Yet, the direct role TNFR2-specific activation plays in the absence of XIAP is unknown. We found TNFR2-specific activation leads to cell death in xiap−/− myeloid cells, particularly in the absence of the RING domain. RIPK1 kinase activity downstream of TNFR2 resulted in a TNF/TNFR1 cell death, independent of necroptosis. TNFR2-specific activation leads to a similar inflammatory NF-kB driven transcriptional profile as TNFR1 activation with the exception of upregulation of NLRP3 and caspase-11. Activation and upregulation of the canonical inflammasome upon loss of XIAP was mediated by RIPK1 kinase activity and ROS production. While both the inhibition of RIPK1 kinase activity and ROS production reduced cell death, as well as release of IL-1β, the release of IL-18 was not reduced to basal levels. This study supports targeting TNFR2 specifically to reduce IL-18 release in XLP-2 patients and to reduce priming of the inflammasome components.The pediatric immune deficiency X-linked proliferative disease-2 (XLP-2) is a unique disease, with patients presenting with either hemophagocytic lymphohistiocytosis (HLH) or intestinal bowel disease (IBD). Interestingly, XLP-2 patients display high levels of IL-18 in the serum even while in stable condition, presumably through spontaneous inflammasome activation. Recent data suggests that LPS stimulation can trigger inflammasome activation through a TNFR2/TNF/TNFR1 mediated loop in  Soluble and membrane TNF can bind and activate TNF receptor 1 (TNFR1) while only the membrane bound form triggers TNFR2 activation2. The outcome of TNF/TNFR1 signaling can range from production of other cytokines, proliferation, survival and differentiation. While activation of TNFR1 does not normally lead to cell death, the capacity for TNFR1 to induce apoptosis or necroptosis is swayed by the ubiquitylation and phosphorylation of RIPK1 and the activation of pro-survival signals mediated by NF-κB and MAPK pathways5. Activation of complex II is blocked by cFLIP, which is transcribed upon TNF/TNFR1 activation, preventing caspase-8 activity. In the absence or inhibition of caspase-8 activity, necroptosis ensues through RIPK1 kinase activity, RIPK3 and MLKL necrosome activity7. Phosphorylation of MLKL causes a conformational change, allowing for pore formation and the release of intracellular components, as well as damage-associated molecular patterns (DAMPs)8.Full length tumor necrosis factor (TNF) is a membrane bound protein, where the extracellular domain can be cleaved by TNF converting enzyme (TACE) to release a soluble form9 a complex is formed that consists of TRAF2, cIAP1, cIAP2 and TRAF310. The activation leads to the degradation of cIAP1 and TRAF2, and signals through the non-canonical NF-κB pathway11. Due to the absence of a death domain, TNFR2 is considered to be mainly involved in survival and maturation of immune cells. Yet, previous data suggest that in some tumor cell lines, TNFR2 can regulate cell death by the loss of cIAP1 and TRAF2, causing production of TNF and thereby TNFR1 activation12. In addition, immortalized macrophages died in response to TNFR2 via necroptosis when caspase activity was inhibited14.The expression of TNFR2 on cells of the immune system and endothelial cells is highly regulated. Upon binding of membrane-bound TNF to TNFR217. The stimulation of TLRs upregulate inflammasome components such as NLRP3, thus priming the cell19. A secondary stimulus is required to activate the inflammasome in vitro. When caspase-1 is cleaved and activated, downstream targets IL-1β and gasdermin D are cleaved. Cleaved gasdermin D forms a pore like structure that facilitates the release of cleaved IL-1β, IL-18 and the osmotic lysis of the cell21. In response to TLR stimulation, xiap−/− macrophages showed increased inflammasome activation compared to wildtype through the activation of TNFR2 and subsequent degradation of cIAP1/TRAF222.In response to toll-like receptor (TLR) activation, XIAP deficient myeloid cells undergo TNF-dependent cell death, resulting in the activation of the inflammasome through RIPK3/caspase-8/caspase-122. Instead, we found that TNFR2 activation acts as a signal 1 for priming the inflammasome in primary macrophages independent of genotype, and the combination of XIAP loss and TNFR1 activation plays a role as signal 2 for activation. Interestingly, cell death was blocked by RIPK1 kinase inhibitor as well as reactive oxygen species (ROS) scavengers, and while many of the pro-inflammatory cytokines returned to baseline, IL-18 required genetic mutation of RIPK1 kinase to reduce to baseline levels. Thus, our work separates a key cytokine implicated in the etiology of XIAP deficient patients  from cell death. Taken together, we discovered a novel role of XIAP to inhibit TNFR2 induced inflammatory pyroptosis, adding additional complexity to treat XLP-2 patients.We sought to understand what function TNFR2 played in the absence of XIAP when normally it would not be expected to cause cell death in primary macrophages. Specific TNFR2 stimulation alone in XIAP deficient macrophages resulted in cell death due to the lack of E3 ligase activity. RIPK1 kinase activity was required for both the TNF production from TNFR2 stimulation, as well as the TNFR1 mediated cell death. Surprisingly, neither necroptosis nor apoptosis occurred in comparison to previously published results23  from xiap/−− bone marrow   showed TNFR1 mediated cell death was induced by soluble TNF produced by XIAP deficient macrophages  Fig. . In agreath Fig. . Co-lossath Fig. . To deteges Fig. .Fig. 2TN22, we assayed for degradation of TRAF2 in response to TNC-TNF over time in wildtype and xiap−/− macrophages. Degradation of TRAF2 occurred by 4 h in both wildtype and xiap−/− macrophages  or ciap1LysMcreciap2−/− (ciap1LCciap2/−−) macrophages with TR1-TNF and/or TNC-TNF. Interestingly, despite sufficient loss of cIAP1 in ciap1LCciap2−/− and xiap/−−ciap1LC macrophages , a kinase inactive mutant28. Consistent with Necrostatin-1s, macrophages from xiap/−−ripk1KD/KD mice were insensitive to TNC-TNF induced cell death  was prolonged compared to wildtype macrophages. Prolonged phosphorylation of p65 was also detected in xiap−/−tnfr1−/− treated with TNC-TNF while xiap−/−tnfr2−/− macrophages did not show any activation of NF-κB and MAPK pathways, further confirming the specificity of TNC-TNF for TNFR2 and that the signaling changes are independent of TNFR1  and traf1 upregulated by TNC-TNF in our RNAseq analysis  and birc3 (cIAP2) is consistent in xiap−/− and xiap−/−tnfr1/−− macrophages . These results suggest that TNFR2 activation not only acts as a signal 1 for inflammasome priming but also induces the expression of cytokines and chemokines similar to TNFR1.Consistent with othersges Fig. . The logxiap−/−, xiap−/−tnfr1/−− and xiap−/−tnfr2−/− macrophages with TNC-TNF overnight and assayed the supernatant as well as the lysate for inflammasome activation. We found that indeed upon TNC-TNF stimulation, NLRP3 and caspase-11 were upregulated  with TNC-TNF. The loss of caspase-1 activation resulted in reduced levels of IL-1β, IL-18, and IL-6 in the supernatant of treated ges Fig. . Using xall Fig. .xiap−/− macrophages. However, in xiap−/−tnfr1−/− macrophages, no cleavage was detected  involved in cell death has been previously reported in cancer cell lines for FN14, CD40, TNFR2, and CD30, acting through a TNF/TNFR1 axis40. In response to NOD2 stimulation, both mutations in the BIR and RING domain led to a loss in NF-kB41. By contrast, our data shows that TNFR2 induced cell death occurs in the absence of E3 ligase activity of XIAP. Whether the loss of E3 ligase activity of XIAP relates to the ROS production is unknown. Further studies in identifying what substrate(s) of XIAP will prevent TNFR2 induced cell death would be beneficial in the treatment of XLP-2 patients.TNFR2-specific activation mediated NOD2 and RIPK2 upregulation, suggesting that TNF may act as a primer for the detection of intracellular pathogens. Indeed, a lack in inflammation has been identified in XLP-2 patients in response to MDP/NOD2 activation through loss of RIPK2 ubiquitylation by XIAP and cIAPs43. XLP-2 is associated with activated macrophages and lymphocytes and overexpression of pro-inflammatory cytokines including TNF during an HLH episode44. During viral infections, the increased TNF presence would trigger TNFR1 and TNFR2. Our study provides evidence that inhibition of cell death may not result in reduction of key inflammasome cytokines such as IL-18 in XLP-2 patients and that there is differential regulation of IL-1β in comparison to IL-18 as recently published45. Contrary to most situations, targeting TNFR2 with blocking antibodies can be of potential therapeutic interest in XIAP deficient patients to limit inflammation.Our findings are of therapeutic interest as XLP-2 patients have increased inflammasome related cytokines. XIAP deficient patients are characterized with multi-organ inflammation triggered by viral infections, as well as intestinal bowel diseaseXiap−/−, ciap1−/−, ciap2−/−, and ciap1LCciap2−/− mice were a kind gift from J. Silke from WEHI and were previously described47. These strains were embryo transferred and maintained in an SPF facility in Zurich. Tnfr1−/− and tnfr2−/− mice were a kind gift from A. Fontana and A. Aguzzi, respectively48, and were crossed to xiap−/− mice to generate xiap−/−tnfr1−/− and xiap−/−tnfr2−/− mice. Ripk1K45A/K45A (ripk1KD/KD) mice were a gift from GlaxoSmithKline and were crossed to xiap−/− mice to generate xiap−/−ripk1KD/KD mice. All mice used in this study were back-crossed to C57BL/6 mice. All animal experiments were performed at the University of Zurich under the ethical license 186/2015. Xiap−/− and xiap−/−nlrp3−/− mice were a kind gift from RA Marsh31. Xiap−/−tnf−/−, xiap−/−ripk3−/−, xiap−/−mlkl−/− and xiapΔRING mice were a kind gift from P. Jost and housed at the Technical University of Munich. Experiments were conducted in accordance with GSK policies on the care, welfare, and treatment of laboratory animals.6 cells/mL into the desired tissue culture plates . TNFR1-Fas and TNFR2-Fas expressing mouse fibroblasts were obtained from Anja Krippner–Heidenreich25 and were cultured in DMEM containing 1 g/L glucose, 1% (v/v) penicillin/streptomycin/glutamine and 10% FBS. HoxB8 progenitor cells were cultured in RPMI 1640 media with 10% (v/v) heat-inactivated FBS, 1% (v/v) penicillin/streptavidin, 7% (v/v) SCF from CHO/SCF conditioned medium, and 0.1 µM 4-hydroxytamoxifen 17. To differentiate granulocytes from the HoxB8 progenitors, cells were washed twice with PBS and re-suspended at a concentration of 2.5 × 104 cells/mL in media without 4-OHT and differentiated for 5 days.To generate bone marrow-derived macrophages (BMDMs), bone marrow was obtained from the tibia and femur of 6–12-week-old mice. Cells were cultured on petri dishes for 5 days in Dulbecco’s modified Eagle medium  containing 1 g/L glucose, 1% (v/v) penicillin/streptomycin/glutamine (Gibco), 10% FBS (SeraGlobe) and supplemented with 20% (v/v) L929 mouse fibroblast conditioned medium. On day 5, cells were harvested and seeded at 1 × 1049. Endotoxin levels were tested and removed . TNF variants were used at 100 ng/mL. Inhibitors were used at the following concentrations: Birinapant , Compound A , multimeric TNF  Necrostatin-1s , Q-VD-OPH , ZVAD-fmk , VX-765 , BHA , Nigericin  and ATP .To produce the agonistic TNFR2 ligands, pCR3 Fc-Flag-TNFR2-specific nonameric murine TNF variant (TNC-TNF), trimeric pCR3 Flag-TNC-TNF or pCR3 Fc-Flag human TNFR1 (TNFR1-TNF), were transfected into 293t cells and purified as previous describedThe following antibodies were used for flow cytometry: CD11b-PeCy7 , F4/80 , and fixable viability dye (eBioscience). The following antibodies were used for western blotting: phospho-p65, NF-κB2, phospho-ERK, phospho-JNK, phospho-p38, total ERK, total JNK, and total p38 from Cell Signaling Technologies. Total p65 was purchased from Santa Cruz. TRAF2, gasdermin D and caspase-11 were purchased from Abcam, caspase-1 from Adipogen and IL-1β from RnD. cIAP1 was purchased from Human Atlas. RIPK1 and XIAP was purchased from BD Biosciences. Secondary antibodies for western blotting such as donkey anti mouse/rabbit/rat IgG conjugated to HRP are from SouthernBiotec, the donkey anti goat IgG was purchased from Santa Cruz. The neutralizing antibody against TNF  was purchased from BioLegend.6 cells/mL. After treatment, cells were trypsinized, washed once and re-suspended in HBSS containing 1 µg/mL propidium iodide (PI) or stained with fixable viability dye (eBioscience). Cells were then assessed for cell death by flow cytometry on a FACS Canto II. Data were analyzed by FlowJo software, version 10.2. Alternatively, cells were directly incubated in the presence of 5 µg/ml propidium iodide and assessed for viability by acquiring both phase contrast and red fluorescence images at 2 h intervals at ×10 magnification over 24 h using the IncuCyte. Confluency and PI fluorescence were measured and analyzed using the IncuCyte Zoom Software (Version 2016A).Cells were seeded at a density of 1 × 10+F4/80+ cells were separated using a FACSMelody 3 L machine.To isolate primary macrophages from mouse bone marrow, CD11bCell lysates were treated and lysed in DISC lysis buffer  glycerol, 1% (v/v) Triton X-100, with protease and phosphatase inhibitors) and incubated with 0.5 mM DEVD-AMC. Results were normalized to the protein concentration, that was calculated by BCA (Pierce) according to manufacturer’s instructions.Multiplex cytokine analysis  was performed according to the manufacturer’s instructions and assayed on a Bio-Rad Bioplex machine.Supernatant was collected when indicated and precipitated using 4% trichloroacetic acid (TCA) before being pelleted in acetone, boiled and run like lysates described in the following. Cells were lysed using DISC lysis buffer  glycerol, 1% (v/v) Triton X-100, with protease and phosphatase inhibitors). The insoluble fraction of the lysate was pelleted by centrifugation and removed. Lysates were boiled and run on 4–12% Bis-Tris Gel NuPAGE using MOPS buffer (Invitrogen). Proteins were then transferred onto PVDF-membrane  using the Trans-Blot® Turbo™ Transfer System (Bio Rad) or the Pierce™ Power Blotter (Thermo Scientific), both according to the manufacturer’s instruction. After blocking with PBST containing 5% (w/v) skim milk, membranes were incubated with the indicated primary antibody in either PBST containing 5% skim milk or 5% (w/v) BSA  for at least 1 h at RT or overnight at 4 °C. Protein level expression was acquired using WesternBright ECL (Advansta) and Amersham Hyperfilm ECL .RNA was isolated using GENEzol Reagent (Geneaid) according to the manufacturer’s instructions. cDNA was produced using MultiScribe™ Reverse Transcriptase and SYBR Green qPCR master mix (Thermo Fisher Scientific) was used for running the qPCR. Melting curves showed that single products were formed. The following primers were used: TNF ; B2M . Relative standard curve analysis was performed using the housekeeping gene B2M and unstimulated samples were used as a calibrator for fold-change.https://github.com/csoneson/ARMOR), including read mapping against the mouse reference genome (Ensembl_GRCm38.90) using STAR50 and sorting/indexing with samtools51. Isoform-level expression estimation using salmon52 and gene-level differential expression (DE) analysis was performed using edgeR53 with separate contrasts for each genotype (treated with TNC-TNF vs. untreated). Differential expression t–statistics used in the geneset analyses were computed using limma-voom54.Cells were stimulated with 100 ng/mL of TNC-TNF for 2 h and RNA was extracted using GENEzol Reagent (Geneaid) and cleaned using PureLink RNA Mini Kit (Invitrogen) according to the manufacturer’s instructions. Libraries were prepared and sequenced at the Functional Genomics Center Zurich . RNA-Seq data was processed through a standard workflow  and Prism 7 (GraphPad Software). Significance between genotypes and treatments was assessed by Student-Supplemental Figure 1Supplemental Figure 2Supplemental Figure 3Supplemental Figure 4Supplemental Figure 5Supplemental Figure 6Supplementary figure legendsSupplemental Dataset 1Supplemental Dataset 2


Two anti-TNF-α VNAR formats, a tandem multivalent trimer, D1-BA11-C4 and an Fc-fused quadrivalent D1-Fc-C4 (Quad-X™) construct were tested in a clinically relevant, preclinical mouse efficacy model of polyarthritis (Tg197) and compared to the commercial anti-TNF-α “best in class” therapy, Adalimumab (Humira®). Both VNAR formats bind and neutralize TNF-α through an epitope that appears to be different from those recognized by other anti-TNF biologics used clinically. All doses of Quad-X™, from 0.5 to 30 mg/kg, significantly blocked the development of polyarthritis. At 0.5 mg/kg Quad-X™, the arthritis score was improved by 76% and the histopathology score by 63%. At 3 mg/kg Quad-X™, control of disease was almost complete at 90% (arthritis) and 88% (histopathology). In marked contrast, 1 mg/kg Humira® saw profound disease breakthrough with scores of 39 and 16% respectively, increasing to a respectable 82 and 86% inhibition at 10 mg/kg Humira®. We have previously reported the superior potency of anti-TNF-α VNARs in vitro and in these studies translate this superiority into an in vivo setting and demonstrate the potential of VNAR formats to meet the requirements of next-generation anti-TNF-α therapies.Tumor necrosis factor-alpha (TNF-α), an established pro-inflammatory cytokine plays a central role in the induction and progression of several chronic inflammatory and autoimmune diseases. Targeting TNF-α as a treatment modality has shown tremendous success, however there are several limitations associated with the current anti-TNF-α biologic drugs including: immunogenicity, life-threatening infections, resistance to treatment, complexity of manufacture and cost of treatment. Here, we report the  The monoclonal antibody, Adalimumab (Humira®) is the most widely used and commercially successful TNF-α antagonist monoclonal antibody  is a pleiotropic cytokine with beneficial functions in immune regulation and host defense, but when expressed at high levels, is also implicated in a number of human diseases. TNF-α is a critical mediator of the autoimmune process, playing a key role in several inflammatory diseases ranging from rheumatoid arthritis, inflammatory bowel diseases, psoriasis, and uveitis. Rheumatoid arthritis (RA) is a common, chronic inflammatory disorder that causes progressive articular destruction and is associated with comorbidities in vascular, metabolic, bone and even the psychological well-being of sufferers , 2. Affeantibody –7. Howevantibody –16. Somevia a more traditional route using IgG (mouse or human) Fc domains (The Variable New Antigen Receptors (VNARs) are the smallest (11 kDa) naturally occurring independent binding domains in the vertebrate kingdom –19. They domains –26. Thesin vitro activity  previously reported as having potent activity  retains via the D1 and C4 domains molecularly fused with a validated, clinical-study ready, serum half-life extension humanized VNAR (soloMER™) domain, BA11 (NDure™) targeting serum albumin (4Ser)4 linkers and contain a C-terminally fused 6x Histidine tag to facilitate purification. The Quad-X™ construct (4Ser)2 fusing the first anti-hTNF-α VNAR domain (D1) to the hinge of a wild-type human IgG1 Fc region, and a longer (Gly4Ser)4 linker fused to the C-terminal of an Fc CH-3 linking to a second anti-hTNF-α VNAR domain (C4). Purification of this construct is via the human IgG1 Fc, using Protein-A affinity chromatography. Both constructs were expressed in suspension-adapted CHO K1 cells.The rational design and reformatting of the two anti-hTNF-α VNAR test articles (D1-BA11-C4 and D1-Fc-C4 (Quad-X™) has been described in a recent publication . D1-BA11 albumin . The VNAonstruct , a 105 kELISA plates were coated with human, mouse, rat, dog, cynomolgus, rabbit, pig TNF-α and human TNF-β at 1 μg/ml concentration in PBS. Plates were incubated for 1 h at 37°C, then blocked with 2% Milk-PBS. Plates were washed 3x with PBS and 0.001% (v/v) Tween-PBS (PBST). Anti-TNF-α VNAR D1 and C4, VHH TNF30 and Humira® were added to designated ELISA wells at a 5 μg/ml top concentration with 2-fold serial dilution. Plates were incubated at room temperature for 1 h, washed as described above. TNF-α binding activity was detected using appropriate secondary antibodies; VNARs D1 and C4, and VHH TNF30 were detected using anti-poly-histidine-HRP, and Humira® was detected using an HRP-conjugated anti-human IgG1 Fc antibody.in vitro TNF-α neutralization assay in a mouse fibrosarcoma (L929) cell line was conducted as described in Ubah et al. , mouse TNFα (0.05 ng/ml), cynomolgus TNF-α (0.15 ng/ml), pig TNF-α (100 ng/ml), canine TNF-α (0.5 ng/ml), and human TNF-β (0.5 ng/ml). A neutralization screen was not performed when all the test anti-TNF-α domains  failed to bind a target species TNF-α, e.g., rat and rabbit TNF-α.An h et al. . LD50 doAggregation propensity of the reformatted VNAR test articles was analyzed using analytical size-exclusion chromatography (SEC) and an Agilent 1200 series HPLC system with a ZORBAX GF 250 9.4 × 250 mm 4 μm column containing phosphate buffered saline pH 7.4 as the mobile phase. VNAR D1-BA11-C4 and Quad-X™ were loaded onto columns at 6 mg/ml from stock concentrations of 40 mg/ml. Purity of the test VNAR protein samples was also assessed by SDS-PAGE electrophoresis of the reduced VNAR protein samples in a MES buffer system (Invitrogen).C57BL/6 and Tg197 mice were used for the maximum tolerated dose (MTD) and polyarthritis prevention studies, respectively and were provided by Biomedcode Hellas SA, Greece. All reported animal studies were conducted at the Biomedcode SA test facility. All procedures conformed to the Presidential Decree No 56/2013 Government Gazette No A' 106 applicable in Greece (EEC Directive 2010/63/ECC) and were approved by the directorate of Agricultural and Veterinary Policy (DAVP) of the Attica Region. Approved license Protocol No. 3798/3-07-2017.In vitro functional activity of these expressed proteins was confirmed  were produced recombinantly in suspension-adapted CHO K1 cells by Evitria AG, Switzerland. onfirmed , and “saAnti-hTNF-α Quad-X™ was evaluated for safety and tolerability in healthy mice. A pool of 6-week-old C57BL/6 mice were allocated to 4 groups (G1-G4), each consisting of 10 gender and age matched mice, based on an equal mean group body weight distribution. On days 0 and 3, animals received subcutaneous administration of either vehicle , 3 mg/kg (G2), 10 mg/kg (G3), or 30 mg/kg (G4) of anti-hTNF-α Quad-X™. Treated animals were monitored daily for 7 consecutive days to assess any possible adverse effects by recording animal viability, overall appearance, body function, behavior, and body weight. Water and food intake were also assessed at 2 different time points during the study. At the end of the clinical monitoring period, mice were sacrificed, and gross necropsy determined. The heart, liver, spleen and kidneys from all experimental animals were collected and weighed to determine organ to body weight ratios.The transgenic Tg197 mouse model was used to investigate the efficacy of the anti-hTNF-α VNAR D1-BA11-C4 and Quad-X™ formats and compared to the positive control article Humira®, as described in Keffer et al. . The Tg1This study was conducted using two independent experiments. In the first, 5 groups of 8 sex and age matched mice (4♂/4♀) were assigned to one of five treatment regimens. PBS treatment, three different dosing regimens of VNAR Quad-X™ and one dosing regimen of Humira®. Doses of 3, 10, 30 mg/kg (Quad-X™), and 10 mg/kg (Humira®) were administered subcutaneously twice weekly, starting at week 3 after birth until 10 weeks of age.In the second study, 7 groups of 8 mice each (4♂/4♀) were assigned to one of seven treatments regimens of twice weekly subcutaneous administration. VNAR Quad-X™ was dosed at 0.5, 1, and 3 mg/kg; VNAR D1-BA11-C4 at 30 mg/kg; and Humira® at 1 and 3 mg/kg.in vivo arthritis scoring system  were sacrificed prior to the first dose administration. An g system  was applAnkle joints were fixed in 4% aqueous formaldehyde solution overnight at room temperature, demineralised in EDTA decalcification solution (13% EDTA in 0.1 M sodium phosphate buffer) at room temperature for 30 days and placed in PBS at 4°C until required for further processing. Samples were paraffin embedded in the sagittal plane, and paraffin blocks sectioned. Blocks were stained with Haematoxylin and Eosin (H&E) staining and evaluated by light microscopy for histopathological hallmarks of arthritis in accordance with a standard histopathology scoring system , 30. TheIn the MTD study, during the necropsy procedure and organ collection weighing, the right kidneys of two mice (one in 10 mg/kg Quad-X™ and the other in the 30 mg/kg Quad-X™ treatment group) were not recovered, therefore no weight of the right kidney was recorded. There were no other study amendments or deviations, and no deaths were reported in both the MTD and Efficacy Preclinical studies.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Statistical analyses were performed using GraphPad Prism 6 software. Statistical tests applied to each data set are specified in each figure legend. Statistically significant differences were noted as: in vivo use. Both VNAR D1-BA11-C4 and Quad-X™ eluted as single peaks and showed no signs of aggregation or impurities. In addition, SDS-PAGE analysis of 5 μg VNAR D1-BA11-C4 and Quad-X™ confirmed the SEC analysis with no evidence of degradation or impurities observed  showed positive reactivity to only human, dog and cynomolgus TNF-α. There was no binding seen with any of these VNAR domains to rat, mouse, rabbit, pig TNF-α or closely related human TNF-β . These bClinically available anti-TNF-α therapies including Infliximab (Remicade®) a mouse-human IgG1-kappa, Certolizumab (Cimzia®) a PEGylated Fab fragment of a humanized mAb, Golimumab (Simponi®) a fully human IgG1-kappa mAb, and Etanercept (Enbrel®) a fusion protein of human TNF receptor 2, all show restricted TNF-species cross-reactivity with recognition profiles different from those determined for the anti-TNF-α VNARs. Infliximab binds and neutralizes only human TNF-α and chimpanzee; Etanercept binds and neutralizes human, cynomolgus, mouse TNF-α and human TNF-β; Certolizumab binds and neutralizes human and cynomolgus TNF-α, and Golimumab binds and neutralizes human, dog, cynomolgus, and rabbit TNF-α  32–34).–34.32–34VNAR multivalent tandem and VNAR-Fc constructs have previously been successfully administered in several animal models including non-human primates, with no drug-associated toxicities reported , 26. Howin vitro neutralizers of hTNF-α  which are potent (low pM) f hTNF-α  were tesf hTNF-α , 35, 36.In vivo arthritis and histopathology scores were inhibited by an average 88% and 86% respectively, across all anti-hTNF-α VNAR doses tested, with 0.5 mg/kg Quad-X™ inhibiting in vivo arthritis and histopathology scores at 76% (p = 0.0001) and 63% (p < 0.0002), 88% (p < 0.0001) and 85% (p < 0.0001) with 1 mg/kg Quad-X™, and 90% (p < 0.0001) and 88% (p < 0.0001) inhibition respectively with 3 mg/kg Quad-X™. Humira® inhibited arthritis and histopathology scores by an average of 84 and 76%, respectively at the 3 & 10 mg/kg dosing regimens, and the disease score inhibition at 1 mg/kg Humira® saw significant breakthrough of disease with values of only 39% (p = 0.923) and 16% (p = 0.999) for in vivo arthritis and histopathology scores, respectively. D1-BA11-C4 at 30 mg/kg dosing inhibited in vivo arthritis and histopathology scores by 90% (p < 0.0001) and 89% (p < 0.0001) respectively. The comparative examination of the arthritis inhibitory effects of Quad-X™ and Humira® at 1 mg/kg and 3 mg/kg dose level revealed that they were statistically undifferentiated in terms of their body weight gain measurement, but the in vivo arthritic and histopathological evaluations revealed that Quad-X™ displayed, with statistical significance, superior efficacy to Humira®. By, the end of study (week 10 of age) mean body weight for the 1 mg/kg Humira® treated mice group was 19.9 ± 0.9 g compared to 17.7 ± 1.3 g (PBS treated group) (p = 0.768). Mean body weight in the 0.5 mg/kg and 1 mg/kg treated groups were 22.5 ± 1.6 g (p = 0.093) and 23.9 ± 1.3 g (p = 0.017) respectively. The average body weight of a healthy 10-week-old mouse is ≈ 25 g  and 20 g . The study 3-week-old control mice, week 3 mean group body weight was 7.3 ± 0.4 g. When translated into a human setting the effect of different doses on mean mouse body weight has been shown to correlate with both a decrease in joint pain levels and an increase in energy levels seen for RA patients.After 14 doses administered twice weekly for a period of 7 weeks, the results of the first study showed that all doses of Quad-X™ and D1-BA11-C4 at 30 mg/kg (second study) blocked the development of disease and sustained increase in body weight , 4, 5. IA lower incidence of histopathology lesions at week 10 was observed in all Quad-X™ and D1-BA11-C4 treatment arms. These outcomes were statistically differentiated from the 3-week old control mice as well as the untreated (PBS treated) mice. Evidence of cell infiltration, bone erosion and cartilage degradation seen in 3-week-old and PBS-treated mice groups was present in 1 and 3 mg/kg Humira® treated mice .in vivo efficacy study was based on the amount of drug in milligram per body weight of animal in kilograms (mg/kg), therefore did not correct for molecular size differences seen between formats . In previously published in vitro efficacy experiments, an equimolar dosing regimen was adopted in an effort to correct for molecular size differences, with the VNAR Quad-X™ demonstrating 10x superior hTNF-α neutralizing potency when compared to Humira®  or 40 kDa (trimer) using NDure™  when compared with conventional anti-TNF-α biologics in the clinic , we speculated that these anti-TNF-α domains (VNAR D1 and C4), forming the backbone of the D1-BA11-C4 and D1-Fc-C4 Quad-X™ constructs, may be interacting with unique and previously unseen “recessed” epitopes on the TNF-α molecule, in stark contrast to the epitopes recognized by the planar paratopes of the commercially available biologics including even VHH , 44–47. Ⓡ (and here the Quad X™) are unable to bind and neutralize, with any therapeutically relevant potency, rodent TNF-α. It is only with the recent development of new transgenic disease models  carrying a 3'-modified UTR human TNF-α gene construct allows deregulated soluble human TNF-α gene expression er birth , 51, 52.ar risks –56. Whilar risks –59. Whilar risks .in vivo efficacy of Quad-X™ and D1-BA11-C4 in a Tg197 transgenic human TNF-α polyarthritis disease model reinforces the validity of our previously reported potent in vitro neutralization data which saw an order of magnitude improvement over Humira®  and were approved by the directorate of Agricultural and Veterinary Policy (DAVP) of the Attica Region. Approved license Protocol No. 3798/3-07-2017.in-silico design and preliminary expression of all anti-TNF VNAR constructs described in this publication. CB led the science team at Elasmogen Limited and co-supervised this project alongside AP. Also, CB and AP reviewed the manuscript.OU, scientific lead on this paper, conducted the majority of the scientific work and wrote the manuscript. JS assisted in the rational Authors affiliations are clearly stated including those affiliated with the biotechnology company, Elasmogen Limited.


E. coli, mostly in the form of inclusion bodies. The solubilization and refolding of GST-hD2 inclusion bodies was achieved with the addition of 4 M urea and subsequent dialysis to recover the fusion protein in soluble form. Then the soluble GST-hD2 was purified by affinity chromatography through immobilized glutathione. The GST pull-down experiment showed a positive interaction between GST-hD2 and TNF-α protein. Moreover, the results of an MTT assay showed that the purified GST-hD2 has TNF-α neutralizing activity (Kd of 1.03 nM) and hence hD2 has the potential to be developed into a therapeutic agent. However, more investigation is needed to elucidate the potential of in-vivo TNF-α neutralizing activity of hD2 in comparison to other anti-TNF-α antibodies.The pro-inflammatory cytokine, TNF-α, which plays a major role in the development and persistence of diseases such as Crohn’s disease, psoriasis, psoriatic arthritis, and rheumatoid arthritis, is the basis for the use of anti-TNF-α therapies. The neutralization of TNF-α or blockage of its binding to the corresponding receptor has mainly served as a therapeutic strategy against some inflammatory diseases. This study aimed to investigate the production of a humanized single chain antibody (scFv) against TNF-α. Therefore, a murine monoclonal antibody, D2 mAb, was selected for humanizing by the complementarity determining region (CDR)-grafting method. Briefly, the replacement of the CDRs from D2 mAb with the specific human single chain scaffold led to the production of a novel humanized single chain fragment variable mAb against human TNF-α (hD2). The subsequent cloning of hD2 into a suitable expression vector, pGEX-6P-1, resulted in the expression of a 52-kDa GST-fusion protein in  Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine, and its abnormal over-expression causes many inflammatory or autoimmune diseases . TNF-α nVarious strategies including blocking of TNF-α mRNA synthesis, inhibition of its post-translational processing, and blocking of the activation of TNF-α receptors have been used to inhibit TNF-α biosynthesis . The utiHowever, a number of studies have reported some restrictions in the therapeutic uses of TNF-α inhibitors, including low stability, high production expenses, considerable treatment costs, and undesired side effects -7, as weH and VL are joined together with a short flexible peptide linker  antibodies in which the genes encoding for Ve linker . Occasioe linker . In compe linker .The need for new TNF-α-blocking agent(s) with higher binding affinity and better neutralizing activity is always sought, because it provides the possibility of using antibodies at lower doses, with less immunogenicity, and for a wider range of patients afflicted with TNF-α-related diseases.The aim of the present study is to develop a humanized single chain antibody with anti-TNF-α activity suitable for the development of new therapeutic agents useful in inflammatory diseases. ReagentsBamHI, and EcoRI were provided from Fermentas (Lituania).Acrylamide, N,N′-methylene-bis-acrylamide, 2-mercaptoethanol, and Dimethyl sulfoxide (DMSO) were prepared from Merck (Germany). Tris-HCL, NaCl, yeast extract, Peptone, Isopropyl β-D-Thiogalactopyranoside (IPTG), Agar, Triton X-10, Triton X-100, urea, N,N, N′,N′-tetramethylethylenediamine (TEMED), Dithiothreitol (DTT), phenylmethylsulfonyl fluoride (PMSF), and trypsin were purchased from AppliChem (Germany). Ni-Sepharose 4B was obtained from GE Healthcare Life Sciences (Sweden). Agarose was from Invitrogen (UK). Plasmid mini extraction kit was purchased from Roche (Germany). -2,5-diphenyltetrazolium bromide (MTT), fetal bovine serum (FBS), and RPMI medium was purchased from Sigma (USA). All chemicals and reagents were of molecular biology grade.PCR master mix, Selection of anti-TNF alpha monoclonal antibodyTo humanize an efficient murine anti-TNF-α antibody, the selection was carried out based on searching through the known antibodies  with higPredicting antibody complementarity determining regionsThe IMGT/V-QUEST program (version 3.3.1) was used to predict the complementarity determining regions (CDRs) on both heavy and light chains of the selected antibody . In addiDesign and Synthesis of human anti-TNF-α scFv encoding geneE. coli genes (BamHI and EcoRI) were introduced at 5′- and 3′-UTR of the fragment, respectively. Sequences were checked to ensure the lack of formation of stem–loop structures and internal sequence similarities. The DNA sequence design was performed using BioEdit software . A humanized version of the selected anti-TNF-α scFv  was desili genes  and takiE. coli DH5α for amplification. The amplified DNA was then used to verify the accuracy of the designed scFv encoding gene by PCR, restriction enzyme digestion pattern, and sequencing by Bioneer, South Korea.The DNA sequence was synthesized and cloned into pGEX-6P-1 vector by Eurofins Genomics, Germany (http://www.eurofinsgenomics.eu/en/). The received construct was transformed into Expression of the synthesized human anti-TNF-α scFv in E. coliE. coli BL21 (DE3) cells for the expression of scFv as a GST-fusion protein. The transformants were grown on LB agar supplemented with ampicillin  at 37 °C overnight and agitated at 180 rpm. The cultures were then diluted 1:100 with fresh LB medium plus antibiotic, and grown to OD600 value of 0.6 at 37 °C. The expression of the fusion protein was induced by the addition of IPTG at a concentration of 0.5 mM at 20 °C. The cells were harvested at intervals of 1, 3, 6, and 24 h after induction. The harvested cells were re-suspended in 10 mM Tris-HCl (pH 8.0) and lysed by sonication. The cellular debris was pelleted by centrifugation at 12000 g for 15 min. Samples from both supernatant and pellet were analyzed by electrophoresis on a 12% SDS-PAGE under reducing conditions after staining with coomassie brilliant blue  were re-suspended in the extraction buffer  containing 1% Triton X-100 and 1 M urea per gram cell wet weight and incubated at room temperature for 5 min. The cell lysate was centrifuged again at the above-mentioned condition, and the pellet was re-suspended in 10 mL wash buffer. Subsequently, it was centrifuged at 4 °C for 30 minat 15,000 15,000 g , 18. Affinity purification of GST-hD2Purification of the refolded fusion protein was achieved using Glutathione Sepharose 4B bulk matrix  according to the manufacturer′s instructions .Pull down assayg for 2 min at 4 °C, and the supernatant was discarded. Then, beads were washed four times with 1 mL of ice-cold GST wash buffer consist of 50 mM Tris (pH 8.0), 100 mM NaCl and 0.1% 2-mercaptoethanol and then were centrifuged as before for 1 min at 4 °C and the supernatant was discarded. Samples of beads from each step were collected and analyzed by SDS-PAGE to determine the association between the fusion protein and TNF-α (The TNF-α (the probe) and GST-hD2 fusion proteins (the target) are incubated together with glutathione-agarose beads and then the complex (TNF-α-GST-hD2) was recovered from the beads and analyzed using SDS-PAGE experiment. Briefly, 25 µg of the fusion protein was incubated with 25 µg of TNF-α and 50 µL of a 50% slurry of glutathione-agarose beads previously equilibrated with equilibration buffer (50 mM Tris (pH 8.0), 100 mM NaCl, 1.4 mM PMSF, 0.1% 2-mercaptoethanol, and 1% Triton X100) for 2 h at 4 °C, while mixing by inverting at cold room. The mixture was centrifuged at 13,000 nd TNF-α . MTT assay5 cells mL-1 and incubated for 20 h. Also several dilutions of GST- hD2 were prepared in medium containing actinomycin D (10 µg mL-1) and TNF-α (2 ng mL-1) and incubated at 37 °C for 2 h. After removing of the supernatants of the cultured L929 cells, different concentrations of GST- hD2 were added to the wells. Then the cells were incubated at 37 °C for 24 h, and the supernatants were removed again. To each well, MTT at 5 mg mL-1 concentration was added and incubation was continued at room temperature for 4 h. After removing supernatant, the solubilization buffer (Sorensen buffer 12.5% and DMSO 87.5%) was added to each well with shaking for 40 min at 25 °C. The plate was read in ELISA Reader for measuring OD in 570 nm (background was read at 630 nm wavelength)  were seeded in 96-well plates at 1×10elength) . Blank cSelection of anti-TNF-α mAb and determining its CDR regionsAs the aim of the current study was to prepare a humanized scFv antibody against TNF-α, the candidate antibodies were limited to those which were not originated from human. On the other hand, the strategy adopted in this work was to make a decision based on TNF-α neutralizing activity for the antibodies observed in biological assay. Design, Synthesis and Expression of hD2 encoding gene4Ser)3 linker, and variable light chain, respectively as expected.In order to construct the humanized D2 scFv, CDR grafting technique was used. To this end the sequences of CDRs from the mD2 mAb were used to replace the CDRs of the specific human single chain framework represented by Tomlinson I+J sequence . The desE. coli BL21 (DE3) cells was produced recombinant bacterial colonies which were resistant to ampicillin and capable to express GST-hD2 fusion protein. The SDS-The transformation of pGEXhD2 into Pull down assayThe result of pull down experiment was shown in TNF-α cytotoxicity neutralizing activity of purified hD2 scFv-1 of hD2 scFv antibody, in the presence of TNF-α (2 ng mL-1) for 24 h. The results showed that the L929 cells death was completely occurred at 2 ng mL-1 of TNF-α. Twenty seven micrograms per milliliter of hD2 could completely neutralize TNF-α (2 ng mL-1) mediated cytotoxicity in L929 cells. The results showed that anti-TNF-α hD2 scFv protects cells form TNF-α cytotoxicity in a dose-dependent manner  to develop a humanized version of mD2 scFv (named hD2) with the sequence shown in n bodies .In this work, salts and unwanted proteins of the host cell were eliminated by a two-step wash procedure. Removing these impurities and obtaining the IBs with high recovery and purity is required because impurities interfere with the refolding and significantly affect the yield and purity of the process. We used 1% Triton X-100 to solubilize the components of the bacterial cell wall that contaminate the inclusion-body preparation, and we used 1 M urea to remove any residual cell debris. In addition, we found that this method provided the most effective means for the refolding of aggregated proteins when the refolding procedure was carried out with a gradually decreasing concentration of urea in the extraction buffer. Thus, optimization of the early stages of the downstream process will impact the yield of the overall process and the purity of the final product. in-vivo TNF-α neutralizing activity of hD2 compared to other anti-TNF-α antibodies.Glutathione-S-transferase (GST) fusion proteins have a range of applications in the detection, isolation and purification of recombinant proteins. A GST pull-down experiment also is used to confirm suspected interactions between a probe protein and a known protein. In the present study, the pull down assay showed the positive interaction between GST-hD2 and TNF-α, suggesting an affinity between the bait and predator proteins. Moreover, the results of an MTT assay showed that the purified GST-hD2 has TNF-α neutralizing activity and hence hD2 may be regarded as a promising antibody to be converted into a therapeutic agent. However, more investigation is needed to elucidate more precisely the potential of


Tumor necrosis factor-α (TNF-α) immunotherapy controls the progression of human cervical cancer. Here, we explored the detailed molecular mechanisms played by melatonin in human cervical cancer (HeLa cells) death in the presence of TNF-α injury, with a particular attention to the mitochondrial homeostasis.HeLa cells were incubated with TNFα and then cell death was determined via MTT assay, TUNEL staining, caspase ELISA assay and western blotting. Mitochondrial function was detected via analyzing mitochondrial membrane potential using JC-1 staining, mitochondrial oxidative stress using flow cytometry and mitochondrial apoptosis using western blotting.Our data exhibited that treatment with HeLa cells using melatonin in the presence of TNF-α further triggered cancer cell cellular death. Molecular investigation demonstrated that melatonin enhanced the caspase-9 mitochondrion death, repressed mitochondrial potential, increased ROS production, augmented mPTP opening rate and elevated cyt-c expression in the nucleus. Moreover, melatonin application further suppressed mitochondrial ATP generation via reducing the expression of mitochondrial respiratory complex. Mechanistically, melatonin augmented the response of HeLa cells to TNF-α-mediated cancer death via repressing mitophagy. TNF-α treatment activated mitophagy via elevating Parkin expression and excessive mitophagy blocked mitochondrial apoptosis, ultimately alleviating the lethal action of TNF-α on HeLa cell. However, melatonin supplementation could prevent TNF-α-mediated mitophagy activation via inhibiting Parkin in a CaMKII-dependent manner. Interestingly, reactivation of CaMKII abolished the melatonin-mediated mitophagy arrest and HeLa cell death.Overall, our data highlight that melatonin enhances TNF-α-induced human cervical cancer HeLa cells mitochondrial apoptosis via inactivating the CaMKII/Parkin/mitophagy axis. Human cervical cancer is the most frequent primary malignancy in woman’s uterus, which accounts for 90–95% of uterus neoplasms based on recent studies , 2. NowaSeveral tumor physiological activities are closely modulated by mitochondrion, such as energy production, precise control of ROS metabolism, calcium flux modification, tumor growth/division, cancer movement, and caspase-9-related programmed cell death (apoptosis) –13. PrevMelatonin has several beneficial effects on body physiological processes such as sleep disorders, liver lipid metabolism, and cardiac ischemia/reperfusion injury , 23–25. ® CCL-2™), were cultured under in F12 medium  containing 10% FBS at 37 °C with 5% CO2. In the present study, 10 ng/ml TNF-α for 12 h to mediate HeLa cell death based on a previous study [HeLa cells, purchased from American Type Culture Collection . Subsequently, the cells were cultured in the dark for 4 h, and DMSO was added to the medium. The OD of each well was observed at A490 nm via a spectrophotometer  . TUNEL aCells were plated on glass slides in a 6-well plate at a density of 1 × 106 cells per well. Subsequently, cells were fixed in ice-cold 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100, and blocked with 2% gelatine in PBS at room temperature . The celThe siRNA against Parkin was obtained from GenePharm . Meanwhile, transfection was performed using Lipofectamine 2000  following the manufacturer’s instructions . After 6Total protein was extracted by RIPA , and the protein concentration of each sample was detected with a bicinchoninic acid (BCA) kit . Deionized water was added to generate 30-µg protein samples for each lane. A 10% sodium dodecyl sulphate (SDS) separation gel and concentration gel were prepared. The following diluted primary antibodies were added to the membrane and incubated overnight: Complex III subunit core , complex II , complex IV subunit II , Parkin , p-CaMKII , CaMKII , ATG5 , Beclin1 , LC3II , Tom20  overnight at 4 °C. The membranes were washed three times with phosphate-buffered saline (PBS) (5 min each time), supplemented with horseradish peroxidase (HRP)-marked second antibody , oscillated and incubated at 37 °C for 1 h. After incubation, each membrane was washed three times with PBS (5 min for each time) and reacted with enhanced chemiluminescence (ECL) solution  at room temperature for 1 min; then, the extra liquor was removed, and the membranes were covered with preservative film . Each me2 staining for 25 min at 37 °C in the dark [2. The change in fluorescence intensity was measured by a fluorescence microscope according to the previous study. Then, the mPTP opening was measured [Mitochondrial membrane potential was measured with JC-1 assays . Cells were treated with 5 mM JC-1 and then cultured in the dark for 30 min at 37 °C. Subsequently, cold PBS was used to remove the free JC-1, and DAPI was used to stain the nucleus in the dark for 3 min at 37 °C. The mitochondrial membrane potential was observed under a digital microscope . In the mPTP opening assay, cells were cultured and then incubated with calcein-AM/CoClthe dark . Subsequmeasured .Cell suspensions were collected. The liquor  was collected, centrifuged for 2 min with the supernatant removed, supplemented with the ROS probe DCFDA, incubated at room temperature for 10 min, centrifuged, and washed with PBS . The celCellular glutathione (GSH), glutathione peroxidase (GPx) and SOD were measured via ELISA assay according to the manufacturer’s instructions. Cellular lactate production in the medium was measured via a lactate assay kit  according to a previous study. The cancer glucose uptake rate was detected via a glucose absorption assay kit  .The extracellular lactate was measured using the cell culture medium with lactate assay kit . Intracellular glucose was measured using cell lysates with glucose assay kit . ATP levels were measured using an ATP assay kit . The uptake of glucose, the production of lactate, and the levels ofATP were all measured according to the manufacturer’s instruction.All experimental data were analyzed using SPSS Statistics software 19.0 . Repeated measures analysis of variance (ANOVA) was used to compare the escape latency among groups. Other data were compared using one-way ANOVA. Data are presented as the mean ± SEM. A value of P < 0.05 was considered significant.To investigate the synergistic effect of melatonin in TNF-α-mediated HeLa cells damage, cell viability was firstly evaluated via an MTT assay. As shown in Fig. To examine the synergistic mechanism exerted by melatonin in the setting of TNF-α-triggered HeLa cells death, mitochondrial function and caspase-9 death signaling were evaluated . MitochoMitochondrial energy stress seems to be the early feature of mitochondrial death, which is also a decisive factor to modulate cancer death , 52. ConMitophagy seems to be a defensive mechanism to eliminate mitochondrial damage. Accordingly, we asked whether melatonin enhanced the vulnerability of HeLa cells to TNF-α-mediated mitochondrial apoptosis via inhibiting mitophagy. Based on this, we firstly observed the mitophagy activity under melatonin and TNF-α treatment. As shown in Fig. Next experiments were performed to analyze the underlying signal pathway modulating mitophagy under TNF-α treatment. At the molecular levels, Parkin is a classical mitophagy receptor and has been found to be associated with cancer progression , 54. WesFurthermore, we explored the upstream signals for the Parkin activation. According to a previous study, the Parkin could be activated via the calcium/calmodulin-dependent protein kinase II (CaMKII) , 56. In Based on our results, we confirmed that human cervical cancer HeLa cell death was triggered by TNF-α via apoptosis. Interestingly, melatonin co-treatment could further amplify TNF-α-initiated apoptosis with further exacerbating mitochondrial stress and launching the caspase-9 mitochondrial death signaling. Besides, we also found that cellular ATP depletion and mitochondrial bioenergetics stress were also modified by melatonin in the presence of TNF-α. Further, our data answered the molecular mechanism by which melatonin facilitated TNF-α-associated mitochondrial malfunction and HeLa cell death. Mitophagy, a mitochondrial protective system, was activated by TNF-α whereas melatonin treatment inhibited mitophagy activity, increasing the TNF-α therapeutic response. Mechanistically, melatonin inhibited CaMKII activation induced by TNF-α, leading to the downregulation of Parkin and mitophagy arrest. As far as we known, our study for the first time to affirm that melatonin could be used as an effective adjuvant to elevate the cancer-killing effects exerted by cytokine-based immunotherapy.Here, we demonstrated that melatonin could enhance TNF-α-based therapeutic efficiency via augmenting cellular apoptosis. Mechanistically, the therapeutic target of melatonin and TNF-α is mitochondrion. On one hand, melatonin triggered the mitochondria-related apoptosis signal. Besides, melatonin also mediated excessive ROS accumulation and elevated mPTP opening rate. Moreover, the pro-apoptotic factor cyt-c was liberated into the nucleus, resulting into the elevation of caspase-9/3 activities. In addition to the activation of mitochondrial death, melatonin supplementation was also connected with mitochondrial energy stress via reducing the levels of mitochondria respiratory complex. Through the above two reasons, melatonin enhanced the HeLa cell damage in response to TNF-α treatment. Thereby, we conclude that mitochondrion is the therapeutic target to intervene HeLa cells survival, providing basic research evidences for the clinical application for the use of melatonin as an effective tool to inhibit the cancer progression. Considering that melatonin is an endogenous indolamine, the combination of melatonin and immunotherapy would be useful in the tumor treatment in clinical practice.Mitophagy could consume the damaged mitochondria via lysosome, finally relieving mitochondrial stress and maintaining mitochondrial quality , 57. OneWe determined that CaMKII/Parkin pathways were the upstream signal controlled by TNF-α. Melatonin disturbed mitophagy through repressing the CaMKII/Parkin pathways. In the process of mitophagy, several factors have been linked to mitophagy activation such as Parkin, FUNDC1, BNIP3, Mfn2 and Drp1. FUNDC1- and BNIP3-related mitophagy are primarily found in the cardiac ischemia reperfusion , 18. MfnCollectively, we reported the synergistic effect of melatonin to augment cervical cancer apoptosis induced by TNF-α. Melatonin enhanced the therapeutic sensitivity of cervical cancer to TNF-α via targeting mitophagy. This finding offers a new insight into the crosstalk between CaMKII/Parkin/mitophagy axis and TNF-α resistance in cervical cancer.


Yet, its role in cancer progression is still a matter of debate. Here, we will (i) summarize key findings linking TNF to the promotion or inhibition of tumor progression, (ii) attempt to reconcile some of the contradictory findings, and (iii) describe the scientific rationale for improving the efficacy of immune checkpoint inhibitors via ligation of the TNFR1  in co-culture experiments. The authors showed that anti-PD-1 therapy slowed the progression of MC38Ova tumors in perforin-deficient animals. This observation suggests that anti-PD-1 treatment could boost CD8+ tumor-infiltrating T lymphocytes (TILs) to use perforin/granzyme-independent cytotoxic mechanisms to impede cancer progression. However, direct implication of TNF in tumor growth control is lacking and would require evaluation of this phenomenon upon anti-PD-1/anti-TNF combination therapy. A recent study showed that tumor cell death following TNF production by CD4+ TILs during ACT required co-treatment with chemotherapeutic agents in murine models of lymphoma as well as colorectal and mammary carcinoma  considerably improved the prognosis for metastatic melanoma patients with an overall survival of 58% at 3 years when treated with a combination of anti-PD-1 (Nivolumab), and anti-CTLA4 (Ipilimumab) . However+ TILs likely related to reduced AICD. Under these settings, anti-PD-1 therapy promoted the expression of TIM-3, a secondary immune checkpoint molecule at the surface of CD8+ TILs as well as PD-L1 on dendritic cells, and TILs , held at the Toulouse Oncopole (promoter: Claudius Regaud institute) funded by Bristol-Myers Squibb (BMS), and directed by Prof N. Meyer, oncodermatologist at the Toulouse cancer center. In TICIMEL, which started in December 2017, 30 patients with metastatic melanoma are for the first time simultaneously treated with TNF blocking antibodies (Infliximab or Certolizumab), and ICI (Nivolumab and Ipilimumab). The primary objective of TICIMEL is to assess the incidence of DLT, evaluated 12 weeks after treatment induction. The secondary objectives are to evaluate (i) the safety and tolerability, (ii) the progression-free survival, and (iii) the objective response rate. For the pharmacodynamic aspects, the systemic as well as the tumor-associated immune activation will be monitored before and during the treatment cycles.In line with our preclinical findings, Melero, and co-workers recently published an interesting study in mouse colon cancer models, indicating that TNF blockers (anti-TNF or Etanercept) not only improves the anti-tumor effect of ICI  but also reduce irAEs . TNF bloTo summarize, TNF implication in cancer development is rather complex with a balance between high, and low levels of TNF having opposing effects on tumor growth. Finding ways to exploit this balance has the potential to help promoting anti-tumor immune responses and improving the efficacy of existing anti-cancer therapies, especially immunotherapies.AM wrote the paper. CC, TL, NA-A, NM, and BS edited the paper.NM has worked as an investigator and/or consultant and/or speaker for: BMS, MSD, Amgen, Roche, GSK, Novartis, Pierre Fabre. BS has worked as an investigator and consultant for BMS. The remaining 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.


Interferon (IFN) β and Tumor Necrosis Factor (TNF) are key players in immunity against viruses. Compelling evidence has shown that the antiviral and inflammatory transcriptional response induced by IFNβ is reprogrammed by crosstalk with TNF. IFNβ mainly induces interferon-stimulated genes by the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway involving the canonical ISGF3 transcriptional complex, composed of STAT1, STAT2, and IRF9. The signaling pathways engaged downstream of the combination of IFNβ and TNF remain elusive, but previous observations suggested the existence of a response independent of STAT1. Here, using genome-wide transcriptional analysis by RNASeq, we observed a broad antiviral and immunoregulatory response initiated in the absence of STAT1 upon IFNβ and TNF costimulation. Additional stratification of this transcriptional response revealed that STAT2 and IRF9 mediate the expression of a wide spectrum of genes. While a subset of genes was regulated by the concerted action of STAT2 and IRF9, other gene sets were independently regulated by STAT2 or IRF9. Collectively, our data supports a model in which STAT2 and IRF9 act through non-canonical parallel pathways to regulate distinct pool of antiviral and immunoregulatory genes in conditions with elevated levels of both IFNβ and TNF. Interferon (IFN) β plays a critical role in the first line of defense against viruses, through its ability to induce a broad antiviral transcriptional response in virtually all cell types . IFNβ alIFNβ typically acts through binding to the IFNAR receptor (IFNAR1 and IFNAR2) leading to the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway involving JAK1- and Tyk2-mediated phosphorylation of STAT1 and STAT2, and to a lesser extent other STAT members in a cell-specific manner ,8. PhospDUOX2 gene belongs to the category of delayed genes that are remarkably induced to high levels in response to the combination of IFNβ and TNF in lung epithelial cells [DUOX2 expression required STAT2 and IRF9 but not STAT1, suggesting that STAT2 and IRF9 activities might segregate in an alternative STAT1-independent pathway that could be involved in gene regulation downstream of IFNβ and TNF [Synergism between IFNβ and TNF was shown to enhance the antiviral response to Vesicular Stomatitis Virus (VSV), Myxoma virus, and paramyxovirus infections ,13,14. Gal cells . We founIn the present study, we aimed to fully characterize the transcriptional profile of the delayed response to IFNβ and TNF that occurs independently of STAT1 and evaluate the role of STAT2 and IRF9 in the regulation of this response. We found that the costimulation by IFNβ and TNF induces a broad set of antiviral and immunoregulatory genes in the absence of STAT1. We also report the differential regulation of distinct subsets of IFNβ− and TNF-induced genes by STAT2 and IRF9. While IFNβ and TNF act in part through the concerted action of STAT2 and IRF9, specific sets of genes were only regulated by either STAT2 or IRF9. Altogether, our findings uncovered non-canonical STAT2 and/or IRF9-dependent pathways that coexist to regulate distinct pools of antiviral and immunoregulatory genes in a context of IFNβ and TNF crosstalk.A549 cells  were grown in F-12 nutrient mixture (Ham) medium supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS) and 1% L-glutamine. The 2ftGH fibrosarcoma cell line and the derived STAT1-deficient U3A cell line, a generous gift from Dr. G. Stark, Cleveland, USA , were grThe sequences of non-targeting control (Ctrl) and STAT2- and IRF9-directed RNAi oligonucleotides  have previously been described in . U3A celCells were lysed on ice using Nonidet P-40 lysis buffer as fully detailed in . Whole-cMX1, IDO, APOBEC3G, CXCL10, NOD2, PKR, IRF1, IFIT1 and IL8 or TaqMan Gene Expression Assays (Life Technologies-Thermo Fisher) for DUOX2, IFI27, SERPINB2, IL33, CCL20, ISG20. Sequences of oligonucleotides and probes used in PCR reactions are described in Total RNA was prepared using the RNAqueous-96 Isolation Kit  following the manufacturer’s instructions. Total RNA (1 µg) was subjected to reverse transcription using the QuantiTect Reverse Transcription Kit . Quantitative PCR were performed using either Fast start SYBR Green Kit  for ® dsDNA Assay Kit  and the Kapa Illumina GA with Revised Primers-SYBR Fast Universal kit (Kapa Biosystems). Average size fragment was determined using a LabChip GX  instrument. Massively parallel sequencing was carried out on an Illumina HiSeq 2500 sequencer . Read counts were obtained using HTSeq. Reads were trimmed from the 3’ end to have a Phred score of at least 30. Illumina sequencing adapters were removed from the reads and all reads were required to have a length of at least 32bp. Trimming and clipping was performed using Trimmomatic [http://broadinstitute.github.io/picard/). For all samples, the sequencing resulted in more than 29 million clean reads (ranging from 29 to 44 million reads) after removing low quality reads and adaptors. The reads were mapped to the total of 63,679 gene biotypes including 22,810 protein-coding genes. The non-specific filter for 1 count-per million reads (CPM) in at least three samples was applied to the reads and 14,254 genes passed this criterion.Total RNA prepared as described above was quantified using a NanoDrop Spectrophotometer ND-1000  and its integrity was assessed using a 2100 Bioanalyzer . Libraries were generated from 250 ng of total RNA using the NEBNext poly(A) magnetic isolation module and the KAPA stranded RNA-Seq library preparation kit , as per the manufacturer’s recommendations. TruSeq adapters and PCR primers were purchased from IDT. Libraries were quantified using the Quant-iT™ PicoGreenmmomatic . The filp < 0.05, false discovery rate (FDR) < 0.05.Differential transcripts analysis. A reference-based transcript assembly was performed, which allows the detection of known and novel transcripts isoforms, using Cufflinks , merged p value < 0.05 were considered significantly enriched.Enrichment of gene ontology (GO). GO enrichment analysis amongst differentially expressed genes (DEGs) was performed using Goseq  against newFC = oldFC − ε (categoryFC). The parameter Clustering of DEGs. We categorized the DEGs according to their response upon silencing of siSTAT2 and siIRF9; categories are listed as A to I . Then to determine relationship between these categories, we calculated the distance of centers of different categories. For each gene, we transformed siSTAT2 and siIRF9 FC to deviation from the mean FC of the category the respective gene belongs to using the equation: tmod package in R [tmod package -GFP  at a multiplicity of infection (MOI) of 5 for 1 h in serum-free medium (SFM). Cells were then washed twice with SFM and further cultured in DMEM medium containing 2% HI-FCl. The supernatants were harvested at 12 h post-infection and serial dilutions were used to infect confluent Vero cells (ATCC) for 1 h in SFM. The medium was then replaced with 1% methylcellulose in DMEM containing 10% HI-FCl. Two days post-infection, GFP-positive plaques were detected using a Typhoon Trio apparatus and quantified using the ImagequantTL software .U3A or U3A-STAT1 cells at 90% confluency were cotransfected with 100 ng of one of the following CXCL10 promoter containing firefly luciferase reporter plasmids , CXCL10p values: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) or p < 0.0001 (****). Differences with a p-value < 0.05 were considered significant. Statistical analysis of the RNASeq data is described in the Bioinformatics analysis section above.Statistical analyses of qRT-PCR and luciferase assay results were performed using the Prism v7 or v8 software  using the tests indicated in the figure legends. Statistical significance was evaluated using the following http://www.ncbi.nlm.nih.gov/geo) under accession number GEO: GSE111195.The entire set of RNAseq data has been submitted to the Gene Expression Omnibus (GEO) database −, STAT2- or IRF9-RNAi and further left untreated or stimulated with IFNβ (1000 U/mL) + TNF (10 ng/mL) for 24 h C. Efficip < 0.05, FDR < 0.05) in IFNβ + TNF vs. NS. Among these, 482 DEGs were upregulated and 130 were downregulated  upon IFNβ + TNF stimulation in the absence of STAT1, comparison between non-stimulated (NS) and IFNβ + TNF-stimulated control cells was performed A. In totp < 10−10) and MF, are depicted in To identify the Biological Processes (BP) and Molecular Functions (MF) induced by IFNβ + TNF independently of STAT1, we further analyzed the upregulated DEGs. The top 40 upregulated DEGs are shown in To gain deeper insight into the relevance of the STAT1-independent IFNβ + TNF-induced transcripts towards immunological and host defense responses, we conducted a modular transcription analysis of upregulated DEGs against 606 immune-related functional modules. These modules were previously defined from co-clustered gene sets built via an unbiased data-driven approach as detailed in the material and methods section ,31. STATGO enrichment and modular transcription analyses were suggestive of an antiviral response being induced by IFNβ + TNF. To demonstrate the physiological relevance of STAT1-independent gene expression, we evaluated the capacity of IFNβ + TNF to restrict virus replication in the absence of STAT1. Previous study has shown that infection by Vesicular Stomatitis Virus (VSV) is sensitive to IFNβ, but not to TNF, in 2ftGH-derived cells . Thus, Up < 0.05, FDR < 0.05) downregulated or upregulated upon silencing of STAT2 , truncated (376bp containing only the ISRE (3) site) or mutated (972bp containing a mutated ISRE (3) site) CXCL10 promoter luciferase (CXCL10prom-Luc) reporter constructs  and (2) sites, but was significantly impaired when the ISRE (3) site was mutated  and/or ISRE (2) sites and the proximal ISRE (3) site  site B. AltogePrevious studies have described that IFNβ and TNF synergize to elicit a specific delayed transcriptional program that differs from the one induced by either cytokine alone ,36. The CXCL10 promoter exhibits partial dependence on STAT1 support a model in which a canonical ISGF3 pathway is engaged downstream of the costimulation. Importantly, it also implied the existence of a STAT2- and/or IRF9-dependent transcriptional response occurring in the absence of STAT1. The human STAT1-deficient U3A cell model offered a unique opportunity to specifically pinpoint this STAT1-independent response. In this model, genome wide RNA sequencing highlighted that the transcriptional program induced by IFNβ + TNF in the absence of STAT1 encompasses a wide range of immunoregulatory and antiviral functions. The functional relevance of this response was confirmed by the observation that the treatment with IFNβ + TNF induced an antiviral state capable of restricting VSV replication in the absence of STAT1. This points to a significant role of the STAT1-independent pathway in the establishment of the antiviral state induced by the synergistic action of IFNβ and TNF that enhances the restriction of VSV  but not on IRF9, (Categories D and E) is a rare genome wide demonstration of gene regulation by STAT2 independently of STAT1 and IRF9. Previous reports have identified ISGF3-independent, STAT2-dependent genes but the association with IRF9 was not formally excluded ,45,46,47ISGF3-independent functions of IRF9 have been proposed based on the study of IRF9 deficiencies ,48. HoweAPOBEC3G that is amongst the STAT2- and IRF9- independent genes is not induced by TNF alone . All transcriptional modules were enriched in this category pointing to a major role of this pathway in the establishment of a host defense and immunoregulatory response. The STAT2 and IRF9 independent genes does not solely reflects induction by TNF alone. For instance, NF alone A. While NF alone , we did NF alone  or qRT-PThis study provides novel insight into the molecular pathways leading to delayed antiviral and immunoregulatory gene expression in conditions where elevated levels of both IFNβ and TNF are present. Altogether our results demonstrate that in addition to the engagement of an ISGF3-dependent canonical response, a broad transcriptional program is elicited independently of STAT1, and support a model in which STAT2 and IRF9 contribute to the regulation of this response through non-canonical parallel pathways involving their concerted or independent action .Consistent with accumulating evidence , these d


Rheumatoid arthritis (RA) is an autoimmune disease characterized by persistent synovial inflammation. The major drivers of synovial inflammation are cytokines and chemokines. Among these molecules, TNF activates fibroblast-like synoviocytes (FLSs), which leads to the production of inflammatory mediators. Here, we show that TNF regulates the expression of the transcription factor interferon regulatory factor 1 (IRF1) in human FLSs as well as in a TNF transgenic arthritis mouse model. Transcriptomic analyses of IRF1-deficient, TNF-stimulated FLSs define the interferon (IFN) pathway as a major target of IRF1. IRF1 expression is associated with the expression of IFNβ, which leads to the activation of the JAK-STAT pathway. Blocking the JAK-STAT pathway with the Janus kinase inhibitor (JAKinib) baricitinib or tofacitinib reduces the expression of IFN-regulated genes (IRGs) in TNF-activated FLSs. Therefore, we conclude that TNF induces a distinct inflammatory cascade, in which IRGs are key elements, in FLSs. The IFN-signature might be a promising biomarker for the efficient and personalized use of new treatment strategies for RA, such as JAKinibs. Thomas Karonitsch at the Medical University of Vienna, Austria, and colleagues showed that activation of the protein interferon regulatory factor 1 (IRF-1) in human synoviocytes promoted the expression of genes that contribute to sustained synovial inflammation in rheumatoid Arthritis (RA). They found that the Januse kinase (JAK) inhibitors baricitinb and tofacitinib, which are both approved for the treatment of RA, prevented the activation of IRF1 regulated proinflammatory genes. Their findings unvover a potential role for IRF1 activated genes as a biomarker to predict the treatment response to RA therapies and moreover highlight the benefit of targeting inflammatory pathways in synoviocytes for the treatment of RA. Cytokine- and chemokine-mediated crosstalk between activated immune cells and FLSs sustains local inflammation and imprints disease-specific cellular signatures3. Whole-genome expression profiling of rheumatoid synovial tissue samples depicts a potential role for interferon-regulated genes (IRGs) in the pathogenesis of RA. Among the IRGs, several genes that drive RA disease progression, such as the transcription factor (TF) STAT1, the chemokines CXCL9 and CXCL10, and the cytokine TNFSF13B, which is also called B-cell activating factor (BAFF), can be found5. The overexpression of IRGs can be detected in up to 65% of RA patients6. Individuals with an interferon (IFN) signature have a significantly higher risk of developing RA7, underlining the major role of IRGs in the pathogenesis of RA. The IFN signature has also been discussed as a potential biomarker, since clinical studies have demonstrated strong associations between IRGs and the clinical response to biological disease modifying antirheumatic drugs (bDMARDS)11. In RA patients, it has been shown that TNF is a strong inducer of IRGs12. Especially in FLSs, we previously demonstrated that the TNF-induced signature contains more than 50% of the IRGs13. However, the signaling circuit that allows TNF to induce the expression of IRGs in FLSs is largely unknown.Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that primarily affects the synovium of diarthrodial joints. Rheumatoid synovitis is characterized by the expansion of resident fibroblast-like synoviocytes (FLSs) and infiltration of immune cells into the synovial membrane15. Since IRF1 knockdown prevents murine collagen-induced arthritis16, and IRF1 is expressed in activated FLSs17, we hypothesized that IRF1 might contribute to the TNF response in RA-FLSs. Through a comprehensive approach encompassing transcriptomics and biochemistry, our study indeed revealed that IRF1 specifically drives the IFN signature in RA-FLSs via STAT1. Accordingly, blocking the JAK-STAT pathway with a JAK inhibitor (JAKinib), baricitinib or tofacitinib, suppressed the TNF-induced expression of proinflammatory IRGs. Thus, our study contributes novel insights into the synovial response to TNF and shows that JAKinibs target the TNF-induced IFN response in RA.Previous studies highlighted a key role for the TF IRF1 in TNF-induced gene expression in monocytes and endothelial cells18) or osteoarthritis (OA) patients undergoing joint replacement or synovectomy. All patients provided written informed consent prior to synovial tissue donation. This study was approved by the ethics committee of the Medical University of Vienna.Synovial tissue samples were obtained from RA (fulfilling the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) classification criteria for RA19). Mice were maintained under conventional housing conditions . All experiments were performed with female mice. Age-matched nontransgenic female littermates were used as controls. All experiments were approved by the local ethical committee and the Federal Ministry of Science, Research and Economics.TNF transgenic mice that overexpress human TNF  were originally generated by the group of George Kollias  littermates. Hind paws were fixed in 7% formaldehyde for 6 h, followed by decalcification in 14% EDTA buffer (pH 7.2) for 4–6 days. Paraffin-embedded sections were used for immunohistochemical staining for synovial IRF1 expression using the antibodies and protocol mentioned above.FLS single cell suspensions were obtained by digesting minced synovial tissue samples with collagenase type II (Merck). FLSs were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum , 1% penicillin/streptomycin (P/S), and nonessential amino acids (both Thermo Fisher Scientific). FLSs beyond passage 4 were used. The following cytokines and inhibitors were used as indicated: TNF , baricitinib  and tofacitinib .FLSs were lysed with RIPA buffer (Thermo Fisher Scientific) supplemented with the Halt™ phosphatase inhibitor cocktail (Thermo Fisher Scientific) and a protease inhibitor mix (Sigma-Aldrich). The protein lysates were fractioned on polyacrylamide gels, followed by electrotransfer to nitrocellulose membranes, which were blocked with either 5% BSA or 5% nonfat dry milk and then incubated with primary antibodies . After an incubation with HRP-conjugated secondary antibodies , specific bands were detected with the BIORAD Clarity ECL Western substrate. Reprobing was performed using ReBlot Plus Strong Solution (Merck).20. FLSs were resuspended in ice-cold Matrigel Matrix (BD Biosciences). The cell/ECM suspension was placed on Poly-HEMA-coated culture dishes (Sigma-Aldrich) and overlaid with culture medium , 0.125% bovine serum albumin , 0.008 g ascorbic acid (Thermo Fisher Scientific), 1% NEAA (Thermo Fisher Scientific) and 1% P/S). At the time points indicated, the micromasses were fixed in paraformaldehyde and then embedded in paraffin. Paraffin-embedded sections were treated with a citrate buffer  or Tris-EDTA . To reduce nonspecific protein binding, the sections were incubated with the goat serum. The following antibodies were used: anti-IRF1  and anti-BAFF (Enzo Life Sciences). Nonimmune immunoglobulins of the same isotype and concentration as the primary antibody  served as controls.Micromass organ cultures were prepared as previously described21.FLSs were cultured in Opti-MEM (Thermo Fisher Scientific) and transfected with SMARTpool: ON-TARGETplus siRNA pools (Horizon Discovery) using Lipofectamine (Thermo Fisher Scientific)RNA was isolated from RA-FLSs using an RNeasy purification kit (Qiagen) according to the manufacturer's protocol. Total RNA was isolated from the frozen front paw tissue of hTNFtg and wild-type littermates by mechanical homogenization using a standard Trizol purification protocol.CXCL9 FW: ATCAGCACCAACCAAGGGACT, RV: GCTTTTTCTTTTGGCTGACCTG; CXCL10 FW: ATTTGCTGCCTTATCTTTCTG, RV: TCTCACCCTTCTTTTTCATTGTAG; CXCL11 FW: GAAGGATGAAAGGTGGGTGA, RV: AAGCACTTTGTAAACTCCGATG; TNFSF13B: FW: GGAGAAGGCAACTCCAGTCAGAAC, RV:CAATTCATCCCCAAAGACATGGAC; and GAPDH FW: TGATGACATCAAGAAGGTGGTGAAG, RV TCCTTGGAGGCCATGTGGGCCAT.RNA was reverse transcribed into cDNA using an Omniscript RT kit (Qiagen). RNA concentrations were determined using a Nanodrop spectrophotometer. qPCR was performed using a Fast Start SYBR Green I kit (Roche). The results were quantified by the 2−ΔΔC(t) method, using GAPDH expression levels for normalization. The primer sequences for human primers were as follows: IRF1 FW: CCCACAGAAGAGCATAGCAC, RV: AGCAGTTCTTTGGGAATAGG.The following mouse primers were used: GAPDH FW TGGCATTGTGGA AGGGCTCATGAC, RV: ATGCCAGTGAGCTTGCCGTTCAGC; and RNA was isolated as described above. The amount of total RNA was quantified using a Qubit Fluorometric Quantitation system (Thermo Fisher Scientific), and the RNA integrity number (RIN) was determined using the Experion Automated Electrophoresis System (Bio-Rad). RNA-seq libraries were prepared with a TruSeq Stranded mRNA LT sample preparation kit (Illumina) using both Sciclone and Zephyr liquid-handling robotics (PerkinElmer). Library concentrations were quantified with the Qubit Fluorometric Quantitation system (Thermo Fisher Scientific), and the size distribution was assessed using the Experion Automated Electrophoresis System (Bio-Rad). For sequencing, samples were diluted and pooled into NGS libraries in equimolar amounts. Expression-profiling libraries were sequenced with Illumina HiSeq 3000/4000 instruments in the 50-base-pair-single-end mode.http://github.com/wtsi-npg/illumina2bam) to generate unaligned BAM files. Sequence reads were mapped onto the human genome release hg38 (GRCh38) with Ensembl transcript annotation version 87 using tophat version 2.1.122 with bowtie version 2.2.923. Reads were counted with featureCounts24. Gene expression values (reads per kilobase exon per million mapped reads (RPKM)) were calculated with Cufflinks version 2.225. The differential expression between two paired sample groups was calculated with edgeR26. The filtering for differentially expressed genes was performed with a p-value of 0.05 (FDR corrected) and minimal fold-change of 2. For GO enrichment analysis of a gene set, GOstats version 2.46.027 was used. Cytoscape version 3.6.0 and the plugin Enrichment map (v3.0.0) were used for network visualization28.Raw sequencing data were processed with Illumina2bam  patients , chemokines , tissue-degrading enzymes (MMP1 and MMP10) and genes  that are associated with synovial inflammation. As highlighted in Fig. RSAD2 and IFIT2; the proinflammatory CXCR3-binding chemokines CXCL9, CXCL10, and CXCL11; the B-cell activating factor TNFSF13B (BAFF) and IFNβ, represent IRGs.To address the genome-wide contribution of IRF1 to the TNF-mediated response in RA-FLSs, we transfected RA-FLSs with siRNA pools targeting IRF1. The transfected RA-FLSs were stimulated with TNF for 3 h Fig. . TranscrConsistently, molecular function enrichment analyses of the 121 TNF-regulated, IRF1-dependent genes revealed an overrepresentation of pathways involved in IFN and viral responses Fig. . OverallCXCL11 and TNFSF13B. As a control, we determined the expression of RIPK2, which is induced without new protein synthesis. As expected, RIPK2 transcription was not affected by CHX treatment. These data support the importance of the TNF-driven de novo synthesis of IRF1 for the regulation of IRGs.To confirm the RNA sequencing data, we analyzed the expression of IFN-dependent chemokines and TNFSF13B. Consistent with the transcriptomic data, we observed diminished expression of CXCL9, CXCL10, CXCL11 and TNFSF13B in TNF-treated, IRF1-silenced FLSs on the mRNA level as assessed by qPCR Fig.  and on t15. We therefore performed time-kinetic analyses of IRF1 and p-STAT1 expression in TNF-stimulated FLSs. As shown in Fig. CXCL9, CXCL10, and CXCL11 in the absence of IFNβ FNβ Fig. . SimilarFNβ Fig. . STAT1 pFNβ Fig.  or a bloFNβ Fig. . These d30. Therefore, we tested whether JAKinibs, which are approved for the treatment of RA, can prevent the TNF-induced activation of STAT1 and the expression of downstream target genes. Indeed, both inhibitors diminished the expression of CXCL9, CXCL10, CXCL11, and TNSF13B in TNF-activated FLSs  JAK1 and Tyk2LSs Fig. . CorrespLSs Fig.  in FLSs To explore the effects of JAKinibs on a tissue-like environment, we employed a 3-D synovial culture system. Tissue-bound FLSs exposed to TNF abundantly expressed TNFSF13B, as assessed by IHC. Both baricitinib and tofacitinib inhibited the expression of TNFSF13B, consistent with the results obtained from the 2-D cell cultures Fig. . Togethe31. Various proinflammatory cytokines can affect FLSs. Among proinflammatory cytokines, TNF strongly stimulates FLSs to produce cytokines and chemokines, which augment and perpetuate inflammatory cell recruitment and activation33. Thus, FLSs represent a promising target for the treatment of RA. However, compared with those in other cells, such as macrophages or lymphocytes, the pathways in FLSs that allow their participation in synovial inflammation are poorly defined. In this study, we showed increased IRF1 expression in inflamed human and mouse synovial tissue. Based on these data, we hypothesized that IRF1 is critical for the TNF response in FLSs. Transcriptomics, indeed, depicted IRF1 as a genome-wide activator of the TNF-induced expression signature in FLSs. Specifically, we found that several IRGs implicated in RA pathogenesis are controlled by IRF1. Increased levels of the CXCR3-binding chemokines  can be found in the rheumatoid synovium. These chemokines are thought to sustain leukocyte recruitment to the inflamed synovial tissue. Moreover, CXCL10 has been shown to promote FLS invasiveness in an autocrine/paracrine manner34. Aside from chemokines, TNFSF13B was suppressed in the absence of IRF1. TNFSF13B, also known as BAFF, is abundantly expressed in RA synovial tissue. TNFSF13B is important for B-cell proliferation and differentiation as well as autoantibody production35. All these data underscore the importance of IRF1 as a key TF in synovial inflammation in RA.FLSs are increasingly recognized as major drivers of synovial inflammation, and joint destruction in RA15. Thus, these observations suggest that this distinct pathway exists in the principal cellular components of the stromal tissue compartment. This conclusion is of particular interest, since stromal cells, such as fibroblasts, macrophages and endothelial cells, define the microenvironment in which inflammatory reactions take place. Especially in RA, activated stromal cells contribute to chronic inflammation and tissue damage by orchestrating the continuous recruitment, activation and retention of leukocytes36. Targeting the TNF-IRF1-IFNβ-STAT1 signaling circuit, which propels the expression of leukocyte-recruiting  and leukocyte-activating  inflammatory mediators, would therefore interrupt the persistent vicious crosstalk between immune cells and synovial stromal cells. These data support the concept of IFN-targeted therapies in RA patients. Ongoing phase 2 trials with anifrolumab are currently investigating the potential of type I IFN blockade in RA patients with a high interferon signature (NCT: 034356019). In macrophages, tofacitinib has been shown to suppress TNF-mediated STAT1 activation and chemokine expression37. Tofacitinib inhibits STAT1-driven CXCL10 expression in RA-FLSs38. In this study, we found that JAKinibs that are currently approved for the treatment of RA, namely, baricitinib and tofacitinib, suppress the expression of all three CXCR3-binding chemokines. Intriguingly, we found that the JAKinibs suppressed the expression of TNFSF13B, indicating that JAKinibs target B-cell-FLS crosstalk, which might therefore provide an additional mode of action. Our findings also provide evidence for a hitherto unrecognized quality of JAKinibs, namely, partial interference with TNF signaling pathways.Our biochemical studies revealed that IRF1 expression represents the first step of a temporally defined signaling circuit that almost exclusively controls the TNF-induced IFN response in FLSs. Knocking down IRF1 expression prevented the TNF-induced transcription of IFNβ and the subsequent activation of the TF STAT1. Consistently, both the lack of IFNβ and the loss of IFNAR function prevented the TNF-induced phosphorylation of STAT1. Our studies reveal a TNF-based signaling circuit that has also been observed in other cell types. In both macrophages and endothelial cells, TNF stimulation results in the expression of IRF1 and IFNβ to sustain the expression of proinflammatory chemokines via STAT1Together, these studies highlight the importance of the TNF-driven, IRF1-mediated regulation of the IFN pathway as a major contributor to FLS-mediated inflammation in RA. Considering the high number of nonresponders to disease-modifying antirheumatic drugs (DMARDS), a better understanding of the pathomechanisms and the informed use of biomarkers, such as the IFN signature, will ultimately lead to a more stratified treatment approach for RA patients.Supplementary Table 1Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4