Antibodies against programmed cell death-1 (PD-1) have considerably changed the treatment for melanoma. However, many patients do not display therapeutic response or eventually relapse. Moreover, patients treated with anti-PD-1 develop immune-related adverse events that can be cured with anti-tumor necrosis factor α (TNF) antibodies. Whether anti-TNF antibodies affect the anti-cancer immune response remains unknown. Our recent work has highlighted that TNFR1-dependent TNF signalling impairs the accumulation of CD8+ tumor-infiltrating T lymphocytes (CD8+ TILs) in mouse melanoma. Herein, our results indicate that TNF or TNFR1 blockade synergizes with anti-PD-1 on anti-cancer immune responses towards solid cancers. Mechanistically, TNF blockade prevents anti-PD-1-induced TIL cell death as well as PD-L1 and TIM-3 expression. TNF expression positively correlates with expression of PD-L1 and TIM-3 in human melanoma specimens. This study provides a strong rationale to develop a combination therapy based on the use of anti-PD-1 and anti-TNF in cancer patients. Most melanoma patients do not respond to anti-PD1 therapy. Here, the authors show that TNFα blockade synergizes with anti-PD-1 by preventing anti-PD-1-induced CD8+ T cell death and TIM-3 expression on such cells. Whereas TNF was first identified as a cytotoxic soluble factor4, mounting evidence indicates that TNF facilitates the accumulation and/or biological activity of regulatory T lymphocytes (Tregs)12, regulatory B lymphocytes (Bregs)8 as well as myeloid-derived suppressor cells (MDSC)9, which are key negative modulators of the immune response. Moreover, TNF triggers activation-induced cell death (AICD) of CD8+ T cells13 and impairs the tumor infiltration by CD8+ T lymphocytes14. CD4+ tumor-infiltrating lymphocytes (TILs) in melanoma produce TNF, which inhibits cytotoxic CD8+ T-cell responses15. In an adoptive CD8+ T-cell transfer protocol, TNF elicited melanoma dedifferentiation, promoting immune escape and melanoma relapse16. More recently, TNF has been shown to enhance the expression of programmed cell death ligand 1 (PD-L1) in cancer cells17, including melanoma15, triggering immunosuppression.Tumor necrosis factor α (TNF) plays a dual role in oncoimmunology18. However, 60–67% of patients do not respond to anti-PD-120, and a significant proportion of responders experience tumor relapse within two years after treatment induction21. One of the documented cause of this relapse was shown to be the expression of secondary immune checkpoints such as T-cell Immunoglobulin and Mucin domain-containing protein 3 (TIM-3)22. Anti-PD-1 treatment is associated with increased TNF gene expression in melanoma samples from metastatic melanoma patients23. In this context, the contribution of TNF to the anti-melanoma immune response is unknown. In addition, responders develop severe immune-related adverse events (irAEs) with a frequency close to 12%20. Whereas anti-TNF antibodies are used to cure irAEs associated with immunotherapies 24, whether TNF blockade affects the anti-melanoma immune response triggered by immunotherapies remains to be determined.Emerging immunotherapies that target immune checkpoints, like anti-PD-1 (programmed cell death-1), have radically changed our strategy to fight melanomaHerein, we have evaluated the consequences of blocking the TNFR1-dependent TNF signalling on anti-PD-1 efficacy in experimental cancer. We provide evidence for the first time that TNF impairs the response to anti-PD-1 not only in mouse melanoma but also in two other experimental cancers . Mechanistically, TNF induced upon anti-PD-1 treatment triggers (i) the expression of both PD-L1 and TIM-3 on CD4+ and CD8+ TILs and (ii) AICD in CD8+ TILs, which co-express PD-1 and TIM-3. Consequently, TNF blockade/deficiency prevents PD-L1 and TIM-3 expression as well as AICD in CD8+ TILs, overcoming the resistance to anti-PD-1 in experimental melanoma.5 melanoma cells. When tumors became detectable , mice received the first out of three injections of anti-PD-1 blocking antibody or isotype control. In agreement with previous observations14, B16K1 tumor growth   and TNF-deficient mice were bilaterally injected with 3.10th Figs.  and animth Figs.  were sigth Figs. . Remarkath Figs. . Moreoveth Figs. . Similar6) Figs. . Of inte6) Figs.  rejected6) Figs. .Fig. 1TN26. Collectively, our data indicate that the TNFR1-dependent TNF signalling impairs the therapeutic benefit of PD-1 blockade.To extend our observations to another cancer model, we tested the effect of anti-PD-1 blocking antibodies in WT, TNF- and TNFR1-deficient mice injected with Lewis lung carcinoma (LLC) cells, which expressed MHC-I at low levels  of TIM-3 staining, which reflects its expression on TILs, was significantly increased on CD8+ and CD4+ TILs following anti-PD-1 administration in WT mice  positively and significantly correlated with that of HAVCR2 (encoding TIM-3), PDCD1LG1 (encoding PD-L1) and PDCD1LG2 (encoding PD-L2)  Fig. . Our anaD-1 Fig. .Fig. 7TNThus, along with our preclinical data, those observations on human melanoma suggest that TNF potently induces the expression of PD-L1, PD-L2 and TIM-3 in melanoma upon anti-PD-1 therapy.33. However, a high proportion of patients affected by cancers that are described as “immunogenic” still poorly respond to these therapies. As opposed to the “primary resistance”, these resistance mechanisms are defined as “adaptive immune resistance” and “acquired resistance”34. One documented cause for this phenomenon is the increased expression of secondary immune checkpoint molecules such as TIM-3 that dampen the anti-tumor immune response22. This study provides the first evidence that TNFR1-dependent TNF signalling constitutes a potent immune escape mechanism in a context of T-cell-inflamed tumor microenvironment, conferring resistance to anti-PD-1.One of the limitations to the successful use of anti-PD-1 to treat cancer patients is the requirement for tumors to be pre-infiltrated with immune cells and more specifically TILs33. This phenomenon was also associated with a reduced expression of PD-L1 on DCs and TILs, in agreement with recent papers demonstrating the role of TNF in PD-L1 stabilization23 and expression15. Accordingly, we observed a strong positive correlation between TNFA and PDCD1LG1 expression in human melanoma specimens. Reduction of PD-L1 likely contributes to PD-1+ TIL accumulation in TNF-deficient mice. Infiltration of human melanoma tumors by CD8+ T cells, which co-express PD-1 and CTLA-4 at high levels and produce IFN-γ efficiently but not TNF, has been proposed as a good predictive marker of anti-PD-1 therapy35. The latter study suggested that TNF is unlikely required for, but rather impairs, the therapeutic response to anti-PD-1, a conclusion that agrees with our findings.We show here that TNF blockade leads to an increased content of CD8+ TILs, which is predictive of the response to anti-PD-1 in melanoma patients37. Our study demonstrates that TNF potently induced TIM-3 expression on CD8+ TILs co-cultured with autologous melanoma cells from metastatic melanoma patients. We also found that the levels of the transcripts coding for TNF and TIM-3 positively correlate in human melanoma biopsies from the TCGA data bank as well as in tumors from anti-PD1-treated melanoma patients23, further reinforcing the close molecular link between TNF and TIM-3 expression. Of note, TNF blockade or deficiency did not impair basal expression of TIM-3 on TILs indicating that other cytokines, such as IL-2, IL-7, IL-15, IL-21 or IL-2738, favor TIM-3 expression. Whereas the inhibition of TIM-3 expression by TILs upon anti-PD-1 therapy may contribute, at least in part, to the synergism of PD-1 and TNF blockade, additional mechanisms are likely involved. Indeed, as reported by others, TNF can trigger melanoma dedifferentiation as well as expression of the Ezh2 histone methyltransferase and the CD73 ectonucleotidase in melanoma cells, all of those events conferring resistance to immunotherapy40. Herein, we noticed that anti-PD-1 therapeutic effect was dramatically enhanced in TNFR1-deficient mice, indicating that TNFR1-dependent TNF signalling in the tumor microenvironment also critically contributes to anti-PD-1 therapy resistance. As a matter of fact, TNF-induced TIM-3 expression on activated T cells was impaired by TNFR1 deficiency.We also identified TNF as a factor required for anti-PD-1-induced TIM-3 expression on TILs, which critically impairs the therapeutic response to anti-PD-1 in mouse and human cancers11. In our study however, anti-PD-1 treatment induced TNF-dependent up-regulation of TIM-3, but not PD-1, on activated CD4+ and CD8+ TILs. To evaluate in more details the phenotype of the TIM-3+ TILs, we monitored IFN-γ and granzyme B expression in CD8+ TILs. TIM-3 expressing CD8+ TILs did not exhibit an exhaustion phenotype but rather potently produced IFN-γ and granzyme B under our experimental conditions. This indicates that, 3 days after a single anti-PD-1 injection, TIM-3 reflects the activation of TILs. It is likely, however, that TIM-3 upregulation triggers TIL exhaustion at later time points in WT mice, as reported by others36. The complete tumor regression in most of the TNF-deficient mice precluded analysis of TILs at later time points under our experimental conditions. Importantly, TIM-3+, but not TIM-3-,CD8+ TILs died upon anti-PD-1 therapy in a TNF-dependent manner, strongly supporting the notion that TNF triggered AICD in CD8+ TILs.In the context of a chronic viral infection, TNF induced CD4+ T cell dysfunction, also called T cell exhaustion, through PD-1 up-regulation41. Strikingly, anti-TNF reduced CD8+ TIL cell death upon anti-PD-1 therapy, further emphasizing the pivotal role of TNF in AICD of CD8+ T cells, in agreement with a previous study13. The TNF-induced up-regulation of TIM-3 and PD-L1 may also contribute to AICD of CD8+ TILs in TNF-proficient animals. Indeed, cell death specifically occurred in PD-1+ TIM-3+ CD8+ TILs upon anti-PD-1 therapy in WT animals.Whereas TNF deficiency modified neither CD8+ and CD4+ TIL proliferation nor chemokine expression in tumors, it reduced cell death of CD8+ and CD4+ TILs. This was associated with an increased plasma membrane permeability, reflecting the permanent loss of plasma membrane barrier function and indicates that TILs indeed died, according to recent criteria for cell death5. Here, we provide evidence that TNF is unlikely a key cytotoxic molecule in three murine cancer models upon anti-PD-1 therapy but rather compromises CD8+ TIL survival. Moreover, the loss of TNF did not affect the expression of granzyme B, another key effector molecule of CD8+ T-cell mediated cytotoxicity. Instead of being cytotoxic towards melanoma cell lines in vitro, TNF was shown to trigger melanoma dedifferentiation, contributing to inflammation-induced immunoediting processes and immune escape16.TNF is considered to be an effector molecule of the cytotoxic T lymphocytes in anti-cancer immune response24. Our study shows that anti-PD-1 triggers TNF production, which, in turn, potently impairs CD8+ TIL response. Co-administering anti-PD-1 and anti-TNF may facilitate a sustained CD8+ T cell-dependent immune response by attenuating TNF-dependent AICD of CD8+ TILs and expression of TIM-3 and PD-L1. Taken together, our results provide the first proof-of-concept of combining anti-PD-1 and anti-TNF to fight melanoma and putatively other cancers. An ongoing phase 1b clinical trial (NCT03293784) will evaluate the safety and tolerance of the combination of immune checkpoint inhibitors and anti-TNF in metastatic melanoma patients.Anti-TNF antibodies, such as infliximab, are already used in the clinic to efficiently cure some irAEs associated with anti-PD-1 or anti-CTLA-4. However, these are neither concomitantly nor systematically administered with anti-PD-1 but only after discontinuation of immunotherapy in patients with severe immune-related colitis−1), anti-TNF , anti-TIM-3  and isotype control antibodies  were purchased from BioXcell; anti-TNFR1  was from Ozyme. Additional antibodies used in this study were anti-mouse CD45 , anti-mouse Thy1 , anti-mouse CD8 , anti-mouse CD4 , anti-mouse TIM-3 , anti-mouse LAG3 , anti-mouse PD-1 , anti-mouse CTLA-4 , anti-mouse TIGIT , anti-mouse CD11c , anti-mouse CD3 , anti-mouse PD-L1 , anti-mouse PD-L2 , anti-mouse Ki67 , anti-human TIM3  and anti-human CD8 . Cell suspensions were incubated with CD16/CD32 blocking antibodies  prior to incubation with fluorochrome-conjugated antibodies. For analysis of live and dead cells, the LIVE/DEAD fixable Aqua reagent (Invitrogen) was used.Anti-PD-1  and were authenticated in February 2012 by the Leibniz-Institut DSMZ GmbH. To guarantee cell line authenticity, the B16K1 cell line was used for a limited number of passages and tested for the expression of melanocyte-lineage proteins such as tyrosinase-related protein 2 (TRP2). Medium was changed every 2–3 days. Lewis lung carcinoma (LLC) and 4T1 breast cancer cells were from ATCC; they were not further authenticated but cultured for a limited number of passages in DMEM medium containing 10% FCS. Cell lines were tested for the absence of mycoplasma contamination by PCR.B16K1 is a genetically modified cell line generated from B16F10 cells, and that stably expresses the MHC-I molecule H-2Kb, known to stimulate CD8+ T cell-dependent immune responsesTNF-deficient (#5540) and TNFR1-deficient (#2818) C57BL/6 mice were purchased from Jackson laboratories. CD8-deficient C57BL/6 mice were a gift from Prof. J. van Meerwijk . C57BL/6 and Balb/c mice were from Janvier and Envigo, respectively. Mice had unrestricted access to food and water and were kept on a 12-h light/dark cycle under specific pathogen-free conditions at the CRCT animal facility (US006 CREFRE—Inserm/UPS), which is accredited by the French Ministry of Agriculture to perform experiments on live mice (accreditation number A-31 55508). All experimental protocols were approved by the local ethic committee  and are in compliance with the French and European regulations on care and protection of laboratory animals. For each experimental condition, at least 5 female 6–12-week-old mice were used.5 or 1 × 106 B16K1 and 4 × 105 LLC cells were intra-dermally and bilaterally injected in WT, TNF-deficient and TNFR1-deficient mice. Anti-PD-1 (10 mg kg−1) or isotype control (10 mg kg−1) was next intraperitoneally injected at days 6, 10 and 13 after B16K1 graft. Anti-TNF44 (10 mg kg−1) was inoculated at days 6, 10, 13 and 16 and anti-TNFR144 (10 mg kg−1) at days 6 and 10. Alternatively, mice were injected with relevant isotype control antibodies (10 mg kg−1). Tumor volumes were calculated using a caliper at the indicated days with the formula: tumor volume = 0.52 × length × width2.3 × 103), according to national and international policies.The overall survival was estimated taking into account the time of natural death of animals or, alternatively, the time of sacrifice when tumors reached 10% of body weight .Protocol 1: C57BL/6 WT mice were bilaterally grafted with 3 × 10Protocol 2: Similar experiment was performed as for protocol 1 with antibody injections carried out at days 13, 16, and 19.Experiments were performed to evaluate the contribution of TIM-3 in mediating the synergism observed upon anti-PD-1 and anti-TNF injections in two experimental settings:5 4T1 cells were orthotopically injected into the mammary fat pad of Balb/c mice. Mice were next injected with isotype control, anti-PD-1, anti-TNF or the combination of anti-PD-1 and anti-TNF (10 mg kg−1 of each antibody) at days 6, 9 and 13. Tumor volume was calculated using a caliper at the indicated days with the formula: Tumor volume = 0.52 × length × width2.1 × 106 B16K1 cells were intra-dermally injected in WT and TNF-deficient mice. At day 7, mice were injected with anti-PD-1. At day 10, mice were sacrificed and tumors were collected, weighed and digested with the Tumor Dissociation Kit, mouse (Miltenyi). Cells were stained with the indicated antibodies and LIVE/DEAD reagent before flow cytometry analysis (BD LSRFortessa X-20).1.106 B16K1 cells were intra-dermally injected in WT, TNF-deficient and CD8-deficient mice. Anti-PD-1 (10 mg kg−1) or vehicle (PBS) was next intraperitoneally injected at day 7. At day 10, mice were sacrificed and tumors were collected and dissociated using a Precellys Evolution tissue homogenizer (Bertin technologies) at 8000 rpm for 2 cycles of 30 s in vials containing ceramic balls. RNA was purified using the RNeasy Midi Kit (Qiagen). cDNA was prepared with SuperScript II reverse transcriptase (Thermofischer) using 1 μg of total RNA from each sample. qPCR was performed using SYBR Green Master Mix (Takara) and primers for transcripts encoding murine β-actin, HPRT, TNFα, IFN-γ and chemokines (Qiagen).10−1). Activated CD8+ T cells were incubated for 48 h in the presence of either recombinant murine or human TNF (Peprotec) and TIM-3 expression was then evaluated by flow cytometry.Murine naive CD8+ T cells were purified from the spleen of naive WT or TNFR1-deficient mice using a mouse naive CD8+ T cell purification kit (Miltenyi Biotec). Human mononuclear cells were obtained from the peripheral blood of healthy donors  after informed, signed consent. Human naive CD8+ T cells were isolated using a human naive CD8+T cell purification kit (Miltenyi Biotec). Murine and human T cells were activated with anti-CD3 and anti-CD28-coated beads (Life Technologies) and anti-CD3, anti-CD28 and anti-CD2 coated beads (Miltenyi), respectively, in the presence of IL-2  separation. Cells at the interphase ring were collected and plated for expansion in DMEM (Lonza) containing 10% FCS, 2 mM L-glutamine (Sigma), 50 µM 2-β-mercaptoethanol (Sigma), 10 mM Hepes (Gibco), 10 U ml−1 Penicillin (Sigma), 0.1 mg ml−1 Streptomycin (Sigma) and 6,000 U ml−1 human recombinant IL-2 (Proleukin) for 10 days before being frozen down.Protocol was approved by “CPP du sud-ouest et outre-mer IV” (Limoges). Informed, signed consents from metastatic melanoma patient 1 and patient 2 were obtained. Patient 1 was affected with metastatic acrolentiginous melanoma and primary tumor sample was collected before therapy induction. We obtained from patient 2, who was diagnosed with metastatic cutaneous melanoma and treated with Nivolumab and Ipilimumab, a biopsy of one abdominal metastasis. Tumor samples from the two metastatic melanoma patients were subjected to mechanical dilacerations followed by filtration through a 40 µm pore strainer. The stroma vascular fraction was then plated overnight to allow for the adhesion and expansion of tumor cells in DMEM 10% FCS. The next day, non-adherent cells were subjected to Ficoll , of autologous melanoma cells at a ratio of 20:1 for 48 h in the presence of 200 U mlhttps://genomecancer.ucsc.edu). The analysis population consisted of 342 patients with distant metastasis for whom RNAseq and clinical data overlap. No formal sample size calculation was performed concerning TCGA analysis. Gene expression was measured experimentally using the Illumina HiSeq 2000 RNA Sequencing platform and log2(x + 1) transformed. Alternatively, TNF expression was analysed in melanoma biopsies from patients treated with anti-PD-1 . In vivo and in vitro experiments were monitored in a non-blinded fashion, no method of randomization was used and usually no sample was excluded for the analysis. For the analysis of TILs, tumors in which the number of cells was too small were excluded from the analysis. Global growth curves and, when indicated, flow cytometry data were analysed using a Two-way Anova test with Tukey multiple comparison test.Each study was designed to use the minimum number of mice or samples required to obtain informative results and sufficient material for subsequent studies. No specific statistical test was used to predetermine the sample size. For animal experimentation, we used at least 5 mice and experiments were typically performed twice, unless otherwise stated in the figure legends. Statistical significance of difference between groups was evaluated by using the Graph-Pad Prism 7 software. Briefly, we tested whether the values come from a Gaussian distribution using a D’agostino-Pearson omnibus normality test (for sample sizes over 7) or the Shapiro Wilk (for samples sizes under 7). When passing the normality test, a two-sided Student’s The data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary InformationPeer Review File


IH) in vein grafts. Mesenchymal stem cells (MSCs) can home to injured intima and promote endothelial repair. However, MSC apoptosis is increased accompanied by decreased functional activity under oxidative stress. Thus, we investigate whether tumour necrosis factor‐α (TNF‐α) can promote the survival and activity of MSCs under oxidative stress to reduce IH more effectively, and establish what role the NF‐κB pathway plays in this. In this study, we preconditioned MSCs with TNF‐α (TNF‐α‐PCMSCs) for 24 hrs and measured the activation of the IKK/NF‐κB pathway. EdU and transwell assays were performed to assess proliferation and migration of TNF‐α‐PCMSCs. Apoptosis and migration of TNF‐α‐PCMSCs were evaluated in conditions of oxidative stress by analysis of the expression of Bcl‐2 and CXCR4 proteins. TNF‐α‐PCMSCs were transplanted into a vein graft model, so that cell homing could be tracked, and endothelial apoptosis and IH of vein grafts were measured. The results demonstrated that TNF‐α promotes proliferation and migration of MSCs. Furthermore, survival and migration of TNF‐α‐PCMSCs under oxidative stress were both enhanced. A greater number of MSCs migrated to the intima of vein grafts after preconditioning with TNF‐α, and the formation of neointima was significantly reduced. These effects could be partially abolished by IKK XII (NF‐κB inhibitor). All these results indicate that preconditioning with TNF‐α can promote survival and migration of MSCs under oxidative stress via the NF‐κB pathway and thus attenuate IH of vein grafts.The oxidative stress caused by endothelial injury is involved in intimal hyperplasia ( Autologous vein grafts are commonly used for myocardial revascularization around the world. Disappointingly, several lines of evidence suggest that approximately 40–50% of these vein grafts ultimately result in stenosis and even occlusion MSCs are mobilized to peripheral blood from the bone marrow and then home to injured vascular intima to promote endothelial repair It was reported that up‐regulation of the nuclear factor of kappa B (NF‐κB) pathway can promote anti‐apoptosis of cells under oxidative stress TNF‐α is an important pro‐inflammatory cytokine. The NF‐κB pathway can be activated by a large variety of factors, including cytokines and stress stimuli via the NF‐κB pathway could enhance the migration of MSCs under oxidative stress.It is widely accepted that the SDF‐1α/CXCR4 axis has a role in mediating the migration of stem cells or tumour cells, one which can be inhibited by the CXCR4 antagonist, AMD3100 In this study, we investigated the effect of the TNF‐α on a variety of activities of MSCs, such as survival, migration and secretion. The findings may shed light on a possible role of the NF‐κB pathway in prevention of IH in vein grafts with the help of MSCs.2. Cell purification was achieved by washing with phosphate‐buffered saline (PBS) and changing the culture medium after 48 hrs. Cells at passage 3 to 4 were used in the experiments. Before transplantation, cells were labelled with chloromethylbenzamido dialkylcarbocyanine (CM‐Dil) for cell tracking in vivo according to the manufacturer's instruction .MSCs were extracted from the femurs of young Wistar rats  under anaesthesia with 1% pentobarbital sodium (40 mg/kg). The culture medium was ɑ‐MEM  with 10% foetal bovine serum (FBS) . The bone marrow was flushed with culture medium and then cultured at 37°C in 5% CO2O2 (200 μM) for 12 hrs.MSCs were preconditioned with TNF‐α (50 ng/ml)  for 24 hrs. Treatment with IKK XII   and/or SB203580   was carried out prior to TNF‐α treatment for 30 min. to determine the role of NF‐κB or p38 MAPK pathway. To investigate the survival and migration of TNF‐α‐ and/or IKK XII‐preconditioned MSCs under oxidative stress, medium was exchanged after 24 hrs of chemical pre‐treatment, and then, MSCs were treated with H8/ml × 0.2 ml) were transplanted through the caudal vein after grafting. Experimental rats were randomly divided into five groups: (i) sham group (skin incision without vein grafting), n = 12; (ii) untreated group (vein grafting without MSC transplantation), n = 12; (iii) Non‐PCMSC group (vein grafting and treatment with non‐preconditioned MSCs), n = 24; (iv) TNF‐α‐PC MSC group (vein grafting and treatment with TNF‐α‐preconditioned MSCs), n = 24; and (v) IKKXII+TNF‐α‐PCMSC group (vein grafting and treatment with IKK XII +TNF‐α‐preconditioned MSCs), n = 24.Adult male Wistar rats weighing 250–300 g were used in this experiment. The left external jugular vein (LEJV) was inserted into the left common carotid artery (LCCA) in the same animal. Vein grafting was performed with the cuff technique In each MSC transplantation group, 12 rats were humanely killed 1 week after operation for evaluation of endothelial apoptosis using a TUNEL assay, and MSC homing using fluorescence microscopy. All remaining rats were killed 4 weeks after the operation. Van Gieson staining was performed to evaluate IH. All of the study protocols were approved by the Animal Care Committee of Qilu Hospital of Shandong University. All animals were cared according to the Guide for the Care and Use of Laboratory Animals.Flow cytometry was used to identify the phenotypes of the cultured MSCs. MSCs at passage 3 were incubated with anti‐CD29, anti‐CD44, anti‐CD45, anti‐CD90  or anti‐CD34 . Antibodies of the corresponding isotype served as the negative control. To detect CXCR4 expression on the cellular surface, cells were stained with mouse anti‐rat CXCR4 antibody  or the isotype control. The secondary antibody was donkey anti‐mouse IgG PE .Protein samples were loaded onto polyacrylamide gels (8–10%) for electrophoresis. The protein was then transferred onto a polyvinylidene fluoride (PVDF) membrane. Each membrane was blocked in tris‐buffered saline with Tween 20 (TBST) containing 5% non‐fat dry milk for 1 hr at room temperature and then incubated at 4°C overnight with one of the following primary antibodies: anti‐IKK‐α ; anti‐IKK‐β ; anti‐p‐IKK‐α/β (Ser176/180) ; anti‐NF‐κB‐p65 ; anti‐p‐NF‐κB‐p65 (S536) ; anti‐p38MAPK ; anti‐p‐p38MAPK (Thr180/Tyr182) ; anti‐CXCR4 ; anti‐Bcl‐2 ; or anti‐GAPDH . This was followed by incubation in peroxidase‐conjugated AffiniPure goat anti‐rabbit or mouse IgG (H+L)  for 1 hr at room temperature. Immunoreactivity was detected by chemiluminescence.Cell cycle was detected using Cycletest Plus DNA Reagent Kit  according to the manufacturer's instruction. To quantify the apoptosis of the MSCs, flow cytometry was performed with an annexin V‐FITC/PI apoptosis detection kit (BD Biosciences).3 cells per well. Following overnight incubation, cells were treated with different reagents for 12–24 hrs, and 10% CCK‐8 solution was then added to each well. The absorbance of the supernatant was measured at a wavelength of 450 nm after the cells were incubated for 2 hrs at 37°C.Cell viability was assessed using a Cell Counting Kit‐8 (CCK‐8) according to the manufacturer's protocol . MSCs were seeded onto 96‐well plates at a density of 6 × 10Cell proliferation was assessed using an EdU DNA Cell Proliferation Kit according to the manufacturer's protocol . The nuclei of cells in proliferation state showed red. The proliferation rate = the number of cells in proliferation state (red)/the number of total cells (purple).Preconditioned MSCs cultured on glass slides were fixed with 4% paraformaldehyde for 20 min. and permeabilized with 0.5% Triton X‐100 for 20 min. Cellular proteins were detected with primary antibodies against p‐NF‐κB‐p65 and CXCR4. FITC‐conjugated goat anti‐rabbit and PE‐conjugated donkey anti‐mouse secondary antibodies were used. Nuclei were stained using 4′, 6′‐diamidino‐2‐phenylindole (DAPI) (Sigma‐Aldrich). Coverslips were mounted with FluorSave Reagent . Images were acquired using fluorescence microscopy.5/ml) was seeded in the upper chamber and 600 μl complete medium with SDF‐1a  was placed into the lower well to induce cell migration. Following 12‐hrs incubation, cells on the upper side of the membrane were removed using cotton swabs. Cells on the bottom surface of the membrane were stained with 0.1% crystal violet and counted in five randomly selected microscopic fields (×200).Migration assay was performed with a transwell  containing a polycarbonate membrane filter (8 μm pore size). 200 μl preconditioned MSCs  concentrations in the serum were measured to assess the level of oxidative stress using an ELISA kit . MSCs were preconditioned for 24 hrs, and the culture medium of each well was collected. VEGF and HGF protein concentrations in each well were quantified using an enzyme‐linked immunosorbent assay (ELISA) kit (ExCellBio) according to the manufacturer's instructions.Rat blood samples were obtained 1 day and 1 week after vein grafting, and serum was isolated from each tube after centrifugation at 500 × The apoptosis of endothelial cells in vein graft was observed using a TUNEL Andy Fluor™ 488 Apoptosis Detection Kit . Endothelial cells were labelled according to the manufacturer's instructions. Stained sections were observed using fluorescent microscopy. We counted the nuclei of vascular endothelium . The nuclei of apoptotic cells showed green. The apoptosis rate = the number of apoptotic cells (green)/the number of total cells (purple).Vein grafts were fixed in 4% paraformaldehyde and then embedded in paraffin. The paraffin specimens were sectioned into 4‐μm‐thick slices. Briefly, sections were prepared by dewaxing, staining and then dehydration. The morphology of the vascular intima was observed by light microscopy.anova) followed by Dunnett's multiple comparison tests. Values of P < 0.05 were considered statistically significant. All analyses were performed with SPSS19.0 software .All values were presented as mean ± S.D. Data were analysed using one‐way analysis of variance  serve as an important barrier, protecting against monocyte adhesion and the proliferation of vascular smooth muscle cells (VSMCs). The apoptosis of ECs will destroy the vascular barrier and expose VSMCs to the blood flow directly, leading to the adhesion and activation of inflammatory cells MSCs can be mobilized after vascular injury, participating in the remodelling processes of the injured vasculature. MSCs exhibited a strong capacity for adhesion to repair vessel wall after injury. On adhesion, a substantial proportion of MSCs differentiated into ECs in the neointima In theory, up‐regulation of the NF‐κB pathway could promote survival of MSCs The percentage of cells in S phase of cell cycle was significantly increased in the TNF‐α group, and the effect can be reversed by IKK XII. We speculate that activation of the NF‐κB pathway could promote cell proliferation. The EdU assay also showed that TNF‐α can significantly enhance the proliferation, supporting the above results. In addition, we have confirmed that the NF‐κB pathway plays the leading role in regulating cell viability by SB203580 antagonizing the p38‐MAPK pathway.via the NF‐κB pathway, consistent with a previous report TNF‐α‐PCMSCs.With TNF‐α increasing p‐NF‐κB‐p65, the CXCR4 was up‐regulated as well. Pre‐treatment with IKK XII abolished the overexpression of CXCR4. We considered that TNF‐α promotes CXCR4 expression and enhances migration of MSCs 2O2, the viability of MSCs was markedly decreased, accompanied by a significant increase in apoptosis. Annexin V‐FITC/PI stain indicated that TNF‐α preconditioning reduced the degree of apoptosis, and an increase in anti‐apoptotic protein Bcl‐2 was observed in the TNF‐α group as well. The anti‐apoptotic effect of TNF‐α preconditioning may be related to the up‐regulation of Bcl‐2. TNF‐α also elevated the expression of CXCR4, while oxidative stress down‐regulated it, suggesting that TNF‐α‐PCMSCs would experience increased rates of migration under oxidative stress. These results confirm that TNF‐α reduces apoptosis and improves migration of MSCs under oxidative stress via the NF‐κB pathway.Following treatment with 200 μM HTNF‐α‐PCMSCs could significantly delay apoptosis of endothelial cells caused by oxidative stress. We speculated that the protective effect of TNF‐α‐PCMSCs on endothelial cells may be related to the secretion of pro‐survival factors, such as VEGF and HGF TNF‐α‐PCMSCs, confirming our speculation. Kwon YW applied conditioned medium derived from TNF‐α‐treated MSCs to treat the limb ischaemia and found that IL‐6 and IL‐8 secreted by MSCs could promote homing of endothelial progenitor cells and angiogenesis An ELISA test of MDA confirmed the existence of oxidative stress caused by vein grafting. And the TUNEL assay revealed that TNF‐α‐PCMSCs migrated to the intima of the vein graft was significantly increased, and this increase was halted by IKK XII. In addition, the neointima of the TNF‐α‐PCMSC group was also thinner than Non‐PCMSC group. These data confirm that TNF‐α‐PCMSCs can more efficiently migrate to the damaged vascular intima and better prevent IH in vein graft. The therapeutic effect may come from the synthetic action of enhanced anti‐apoptosis and ameliorative migration of MSCs.The number of In summary, we have demonstrated that TNF‐α preconditioning can promote survival and migration of MSCs under oxidative stress and promote an increase in number of MSCs migrating to vein graft. As a result, apoptosis of endothelial cells is reduced, and IH of vein graft significantly attenuated. Meanwhile, the NF‐κB pathway participates in the regulation process. Functionally improved MSCs therapy for IH will have good prospects. So far, the clinical trials of stem cell therapy for myocardial infarction or spinal cord injury have obtained the very good results. The breakthrough of the trials indicates the stem cell therapy is striding forward greeting the dawn of the new age.The authors confirm that there are no conflict of interests.


The sensitivity and specificity of1H-MRSI and PI-RADS v2 for extra-prostatic extension (EPE)were 64.0% (40%) and 20.0% (48%), and 50.0% (57.1%) and 71.4% (64.3%). Thearea under the ROC curves (AUC) for prediction of high-grade prostate cancerwere 0.65 and 0.61 for PI-RADS v2 and 0.72 and 0.70 when combined with1H-MRSI . For prediction of EPE the AUC were 0.54 and 0.60 for PI-RADS v2 and0.55 and 0.61 when combined with 1H-MRSI (p >0.05).The sensitivity and specificity of 1H-MRSI might improve the discrimination of high-grade prostatecancer when combined to PI-RADS v2, particularly for PI-RADS v2 score 4lesions, but it does not affect the prediction of EPE. Multiparametric magnetic resonance imaging (mpMRI) of theprostate has gained considerable utilization in the setting of newly diagnoseddisease to identify occult, higher-grade or stage elements missed by conventionalbiopsy,4. Moreover, when coupled with real timeultrasonography, fusion mpMRI biopsy has demonstrated superior PCa detection ratescompared with traditional template guided biopsy.Prostate cancer (PCa) is diagnosed in approximately 230,000 men in the United Stateseach year. The AmericanCollege of Radiology, the AdMeTech Foundation, and the European Society ofUrogenital Radiology have partnered and recently presented a new version of theProstate Imaging Reporting and Data System (PI-RADS v2), which integrates results ofT2-weighted (T2W), high b-value diffusion-weighted image (DWI), and dynamic contrastenhanced (DCE) MRI. Proton MRspectroscopic imaging (1H-MRSI), previously an optional tool, was notincluded in the current version of the document. 1H-MRSI has, though,been recognized as a useful non-invasive method for evaluating metaboliccharacteristics of prostatic lesions, yielding identifiable signatures that mayallow for the discrimination of high-grade tumors. However, 1H-MRSI is susceptible tofalse positive related to choline contamination from the seminal vesicles orurethra, or byprostatitis.Furthermore, the ACRIN 6659 study that was published by Weinreb et al. found noadded benefit for 1H-MRSI compared with T2W alone to localize PCa to thegland sextant.With growing integration of mpMRI as an adjunct diagnostic modality, the need tostandardize acquisition protocols and study reporting is evident as it mayfacilitate benchmarks for consistency in both clinical care and research settingsalike1H-MRSI for predicting PCa with dominant Gleason pattern≥ 4 or non-organ confined disease at the time of surgery.In this context, we sought to compare the diagnostic performance of PI-RADS v2 withor without 1H-MRSI; radical prostatectomywithin six months of imaging; no treatments between imaging and surgery.The Institutional Review Board approved this retrospective single center study.Informed consent was prospectively obtained from all patients authorizing the use ofclinical data in future studies. Consecutive subjects were identified throughsearches of our Urological Oncological Database, Prostate MR Imaging Database, andelectronic medical records. Inclusion criteria: biopsy-proven PCa; 3-teslaendorectal prostate mpMRI, including 1H-MRSI data. Therefore, 39 men formed the study population. Patientswere clinically risk stratified using the Cancer of the Prostate Risk Assessmentscore (CAPRA). CAPRA is aneasy to calculate validated nomogram that predicts outcomes across multipletreatment approaches and it predicts an individual’s likelihood of metastasis,cancer-specific mortality, and overall mortality. The score is calculated usingpoints assigned to: age at diagnosis, PSA at diagnosis, Gleason score of the biopsy,clinical stage and percent of biopsy cores involved with cancer. Three categorieswere assigned: low (scores 0-2), intermediate (scores 3-5), and high risk (scores6-10).Forty patients seen between January 2013 and December 2014 fulfilled these criteria,but one was excluded because of a hip replacement that distorted the.Scans were acquired on a 3-tesla scanner  usingthe body coil for excitation and an endorectal coil  filled with perfluorocarbon  and aphased-array coil for reception. Images were post-processed to compensate forthe reception profile of the endorectal coilThe protocol included:- Oblique axial T2W high-resolution 2D FSE MR images . Acquisition time = 4 min 1 s.- Axial CUBE T2W 3D FSE MR images . Coronal and sagittal reformats were generated. Acquisitiontime = 4 min 42 s.- Two 2D single-shot EPI SE high b-value DWI acquisitions . Acquisition times = 3min 52 s and 4 min 29 s. Two ADC maps were reconstructed from eachacquisition.3/susing a power injector, followed by a 20 cm3 saline bolus atthe same rate, 5 min acquisition, temporal resolution = 10 s).Acquisition time = 4 min 58 s.- Oblique axial T1-weighted 3D spoiled gradient echo dynamically contrastenhanced MR images  withspectral-spatial pulses for the two 180º excitation pulses, andouter-voxel saturation pulses . A PRESS volume was selected usingthe oblique axial T2W images that incorporated the entire prostate whileminimizing inclusion of the rectum and peri-prostatic lipids. The PRESSvolume was shimmed using an automated phase mapping algorithm, followedby manual shimming of the x, y and z gradients until a water line-widthof ≤ 12 Hz was obtained. An interleaved flyback echo-planarspectroscopic readout with a spectral bandwidth of 1012 Hz was used inthe left-right dimension. Acquisition time = 7 min 50 s.- 3D 1H-MRSI data were processed using custom processingsoftware. The rawdata acquired with the modified PRESS incorporating the flyback echo-planarreadout trajectory were reordered as previously described and processed in the samemanner as the conventional 4D 1H-MRSI dataset. The spectral data wereapodized with a 2-Hz Lorentzian function in the frequency domain, with nofiltering in the spatial dimensions. Data were Fourier transformed in the timedomain and in three spatial domains. Spectral phase, baseline, and frequencycorrections were iteratively made and metabolite peak areas calculated aspreviously described. The3D 1H-MRSI spectral arrays and associated metabolite peak area ratioswere overlaid on the corresponding transverse T2W images using the open-sourcespectral processing package SIVIC.The - Axial T1-weighted FGRE MR images .Acquisition time = 2 min 45 s.1H-MRSI and 2 yearsof experience with PI-RADS v2, i.e. since its initial publication), unaware ofthe clinical and pathologic data, independently reviewed all scans on a PACSworkstation  in a single session. To mimicclinical practice, the radiologist could review the T2W, DWI, and DCE sequencesin any order, alone or in conjunction. The radiologists had no access to1H-MRSI images at this stage. Up to four suspicious foci wereidentified and PI-RADS v2 scores assigned to each (.Two radiologists (8 and 5 years of experience with  to each (7).1H-MRSI score to all suspicious lesions previously assigned a PI-RADSv2 score, i.e. lesions that received a PI-RADS v2 score ranging from 3 to 5. Allusable voxels were scored using the five-point scale based on the area ratio ofthe citrate and choline peaks  or positive (score 4 or 5).The primary outcomes were the predictions of high-grade PCa, defined as Gleasonscore ≥ 4+3, and high-stage disease, defined as extra-prostatic extension(EPE) (≥ T3A) at radical prostatectomy on a per patient basis. In theevent of multiple lesions, only the index lesion was considered for analyses.The index lesion was defined as the lesion with the highest overall PI-RADSscore. If two or more lesions received the same score, the index lesion was theone associated with clear EPE. If none of the lesions demonstrated EPE, theindex lesion was the largest one. We assessed the sensitivity, specificity,negative predictive value (NPV), positive predictive value (PPV), and accuracyof the overall PI-RADS v2 score and 1H-MRSI score; and those to the area under the ROC curve derivedfrom the multivariate models that included the overall PI-RADS v2 and1H-MRSI scores. As mentioned above, if more than one lesion wassuspected on mpMRI, only the index lesion was utilized in the analyses.We compared the areas under the receiver operating characteristic (ROC) curve ofunivariate logistic regression models that included the overall PI-RADS v2 scoreor 1H-MRSI scores werecalculated utilizing a weighted kappa score (weights = 1 / 1 - 1 / 0.25 - 0.25 -1 / 0 - 0 - 0.75 - 1 / 0 - 0 - 0.5 - 0.75 - 1).Interobserver agreement of overall PI-RADS v2 and P values < 0.05 were considered statisticallysignificant.All analyses were performed using Stata version 13.1 .The median age was 65 years (interquartile range (IQR), 11). The median PSA atdiagnosis was 6.8 ng/mL . CAPRA classified 13 men (33.3%) with low, 21(53.9%) with intermediate, and 5 (12.8%) with high clinical risk. Thirty-seven men(94.8%) had clinically organ-confined disease at diagnosis; and 28 (71.8%) hadbiopsy Gleason score ≤ 3+4. The complete clinical, demographic and pathologiccharacteristics are presented in At prostatectomy, 5 men (12.8%) had Gleason score 3+3, 20 (51.3%) had Gleason score3+4, 12 had Gleason score 4+3 (30.8%), 1 (2.6%) had Gleason score 4+4, and 1 (2.6%)had Gleason score 5+4. Disease was organ-confined (T2C or less) in 19 (48.7%).Twenty men (51.3%) had extra-prostatic extension.1H-MRSI scores were 0.62  and 0.46 ,respectively.1H-MRSI (assigned to a suspicious lesion) to predictGleason pattern ≥ 4+3 was higher than the specificity of the overall PI-RADSv2 score . For thedetection of stage ≥ T3a, the use of 1H-MRSI scores to furthercharacterize suspicious lesions led, for reader 1, to an increase in sensitivity(64% versus 20%) associated with a decrease in specificity (50% versus 71.4%). Noclear differences were seen for reader 2. The performance characteristics areoutlined in The specificity of 1H-MRSIto PI-RADS v2 improves the prediction of high-grade PCa when lesions arecharacterized as PI-RADS v2 score 4. There were no statistically significantdifferences between the AUC of overall PI-RADS v2  and 1H-MRSI  for either reader.The AUC of overall PI-RADS v2 combined with 1H-MRSI was significantlyhigher than the AUC of overall PI-RADS v2 alone for reader 1 , but not for reader 2 .Analysis of the shape of the ROC curves shows that the addition of 1H-MRSI , and their combination for either reader.For the discrimination of stage ≥ T3a, there were no statistically significantdifferences between the AUC of overall PI-RADS v2 , 1H-MRSI to PI-RADS v2 mightimprove the detection of PCa with Gleason pattern ≥ 4+3, in particular ofPI-RADS v2 score 4 lesions; however, it does not seem to increase the detection ofhigh stage (≥ T3a) disease.Our results suggest that the addition of 1H-MRSI.Yet, the PI-RADS Steering Committee encourages “the continued development ofpromising MRI methodologies”, including 1H-MRSI, and state that thesetechnologies will be considered for inclusion in future versions, pending new data.While the PI-RADS v2 document does not provide specific reasons for not including1H-MRSI, it is known that it is a complex technique with limitedacceptance outside specialized centers due to its long acquisition time, need forlocal expertise, and general reliance on endorectal coil imaging. Yet,1H-MRSI warrants continue attention; new hardware and softwaredevelopments may make it more manageable.Different from its initial version, PI-RADS v2 does not include 1H-MRSI improves tumor localization,18, volume estimation,20,staging, tissuecharacterization, andidentification of recurrent disease after therapy,24. Amulticenter study showed that positive MR spectroscopy findings are likely toreflect higher tumor grade and/or volume. These studies, though, do have limitations, and thereare, also, those with less encouraging results; the ACRIN study published in 2009,for example, showed no difference of accuracy when comparing combined T2W and1H-MRSI and T2W alone.Based on previous studies, 1H-MRSI, however, led to a 50% increase in specificity, andmight at least in some cases help to identify men with high-grade PCa. Our resultsshowed this is particularly true when a lesion receives a PI-RADS v2 score of 4.These results are aligned to those of Giusti et al., who showed that metabolicratios correlate with Gleason scores, and they are similar to those of a meta-analysis in which1H-MRSI had a higher specificity than T2W and increased thespecificity of the combination of T2W and DWI. While the comparison of overall AUCs  found an improvement of discrimination between men with andwithout PCa Gleason pattern ≥ 4+3 using the combined PI-RADS v2 and1H-MRSI for one reader only, the assessment of the shape of thecurves shows a clear separation between the lines of the ROC curves of PI-RADS v2alone and PI-RADS v2 combined with1H-MRSI at the segment that includesonly PI-RADS v2 score 4 lesions for both readers. It is possible that thisdiscrepancy in results is due to differences in readers’ experience.1H-MRSI is a complex technique and interpretation can be challenging. Itis important to make note of this fact, as these same challenges are likely to befound at other sites that lack radiologist with experience with1H-MRSI.Our results show that the overall PI-RADS v2 score is very sensitive to detectGleason pattern ≥ 4+3, but its specificity is very low. This suggests PI-RADSv2 is a good option to detect the disease, but not necessarily to characterize it.The use of 1H-MRSI might explain why it did improve thedetection of EPE, as EPE is typically detected on anatomical images. The overallPI-RADS v2 score, however, does include an anatomical assessment. Furthermore, bothreaders assigned an overall PI-RADS score of 4 or 5 to more than 80% of thesesuspicious lesions, and an overall PI-RADS v2 score of 5, at least in someinstances, characterizes definite EPE. An increase in specificity after utilizing 1H-MRSImight, therefore, not have been expected. Similarly, because 1H-MRSI wasapplied after the detection of a suspicious lesion using PI-RADS v2, its sensitivityis a direct reflection of this initial detection. Accordingly, it would be expectedthat positive 1H-MRSI results would have a greater impact on loweroverall PI-RADS v2 scores. In this study we opted for analyzing only the indexlesion, less than 20% of which received a score of 3, likely explaining the lack ofbenefit of 1H-MRSI. It remains, thus, unknown if 1H-MRSI wouldhave affected cases presenting with these indeterminate lesions.The metabolic nature of 1H-MRSI toPI-RADS v2, but a few authors tested it with its previous version with variousresults. The studies of Khalifa et al. and Panebianco et al. suggested 1H-MRSI improved characterization ofPCa and support our results. Yet, the results of Platzek et al. and, more recently, Polanec etal. found that1H-MRSI did not increase the detection and nor improved the gradingof PCa. While several possible explanations exist for these discrepancies, theexercise of explaining them is likely not warranted, as the first version of PI-RADSis quite different from PI-RADS v2 and should no longer be utilized. More important,perhaps, is to recognize that considerable interest exists in optimizing theidentification high grade or stage disease among men with clinically localized PCaas such determinations may improve management decisions. And that other imagingtechniques, including 1H-MRSI, may be helpful.We did not find other studies evaluating the incorporation 1H-MRSI in improveddelineation of PCa outcomes. Third, PI-RADS was designed with the intent to improvethe detection of tumors with Gleason score ≥ 3+4, and not ≥ 4+3 as weproposed. This could, perhaps, explain the low specificity of PI-RADS v2 found inthis study. More important, though, is that we may have overestimated the diagnosticperformance of both PI-RADS v2 and 1H-MRSI due to spectrum bias. Spectrumbias refers to the fact that it is usually easier to detect advanced disease thanearly-stage disease, as subtle abnormalities can be hard to distinguish from normalfindings. This typically leads to a higher diagnostic accuracy when a study includesin a population with advanced disease than when the subjects have less severedisease. We opted for characterizing as high-grade tumors only tumors with Gleasonscore ≥ 4+3 because many institutions consider men with Gleason 3+4 ascandidates for active surveillance, while a Gleason score ≥ 4+3 isuniversally considered an indication for definitive therapy.This study has limitations. First, this was a retrospective study with thelimitations inherent to this type of design. The population studied was highlyselected and included only men who had endorectal mpMRI and radical prostatectomy.We, therefore, probably incurred selection bias and our patients may not fullyrepresent all men with PCa. This is illustrated by the fact most of our lesions werecharacterized as PI-RADS v2 4 and 5, as men with lower scores are less likely tohave cancer and to undergo surgery. However, we considered the need for an adequatestandard of reference more important than the limited generalizability. One possibleoption to prostatectomy is MR-guided biopsy, which can be performed in-bore or byfusion with ultrasound. Accordingly, some of our results, in particular the positiveand negative predictive values, do not apply to all men with suspected PCa nor toall men who are under active surveillance and typically have low-grade low-volumedisease. Second, we examined endpoints of high-grade and/or non-organ confineddisease, but not more distant oncologic endpoints including biochemical recurrenceor metastatic progression. Prospective studies with extended follow up may bewarranted to definitively evaluate the role of 1H-MRSI might improve the discrimination of pathologicalGleason score ≥ 4+3 when added to the PI-RADS v2, in particular for lesionsthat receive a score of 4, but it does not affect the prediction of PCa stage≥ T3a.In summary,


Recent studies have suggested that the tumor necrosis factor-α (TNF-α) pathway is a potential target for the management of osteoarthritis (OA). Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is essential in several cytokine-mediated cascades, including the TNF-α, interleukin-1 (IL-1), and TGF-β pathways. The role of TAK1 in synovial tissue in OA is not fully understood. Using synovial cells harvested from OA patients during surgery, we investigated whether TAK1 inhibition suppresses production of TNF-α-induced extracellular matrix degrading enzymes and expression of pain-related molecules.Synovial tissues were harvested from ten subjects with radiographic evidence of osteoarthritis (OA) during total knee arthroplasty. Synovial cells were cultured and stimulated with control (culture media), 10 ng/mL human recombinant TNF-α, or 10 ng/mL TNF-α and 10 μM of the TAK1 inhibitor (5Z)-7-oxozeaenol for 24 h. Real-time polymerase chain reaction (PCR) analysis was used to monitor expression of mRNA of the extracellular matrix degrading enzymes matrix metalloproteinase-3 (MMP-3) and a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 4 (ADAMTS-4); and of the pain-related molecules cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1), and nerve growth factor (NGF). MMP-3 and NGF protein concentrations in cell supernatant were measured by enzyme-linked immunosorbent assay (ELISA). COX-2, mPGES-1 and ADAMTS-4 protein expression was also evaluated by western blotting.p < 0.05) and protein expression . Treatment with the TAK1 inihibitor (5Z)-7-oxozeaenol reduced ADAMTS-4 and MMP3 mRNA  and protein expression  in OA synovial cells. COX-2, mPGES-1 and NGF mRNA  and protein expression  were increased by TNF-α. (5Z)-7-oxozeaenol treatment reduced mPGES1 and NGF mRNA  and protein .TNF-α stimulated increases in ADAMTS-4 and MMP3 mRNA (2.0-fold and 1.6-fold, respectively, TAK1 plays an important role in the regulation of TNF-α induced extracellular matrix degrading enzymes and pain-related molecule expression. TAK1 may be a potential target for therapeutic strategies aimed at preventing osteoarthritis progression and pain. Osteoarthritis (OA), the most common joint disease in humans, is characterized by cartilage destruction. Its main symptoms are joint stiffness and pain with associated joint space narrowing. OA severely limits physical activity and greatly impairs quality of life. The identification of molecules that inhibit OA progression and relieve pain is key to OA treatment.Synovial tissues produce inflammatory cytokines and contribute to OA pathogenesis –3. SeverTransforming growth factor (TGF)-β-activated kinase 1 (TAK1) is a member of the mitogen-activated protein kinase (MAPK) family . TAK1 isWe investigated whether TAK1 inhibition suppresses production of TNF-α-induced extracellular matrix degrading enzymes and expression of pain-related molecules.Human recombinant TNF-α was purchased from Biolegend  and (5Z)-7-oxozeaenol was purchased from Sigma .2 (range 22.6–31.4). A sample of synovial tissue was harvested from the suprapatellar pouch of each operated knee during total knee arthroplasty surgery. Informed consent for participation in this study was obtained from each patient on the day before surgery.A total of ten participants with radiographic knee OA  underwent total knee arthroplasty at our institution. The study included 3 men and 7 women aged 60–89 years  with a mean ± SD body mass index (BMI) of 26.3 ± 2.6 kg/m184/187) . After washings twice in PBS, the sections were incubated for 10 min at room temperature with biotinylated anti-rabbit IgG (Nichirei). Subsequently, the sections were washed twice in PBS and incubated with horseradish peroxidase (HRP)-conjugated streptavidin for 5 min. Peroxidase activity was revealed by 3,3′-diaminobenzidine (Nichirei) and the sections were counterstained with Mayer’s hematoxylin.To determine the localization of phosphorylated TAK1 (p-TAK1) and TNF-α, the paraformaldehyde-fixed synovial tissue samples were embedded in paraffin and sliced into 3-μm thick sections. The sections were deparaffinized with xylene for 1 h, hydrated in serial dilutions of ethanol  and then rinsed in distilled water. For antigen retrieval, deparaffinized sections were immersed in 10 mM sodium citrate buffer pH 6.0 and maintained at 98 °C for 30 min. After cooling at room temperature, endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol for 15 min. The slides were washed in phosphate-buffered saline (PBS) and incubated with 10% goat serum  at room temperature. Subsequently, the sections were incubated overnight at 4 °C with rabbit polyclonal primary antibody against p-TAK1 , 10 ng/mL human recombinant TNF-α, or 10 ng/mL TNF-α and 10 μM (5Z)-7-oxozeaenol for 30 min or 24 h. The concentration of 10 μM (5Z)-7-oxozeaenol was determined based on previous studies -1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine dye reagent  and an NGF ELISA kit (R&D Systems).To investigate ADAMTS-4, COX-2, and microsomal prostaglandin E2 synthase 1 (mPGES1) protein expression, cells harvested from five patients were stimulated with TNF-α, or 10 ng/mL TNF-α and 10 μM (5Z)-7-oxozeaenol, for 24 h. Using methodology described previously , synoviap < 0.05 was considered statistically significant. Statistical analyses were performed using commercial software .Differences between the untreated and treated synovial cells were compared using one-way ANOVA with Fisher’s least significant difference test. A To investigate the localization of p-TAK1, immunohistochemical analysis was performed. Immunoreactivity with p-TAK1 was observed in the synovial lining cells of OA patients . MMP-3 protein levels were also significantly increased in synovial cell culture supernatant in the presence of exogenously added TNF-α (2.0-fold) and this expression was reduced by (5Z)-7-oxozeaenol  and that this expression was reduced by (5Z)-7-oxozeaenol  and were reduced by TNF-α with added (5Z)-7-oxozeaenol (Table p < 0.05). However, TNF-α-induced COX-2 protein elevation was not significantly reduced by (5Z)-7-oxozeaenol (p = 0.065). NGF concentration in cell culture supernatant was also significantly increased by TNF-α  and decreased by added (5Z)-7-oxozeaenol (Table p < 0.05).Protein expression of COX-2 and mPGES1 also increased in synovial cells in the presence of exogenously added TNF-α  and mPGES1 protein expression were significantly reduced by (5Z)-7-oxozeaenol -7-oxozeaenol reduced expression of the TNF-α-induced extracellular matrix degrading enzymes MMP-3 and ADAMTS-4 and the pain-related molecules mPGES-1 and NGF in the synovial tissues of OA patients.MMP-3 and ADAMTS-4 have degradative effects on the extracellular matrix, and it has been suggested that they function as important inflammatory mediators in OA , 23–26. COX-2 and mPGES-1 enzymes are expressed in synovial lining cells. Prostaglandin E2 (PGE2) levels are elevated in inflamed synovial tissue , 27, 28 TNF-α-upregulated-COX-2 and mPGES1 mRNA expression significantly decreased in the presence of (5Z)-7-oxozeaenol consistent with a previous report, where hydrogen peroxide-induced COX-2 elevation suppressed 5 μM (5Z)-7-oxozeaenol in bovine synovial fibroblasts. Although COX-2 protein expression was not completely suppressed, phosphorylation of p38 MAPK was reduced below baseline levels. Further investigations into the regulation of COX-2 by TAK1 in synovial cells are needed.NGF also plays an important role in OA pain , 33–36, Several limitations of the present study warrant mention. First, we evaluated a single concentration of TNF-α at one time point. Second, this was an in vitro study. It remains to be determined if TAK1 inhibition will reduce OA progression and pain in vivo. Finally, we showed that TAK1 inhibition reduced MMP-3 and ADAMTS-4 levels, however, it remains to be determined whether blockage of TAK1 inhibits degradation of ECM proteins. This may be elucidated by investigating whether cell supernatants degrade purified extracellular matrix using an overlay assay .In conclusion, TAK1 upregulates TNF-α-induced extracellular matrix-degrading enzymes and pain-related molecule expression in OA synovial tissues. These properties suggest that TAK1 in synovial tissue may be a potential target for therapeutic strategies for osteoarthritic progression and pain.


Systemic as well as mucosal IgG and IgA antibodies in serum and intestinal and nasal secretions were measured. As expected, strong serum IgG, IgG1, and IgG2a antibodies as well as a dose sparing effect were induced by both Al(OH)3 and MPLA, but no mucosal IgA antibodies were detected. In contrast, IN immunization with GII.4 VLPs without an adjuvant induced systemic as well as mucosal IgA antibody response. These results indicate that mucosal delivery of NoV VLPs is needed for induction of mucosal responses.Norovirus (NoV) is a main cause of acute gastroenteritis across all ages worldwide. NoV vaccine candidates currently in clinical trials are based on noninfectious highly immunogenic virus-like particles (VLPs) delivered intramuscularly (IM). Since NoV is an enteric pathogen, it is likely that mucosal immunity has a significant role in protection from infection in the intestine. Due to the fact that IM delivery of NoV VLPs does not generate mucosal immunity, we investigated whether NoV genotype GII.4 VLPs coadministered with aluminum hydroxide (Al(OH) It infeA (MPLA) . AlternaA (MPLA) , 8. DespA (MPLA) –12.At present, there are only a few adjuvants approved for human use, and none for mucosal delivery , 14. Alu3 or MPLA, has an effect on generation of NoV-specific mucosal immunity.We have recently shown that NoV GII.4 VLPs induce protective IgA antibodies in mucosal lavages of mice immunized via intranasal (IN), but not IM, route . Here, wNoV GII.4-1999 (reference strain accession number AF080551) VLPs used for immunization and as antigen in immunological assays were produced by recombinant baculovirus technology in Sf9 insect cells and purified as described in detail elsewhere [μg dose of GII.4 VLPs alone or formulated with 100 μg of Al(OH)3  or 5 μg of MPLA from S. minnesota R595 (InvivoGen). In addition, two groups of mice received a combination vaccine [μg GII.4 VLPs via IM and IN delivery. Mice administered IM or IN with a carrier (sterile PBS only) served as control groups. Immunizations were performed under general anesthesia induced with a mixture of ketamine  and medetomidine .Female 7-week-old BALB/c OlaHsd mice   were immunized IM two times (at study weeks 0 and 3) with 0.3  vaccine  containiTo test the kinetics of the antibody responses in sera, tail blood samples (diluted 1 : 200 in PBS at the time of collection) were collected at study weeks 0  and 3. Mice were sacrificed at study week 5 by decapitation, when whole blood, intestinal secretions (feces), nasal washes (NWs), and mesenteric lymph nodes (MLNs) were collected. Preparation of blood samples and lymphoid tissues was conducted according to the previously published procedures , except 2 1420; PerkinElmer, Waltham, MA). A sample was considered positive if the OD490 was above the cut-off value (mean OD490 + 3 × SD of the control mice and OD490 > 0.1). The end-point titer was defined as the reciprocal of the highest dilution with an OD490 above the cut-off value. For negative samples with the OD490 below the cut-off limit, a half of the starting dilution was assigned for the titer.Serum samples of individual mice were serially diluted two-fold from 1 : 200 (for IgG) or 1 : 20 (for IgA) and tested in ELISA for the presence of NoV GII.4-specific IgG, IgG1, IgG2a, and IgA antibodies as described elsewhere , 21. Fec6 cell/ml) from immunized mice were stimulated in vitro [μg/ml of GII.4 VLPs or culture medium  only. After incubation at 37°C for 10 days, supernatants were collected and stored at −20°C until analyzed in ELISA for anti-GII.4 IgG and IgA antibodies as described above.Group-wise pooled MLN cells . The statistically significant difference was defined as p ≤ 0.05.Fisher's exact test was employed to assess the intergroup differences in the IgG and IgA end-point titers. The Mann–Whitney μg of GII.4 VLPs alone or 0.3 μg dose combined with Al(OH)3 or MPLA. For comparison, one group of mice received 10 μg GII.4 VLPs via IN delivery. IM immunization with 0.3 μg of GII.4 VLPs did not elicit a significant serum IgG response , whereas coadministration of 0.3 μg VLPs with Al(OH)3 or MPLA resulted in robust (GMTs > 5log10) NoV GII.4-specific IgG levels  was observed in the magnitude of the responses induced by 0.3 μg dose of VLPs with either of the adjuvants and 10 μg dose of VLPs via IM or IN route.Effect of two commonly used adjuvants on NoV GII.4-specific serum IgG and IgA antibody responses was investigated by immunizing the experimental mice twice IM with 0.3 or 10  Figures . Also, I Figures . No signμg dose formulated with Al(OH)3  or MPLA   IgA response  compared with IM administration of the adjuvanted VLP formulations 3 or MPLA as well as 10 μg dose of VLPs alone resulted in comparable IgG responses (p = 0.679) (p = 0.176), as observed at week 5 (490 < 0.1) during the study period (To study the effect of Al(OH)= 0.679) . The sect week 5 . Controly period .p = 0.122) induced by 0.3 μg of VLPs in the presence of Al(OH)3 or MPLA or 10 μg of VLPs alone (3 adjuvanted group having significantly lower titers compared to other groups (p = 0.039). End-point titer IgG1/IgG2a (Th2/Th1) ratio was 2 : 1 for mice immunized with a combination of VLPs and MPLA and 10 : 1 for VLPs and Al(OH)3. No GII.4-specific IgG subtype antibodies were detected in sera of control mice   and IgG2Ps alone . In contPs alone , Al(OH)3 Figures .3 or MPLA induced antibodies at mucosal surfaces, 10% fecal suspensions and NW samples of experimental animals were tested for the presence of anti-GII.4 IgG and IgA antibodies. As expected, mice immunized IM with 0.3 μg dose of GII.4 VLPs alone did not develop intestinal IgG antibodies (p = 0.277) in the intestines (In order to investigate if NoV VLPs coadministered with Al(OH)tibodies . Insteadtestines .μg dose with Al(OH)3  or MPLA  (p = 0.023) was elicited by mucosal IN delivery of 10 μg of VLPs  , but fecal IgA response was detected in 1/5 and 2/5 mice after coadministration of 0.3  = 2–15) , corrobo = 2–15) . Signifi= 12–53) .p = 0.902) were induced by 0.3 μg of VLPs in the presence of Al(OH)3  or MPLA  via IM delivery, or by 10 μg of VLPs alone via IM  or IN delivery   . In cont = 4–33) .3 and MPLA immunized mice were tested for the production of IgG and IgA antibodies. Cells from mice receiving VLPs in the presence of Al(OH)3 or MPLA via IM delivery responded with considerable IgG production to in vitro stimulation with GII.4 VLPs  alter migration patterns of dendritic cells in vivo and promote induction of mucosal responses to codelivered antigens as a consequence of antigen-loaded dendritic cells migrating to both draining and nondraining lymph nodes [Protection against pathogens at mucosal surfaces is largely dependent on secretory IgA effectively induced by IN immunization , 26. Becph nodes . Therefo3, being effective in inducing high systemic antibody responses [The current NoV VLP vaccine candidate in the most advanced phases of clinical trials is administered IM in a formulation with MPLA and/or Al(OH)esponses . After tesponses . Mucosalesponses . In gnotesponses . Severalesponses , 11, 12.esponses , 34.3 and MPLA promoted >30-fold dose sparing above nonadjuvanted dose  and induced considerable levels of IgG1, a marker of Th2 response, as well as IgG2a, a marker of Th1 response. Although MPLA promotes a Th1 bias [3 stimulated Th2-biased response, which is in concordance with previous observations [3 and MPLA work similarly for IM delivered NoV VLPs in terms of the dose sparing, kinetics, and generation of systemic IgG antibodies.In this study, high levels of serum IgG antibodies were induced by both adjuvants. Al(OH)Th1 bias , 35, ourrvations , 36, 37.rvations , our dat3 or MPLA adjuvant. Similarly, Bessa and colleagues have detected IgG, but not IgA, ASCs in MLN after subcutaneous immunization of mice with a vaccine platform based on VLPs [Our recent study demonstrated that IgA levels in mucosal tissues correlated with blocking activity, suggesting that IgA, but not IgG, was the main antibody neutralizing NoV on the mucosal surfaces . In conc on VLPs .3 or MPLA used. Others have shown that parenteral delivery  of vaccine antigens with mucosal adjuvant dmLT (a detoxified form of the heat-labile enterotoxin of E. coli) promotes mucosal immunity [Our results clearly demonstrate the lack of the mucosal IgA antibody generation in naïve animals after IM delivery of NoV VLPs regardless of the adjuvants Al(OH)immunity , likely immunity , althougimmunity , or at l3 or MPLA does not induce significant mucosal IgA antibodies in mice. Instead, IN immunization with GII.4 VLPs alone elicited NoV-specific mucosal immunity. These results underline the importance of mucosal delivery route in induction of potent mucosal responses.The present study demonstrates that IM administration of NoV GII.4 VLPs formulated with Al(OH)


Clavibacter michiganensis subsp. nebraskensis (Cmn) and has spread in recent years throughout the Great Plains, posing a threat to production. The genetic basis of plant resistance is unknown. Here, a simple method for quantifying disease symptoms was developed and used to select cohorts of highly resistant and highly susceptible lines known as extreme phenotypes (XP). Copy number variation (CNV) analyses using whole genome sequences of bulked XP revealed 141 genes containing CNV between the two XP groups. The CNV genes include the previously identified common rust resistant locus rp1. Multiple Rp1 accessions with distinct rp1 haplotypes in an otherwise susceptible accession exhibited hypersensitive responses upon inoculation. GW provides an excellent system for the genetic dissection of diseases caused by closely related subspecies of C. michiganesis. Further work will facilitate breeding strategies to control GW and provide needed insight into the resistance mechanism of important related diseases such as bacterial canker of tomato and bacterial ring rot of potato.Goss's wilt (GW) of maize is caused by the Gram-positive bacterium  Clavibacter michiganensis subsp. nebraskensis (Cmn), the causal agent of GW, is a Gram-positive bacterium (Phylum Actinobacteria) and a vascular pathogen of maize, causing systemic wilting on young plants and leaf blight at all stages of plant growth , was first identified in 1969  family. Unequal crossovers generate gene duplications, gene losses, and genes with new phenotypic characteristics, and, ultimately, yielding lines with diverse haplotypes at the rp1 region  that were subjected to disease phenotyping, including lines from the Maize 282 Association Panel  medium at 28°C for 2–3 days. Maize plants were grown in the greenhouse at 28°C with a 14 h photoperiod. The third leaf of three-leaf maize seedlings was inoculated by cutting at 2 cm from the tip with scissors dipped in bacterial inoculum of optical density of 0.55–0.60 at 600 nm. Lengths of lesion were measured from the cut surface at the tip to the distal-most position on the leaf that exhibits a gray, chlorotic or water-soaked lesion at 13 days post-inoculation (DPI). Common lines were used in different batches to examine batch effects.N = 37) or highly S lines (N = 44) were used for the XP-CNV analysis.The lesion phenotyping protocol was used to inoculate the third leaf of three-leaf seedlings. An exception was made for seedlings  that had not developed to the desirable stage on the inoculation day. For those cases, the second or fourth leaves were inoculated instead. From phenotyping data, a statistically significant but weak association was found between seedling heights, from soil surface to the top node of plants, and lesion lengths. To generate comparable lesion phenotypes among maize genotypes, a linear model to obtain best linear unbiased estimation (BLUE) was applied. The model used raw lesion lengths measured at 13 DPI and genotype as response variable and explanatory variable, respectively. Seedling height, inoculation leaf, and batch were used as other covariates in the model. The BLUE values of lesion lengths were used to represent host resistance levels. Maize genotypes with estimated lesions less than 9 cm were considered as highly resistant (R) genotypes, while those with lesions higher than 22 cm or those with lesions higher than 20 cm and 80% inoculation leaves showing whole leaf wilting were considered as highly susceptible (S) genotypes. Based on genetic relations inferred from genotypes, some highly R and S lines were removed to reduce the genetic redundancies from closely related lines. Selected highly R  was used to filter alignments to ensure that reads were uniquely aligned with high confidence. Specifically, each alignment was required to have the insert between 150 and 800 bp, at least 50 bp alignment length, at least 96% identity, and at most 4% unaligned percentage of a read length. The samtools software  was used to trim adaptor sequences and low quality sequences  was used to count read depth for each gene  using the “union” mode. The generalized linear model, implemented in the DESeq2 package (version 1.4.5), for read counts of genes with at least 20 total reads from all four samples was used for testing the null hypothesis that no difference in read depth between highly R pools and highly S pools  and the B73Ref2 annotation at maizesequence.org.Gene annotation was downloaded from the annotation at Phytozome  in the GOSeq enrichment test  vs. Dn-CNV genes (fewer copies in R pools), and vice versa. The randomly resampling method  on the CFX96™ real time system (Bio-Rad). Primer efficiencies were measured and relative copy number was calculated using the comparative Ct method  were performed as described previously  .To establish a rapid and reproducible method for quantification of GW, the effects of growth stages and leaf positions on lesion expansion were first tested on seedlings of a maize inbred line Mo17. The results indicated that three-leaf seedlings overall exhibited longer lesion expansion compared to four-leaf seedlings in the same time period, allowing for greater potential variation in symptoms  with at least 20 total reads from all the four samples  was subjected to a statistical test for the null hypothesis of no difference in sequencing depths between the R and S pools  sequencing. In total, ~50.3x (125.7 Gb) and 49.6x (123.9 Gb) sequences were generated for the R and S pools, respectively. WGS sequencing data were aligned to the B73 reference genome and uniquely mapped reads were used for determining read depths per gene. We employed a novel approach, XP-CNV, to examine CNV between the R and S pools through comparing genic read depths. Each of the genes  of sequencing depths in R pools vs. S pools among all Up-CNV genes , and one of top Up-CNV genes, displayed a 2.4x fold change in sequencing depths of R pools vs. S pools . Other GO terms associated with protein kinase, electron carrier, ion binding and heme binding, although not significantly overrepresented, occurred at least three times in Up-CNV but were absent in Dn-CNV.Besides rp1 with disease resistance. The 239 lines are a subset of 615 lines whose disease resistance levels (lesion lengths) were phenotyped in this study. For each rp1 gene, the correlation of lesion lengths with normalized read counts of the gene were examined. All four rp1 genes identified as Up-CNV genes showed negative correlations with susceptibility as determined by lesion length     using a 1.7kb rp1 probe to examine the overall rp1 FISH signals  Figure . Eight Rs Figure . Thus, iRp1 haplotypes, containing various rp1 copies and different rp1 genes , Rp1-IG or Rp1-JC13, showed high levels of resistance in comparison to H95, with 61.6% (p = 2.4e-6), 64.9% (p = 1.4e-11), and 47.5% (p = 2.8e-10) reduction in lesion length, respectively , 33.8% (p = 7.3e-6), and 29.4% (p = 1.2e-3) reduction in lesion length, respectively. Both Rp3 and Rp5 accessions did not show enhanced resistance. Quantification of disease resistance of these rp1 haplotypes corroborated the association between the rp1 locus and GW resistance.Multiple y Figure . All thry Figure . Some RpClavibacter, in any system, is poorly understood. This study examined the genetic basis of the emerging maize Gram-positive bacterial disease GW. We applied XP-CNV to identify GW-associated CNV and discovered an associated locus in the region of the R locus rp1 to Puccinia sorghi, the causal agent of common leaf rust  includes CNV and other SV types such as inversions and rearrangements, and only CNV was examined in this study. It is still challenging to reliably genotype SV other than CNV in a population scale. However, the advance of longer read sequencing and bioinformatics algorithms has improved the calling accuracy and reliability . Alternatively, toxins or other non-proteinaceous compounds may trigger the HR. The revelation of the association between the rp1 locus and GW provides a system to elucidate molecular interactions between hosts and bacterial species of C. michiganensis.Through XP-CNV, we identified the association between the rp1 lines; YH, FW, and SL wrote the manuscript with the input from ZP, AR, TD, JF, SH, and HW. All authors read and approved the final manuscript.YH, JR, FW, AR, and SL designed experiments; YH, HW, JF, and SL analyzed data. YH, JR, ZP, AU, HL, and TD performed wet-lab experiments; SH maintained and provided The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer KM and handling Editor declared their shared affiliation.


In-depth chromatin immunoprecipitation analysis unravelled that decreased enrichment of H3K27me3 on Tnf promotor, resulting in TNF elevation in Kupffer cells from liver failure mice. Nuclear factor kappa B (NF-κB) and protein kinase B (Akt) signalling pathways were activated upon lipopolysaccharide stimulation, but attenuated by using GSK126, accompanied with decreased TNF in vitro. In conclusion, EZH2 and H3K27me3 contributed to the pathogenesis of liver failure via triggering TNF and other indispensable proinflammatory cytokines. EZH2 was to modify H3K27me3 enrichment, as well as, activation of the downstream NF-κB and Akt signalling pathways.Acute-on-chronic liver failure is mainly due to host immunity self-destruction. The histone H3 lysine 27 (H3K27) trimethylating enzyme, enhancer of zeste homolog 2 (EZH2) mediates epigenetic silencing of gene expression and regulates immunity, also involves pathogenesis of several liver diseases. The current study was to determine the role of methyltransferase EZH2 and its catalysed H3K27 trimethylation (H3K27me3) in liver failure, and to further investigate the potential target for liver failure treatment. EZH2 and its catalysed H3K27me3 were determined in peripheral blood mononuclear cells (PBMC) from liver failure patients and Kupffer cells from experimental mice. Furthermore, GSK126 (an inhibitor for EZH2 trimethylation function) was applied in liver failure mice in vivo, and lipopolysaccharide-stimulated mononuclear cells in vitro. EZH2 and H3K27me3 were significantly upregulated in human PBMC from liver failure patients or murine Kupffer cells from the liver failure animals, respectively. GSK126 ameliorated disease severity in liver failure mice, which maybe attribute to down-regulate circulating and hepatic proinflammatory cytokines, especially TNF  Despite extensive intervention, the short-term mortality in liver failure patients is unacceptably high, mainly due to inadequate response to conventional treatment and lack of opportunity for liver transplantation2. Host immunity, leading to secretion of inflammatory cytokines and activation of excessive immune cascade, are accompanied with massive hepatic injury during the early episodes of ACLF3. Inflammatory cytokines, e.g., tumour necrosis factor (TNF), progressively increase in relation to the severity and prognosis of ACLF5. However, the precise underlying mechanism of ACLF, particularly how these proinflammatory cytokines is regulated by host immunity remains to be explored.Acute-on-chronic liver failure (ACLF) is a severe deterioration of liver function accompanied with massive hepatocyte necrosis/apoptosis and recruitment of hepatic inflammatory cells, leading to multi-organ dysfunction6. Emerging evidence suggests that epigenetics play vital roles in modulation of immunity8. Targeting at epigenetic modifications, novel therapeutic strategy for regulation of immune response during liver failure are definitely urgent for patients suffering from fatal liver failure and multi-organ dysfunction.Epigenetics, including DNA methylation and histone modifications, mainly regulate gene expression without altering gene sequence9. EZH2-catalysed trimethylation of H3K27 (H3K27me3) is overexpressed in many cancers with poor prognosis11. EZH2/H3K27me3 also involves in liver diseases, including nonalcoholic fatty liver diseases, liver fibrosis and hepatitis B13. Furthermore, methyltransferase EZH2 regulates T cells proliferation and differentiation, contributes to autoimmune diseases and alloimmunity15. However, the role of EZH2/H3K27me3 in host immunity, particularly in regulating inflammation during liver failure remains to be explored.The histone H3 lysine 27 (H3K27) trimethylating enzyme, enhancer of zeste homolog 2 (EZH2) is an indispensable histone methyltransferase contributing to silence of the target genes via histone modificationsIn the current study, it was investigated the potential role of EZH2/H3K27me3 during the development of liver failure. Moreover, the underlying mechanism involved EZH2-catalysed H3K27me3 in the liver failure was explored at molecular level via modification of methyltransferase activity of EZH2 in vivo. Our data suggest that there was a correlation between increased EZH2/H3K27me3 and upregulated TNF in liver failure. Such data may provide useful information for both basic research and clinical precision medicine with specific target(s).16. Although variation in definitions, the prevalence of ACLF is the highest among liver failure in hospitalized patients17. Recently numerous clinical studies demonstrate that immune response plays an important role in the occurrence and development of ACLF2. PBMC includes monocytes and lymphoblasts, and could reveal the immunity in many organs and various diseases18. To investigate the role of EZH2-mediated H3K27me3 in pathogenesis of liver failure, the levels of EZH2 and H3K27me3 in PBMC from ACLF patients and HC were determined using western blot. EZH2 or H3K27me3 in PBMC from ACLF patients was 2- or 10-fold higher than that from HC (P < 0.001)  Fig. . The inc01) Fig. .Fig. 1EZ+ cells were increased in the liver failure patients, some of them were closely related to CD68+ cells (Kupffer cells), accompanied with substantial increased infiltrating cells, compared to those from HC. Similarly, intrahepatic H3K27me3+ cells were observed in these liver failure patients /D-GalN-induced liver failure was established, which might be a reasonable tool to explore acute liver inflammation and mimic the acute deterioration period of ACLF. EZH2 and H3K27me3 in Kupffer cells from mice liver were increased significantly (P < 0.05) in liver failure mice following LPS/D-GalN injection   Fig. . Similarved Fig. . Signifi20. Inhibition of EZH2-catalysed H3K27me3 in vivo was carried out using GSK126 to determine the function of EZH2 and H3K27me3 during liver failure in mice model. All of the vehicle (DMSO)-treated liver failure animals were died within 6 h post-LPS/D-GalN injection. In contrast, GSK126 treatment effectively prolonged survival time and improved survival rate (P < 0.001)   Fig. . Accordi01) Fig. . Moreove01) Fig. . Such re4. To explore the mechanism by which inhibiting methyltransferase activity of EZH2 protected mice from liver injury, proinflammatory cytokines during liver failure were examined.Previous studies have reported that proinflammatory cytokines play critical roles during liver failureTnf, interleukin (Il)-1β, and Il-6 mRNA were determined from these induced liver failure mice with or without GSK126 pre-treatment. Hepatic Tnf mRNA was 15-fold increase in liver failure mice with the vehicle (DMSO) treatment, compared to that from non-liver failure animals , compared to that from the vehicle-treated liver failure mice.Hepatic als Fig. . Increasove Fig. . Serum Tove Fig. . HoweverTo further investigate the effect of GSK126 on TNF in vitro, mRNA expression was determined in LPS-stimulated PBMC from HC treated with GSK126 or vehicle (DMSO). It was observed that TNF mRNA was increased 2.5- or 2-fold at 1 or 3 h, respectively, in the LPS-challenged PBMC with vehicle treatment Fig. . As expe21. We further examined affinity of EZH2 and H3K27me3 on Tnf promoter in mice Kupffer cells. Eight pairs of primers were designed to amplify 100–150 bp around the regions indicated . The data suggest that decreased H3K27me3 enrichment on Tnf promoter region contributes to Tnf transcription.TNF is a major contributor to the inflammatory process in liver failure and secreted mainly by Kupffer cells during liver failureted Fig. . Only foent Fig. , but notlls Fig. , suggestme3 Fig.  from bot23. Interestingly, H3K27me3 enrichment on promoter region of Tgf-β1 was induced significantly upon liver failure  effectively attenuates the TNF-mediated necrosis/apoptosisure Fig. .P < 0.05) following LPS induction, respectively.To further uncover the role of EZH2/H3K27me3 in regulating TNF in immune cells, EZH2 and H3K27me3 were measured in the healthy murine mononuclear cells and PBMC from HC following LPS stimulation Fig. a, b. EZHThe efficacy of GSK126 in suppressing EZH2-catalysed H3K27me3 in the mononuclear cells were measured. EZH2 was not affected significantly, but H3K27me3 was decreased by >30%  and degradation of NF-κB inhibitor-α (IκB-α) are markers for activation of the canonical NF-κB pathway25. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a critical upstream molecule for blockade of Akt pathway26. The indicators of Akt signalling pathways were observed in GSK126-treated murine MNC or PBMC from HC, compared to the vehicle-treated monocytes. As shown in Fig. It has been demonstrated that there is a direct involvement of Akt pathway in LPS-activated NF-κB pathways and other inflammatory moleculesvia the H3K27me3 enrichment, as well as NF-κB and Akt signalling pathways.In the current study, there was a correlation between elevated EZH2/H3K27me3 production and upregulated proinflammatory cytokines during the development of liver failure. Furthermore, inhibitor GSK126 for EZH2-catalysed H3K27me3 ameliorated liver injury and improved survival in liver failure mice, via suppressing TNF and other indispensable proinflammatory cytokines. At the early phase of liver failure, EZH2/H3K27me3 promote the expression of proinflammatory cytokines, which was 27. Recently, EZH2/H3K27me3 has been shown to exert the essential role in maintained T cells differentiation and polarization for promoting immune response in alloimmunity28. This is in line with our data, showing that EZH2/H3K27me3 were upregulated with progression of liver injury and inflammation. It invites speculation that EZH2/H3K27me3 is involved in liver failure via regulating immune activation during liver failure.EZH2-mediated gene repression via H3K27me3 promotes immune response in hepatocellular carcinoma by regulation of CXC chemokine receptors29, modification of EZH2 becomes a hotspot in the development of pharmaceutical agents. 3-deazaneplanocin A (one of EZH2 inhibitors) reactivates the EZH2-silenced genes via degradation of the PRC2 complex, which causes some unexpected effects and is limited in clinical application30. Focusing on methyltransferase activity of EZH2 and the histone modification, GSK126 is selected by its highly selectivity in inhibiting EZH2 methyltransferase via competing with S-denosyl-methionine but without degradation of EZH220. Such effect of GSK126 was also confirmed in human PBMC and murine splenic cells in the current study.As EZH2/H3K27me3 has been linked to many diseases31. These reports are consistent with our study, demonstrating that inhibition of EZH2-catalysed H3K27me3 using GSK126 significantly attenuated liver injury and impaired inflammatory cells infiltration, which might partially contribute to the reduced proinflammatory cytokines. Additionally, it was found that GSK126 reduces TNF expression and other proinflammatory cytokines directly in human PBMC in the current study. Collectively, our data suggest that EZH2 enhances some proinflammatory cytokines production upon stimulation during liver failure. Such data also provides compelling evidence to support potential clinical application of the targeting in EZH2 methyltransferase for liver failure.Epigenetic modification is crucial in the regulation of inflammatory cytokine production. Suberoylanilide hydroxamic acid, one of histone deacetylase inhibitors, shows satisfactory anti-inflammatory effects via reducing the production of proinflammatory cytokines in acute graft-versus-host diseaseTnf promotor in Kupffer cells following liver failure, which is supported by others, showing TNF gene is regulated under H3K27me3 in breast cancer cells32. Tnf is regulated by coordinated actions of histone modifications33, our result confirmed that H3K27me3 is one of the predominate epigenetic components in Tnf at transcriptional level. On other hand, EZH2 acts as a gene activator in some cases via directly locating the promotor of target gene, in addition to the canonical role of EZH2 as a gene silencer9. Interestingly our data showed that no obvious affinity of EZH2 in Tnf promotor was observed in both normal and liver failure mice. Compared to directly binding on Tnf promotor as a transcription factor, EZH2 regulated TNF in liver failure might be largely due to its methylation activity and its downstream product (H3K27me3).Notably, using GSK126 inhibiting EZH2/H3K27me3 directly in vitro significantly reduced TNF in the current study, which raises an interesting question: how do EZH2 and H3K27me3 modulate the proinflammatory cytokines, especially TNF during liver failure. It was observed that H3K27me3 enrichment was reduced in Tnf promotor decreased. The explanation for such discrepancy might be that H3K27me3 enrichment increased on some other gene promotors to silencing the target genes. It was further investigated H3K27me3 enrichment on promotor of other genes, especially Tgf-β1 which protects various parenchymal cells from TNF cytotoxicity23. It is consistent with Mondal et al., indicating EZH2/H3K27me3 regulated TGF-β1 and the related genes34. We speculated that Tgf-β1 represented one of many genes which were silenced via the H3K27me3 enrichment on their promotors during liver failure. It might be an epigenetic mechanism in which EZH2/H3K27me3 promoted TNF-mediated liver failure and GSK126 could improve the liver inflammation.During liver failure, the global EZH2-catalysed H3K27me3 increased while its enrichment on via suppressing LRRC33 in LPS-stimulated macrophages in vivo and in vitro35. TLR pathways may affect the induction of many immediate early genes involved in the proinflammatory cytokines during the liver failure32. Furthermore, NF-κB pathway, an important part of TLRs pathways, plays pivotal roles in TNF36. In addition, Akt signalling pathway is closely related to immune regulation following activation of TLRs pathway37. This is further supporting our current study, showing GSK126 suppresses LPS-activated signalling pathways and subsequently promoting inflammatory cytokines.Finally, the linkage between EZH2/H3K27me3 and other possible signalling pathway(s) in immunity was also explored. EZH2-mediated gene silence promotes Toll-like receptors (TLRs) Overall, our data suggested that EZH2/H3K27me3 promoted the development of liver failure, which was ameliorated using GSK126 via modulation of TNF and overall inflammation. Moreover, TNF was manipulated with the histone modification of H3K27me3, as well as the downstream signalling pathways of NF-κB and Akt. Thus, our data provided compelling evidence to support potential clinical application of pharmacological targeting of epigenetic modifications, specifically for treatment of ACLF.16 (n = 10) and healthy controls . Characterization was based on Asian Pacific Association for the Study of the Liver criteria16. The study was approved from the Human Ethics Committee, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. The written consent form was obtained from each subject.A total of 20 subjects were enrolled between June 2016 to May 2017 in Department of Infectious Diseases, Ruijin Hospital, Shanghai, China, including hepatitis B virus-related acute-on-chronic liver failure patientsthe Institutional Animal Care and Use Committees of Ruijin Hospital.C57BL/6 female mice, 10-week old with body weight of 18–20 g, were injected intraperitoneally with 10 μg/kg of LPS  and 700 mg/kg D-galactosamine   dissolved in 200 μl PBS for induction of liver failure. For the indicated experiments, these mice were pre-treated with intraperitoneal injection of vehicle (5% DMSO) or 10 mg/kg GSK126  dissolved in 200 μl 5% DMSO for 2 h, and then subjected for liver failure induction. The harvested serum and liver tissues were analysed at the indicated time points. Kupffer cells isolation were also perform as described in 38, and exacted for total RNA and then subjected for reverse transcription-PCR39. Murine serum TNF was also analysed using ELISA  were heat-induced epitope retrieval, using sodium citrate buffer (pH 6) for 5 min. The sections were incubated with primary antibodies mixture of rabbit anti-EZH2 antibody  and mouse anti-CD68 antibody  for 8 h incubation at 4 °C. Subsequently, antibodies mixture of peroxidase AffiniPure goat anti-rabbit IgG41 and alkaline phosphatase AffiniPure goat anti-mouse IgG41 were followed for 30 min at room temperature. Finally, EZH2 or CD68 was detected, using 3,3′-diaminobenzidine  or alkaline phosphatase-red (Shanghai ZuochengBio). Similar double staining method was applied for H3K27me3  and CD68 staining (as described above).Double staining of EZH2 and CD68 was performed as describedFrozen sections (8 μm) were applied in the murine tissues for co-localization of F4/80 with EZH2 or H3K27me3 using double immunofluorescence. Briefly, incubation with blocking solution (containing goat serum) at room temperature for 1 h was performed, followed by an overnight incubation at 4 °C with the mixture of anti-EZH2 antibody  and anti-F4/80 antibody , or anti-H3K27me3 antibody  and anti-F4/80 antibody , respectively. Incubation with mixtures of Alexa Fluor 488-conjugated secondary antibody  and Alexa Fluor 555-conjugated secondary antibody  for 1 h at room temperature in the dark was performed, followed by a 5 min incubation of DAPI at room temperature in the dark.38.Liver tissues were collected and then fixed in 10% formalin and embedded in paraffin were subjected for hematoxylin–eosin staining as previously described42.Using image analysis software ImagePro Plus 7.2 (Diagnostic Instruments), the intrahepatic bleeding was determined, using necrosis area and bleeding area versus total hepatic are in the field, respectively. The infiltrating cells were counted, as describedThe sections were subjected for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays using colorimetric TUNEL apoptosis assay kit (Beyotime) according to the instruction from the manufacturer. It was detected using DAB for visualization.TM A kit (Millipore), following the instructions from the manufacturer. The antibodies of EZH2 and H3K27me3 used in the ChIP assay were both purchased from Cell Signalling Technology. The purified DNA fragments were amplified for PCR. The used primers were listed in the ChIP analysis was applied for these isolated Kupffer cells using EZ-Magna ChIP6 cells/ml with LPS (1 μg/ml). For the indicated experiments, these cells were pre-treated with 2 μl/ml DMSO or 10 nM/ml GSK126 (dissolved in DMSO) 1 h prior to LPS stimulation.Peripheral blood mononuclear cells (PBMC) from HC human subjects and splenic mononuclear cells (MNC) from normal mice were obtained using Ficoll density gradient separation, and maintained in complete medium which contained 10% foetal bovine serum and RPMI1640 medium (Gibco). Then these cells were plated in 12-well plates as a density of 1 × 10t tests for significant differences. In addition, log-rank tests were performed for survival analysis. All experiments had been repeated for at least three times. P < 0.05 was set as statistical significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001.All results are described as means ± SEM, which were further calculated by two-tailed, unpaired or paired Student’s supplementary figure legends