Vasodilatory shock in sepsis is caused by the failure of the vasculature to respond to vasopressors, which results in hypotension, multiorgan failure, and ultimately patient death. Recently, it was reported that CPI-17, a key player in the regulation of smooth muscle contraction, was downregulated by lipopolysaccharide (LPS) in mesenteric arteries concordant with vascular hypocontractilty. Vasodilatory shock in sepsis is caused by the failure of the vasculature to respond to vasopressors, which results in hypotension, multiorgan failure, and ultimately patient death. Recently, it was reported that CPI-17, a key player in the regulation of smooth muscle contraction, was downregulated by lipopolysaccharide (LPS) in mesenteric arteries concordant with vascular hypocontractilty. While Sp1 has been shown to activate CPI-17 transcription, it is unknown whether Sp1 is involved in LPS-induced smooth muscle CPI-17 downregulation. Here we report that tumor necrosis factor (TNF) was critical for LPS-induced smooth muscle CPI-17 downregulation. Mechanistically, we identified two GC boxes as a key TNF response element in the CPI-17 promoter and demonstrated that KLF4 was upregulated by TNF, competed with Sp1 for the binding to the GC boxes in the CPI-17 promoter, and repressed CPI-17 transcription through histone deacetylases (HDACs). Moreover, genetic deletion of TNF or pharmacological inhibition of HDACs protected mice from LPS-induced smooth muscle CPI-17 downregulation, vascular hypocontractility, hypotension, and mortality. In summary, these data provide a novel mechanism of the transcriptional control of CPI-17 in vascular smooth muscle cells under inflammatory conditions and suggest a new potential therapeutic strategy for the treatment of vasodilatory shock in sepsis. Sepsis is a life-threatening disease with a high mortality rate of up to 50% affecting over 750,000 patients in the United States . Sepsis Several potential mechanisms have been proposed for vasodilatory shock: (i) excessive production of nitric oxide (NO) by induction of inducible nitric oxide synthase (iNOS), (ii) increased levels of prostacyclin by cyclooxygenase 2, (iii) elevated peroxynitrite by superoxide, (iv) activation of vascular ATP-sensitive and calcium-activated potassium channels, and (v) deficiency in corticosteroid and vasopressin . However2+ increase, and phenylephrine (PE) in a mouse lipopolysaccharide (LPS) model of sepsis  may also play a pivotal role in vasodilatory shock. Progress made in the last decades has established a smooth muscle contractile machinery composed of a contractile unit (smooth muscle myosin and actin) and regulatory proteins, including myosin light chain kinase (MLCK), protein kinase C (PKC), small GTPase Rho-A, Rho kinase (ROCK), myosin phosphatase targeting subunit 1 (MYPT1), and protein kinase C-potentiated phosphatase inhibitor of 17 kDa (CPI-17) , 7. Amon7–9–There are limited studies on the regulation of CPI-17 at the transcriptional level, which is in sharp contrast to its posttranslational modifications  that has been extensively studied . More rein vivo (mice), ex vivo (organ culture), and in vitro (cell culture). Moreover, we identified the two proximal GC boxes in the CPI-17 promoter as a key cis TNF response element critical for TNF-induced CPI-17 transcriptional suppression. Mechanistically, we demonstrated that the transcription factor Krüppel-like factor 4 (KLF4) competed with Sp1 for binding to the same GC boxes in the CPI-17 promoter thus suppressed Sp1-mediated CPI-17 transcription via histone deacetylases (HDACs). Finally, we showed that genetic deletion of TNF or pharmacological inhibition of HDAC protected mice from LPS-induced CPI-17 downregulation, vascular hypocontractility, hypotension, and mortality.Here we report that vascular hypocontractility and hypotension were associated with a selective CPI-17 downregulation in mesenteric arteries from mice injected with LPS. We further demonstrated that tumor necrosis factor alpha (TNF), a pleiotropic inflammatory cytokine, mimicked LPS in downregulation of CPI-17 in VSMCs To study vasodilator shock in sepsis, C57BL/6 mice were injected intravenously with LPS (10 mg/kg of body weight). The effect of LPS on BP was determined by telemetry. Consistent with that in patients with sepsis , hypoten20), and CPI-17, CPI-17 was selectively downregulated by LPS relative to that in the control mice injected with saline  to induce vasodilatory shock as previously described . ConsistFourth, to investigate whether TNF can directly induce CPI-17 downregulation, aortas were isolated from C57BL/6 mice and cultured in Krebs solution with or without TNF (10 ng/ml) for 48 h. We chose 10 ng/ml of TNF in aortic organ culture studies because the similar concentration of TNF has been reported in serum from mice 1.5 h after LPS injection . Consistin vivo. Primary rat aortic VSMCs were starved in fetal bovine serum (FBS)-free medium for 24 h and then treated with TNF (10 ng/ml) in fresh FBS-free Dulbecco modified Eagle medium (DMEM) for 6, 12, 24, and 48 h. The total CPI-17 (CPI-17-T) and phosphorylated CPI-17 (CPI-17-P) expressions were determined by Western blotting using antibodies specific for CPI-17-T and CPI-17-P (T38) as previously described  for 3, 6, and 24 h. The effect of TNF on CPI-17 mRNA expression was determined by real-time PCR. As shown in cis TNF response element critical for TNF-induced CPI-17 transcriptional suppression, a series of 5′-end-truncated CPI-17 promoter-luciferase constructs were generated by PCR cloning and were transfected with a pRL Renilla luciferase control vector into rat aortic VSMCs treated or not treated with TNF . The effect of TNF on CPI-17 promoter activity was determined by a modified dual-luciferase assay . To identify a key se assay .In the absence of TNF, the bp −792 construct exhibited a promoter activity 115-fold over that achieved by the pGL3-basic vector . Progrescis TNF response element critical for TNF to suppress CPI-17 transcription.In the presence of TNF, the bp −792 promoter activity was decreased by 46% . Progrescis TNF response element in the CPI-17 promoter, the bp −792 promoter was analyzed by Genomatix MatInspector to search potential transcription factor binding sites. The resultant analysis predicted 160 putative transcription factor binding sites in the bp −792 promoter (data not shown), including the three GC boxes located between bp −92 and −27 of the CPI-17 promoter  in the bp −92 construct was changed to adenine by site-directed mutagenesis . The wilIt was noted that the basal promoter activity of the bp −92 construct was considerably lower than that of less truncated constructs . To veriGenomatix MatInspector also predicted a proximal NF-κB binding site in the CPI-17 promoter . NF-κB iIt has long been known that Sp1 can bind to GC boxes in many gene promoters to regulate gene expressions in a promoter-, cell-, and, tissue-specific manner . In partConsistent with the previous report , Sp1 bou32P-labeled probes containing GC box 1, 2, or 3 to form protein-DNA complexes. In the absence of TNF, nuclear extracts bound to all three probes to form protein-DNA complexes  were incubated with 32P-labeled probe 2 or 3. Compared to the controls without antibody addition . Mithramycin A is an antibiotic with the ability to compete with Sp1 for binding to GC-rich DNA and has been shown to selectively inhibit Sp1  in vivo . In the  in vivo . In cont in vivo , mithramTo more specifically define the role of Sp1 in TNF-induced CPI-17 transcriptional suppression, we investigated whether downregulation of Sp1 by small interfering RNAs (siRNAs) affects TNF-induced CPI-17 transcriptional suppression. As shown in To define the mechanism by which TNF inhibits the binding of Sp1 to the proximal GC boxes in the CPI-17 promoter, we explored the possibility that TNF decreases Sp1 nuclear accumulation and thereby attenuates the binding of Sp1 to the proximal GC boxes in CPI-17 promoter, thus suppressing CPI-17 transcription. Surprisingly, there was no difference in the levels of Sp1 nuclear accumulation between the cells treated and not treated with TNF , Genomatix MatInspector predicted several transcription factors putatively binding to the GC boxes in the CPI-17 promoter (data not shown). Among them, KLF4, in addition to Sp1, was particularly interesting, since KLF4 and Sp1 are in the same family of transcription factors that can bind to the GC boxes in the gene promoters with different sequence binding preferences and affinities and can function as activators or repressors depending on which promoter they bind and the coregulators with which they interact . Thus, wTo test this hypothesis, we first determined whether KLF4 responds to TNF to be upregulated in cultured rat aortic VSMCs treated with TNF (10 ng/ml) for 3, 6, 12, 24, and 48 h. Representative blots  and quanTo directly test whether TNF-induced KLF4 protein upregulation causes TNF-induced CPI-17 transcriptional suppression, we investigated whether downregulation of KLF4 by siRNA affects TNF-induced CPI-17 mRNA suppression in cultured rat aortic VSMCs. As shown in To define the mechanism by which KLF4 participates in TNF-induced and Sp1-mediated CPI-17 transcriptional suppression, KLF4- and Sp1-bound chromatins were pulled down by antibodies specific for KLF4 and Sp1, respectively, in mouse aortic VSMCs treated or not treated with TNF . The effect of TNF on the binding of KLF4 and Sp1 to the proximal GC boxes in the CPI-17 promoter was evaluated by ChIP-PCR . ConsistTo ensure the specificity of the ChIP assay, two controls were included. First, a nonspecific control antibody ensured the specificity of Sp1 and KLF4 antibodies  as described previously (50) of KLF4 was 28.98 ng, indicating that KLF4 has a higher binding affinity for GC box 2 than Sp1.To test whether upregulation of KLF4 by TNF can compete with Sp1 for binding to the same GC boxes in the CPI-17 promoter, we used purified KLF4 and Sp1 proteins and carried out an eviously . A biotieviously . The halTo investigate whether upregulation of KLF4 by TNF caused CPI-17 transcriptional suppression, rabbit aortic VSMCs were transfected with various concentrations of human KLF4 cDNA , and the effect of upregulation of KLF4 on CPI-17 mRNA expression was determined by real-time PCR. Rabbit aortic VSMCs were used because they have a relatively higher transfection efficiency than the rat or mouse aortic VSMCs. The same amount of plasmid DNA (pcDNA3.1KLF4 + pcDNA3.1 = 2 μg) was used for each sample to ensure similar transfection efficiencies. As shown in To investigate the physiological significance of the signaling pathway that we defined in cell culture and a test tube, we determined whether blockade of TNF/KLF4/SP1/CPI-17 signaling protects mice from LPS-induced hypotension. We first investigated whether genetic deletion of TNF protects mice from LPS-induced hypotension. TNF knockout (TNF-KO) and WT control mice were injected intravenously with LPS (10 mg/kg), and systolic BP (SBP), diastolic BP (DBP), mean arterial pressure (MAP), pulse pressure (PP), and heart rate (HR) were measured by telemetry before and after LPS injection. Before LPS injection 0 h in , there wd-galactosamine . d-GalNAc is an LPS-sensitizing reagent. In the presence of d-GalNAc, a lower dose of LPS can potently and rapidly induce septic endotoxin shock and animal death that are entirely dependent upon TNF  plus upon TNF , 33. As d-GalNAc .d-GalNAc. As expected, smooth muscle contractile responses to angiotensin II , serotonin , and PE (10 μM) were reduced in mesenteric arteries from mice injected with LPS plus d-GalNAc compared to that in WT control mice  that was sufficient to inhibit CPI-17 transcription . Cells win vitro finding that KLF4 represses CPI-17 transcription through HDACs, C57BL/6 mice were injected intraperitoneally with SAHA (50 mg/kg) before being injected intraperitoneally with LPS (0.5 mg/kg) plus d-GalNAc (100 mg/kg). The effect of SAHA on hypotension induced by LPS plus d-GalNAc was determined by telemetry. As shown in d-GalNAc. Consistent with these data, mortality induced by LPS and d-GalNAc was also significantly reduced in mice treated with SAHA compared to that in the control mice treated with a vehicle (DMSO).To investigate the physiological relevance of our cis TNF response element (GC boxes) in the CPI-17 promoter critical for TNF-induced and KLF4- and Sp1-mediated CPI-17 transcriptional suppression in VSMCs. Mechanistically, we demonstrated that KLF4 is upregulated by TNF, competes with Sp1 for the binding of the same GC boxes in the CPI-17 promoter, and represses CPI-17 transcription via HDACs  and fix the transcriptional start site at TATA-less promoters . KLF4, oin vivo . Interesin vitro mediates TNF-induced CPI-17 mRNA downregulation occurs in vivo remains to be determined. There are likely enhancer elements elsewhere within or beyond the gene that confer tight transcriptional control of CPI-17. Sequence analyses by Dippold and Fisher, who identified conserved putative enhancer sequences in introns 2 and 3 of the CPI-17 gene, support this possibility  may compound the experimental results because of possible chemical interactions. Finally, the current study is not sufficient to exclude a role for NF-κB in TNF-induced transcriptional suppression since the current study investigated only one of the putative NF-κB binding sites in the CPI-17 promoter in TNF-induced transcriptional suppression.The current study has several limitations. First, this study has not established that downregulation of CPI-17 is the mechanism by which LPS or TNF mediates vasodilatory shock, hypotension, and mortality. Further studies on CPI-17 knockout mice injected with LPS or TNF may help to address this critical issue. Second, whether the minimal CPI-17 promoter tested sibility . Third, upon TNF , 33, thuupon TNF , and thuTNF knockout mice (TNF-KO) and wild-type (WT) C57BL/6J mice were purchased from the Jackson Laboratory. All mice were housed at an animal care facility at the University of Kentucky. Only male mice at 12 to 14 weeks of age were used. All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Kentucky.Escherichia coli O111:B4; Sigma-Aldrich; 10 mg/kg of body weight in 100 μl of saline for 6 or 16 h). For TNF injection, mice were injected intravenously with TNF . For low-dose LPS plus d-galactosamine , mice were injected intraperitoneally with LPS  plus d-GalNAc . For suberoylanilide hydroxamic acid (SAHA), mice were injected intraperitoneally with SAHA .As explicitly described in Results, LPS, TNF, and other compounds were administered to mice by intravenous or intraperitoneal injection as follows. For high-dose LPS, mice were injected intravenously with LPS  to determine escribed , 45, 46.The procedures for RNA extraction, cDNA synthesis, and real-time PCR were performed as previously described , 45–48. 45–45–Aortas were isolated from C57BL/6 mice. After dissection to remove fat and adventitial tissues, aortas were cut into 3-mm segments. Endothelial cells were mechanically removed. Aortic segments were incubated with purified recombinant mouse TNF  for 48 h in Krebs-Ringer bicarbonate buffer containing penicillin and streptomycin. The incubation buffer containing TNF was replaced every 12 h to ensure the efficacy of TNF.The procedures for isolating and culturing aortic VSMCs from mice, rats, and rabbits were conducted as previously described , 26, 47.A bacterial artificial chromosome (BAC) clone containing the mouse CPI-17 promoter was purchased from Invitrogen and was used as a PCR template to amplify the bp −792 promoter. After sequencing , the bp −792 promoter was used as a PCR template to amplify the bp −592, −392, −203, and −92 promoters. The PCR primers to amplify all of these promoters are described in Table S2. The bp −27 promoter was synthesized by Integrated DNA Technologies (IDT). All CPI-17 promoters were subcloned into a pGL3-basic vector (Promega) as previously described , 26, 47.A QuikChange II site-directed mutagenesis kit (Agilent Technologies) was used to generate mutations in the CPI-17 promoter. The PCR primers used for generating mutations of GC boxes 1, 2, and 3 and NF-κB in the CPI-17 promoters are described in Table S2. All mutations were verified by DNA sequencing.Renilla luciferase control vector  using Lipofectamine-Plus reagent (Life Technologies). After TNF treatment , CPI-17 promoter activity was assayed by a modified dual-luciferase enzyme assay as described previously  or nonspecific siRNA duplex (Qiagen) using INTERFERin reagent (Polyplus-transfection Inc.) for 48 h. The specificity and efficiency of siRNA downregulation of Sp1 and KLF4 were determined by Western blotting.32P-labeled double-stranded DNA probes corresponding to WT or mutant GC boxes 1, 2, and 3 in the CPI-17 promoter. To ensure the specificity of the assay, excess unlabeled probes (100:1) were added to the reaction mixture. To determine whether Sp1 binds to the GC boxes, 0.5 or 1 μg of anti-Sp1 antibody (Santa Cruz Biotech) was included in the reaction mixture. The WT and mutant probes used for EMSA are described in Table S2. All probes were synthesized by IDT and labeled with [γ-32P]ATP  by T4 kinase (New England BioLabs). Reaction mixtures were analyzed on 6% nondenaturing polyacrylamide gels, followed by autoradiography.Electrophoretic mobility shift assay (EMSA) was conducted as previously described . BrieflyThe chromatin immunoprecipitation (ChIP) assay was conducted as previously described . Mouse pA DNA-protein interaction enzyme-linked immunosorbent assay (DPI-ELISA) was conducted as previously described . Brieflyin vitro experiments from vessel excision was usually 6 h.The procedures for mesenteric artery isometric contraction measurement were initially reported by Horiuti  and condt test. For a comparison of 1 parameter among multiple samples, statistical analysis was performed using 1-way analysis of variance (ANOVA) with a Newman-Keuls posttest. For comparison of multiple parameters, statistical analysis was performed using 2-way ANOVA with repeated measures and Bonferroni’s posttest. A P value of <0.05 was considered significant. A P value of ≥0.05 was considered nonsignificant (ns).All data were expressed as means ± standard errors of the means (SEM). For a comparison of 1 parameter between 2 samples, statistical analysis was performed using 2-tailed, unpaired Student’s


In the process of autophagy, a newly synthesized isolation membrane (IM) is developed to sequester degradation targets and eventually the IM seals, forming an autophagosome. One of the most poorly understood autophagy‐related proteins is Atg2, which is known to localize to a contact site between the edge of the expanding IM and the exit site of the endoplasmic reticulum (ERES). Recent advances in structural and biochemical analyses have been applied to Atg2 and have revealed it to be a novel multifunctional protein that tethers membranes and transfers phospholipids between them. Considering that Atg2 is essential for the expansion of the IM that requires phospholipids as building blocks, it is suggested that Atg2 transfers phospholipids from the ERES to the IM during the process of autophagosome formation, suggesting that lipid transfer proteins can mediate  Atgautophagy‐relatedEMelectron microscopyERendoplasmic reticulumERESER exit siteE‐Sytsextended synaptotagminsERMESER–mitochondria encounter structure complexIMisolation membraneLPSlipopolysaccharideLTlipid transferLUVlarge unilamellar vesicleMTmembrane tetheringNRN‐terminal regionPASpreautophagosomal structurePEphosphatidylethanolaminePI3Kphosphatidylinositol 3‐kinasePI3Pphosphatidylinositol 3‐phosphatePROPPINβ‐propellers that bind polyphosphoinositidesSMPsynaptotagmin‐like mitochondrial lipid‐binding proteinSpSchizosaccharomyces pombeSUVsmall unilamellar vesicleWIPIWD repeat domain phosphoinositide‐interacting proteinsde novo organelle biogenesis.Saccharomyces cerevisiae, this process starts with the nucleation of an isolation membrane (IM) in the vicinity of a vacuole. The cup‐shaped IM expands and encapsulates various cellular components, including biomolecules such as proteins and nucleic acids, and organelles such as mitochondria, endoplasmic reticulum (ER), and nucleus.de novo double‐membrane organelles.Autophagy is a highly conserved catabolic pathway in eukaryotes and occurs by a process of In budding yeast, dozens of autophagy‐related (Atg) proteins regulate the steps of autophagy through interaction with membranes.During the expansion of the IM, the Atg2–Atg18 complex localizes to the edge of the IM in a process dependent on PI3P and Atg9. The PROPPIN (β‐propellers that bind polyphosphoinositides) protein Atg18 recognizes PI3P for the recruitment of the Atg2–Atg18 complex to the IM,S. cerevisiae autophagy‐defective mutants led to the identification of 13 apg genes required for autophagy, including apg2, later renamed to atg2.atg2 gene was cloned, and the essential role of the gene product, Atg2 protein, in autophagosome formation was established.In 1993, the screening of Although structural studies on the core Atg proteins have significantly progressed in the last 15 years,S. cerevisiae Atg2 as well.in vivo.In parallel with the structural studies, the biochemical studies of Atg2 using purified proteins began to unveil the specific functions of Atg2. Purified ATG2B was found to bind to liposomes in a PI3P‐independent manner, in contrast to purified WIPI4 that requires a relatively large amount (∼15%) of PI3P for liposome binding.Schizosaccharomyces pombe Atg2 (SpAtg2NR), which contains an evolutionarily conserved region termed Chorein_N, was recently crystallized and its crystal structure was determined.NR comprises a helical region and a twisted β‐sheet, which fold into a globular structure with a large hydrophobic cavity. Mixing SpAtg2NR and PE resulted in the formation of a SpAtg2NR–PE complex, whose structure was determined at 2.7 Å resolution  domains, despite little topological similarity between them. The SMP domain is found in proteins, such as the ER–mitochondria encounter structure (ERMES) complex and extended synaptotagmins, both of which are localized at membrane contact sites and appear to transfer phospholipids between organelles.NR showed high structural similarity to the N‐terminal region of Vps13, which also contains Chorein_N.To date, although the full‐length Atg2 has not been crystallized, the N‐terminal region of LT assays using liposomes that contain fluorescently labeled phospholipids have demonstrated that Atg2 transfers phospholipids.In vitro LT activity was also confirmed for human ATG2ANR binds to a variety of phospholipids, including PE, phosphatidylcholine, and phosphatidylserine with low specificity.NR is much lower than that of the full‐length Atg2, implying that a region other than Atg2NR is required for efficient phospholipid transfer. Together with the observation that Atg2NR is located at the one end of the rod‐like architecture and Atg2 tethers two membranes using both ends, it is proposed that Atg2NR functions as an extractor of phospholipids from one membrane. The substitution of the conserved basic residues at the surface of Atg2NR with acidic residues caused significant reduction in the MT and LT activities of Atg2.NR are not conserved in Vps13, a chimeric Atg2 involving the Chorein_N of Vps13, instead of the intrinsic Chorein_N, exhibited the same autophagic activity as wild‐type Atg2.VPS13A, which are found in Chorea‐acanthocytosis patients, are located in the Chorein_N region,Atg2NR, which has been predicted to contain five repeated structures.NR and the five repeated structures constitute a successive hydrophobic groove that functions as a pathway for phospholipids; phospholipids move from ERES to the IM by sliding the hydrophobic acyl chains along the hydrophobic groove while exposing the hydrophilic head groups to the cytosol (Fig. How does Atg2 transfer phospholipids from ERES to the IM? Because the MT activity is indispensable for the LT activity of Atg2, a well‐known LT system, in which LT proteins shuttle between organelles,sol Fig. , right. NR, and recognizes PI3P existing in the IM.NR toward ERES, possibly allowing Atg2NR to interact with ERES but not with IM and extract phospholipids only from ERES.To expand the IM, Atg2 must mediate a unidirectional transfer of phospholipids from ERES to IM. Atg18 and Atg9 might determine the flow direction of the phospholipids by fixing the orientation of Atg2: Atg18 binds to one end of Atg2, which is opposite to Atg2de novo autophagosome biogenesis.Autophagosomes, 0.3–0.9 μm in size, are formed within 10 min in yeast.in vivo visualization of phospholipid movement from the ER to IM via Atg2 and atomic resolution structural determination of the full‐length Atg2 will provide further mechanistic insights into Atg2 function solving one of the biggest questions in autophagy: the mechanism of de novo autophagosome biogenesis.Recent advances in structural biology and biochemical studies have shed light on the molecular functions of Atg2, one of the most poorly understood proteins in the field of autophagy. Thus, the finding that Atg2 is a novel type of phospholipid transfer protein, bridging two membranes and at the same time transferring phospholipids between them, is unexpected and provides a novel model of autophagosome formation: LT proteins supply phospholipids from membrane source organelles as building blocks for autophagosome formation. Although technically difficult,


Mkk4, Mkk7, and both Mkk4 and Mkk7 were generated. Immunohistochemistry was used to study the distribution and structure of retinal cell types and to assess RGC survival after optic nerve injury ). Adult Mkk4- and Mkk7-deficient retinas had all retinal cell types, and with the exception of small areas of disrupted photoreceptor lamination in Mkk4-deficient mice, the retinas of both mutants were grossly normal. Deficiency of Mkk4 or Mkk7 reduced JNK signaling in RGCs after axonal injury and resulted in a significantly greater percentage of surviving RGCs 35 days after CONC as compared to wild-type controls . Combined deficiency of Mkk4 and Mkk7 caused failure of optic nerve formation, irregular retinal axonal trajectories, disruption of retinal lamination, clumping of RGC bodies, and dendritic fasciculation of dopaminergic amacrine cells. These results suggest that MKK4 and MKK7 may serve redundant and unique roles in molecular signaling important for retinal development and injury response following axonal insult.The mitogen-activated protein kinase (MAPK) pathway has been shown to be involved in both neurodevelopment and neurodegeneration. c-Jun N-terminal kinase (JNK), a MAPK important in retinal development and after optic nerve crush injury, is regulated by two upstream kinases: MKK4 and MKK7. The specific requirements of MKK4 and MKK7 in retinal development and retinal ganglion cell (RGC) death after axonal injury, however, are currently undefined. Optic nerve injury is an important insult in many neurologic conditions including traumatic, ischemic, inflammatory, and glaucomatous optic neuropathies. Mice deficient in  In the retina, MAPK signaling plays a role in retinal formation and axonal injury-induced retinal ganglion cell (RGC) death12. The MAPK, c-Jun N-terminal kinase (JNK), is regulated by two upstream MAP2Ks: MKK4 and MKK714. The specific requirements of MKK4 and MKK7 in retinal development and neurodegeneration, however, are currently undefined.The mitogen-activated protein kinase (MAPK) pathway is involved in development, neurodegeneration, and the immune response15. In the central nervous system, MKK4 and MKK7 and their downstream effector molecules, the JNKs (JNK1–3), play important roles in both development and maintenance of neural structures. MKK4, MKK7, and the JNKs contribute to the regulation of cellular organization and axonal migration through both overlapping and non-redundant mechanisms20. JNK signaling has also been shown to contribute to multiple aspects of retinogenesis such as progenitor cell proliferation21. The exact contributions of MKK4 and MKK7 to retinal development, however, remain largely unexplored. In the adult, multiple MAPK members have been shown to be key mediators of the apoptotic injury response and RGC death after axonal injury. Specifically, JNKs and their canonical downstream effector molecule, the transcription factor JUN, are important for RGC death after mechanical- and ocular hypertension-induced axonal injury24. Despite this known involvement, the critical molecular events leading from axonal injury to RGC death are not fully defined. Determining the molecular mechanisms of RGC pro-death signaling after axonal injury is necessary for understanding the molecular underpinnings of diseases such as glaucoma and traumatic optic neuropathies which result in RGC loss.MKK4 and MKK7 are required for normal developmentMkk4 and Mkk7, we demonstrate their redundant and unique roles in retinal development and injury signaling.The importance of JNK signaling for both RGC development and response to axonal injury is well established, but little is known regarding the role of the MAP2Ks upstream of JNK in these processes. Selectively targeting these upstream MAPKs may allow us to define the specific pathological signaling pathway that activates pro-death JNK activation in RGC axons after an insult. Furthermore, understanding the contribution of MKK4 and MKK7 to the injury response and to JUN activation in RGCs will likely have implications for other diseases or traumas involving axonal injury. Here, using conditional null alleles of Mkk4 and Mkk726 were maintained on the C57BL/6J background. To generate animals deficient in Mkk4 or Mkk7, mice carrying the respective floxed allele were bred to animals carrying Six3cre which is first expressed in the optic cup between E9.0 and 9.527. Animals carrying the Cre recombinase and one Mkk4 or Mkk7 floxed allele were intercrossed to generate animals: (1) carrying Cre recombinase and two copies of either Mkk4 or Mkk7 floxed alleles, referred to as Mkk4 deficient  and Mkk7 deficient  respectively and (2) animals without floxed alleles and/or without Cre recombinase, referred to as wild-type controls . Animals deficient in both Mkk4 and Mkk7 were generated by breeding animals carrying the floxed alleles and Six3cre together to generate Six3cre+; Mkk4f/f; Mkk7f/f animals .All experiments were conducted in adherence with the Association for Research in Vision and Ophthalmology’s statement on the use of animals in ophthalmic and vision research and were approved by the University of Rochester’s University Committee on Animal Resources. Animals were housed on a 12-h light and dark cycle and received chow and water ad libitum. Floxed alleles of 7. For optic nerve morphological analysis, 8 additional animals per genotype were perfused with 4% PFA. Brains were then dissected free and the optic nerves were photographed using a stereomicroscope.Eyes used to evaluate retinal morphology were processed in 2.5% glutaraldehyde and 2% paraformaldehyde (PFA) for 24 h at 4 °C. Eyes were then dehydrated with a series of ethanol washes, embedded in Technovit (Electron Microscopy Services), sectioned at 2.5 μm, and stained with Multiple Stain Solution . Eyes to be processed for immunohistochemistry and retinal flat mounts were harvested and processed in 4% PFA for 2 h at room temperature prior to storage in 1 M phosphate-buffered saline (PBS). The anterior segment of the eye was dissected away and the posterior segment of the eye was processed for cyrosectioning (14 μm sections) or whole retina flat mounts as previously describedFor immunohistochemical staining on cryosections, sections were blocked in 10% horse serum with 0.1% Triton X in 1× PBS for 3 h at room temperature and then incubated in primary antibody overnight at 4 °C. Primary antibodies included: goat anti-SOX2 , goat anti-CHaT , rabbit anti-calretinin , mouse anti-calbindin-D-28K , rabbit anti-PKCα , and rabbit anti-pJNK . Cryosections were washed in PBS and incubated in Alexafluor-conjugated secondary antibodies (Invitrogen) for 2 h at room temperature. Cyrosections were then washed, counterstained with 4′,6-diamidino-2-phenylindole (DAPI), and mounted in Fluorogel in TRIS buffer (Electron Microscopy Sciences). A complete list of antibodies used is provided in Table 28. Primary antibodies included: mouse anti-TUJ-1 , goat anti-BRN3b , rabbit anti-TH (1:1000 Millipore), rabbit anti-pJUN , rabbit anti-cCASP3 , and rabbit anti-Neurofilament light chain . Whole retinas were then washed in 1× PBS and incubated in Alexafluor-conjugated secondary antibodies for 2 days at room temperature. Whole retinas were then mounted RGC side up in Fluorogel in TRIS buffer. RGCs were quantified in eight equally spaced 40× fields taken approximately 220 μm from the peripheral edge of the retina as previously described29. Quantification was completed using the cell counter tool in ImageJ. The area of fasciculations in Mkk7-deficient mice was determined by tracing the outside edge of each fasciculation using the outline tool in Axiovision software (Carl Zeiss).For immunohistochemistry on flat mounts, floating retinas were blocked in 10% horse serum with 0.3% Triton X in 1× PBS overnight on a shaker at 4 °C and then incubated in primary antibody for 3 days at 4 °C as previously describedMkk4- and Mkk7-deficient mice were completed as previously described7. Nanodrop was used to determine protein concentration from retinal lysates and 30 μg of protein was loaded into each well. Blots containing rabbit anti-pJUN  and rabbit anti-GAPDH  were incubated overnight at 4 °C. A chemiluminescent kit  was used to detect immunoreactive bands followed by densitometric analysis using Quantity One Software (Bio-Rad). Western blots for MKK4 in Mkk4-deficient mice, MKK7 in Mkk7-deficient mice, and pJNK in Mkk4/Mkk7-deficient animals were performed as previously described8. Briefly, retinas were placed in 100 μl ice-cold lysis buffer after dissection  and protease/phosphatase inhibitor cocktail ). Tissue was sonicated  and spun down in a microcentrifuge. Then, 10 μl of supernatant was boiled for 10 min with 10 μl of 2× Laemmli loading buffer (Bio-Rad) and then run on a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel. Membranes were treated with the Qentix Western Blot Signal Enhancer kit  after they were transferred to a polyvinylidene difluoride (PVDF) membrane and then blocked and probed overnight at 4 °C with primary antibodies: rabbit anti-pJNK , rabbit anti-MKK4 , rabbit anti-MKK7 , or rabbit anti-GAPDH . Membranes were then washed and treated with secondary antibody: horseradish peroxidase-conjugated anti-rabbit IgG . A chemiluminescence kit  was used to detect immunoreactive bands prior to exposure using either film or digital detection equipment (Azure Biosystems c500). Occasionally, membranes were stripped  after development and treated with another primary antibody. ImageJ was used to perform densitometric analyses to determine the relative abundance of protein expressed relative to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading controls.Western blots for pJUN in WT and 30. Briefly, animals were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine, the optic nerve was surgically exposed, and then crushed for 5 s with a pair of self-closing forceps immediately behind the globe. A cohort of eyes underwent sham surgery, in which the optic nerve was exposed but not crushed. These eyes along with eyes that had not been manipulated served as experimental controls. Animals were harvested after 2 h, 1 day, 5 days, or 35 days.Controlled optic nerve crush (CONC) was performed as previously describedt-tests were used to compare differences across two groups. A one-way analysis of variance followed by the Bonferroni post hoc test for group comparisons was used to compare differences across more than two groups at a single time point. A P value < 0.05 was considered statistically significant. Means ± SEM are displayed in graphs.At least three retinas of each genotype were analyzed for all experimental conditions. Experimenters were masked to genotype and experimental condition for all quantification of RGC counts. Unpaired Student’s Mkk4 or Mkk7, Six3cre was used to recombine floxed alleles of either Mkk4 or Mkk7 with efficient deletion of both Mkk4 and Mkk7  and calretinin were used to label amacrine cell bodies in the inner nuclear layer and synaptic strata in the inner plexiform layer32. Amacrine cells and inner plexiform lamination in both Mkk4- and Mkk7-deficient retinas appeared to develop normally . Furthermore, while no gross alteration was observed in Mkk4-deficient RGCs, Mkk7 deficiency resulted in sporadic clumping and axonal fasciculation . To determine whether the decreased RGC density in adult Mkk4- and Mkk7-deficient retinas was due to an early developmental defect, flat mounts were examined at P0, a time point subsequent to RGC birth and determination33. Retinas were stained for BRN3B (POU4F2), another marker for RCGs35. RGC cell counts in both mutant mice were normal at this age, suggesting the correct amount of RGCs were born but died at a later stage .RGC cell body clumping and axonal fasciculation was observed in 100% of nas Fig. . Areas o36. Down syndrome cell adhesion molecule (DSCAM) has also been implicated in JNK1–Netrin-1-mediated neurite growth, and its loss of function leads to clustering of both RGC axons and cholinergic amacrine cell dendrites37. To examine whether the same amacrine cell phenotype resulted from Mkk4 or Mkk7 deficiency, additional retinal flat mounts were stained for tyrosine hydroxylase (TH), which labels a subset of dopaminergic amacrine cells. Similar to Dscam mutant mice, deficiency of Mkk7 resulted in fasciculation of dopaminergic amacrine cell dendrites .JNK1 is required for Netrin-1-induced axon outgrowth in the spinal cordtes Fig.  which wa41. MKK4 and MKK7 have been previously reported to serve non-redundant functions in vivo44. For example, activation of MKK4 but not MKK7 triggers neuronal death following oxidative stress45 while MKK7 is required for JNK activation caused by pro-inflammatory cytokines (such as tumor necrosis factor-α and interleukin-1)15. Together, these results suggest activation of JNK after axonal injury may be preferentially controlled by a single MAP2K. To test this possibility, JNK–JUN signaling was evaluated in the Mkk4- and Mkk7-deficient animals. Both JNK and JUN are activated following optic nerve injury47. Expression of pJNK was observed in RGC axons entering the optic nerve head and pJUN-positive RGCs were present in WT and Mkk4- and Mkk7-deficient mice -deficient retinas after CONC; however, detectable levels of the protein were found in both cohorts  at 5 days after CONC as compared to controls even after correcting for the decreased number of RGCs in Mkk4- and Mkk7-deficient retinas . This protection was not as robust as that previously observed in Jun-deficient mice (~75%) at the same time point, suggesting both Mkk4 and Mkk7 can activate JNK–JUN-dependent RGC death after axonal injury.The MAPK signaling family has been previously shown to be an important pro-apoptotic signaling pathway after axon injury nas Fig. . In addiMkk4 or Mkk7 alone provided significant protection to RGCs after mechanical axon injury; however, neither deficiency by itself afforded complete protection to RGCs. As MKK4 and MKK7 are the only known molecules acting directly upstream of JNK, MKK4 and MKK7 appear to have redundant roles in RGCs after CONC43. Moreover, as either deficiency alone did not cause severe retinal dysgenesis during development, removing both genes, and therefore any possible genetic redundancy, could allow for the role of MAP2Ks in axonal injury-induced RGC death to be tested. To examine these possibilities, retinas deficient in both Mkk4 and Mkk7 were generated using Six3cre. JNK signaling, as measured by pJNK protein levels, was reduced by 86.7% in dual Mkk4/Mkk7-deficient mice 8. Mkk4/Mkk7-deficient mice had highly atrophic optic nerves  which was more prevalent and severe than the phenotype observed in Mkk7-deficient retinas  and sonic hedgehog (SHH)-mediated control of paired-like homeobox transcription factor (PAX2), which contributes to closure of the optic fissure52. Netrin-1 also contributes to optic nerve head formation as deficiency of Netrin-1 leads to failure of RGC axons to exit the eye leading to optic nerve hypoplasia53. Netrins serve as important axonal guidance cues in the nervous system and contribute to proper axon outgrowth and pathfinding54. JNK1 has been shown to be required for netrin-1 signaling and inhibition of JNK1 reduces netrin-1-dependent axonal projections and pathfinding in other neural systems36. DSCAM is a netrin-1 receptor necessary for neurite arborization and prevention of abnormal neural fasciculations56. Axonal fasciculations and ectopic photoreceptor phenotypes similar to those in Mkk4/Mkk7-deficient retinas were observed in Dscam-deficient retinas57. Proper fasciculation is necessary for RGC axon pathfinding; however, as axons approach deeper targets in the brain, they must disassociate from their neighbors58. It is possible in Mkk4/Mkk7 mutants, the extrinsic cues directing axon divergence are disrupted, resulting in abnormal fasciculation in the retina. Jnk2/3-deficient mice and Jun-deficient mice do not have aberrant lamination or optic nerve head dysgenesis, which further supports the idea that morphologic differences observed in the Mkk4/Mkk7-deficient mice are likely due to JNK1 or JUN-independent signaling mechanisms59. Future study to evaluate the downstream signaling mechanisms of MKK4 and MKK7 will likely reveal key contributors to retinal and optic nerve development. Despite the lack of optic nerve formation in the Mkk4/Mkk7-deficient animals, RGCs survive within the retina. Normal pathfinding and neurotropic support is essential for RGC survival, and thus RGC survival in the absence of optic nerve formation may be due to decreased pro-apoptotic signaling as JNK–JUN signaling contributes to both pro-survival and pro-apoptotic signaling65.In contrast to the 41. Similar to other MAPK family members, single deficiency of Mkk4 or Mkk7 provides significant protection to RGCs after axonal injury. The level of protection observed, however, does not phenocopy deficiency of other MAPK signaling molecules, supported by the significant, but not complete, reduction of pJUN. Deficiency of the upstream MAP3K, Dlk, and downstream targets, Jnk2/3 and Jun, provided greater protection to RGCs after CONC than that observed in this study, suggesting MKK4 and MKK7 likely have redundant roles in pro-apoptotic signaling after axonal injury66.Pro-apoptotic MAPK signaling is also an important component of molecular signaling after axonal injury in RGCs67. Activation of p38 occurs after axonal injury in both neurons and glia70. Inhibition of p38 signaling has been shown to provide mild protection to RGC somas and reduce axonal transport deficits following axonal injury69. Activation of the p38 arm of MAPK signaling can also directly activate Ddit3 (DNA damage inducible transcript 3) which encodes the protein CCAAT/enhancer binding homologous protein (CHOP)/GADD153, a key mediator of endoplasmic reticulum (ER) stress72. CHOP has been independently shown to be important for pro-apoptotic signaling after axonal injury74. In order to understand the molecular signaling cascade contributing to RGC death after axonal injury, it will be necessary to parse apart the downstream signaling contributions of MKK4 and MKK7.While both MKK4 and MKK7 are known to activate the JNKs, MKK4 additionally regulates p38Mkk4/Mkk7 deficiency in a temporally controlled conditional knockout animal  to determine if deficiency of these molecules together might provide greater protection to RGCs after injury than either alone. It is tempting to consider that dual deficiency of Mkk4/Mkk7 may be more protective of RGCs than deficiency of other downstream targets alone, as combined deficiency of Mkk4/Mkk7 may alter multiple molecular signaling pathways that have been previously shown to be important for RGC death including JNK, p38, and ER stress signaling75.Future studies should also evaluate combined 77. Analysis of the transcriptome of MKK4 and MKK7 is necessary to identify specific downstream targets. Such inquiry would determine if the downstream effectors leading to developmental phenotypes in deficient animals are independent from those downstream effectors mediating pro-apoptotic signaling in RGCs after optic nerve crush.Future investigation of the unique and overlapping roles of MKK4 and MKK7 must consider the downstream targets of their activation. MKK4 and MKK7 are known to preferentially target different amino acid residues on the JNKs. MKK4 targets the tyrosine residue while MKK7 preferentially phosphorylates the threonine residueMkk4 and Mkk7 offer mild protection for RGCs against optic nerve injury, though there was likely genetic compensation allowing for pro-apoptotic signaling to proceed. Due to the severity of developmental defects in the double-deficient mice, possible additive protection following mechanical insult was not assessed. Additional experiments employing an inducible double Mkk4/Mkk7 knockout would allow for proper optic nerve formation and subsequent analysis of RGC survival following injury. This would define the aggregate role of these MAP2K molecules and the specificity of the cellular response in a glaucomatous-relevant injury model.MKK4 and MKK7 are members of the MAPK pathway important for both retinal development and the injury response following axonal insult. While we have shown MKK4 and MKK7 are required for maintaining RGC survival, future studies should examine the cellular and molecular mechanisms underlying the observed postnatal dropout in single-deficient animals. Single deficiency of Supplemental Material


Toxoplasma gondii together with influenza matrix protein 1 (M1) as a core protein. We also generated three VLPs expressing IMC, ROP18, or MIC8 together with M1 for combination VLPs (TG1/TG4/TG6). A total of four kinds of VLPs generated were characterized by TEM. Higher number of VLPs particles per μm2 were observed in multi-antigen VLPs compared to combination VLPs. Mice (BALB/c) were intranasually immunized with multi-antigen VLPs or combination VLPs and challenged with T. gondii tachyzoites (GT1) intraperitoneally. Compared to combination VLPs, multi-antigen VLPs showed significantly higher levels of CD4+ T cell, and germinal center B cell responses with reduced apoptosis responses, resulting in significant reduction on parasite burden. These results indicate that higher efficacy of VLPs generated by multi-antigen VLPs can induce significant reduction of parasite burden and better survival of mice than that by combination VLPs, providing important insights into vaccine design strategy for VLPs vaccine expressing multiple antigenic proteins.Virus-like particle (VLP) as a highly efficient vaccine platform has been used to present single or multiple antigenic proteins. In this study, we generated VLPs  in insect cells co-infected with recombinant baculoviruses presenting IMC, ROP18, and MIC8 of  VLPs represent safe vaccine platforms since VLPs are composed of viral structural proteins without requiring the presence of the genome. In addition, VLPs are highly immunogenic, even in the absence of adjuvants compared to soluble subunit protein vaccines , 2. VLPs B cells . ToxoplaT. gondii VLP vaccines can provide protection against T. gondii ME49 strain or GT1 strain using mouse models (T. gondii inner membrane complex (IMC) or microneme protein 8 (MIC8) proteins were expressed on the surface of VLPs. These proteins were selected because they are critical for the proliferation and invasion of host cells. These studies indicate that VLP vaccine immunizations can induce serum antibody responses or serum neutralizing activity and T cell responses to show complete protection . Multi-protein vaccines might induce immunity against multiple antigenic targets, multiple strain variants, and/or even multiple pathogens. High immunity induced by multiple antigenic proteins expressed by VLPs could be promising alternative screening strategy.Previous studies have shown that e models , 6. In tst cells –9. ROP18nfection . Recombiinvasion . These sThere are two ways to generate VLPs containing multiple antigenic proteins. One is through combination VLPs in which VLPs expressing three antigens can be generated by combining three VLPs with each VLP produced by infecting insect cells with baculoviruses expressing single antigenic protein. Another is through multi-antigen VLPs that can be generated by infecting insect cells simultaneously with three baculoviruses with each baculovirus expressing a single antigenic protein (multi-antigen VLPs). The process in producing multi-antigen VLPs is much easier and faster than that from combination VLPs. Thus, in this study, we evaluated and compared protective immune responses induced by multi-antigen VLPs with combination VLPs expressing IMC, MIC8, and ROP18 proteins. Multi-antigen VLPs showed better protection than combination VLPs by inducing significantly higher levels of T cell and germinal center B cell responses with reduced apoptosis responses, resulting in significant reduction of parasite burden.All animal experiments and husbandry involved in these studies were conducted under the guidelines of Kyung Hee University IACUC (permit number: KHUASP (SE)−16–157). All animal procedures performed in this study were reviewed, approved, and supervised by Kyung Hee University IACUC. The research staff was trained in animal care and handling. They have received the certificate of completion for Animal Welfare & Ethics Course (K-2015-18060371) form CITI. All surgery was performed under isoflurane anesthesia (BSL2). All efforts were made to minimize animal suffering.T. gondii GT1 strains were maintained in female BALB/c obtained from KOA Tech  as described previously  or oral passage (ME49) in Balb/C mice as described (Spodoptera frugiperda Sf9 cells used for production of recombinant baculovirus (rBV) and virus-like particles were maintained in serum-free SF900 II medium  in spinner flasks at 27°C and 130–140 rpm. T. gondii (ME49)–infected sera from mice were used. Horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin IgG was purchased from Southern Biotech . Monoclonal mouse anti-M1 antibody was purchased from Abcam .eviously , 12, 13.escribed . SpodoptT. gondii IMC, rhoptry protein 18 (ROP18), microneme protein 8 (MIC8), and influenza matrix protein 1 (M1) genes were clones as described previously , ROP18 , MIC8 , or M1  gene in pFastBac vector were confirmed by DNA sequencing. Recombinant plasmids were transformed into DH10-Bac. Recombinant baculoviruses expressing IMC, ROP18, MIC8, or M1 were produced as described previously   . Three gn = 20 per group) to receive multi-antigen VLPs or combination VLPs. For multi-antigen VLPs (TG146) immunization, 60 μg of multi-antigen VLPs per mouse were used to intranasally (IN) immunize mice at weeks 0 and 4. For combination VLPs (TG1/TG4/TG6), three VLPs expressing IMC, ROP18, or MIC8 were combined at a ratio of 1:1:1 . At 4 weeks after the 2nd immunization, mice were challenged with 1 × 103 tachyzoites of the GT1 strain by intraperitoneal (IP) injection. Ten mice from each group were sacrificed at 7 days post-challenge. Ascites of abdominal cavity and spleen samples were collected. The remaining mice (10 mice in each group) were observed daily to monitor changes in body weight and survival rates until death. Mice that lost 20% in body weight were humanly euthanized.BALB/c mice  were randomly divided into different experimental groups (T. gondii (ME49)—infected sera were used as a positive control. T. gondii-specific IgG antibody was determined by enzyme-linked immunosorbent assay (ELISA) as described previously  per well at 4°C overnight. Then 100 μL of serum samples (diluted 1:100 in PBST) per well were incubated in the plates for 2 h at 37°C as primary antibody response. HRP-conjugated goat anti-mouse IgG in PBST  were used to determine T. gondii-specific IgG response.Mice sera were collected from all groups at 4 weeks after prime and boost immunization. Sera from naïve mice were used as a negative control, and sera from eviously . BrieflyT. gondii (GT1) at 37°C for 1 h. The mixture of tachyzoites and serum was used to intraperitoneally (IP) infect naive mice (10 mice in each group). The mixture of tachyzoites and PBS was used as control. At 7 days after infection, tachyzoites of T. gondii were collected from the abdominal cavity of mouse and counted by hemocytometer chamber under microscopy  and germinal center B cells (GC) from splenocytes of mice on day 7 after challenge infection were analyzed by flow cytometry. Briefly, 1 × 106 splenocytes (each tube) in staining buffer  were incubated at 4°C for 15 min with Fc Block . For surface staining, cells were incubated with surface antibodies  at 4°C for 30 min. Splenocytes were washed with staining buffer and fixed with 4% paraformaldehyde at 4°C for 30 min before acquisition using a BD Accuri C6 Flow Cytometer . Data were analyzed using C6 Analysis software .The population of T cell  and analyzed with C6 Analysis Software .To determine apoptosis in splenocytes, Annexin-V and PI were stained using BD Apoptosis Detection Kit I . Splenocytes were collected at 7 days post-challenge. Then 1 × 10P < 0.01 was considered to be significant.The data were analyzed statistically using the ANOVA with multiple comparison test of PC-SAS 9.3 . A T. gondii IMC, ROP18, or MIC8 together with influenza M1 were generated  or combination VLPs (D). T. gondii VLPs had a spherical morphology with a size of 40–120 nm .Multi-antigen VLPs or combination VLPs expressing d Figure  and elecd Figure . Resultsm Figure , 17, 18.T. gondii-specific IgG antibody induced by multi-antigen VLPs or combination VLPs immunizations, we measured these levels in mice sera upon prime and boost after IN immunizations  group .To evaluate the level of  Figures . Multi-a*P < 0.01) or multi-antigen VLPs were significantly reduced (*P < 0.01) compared to those in controls (non-immunized naïve sera and PBS). There was no significant difference in parasite neutralizing ability between sera from multi-antigen VLPs and combination VLPs groups.Neutralizing activity afford by prior vaccination has long been considered the main mechanism of protection . In this+ T cells and CD8+ T cells  or combination VLPs (TG1/TG4/TG6+Cha) (*P < 0.01) immunized mice compared to naïve challenge (Naïve+Cha) control mice. Significantly higher levels of CD4+ T cell responses were also found in multi-antigen VLPs compared to those in combination VLPs (*P < 0.01). However, there was no significant difference in CD8+ T cell responses between multi-antigen VLPs and combination VLPs. As shown in Figure *P < 0.01).To determine T cell and B cell responses in immunized mice upon challenge, flow cytometer analysis was performed using mouse spleen cells. As shown in Figures s Figure  were fouT. gondii is highly pathogenic. It induces high level of apoptosis, leading to widespread parasite dissemination and rapid death of mice (*P < 0.01) while multi-antigen VLPs (*P < 0.01) or combination VLPs (*P < 0.01) resulted in significantly lower levels of apoptotic responses. Compared to combination VLPs, multi-antigen VLPs showed significantly lower level of apoptotic responses (*P < 0.01). of mice . Mouse sT. gondii GT1 strain (1 × 103 tachyzoites) at 4 weeks after boost by IP injection. Survival rates and protection were determined. As shown in Figure T. gondii were collected from abdominal cavities of mice and counted. Mice immunized with multi-antigen VLPs or combination VLPs significantly inhibited parasite replication compared to non-immunized control (Naïve+Cha) . Multi-antigen VLPs showed higher inhibition of parasite replication compared to combination VLPs (*P < 0.01).To determine the protective efficacy of VLPs vaccine, immunized, and control mice were challenged with lethal T. gondii has a complex and variety of invasion pathways for attaching and invading host cells. Although several invasion factors of T. gondii involved in invasion of host cells have been identified as vaccine candidate antigens, those capable of inducing a strong protective immunity are limited  induced by serum neutralizing activity to obtain more convincing results. Both multi-antigen VLPs and combination VLPs showed higher levels of neutralizing activity compared to naïve control. No significant difference in parasite neutralizing ability between multi-antigen VLPs and combination VLPs were determined. However, interestingly, higher levels of T. gondii-specific IgG antibody responses in sera from multi-antigen VLPs vaccination than those in sera from combination VLPs were found, indicating non-neutralizing antibodies from multi-antigen VLPs might be involved in reduction of parasite burden. In the current study, significantly higher levels of CD4+, CD8+ T cell, and B cell responses were observed. Importantly, multi-antigen VLPs showed higher levels of responses compared to combination VLPs, indicating that higher density or higher purity of multi-antigen VLPs might contribute to higher immunogenicity and better protection compared to combination VLPs.Neutralizing antibodies play an important role in protection against viral infection in vivo –27. In tT. gondii GT1 is highly pathogenic and uniformly lethal. GT1 infections by IP route can lead to complete destruction and cell death in the spleen, resulting in remarkable high levels of IFN-g production and death of mice ( of mice . In thisT. gondii VLPs expressing multiple antigenic proteins.In summary, multi-antigen VLPs showed high quality of particles and significantly saved time for VLPs preparation compared to combination VLPs. Higher quality of multi-antigen VLPs induced higher T and B cell responses and significantly reduced apoptotic responses, resulting in significant higher inhibition of parasite replication and increased survival rate compared to combination VLPs. These results provide very important insights into vaccine design strategy for S-HL carried out the molecular genetic studies, participated in the sequence alignment, prepared VLPs, animal experiment and drafted the manuscript. H-JK and D-HL carried out experiment. F-SQ carried out the experiment design, analysis and interpretation of data and drafted the manuscript. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Myelodysplastic syndromes represent a heterogeneous group of age-associated haematopoietic diseases, which originate from changes in the function of haematopoietic stem cells (HSC). The conditions are referred to as clonal neoplasms and are characterised mainly by abnormal blood cell maturation plus a high propensity of progression to acute myeloid leukaemia (AML) . At presDNMT3A, TET2, ASXL1, TP53 and SF3B, might be important for initiating the disease.One aspect of MDS that remains unclear is which genetic mutations arising in the HSC population contribute significantly to disease pathogenesis. Over the last decade, next generation sequencing studies have shed light on this by investigating the clonality of MDS and made three main observations , 3, 4, 5SF3B, mutations in these genes are often acquired during normal healthy ageing, making their use for diagnostic purposes difficult. Therefore, there is an unmet clinical need for the identification of new markers with diagnostic and prognostic value. In particular, identification of molecular markers that affect disease severity would allow the stratification of patients to potentially inform choice of treatment. To this end, our recent work has identified the RNA expression of the MYBL2 gene in HSC as a critical determinant of disease prognosis and DNA double-strand break (DSB) repair in MDS patients [Although these studies identify commonly mutated genes in MDS, insight into the functional consequences of these mutations is often lacking. Furthermore, and with the exception of patients .MYBL2 expression in CD34+ cells from MDS patients correlates with a poor prognosis even in patients with a normal karyotype, suggesting that this could contribute to disease pathology [MYBL2 function plays a key role in the development and progression of MDS.MYBL2 is a transcription factor known to be important for maintaining genome integrity, and is located within the minimal commonly deleted region of the long arm of chromosome 20 (del20q), a frequent chromosomal abnormality present in MDS. We and others have shown previously that reduced athology , 8. Thisathology , 8. Take+ cells from MDS patients exhibit a significant reduction in DNA repair gene expression [We have further investigated the mechanisms by which MYBL2 function could contribute to MDS development and uncovered an important role for MYBL2 in DNA DSB repair in HSC . HSC obtpression , in partMYBL2 levels exhibited a significant reduction in the expression of DNA repair genes present in the reactome DNA repair pathway  [+ cells with low MYBL2 expression failed to efficiently repair DNA DSBs when compared to those with high MYBL2 levels [MYBL2 levels and DNA repair capacity indicates for the first time, that the poor prognosis associated with low MYBL2 expression may arise from increased genome instability and the acquisition of mutations/chromosomal rearrangements that facilitate disease progression.We also observed that MDS patients with low 5, MDC1) , indicat2 levels . This coMYBL2 levels to support DNA DSB repair in HSC, providing a molecular basis for the clinical phenotype. Our study has highlighted that MYBL2 RNA expression could be used as a novel predictor of DNA repair capacity in CD34+ cells from MDS patients, and measuring this single gene enables the association of a wider and distinctive DNA repair gene signature. This could be a powerful clinical biomarker to allow better patient stratification, specifically to inform decisions regarding patient selection for transplantation or treatments which target DNA repair. MDS remains a complex and heterogeneous disease which will always require comprehensive genetic analysis. However, if in the future we can profile functional genetic changes similarly to low MYBL2 expression, this will significantly improve our understanding, diagnosis and treatment of the disease in the ever-increasing ageing population.Given our findings, we suggest that HSC with a compromised MYBL2-dependent DDR can promote MDS development by facilitating telomere instability and inefficient repair of physiological DSBs. Taken together, these processes are known to contribute to oncogenic transformation in the HSC population, thus contributing to the pathology of MDS. Furthermore, the accumulation of incorrectly repaired DSBs in premalignant cells with sustained low levels of MYBL2 protein may also confer an increased susceptibility to disease progression. Overall, our work identifies the requirement of correct


Tumour necrosis factor (TNF) is a cytokine belonging to a family of trimeric proteins; it has been shown to be a key mediator in autoimmune diseases such as rheumatoid arthritis and Crohn’s disease. While TNF is the target of several successful biologic drugs, attempts to design small molecule therapies directed to this cytokine have not led to approved products. Here we report the discovery of potent small molecule inhibitors of TNF that stabilise an asymmetrical form of the soluble TNF trimer, compromising signalling and inhibiting the functions of TNF in vitro and in vivo. This discovery paves the way for a class of small molecule drugs capable of modulating TNF function by stabilising a naturally sampled, receptor-incompetent conformation of TNF. Furthermore, this approach may prove to be a more general mechanism for inhibiting protein–protein interactions. While biologics have been successfully applied in TNF antagonist treatments, there are no clinically approved small molecules that target TNF. Here, the authors discover potent small molecule inhibitors of TNF, elucidate their molecular mechanism, and demonstrate TNF inhibition in vitro and in vivo. Nonetheless, the use of biologics remains restricted to a subset of the potential patient population, as immunogenicity2, supply chain complexity3 and health economics restrict their application. It is clear that many more patients could benefit from a small molecule drug with the efficacy of the anti-TNF biologics.TNF antagonist treatments have been one of the success stories of recent years. Biologics such as infliximab, etanercept, adalimumab, golimumab and certolizumab pegol have proved efficacious in the clinic and have a well-documented mode of action and side-effect profile304 targets TNF directly and has been shown to inhibit activity by destabilising the TNF trimer, resulting in a dimer that is no longer able to interact with receptor4. Although this strategy has so far not led to molecules capable of being developed, work is continuing in this area7. The TNF trimer may also be induced to disassemble by aggregating small molecules8. These, however, behave as promiscuous aggregators9 and the inhibition can be reversed with detergent, a hallmark of promiscuous aggregators. Approaches have also been described using natural products such as Japonicone A10, and others have targeted the receptor rather than the TNF ligand12. The status of small molecule inhibitors of TNF has been reviewed in a number of articles14.The compound SPD-16. The potency of TNF for TNF receptor 1 (TNFR1) is picomolar and despite the interest in the field generated by the success of the biologics, no orthosteric small molecule inhibitors have been described. We believe that disrupting the interaction allosterically17 through a region of TNF that is not in direct contact with its receptor may offer advantages that make this an attractive way to block its mechanism of action. In this paper, we describe the discovery and characterisation of a way in which antagonism of TNF signalling is achieved in such a manner with low-molecular-weight compounds. This finding has the potential to broaden the availability of the therapeutic benefits of TNF inhibition.Small molecule modifiers of protein-protein interactions are generally described as orthosteric or allosteric. The former compete directly with the two proteins for binding. There are a number of examples described in the literature6786  using surface plasmon resonance (SPR) measurement on a Biacore 4000. The fragments were passed over the Biacore chip surface coated with immobilised TNF and screened at a concentration of 250 μM to detect weak binders. This screen identified UCB-786 Fig.  and an ains Fig. .Fig. 1Fr6876 for TNF by SPR. Association rate constants (ka) for typical small molecules are limited by the rate of diffusion and are commonly higher than 106 M−1s−118 but here the ka proved to be remarkably slow (923 M−1s−1). Conversely, the dissociation rate constant of this compound was also slow, particularly for a fragment (kd of 0.02 s1) resulting in a KD (kd/ka) of 22 μM. The slow kinetic profile is unusual for molecules of this size and the resulting curve   and π stacking of the benzimidazole with Y59C. The 2,5-dimethylbenzyl moiety of UCB-6876 filled a hydrophobic pocket, assigned as Site 2, with compound TNF interactions composed of Y59A, Y119B and L57B .Attempts were then made to obtain an X-ray co-crystal structure by soaking apo-TNF crystals with compound and conducting co-crystallisation trials, also in the presence of the compound. In the soaking studies the presence of UCB-SU) Fig.  and cleamer Fig. . The fol57B Fig. . It is aThe asymmetry of the compound-bound TNF trimer results in minor distortion at two of the receptor-binding sites and a more significant change at the AC binding site . There is a clear opening of the AC site because of monomer A twisting and tilting down and away from monomer C Fig. . By alig6876 are unlikely to induce a large quaternary conformational change as seen here. Also, the UCB compounds bind significantly faster than the timescale suggested for subunit exchange4. An enhanced molecular dynamics simulation19 was used to investigate the conformational transition between the symmetrical apo-TNF and the asymmetrical, open conformation of the TNF trimer  analysis20 and stabilised by UCB-6876. This model supports our hypothesis that the compounds can bind to and stabilise a naturally sampled transient TNF trimer conformation. That UCB-6876 binds to a low abundance form of TNF is consistent with the slow on-rate observed by SPR, since the association rate constant is dependent on binding site access and concentration. As indicated by the crystal structure, once bound it is completely buried between the TNF monomers, explaining the slow dissociation rate.We were interested in the mechanism of action of these inhibitors and hypothesised that small molecules such as UCB-21, and these have been shown to exchange with monomers from a different TNF molecule23.We confirmed that the compounds stabilised the TNF trimer by investigating their effect on the process of TNF monomer exchange. At low physiological concentrations, the trimer can dissociate into monomers22. We first showed that incubating a mixture of mouse and human TNF quickly produced heterotrimers consisting of different ratios of mouse monomer and human monomer  showed that the appended pyridyl group occupied the MPD pocket. The pyridyl nitrogen formed an H-bond with Y119A, accounting for the increase in potency  compared with TNF in the presence of DMSO alone   to test if asymmetric TNF was compromised in its ability to bind TNFR1.5307 prior to incubation with a 3.2-fold excess of receptor. The compound had little effect on the migration of hTNF alone  the major species was the two-receptor bound form  with one, two or three receptors bound was first characterised by titrating hTNFR1 with hTNF , which is distal to the TNF:TNFR1 signalling complex25. After TNF stimulation alone, increased levels of RIP were recruited to TNFR1, with a concomitant increase in phosphorylation of NF-κB  after TNF stimulation  and a geometric mean IC50 of 120 nM,  using human and mouse TNF, respectively  thus forming a ‘gateway’ to the pocket. Accordingly, we mutated Leu57 of TNF to phenylalanine, mimicking other family members, to which our compounds did not bind in a thermal melt assay. The mutated TNF (L57F) was equally active as wild-type TNF in a HEK assay 29 to inhibit human TNF, and etanercept to inhibit murine TNF. Following oral administration, UCB-9260 showed a dose-dependent inhibition of neutrophil recruitment, measured as CD45+GR1+ cells by flow cytometry with significant inhibition obtained at 30 mg/kg of human TNF  showed profound inhibition of the clinical score , 6 h after the LPS challenge (therapeutic dosing), also showed a significant reduction of the clinical score  model uses a combination of an anti-collagen antibody cocktail and lipopolysaccharide (LPS) to produce rheumatoid arthritis-like symptoms in the mouseore Fig. . UCB-926ore Fig. . StatistWe have discovered orally active small molecules that inhibit TNF activity both in vitro and in vivo. The compounds bind to a pocket in the centre of the TNF trimer formed by the movement of the TNF monomers, stabilising the distorted trimer, which leads to reduced signalling through TNFR1. Analytical size exclusion suggests that the asymmetric trimer only binds to two receptors, consistent with the structural data showing significant distortion at one receptor binding interface. Rather than inducing a conformational change, we believe that the compounds bind to TNF through a process that is largely governed by conformational selection as suggested by our molecular dynamic (MD) simulations Fig.  and DEER31. Indeed, the disruption of PPIs has been considered difficult by medicinal chemists for many years and there are few examples in the literature32. The surface between proteins is typically large, flat and featureless. Accordingly, many groups choose to target these interactions using relatively large molecules based on peptides or scaffolds that mimic peptides. Other approaches, however, have been described, such as targeting ‘hot spots’ in the binding site that appear to contribute most of the binding energy, making smaller molecules a viable alternative33. Notably, so-called fracturing of CD40L, a member of the TNF superfamily, using a relatively large molecule retained the trimeric assembly34. Indeed, there is some overlap between where this compound binds on CD40L and the TNF binders described here. Furthermore, stabilisation of protein complexes by binding naturally occurring small molecules at the interface has also been described35, and it is interesting to speculate that there could be naturally occurring molecules that specifically modulate the function of TNF. Most importantly, this work suggests a method of discovering inhibitors using protein stabilisation of a non-active conformation as an end point, which could be facilitated by an antibody that holds the target protein in the desired inactive conformation36. Here we demonstrate that fragment-based drug discovery can be used to provide starting points for orally active compounds that modulate protein-protein interactions.Inhibiting protein-protein interactions (PPIs) with small molecules has been described as reaching for the ‘high hanging fruit’Small molecules offer several advantages over biologics, including cost, the convenience of oral delivery and shorter half-lives, which benefit patients requiring drug holidays. In addition, they can be designed to diffuse across the blood-brain barrier, making them suitable for targeting central nervous system disorders. The possibility of being able to target TNF-driven neuroinflammation, for example, is particularly interesting.From our experience with TNF, it seems that orally available, bona fide small molecules, capable of stabilising favoured, naturally sampled conformations of target proteins, offer an attractive and chemically tractable approach to tackling protein-protein interactions.BIA  uses SPR to measure the binding of compounds to a protein immobilised on a chip surface. For this purpose a Biacore A100/4000  was used. A CM5 sensor was immobilised via amine coupling on flow cell 1 with human Fc antibody domain  and TNFR-Fc captured (2.5 kRU), flow cell 2 TNF (11 kRU), flow cell 3 was left blank (for non-specific binding), flow cell 4 TNFR-Fc (10 kRU), and flow cell 5 goat anti-human Fc 16 kRU). The sensor surface was then equilibrated in HBS-P  with 5% DMSO for at least 5 h. Compounds were diluted from 100 mM stocks into DMSO matched buffer and then flowed over the immobilised flow cells at a concentration of 250 µM at the maximum flow rate of 30 μL/min. Background subtraction binding curves were analysed using the Biacore A100 BIA evaluation software version 1.1 following standard procedures.D) of compounds. TNF (VCID 2043 Beryllium) was immobilised to approximately 5 kRU as described above and equilibrated for at least 5 h in HBS P + buffer with 5% DMSO. UCB-6876 was serially diluted from 187.5 µM, flowing over the immobilised TNF at 50 µL/min for 3 min before allowing full dissociation under flow for at least 6 min. Kinetic constants were obtained from multicycle data that had been double-referenced, the data fitted and then analysed using the Levenberg Marquardt algorithm in the BIA Evaluation software . Compounds UCB-5307 and UCB-9260 were titrated from 30 µM with three-fold serial dilutions to 0.37 μM and flowed over approximately 1.5 kRU of linked TNF with an n-terminal HKH tag (Beryllium) in HBS P buffer with 5% DMSO at 100 μl/min. The compounds were allowed to dissociate for 2 h under flow. Kinetic constants from single-cycle data were obtained  and compound characterisation details can be found in Surface plasmon resonance using a Biacore T100/T200 was used to measure the association rate constant (ka), the dissociation rate constant (kd) and the affinity  and 3′ TGATAAGCTT (HindIII is underlined), such that two stop codons follow the C-terminal residue. The final gene insert was then synthesised by DNA 2.0 and delivered in a shuttle vector. Following synthesis, the gene insert was digested with BamHI and HindIII and subcloned to vector pEMB54, which is an ampicillin-resistant, arabinose-inducible vector with pMB1 origin of replication and 6XHis-Smt3 under the PBAD promoter, followed by a multiple-cloning site which includes BamHI followed by HindIII. After BamHI/HindIII cloning into pEMB54, gene inserts are fused in-frame with the 6XHis-Smt3 sequence. Following digestion of both pEMB54 and CID2043 inserts with BamHI/HindIII, both were gel-purified, the inserts ligated into the vector and the ligation transformed to chemically competent TOP10 cells. One transformant was mini-prepped and submitted for DNA sequencing of the Open Reading Frame. CID7210 (see source data for plasmid sequence) was cloned in a manner similar to CID2043, and consisted of a triple tandem fusion of the TNF ECD (‘TNF Trimer’), wherein Uniprot P01375 77–233 was followed by 85–233, with an SG linker between the TNF monomers. This gene  was optimised in GeneComposer with identical BamHI/HindIII adaptors, synthesised by DNA 2.0 and subcloned into final expression vector pEMB116, which is the same as pEMB54 except the N-terminal tag is HKH instead of 6XHis-Smt3. CID8703 was cloned via Quikchange mutagenesis (Agilent) using standard protocols, using CID2043 as a template.Human TNF  was codon-engineered in-silico using GeneComposer™ for E. coli cells. A starter culture containing 100 µg/mL  ampicillin (Teknova) was inoculated with a single colony and grown for 16 h at 37 °C. This was then transferred to 8 L of Terrific Broth (Teknova) containing 100 µg/mL  ampicillin and grown to OD600 = 0.6. Protein expression was induced by adding arabinose to a final concentration of 0.1% (VWR) and grown for 16 h at 25 °C. The cells were harvested by centrifugation (Beckman) at 6240×g for 15 min and the pellets were collected and stored at −80 °C.Briefly, the target-specific vector was transformed into TOP10 g for 30 min at 4 °C (Beckman) and filtered with a 0.2 µm bottle-top filter . The supernatant was applied to two 5 mL Ni2+ charged HiTrap Chelating HP  columns and the protein eluted with a 500 mM imidazole gradient over 20 column volumes. The fractions of interest were pooled and the His-Smt tag was removed via cleavage with Ubiquitin-like-specific protease 1 (Ulp-1) while dialysing against 2 L of 25 mM Tris pH 8.0 and 200 mM NaCl overnight at 4 °C using 3.5 kDa MWCO snakeskin dialysis tubing. The affinity tag was removed by applying the digested pool over two 5 mL Ni2 + charged HiTrap Chelating HP columns. The flow-through contained the cleaved protein of interest. The protein was concentrated for size exclusion chromatography via centrifugal concentration  to 13.5 mg/mL for injection over a HiPrep 16/60 Sephacryl S-100 HR  in 10 mM HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), pH 7.5 and 150 mM NaCl. Fractions of interest were pooled and concentrated via centrifugal concentration  to 20 mg/mL, aliquoted and stored at −80 °C.Cells were resuspended 1 g:4 mL in 25 mM Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) pH 8.0 (Teknova), 200 mM NaCl (Teknova), 0.02% 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (JT Baker), 50 mM L-arginine (Sigma), 500U of benzonase (Novagen), 100 mg lysozyme (Sigma) and one complete EDTA-free protease inhibitor tablet (Roche). The cells were lysed via sonication (Misonix) and clarified via centrifugation at 142,000×Purified human TNF was diluted to 4–7 mg/mL in 10 mM HEPES pH 7.5, 150 mM NaCl buffer followed by overnight incubation at 4 °C with 0.5 mM compound (1–2 molar excess). The TNF-compound complex was crystallised by sitting drop vapour diffusion by mixing 0.5 μL of complex with 0.5 μL of 13% MPD, 13% PEG 1000, 13% PEG3350, 0.1 M HEPES, pH 8.0. Crystals were harvested for data collection ~2 weeks after initial set-up. Crystals were cryo-protected in ethylene glycol and/or vitrified directly in liquid nitrogen for data collection.6876 and UCB-9260 were collected at the Stanford Synchrotron Radiation Lightsource (SSRL) beam line 7–1 (ADSC Quantum-315R CCD X-ray detector). The dataset for human TNF complexed with UCB-5307 was collected at the Advanced Light Source (ALS) beam line 5.0.3 (ADSC Quantum-315R CCD X-ray detector). Diffraction data were reduced and scaled with XDS/XSCALE37. The structure was solved by molecular replacement using Phaser with input model of 1TNF. Iterative manual model building using Coot 38 and Refmac 39 continued until R and Rfree converged. Model quality was validated using Coot and MolProbity40. Structures were validated using Molprobity prior to deposition in the Protein Data Bank 42. Statistics for each crystal structure are provided in Supplementary Table The dataset for human TNF complexed with UCB-m/z; spray voltage 1.5 kV; capillary temperature 150 °C; S-lens RF 200; source DC offset 25 V, injection flatapole 7 V; inter flatapole 6 V; bent flatapole DC 6 V; EMR mode on. Data were processed using ThermoFisher Protein Deconvolution 3.0.Mouse and human TNF protein were made up to a concentration of 2 mg/mL  and desalted and buffer-exchanged using Zeba columns. The protein was then diluted to 20 μM in ammonium acetate buffer. An aliquot of each was mixed 1:1 and analysed at various time points. Human TNF was incubated  with compound (20 μΜ), 4% DMSO in 20 mM ammonium acetate) and mixed with mouse TNF (1:1) and tested at various time points using an Advion Nanomate (for sample introduction by nanospray) and ThermoFisher Exactive Plus EMR Mass Spectrometer. The following settings were utilised on the mass spectrometer: scan range 1000 to 8000 43 database structure prediction software. An error in the amino acid sequence of TNF structure 1TNF was detected within this process. In all three monomers of PDB structure 1TNF residue 143 is a leucine; this is in contrast to sequence data for TNF stored in Uniprot44 where this residue is an aspartate. For this study residue 143 of 1TNF was mutated to an ASP. Maestro45 was then used to add all other missing atoms and pick sensible protonation states for titratable residues, ensuring that all homotrimers remained symmetric.Missing backbone atoms were added using FREAD45 Amber force field was employed for all protein parameters, set up using the LEaP software from AmberTools 1.446. The TIP3P47 water model was used in a solvating cube along with neutralising sodium chloride ions at an approximate concentration of 0.15 M. NAMD 2.748 was used to carefully equilibrate for temperature and pressure . ACEMD49 was then used for a 1 ns structural equilibration and when patched with Plumed 1.350, for all metadynamics simulations. ACEMD simulations used a 4 fs time step throughout. All simulations used particle mesh Ewald51 for long-range electrostatics with appropriate potential truncation and smoothing.The ff99SB−1. The bias-factor was set to 15. Gaussian widths were set on a system basis using the last 200 ps of a 500 ps test run to estimate average fluctuation seen in 1 ps for each CV. A Gaussian width was chosen to be less than half of the average 1 ps fluctuation. Potential walls were employed to stop the opening subunits from dissociating. In these cases the inversion conditions of Crespo et al. were used to limit any error in the FES52. Wall and inversion parameters were chosen in line with the Gaussian widths being used. Convergence was pre-defined as a Gaussian height <100 times smaller than the initial value, convergence of any FE difference between open and closed states and free movement across any energy barriers. Hence, once a significant low energy region is found away from the apo (symmetric homotrimer state) region, a return to the apo region was deemed necessary.Initial metadynamics simulations used two CVs based on a quaternary torsion and a distance  and 5 µL of TNF (at 1 mg/mL), 37.5 μL PBS, pH 7.4 and 2.5 μL of compound (at 2 mM in DMSO). The mixture (10 µL) was dispensed in quadruplicate into a 384 PCR optical well plate and was run on a 7900HT Fast Real-Time PCR System (Agilent Technologies). The PCR System heating device was set at 20 °C to 99 °C with a ramp rate of 1.1 °C min−1; fluorescence changes in the wells were monitored by a CCD device. The fluorescence intensity increase was plotted as a function of temperature and the Tm calculated as the midpoint of this denaturation curve (determined as the point of inflection).A fluorescence probed thermal denaturation assay was performed to assess the effect of the compounds on the thermal stability of TNF (or other members of the TNF family) as a measure of compound binding. The reaction mixture contained 5 µL of 30× SYPRO5307 at a final concentration range from 90 μM to 690 μM, maintaining a constant DMSO concentration of 1%. The compound-bound hTNF was incubated for 1 h at 22 °C with hTNFR1 at a 3.2-fold molar excess over homotrimers . Identical samples were prepared with DMSO only, giving a final DMSO concentration of 0.25% for both sets of samples. Alternatively, a fully assembled hTNF/hTNFR1 complex (3 receptors per homotrimer) was formed by incubating hTNF with a 3.2-fold molar excess of hTNFR1 for 1 h at 22 °C. The TNF/TNFR1 complex was then incubated overnight at 4 °C with UCB-5307 at a final concentration range from 90 µM to 690 µM. Samples were subject to analytical size exclusion using HPLC. Injection volumes of 50 µL were separated on a TSK G3000SW L × I.D. 30 cm × 7.5 mm column (10 µm particle size) pre-equilibrated in 10 mM HEPES, pH 7.5, 150 mM NaCl.A stock of compound bound to human (h) TNF was prepared by pre-incubating hTNF  overnight at 4 °C with UCB-9260 (10 µM) or anti-TNF  for 1 h at 37 °C, and then incubated for 5 min at 37 °C with 50 × 106 Jurkat cells. Cells were then lysed for 1 h in cold lysis buffer , and cell supernatant harvested. TNFR1 immunoprecipitation was performed by incubating supernatant with 5 µg anti-TNFR1 antibody  overnight while rotating at 4 °C, followed by the addition of 20 µL protein G sepharose beads for 1 h rotating at 4 °C. An isotype control sample was incubated with 5 µg goat IgG, instead of anti-TNFR1 antibody to assess how much unspecific binding to the beads occurs during immunoprecipitation. Beads were subsequently washed 3× in lysis buffer, and then the supernatant removed and SDS-PAGE reducing sample buffer added to beads and samples boiled for 10 min. Sample (15 µL) was then loaded on a 4–12% Bis-Tris gel and run for 65 min at 200 V in MOPS buffer. Gels were then transferred to nitrocellulose membrane using iBlot (Invitrogen) and blocked in 5% milk in TBS/0.05% Tween (blocking buffer) for 1 h. RIP-1 ubiquitination  was then detected by Western blotting using anti-mouse-HRP . pNFκB  and GAPDH  were measured in the total cell lysates by Western blotting using anti-rabbit HRP . Blots were developed by detecting chemiluminescence using ImageQuant .TNF (25 ng/mL) was pre-incubated with UCB-TM CD40L SEAP  reporter cell line used to determine TNF activity . Compounds were titrated in DMSO and pre-incubated with TNF at a concentration giving half maximal response (10 pM for human or 20 pM for mouse) or the anti-human TNFR1 agonist antibody (R&D Systems. #AF 225) for 1 h. Cells were added to the compound/stimulus mixture and further incubated for 18 h. SEAP was measured using the colorimetric substrate HEK-Blue DetectTM or QUANTI-BlueTM from InvivoGen. Percentage inhibitions for compound dilutions were calculated between a DMSO control and maximum inhibition . The IC50 was determined as the point of inflexion between the fitted minimum and maximum of the compound dose response curve, calculated using 4PL fitted curve. Mean IC50s are reported as a mean of at least two experiments. Dose response curves for TNF and the anti-human TNFR1 agonist antibody are shown in Supplementary Fig. Stimulation of HEK-293 cells by TNF leads to activation of the NF-κB pathway. The HEK-Blue18. Compounds were titrated in DMSO, and then pre-incubated with human or mouse TNF for 1 h. L929 cells  in media containing actinomycin D (2 µg/mL) were added to the TNF inhibitor mixture and incubated for 18 h. L929 cytotoxicity was measured by the addition of CellTitreGlo (Promega). Luminescence was measured after an incubation of at least 20 min in the dark. Percentage inhibition was calculated between unstimulated wells , and wells with TNF, DMSO and actinomycin D . The IC50 was calculated as the point of inflexion for a 4PL fitted curve. Mean IC50s are reported as a mean of at least two experiments. Cells were described as mycoplasma-free when purchased and were tested using Mycoalert (Lonza) after each batch of assay vial preparation.TNF (human or mouse) kills L929 cells which have been compromised by incubation with a sub-lethal concentration of actinomycin D, and this effect is inhibited with TNF biologicsn = 4; vehicle n = 8; UCB-9260 10, 30 and 300 mg/kg n = 6 and 100 mg/kg n = 8; CDP571 n = 5 mice and mouse: PBS n = 3; vehicle n = 8; UCB-9260 10 mg/kg n = 7, 30 mg/kg n = 6, 100 and 300 mg/kg n = 8; CDP571 n = 5 mice, from Charles River UK) were dosed orally with vehicle  or compound, 30 min prior to intraperitoneal administration of mouse (0.05 µg/kg) or human (0.03 or 1 µg/kg) TNF diluted in PBS. Anti-TNFα biologics, CDP57121 (10 mg/kg) and etanercept (30 mg/kg), diluted in PBS, were dosed intravenously prior to human and mouse TNF challenge. Two hours after the TNF challenge, mice were sacrificed and the peritoneal cavity lavaged with 3 mL of FACS buffer. An aliquot (0.3 mL) of lavage fluid was used to assess the number CD45+GR1+ cells by flow cytometry. Briefly, the cells were treated with ACK lysis buffer, washed and suspended in FACS buffer prior to staining with FITC anti-mouse CD45 and PE anti-mouse GR1. Data are shown as mean ± standard error of the number of CD45+GR-1+ cells. Statistical significance  was determined using one-way ANOVA with Dunnett’s multiple comparison test.All in vivo studies were reviewed by an internal Ethical Review Body (ERB) and conducted in accordance with the Animals (Scientific Procedures) Act 1986, EU Directive 2010/63/EU. Adult male Balb/c mice  received 4 mg of anti-collagen type II antibody cocktail intraperitoneally, which was followed 24 h later with 25 µg of lipopolysaccharide (LPS) intraperitoneally. Dosing with vehicle (30% cyclodextrin po bid) or UCB-9260 (150 mg/kg po bid) was initiated 6 h post-LPS administration. Ab501  was administered intravenously 6 h post-LPS administration followed by three further doses administered subcutaneously at 48-h intervals. Animals were scored for signs of arthritis and weighed daily. The following scoring system was used for signs of arthritis: 0-no arthritis, 1-wrist/ankle affected, 2-wrist/ankle and pad affected and 3-wrist/ankle, pad, and digits affected. Data shown as the mean ± standard error of arthritis clinical score for the treatment group. Statistical significance  was determined using one-way ANOVA with Dunnett’s multiple comparison test.Adult male Balb/c mice  Act 1986, EU Directive 2010/63/EU. Animals were kept under a light/dark cycle of 12/12 h and had access to food and water ad libitum.n = 5; vehicle n = 8; UCB-9260 10, 30, 100 and 300 mg/kg n = 8; Ab501 n = 3 mice, from Charles River UK) were dosed intravenously with 30 mg/kg of Ab501 (in-house anti-mouse TNF antibody) diluted in PBS 1 h prior to zymosan challenge. Vehicle (30% cyclodextrin) or UCB-9260 at 10, 30, 100 and 300 mg/kg was dosed orally at the time of zymosan challenge. Zymosan (R&D Systems) was administered by intraperitoneal injection at a concentration of 1 µg per mouse in PBS (100 µL). One hour post oral administration of UCB-9260, mice were bled via the tail to analyse exposure levels of the compound. Four hours after the zymosan challenge, mice were sacrificed and the peritoneal cavity lavaged with 3 mL of FACS buffer. An aliquot (0.3 mL) of lavage fluid was used to assess the number of CD45+GR1+ cells by flow cytometry. Briefly, the cells were treated with ACK lysis buffer, washed and suspended in FACS buffer prior to staining with FITC anti-mouse CD45 and PE anti-mouse GR1 (BD Biosciences). Data are shown as mean ± standard error of the number of CD45+GR-1+ cells. Statistical significance  was determined using one-way ANOVA with Dunnett’s multiple comparison test.Adult male Balb/c mice (aged 6–8 weeks, PBS Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Reporting Summary