TNF is not only a major effector molecule of PAMP/DAMP-activated macrophages, but also regulates macrophage function and viability. We recently demonstrated that TNFR2 triggers necroptosis in macrophages with compromised caspase activity by two cooperating mechanisms: induction of endogenous TNF with subsequent stimulation of TNFR1 and depletion of cytosolic TRAF2-cIAP complexes. Here we show that TNFR2 activation in caspase-inhibited macrophages results in the production of endogenous TNF and TNFR1 stimulation followed by upregulation of A20, TRAF1, IL-6, and IL-1β. Surprisingly, TNFR1-mediated induction of IL-6 and IL-1β was clearly evident in response to TNFR2 stimulation but occurred not or only weakly in macrophages selectively and directly stimulated via TNFR1. Moreover, TNFR2-induced TNFR1-mediated gene induction was largely inhibited by necrostatin-1, whereas upregulation of A20 and TRAF1 by direct and exclusive stimulation of TNFR1 remained unaffected by this compound. Thus, treatment with TNFR2/ZVAD enables TNFR1 in macrophages to stimulate gene induction via a pathway requiring RIPK1 kinase activity. TNFR2/ZVAD-induced production of IL-6 and IL-1β was largely blocked in necroptosis-resistant MLKL- and RIPK3-deficient macrophages, whereas induction of A20 and TRAF1 remained unaffected. In sum, our results show that in caspase-inhibited macrophages TNFR2 not only triggers TNF/TNFR1-mediated necroptosis but also TNF/TNFR1-mediated RIPK3/MLKL-dependent and -independent gene induction. Activated macrophages not only act as phagocytes of pathogens and dead cells but also connect innate immunity with adaptive immunity by antigen presentation to T-helper cells and release of pleiotropic cytokines such as interferons and tumor necrosis factor (TNF). The latter is not only a major effector molecule of activated macrophages but also retroacts on this cell type resulting in enhanced and sustained activation. Dependent on the circumstances, TNF can also elicit cytotoxic effects in macrophages15.Macrophages can be activated by a variety of pathogen- and damage-associated molecular patterns (PAMPs and DAMPs) and are of major importance for innate immunity and tissue repair16. Both forms of TNF bind to two receptors of the TNF receptor superfamily (TNFRSF), TNFR1 and TNFR2. Although binding of soluble TNF by TNFR1 is fully sufficient for receptor activation, soluble TNF poorly stimulates TNFR2 signaling despite high-affinity binding16. In contrast, membrane TNF activates TNFR1 and TNFR2 with high efficiency. TNFR1 and TNFR2 are representatives of two distinct subgroups of the TNFRSF. TNFR1 is a member of the death receptor subgroup with the ability to stimulate apoptotic and/or necroptotic signaling18. However, TNFR1-induced cytotoxic signaling is normally masked by TNFRSF1A associated via death domain (TRADD)-mediated recruitment of TRAF2-cIAP1 and TRAF2-cIAP2 complexes, and the ability of TNFR1 to stimulate the classical nuclear factor-κB (NFκB) pathway, which controls the expression of various anti-apoptotic proteins18. TNFR2 belongs to the TRAF-interacting subgroup of the TNFRSF. The receptors of this subgroup are characterized by the ability to directly recruit TRAF adapter proteins, especially TRAF2, by virtue of short peptide motifs19. TNFR2 activates the classical and alternative NFκB pathway, but in contrast to TNFR1 it is not directly linked to cytotoxic pathways20. Interestingly, TNFR2 elicits cytotoxic activity by two indirect mechanisms: first, by triggering NFκB-dependent production of TNF and subsequent activation of the death receptor TNFR1, and second by depletion/degradation of the anti-cytotoxic TRAF2-cIAP1 and TRAF2-cIAP2 complexes, and limiting their availability for TNFR116. Indeed, early on, we were able to show that TNFR2 enhances TNFR1-induced activation of caspase-8 and apoptosis16 and in recent years we and others showed that TNFR2-induced TRAF2-cIAP1/2 depletion also sensitizes for death receptor-induced necroptosis14.TNF is initially expressed as a type II transmembrane protein. A soluble form of TNF furthermore originates from transmembrane TNF by proteolytic processing14.Macrophages express TNFR1 as well as TNFR2, and are important producers of TNF upon exposure to PAMPs and DAMPs. In addition, both TNF receptors are able to induce the production of TNF in macrophages. In view of this complex interplay, it is not surprising that the molecular mechanisms of TNF signaling in macrophages are still poorly understood. We recently showed that TNFR2 induces necroptosis in macrophages compromised for caspase-8 activation by autocrine TNF production along with depletion/degradation of TRAF2-cIAP1/2 complexesHere we investigated the effect of TNFR2 stimulation on NFκB-regulated factors. In contrast to TNFR1, TNFR2 induced NFκB-regulated genes preferentially when caspase activation was inhibited. Similar to TNFR2-induced necroptosis, TNFR2-induced gene induction largely occurred via endogenous TNF and subsequent TNFR1 stimulation in an RIPK1 kinase inhibitor-sensitive manner. Intriguingly, although TNFR2-induced interleukin (IL)-6 and IL-1β induction were co-mediated with necroptosis by RIPK3 and MLKL, the upregulation of TRAF1 and A20 bifurcated from necroptotic signaling upstream of RIPK3 but downstream of TNFR1-induced RIPK1 phosphorylation. Thus, in caspase-inhibited macrophages, TNFR2 unmasks a yet not recognized TNFR1-associated NFκB signaling pathway requiring RIPK1 kinase activity.21. TNC-sc(mu)TNF80 is not only highly specific for TNFR2 but, due to its oligomeric nature, also overcomes the poor activity of soluble TNF trimers on TNFR221. Unexpectedly, inhibition of RIPK1 kinase activity using necrostatin-1 strongly reduced TNC-sc(mu)TNF80-induced TNFR2-mediated transcription of the NFκB target genes Tnf, Cflar, A20, and Traf1 in the presence of carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (ZVAD) TNF80, a fusion protein of the small trimerization domain of tenascin-C and three amino acid linker connected protomers of a TNFR2-specific mutant of soluble mouse TNFAD) Fig.  SimilarlAD) Fig. , whereasAD) Fig. . Loss ofAD) Fig. . Selectible Fig. . Collect14. Thus, TNF and TNFR1 may also have a key role in TNFR2-induced gene transcription in caspase-inhibited macrophages. Deficiency in TNF or TNFR1 abrogated TNFR2-induced expression of A20 TNF80 but not human TNF triggered RIPK1 phosphorylation TNF8021. These investigations came up with two puzzling observations: first, TNFR2-induced expression of TNF and other NFκB-regulated factors was strongly enhanced by caspase inhibition TNF80-induced signaling. However, there are also yet not explainable differences between ZVAD/TNC-sc(mu)TNF80- and ZVAD/LPS-induced RIPK1 kinase activity-dependent gene induction. Although in the case of ZVAD/LPS gene induction required RIPK3 but not MLKL24, in the case of ZVAD/TNC-sc(mu)TNF80 stimulation both molecules were largely dispensable for induction of A20 and TRAF1  triggers a similar mode of gene inductionAF1 Fig.  but wereAF1 Fig. . Thus, dThe RIPK1 kinase activity-dependent mode of gene induction of TNFR1 requires caspase inhibition to become evident, although there was no obvious apoptosis induction in TNF-treated macrophages. Thus, it appears possible that low/tonic caspase activity, insufficient to trigger apoptosis, constitutively inhibit this mode of TNFR1 signaling. Another future challenge is therefore to identify the physiological conditions under which TNFR1-induced RIPK1 kinase activity-dependent gene induction gains relevance in macrophages and whether this mode of TNFR1 signaling is also operative in other cell types.25 using femurs of C57Bl/6 wild-type mice and TNFR1-, TNFR2-, TNF-, MLKL-, and RIPK3-knockout mice and lentivirus with estrogen-regulated HoxB8 expression. The required estrogen-regulated expression plasmid 3HAERHBH-HoxB8 was a kind gift of Hans Haecker . The bones of RIPK3- and MLKL-knockout mice were kindly provided by Philip Jost  Immortalized MPCs were expanded in MPC medium (Optimem (first 3 days) or RPMI 1640 (after 3 days) supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) l-glutamine, 30 µM β-mercaptoethanol with 1 µM estradiol (Sigma) and 4% (v/v) SCF containing cell culture supernatant derived of SCF-producing CHO cells). MPC medium was changed two to three times per week. For macrophage differentiation, MPCs were washed three times with warm 10% FCS (v/v) supplemented phosphate-buffered saline (PBS). Cells (10 × 106) were subjected to uncoated cell culture plates in macrophage differentiation medium  FCS, 1% (v/v) l-glutamine, 30 µM β-mercaptoethanol, 10% (v/v) conditioned cell culture supernatant of macrophage colony-stimulating factor-producing L929 cells. Medium was replaced once after 3 days and after 2 additional days cells were collected by scraping with a rubber policeman and analyzed by flow cytometry for cell surface expression of CD11b and F4/80 to control for the success of macrophage differentiation. For viability assays and evaluation of cytokine induction, macrophages were seeded in 96-well plates (45 × 103 per well) and were stimulated the next day. For western blotting and quantitative PCR (qPCR) experiments, macrophages were seeded in six-well plates (1 × 106 per well).Macrophages were differentiated from stem cell factor (SCF)-ER-Hoxb8 immortalized murine myeloid progenitor cells (MPCs). The latter were generated essentially as described by Wang et al.Antibodies used in this study were from Miltenyi Biotec, Bergisch Gladbach, Germany , Cell Signaling Technology, Beverly, MA, USA , rabbit polyclonal IgG, #31122; anti-TRAF2, rabbit polyclonal IgG, #4724), Santa Cruz, USA , Enzo , BD Bioscience , Abcam, Cambridge, UK (anti-p-RIPK3(S232), rabbit polyclonal, #ab195117) and NeoMarkers, Fremont, USA .21. In brief, HEK293 cells were transiently transfected with an expression plasmid encoding TNC-sc(mu)TNF80 and after 5–7 days supernatants were collected. By help of the FLAG tag contained in TNC-sc(mu)TNF80, the protein was purified from the pooled supernatants by affinity chromatography on anti-FLAG mAb M2 agarose according to the recommendations of the supplier . Purified TNC-sc(mu)TNF80 were regularly controlled for potential LPS contaminations acquired during purification and/or storage using the PierceTM LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher Scientific). If required, LPS was removed with the Pierce™ High Capacity Endotoxin Removal Resin.The pan caspase inhibitor ZVAD was purchased from Thermo Fisher Scientific Waltham, MA, USA (Bachem #N-1510.0025) and the RIPK1 inhibitor necrostatin-1 was from Biomol, Hamburg, Germany (#AP-309). Human TNF was a kind gift from Professor Daniela Männel . Production, purification, and properties of the agonistic murine TNFR2-specific nonameric murine TNF variant TNC-sc(mu)TNF80 have been described elsewhere6) were stimulated with the reagents of interest for 7 h. After washing the cells twice with ice-cold PBS RNA was prepared with the RNeasy mini kit  following the recommendations of the supplier. RNA (2 µg) were transcribed into DNA using the high-capacity cDNA reverse transcription kit from Applied Biosystems  and mRNA levels were quantified using an ABI Prism 7900 sequence detector (Applied Biosystems) and TaqManTM gene expression assays (Applied Biosystems) for mouse Cflar (Mm01255578_m1), Tnfaip3 (Mm00437121_m1), Traf1 (Mm00493827_m1), Tnf (Mm00443258_m1), and Hprt1 (Mm00446968_m1). Reactions were performed in technical triplicates for each sample of an experiment. Expression levels of mRNAs were calculated by help of the SDS 2.1 software (Applied Biosystems) and were finally normalized to the housekeeping gene Hprt1.Macrophages  and resuspended in 4 × Laemmli sample buffer (8% (w/v) SDS, 0.1 M dithiothreitol, 40% (v/v) glycerol, 0.2M Tris, pH 8.0) supplemented with phosphatase inhibitor cocktail II . Cell lysis was enhanced by sonification (ten pulses) and heating . Lysates were cleared from remaining insoluble debris by centrifugation  and subjected to SDS-polyacrylamide gel electrophoresis. After separation proteins were transferred from the gel to nitrocellulose by wet blotting for 90 min. Remaining free binding sites on the nitrocellulose were blocked by overnight incubation at 4 °C in Tris-buffered saline with 0.1% (v/v) Tween 20 and 5% (w/v) dry milk. After washing once with TBST, the nitrocellulose membrane was incubated with the primary antibodies of interest . After washing twice again in TBST, the nitrocellulose membrane was incubated with an appropriate horseradish peroxidase-conjugated secondary antibody  and the antigen–antibody complexes were finally detected by help of the ECL western blotting detection reagents and analysis system (Thermo Fisher Scientific).3/well) in 100 µl culture macrophage differentiation medium. The following day, macrophages were stimulated in triplicates with the reagents of interest for 36 h. Finally, either cell viability was determined using the MTT assay or upregulation of IL-6 and Il-1β production was measured by enzyme-linked immunosorbent assay (ELISA) of supernatants . In each cell viability experiment, one triplicate of cells was treated with a cytotoxic cocktail containing 1 µg/ml Fc-CD95L, 20 µM CHX, and 1% (w/v) sodium azide, which triggers complete cell death. The triplicate of cells challenged with the cytotoxic mixture defined 0 % viability and the triplicate of untreated cells defined 100% viability. All other viability measurements were normalized against these values. The averages of the technical triplicates were considered as a data point for further statistical evaluation .Macrophages were seeded in 96-well tissue culture plates (45 × 10


Although disease in a majority of rheumatoid arthritis (RA) patients is often initially limited to one or a few joints, currently approved medications including anti-tumor necrosis factor-α antibody (α-TNF) are injected systemically. Given that α-TNF systemic injection typically does not cure RA and involves risk of treatment-related adverse events, one possible approach to enhance therapeutic efficacy and reduce α-TNF systemic exposure is to retain the antibodies in arthritic joints after local administration. The aim of this study was to evaluate the approach of conferring extracellular matrix (ECM) binding affinity to α-TNF antibodies in a RA model.123-144). The binding activity of PlGF-2123-144-conjugated α-TNF (PlGF-2123-144-α-TNF) against ECM proteins was assessed by ELISA and by immunostaining on human cartilage specimens. The effect of conjugation on antibody function was assessed as a neutralizing activity against osteoclast differentiation. Retention at the injection site and therapeutic efficacy of PlGF-2123-144-α-TNF were tested in a collagen antibody-induced arthritis (CAIA) model in the mouse.α-TNF was chemically conjugated with a promiscuous ECM-binding peptide derived from placenta growth factor 2 (PlGF-2123-144 peptide conjugation conferred α-TNF with affinity to ECM proteins without impairment of antigen recognition. PlGF-2123-144-α-TNF locally injected at a paw in the CAIA model was retained for at least 96 h at the injection site, whereas unmodified α-TNF was dispersed rapidly after injection. Local treatment with unmodified α-TNF did not suppress the arthritis score relative to isotype controls. By contrast, local administration of PlGF-2123-144-α-TNF suppressed arthritis development almost completely in the treated paw even at a 1000× lower dose.PlGF-2These data demonstrate that retention of α-TNF in arthritic joints can suppress arthritis development and enhance therapeutic efficacy. This simple bioengineering approach of ECM-binding peptide conjugation offers a powerful and clinically translational approach to treat RA. Rheumatoid arthritis (RA) is a chronic, progressive autoimmune disease characterized by the formation of hyperplastic synovial pannus tissue, which causes joint destruction , 2. BiolDirect intra-articular administration of drugs is a reasonable approach to enhance their local efficacy and reduce systemic adverse effects and is relevant for the treatment of monoarthritis and oligoarthritis. However, drugs injected into the joint are rapidly cleared via lymphatics or sub-synovial capillaries in the absence of a retention strategy . In addi123–144) has an exceptionally high affinity for multiple ECM proteins [123–144 peptide to immune checkpoint blockade and immune agonist antibodies enhanced antibody retention within tumors when injected near the tumor, improving therapeutic efficacy and safety by limiting systemic exposure [Heparin-binding domains (HBDs) derived from growth factors (GFs) bind to HBDs derived from a variety of extracellular matrix (ECM) proteins –22. By sexposure , 25.123–144 peptide for RA therapy. We tested whether the enhanced retention of α-TNF in tissue surrounding the joints would improve its local efficacy against arthritis development.Here, we have hypothesized that utilizing ECM-binding technology for prolonged tissue retention would improve the ability of an anti-inflammatory cytokine antibody to effectively suppress RA development. We engineered α-TNF conjugated with PlGF-2123-144-α-TNF was performed as described previously [4 crosslinker  for 30 min at room temperature. Unreacted crosslinker was removed using a Zeba spin desalting column (Thermo Fisher Scientific), and then 15-fold molar excess of PlGF-2123-144 peptide (RRRPKGRGKRRREKQRPTDCHL) was added and reacted for 1 h at room temperature for conjugation to the thiol moiety on the C residue. The peptide had been synthesized with > 95% purity by Genscript .The synthesis of PlGF-2eviously . Rat antSodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 4–20% gradient gels  after α-TNF was reduced with 10 mM DTT. After electrophoresis, gels were stained with SimplyBlue SafeStain (Thermo Fisher Scientific) according to the manufacturer’s instruction. Gel images were acquired with the ChemiDoc XRSþ system (Bio-Rad).Antibodies were analyzed by Matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS) using a Bruker Ultraflextreme MALDI TOF/TOF instrument. All spectra were collected with acquisition software Bruker flexControl™ and processed with analysis software Bruker flexAnalysis™. First, a saturated solution of the matrix, α-cyano-4-hydroxycinnamic acid , was prepared in 50:50 (v/v) acetonitrile:(1% trifluoroacetic acid in water) as a solvent. The analyte in phosphate-buffered saline  and the matrix solution (25 μL) were then mixed, and 1 μL of that mixture was deposited on the MTP 384 ground steel target plate. The drop was allowed to dry in a nitrogen gas flow, which resulted in the formation of uniform sample/matrix co-precipitate. All samples were analyzed using the high mass linear positive mode method with 5000 laser shots at a laser intensity of 75%. The measurements were externally calibrated at three points with a mix of carbonic anhydrase, phosphorylase B, and bovine serum albumin.Ninety-six well Enzyme-linked immunosorbent assay (ELISA) plates  were coated with recombinant human ECM proteins (10 μg/mL): fibronectin (MilliporeSigma), decorin , collagen I (MilliporeSigma), collagen II (MilliporeSigma), collagen III (MilliporeSigma), or collagen IV (MilliporeSigma); or 1 μg/mL of recombinant murine TNFα  in PBS overnight at 37 °C, followed by blocking with 2% BSA in PBS with 0.05% Tween 20 (PBS-T) for 1 h at room temperature. Then, wells were washed with PBS-T and further incubated with test antibody (100 μg/mL each) for 1 h at room temperature. After three washes with PBS-T, wells were incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated antibody against rat IgG . After washes, bound antibodies were detected with tetramethylbenzidine substrate by measurement of absorbance at 450 nm with the subtraction of absorbance at 570 nm.123-144-α-TNF, 5 μg/mL of rabbit anti-human collagen II antibody (Abcam), or 25 μg/mL of mouse anti-human decorin antibody  were used as primary antibodies. After incubating with the fluorescently tagged secondary antibodies, slides were covered with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). The images were scanned with a Pannoramic digital slide scanner  and analyzed using a Pannoramic Viewer software (3DHistech).Frozen sections of cartilage from an osteoarthritis patient were purchased from OriGene Technologies . The sections were blocked with 2% FBS in PBS overnight at room temperature. The sections were incubated with primary antibodies for 3 h at room temperature. Either 50 μg/mL of rat α-TNF or equimolar PlGF-22 atmosphere.The murine macrophage-like cell line RAW264.7, an osteoclast precursor, was obtained from the American Type Culture Collection . Cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% FBS (Invitrogen), 100 U/mL of penicillin , and 100 μg/mL of streptomycin  at 37 °C in a humidified 5% CO6 cells per well and cultured for 7 days in the presence of 50 ng/mL of recombinant mouse TNFα , 10 ng/mL of recombinant mouse receptor activator of nuclear factor κΒ ligand , and 50 to 1000 ng/mL of test antibody. The cells were then washed with PBS and stained using Acid Phosphatase, Leukocyte (TRAP) kit (MilliporeSigma) according to the manufacture’s instruction. TRAP-positive multinucleated cells having three or more nuclei were considered as osteoclasts, and their number was counted in each well. Percent inhibition was calculated from a ratio of the number of osteoclasts in each treatment to that in the untreated control. The 50% inhibitory concentration (IC50) values were obtained by nonlinear regression analysis .RAW264.7 cells were seeded on 60-mm dishes at a density of 3 × 10Balb/c and athymic nude mice at 8 to 9 weeks of age were obtained from the Jackson Laboratory . Experiments were performed with approval from the Institutional Animal Care and Use Committee of the University of Chicago .123-144 peptide , 100 μg of control IgG , 100 μg of unmodified α-TNF, or 0.1 or 1 μg of PlGF-2123-144-α-TNF. Joint swelling in the left and right hind paws was scored daily as described elsewhere [Arthritis was induced in Balb/c mice by intraperitoneal injection of anti-collagen antibody cocktail at 1.5 mg  on day − 3, followed by intraperitoneal injection of LPS at 50 μg (Chondrex) on day 0. On the day of LPS injection, mice were injected at the left hind footpad with PBS, 15 μg of PlGF-2lsewhere . The lef123-144 peptide that was labeled with Cy7 in its N-terminus was chemically synthesized with > 90% purity by Thermo Fisher Scientific. Cy7-labeled PlGF-2123-144 was conjugated to α-TNF as described above. The Cy7 labeled antibody at a dose of 20 μg was injected at the left hind footpad of CAIA mice or athymic nude mice. The fluorescence level was measured using the Xenogen IVIS Imaging System 100  under the following conditions: f/stop: 2; optical filter excitation 745 nm; excitation 800 nm; exposure time: 5 s; small binning. Retention rates of the injected antibody immediately after the injection and that at 168 h after the injection were defined as 100% and 0%, respectively.α-TNF was fluorescently labeled using sulfo-Cyanine 7 (Cy7) NHS ester  according to the manufacture’s instruction. PlGF-2123-144-α-TNF or 100 μg of unmodified α-TNF. Blood was collected from the tail vein with a heparinized capillary at 24, 48, 72, and 96 h after the injection, and plasma was obtained by centrifugation. The injected antibodies in plasma were measured by ELISA as described above.CAIA mice received 1 μg of PlGF-2P < 0.05 was considered statistically significant. Binding activities against ECM proteins were analyzed using Student’s t test for comparisons between PlGF-2123-144-α-TNF and unmodified α-TNF. The retention effect of PlGF-2123-144-α-TNF was analyzed in the area under the percent of retention at the injection site-time curve from 0 to 96 h (AUC0-96h) compared with unmodified α-TNF using Student’s t-test. The AUC0-96h of plasma concentration was analyzed using Student’s t-test for comparisons between PlGF-2123-144-α-TNF and unmodified α-TNF. To compare the efficacy of PlGF-2123-144-α-TNF with unmodified α-TNF, the data on day 6 were analyzed using Tukey’s multiple comparison test.Statistical analyses were performed using GraphPad Prism software, and 123-144 peptide was covalently conjugated with α-TNF using a crosslinker. SDS-PAGE revealed that the molecular weights of both the light and heavy chains of α-TNF were increased  as measured by MALDI-TOF MS  of unmodified α-TNF and PlGF-2123-144-α-TNF were 98 (87–113) ng/mL and 128 (108–155) ng/mL, respectively. These data indicate that PlGF-2123-144 conjugation provided α-TNF with affinity against ECM proteins, with limited impairment of its function.We tested the effect of the PlGF-2.7 cells . RAW264.123-144-α-TNF at the injection site was determined by in vivo bio-distribution analysis in CAIA mice. Fluorescently labeled α-TNF or PlGF-2123-144-α-TNF was injected at the left hind footpad of CAIA mice on the day following arthritis induction by LPS. Whole-body fluorescence was measured immediately after the injection, and 0.5, 1, 2, 3, 24, 48, 96, and 168 h after the injection , unmodified α-TNF (100 μg), or PlGF-2123-144-α-TNF (1 μg) was injected at the left hind footpad of CAIA mice (Fig. 123-144-α-TNF; however, suppressed arthritis development almost completely in the treated paw even at a 100× lower dose than unmodified α-TNF. PlGF-2123-144-α-TNF did not suppress arthritis development in the untreated paw, indicating its localized efficacy. We also confirmed that PlGF-2123-144 peptide in itself did not suppress arthritis development (Additional file 123-144-α-TNF was not detected in plasma (Additional file 123-144-α-TNF at 10× further lower dose. PlGF-2123-144-α-TNF even at 0.1 μg completely suppressed the arthritis score at the treated paw (Fig. 123-144-α-TNF induces robust improvement of RA therapy without systemic exposure.To examine the therapeutic efficacy of PlGF-2ice Fig. . The artpaw Fig. a, and jopaw Fig. b. These 123-144 peptide conjugation enhanced the local efficacy of α-TNF by at least 1000× based on dose-comparison and that the therapeutic efficacy was robust. This demonstrates that PlGF-2123-144 peptide conjugation enables α-TNF to be retained at joints to block TNFα signaling thoroughly and enhance its potency. Significant reduction of the effective dose and corresponding systemic exposure of locally-administered PlGF-2123-144-α-TNF suggests that this technology may help avoid unnecessary exposure of α-TNF to the whole body, with a corresponding reduction in adverse events.Interest in clinical translation of intra-articularly administered therapies is growing rapidly . Systemi123-144-α-TNF showed high efficacy when injected at the paw. Since the mouse paw contains many joints and disease scoring in a single joint over time is difficult, in this study we injected drugs nearby the joint space. PlGF-2123-144-α-TNF binds strongly to ECM proteins including collagen II, i.e., major components of cartilage and synovium, and was retained at the injection site. Therefore, these data support the concept that that PlGF-2123-144-α-TNF could bind to ECM in cartilage and the synovium before draining from lymphatic vessels even when PlGF-2123-144-α-TNF is injected intra-articularly into the joint, although further evaluation of intra-articular injection using larger animals is warranted.PlGF-2Various technologies have been proposed to achieve prolonged retention of locally injected drugs, such as particle- or liposome-based drug delivery systems and modification by polypeptide conjugation . Althoug123-144 peptide, which naturally exists in the human body as a domain on PlGF, limiting the possibility of recognition by the immune system. It was confirmed that the PlGF-2123-144 peptide-conjugated antibody was capable of binding with human ECM proteins and human specimens. Also, the PlGF-2123-144 peptide can be conjugated to antibodies with a simple chemical reaction. The advantage of this feature is in the simplicity in production, in that it is possible to work with antibodies for which production has already been optimized. The PlGF-2123-144 peptide conjugation synthesis reaction for antibodies can be accomplished using chemistry that is analogous to PEGylation of proteins. Indeed, the same chemical reaction is used in antibody-drug conjugates, such as in the production of trastuzumab emtansine. These features may facilitate the development of ECM binding-drug therapy to overcome the barriers to clinical translation.For clinical translation of ECM-binding anti-inflammatory bio-therapeutics, an advantage also lies in the use of the PlGF-2We have found that installing an ECM-binding affinity to α-TNF enables it to be retained at the injection site. Moreover, ECM-binding α-TNF showed robust therapeutic efficacy in a mouse model of RA at a very low dose. This simple approach of generating an engineered ECM-binding α-TNF bio-therapeutic may hold potential for clinical translation as a local anti-inflammatory therapeutic in RA.Additional file 1:Figure S1. Retention of PlGF-2123-144-α-TNF at the injected paw of athymic nude mice. Figure S2. Effect of PlGF-2123-144 peptide locally injected around joints of the CAIA model. Figure S3. Changes in plasma concentrations after local injection to CAIA mice.


Tumor necrosis factor (TNF)-alpha was originally identified in the 1970s as the serum mediator of innate immunity capable of inducing hemorrhagic necrosis in tumors. Today, a wide spectrum of biological activities have been attributed to this molecule, and clinical translation has mainly occurred not in using it to treat cancer, but rather to inhibit its effects to treat autoimmunity. Clinical trials utilizing systemic TNF-alpha administration have resulted in an unacceptable level of toxicities, which blocked its development. In contrast, localized administration of TNF-alpha in the form of isolated limb perfusion have yielded excellent results in soft tissue sarcomas. Here we describe a novel approach to leveraging the potent antineoplastic activities of TNF-alpha by enhancing activity of locally produced TNF-alpha through extracorporeal removal of soluble TNF-alpha receptors. Specifically, it is known that cancerous tissues are infiltrated with monocytes, T cells, and other cells capable of producing TNF-alpha. It is also known that tumors, as well as cells in the tumor microenvironment produce soluble TNF-alpha receptors. The authors believe that by selectively removing soluble TNF-alpha receptors local enhancement of endogenous TNF-alpha activity may provide for enhanced tumor cell death without associated systemic toxicities. Streptococcus pyogenes and Serratia marcescens, was demonstrated to possess therapeutic activity and became widely used in the USA prior to the advent of chemotherapy and radiotherapy. Today, such “Coley’s toxins” are limited due to lack of controlled clinical trials and FDA approvals. In the 1960s, attempts to identify the molecular mechanisms by which Serratia marcescens induced tumor regression led to the discovery of a “factor” in the sera of treated mice [The history of TNF-alpha is very closely related to the history of tumor immunotherapy. In the early 1900s, the New York physician William Coley observed that various cancer patients would enter remission after experiencing bacterial infections. In a brave set of experiments, Dr. Coley began emulating bacterial infections by purposely administering various combinations of pathogens to patients. One of these mixtures, containing ted mice . It was Molecular analysis led to cloning of the cDNA and revealed the molecule was comprised of 233 amino acids with a leader sequence of the first 76 amino acids . InteresIt was found that administration of TNF-alpha in quantities approximating endogenous levels that were observed in response to endotoxin resulted in hypotension, metabolic acidosis, hemoconcentration, and death due to respiratory arrest within minutes to hours. This sequence resembled sepsis associated symptomology. Hyperglycemia and hyperkalemia were also observed after infusion. At necropsy, diffuse pulmonary inflammation and hemorrhage were apparent on gross and histopathologic examination, along with ischemic and hemorrhagic lesions of the gastrointestinal tract, and acute renal tubular necrosis –11. ThusTNF-alpha is found in a soluble and membrane bound form. The soluble plasma form of TNF-alpha is cleaved from the membrane forms by a metalloproteinase termed TNF-alpha-converting enzyme (TACE) which belongs to the ADAMs family of disintegrins , 13. SolThe activity of TNF-alpha is mediated through two cell surface receptors, TNF-R1 (p55) and TNF-R2 (p75) that differ in their signaling activity. TNF-R1 is usually pro-apoptotic whereas TNF-R2 is usually anti-apoptotic . TNF-R1 While TNF-R1 is expressed on various tumor cells , and tumThe effects of systemically administered TNF-alpha are blocked by soluble receptors, sTNF-R1 and sTNF-R2, that are released into the plasma . These aThe concentration of the soluble receptors increases following exposure to TNF that is produced after infections or upon administration of recombinant TNF as a function of the natural TNF buffering system to control runaway cytokine response . These rApproximately 28% of cancers are susceptible to direct cell killing mediated by soluble TNF. The anti-tumor activity of TNF-alpha is now well established and can be mediated through a variety of mechanisms including: 1) Cellular apoptosis by binding to tumor cell surface receptors; (2) T-effector cell activation (macrophage and NK cells) by blocking T-Reg cells that are immune suppressors , 55; (3) CellularAs the most pleiotropic of cytokines, TNF-alpha contributes to both inflammation during infections and anti-inflammatory/tissue repair processes after clearance of infections . Its effThe anti-tumor effects of TNF-alpha have been demonstrated on primary tumors with significant pancancer effects through vascular destruction and tumor necrosis . This efTumors can be categorized into three major types regarding their response to chemotherapy immune modulators: “hot”, “cold” and “intermediate”. Hot tumors contain a plethora of cell infiltrates whereas cold tumors have relatively few. Intermediate tumors are types that fall in between. Clinically, hot tumors tend to respond well to therapy, whereas cold tumors are resistant. Intermediate tumors may respond at first but then become resistant to therapy.With few exceptions, tumors are dependent on neovascularization and in theory share a common susceptibility to TNF induced vascular disruption. In sufficient amounts, the global effect of TNF is predictably rapid, dependent on pertussis toxin G-protein inhibition and stimulation of the release of protein S from the tumor endothelium to promote vascular modulation, and induces fibrin accumulation with clotting and enhancement of permeability leading to necrosis . This ef2 and escalated to 16 × 10 [2. Fever, rigors, nausea and vomiting, and anorexia toxicities where found to be non-dose-dependent; whereas hypotension, leukocytosis, thrombocytopenia and transient elevation of transaminases (SGOT and SGPT) where dose-dependent. Disseminated intravascular coagulopathy (DIC), a classical symptom of sepsis, was observed at the highest dose. The authors concluded that the maximum tolerated dose was 12 × 10 [2 [Initial clinical trials of TNF-alpha utilized systemic administration. Phase 1 studies all reported sepsis-associated symptoms as dose-limiting toxicities . For exa 16 × 10  units/m2 12 × 10  units/m2 × 10 [2 . Other s × 10 [2 –84. Part × 10 [2 .Given the inability to translate the profound anti-tumor effects observed in animal studies to human studies, some researchers have explored localized administration of TNF-alpha in hopes of avoiding adverse effects associated with systemic use. One of the first reports describing localized administration of TNF-alpha was Kahn et al. who treated 27 patients suffering from Kaposi’s sarcoma. Intratumoral administration reduced the cross-sectional area in 15 of 16 injected cancer lesions and caused the complete disappearance of three lesions . The notOne clinically successful utilization of localized TNF-alpha therapy is its use  in isolated limb perfusion (ILP) protocols. Early studies demonstrated synergy between TNF-alpha and melphalan in animal models of ILP , 92. TheAnother clinical success of localized TNF-alpha administration is in liver metastasis where isolated hepatic perfusion (IHP) may be performed. IHP was first clinically applied almost 50 years ago  and offeIn the light of their pro-tumor activity, the removal of soluble TNF-Rs seemed to be a logical step toward the development of an effective anti-cancer therapy , 53. To Overall, 50–60% of the patients treated were observed to have either stable disease or partial responses by RECIST Criteria during treatment with one patient having experienced a complete response with clearance of metastases. In over 300 treatments, throughout the study, the LW-02 devices appeared to be safe with no adverse events that were attributable to their use. The quality of life of the patients was effectively maintained during the treatment regimens and a significant life extension was observed based on initial prognoses.In humans, few treatment options are available for stage IV patients that are unresponsive to standard treatments. Thus, subtractive therapies may be an effective alternative to fill this gap.There is also potential for its use: 1) In combination therapies with cytotoxic drugs that induce or are enhanced in combination TNF-alpha (Table  In combiTNF-alpha is a fundamental molecule in various aspects of immunology. Original efforts at therapeutic applications failed due to systemic toxicities. In the new era of cancer immunotherapy, a promising research direction is augmentation of endogenous TNF-alpha activity through removal of its soluble receptors.


Heart failure with preserved ejection fraction (HFpEF) is characterized by signs and symptoms of heart failure in the presence of a normal left ventricular ejection fraction. Although it accounts for up to 50% of all clinical presentations of heart failure, there are no evidence-based therapies for HFpEF to reduce morbidity and mortality. Additionally there is a lack of mechanistic understanding about the pathogenesis of HFpEF. HFpEF is associated with many comorbidities  and is coupled with both cardiac and extra-cardiac abnormalities. Large outcome trials and registries reveal that being obese is a major risk factor for HFpEF. There is increasing focus on investigating the link between obesity and HFpEF, and the role that the adipose tissue and the heart, and the circulating milieu play in development and pathogenesis of HFpEF. This review discusses features of the obese-HFpEF phenotype and highlights proposed mechanisms implicated in the inter-tissue communication between adipose tissue and the heart in obesity-associated HFpEF. Heart failure (HF) is a clinical syndrome caused by structural and functional abnormalities in the heart that impair the ability of the ventricles to fill or eject blood. The cardinal manifestations of HF are breathlessness, dyspnea and fatigue, which may lead to limited effort tolerance; and fluid retention, thus resulting in pulmonary congestion and/or peripheral edema , 2. HF iSegregating patients with HF by left ventricular (LV) ejection fraction (EF) is an important phenotypic marker as it indicates unique pathophysiological mechanisms and thus subsequent responses to therapy –8. PatieIn contrast to HFrEF, there are no evidence-based therapies, to date, which have shown improved outcomes in HFpEF , likely Obesity has reached epidemic proportions worldwide and is a major comorbidity in HFpEF patients –25. The There is an increasing appreciation of the complex connection between the adipose tissue and the heart, which highlights the importance of the heart-adipose-axis in the pathogenesis of cardiovascular disease and specifically HF . Howeveraka adipokines, such as leptin or adiponectin, which elicit a variety of local and systemic responses  at peak exercise, and any factor that limits peak VO2, by impeding O2 delivery and/or utilization, can cause exercise intolerance  which are then processed to their biologically active forms ANP and BNP, and biologically inactive N-terminal proANP (NT-proANP) and NT-proBNP forms . Of thesThe inverse relationship between circulating cardiac BNP and obesity (defined by BMI) is termed the “natriuretic handicap” and has been described in both healthy subjects and patients with HF , 128. ItWhite adipose tissue was previously thought to only function as an energy storage unit with limited metabolic activity, and human brown adipose tissue to be active only in infants before disappearing in childhood. It is now known that both, white and brown adipose tissues have in highly active roles in metabolic regulation –143. We Metabolic disorders such as obesity and type 2 diabetes are associated with dysregulation of the natriuretic peptide system , 155. ThThere is also interplay between natriuretic petides released from the heart and adipokines released by adipose tissue. ANP decreases the secretion of leptin in cultured human subcutaneous adipose tissue  and isolHFpEF is a major public problem that is increasing in prevalence yet lacking in evidence-based therapies. A more tailored approach in HFpEF is needed to investigate the pathophysiological mechanisms that underlie this syndrome. Obesity-associated HFpEF is an important sub-phenotype of HFpEF, with evidence supporting crosstalk between the heart and the adipose tissue. Thus, the ability to modulate the signaling pathways that regulate adipose tissue and the heart in HFpEF might have clinical implications and be translated into effective therapies for HFpEF, particularly obesity-associated HFpEF.FS and MV-M discussed and conceived the outline of the manuscript. AO, RO, and MV-M drafted the initial version of the manuscript. FS and MV-M reviewed the manuscript. All authors 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.


To survive a predator’s attack successfully, animals choose appropriate actions from multiple escape responses. The motor performance of escape response governs successful survival, which implies that the action selection in escape behaviour is based on the trade-off between competing behavioural benefits. Thus, quantitative assessment of motor performance will shed light on the biological basis of decision-making. To explore the trade-off underlying the action selection, we focused on two distinct wind-elicited escape responses of crickets, running and jumping. We first hypothesized a trade-off between speed and directional accuracy. This hypothesis was rejected because crickets could control the escape direction in jumping as precisely as in running; further, jumping had advantages with regard to escape speed. Next, we assumed behavioural flexibility, including responsiveness to additional predator’s attacks, as a benefit of running. The double stimulus experiment revealed that crickets running in the first response could respond more frequently to a second stimulus and control the movement direction more precisely compared to when they chose jumping for the first response. These data suggest that not only the motor performance but also the future adaptability of subsequent behaviours are considered as behavioural benefits, which may be used for choosing appropriate escape reactions. Numerous studies in behavioural economics have revealed that this decision-making is based on the trade-off between the amount of expected benefit and effort for the chosen behaviour10. Thus far, most studies on animals have focused on the relationship between the choice in a learned task and the expected value of rewards such as food intake resulting from their reactions13. In this context, the amount of benefit is related with neither the motor characteristics nor performance of the chosen behaviour. In contrast, the performance of innate behaviours in natural environments is directly linked to their benefits, such as survival rate against predators15 and mating success rate17. Therefore, quantitative assessment of the performance of selected behaviours is necessary to understand the innate mechanisms of decision-making in animals.It is necessary to choose actions in every situation in daily life. Animals do not always enact the same reaction to a certain stimulus, in that they decide on the appropriate behaviour depending on the situation, external stimulus18 are a universal innate behaviour exhibited by various species, including vertebrates and invertebrates21. The benefits of escape behaviour can be regarded as the characteristics of its performance which make escape more successful. To successfully evade predator attacks, the speed of the escape movement is considered to be one of the most important features22. However, several animal species exhibit multiple types of escape actions; some actions are slower than others29. This suggests that slower escape reactions may have other benefits to compensate for speed. For instance, fish displays the following two different types of escape behaviour: C-turn behaviour triggered by firing of giant nerve cells identified as Mauthner cells (M-cells) and another without M-cell activity. The C-turn allows fish to escape at high speed31, but the stereotypic locomotion of the C-turn is considered a disadvantage because it makes the prey’s movement easily predictable by the predator32. Meanwhile, the slower response not mediated by M-cells is more variable in escape direction than that of the C-turn27, making it harder to predict by predators. Thus, the decision-making of fish for escape behaviour appears to be based on the trade-off between speed and unpredictability. However, little is known on the benefits of the trade-off underlying the decision-making in the context of innate behaviour. To address this question, a quantitative and comprehensive analysis of motor performance in innate behaviours is required.Escape actions that directly affect the survival of animals35. Jumping responses in crickets are similar to those in locusts in which animals escape exclusively in a forward direction with high speed39. These features of jumping seem to be advantageous for a successful escape. However, crickets exhibit running responses more frequently than jumping for escape elicited by an air stimulus40, suggesting that running for escape may have advantages other than speed. Our previous experiments using a treadmill system revealed that crickets run away from the stimulus source in a direction opposite to that of the airflow41. Their direction of movement in the wind-elicited escape response is precisely controlled by, and dependent on, the stimulus angle. The accurate control of movement direction reported in various species42 is critical for escape behaviour to increase distance from the predator. Thus, a trade-off between speed and directional control possibly exists in the cricket escape behaviour: jumping would allow crickets to escape quicker but to control their movement direction less accurately than running.Crickets exhibit several innate escape actions including running and jumping in response to a short air current detected as predator approach using the cercal sensory system43. In this case, it is important for the prey animals to flexibly adapt their behaviour in response to additional attacks. The crickets running away may be able to change their movement in response to additional attacks but jumping ones cannot. To mimic the predator’s chase, we applied two successive air currents and examined the response to the second stimulus that was applied during/after the running or jumping elicited by the first stimulus. Our results proposed that the trade-off in motor performance of the reaction as well as flexibility for taking subsequent actions could be involved in decision-making in escape behaviour.Here, we hypothesized that running and jumping of the cricket would have distinct behavioural benefits such as speed and directional accuracy. To test this hypothesis, we quantitatively assessed several aspects of motor performance of running and jumping and examined distinct benefits of these escape actions. Furthermore, we focused on behavioural flexibility as another important benefit for successful escape. Even if animals successfully avoid the first attack, predators may continue to chase and attack them40. Then, in order to elicit not only running but also jumping for each cricket, a faster stimulus (834 mm/s) was used in this study. Running was observed with a probability of 53%  and jumping was with a probability of 38%  of these linear relationships had similar values (0.840 for running and 0.820 for jumping). Further, we divided all trials into three categories, “behind”, “lateral”, and “front”, based on the stimulus angle and compared the magnitude of movement direction , which was used as the index indicating the variability in directional control. Figure J/RSSR in all shuffled datasets. The RSSJ/RSSR value of the original data, indicated by a black triangle in Fig. To statistically test the variability of movement direction against the stimulus angle, the mean of the residual sum of square  for all plots from the regression line was calculated for each response . These results implied differences in the dependency of the turn angle on the stimulus between running and jumping. To test the variability of the turn angle against the stimulus angle, the RSS/trials for the plots from the regression curves were calculated. The mean RSS/trials for jumping was approximately twice as large as that for running . In this stimulation, the second air-puff was applied before the end of the initial responses; the duration of the initial responses was >250 ms in running and >300 ms in jumping after the first stimulus onset, respectively . In this experiment, the second air-puff was given just after the initial response finished  and jumping (DJ) significantly decreased in “Double 400” compared to that in “Single”. In contrast, that difference between DR and DJ in “Double 100” was not significantly different from that in “Single”  within two weeks after the imaginal moulting were used throughout experiments. They were reared under 12:12-hour light/dark conditions at a constant temperature of 27 °C and could freely access food and water. The guidelines of the Institutional Animal Care and Use Committee of the National University Corporation, Hokkaido University, Japan, specify no requirements for the treatment of insects in experiments. All experiments were conducted during the dark phase at room temperature (26–28 °C).Laboratory-bred adult male crickets  covered with aluminium foil. After the beaker was carefully lifted, an air-current stimulus was immediately applied to the stationary cricket, and the wind-elicited response was recorded by the camera. Based on the video data , the markers on the animal were automatically traced, and several locomotor parameters were measured with motion analysing software . To monitor the entire movement trajectory, we adopted 285.7 × 285.7 mm frame size in 1024 × 1024 pixel resolution, which covered the whole arena.Cricket movement was monitored using a high-speed digital camera  above the experimental arena (ø = 260 mm) throughout experiments Fig. . After aThe air-current stimulus was a short puff of nitrogen gas from a plastic nozzle (ø = 15 mm) connected to a pneumatic picopump . One air-current nozzle was installed on the inside wall of the arena on the same horizontal plane as the animal Fig. . Since tTwo experiments were performed with different stimulations: single- and double stimulation  was used for all statistical analyses. Response probabilities were compared using Wilcoxon signed-rank test. Student t-test was used to compare running and jumping in metric locomotor parameters. For angular parameters, the absolute values (0°−180°) were compared between running and jumping in three different ranges of the stimulus angle, which corresponded to behind (−60° to 60°), lateral (60° to 120° and −60° to 120°), and front (120° to 180° and −120° to −180°) using Student t-test.55, movement direction was considered as non-circular data and linear regression analysis used as follows:Since a strong linear correlation between stimulus angle and movement direction was observed as in previous studiesα) was tested. In contrast, to analyze the relationship between stimulus angle and turn angle that was considered circular data, circular-circular regression analysis was used as follows:Significance of the slope of the regression line (R) and jumping (RSSJ), and the ratio of RSSJ to RSSR was calculated. If the RSSJ/RSSR was larger than 1, it meant that the movement directions in jumping were more varied than those in running. Then, the category of the data, “running” and “jumping”, was shuffled 1,000 times, and the distribution of RSSJ/RSSR for all shuffled data was plotted. If RSSJ/RSSR for the original (non-shuffled) data was outside the range of the mean ± 2SD of the ratio for shuffled data, the difference in variability of the angular parameters between running and jumping was considered significant.Permutation test was adopted to assess the difference in variability between running and jumping in the distribution of movement direction or turn angle against stimulus angle. Initially, the mean value of residual sum of square  was measured for each plot from the regression line or curve in running (RSSR) and jumping (DJ) to their total sum was compared using Wilcoxon signed-rank test with Bonferroni correction for multiple comparisons.In the double stimulus experiment, to compare “Single” with “Double 100” or “Double 400” in total movement distance of individuals, paired t-test with Bonferroni correction for multiple comparisons was used. To determine whether the difference between running and jumping in total movement distance was altered by two stimulations, the ratio of the difference between the distance of running (DSupplementary information


Direct TNF inhibition through identification of potent small molecules is a highly desired goal; however, it is often hampered by severe limitations. Our approach yielded a priority list of 15 NPs as potential direct TNF inhibitors that were subsequently tested in vitro against TNF and RANKL. We thus identified two potent direct inhibitors of TNF function with low micromolar IC50 values and minimal toxicity even at high concentrations. Most importantly, one of them (A11) was proved to be a dual inhibitor of both TNF and RANKL. Extended molecular dynamics simulations with the fully automated EnalosMD suite rationalized the mode of action of the compounds at the molecular level. To our knowledge, these compounds constitute the first NP TNF inhibitors, one of which being the first NP small-molecule dual inhibitor of TNF and RANKL, and could serve as lead compounds for the development of novel treatments for inflammatory and autoimmune diseases.An  These r therapy , psorias therapy .To date, three synthetic antibodies that block the activity of TNF have been reported, namely infliximab, adalimumab, and etanercept . HoweverAccording to a recent report , there a2), solvent-exposed, and are characterized by hydrophobic and electrostatic interactions    and part2) . An effe2) . Moreove2)  and the 2) , the use2) , and cel2) . Moreove2) , and a m2) , which a2) . ImportaDrug discovery based on NP-like scaffolds has rapidly advanced through novel computational approaches . Recent in vitro to assess their inhibitory activity against TNF.In search of plant-origin NPs as direct TNF inhibitors, we combined chemoinformatics techniques, high-throughput virtual screening, and molecular dynamics (MD) simulations with experimental evaluation, ultimately aiming at discovering potent TNF-functioning NP inhibitors. 3,573 pure NPs of plant origin were virtually screened from the MEGxp database, which is one of the largest chemical libraries of NPs available ; the highest scoring compounds were then tested Scheme 1.Our strategy for identifying these novel plant-origin small molecule TNF inhibitors is presented in The initial model of TNF was built from the X-ray co-crystal structure of TNF dimer with SPD304 (PDB code: 2AZ5). All structures were prepared using Molegro’s Molecules and Protein Preparation Wizard . Proper Experiments included a TNF-induced death assay in L929 cells, a measurement of cytotoxicity in L929 cells, and a TNF/TNFR1 ELISA assay. Compounds were tested with respect to TNF using a battery of previously reported assays .4 cells per well and cultured in AMEM medium (GIBCO) containing 10% fetal bovine serum supplemented with 40 ng/ml RANKL (Peprotech) and 25 ng/ml M-CSF (R&D Systems) for 5 days (Bone marrow (BM) cells were collected after flushing out of femurs and tibiae, subjected to gradient purification using Ficoll-Paque , plated in 96-well plates at a density of 6 × 10r 5 days . Compoun4 cells per well and cultured in AMEM medium (GIBCO) containing 10% fetal bovine serum supplemented with 40 ng/ml RANKL (Peprotech) and 25 ng/ml M-CSF (R&D Systems) for 4 days. Then, cells were lysed in ice-cold phosphate buffer containing 0.1% Triton X-100. Lysates were added to 96-well plates containing phosphatase substrate (p-nitrophenol phosphate) and 40 mM tartrate acid buffer and incubated at 37°C for 30 min. The reaction was then stopped with the addition of 0.5 N NaOH. Absorbance was measured at 405 nm on a micro-plate reader . TRAP activity was normalized to total protein which was determined using the Bradford assay (Bio-Rad).In the TRAP activity assay, BM cells were plated in 96-well plates at a density of 6 × 105 cells/well in 96-well plates and incubated with A11 and A25 compounds for 48 h in AMEM containing 10% fetal bovine serum supplemented with 25 ng/ml M-CSF (R&D Systems). After removal of the medium, each well was incubated with 0.5 mg/ml MTT (Sigma–Aldrich) in AMEM serum-free medium at 37°C for 2 h. At the end of the incubation period, the medium was removed and the intracellular formazan was solubilised with 200 μl DMSO and quantified by reading the absorbance at 550 nm on a micro-plate reader . Percentage of cell viability was calculated based on the absorbance measured relative to the absorbance of the untreated control.Cytotoxicity was evaluated for BM cells using the 3--2,5-diphenyltetrazolium bromide (MTT) assay, which measures the ability of viable cells to reduce a soluble tetrazolium salt to an insoluble purple formazan precipitate. BM cells used for MTT assay were seeded at a density of 10Molecular dynamics simulations were performed with our in-house developed EnalosMD suite of programs . A fully(a)Initial model structures were constructed with AmberTools16 . Missing(b)-1 regulated the temperature Analysis of the results  was performed with the cpptraj version of AmberTools.in silico explored 3,573 NPs contained in MEGxp database using a structure-based docking approach. The crystal structure of TNF dimer with SPD304 (PDB code: 2AZ5) was used as the molecular model for our investigation and the compounds were docked into the protein–protein interface. Computational molecular docking studies were performed using MVD  was then further characterized. In dose–response experiments, the small molecules were shown to inhibit human TNF-driven death in L929 cells with an IC50 of 35 ± 3 μM (A11) and 33 ± 2 μM (A25). Both compounds were found to be minimally toxic in these cells (LC50 > 80 μM), in contrast to the published high toxicity of SPD304 (7.5 μM) .Out of the 15 prioritized NPs mentioned above, two emerged as the most promising ones based on (7.5 μM) . An alre50 of 3.3 ± 0.9 μM for A11 and 4.1 ± 1.7 μM for A25. Adalimumab was again used as a positive control eliminating the TNF-TNFR1 binding with a low IC50 of 0.2 nM .Having established that the selected products can obstruct the function of TNF, and given that TNF exerts its functions primarily through interacting with its receptor, TNFR1, an ELISA-based assay was used to quantify effects on this interaction. Both compounds significantly reduced binding of TNF to TNFR1, with an estimated ICParthenocissus tricuspidata and the glycosyloxyflavone analog A25 -6-oxy-4,5-dihydroxy-6-methyloxan-3-yl]oxy-3,4,5-trihydroxyoxan-2-yl]methyl(E)-3-(4-hydroxyphenyl)prop-2-enoate) is an NP that has been isolated from Ginkgo biloba . Except being isolated from natural sources, A11 can also be synthesized through a selective functionalization procedure as described by Rodrigues et al.  constitute structures that are ideal binders to the concave hot-spot area of the protein -1,7-Bis-2,6,8,12-tetrakis(4-hydroxyphenyl)-1,2,6,6a,7,8,12,12a-octahydrofuroindenoindeno furan-5,11-diol) is an NP that has been isolated from s et al. . Compouns et al. . Indeed, protein . It has  protein .In comparison to SPD304, NPs A11 and A25 are predicted by the molecular docking study to occupy a similar region in the binding pocket, and to be relatively hydrophobic and large enough to interact with residues from both subunits of the TNF dimer. Nonpolar residues are predominant in the binding site, which mainly includes glycine, leucine, and tyrosine. Only one HB interaction is observed between compound A25 and Tyr151. Both compounds appear to be situated more closely to subunit A than subunit B and are in close contact with the Leu120-Gly121-Gly122 β-strand of subunit A. The lack of salt bridges or extended HB interactions indicates the hydrophobic character of A11 and A25 binding as also observed with SPD304. The docked SPD304 conformation reproduced its crystal form, with an RMSD of 0.67 Å between the two structures. The docking score of SPD304 binding to TNF was calculated to be -171.08 (arbitrary units), and compounds A11 and A25 showed a binding score of -195.76 and -180.19, respectively, thus suggesting a strong interaction between the compounds and the TNF dimer. The high inhibitory potency of A11 and A25 against TNF was also indicated by our recently developed TNF model, released through the Enalos Cloud platform . After sFigure 4). Moreover, using a quantitative assay that measures TRAP activity, A11 inhibited RANKL-induced osteoclastogenesis in a dose-dependent manner, displaying an IC50 of 3.42 ± 0.45 μM . Furthermore, in order to exclude the possibility that inhibition of A11 on TRAP activity was due to cytotoxicity, the viability of BMMs was tested through the MTT assay. A11 displayed an LC50 of 44.76 ± 4.61 μM , suggesting that it affects osteoclastogenesis without interfering with cell viability. On the other hand, A25 had no effect either on osteoclastogenesis or BMM viability (LC50 > 100 μM) .Receptor activator of nuclear factor kappa-B ligand, another TNF superfamily member, is the main regulator of osteoclast formation and bone resorption . We evalWe subsequently investigated the binding of A11 to RANKL using the proposed molecular scaffolds in a structure-based approach. For this purpose, we employed the jFATCAT pairwise structure alignment algorithm  to alignFigure 6).Additionally, we employed our recently developed EnalosMD suite to perform extended MD simulations for A11 and A25 in complexes with TNF and RANKL. EnalosMD automates the preparation of any ligand-protein system and performs MD calculations in a way that minimal effort by the user is required. This application provides a powerful way to perform robust MD calculations with unprecedented speed and easiness regarding the construction of the initial model structure. Therefore, we carried out four 1000 ns-long MD runs to identify structural and energetic properties of the complexes that may further elucidate the mode of action of the two compounds. EnalosMD offers optimal performance by combining several computational programs and functionalities . However, during the first 200–250 ns of A25–RANKL complex simulation, a noticeable conformational change of A25 stabilized the molecule in a new orientation with respect to the binding site of RANKL . This conformational change may have induced great flexibility to B chain terminal residues Tyr187–Asp189 as denoted by further fluctuation calculations . Therefore, the experimentally observed lower affinity of A25 against RANKL compared to A11 may be rationalized through the A25-induced destabilization of the terminal region of monomer B. Average conformations of A11 and A25 into their protein targets, along with protein residues that are involved in dominant HB interactions with the compounds are shown in Figures 9, 10. The sole interaction between A25 and Tyr151, which was shown after docking calculations in TNF complex is also observed by the MD runs, however, it is complemented by three significant interactions from chain A .The MD results showed that protein structures early stabilized during the simulations in all complexes with RMSD values that do not exceed 3 and 4 Å in TNF and RANKL complexes, respectively  were shown to have ICAll animal procedures were approved and carried out in strict accordance with the guidelines of the Institutional Animal Care and Use Committee and the Region of Attica Veterinarian Office.AA and GK conceptualization, funding acquisition, methodology, project administration, and supervision. GM, EN, VR, DP, GL, ED, AA, and GK data curation, formal analysis, investigation, resources, validation, visualization, writing – original draft, and writing – review and editing. GM, GL, and AA software.GL and AA are affiliated with NovaMechanics Ltd., a drug design company. 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.


TNF locus to generate a novel transgenic mouse, the hTNF.LucBAC strain. In vitro stimulation of hTNF.LucBAC cells of different origin revealed a cell specific response to stimuli demonstrating the integrated construct’s ability as a proxy for inflammatory gene response. Lipopolysaccharide was the most potent luciferase inducer in macrophages, while TNF was a strong activator in intestinal organoids. Lipopolysaccharide-induced luciferase activity in macrophages was downregulated by inhibitors of NF-κB pathway, as well as by Interleukin-10, a known anti-inflammatory cytokine. Moreover, the transgene-dependent luciferase activity showed a positive correlation to the endogenous murine soluble TNF secreted to the culture medium. In conclusion, the hTNF.LucBAC strain is a valuable tool for studying and screening molecules that target TNF synthesis and will allow further functional studies of the regulatory elements of the TNF locus.Tumour necrosis factor (TNF) is a key cytokine during inflammatory responses and its dysregulation is detrimental in many inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. Here, we used a bacterial artificial chromosome (BAC) construct that expresses luciferase under the control of the human  Uncontrolled release of biologically active TNF is linked to development of inflammatory and autoimmune diseases like rheumatoid arthritis, inflammatory bowel disease (IBD), psoriasis and ankylosing spondylitis1. At the DNA level, the coding region of human TNF gene is located within TNF/LT locus, together with the Lymphotoxin-α (LTA), and Lymphotoxin-β (LTB) genes2. The TNF gene is one of the immediate early genes and there is a wide range of stimuli that activate its transcription. Such examples are the calcium-dependent pathway3, bacteria (such as Mycobacterium tuberculosis) and viruses5, osmotic stress6 and TNF itself 7. The transcription of the TNF gene is regulated by the enhanceosome, a higher-order protein structure, which in the case of the TNF promoter, assembles in a cell-type and stimulus-specific manner8. For example, calcineurin phosphatase mediates the induction of human TNF in stimulated T- and B-lymphocytes3. However, in macrophages, lipopolysaccharide (LPS) induces human TNF production by activation of extracellular signal-related kinase (ERK) activity9. The NFAT, ATF-2/Jun, Ets/Elk, and Sp1 protein along with CEB/p300 play a crucial role in regulation of TNF expression10. Moreover, there is a number of constitutive and inducible DNase I hypersensitive sites (HSs), which have been mapped across the TNF/LT locus. Barthel and colleagues showed that intron 3 in the human TNF gene contains a constitutive HSs site in Jurkat T cells, but not in THP-1 monocytic cells, demonstrating cell-type specificity of different regulatory regions11. Therefore, the human TNF gene has multiple transcription-binding sites that act in a cell-type and stimulus-specific fashion. In 1991, the first human TNF transgenic mouse was reported and revealed a major impact of TNF in chronic inflammatory polyarthritis12. Since then, many studies have utilised TNF transgenic mice in order to evaluate the pathways in rheumatoid arthritis13 and many studies focused on anti-TNF therapies14. Due to complex genomic organisation and regulation of the TNF gene15, it is vital that any reporter system should include all potential regulatory elements, and in the correct context to faithfully recapitulate and accurately report on TNF gene activation in different cell-types. Reporter constructs are created by placing a suitable genetically encoded reporter gene under the control of the regulatory elements of the gene of interest. Classically, plasmids and viruses have been widely used, but are limited by the size of genetic material that can be included. However, bacterial artificial chromosomes (BAC) encompass large (~150–200 kb) genetic regions, that can be propagated and manipulated in vitro by recombineering strategies to integrate a reporter gene16. The TNF genetic region is complex with respect to gene activation, thus the use of a BAC would ensure the inclusion of all such elements. Furthermore, the use of a human BAC on a mouse background allows for the study of human gene activation in a surrogate physiological system. Genetically encoded reporters, such as luciferases and fluorescent proteins, are powerful tools to detect gene expression, providing both accurate and quantifiable dynamic measurement of activity in gene expression. Firefly  luciferase is particularly suitable for use in vivo due to its short half-life (40 min for destabilised Luc), high signal to noise ratio, high dynamic range and favourable pharmacokinetics of its luciferin substrate. The emission spectrum is shifted slightly towards red, often enabling direct in vivo assay17. Moreover, it is sensitive reporter with a wide dynamic range, a short-assay time and is non-radioactive. In this study, we have generated a reporter mouse by utilising a BAC that carries the firefly luciferase coding sequence under the control of the human TNF promoter. This system allowed us to directly measure human TNF promoter activity in ex-vivo and in vitro approaches.Tumour necrosis factor (TNF) is an important pro-inflammatory cytokine produced by the majority of the cells of the immune system. It plays a crucial role in homeostasis and disease pathogenesisTNF is located within TNF/LT locus, together with the lymphotoxin-α (LTA), and lymphotoxin-β (LTB) genes. Several transcription factor binding sites have been identified in the human TNF promoter, in upstream and downstream sequences , as well as luciferase mRNA (1.76 ± 0.13) occurred at 1 h , as well as luciferase mRNA (1.71 ± 0.18) also occurred at 1 h  that peaked about 3 h post-stimulation , however it was noted that peak fold-change in luciferase mRNA (41.88 ± 7.7) was delayed, occurring at 3 h  differentiated ion Fig. . Transcr3 h Fig. . Neverth3 h Fig. . BMDMs w3 h Fig. ; all p <3 h Fig. . Other s3 h Fig. . Overall4 cells per well, but sensitivity of luciferase detection was increased with a higher cell number  of luciferase production to the soluble TNF secreted to culture medium was 0.97. To rule out possible autocrine effects elicited by secreted TNF in LPS-stimulated hTNF.LucBAC BMDMs, we measured luciferase activity in the absence or presence of neutralizing anti-TNFR1/2antibodies. The results showed that there was little, if no effect of blocking the TNFR1 and TNFR2 pathways  and the proteasome inhibitor MG132 (10 μM), blocked p65 DNA-binding activity in LPS-stimulated hTNF.LucBAC BMDMs; both p < 0.01 ANOVA , which in turn leads to activation of NF-κB and MEK/ERK signalling pathwaysTNF promoter-regulated luciferase activity, hTNF.LucBAC BMDM cultures were pre-incubated in the presence of these inhibitors for 30 min (concentrations as described above), followed by stimulation with 10 ng/mL LPS. Luciferase activity was monitored continuously over a period of 16 h. We observed that MEK inhibition by PD0325901 resulted in ~30% reduction in transgene activation, whereas NF-κB signalling inhibition by BI605906 or MG132 treatment resulted in almost complete inhibition of luciferase activity, all p < 0.01 ANOVA  was able to suppress luciferase activation in a dose-dependent manner in 10 ng/mL LPS-stimulated hTNF.LucBAC BMDMs, with IL-10 alone having no effect on luciferase activity; for all doses p < 0.05 ANOVA  = 0.98 , a well-established, potent negative regulator of TNF in both monocytes and macrophagesOVA Fig. . The calOVA Fig. . The levOVA Fig. . The inh.98 Fig. . The IKK.98 Fig. .Figure 7TNF promoter, and we have examined the response of this transgene in both transfected human cell-lines and primary cells derived from the hTNF.LucBAC transgenic mouse. BACs are large DNA constructs that typically transiently transfect at low efficiencies; however, in two cell-lines tested, SK-N-AS and HCT116, we were able to detect inducible luminescence following stimulation with various ligands that activate the TNF promoter, including TNF 25 and IL-1β (binding IL-1R)26. The TLR1/2 agonist PAM3CSK4 was also able to induce luciferase expression in SK-N-AS cells, but we observed no reporter gene activation with PAM3CSK4 in the HCT116 cell-line. HCT116 cells have been reported to lack expression of the TLR1/2 receptor complex27, which likely explains the absence of reporter activation. These data clearly indicate that the hTNF.LucBAC construct is functional and can report differential TNF gene activation patterns in cultured human cell-lines. Low transfection efficiencies known to occur using large DNA constructs may limit in vitro application to other cell-types, including transfection of primary cells, although other suitable vectors may be applied. Importantly, the BAC approach gave us opportunity to stably integrate the construct into the mouse genome, allowing for dynamic assessment of TNF gene activation in a more physiological context, and for study of a variety of different cell-types via generation of reporter primary cultures.In this study, we have generated a novel hTNF.LucBAC construct and a reporter mouse that expresses luciferase under the control of the human Tnf gene), showing that the luciferase protein expression follows the activation of the hTNF promoter. It has also been reported previously, that peritoneal macrophages and BMDMs have subtly different expression patterns of common cytokine and chemokine receptors, with different gene expression kinetics upon activation with LPS29. Whilst LPS was consistently the strongest effector, we also showed that the hTNF.LucBAC BMDMs could respond to other pro-inflammatory cytokines (including TNF itself) and to other bacterial effectors, such as MDP. Furthermore, using hTNF.LucBAC isolated splenocytes, a mixed population consisting mainly of T- and B-lymphocytes as well as macrophages, we also observed the same rapid induction of luciferase with lower overall fold induction. The latter observation was likely because only B-lymphocytes, which represent 40–50% of total splenocytes, can respond to LPS in such an experimental setting. However, the doses of LPS we used in this study were far lower than those previous reported for in vitro B-lymphocyte activation32 and as a result lack of luciferase induction is to be expected. It is also worth noting that unstimulated splenocyte cultures showed elevated luciferase activity. This is probably because the various cell-types within this mixed population, cannot reach a resting status as they likely interact with each other, elevating basal levels of stimulatory molecules such as IL-12, TNF and IL-6, each of which can enhance TNF promoter activity33.To confirm inducible reporter gene expression in our hTNF.LucBAC mouse model, we successfully derived and screened various tissue cell populations and their response to key pro-inflammatory stimuli. Peritoneal macrophages and BMDMs both demonstrated a strong response to LPS, resulting in rapid induction of luminescence with similar maxima of luciferase activity. BMDMs though, showed a delayed relative luciferase peak activity (peak observed at 3–4 h) compared to peritoneal macrophages (peak observed 2–3 h). This delay in BMDMs was also observed at the transcriptional level of the luciferase gene 35. IBD is a chronic inflammatory condition of the gastrointestinal tract where TNF is a major proinflammatory molecule involved in both development and progression of the disease, and therefore represents an important biological target for drug development36. We therefore derived mouse intestinal crypt stem cells and differentiated them to 3D intestinal epithelium organoid cultures in order to test our reporter system. We observed induction of luciferase activity in hTNF.LucBAC organoid cultures in response to TNF, peaking at about 14 h post-stimulation. Unlike BMDMs, other stimuli such as IL-1β and bacterial products were unable to elicit a reporter response in intestinal organoids. Overall, these results highlight the utility of this transgenic mouse model, where cell-types of interest can be isolated to specifically test stimulation response patterns to physiological stimuli and to pathological changes.A key new technology in biological research has been the development of 3D organoid cultures37, whilst in non-immune cells , the TNF promoter is activated by TNF alone, in an autocrine way, as previously reported38. BMDMs are an ideal model for inflammation research as they can be obtained in high yield, with homogeneity and a long lifespan in culture28, and are strongly responsive to a variety of physiological stimuli. We measured both dynamic luciferase activity in LPS-stimulated hTNF.LucBAC BMDMs and their secretion of soluble murine TNF and found a strong positive linear correlation, suggesting that the regulation of the human TNF-luciferase transgene was indeed similar to regulation of the endogenous murine Tnf promoter. Our hTNF.LucBAC luciferase reporter system therefore allows for measurement of real-time gene activation in living cells as a response to pro-inflammatory stimuli. The reporter data described here, together with our transcription-based and secretion-based end-point assays, supports previous work using transcriptomic approaches39.In BMDMs, the most potent effector ligand was LPS, followed by TNF and MDP, respectively. It is well established that LPS is a major inducer of inflammatory response in immune cells, leading to rapid expression and secretion of pro-inflammatory cytokines40. Activation of transmembrane receptors by external stimuli ultimately signal to the IKK complex. IKK-2 subsequently phosphorylate IκBs, a family of inhibitor proteins that retain NF-κB dimers within the cytoplasm in an inactive state. Phosphorylated IκBs are then targeted for ubiquitination and degradation by the proteasome which releases, NF-κB dimers to translocate into the nucleus and activate their target genes. Inhibitors of NF-κB have been developed targeting this key signalling pathway, and NF-κB has long been thought as a potential therapeutic drug target43. We tested whether BMDMs derived from our mouse model could be used to assay known inhibitors of this pathway. Using potent inhibitors of IKK-2 (BI605906) and the proteasome (MG132), we were able to show significant blockade of the induced luciferase activity and inhibition of p65 DNA-binding activity in LPS-stimulated BMDMs obtained from the hTNF.LucBAC reporter mouse. Our inhibition data is in agreement with that of a previous study which showed similar levels of inhibition of NF-κB DNA-binding by MG132 in rat renal tubular epithelial cell-line stably transfected with the pNF-κB-Luc reporter construct44. Here we also tested the inhibitor PD0325901 which is known to block MEK phosphorylation. Treatment of hTNF.LucBAC BMDMs with PD0325901 showed complete blockade of ERK phosphorylation, decreased LPS-induced luciferase activity and reduced secretion of soluble mouse TNF. Previous studies using this inhibitor and genetic approaches (Map3k8−/− and Map3k8 kinase-dead mice) have revealed that the TPL2/MEK/ERK pathway is involved in the regulation of TNF production in macrophages20.Key regulators of the tumour necrosis factor promoter are members of the NF-κB family, transcription factors that share a highly conserved DNA-binding domain known as the Rel homology domain (RHD)45, and a later study by Smallie et al. also showed that IL-10 could inhibit transcriptional elongation of TNF in LPS-treated human macrophages by blocking RelA recruitment to a sequence downstream of the TNF 3′ untranslated region46. Previous use of an adenovirus-based system to express TNF promoter-luciferase reporters of varying, but limited, sizes (~1000 bp) had shown LPS-induced expression can only be mildly attenuated by IL-1046. Whilst exogenous expression systems such as these are flexible, and do not require the maintenance of a transgenic mouse colony, they suffer from a lack of regulatory context. Our hTNF.LucBAC mouse model however, shows far greater repression of LPS-induced luciferase activity by IL-10 over an extended time period using live cell luminometry. This likely reflects the more physiological situation in which the regulation and function of the human TNF promoter is studied.One of the most potent natural inhibitors of TNF is Interleukin-10 (IL-10). IL-10 is an immunoregulatory cytokine that plays an essential role in suppressing pro-inflammatory molecules such as TNF and other cytokines, including IL-6 and IL-1β. In our study, we observed that IL-10 could indeed supress the transgene-regulated luciferase activation of LPS-stimulated hTNF.LucBAC BMDMs and reduce release of soluble TNF to the culture medium. In support, Denys and colleagues have previously reported that IL-10 could supress TNF production in primary human macrophagesTNF gene activation are maintained. In this study, the construct responded to a variety of known pro- and anti-inflammatory stimulations. This model could easily be applied in screening for pro- and anti-inflammatory molecules in tissue specific ex vivo organotypic culture systems, such as intestinal organoids as demonstrated here. With access to high-sensitivity bioluminescence imaging systems, it is plausible that this model could be used for live in vivo analysis of inflammatory modulators.The use of a BAC as the basis of our reporter system is vital as it ensures all known and as-yet unknown regulatory elements involved in et al. previously generated a NF-κB reporter mouse47. This mouse model has a randomly integrated DNA construct consisting of three NF-κB binding sites from the Ig light chain promoter driving luciferase expression, which served as good reporter of NF-κB activation ex vivo and in vivo. By imaging anaesthetised NF-κB reporter mice, Carlsen and colleagues were able to visualise luminescent reporter signal after immune challenge47. Our interest lies in the regulation of the human TNF gene, which is affected by several signalling pathways and transcription factors. Therefore, the human TNF promoter requires expression from a recombinant BAC to fully contain all critical regulatory sites and in correct positional context. In future experiments it would be interesting to study the dynamics and reporter intensity of our BAC-based system. In conclusion, we have generated a valuable reporter system for studying the regulation of the human TNF promoter in different cell-types and tissues. Future experiments will reveal whether the hTNF.LucBAC mouse strain can be used to study the regulation of TNF synthesis in the context of both health and disease, and to assess potential therapeutic targets/interventions.Carlsen TNF BAC clone  was purified using a Machery-Nagel Nucleobond BAC100 kit and used to transform electro-competent SW102 E. coli . SW102-TNF positive clones were confirmed by pulsed-field gel electrophoresis analysis. Recombineering was performed using seamLess GalK mediated selection/counter selection strategy, as described previously48. In brief, two targeting cassettes designed to excise the first coding exon of the TNF gene were generated to first incorporate the GalK gene within the BAC, positive recombinants derived by selection on minimal media containing galactose, followed by the removal of GalK and integration of the luciferase-polyA coding sequence from pGL3Basic  and counter selection using 2-deoxy-galactose and glycerol. Correctly modified colonies were identified by PCR screening and pulsed-field gel electrophoresis.The human The hTNF.LucBAC construct was purified, linearized by BsiWI restriction enzyme digestion and subjected to Sepharose 4B-CL column purification. Linear DNA was then microinjected into the pronucleus of one-day single cell mouse embryos from B6D2F1 mice (Envigo). Zygotes were cultured overnight, and the resulting 2 cell embryos surgically implanted into the oviduct of day 0.5 post-coitum pseudo-pregnant mice. After birth and weaning, genomic DNA was extracted using RED Extract-N-AmpTM Tissue PCR Kit  and used to genotype pups. Founders with luciferase gene integration were back crossed onto C57Bl/6JOlaHsd and transgene positive mice derived from the founders were used in all subsequent experiments. All experimental procedures and protocols were carried out in accordance to the guidelines of the Animals (Scientific Procedures) Act 1986, and subject to local ethical approval by the University of Manchester Animal Welfare and Ethical Review Board (Project licence number 70/7800).TNF promoter gene by using 250 nmol/L PCR primers as follows: hTNF-F: GGGCAGCTCCAGTGGCTGA and hTNF-R: GGTAGGAGACGGCGATGCGG. The PCR conditions were as set up initial denaturation step at 95 °C for 30 s, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and extension at 68 °C for 60 s. We used a PTC-225 Peltier Thermal Cycler (MJ Research) for all PCR reactions.Genomic DNA extracted using RED Extract-N-AmpTM Tissue PCR Kit  and stored at −20 °C was used for genotyping by Polymerase Chain Reaction (PCR). The PCR product generated was a result of amplification of a region of the human ®; CCL-247™) were cultured in MEM and DMEM respectively, supplemented with 10% v/v foetal calf serum  and 1% w/v non-essential amino acids (Sigma-Aldrich). Both cell-lines were sub-cultured at densities between 80–90%. Cells were routinely tested by PCR to exclude mycoplasma contamination. For transient transfection, BAC DNA was purified  and 3 μg was used to transfect 106 cells in a 10 cm dish using 9.87 µL ExGen500 transfection reagent (following manufacturer’s recommendations). For luminometry, cells were split and 5 × 105 cells were re-seeded in 24 well luminometry plates 24 h prior to stimulation.The human neuroblastoma cell-line SK-N-AS  and human colon carcinoma cell-line HCT116 , and 50 μM β-mercaptoethanol) at 5 × 106 cells per 90 mm bacterial petri dish . After 4d of culture, 10 mL of complete BMDM medium was added, and cells were cultured a further 3d. Non-adherent cells were aspirated, and remaining adherent cells were harvested by incubation with 5 mL of PBS supplemented with 5% v/v FBS and 2.5 mM EDTA. The purity of BMDMs were >95% F4/80 positive as determined by flow cytometry. For protein chemistry experiments, BMDMs were cultured as 1 × 106 cells/well, in 6-well plates in 2 mL medium and they were left to rest overnight. The next day, the cells were stimulated with 10 ng/mL LPS for the indicated time points and at the end of stimulation, they were washed once in cold PBS and lysed in 250 μL RIPA buffer (Sigma) for 15 min on ice. After centrifugation at 10000 × g for 10 min, the clear supernatant was transferred into new Eppendorf tubes and stored at −80 °C until further use. For detection of ERK phosphorylation and total ERK protein, the ERK1/2  ELISA Kit  was used. For detection of p65 DNA-binding activity, the TransAm Flexi NFκB p50/NFκB p65 ELISA kit  was used.Mice were culled by cervical dislocation and resident peritoneal macrophages were harvested following intraperitoneal injection of mice with 4 mL of RPMI medium supplemented with 10% v/v foetal bovine serum (FBS) and subsequent aspiration. Macrophages were purified from the resulting cell suspension by adherence after 3 h of culture at 37 °C and removal of non-adherent cells by washing with phosphate-buffered saline (PBS). Bone marrow-derived macrophages (BMDM) were isolated and prepared as described previouslySingle-cell suspensions were generated from spleen via gentle homogenization through nylon mesh filters . Erythrocytes in spleens were lysed with ammonium chloride potassium (ACK) lysis buffer and the cell pellet was resuspended in DMEM medium supplemented with 10% v/v/FBS, 1% w/v/Penicillin/Streptomycin, 1 mM glutamine and 50 μM β-mercaptoethanol.50. Briefly, proximal small intestine tissue was harvested and flushed with ice-cold sterile PBS to remove luminal contents. Intestine was then opened longitudinally and cut into 1–2 cm pieces followed by washing in cold PBS until the supernatant was clear. Tissue fragments were then incubated with 2 mM EDTA for 30 min with gentle shaking. After removal of buffer containing EDTA, the fragments were resuspended in shaking buffer (43.3 mM sucrose and 59.4 mM sorbitol in PBS) and vigorously shaken for 2 min to ensure crypt release. Crypt-containing supernatant fractions were pooled together, passed through a 70 μm cell strainer, and centrifuged at 200 × g for 10 min at 4 °C. The crypt pellet was resuspended in ice-cold Matrigel  (500 crypts/50 µL Matrigel) containing 50 mg/mL recombinant mouse epidermal growth factor (EGF), 100 ng/mL recombinant mouse Noggin CF and 500 ng/mL recombinant mouse R-spondin 1; all obtained from R&D Systems UK. Using pre-chilled pipette tips, the crypt/matrigel suspension was plated as 50 µL/well in a 24-well plate (Nunc) and incubated for 5–10 min at 37 °C for the Matrigel to polymerize. Subsequently, 0.5 mL culture medium was added per well . Small intestinal organoids were cultured for 7 d and were split once before they were used for experiments at day 10.Small intestinal crypts were isolated as described previouslyTnf (Mm00443258_m1), luciferase (Mr03987587_mr) and hypoxanthine guanine phosphoribosyltransferase . The reactions were run using a LightCycler® 480 qPCR Instrument  and the data were analysed according to the ΔΔCt method51.Freshly isolated peritoneal macrophages and splenocytes, and terminally differentiated BMDMs, were used for transcriptional studies. Cells were either left unstimulated or stimulated with LPS (at 10 ng/mL for macrophages and 1 µg/mL for splenocytes) at various time points up to 6 h. Total RNA was purified using the RNeasy mini kit  and isolated RNA converted to cDNA using the High Capacity RNA-to-cDNA Kit . For real-time PCR reactions, the Taqman Fast Advanced Master mix was used, together with FAM probes for mouse Salmonella enterica serovar Minnesota R595; Alexis Biochemicals, UK). The luciferase activity was measured over time in a CO2 Lumistar Omega luminometer . Unstimulated cells were used as negative control. Recombinant murine cytokines also tested included 0.01–10 ng/mL IL-10 , 20 ng/mL IL-1β , 20 ng/ml Pam3CSK4 (Sigma), 50–100 ng/mL TNF (PeproTech), 20 ng/mL of IL-4, IL-6, IL-13 and IFN-γ (PeproTech). Bacterial N-acetylmuramyl-L-alanyl-D-isoglutamine hydrate ) was tested at 500 ng/mL. NF-κB and ERK signalling pathway inhibitors used included BI605906  and MG132 (Sigma), both used at 10 µM; and PD0325901 (Sigma) used at 100 nM. For TNFR blocking experiments, we used purified anti-mouse CD120a (TNFR1/p55) neutralizing antibody , purified anti-mouse CD120b (TNFR2/p75) neutralizing antibody , anti-human TNF RI/TNFRSF1A neutralizing antibody  and the Armenian Hamster IgG isotype control antibody, Functional Grade (eBioscience) at final concentration of 50 μg/ml.For luciferase assay cells were cultured in 96 well plates  in 0.2 mL medium containing 1 mM luciferin (Promega). Cells were stimulated with 10 ng/mL LPS (Results are expressed as mean ± standard error of mean (SEM). Following assessment for normality and equality of variances, statistical inferences on data were performed using one-way analysis of variance (ANOVA) followed by pairwise comparisons of treatment means using Dunnett’s post-hoc test (vs. unstimulated controls or vs. stimuli used in the inhibition studies). Differences were considered statistically significant when p < 0.05. Luciferase activity represented as area under the curve (AUC) and soluble TNF secreted levels were evaluated using Pearson’s correlation analysis. Statistical analyses were performed using GraphPad Prism 7 Software Statistical Package, La Jolla CA; USA.Supplementary Information