used in the study was generated in our laboratory using purified ORF50(333–691) as an immunogen. Antibodies to FLAG , tubulin , GFP , HA , MDM2 , K8 , ORF45  and actin  were purchased commercially.7) were transfected with pCMV-HA-Ub and pCMV-FLAG-ORF50 or ORF50 mutant constructs. At 16 hr posttransfection, cells were further treated with 5 μM MG132 for 24 h to stabilize the ubiquitinated proteins. Cells were harvested and lysed in 200 μl of the ubiquitination protective buffer  [293T cells (1×10.1% SDS) , 40. Aft293T cells were cotransfected with pCMV-FLAG-ORF50 and pCMV6-XL-MDM2 or vector control. At 19 hr posttransfection, cells were treated with 75 μg/ml cycloheximide (Sigma-Aldrich) to inhibit de novo protein synthesis. At indicated time points after treatment with cycloheximdie, cells were harvested and subjected to immunoblot analysis. Relative levels of F-ORF50 normalized to α-tubulin were quantified by densitometry.293T cells were transfected with pEGFP-C2 or pCMV-GFP-ORF50 for 30 hr, whereas HH-B2 cells were untreated or treated with 3 mM SB for 6 hr, and then incubated with 5 μM MG132 for another 20 hr. Cell samples were harvested and resuspended in radioimmunoprecipitation assay (RIPA) buffer  . ProteinE. coli BL21(DE3). The bacterial cells expressing recombinant proteins were harvested and were lysed in NETN buffer (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% NP-40) [GST-MDM2 fusion proteins and His-tagged ORF50 proteins were expressed in % NP-40) . In the in vitro ubiquitinylation reaction was incubated at 37°C for 90 minutes.The complete reaction mixture (25 μl) contained 100 nM E1 , 2.5 μM E2 (UbcH5B) , 11 μM Ub , 5 mM Mg-ATP , His-ORF50 (120 nM) and GST-MDM2 (100 or 200 nM) in the ubiquitinylation buffer . In some reactions individual components were omitted or substituted as indicated. The MDM2 shRNA is 5’-ATTATCTGGTGAACGACAAAG. For lentiviral transduction, HH-B2 or BC3 cells were infected with the collected lentiviruses in the presence of polybrene at a final concentration of 8 μg/ml. After 24 hr post-infection, HH-B2 and BC3 cells were left untreated or treated with 3 mM SB for another 18 hr.The preparation of shRNA lentiviral particles has been described previously . All RNAS1 FigProtein extracts of HKB5/B5 cells transfected with pCMV-FLAG-ORF50 or pCMV-FLAG-ORF50(KK/EE) were treated with calf intestinal phosphatase (CIP) or lambda protein phosphatase (λPPase). After 1.5 hr incubation at the indicated temperature, the treated cell extracts were analyzed by immunoblotting using anti-FLAG antibody. ORF50-A (110 kDa): a hyperphosphorylated form of ORF50; ORF50-B (90 kDa): a hypophosphorylated form of ORF50.(EPS)Click here for additional data file.S2 Fig293T cells were transiently transfected with the indicated plasmids for 24 hr. Cells were fixed, permeabilized, and probed with anti-FLAG antibody. Images were taken with the same settings on the confocal microscopy. The observed degree of ORF50 abundance in 293T cells is evaluated by “+”. N: nucleus; C: cytoplasm; N/C: both nucleus and cytoplasm.(EPS)Click here for additional data file.S3 FigAfter transfection with the indicated plasmids for 24 hr, cells were harvested, fixed, permeabilized, and probed with anti-FLAG antibody. All images were taken with the same settings on the confocal microscopy. The observed degree of the abundance of ORF50 mutants in 293T cells is evaluated by “+”. N: nucleus; C: cytoplasm; N/C: both nucleus and cytoplasm.(EPS)Click here for additional data file.S4 FigTotal protein lysates of 293T cells that were transfected with plasmids expressing wild-type F-ORF50 or its deletion mutants were prepared for immunoblotting analysis. Each lane in the assay contains 20 μg of lysate protein. An HRP-conjugated anti-FLAG antibody (A) and various HRP-conjugated anti-immunoglobulin antibodies (B-G) were used in immunoblotting analysis. The epitope that is recognized by these secondary antibodies is mapped to the C-terminal region from aa 650 to 691 of ORF50 protein.(EPS)Click here for additional data file.S5 Fig(A and B) Effect of dominant-negative cullin expression on ORF50 stability. 293T cells were cotransfected with pCMV-FLAG-ORF50 and the indicated plasmids expressing dominant negative cullins . All plasmids encoding dominant negaive cullins were gifts from Dr. Wader Haper’s lab . The expression levels of F-ORF50 and dominant negative cullins in cells were determined by immunoblotting using anti-FLAG antibody. (C and D) Effect of UBE3A or GFP-RNF4 expression on ORF50 abundance. Increasing amounts of an UBE3A or GFP-RNF4 expression plasmid were cotransfected with the F-ORF50 expression plasmid in 293T cells. At 24 hr after cotransfection, immunoblotting analysis was carried out to determine the expression of F-ORF50, UBE3A and GFP-RNF4.(EPS)Click here for additional data file.S6 FigAt 24 hr after transduction with lentiviruses expressing sh-Luc or sh-MDM2, cells were then transfected with the plasmid expressing either F-ORF50 or F-590 for another 24 hr. The expression of ORF50 proteins and MDM2 was determined by immunoblotting.(EPS)Click here for additional data file.S7 FigB. subtilis) was used a negative control. (C) Detection of the interaction between His-ORF50 and cellular RBP-Jκ in pull-down assay. Total protein lysates of 293T cells that were transfected with the plasmid expressing FLAG-tagged RBP-Jκ (F-RBP-Jκ) were used in pull-down assay. Cellular F-RBP-Jκ was pulled down by His-ORF50-Ni-NTA resins, but not pulled down by control Ni-NTA resins.(A) Immunoblotting analysis of GST-MDM2 using different antibodies  that recognize either the N-terminal or C-terminal part of MDM2. (B) Immunoblotting analysis of His-ORF50 using different polyclonal rabbit antibodies that recognize the N-terminal ORF50(1–590) or C-terminal ORF50(333–691) region. Both polyclonal anti-ORF50 antibodies were generated in our laboratory. An unrelated His-tagged protein (FenB protein of (EPS)Click here for additional data file.S8 Fig(A) Diagram of the positions of all 25 lysine residues in ORF50. The N-terminal 356-aa region contains 19 lysine residues and is divided into three clusters, including N7, M8 and C4. (B-D) Immunoblotting analysis of K-to-R mutants in 293T cells. Cells were transfected with the indicated plasmids for 24 hr. Cell lysates were then immunoblotted using anti-FLAG antibody. Note that only the mutant with substitutions at residues K152 and K154 (K152/154) shows an elevated protein expression.(EPS)Click here for additional data file.S9 Fig(A and B) Flow cytometric analysis of ORF50 and MDM2 expression in HH-B2 and BC3 cells. The lentiviral vector-mediated MDM2 knockdown was performed in HH-B2 and BC3 cells. At 24 hr after lentiviral infection, HH-B2 or BC3 cells were either untreated or treated with SB for another 18 hr. Cells were subjected to flow-cytometry analysis using anti-MDM2 antibody  and anti-ORF50 antibody. (C and D) Quantitative RT-PCR analysis of ORF50 and MDM2 mRNA levels in HH-B2 and BC3 cells. Total RNAs were isolated from the above treated cells and then were subjected to quantitative RT-PCR analysis as described previously . The rel(EPS)Click here for additional data file.S10 FigThe MDM2-binding motifs found in p53, p73, E2F1 and p63/51 are included in the alignment. Black dots on top indicate the residues in p53 that make contact with MDM2.(EPS)Click here for additional data file.


Through fluorescent labeling of these BM-MDSCs and optical imaging, we could visualize the recruitment and localization of BM-MDSCs in breast tumor-bearing mice in vivo. Furthermore, we were able to demonstrate that BM-MDSCs home to primary and metastatic breast tumors, but have no significant effect on tumor growth or progression. Ex vivo flow cytometry characterization of BM-MDSCs after adoptive transfer demonstrated both organ-and tumor-specific effects on their phenotype and differentiation, demonstrating the importance of the local microenvironment on MDSC fate and function. In this study, we have developed a method to generate, visualize and detect BM-MDSCs in vivo and ex vivo through optical imaging and flow cytometry, in order to understand the organ-specific changes rendered to MDSCs in breast cancer.Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid progenitor cells that are expanded in cancer and act as potent suppressors of the anti-tumor immune response. MDSCs consist of two major subsets, namely monocytic (M-) MDSCs and granulocytic (G-) MDSCs that differ with respect to their phenotype, morphology and mechanisms of suppression. Here, we cultured bone marrow cells with IL-6 and GM-CSF  OI scans were carried out at timepoints indicated and were analyzed using Living Image Software 4 (Perkin Elmer). Regions of interest were drawn on target organs and background areas in ex vivo OI scans to acquire mean values for the calculation of organ/background-ratios.Mice were injected with 150 mg/kg Luciferin (Perkin Elmer) i.p. for bioluminescence (BL) scans. After a 2 minute uptake period, fluorescence  and BL  scans were carried out on an Ivis Spectrum OI system (Perkin Elmer). BL scans were acquired for 20, 60 and 120 seconds and signals were normalized for the photon radiance  and 0.02 mg/mL DNase I (Roche) at 37°C for 45 min and 30 min respectively. Single cell suspensions of lung, spleen, tumor or BM-MDSCs were stained with relevant anti-mouse antibodies in the presence of anti-CD16/32 to block Fc-receptors on ice. Non-viable cells were excluded on the basis of staining with 7-aminoactinomycin or Zombie Yellow (BioLegend). Flow cytometry acquisition and sorting were carried out on a FACS Fortessa 4 and Aria II (BD Biosciences) respectively. Data analysis was performed using FlowJo software (Tree Star). Antibody details listed in 5 cells/mL in PBS and spun onto slides using a Cytospin 3 centrifuge (Shandon) at 500 rpm for 5 minutes. Slides were Diff-Quick stained the next day and images taken using a Leica DMIRB microscope with NIS Elements software.Myeloid cells isolated by FACS from the tumor, lung and spleen were resuspended at 2x10Results are expressed as mean ± SEM and analyzed by one-way ANOVA with Tukey’s multiple comparison test for significance. Kaplan-Meier Survival curves were analyzed using Log-rank (Mantel-Cox) Tests; p values <0.05 were considered significant .+/Ly6Cmed/Ly6G+ G-MDSCs were important in creating an immune-suppressed pre-metastatic niche in breast cancer PTSM or 64Cu-labeled monoclonal antibodies for quantitative positron emission tomography (PET) and iron oxide labeling, or 19F-labeling for magnetic resonance imaging (MRI) [hi/Ly6G-) and compare and contrast their homing, localization, plasticity and differentiation in breast tumor-bearing mice and other cancer types. Furthermore, very little is known regarding the involvement of MDSCs in metastatic tumor microenvironments and for example, in the formation of pre-metastatic niches, in which they have been reported to accumulate and promote metastatic breast tumor outgrowth [In this study, we have also developed a tool that will allow us to address these additional questions. The ls alone , contamils alone . We founng (MRI) –47. Combutgrowth . DetermiS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 FigA) Quantification of radiant efficiency of FL-signal for ex vivo lung, liver and spleen 7 days after i.v., i.c. or i.p. injection of 1x106 DiD-labeled BM-MDSCs into naïve mice as shown in B) Quantification of radiant efficiency of FL-signal for ex vivo lung, liver, spleen and tumor from Luc-PyMT tumor-bearing mice on day 21; 7 days after DiD-BM-MDSCs injection from (TIF)Click here for additional data file.S2 FigA-B) Representative ex vivo images of DiD-BM-MDSC  localization to liver (A) and lung (B) 2 weeks after i.v. injection of DiD-BM-MDSCs into mice with metastatic tumors  from (TIF)Click here for additional data file.S3 FigA) Schematic of treatment regimen for survival analysis after adoptive transfer of BM-MDSCs into tumor-bearing mice. Mice were orthotopically injected with 5x105 PyMT-WT cells into the MFP on day 0, and i.v. injected with 1x106 or 4x106 BM-MDSCs on day 10 and 20. Primary tumor growth was monitored to endstage. B,C) Individual tumor growth curves (B) and Kaplan-Meier survival curves (C) after i.v. injection of 1x106 BM-MDSCs at day 10 and 20 . D,E) Individual tumor growth curves (D) and Kaplan-Meier survival curve (E) for mice injected with 4x106 BM-MDSCs on day 10 and day 20 .(TIF)Click here for additional data file.S4 FigA,B) Individual tumor growth curves (A) and Kaplan-Meier survival curve (B) for mice injected with 5x105 PyMT-WT tumor cells alone (primary tumor n = 5) or co-injected with 5x105 BM-MDSCs in the mammary fat pad (primary tumor + BM-MDSCs n = 7).(TIF)Click here for additional data file.S5 FigA) Schematic of treatment regimen for tumor microenvironment analysis after adoptive transfer of DiD-BM-MDSCs into tumor-bearing mice. Mice were orthotopically injected with 5x105 PyMT-WT cells into the MFP on day 0, and i.v. injected with 1x106 DiD-BM-MDSCs on day 10 and 20. Tumors and organs were harvested and analyzed by flow cytometry at 48 hours (day 22) or 7 days (day 27) after the second BM-MDSC injection. Tumors from mice injected with PyMT-WT cells alone were used as controls. B-D) Flow cytometry analysis of CD3 lymphocyte populations (B) as a percentage of CD45.2, Ly6C and Ly6G myeloid cell populations (C) as a percentage of CD45.2/CD11b cells, and macrophage and dendritic cell populations (D) as a percentage of CD45.2/MHC Class II cells within the primary tumor 48 hours (n = 4 per group) and 7 days (n = 6 per group) after second BM-MDSC injection. Data represented as mean ± SEM. **p<0.01; ***p<0.001.(TIF)Click here for additional data file.S6 FigA-C) Flow cytometry analysis of CD3 lymphocyte (A), CD11b myeloid (B) and macrophage and dendritic cell (C) populations within the spleen of mice injected with 1x106 BM-MDSCs i.v. on day 10 and 20 after PyMT-WT tumor cell injection . Spleens were analyzed 48 hours (day 22) and 5 days (day 25) after second injection of BM-MDSCs. Spleens from PyMT-WT tumor-bearing mice were used as controls. (D-F) Flow cytometry analysis of CD3 lymphocytes (D), CD11b myeloid (E) and macrophage and dendritic populations (F) in the lungs of mice injected with 1x106 BM-MDSCs i.v. on day 10 and 20 after PyMT-WT tumor cell injection . Lungs were analyzed 48 hours (day 22) and 5 days (day 25) after second injection of BM-MDSCs. Lungs from PyMT-WT tumor-bearing mice were used as controls. Data represented as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001.((TIF)Click here for additional data file.S7 FigA) Flow cytometry analysis of CD3 lymphocytes, CD3/CD4 and CD3/CD8 T cells, as well as CD3-/NK1.1+ NK cells from the spleen of tumor-bearing mice (control) and after adoptive transfer of DiD-BM-MDSCs at day 24 (48 hours after second dose of DiD-BM-MDSCs). B) Flow cytometry analysis of the percentage of Foxp3+ cells within the CD3/CD4 T cell population in the spleen at day 24. C) Flow cytometry analysis of CD11b/Ly6C myeloid populations as a percentage of CD45.2+ cells within the spleen at day 24. D) Flow cytometry analysis of DCs and macrophages as a percentage of CD45.2+ cells within the spleen at day 24. E) Representative flow cytometry plots of CD11b/Ly6C populations within the spleen at day 24. For all analyses, control n = 5; DiD-BM-MDSC n = 6. Data represented as mean ± SEM. *p<0.05; n.s. not significant.(TIF)Click here for additional data file.


Genomic profiling of osteoblasts from mandibular and femur/tibia bone marrow revealed deficiencies in several HSC niche regulators among mandibular osteoblasts including Cxcl12. Neural crest derived bone harbors HSCs that function similarly to appendicular HSCs but are deficient in the lymphoid lineage. Thus, lymphoid deficiency of mandibular HSCs may be accounted by putative niche regulating genes. HSCs in craniofacial bones have functional implications in homeostasis, osteoclastogenesis, immune functions, tumor metastasis and infections such as osteonecrosis of the jaw.Hematopoietic stem cells (HSCs) in the endosteum of mesoderm-derived appendicular bones have been extensively studied. Neural crest-derived bones differ from appendicular bones in developmental origin, mode of bone formation and pathological bone resorption. Whether neural crest-derived bones harbor HSCs is elusive. Here, we discovered HSC-like cells in postnatal murine mandible, and benchmarked them with donor-matched, mesoderm-derived femur/tibia HSCs, including clonogenic assay and long-term culture. Mandibular CD34 negative, LSK cells proliferated similarly to appendicular HSCs, and differentiated into all hematopoietic lineages. Mandibular HSCs showed a consistent deficiency in lymphoid differentiation, including significantly fewer CD229 + fractions, PreProB, ProB, PreB and B220 + slgM cells. Remarkably, mandibular HSCs reconstituted irradiated hematopoietic bone marrow  Hematopoietic stem cells (HSCs) undergo self-renewal and differentiate into all blood lineages. HSCs have been studied extensively in bone marrow of mesoderm derived appendicular and axial skeletons with a demonstrated capacity to restore irradiated bone marrow by single clonal progenies35810Cxcl12 from osteoblasts (Col2.3-Cre) depletes early lymphoid progenitors but not HSCs and cellularity in bone marrowin vivoHSCs are maintained and regulated in the microenvironments called niches in which preserve the properties including cells and signal molecules. Cxcl12 (chemokine C-X-C motif) ligand, expressed by stromal niche cell populations, is a key chemokine that regulate HSC functions14151920The mandibles of 8-wk-old C57/B6 mice were dissected to isolate total mononucleated cells following removal of all the teeth and the mandibular condyles. Bone marrow cells from the mandibular and femur/tibia bones were negatively selected by magnetic beads with lineage markers, followed by CD34-/low selection and Sca-1 and c-kit double-positive selection per prior protocols25Assay for long-term culture-initiating cells (LTC-IC) allows a subset of LTC-IC to show their myeloid and lymphoid differentiation potential based on their capacity to produce progenies−CD48+ LSK) cells were detected in the mandible (0.090 ± 0.011) than femur/tibia (0.144 ± 0.009) (+ CD48+) LSK cells were identified in the mandible (0.013 ± 0.001) (− (P1 and P2) subpopulation and significantly less CD229+ population (SLAM family receptors distinguish subpopulations of HSCs and multipotent progenitors28± 0.009) , whereas± 0.001) . Mandibupulation , with a pulation , suggestB-cell precursors are classified into four populations according to their differential expression of multiple cell-surface markers during development in bone marrowTo test whether the lineage bias was contributed by local HSCs or influenced by neural-crest bone marrow niche, we then compared the reconstitution ability of femoral/tibial and mandibular hematopoietic stem/progenitor cells following bone-marrow transplantation. Donor cells were isolated and transplanted into irradiated recipient mice along with same number recipient bone marrow cells. Neural crest-derived mandibular HSCs gave rise to all major hematopoietic lineages in reconstituted bone marrow of irradiated mice including myeloid (Gr-1 + and MAC-1 + ), B cells and T cells, similar to femur/tibia HSCs . MandibuGiven the observed similarities and differences between femur/tibia and mandibular HSCs, we analyzed the expression of Cxcl12, a significant chemokine in HSCs niches and regulating lymphoid differentiation. Cxcl12 expression was confirmed by immunofluorescence staining in bone marrow of both femur/tibia  and mand31A schematic of neural crest-derived vs. mesoderm-derived hematopoietic stem cells is provided as The present findings suggest that neural crest-derived mandible harbors HSCs. HSCs in neural crest-derived bone marrow replenish all hematopoietic lineages, and are able to restore irradiated bone marrow, just as donor-matched mesoderm-derived appendicular HSCs. Postnatal HSCs in the jaw bone corroborate by robust hematopoiesis with long-term, self-renewable HSCs in mouse embryonic head at E12.5−/lowSca-1+ c-kit+ (LSK) cells are subdivided into MPPs (CD150−CD48− LSK), HPC (CD48+ LSK) and HSCs (CD150+ CD48− LSK)333439Mandibular CD34ˉLSK cells not only differentiated into all hematopoietic lineages per CFU assays, but also showed strong potential to the myeloid lineage, comparable to donor-matched tibia/femur CD34ˉLSK cells. In the LTC-IC assay, the myeloid lineage differentiation potential of mandibular HSCs was similar to that of femur/tibia HSCs. We further distinguished subsets of hematopoietic stem/progenitor cells with several SLAM family markers. LineageHematopoietic stem cell niche maintains and regulates HSCs homeostasis and regenerative potential, which consists of cells and signals. Among multiple niche-critical genes, Cxcl12 is an indispensable factor regulating HSCs retention, quiescence and multilineage reconstitution451923All methods were carried out in accordance with relevant guidelines and regulations.Following IACUC approval, 8-wk-old C57/B6 mice were sacrificed with total bone-marrow cells isolated from femurs and tibiae, as well as the mandibles. Col2.3-GFP mice (Jackson Laboratories) expressed enhanced green fluorescent protein gene under the control of 2.3 kb mouse procollagen, type 1, alpha 1(Col1a1) promoter. All experimental protocols were approved by Columbia University IACUC committee.−/lowc-Kit+ Sca-1+ lineage marker (CD34ˉLSK) cells. The following antibodies were used to further characterize the cells: anti-Ter119/Ly-76 (TER-119), anti-CD71 (R17217), CD41 (MW Reg30), CD14 (rmC5-3), F4/80 (BM8), anti-GR1 (8C5), CD11b (M1/70), anti-B220/CD45R (RA3-6B2), CD19 (1D3) and CD150 (TC15-12F12.2). The following HSCs antibodies were used as lineage markers: anti-CD2 (RM2-5), anti-CD3 (17A2), anti-CD5 (53-7.3), anti-CD8 (53-6.7), anti-TER119, anti-B220, anti-GR1, anti-CD150 (TC15-12F12.2), anti-CD48 (HM48-1), anti-CD229 (Ly9ab3) and CD244.2 (2B4). Hematopoietic PreProB progenitors were characterized with the following antibodies: anti-B220, anti-IgM (II/41), anti-CD43 (1B11) and anti-CD24 (M1/69). DAPI was used to exclude dead cells.8-wk-old C57/B6 mice were dissected to isolate total femur/tibia and mandibular bone  mononucleated cells by crushing with a mortar and pestle in Ca2 + and Mg2 + free HBSS with 2% heat-inactivated bovine serum. Cells were dissociated to single cell suspension by filtering through a 70-mm nylon mesh. For CD34ˉLSK cells isolation, lineage + cells were excluded by magnet selectionSorted CD34ˉLSK cells were cultured in StemSpan SFEM medium (Stem Cell Technology) suspended with purified recombinant mouse SCF , human TPO , human Flt-3L  and mouse IL-352533 cells were cultured in methylcellulose culture medium  with recombinant cytokines and incubated at 37 °C as instructed. Colony-forming units in culture were subsequently scored under inverted microscope. Colony-forming unit Erythroid (CFU-E), mature burst-forming unit-erythroid (BFU-E), colony-forming unit (CFU)-granulocyte/macrophage (GM), CFU-granulocyte/erythroid/megakaryocyte/macrophage (GEMM) and pre-B lymphoid (CFU-Pre B) progenitor numbers were defined as previous studies. Then cells were trypsinized and further stained to perform flow cytometry.Colony-forming assay was performed per prior methods−3 Hydrocortisone at 33 °C with medium change every 3 days and concomitant removal of half of the non-adherent cells. Upon 70–80% confluence, cells were irradiated at I500 cGy from a 137Cs r-irradiation source. Cells were then cultured onto the feeder layer and maintained in the same culture medium for 4 wks, followed by separately harvesting pooled adherent and non-adherent fractions for BFU-E, CFU-GM, and CFU-GEMM assays. For lymphoid differentiation, a two-stage culture system was used. Cells were initially seeded onto pre-established irradiated marrow feeders for 4 wks, followed by myeloid culture and removal of non-adherent cells. Adherent cell layer was washed twice with warm RPMI 1640 (Invitrogen) and subjected to RPMI1640 supplemented with 5% fetal calf serum and 50 μmol β-ME  at 37 °C for 1 wk. Trypsinized adherent cells were pooled with their corresponding non-adherent cells for lymphoid colony assay27Bone-marrow cells for feeder layer were flushed by using a 21-gauge needle into cold α-MEM medium  with 2% fetal calf serum. Single-cell suspension was obtained by repeated gentle aspiration and plating. Cells were maintained in MyeloCult M5300 with 10−1 neomycin sulphate and 0.121 gl−1 polymixin B) for 14 days following transplantation and then switched to regular water. Recipient mice were bled from 4 to 16 wks following transplantation to examine donor-derived myeloid, B and T cells in their blood with the following antibodies to analyze donor chimaerism: anti-CD45.1 (A20), anti-CD45.2 (104), anti-GR1 (8C5), anti-Mac1 (M1/70), anti-B220 (6B2) and anti-CD3 (KT31.1).Adult mice were given a minimum lethal dose of radiation using a Cesium 137 GammaCell 40 Irradiator (MDS) to deliver two doses of 540 rad  at least 2 hrs apart. Donor cells were injected into the retro-orbital venous sinus of anaesthetized C57BL/6-SJL (CD45.1) mice as the recipient. Mononucleated cells were counted before transplantation using a haemocytometer. Recipient mice were maintained on antibiotic water  overnight at 4 °C followed by secondary antibodies, Alexa Fluor® 647 Goat Anti-Rabbit IgG (H + L) Antibody .Total RNA was isolated per mRNA Isolation Kit (Invitrogen). The quality of total RNA was verified by an Agilent 2100 Bioanalyzer profile with RIN number >8.0. The extracted mRNAs were isolated and reverse transcribed into cDNA using the mRNA-Seq preparation kit . RNA sequencing was performed at UR Genomics Research Center. Illumina Hiseq platform was used, generating 100bp paired end reads. Differentially expressed genes were selected for network construction abd uploaded into ingenuity pathway analysis (IPA) software. Quantitative RT-PCR (TaqMan) was performed to validate gene expression with targeted mRNA expression normalized to GAPDH.Upon confirmation of normal data distribution, all quantitative data of control and treated groups were treated with two-tailed t-tests with an α level of 0.05. All experiments were repeated at least three times.How to cite this article: Jiang, N. et al. Hematopoietic Stem Cells in Neural-crest Derived Bone Marrow. Sci. Rep.6, 36411; doi: 10.1038/srep36411 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Breast cancer is a heterogeneous disease comprised of four molecular subtypes defined by whether the tumor-originating cells are luminal or basal epithelial cells. Breast cancers arising from the luminal mammary duct often express estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth receptor 2 (HER2). Tumors expressing ER and/or PR are treated with anti-hormonal therapies, while tumors overexpressing HER2 are targeted with monoclonal antibodies. Immunohistochemical detection of ER, PR, and HER2 receptors/proteins is a critical step in breast cancer diagnosis and guided treatment. Breast tumors that do not express these proteins are known as “triple negative breast cancer” (TNBC) and are typically basal-like. TNBCs are the most aggressive subtype, with the highest mortality rates and no targeted therapy, so there is a pressing need to identify important TNBC tumor regulators. The signal transducer and activator of transcription 3 (STAT3) transcription factor has been previously implicated as a constitutively active oncogene in TNBC. However, its direct regulatory gene targets and tumorigenic properties have not been well characterized. By integrating RNA-seq and ChIP-seq data from 2 TNBC tumors and 5 cell lines, we discovered novel gene signatures directly regulated by STAT3 that were enriched for processes involving inflammation, immunity, and invasion in TNBC. Functional analysis revealed that STAT3 has a key role regulating invasion and metastasis, a characteristic often associated with TNBC. Our findings suggest therapies targeting STAT3 may be important for preventing TNBC metastasis. Breast cancer is the most prevalent cancer diagnosed in women worldwide, and is the second leading cause of death by cancer in women , 2. In 2The presence of ER is one of the most important discriminators for diagnosing primary breast tumor subtype . ER-posiTNBCs typically arise from basal cells, and are diagnosed at higher tumor stage and grade, contributing to the aggressive biology of these cancers . TNBC trGenomic studies have reported gene expression signatures characterizing basal TNBC, but lack insight into upstream transcriptional regulators , 13, 14.STAT3 has been widely recognized as an oncogene in various cancers and has been confirmed to be constitutively active in TNBC , 16. TheTo characterize STAT3 binding patterns associated with TNBC, we performed, in replicate, Chromatin Immunoprecipitation followed by massively parallel sequencing (ChIP-seq) in five basal breast cancer cell lines with a STAT3-specific antibody . We idenin vivo in patient tumors.Because human breast cancer cell line models were used to characterize the genome-wide binding patterns of STAT3 in TNBC, we next determined if these genome-wide binding patterns are also seen in primary tissue from TNBC tumors. We performed ChIP-seq on STAT3 in two frozen TNBC patient tumors , and ideThe high similarity of STAT3 binding patterns in TNBC tumors and cell lines led us to further analyze the binding patterns of STAT3 in two TNBC cell lines. Due to the heterogeneity of TNBC breast cancers, we selected two cell lines harboring broad molecular subtypes of TNBC-basal A (HCC70) and basal B MDA-MB-231) tumors. ChIP-seq binding sites for each cell line was integrated with RNA-seq data to determine STAT3 driven gene regulation in TNBC. In HCC70, 20,808 high-confidence STAT3 binding sites were identified  have a STAT3 binding site within 50 kb of their transcriptional start site (TSS)  were identified to have a STAT3 binding site within 50 kb of the gene's TSS , inflammatory response (FDR=2.51×10-04), and immunity (FDR=1.26×10-03) (Table -04), secreted factors (FDR=1.10×10-07), and epithelial to mesenchymal transition (EMT) (FDR=1.17×10-05) (Table -04), matrisome (FDR=8.54×10-04), and complement and coagulation cascades (FDR=8.59×10-04) (Table -06), matrisome (FDR=9.18×10-06), and matrisome associated (FDR=2.64×10-04)  to include matrisome FDR=7.85×100-12, inf4) Table -S4. Inte4) Table -S7. ThesOur results suggest a potential role for STAT3 in cellular invasion and migration in TNBC. We further investigated its role in invasion in HCC70 and MDA-MB-231 cells by siRNA-mediated knockdown of STAT3 followed by matrigel transwell invasion assays. We observed a significant 44% and 42% decrease  in invasion of HCC70 and MDA-MB-231 cells, respectively transfected with the STAT3 siRNA pool  proteins and related ECM proteins) formation , 31. MatSTAT3 has been implicated in various cancers, including breast, ovarian, prostate, and lung cancers, as having a regulatory role in cancer development, including transformation, proliferation, EMT, invasion, and metastasis . HoweverFurther investigation must be conducted to unravel the role of STAT3 in actual metastasis of TNBC tumor cells to a secondary site. Development of TNBC xenograft mouse models followed by STAT3 perturbation will assist in discovering if STAT3 is indeed a therapeutic target for inhibiting metastasis of TNBC tumors. Direct inhibition of STAT3 may potentially improve chemotherapy response and decrease metastasis in TNBC, ultimately improving overall survival of TNBC patients. STAT3 subtype specific gene signatures could serve as potential biomarkers for determining the specific subtype of TNBC tumor and appropriate choice of STAT3 inhibition and chemotherapy for personalized treatment. Additional studies aimed at understanding the pharmacological response of the TNBC subtype specific STAT3 targets are needed to develop personalized therapies for inhibiting this factor.The following cell lines: MDA-MB-231, MDA-MB-157, MDA-MB-468 were cultured in DMEM media  supplemented with 10% fetal bovine serum , 1 mM sodium pyruvate, and non-essential amino acids (Gibco). HCC70 and HCC1143 were cultured in RPMI media (Gibco), supplemented with 10% fetal bovine serum (Hyclone), 10 mM HEPES , 1 mM sodium pyruvate (Gibco), and 4.5 g/l glucose (Sigma-Aldrich). Cells were passaged and harvested at 80% confluence. Prior to harvest, cells were treated for 72hr or 96hr with either 10 μM STAT3 siRNA or an equal concentration of control non-targeting siRNA.For ChIP-seq experiments, protein-DNA complexes were covalently cross-linked by incubating cells in 1% formaldehyde for 10 minutes at room temperature. Cells were incubated with 0.125 M glycine for 5 minutes, to quench cross-linking reaction. Cells were washed and scraped with PBS (pH 7.4) . Cells were lysed with Farnham Lysis Buffer  containing protease inhibitor . Cell lysate was centrifuged at 2000 rpm for 5 minutes at 4°C. The crude nuclear extract contained in the supernatant was stored at -80°C.For RNA-seq, cells were treated with Buffer RL  and stored at -80°C. Cell lysate was collected after addition of 100% molecular biology grade ethanol. Total RNA was extracted using the Norgen Animal Tissue RNA Purification Kit.We used an ON-TARGETplus Human STAT3 siRNA kit from GEHealthcare . This SMARTpool siRNA contains four pooled siRNAs, each targeting a separate region of the STAT3 RNA sequence. We also used ON-TARGETplus Non-targeting siRNA #1 (D-001810-01-05) as a non-targeting control. The siRNA SMARTpool and Non-targeting siRNA target sequences are below:ON-TARGETplus SMARTpool siRNA J-003544-07, STAT3Target Sequence: GAGAUUGACCAGCAGUAUAON-TARGETplus SMARTpool siRNA J-003544-08, STAT3Target Sequence: CAACAUGUCAUUUGCUGAAON-TARGETplus SMARTpool siRNA J-003544-09, STAT3Target Sequence: CCAACAAUCCCAAGAAUGUON-TARGETplus SMARTpool siRNA J-003544-10, STAT3Target Sequence: CAACAGAUUGCCUGCAUUGON-TARGETplus Non-targeting siRNA #1Target Sequence: UGGUUUACAUGUCGACUAAThe siRNA transfection experiments were performed in 6-well and 96-well cell culture plates in triplicate and quintuplicate, respectively, and included the non-targeting control. For transfection, the Lipofectamine RNAiMAX Transfection Reagent (ThermoFisher Scientific) was used. Lipofectamine and siRNAs were prepared per manufacturer's instructions (ThermoFisher Scientific). To each well containing cells, in the 96-well plate experiments (viability), we added 10 ul of the siRNA-transfection reagent mix diluted in Opti-MEM I Reduced Serum Medium (ThermoFisher Scientific); this resulted in 1 pmol siRNA in 0.3 μl Lipofectamine RNAiMAX reagent per well. For 6-well cell culture plate experiments, 250 μl siRNA-transfection reagent mix was added to each well, for a final concentration of 25 pmol siRNA in 7.5 μl Lipofectamine RNAiMAX reagent per well.To confirm knockdown of STAT3 protein levels, SDS-PAGE followed by western blotting was performed. HCC70 and MDA-MB-231 cells were seeded in six-well plates, at a density of 300,000 (MDA-MB-231) or 600,000 (HCC70) cells per well. After 96 hours, cells were harvested via trypsinization followed by centrifugation at 2,000 rpm for 5 minutes. Cell pellets were washed in 1 mL PBS, centrifuged a second time, and stored at -80°C until immunoprecipitation step. Cell pellets were resuspended in 100 μl RIPA buffer containing protease inhibitors, and incubated, on ice, for 30 minutes. Cells were dounced with a syringe to further lyse cells, followed by centrifugation at 14,000 rpm for 10 minutes. The supernatant was collected and protein concentrations were measured using the Qubit Protein Assay kit and Qubit fluorometer (ThermoFisher Scientific). 40 μg protein for each cell line and condition were mixed with an equal volume of Laemelli Buffer  supplemented with 10% Beta-mercaptoethanol (Sigma). Protein samples were heated in a 95-100°C water bath for ten minutes. Protein lysate samples and SeeBlue Plus2 (ThermoFisher Scientific) and Magic Mark XP (ThermoFisher Scientific) protein standards were loaded onto a pre-cast SDS-polyacrylamide gel (Bio-Rad) assembled in a reservoir containing 1X Tris/Glycine buffer (BioRad).The SDS-PAGE gel was run for 1.5 hours at 150V. Proteins were transferred to a nitrocellulose membrane using the iBlot gel transfer device, per the manufacturer's instructions, using program 3 for seven minutes (ThermoFisher Scientific). Following transfer, the protein-containing membrane was placed in blocking reagent (PBS + NF dairy milk + 10% Tween), followed by shaking for 1 hour at room temperature. Blocking reagent was discarded and wash buffer containing antibodies targeting STAT3 (Santa-Cruz) phosphorylated STAT3 (Ser727) , β-actin (loading control)  were added to the membrane and incubated overnight at 4°C, with rotation. The membrane was washed three times, for ten minutes each, with western wash buffer (1X PBS with 0.05% Tween 20). Wash buffer containing anti-rabbit or anti-mouse secondary antibody was added to the membrane and incubated for 1 hour at room temperature. The membrane was again, washed three times for ten minutes. To visualize blotted proteins, SuperSignal West Femto working solution was prepared by mixing equal parts of stable peroxide and luminol/enhancer, per manufacturer's instructions (ThermoFisher Scientific) and incubated with the membrane for 5 minutes. Imaging was conducted using the UVP imaging system.2. Cells were harvested via trypsinization followed by centrifugation at 2,000 rpm for five minutes. Cell pellets were resuspended in 10 ml media and quantified using a hemocytometer. Cells were diluted to a concentration of 40,000 cells/ml in serum free media and seeded onto Corning Biocoat matrigel invasion chambers  as previously described [To determine if STAT3 regulates invasion, 300,000 (HCC70) or 75,000 (MDA-MB-231) cells were seeded into six-well plates and transfected with STAT3 siRNAs and non-targeting siRNAs as described above. Cells were incubated for 72 hours (HCC70) or 96 hours (MDA-MB-231) at 37°C/5%COescribed . StudentCell viability assays were conducted in 96-well cell culture plates. HCC70 and MDA-MB-231 cells were transfected with a siRNA targeting STAT3 or a non-targeting siRNA vehicle control for 96 hours. Cell proliferation was measured using the ATPLite Assay  for cell metabolism.To characterize genome-wide binding patterns of STAT3 in TNBC cell lines, ChIP-seq was performed as previously described . AntibodMeasurement of STAT3 binding in TNBC tumor tissues was performed as previously described .Cells were lysed with Buffer RL (Norgen) containing 10% beta-mercaptoethanol. Cell lysate was collected after addition of 300 μl 100% molecular biology grade ethanol. Total RNA was extracted using the Animal Tissue RNA Purification Kit (Norgen). RNA-seq libraries were prepared from 250 ng total RNA via polyA-selection  followed by transposase-mediated non-stranded library construction . TopHat For each ChIP-seq replicate, peaks were identified and reported using the MACS peak caller program . Motif aDifferential gene expression analysis was performed in the statistical software, R (version 3.2.1) , using DDifferentially expressed genes with a nearby binding site were considered to be direct targets and were used for Hallmark and Canonical Pathway enrichments using Gene Set Enrichment Analysis , 45. StuAll ChIP-seq and RNA-seq datasets have been deposited in GEO (accession number: GSE85579).


Alectinib inhibited the viability of NCOA4-RET-positive EHMES-10 cells, as well as CCDC6-RET-positive LC-2/ad and TPC-1 cells. This was achieved via inhibition of the phosphorylation of RET and induction of apoptosis. Moreover, alectinib suppressed the production of thoracic tumors and pleural effusions in an orthotopic intrathoracic inoculation model of EHMES-10 cells. In vivo imaging of an orthotopically inoculated EHMES-10 cell model also revealed that alectinib could rescue pleural carcinomatosis. These results suggest that alectinib may be a promising RET inhibitor against tumors positive for not only KIF5B-RET and CCDC6-RET, but also NCOA4-RET.Rearranged during transfection (RET) fusion-positive non-small cell lung cancer (NSCLC) accounts for approximately 1–2% of all NSCLCs. To date, RET fusions that involve at least six fusion partners in NSCLC, such as KIF5B, CCDC6, NCOA4, TRIM33, CLIP1, and ERC1, have been identified. Recent clinical trials for RET fusion-positive NSCLC using vandetanib or cabozantinib demonstrated positive clinical response and considerable differential activities for RET inhibitors among fusion partners. Alectinib, an approved ALK inhibitor, is reported to inhibit KIF5B-RET and CCDC6-RET. However, the activity of alectinib with respect to RET with other fusion partners is unknown. In the present study, we investigated the effects of alectinib on NCOA4-RET fusion-positive tumor cells  The rearranged during transfection (RET) gene was discovered in 1985 as an oncogene produced by recombination during the transfection of NIH 3T3 cells with human lymphoma DNA . RET fusRET fusion genes were recently identified in a population of non-small cell lung cancers (NSCLCs) –8. The mAlectinib is a second generation ALK-TKI approved for ALK-rearranged NSCLC . AlthougIn the present study, we investigated the effect of alectinib on NCOA4-RET utilizing native tumor cells with this type of RET fusion. We compared this to tumor cells harboring CCDC6-RET, which were shown to have sensitivity to vandetanib in an earlier clinical trial .The mesothelioma cell line EHMES-10 –19 contaWe examined the effect of phosphorylation of RET and its downstream targets, ERK and AKT. Western blot analyses revealed that alectinib inhibited the phosphorylation of RET, ERK, and AKT in NCOA4-RET-positive EHMES-10 cells and CCDC6-RET-positive LC-2/ad and TPC-1 cells  in NCOA4-RET-positive EHMES-10 cells. This was also observed in CCDC6-RET-positive LC-2/ad and TPC-1 cells Figure . Moreovein vivo. As we previously reported  dye reduction method. Tumor cells  and transferred to polyvinylidene difluoride membranes (Bio-Rad). Membranes were washed three times and then incubated with Blocking One solution  for 1 h at room temperature. The membranes were incubated overnight at 4°C with primary antibodies against anti-RET (D3D8R) (#14698), anti-phospho-RET (Tyr905) (#3221), anti-AKT (#9272), anti-phospho-AKT (Ser473) (#4060), anti-cleaved PARP (#5625), anti-cleaved caspase-3 (#9664), and anti-β-actin (13E5) (#4970) antibodies . Additional antibodies were also used including anti-human/mouse/rat extracellular signal-regulated kinase (ERK) 1/ERK2 (0.2 μg/mL) (AF1576) and anti-phospho-ERK1/ERK2 (T202/Y204) (0.1 μg/mL) (AF1018) from R&D Systems. The membranes were washed three times and then incubated for 1 hour at room temperature with species-specific horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized with SuperSignal West Dura Extended Duration Substrate, an enhanced chemiluminescent substrate . Each experiment was performed independently at least three times.5 cells in 2 mL antibiotic-free medium were plated into each well of a 6-well plate and incubated at 37°C for 24 hours. The cells were transfected with siRNA (250 pmol) or scrambled RNA using Lipofectamine 2000 (5 μL) in accordance with the manufacturer’s instructions (Invitrogen).Duplexed Stealth RNAi (Invitrogen) against RET and a Stealth RNAi-negative control low GC Duplex #3 (Invitrogen) were used for RNA interference (RNAi) assays (DOC. S1). Briefly, aliquots of 1–2 × 10scidHrhr mice  for the study. All animal experiments complied with the Guidelines of the Institute for Laboratory Animals of Advanced Science Research Center at Kanazawa University. For experiments using the pleural carcinomatosis mouse model [6/0.1 mL) through the parietal pleura into the right thoracic cavity. After inoculation, the quantity of tumors was tracked in live mice by repeated noninvasive optical imaging of tumor-specific luciferase activity using the IVIS Lumina XR Imaging System  [We used 6-week-old male SHO-Prkdcse model , an incieda, CA)  or CT sceda, CA) .P < 0.05 was considered significant.Differences between groups were analyzed with one-way analysis of variance (ANOVA). All statistical analyses were performed using GraphPad StatMate 4 .


EGFR T790M mutation accounts for about 40-55% drug resistance for the first generation EGFR kinase inhibitors in the NSCLC. Starting from ibrutinib, a highly potent irreversible BTK kinase inhibitor, which was also found to be moderately active to EGFR T790M mutant, we discovered a highly potent irreversible EGFR inhibitor CHMFL-EGFR-26, which is selectively potent against EGFR mutants including L858R, del19, and L858R/T790M. It displayed proper selectivity window between the EGFR mutants and the wide-type. CHMFL-EGFR-26 exhibited good selectivity profile among 468 kinases/mutants tested (S score (1)=0.02). In addition, X-ray crystallography revealed a distinct “DFG-in” and “cHelix-out” inactive binding mode between CHMFL-EGFR-26 and EGFR T790M protein. The compound showed highly potent anti-proliferative efficacy against EGFR mutant but not wide-type NSCLC cell lines through effective inhibition of the EGFR mediated signaling pathway, induction of apoptosis and arresting of cell cycle progression. CHMFL-EGFR-26 bore acceptable pharmacokinetic properties and demonstrated dose-dependent tumor growth suppression in the H1975 (EGFR L858R/T790M) and PC-9 (EGFR del19) inoculated xenograft mouse models. Currently CHMFL-EGFR-26 is undergoing extensive pre-clinical evaluation for the clinical trial purpose. EGFR is a receptor tyrosine kinase, which upon ligand EGF  binding will result in auto-phosphorylation and subsequently lead to activation of downstream signaling pathways such as RAS/RAF/ERK, PI3K/AKT, and JAK/STAT pathways . Gain-ofin vitro and in vivo anti-NSCLC efficacies in the preclinical models.Recently, Ibrutinib, an FDA approved irreversible BTK kinase inhibitor for MCL, CLL etc, has been found to be moderately active against EGFR T790M mutant and has been evaluated in the clinic , 12. How50s range from 0.0003 μM to 0.013μM) meanwhile kept over 500-fold selectivity over EGFR wt (GI50: 5.4 μM). In addition, it did not exhibit any apparent inhibitory activity against parental BaF3 cells (GI50: >10 μM) indicating no general toxicity. For all of the sensitive mutants, when C797S mutant was introduced, CHMFL-EGFR-26 lost activity significantly (50-10000 folds) which suggested an irreversible binding mode via cysteine 797 residue. Furthermore, the reversible version of CHMFL-EGFR-26, which was generated by saturation of acrylamide to propionamide  and the selectivity window between the EGFR mutants and WT was narrower than CHMFL-EGFR-26. The enzymatic inhibition result of CHMFL-EGFR-26 was detected by SelectScreen techonology (Life Technologies). CHMFL-EGFR-26 showed an IC50 of 19nM against EGFR T790M mutant, 71nM against EGFR WT and 215 nM against EGFR L858R mutant .In order to further confirm this compound's on-target effect, we then examined its effect on the blockage of EGFR auto-phosphorylation in EGFR transformed BaF3 isogenic cell lines. The results demonstrated that upon 4h's CHMFL-EGFR-26 treatment, the phosphorylation of EGFR Y1068 was inhibited with an ECs Figure , suggest50=3.9nM), Her4 (IC50=5.39nM), Her2 (IC50=15.9nM), BTK (IC50=16.3nM), BMX (IC50=38.9nM), JAK3 (IC50=30.6nM) and moderate activity against MEK1 (IC50=131nM). This was not surprising because EGFR, JAK3, HER2, HER4 and TEC family kinases  all share a similar cysteine residue at the identical position analogous to Cys 797 in EGFR in the hinge binding area  in which width (W) is defined as the smaller of the two measurements and length (L) is defined as the larger of the two measurements.Five-week old female nu/nu mice were purchased from the Shanghai Experimental Center, Chinese Science Academy . All animals were housed in a specific pathogen-free facility and used according to the animal care regulations of Hefei Institutes of Physical Science Chinese Academy of Sciences. Prior to implantation, cells were harvested during exponential growth. Five million cells in PBS were formulated as a 1:1 mixture with Matrigel (BD Biosciences) and injected into the subcutaneous space on the right flank of nu/nu mice. Daily oral administration was initiated when tumors had reached a size of 200 to 400 mmIn Situ Cell Death Detection Kit (POD)  according to the manufacturer's instructions.Tumor tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin. Six-micron tissue section were prepared, deparaffinized, dehydrated, and then stained with hematoxylin and eosin (H&E) using routine methods. Commercially available primary antibody to human Ki-67  was used for Ki-67 staining. After heat-induced antigen retrieval, formalin-fixed and paraffin-embedded tumor tissue sections were stained with primary antibody overnight at 4°C. The slides were subsequently incubated with ImmPRES anti-mouse Ig  at room temperature for 30 min, stained with peroxidase substrate 3,3’-diaminobenzidine chromogen (Vector Laboratories), and finally counterstained with hematoxylin. TUNEL staining was assessed using


Interleukin-6 (IL-6) potently inhibits Treg differentiation and its blockade was shown to increase Treg numbers in vivo. Therefore, we investigated whether IL-6 blockade can replace adoptive Treg transfer in irradiation-free allogeneic BMT. Treatment with anti-IL-6 instead of Treg transfer led to multi-lineage chimerism (persisting for ~12 weeks) in recipients of fully mismatched BM and significantly prolonged donor skin (MST 58 days) and heart (MST > 100 days) graft survival. Endogenous Foxp3+ Tregs expanded in anti-IL-6-treated BMT recipients, while dendritic cell (DC) activation and memory CD8+ T cell development were inhibited. Adding anti-IL-17 to anti-IL-6 treatment increased Treg frequencies, but did not further prolong donor skin graft survival significantly. These results demonstrate that IL-6 blockade promotes BM engraftment and donor graft survival in non-irradiated recipients and might provide an alternative to Treg cell therapy in the clinical setting.Transfer of recipient regulatory T cells (Tregs) induces mixed chimerism and tolerance in an irradiation-free bone marrow (BM) transplantation (BMT) model involving short-course co-stimulation blockade and mTOR inhibition. Boosting endogenous Tregs pharmacologically  Long-term allograft survival without the need of permanent immunosuppression remains an important goal in transplantation medicine . In thisin vivo  Tregs (Our group has recently developed a regimen free of myelosuppression that induces chimerism and long-term tolerance through a combination of donor BM transplantation (BMT) and recipient regulatory T cell (Treg) therapy under short-course CB (anti-CD40L and CTLA4-Ig) and of mTOR inhibition –9. Even in vivo  or the cp) Tregs . With rep) Tregs  and thusin vivo dendritic cell (DC) differentiation through STAT3  is a critical player in T cell and B cell immunity , 19. IL-gh STAT3 . Moreoveoduction , 25. Preoduction –28. The oduction –31. Inhioduction . Recentloduction . Furtheroduction –36. Nota regimen . Moreove regimen . Several regimen –41.To date, suitable protocols that achieve allogeneic BM engraftment without the need of cytoreductive therapy are still limited. The combination of CB, mTOR inhibition, and Treg therapy seems to be a promising approach. To increase clinical applicability, we investigated whether anti-IL-6 administration could obviate the need for Treg cell therapy in this non-cytotoxic murine mixed chimerism model.b), BALB/c , and C3H/N  mice were purchased from Charles River Laboratories , housed under specific pathogen-free conditions, and used for experiments between 6 and 12 weeks of age. Congenic B6.SJL-Ptprca Pepcb/BoyJ (CD45.1+) and B10.D2 mice  were purchased from Jackson Laboratory  and bred in our own facility. Skin and heart transplantations were performed under controlled anesthesia with an intraperitoneally (i.p.) injected mixture of xylazin (5 mg/kg) and ketamine (100 mg/kg). All experiments were approved by the local review board of the Medical University of Vienna and the Austrian Federal Ministry of Science, Research and Economy and were performed in accordance with national and international guidelines of laboratory animal care (permission number GZ: BMWFW-66.009/0377-WF/V/3b/2016 and GZ: BMWFW-66.009/0303-WF/V/3b/2016).Female C57BL/6  control] from BALB/c donors by injection into the tail vein together with CB consisting of anti-CD154 mAb , hCTLA4-Ig , and a short-course of rapamycin  (Groups of age-matched C57BL/6 recipients received 20 × 10atories) , 28. Wheatories) , 43.Full thickness tail skin from BALB/c, B10.D2, or C3H/N mice was grafted to recipient flanks  5 weeks after BMT and visually inspected thereafter at short intervals. Grafts were considered to be rejected when less than 10% of the graft remained viable . CervicaSkin and heart grafts were harvested at the end of the experiment and fixed in 7.5% formalin. Paraffin blocks were sectioned and stained with hematoxylin and eosin (H&E) according to standard protocols. Histological slides were then scanned by Aperio ScanScope scanner  and analyzed with ImageScope software. Additionally, grading of skin grafts was performed according to Banff 2007 working classification of skin-containing composite tissue allograft pathology and hearts were scored according to the International Society for Heart and Lung Transplantation (ISHLT) 2005 guidelines for cellular rejection by a pathologist blinded to the experimental background of samples.5 congenic C57BL/6 CD45.1+ responder cells (unseparated splenocytes) were cultured for 5 days in RPMI1640 supplemented with 10% FCS (Linaris), PenStrep , 10 mM Hepes , 1 mM Sodium Pyruvat (Sigma), and 1× non-essential amino acids (Sigma). Responder cells were stimulated with equal numbers of stimulator cells (unseparated splenocytes) from BALB/c  and C57BL/6 (self) or with Medium. Proliferation was measured by staining Ki-67 in responding (CD45.1+) CD4+ and CD8+ T cells after 5 days of culture. For the in vitro suppression assay, a 96-well plate was pre-coated overnight with 1 µg/ml anti-CD3  diluted in PBS. On the next day, 4 × 105 naïve responder CD45.1+ splenocytes (VPD 450 pre-labeled) were cocultured with indicated numbers of Tregs  isolated on day 14 from BMT recipients (receiving CB and rapamycin) either with or without anti-IL-6. Tregs were purified by magnetic bead separation using the MACS CD4+CD25+ regulatory T-cell Isolation Kit (Miltenyi Biotec). Purity of separated population was >90%. After 4 days of incubation responder cell proliferation was assessed by measuring the dilution of VPD450. The percentage of suppression was calculated using the formula: [/% of Tresp. cells alone] × 100.Regarding MLR 5 × 10+ [H2-Dd-BIO (34-2-12)] in different leukocyte lineages {myeloid cells [Mac-1-FITC (M1/70)], B cells [CD19-PE (6D5)], CD4+ cells [CD4-APC-Cy7 (RM4-5)], and CD8+ cells [CD8-PE-Cy7 (53-6.7)]}. CD44-BIO (IM7), CD62L-PE (MEL-14), CD11c-BIO (N418), CD80-PERCP-Cy5 (16-10A1), CD86-FITC (GL-1), MHCII-PE-Cy7 (M5/114.15.2), CD40-PE (3/23), CD3-PerCP-Cy5 (17A2), SAV-APC, CD45.1-BIO (A20), CD45.2-BIO (104), and IgG-PE (Poly4053) were purchased from BioLegend . For intracellular staining, cells were permeabilized with the Foxp3/Transcription Factor Staining Buffer Set from eBioscience according to the manufacturer’s specification. Anti-mouse/rat Foxp3 APC (FJK-16s) and anti-mouse/rat Ki-67 PE-Cy7 (SolA15) were obtained from eBioscience. Flow cytometric analysis was performed with a BD FACS Canto II, and data were analyzed using FlowJo (10.0.8) software.Three-color flow cytometric analysis of multi-lineage chimerism was performed as described previously , 46. Bri5 naïve recipient or donor thymocytes were incubated with 15 µl serum of indicated mice for 30 min at 37°C after heat inactivation of complement as described earlier . Differences between chimerism levels were compared using ANOVA. Error Bars represent SD and statistics were performed with a two-sided Student’s p = 0.017). Among all measured leukocyte lineages, the myeloid population showed the highest chimerism levels over the whole period, peaking at week 2. In contrast, almost all control mice without anti-IL-6 (and without Treg transfer) lost myeloid  chimerism by week 3, or shortly thereafter  , rapamycin and in addition anti-IL-6 (instead of adoptive Treg transfer). All mice receiving anti-IL-6 developed early multi-lineage chimerism (within the myeloid and B cell lineages) that persisted for up to 12 weeks . Noticeably, T cell chimerism appeared in most anti-IL-6 treated mice between weeks 6 and 9 and lasted until week 12 . Third-party skin grafts were rapidly rejected in all groups . To test whether the observed prolongation of BALB/c donor skin graft survival after BMT with anti-IL-6 depends on (transient) BM engraftment, a group of mice was treated with the same anti-IL-6 regimen but received donor splenocytes instead of BM . DST recipients promptly rejected donor skin (MST = 12.5 days), suggesting that BM engraftment is essential for graft prolongation and that DST does not suffice , indicating that donor-specific humoral immunity is prevented in anti-IL-6 treated mice . Donor skin graft survival was significantly prolonged in anti-IL-6-treated mice [median survival time (MST) 58 days, p = 0.0014) but all grafts were eventually rejected  are a barrier to tolerance induction through chimerism , 48. To These results show that anti-IL-6 prolongs donor skin graft survival due to enhanced allogeneic BM engraftment.n = 4, 100 days), whereas 3/4 recipients from the group without anti-IL-6 rejected their grafts   Figure C. Heart ) Figure B.Hence, these data demonstrate that anti-IL-6 treatment leads to long-term acceptance of donor heart grafts in an irradiation-free BMT setting.in vivo boosting of endogenous Tregs, we measured the frequency, number, and proliferation of Foxp3+CD4+ T cells. Anti-IL-6 was associated with significantly increased frequencies of Foxp3 Tregs in blood (on days 14 and 21 post-BMT)  was analyzed by flow cytometry. The number of DCs was significantly reduced in anti-IL-6-treated mice compared to the untreated group  , suggesting reduced DC activation (MHCII and CD40 were similarly expressed in both groups)  remained constant at around 60% (similar to pre BMT levels), whereas it declined in mice not receiving anti-IL-6  remained relatively stable in the anti-IL-6 group and was significantly lower compared to the group without anti-IL-6 with the most pronounced difference at week 3  . To investigate whether the tempered CD8+ TEM development affects the “second-set” allograft response, a second donor skin graft was transplanted 3 weeks after the rejection of the first donor graft. Both groups rejected second donor skin grafts with similar kinetics as first grafts were rejected in untreated mice    with higher levels at all measured time points . Moreover, Treg transfer led to permanent donor skin (vs MST 58 with anti-IL-6) and heart graft survival (similar to anti-IL-6) (The pro-tolerogenic effect of anti-IL-6 occurred only in the context of (transient) BM engraftment, as no such effect was seen when DST with donor splenocytes instead of BM was given. Since heart grafts survived until the end of the observation period while skin grafts were gradually rejected , tissue-specific antigens could be one reason for this difference in outcomes. However, it cannot be ruled out that the heart allografts would have been rejected at later times due to different effector mechanisms accounting for rejection of distinct tissues. mHAg disparities have a profound role in organ transplantation  and our +CD25+ cells from IL-6 deficient mice showed a greater potency to suppress IFN-γ production than wild-type Tregs .All authors have actively contributed to the study. NG and TW designed all experiments, analyzed and interpreted the data, and wrote the manuscript. NG, MM, BM, NP, AF, LU, SM, RH, and MW performed experiments. HR performed histopathological staining and analysis. All authors approved the final version of the manuscript.The author declares that the complete study was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.