Multi-Arm PEG/Peptidomimetic Conjugate Inhibitors of DR6/APP Interaction Block Hematogenous Tumor Cell Extravasation.

The binding of amyloid precursor protein (APP) expressed on tumor cells to death receptor 6 (DR6) could initiate the necroptosis pathway, which leads to necroptotic cell death of vascular endothelial cells (ECs) and results in tumor cells (TCs) extravasation and metastasis. This study reports the first inhibitor of DR6/APP interaction as a novel class of anti-hematogenous metastatic agent. By rationally utilizing three combined strategies including selection based on phage display library, d-retro-inverso modification, and multiple conjugation of screened peptidomimetic with 4-arm PEG, the polymer-peptidomimetic conjugate PEG-tAHP-DRI (tetra-(D-retro-inverso isomer of AHP-12) substitued 4-arm PEG5k ) is obtained as the most promising agent with the strongest binding potency (KD  = 51.12 × 10-9  m) and excellent pharmacokinetic properties. Importantly, PEG-tAHP-DRI provides efficient protection against TC-induced ECs necroptosis both in vitro and in vivo. Moreover, this ligand exhibits prominent anti-hematogenous metastatic activity in serval different metastatic mouse models (B16F10, 4T1, CT26, and spontaneous lung metastasis of 4T1 orthotopic tumor model) and displays no apparent detrimental effects in preliminary safety evaluation. Collectively, this study demonstrates the feasibility of exploiting DR6/APP interaction to regulate hematogenous tumor cells transendothelial migration and provides PEG-tAHP-DRI as a novel and promising inhibitor of DR6/APP interaction for developments of anti-hematogenous metastatic therapies.

Structural feature of TRAFs, their related human diseases and therapeutic intervention.

Several studies have been conducted over the years to unravel the structural information on the receptors that bind to tumor necrosis factor receptor-associated factor (TRAF) and the driving forces for the TRAF/receptor complex. In addition, studies have also been performed to highlight the influence of TRAF malfunctioning and mutations on the development of human disease. However, a holistic study that systematically summarizes the available information and the existing clinical trends towards development of the TRAF-targeting drugs has not been conducted to date. Herein, I reviewed existing research that focused on the structural information of various receptors recognized by the different members of the TRAF family. I also reviewed studies on the different human diseases that occur due to TRAF malfunctioning or mutations as well as the clinical trials undertaken to treat TRAF-associated diseases.

Biologic TNF-α inhibitors reduce microgliosis, neuronal loss, and tau phosphorylation in a transgenic mouse model of tauopathy.

BACKGROUND: Tumor necrosis factor-α (TNF-α) plays a central role in Alzheimer's disease (AD) pathology, making biologic TNF-α inhibitors (TNFIs), including etanercept, viable therapeutics for AD. The protective effects of biologic TNFIs on AD hallmark pathology (Aß deposition and tau pathology) have been demonstrated. However, the effects of biologic TNFIs on Aß-independent tau pathology have not been reported. Existing biologic TNFIs do not cross the blood-brain barrier (BBB), therefore we engineered a BBB-penetrating biologic TNFI by fusing the extracellular domain of the type-II human TNF-α receptor (TNFR) to a transferrin receptor antibody (TfRMAb) that ferries the TNFR into the brain via receptor-mediated transcytosis. The present study aimed to investigate the effects of TfRMAb-TNFR (BBB-penetrating TNFI) and etanercept (non-BBB-penetrating TNFI) in the PS19 transgenic mouse model of tauopathy. METHODS: Six-month-old male and female PS19 mice were injected intraperitoneally with saline (n = 12), TfRMAb-TNFR (1.75 mg/kg, n = 10) or etanercept (0.875 mg/kg, equimolar dose of TNFR, n = 10) 3 days/week for 8 weeks. Age-matched littermate wild-type mice served as additional controls. Blood was collected at baseline and 8 weeks for a complete blood count. Locomotion hyperactivity was assessed by the open-field paradigm. Brains were examined for phosphorylated tau lesions (Ser202, Thr205), microgliosis, and neuronal health. The plasma pharmacokinetics were evaluated following a single intraperitoneal injection of 0.875 mg/kg etanercept or 1.75 mg/kg TfRMAb-TNFR or 1.75 mg/kg chronic TfRMAb-TNFR dosing for 4 weeks. RESULTS: Etanercept significantly reduced phosphorylated tau and microgliosis in the PS19 mouse brains of both sexes, while TfRMAb-TNFR significantly reduced these parameters in the female PS19 mice. Both TfRMAb-TNFR and etanercept treatment improved neuronal health by significantly increasing PSD95 expression and attenuating hippocampal neuron loss in the PS19 mice. The locomotion hyperactivity in the male PS19 mice was suppressed by chronic etanercept treatment. Equimolar dosing resulted in eightfold lower plasma exposure of the TfRMAb-TNFR compared with etanercept. The hematological profiles remained largely stable following chronic biologic TNFI dosing except for a significant increase in platelets with etanercept. CONCLUSION: Both TfRMAb-TNFR (BBB-penetrating) and non-BBB-penetrating (etanercept) biologic TNFIs showed therapeutic effects in the PS19 mouse model of tauopathy.

Collaborative study for the establishment of Etanercept Biological Reference Preparation Batch 1.

Three preparations of the human tumour necrosis factor (TNF) receptor II Fc fusion protein (TNFR II-Fc) Etanercept were formulated and lyophilised at the National Institute for Biological Standards & Control (NIBSC) prior to evaluation in a collaborative study for their suitability to serve as a World Health Organization (WHO) International Standard (IS)/European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for the potency assay of Etanercept. Seven laboratories tested the preparations using an in vitro cell-based bioassay (TNF-α neutralisation) prescribed by the Ph. Eur. monograph on Etanercept (2895). The results of this study indicated that the candidate preparation, coded 13/204, established as the first IS for Etanercept with an assigned potency for TNF neutralisation activity of 10 000 IU per ampoule was also suitable to serve as Ph. Eur. BRP batch 1. The results were compared to those obtained with different cell-based neutralisation assays that were used by further laboratories in the context of establishing the 1st WHO IS for Etanercept. Based on these analyses, preparation 13/204 was adopted by the Ph. Eur. Commission as Etanercept BRP batch 1 with an assigned potency of 10 000 IU per ampoule.

Dermatological Manifestations in Pediatric Inflammatory Bowel Disease.

Background and Objectives: Over the last years, inflammatory bowel disease (IBD) has been reported on a high incidence in pediatric populations and has been associated with numerous extraintestinal manifestations, making its management a real challenge for the pediatric gastroenterologist. Dermatological manifestations in IBD are either specific, related to the disease activity or treatment-associated, or non-specific. This literature review aims to identify and report the dermatological manifestations of IBD in children, the correlation between their appearance and the demographical characteristics, the relationship between these lesions and disease activity, and to highlight the impact of dermatological manifestations on an IBD treatment regime. MATERIALS AND METHODS: A systemic literature review was performed, investigating articles and case reports on dermatological manifestations in children with IBD starting from 2005. A total of 159 potentially suitable articles were identified and after the exclusion process, 75 articles were selected. RESULTS: The most common dermatological manifestations reported in pediatric IBD are erythema nodosum and pyoderma gangrenosum. More rare cases of metastatic Crohn's disease, epidermolysis bullosa acquisita, small-vessel vasculitis, necrotizing vasculitis, leukocytoclastic vasculitis, cutaneous polyarteritis nodosa, and Sweet's syndrome have been reported. Oral manifestations of IBD are divided into specific (tag-like lesions, mucogingivitis, lip swelling with vertical fissures, aphthous stomatitis, and pyostomatitis vegetans) and non-specific. IBD treatment may present with side effects involving the skin and mucosa. Anti-tumor necrosis factor agents have been linked to opportunistic skin infections, psoriasiform lesions, and a potentially increased risk for skin cancer. Cutaneous manifestations such as acrodermatitis enteropathica, purpuric lesions, and angular cheilitis may appear secondary to malnutrition and/or malabsorption. CONCLUSIONS: The correct diagnosis of dermatological manifestations in pediatric IBD is of paramount importance because of their impact on disease activity, treatment options, and a patient's psychological status.

Gut Microbiota Dysbiosis Enhances Migraine-Like Pain Via TNFα Upregulation.

Migraine is one of the most disabling neurological diseases worldwide; however, the mechanisms underlying migraine headache are still not fully understood and current therapies for such pain are inadequate. It has been suggested that inflammation and neuroimmune modulation in the gastrointestinal tract could play an important role in the pathogenesis of migraine headache, but how gut microbiomes contribute to migraine headache is unclear. In the present study, we investigated the effect of gut microbiota dysbiosis on migraine-like pain using broad-spectrum antibiotics and germ-free (GF) mice. We observed that antibiotics treatment-prolonged nitroglycerin (NTG)-induced acute migraine-like pain in wild-type (WT) mice and the pain prolongation was completely blocked by genetic deletion of tumor necrosis factor-alpha (TNFα) or intra-spinal trigeminal nucleus caudalis (Sp5C) injection of TNFα receptor antagonist. The antibiotics treatment extended NTG-induced TNFα upregulation in the Sp5C. Probiotics administration significantly inhibited the antibiotics-produced migraine-like pain prolongation. Furthermore, NTG-induced migraine-like pain in GF mice was markedly enhanced compared to that in WT mice and gut colonization with fecal microbiota from WT mice robustly reversed microbiota deprivation-caused pain enhancement. Together, our results suggest that gut microbiota dysbiosis contributes to chronicity of migraine-like pain by upregulating TNFα level in the trigeminal nociceptive system.

The Ulcerative Colitis Response Index for Detection of Mucosal Healing in Patients Treated With Anti-tumour Necrosis Factor.

BACKGROUND: Surrogate markers that accurately detect mucosal healing [MH] in patients with ulcerative colitis [UC] are urgently needed. Several stool neutrophil-related proteins are currently used as biomarkers for MH. However, the sensitivity and specificity are not sufficient to avoid unnecessary endoscopic evaluations. METHODS: Novel serum neutrophil-related markers (neutrophil gelatinase B-associated lipocalin and matrix metalloproteinase-9 [NGAL-MMP-9 complex], cathelicidin LL-37 and chitinase 3-like 1 [CHI3L1]), together with C-reactive protein [CRP] and neutrophil counts were studied. Serum samples were obtained from 176 anti-tumour necrosis factor [anti-TNF]-treated UC patients (145 infliximab [IFX] and 31 adalimumab [ADM]) at baseline and after a median of 9.5 weeks. All patients had active disease prior to treatment (Mayo endoscopic subscore [MES] ≥ 2), and MH was defined as MES ≤ 1. Serum was also obtained from 75 healthy controls. Binary logistic regression analysis was used to generate the Ulcerative Colitis Response Index [UCRI]. The performance of individual markers and UCRI was tested with receiver operating characteristic analysis. RESULTS: All neutrophil-related markers were significantly higher in active UC patients compared to healthy controls. In the IFX cohort, CRP, NGAL-MMP-9, CHI3L1 and neutrophil count decreased significantly after treatment and all marker levels were significantly lower in healers compared to non-healers following IFX. In the ADM cohort, CRP, NGAL-MMP-9, CHI3L1 and neutrophil count decreased significantly only in healers. UCRI [including CRP, CHI3L1, neutrophil count and LL-37] accurately detected MH in both IFX-treated (area under the curve [AUC] = 0.83) and ADM-treated [AUC = 0.79] patients. CONCLUSIONS: The new UCRI index accurately detects MH after treatment with IFX and ADM. This panel is useful for monitoring MH in UC patients under anti-TNF treatment. PODCAST: This article has an associated podcast which can be accessed at https://academic.oup.com/ecco-jcc/pages/podcast.

TNFRSF receptor-specific antibody fusion proteins with targeting controlled FcγR-independent agonistic activity.

Antibodies specific for TNFRSF receptors that bind soluble ligands without getting properly activated generally act as strong agonists upon FcγR binding. Systematic analyses revealed that the FcγR dependency of such antibodies to act as potent agonists is largely independent from isotype, FcγR type, and of the epitope recognized. This suggests that the sole cellular attachment, achieved by Fc domain-FcγR interaction, dominantly determines the agonistic activity of antibodies recognizing TNFRSF receptors poorly responsive to soluble ligands. In accordance with this hypothesis, we demonstrated that antibody fusion proteins harboring domains allowing FcγR-independent cell surface anchoring also act as strong agonist provided they have access to their target. This finding defines a general possibility to generate anti-TNFRSF receptor antibodies with FcγR-independent agonism. Moreover, anti-TNFRSF receptor antibody fusion proteins with an anchoring domain promise superior applicability to conventional systemically active agonists when an anchoring target with localized disease associated expression can be addressed.

Upside and Downside of Tumor Necrosis Factor Blockers for Treatment of Immune/Inflammatory Diseases.

Tumor necrosis factor (TNF)-α, the most potent proinflammatory cytokine discovered to date, was first isolated in 1984 from human macrophage cells. Initially, it was thought to be a protein that was cytotoxic to tumor cells. But later, it was regarded as an agent that promotes inflammation and other chronic diseases found in humans. Currently, we know that the TNF superfamily (TNFS) has 19 members that perform a wide variety of functions via > 40 TNF receptors. Of TNFS members, TNF-α has been studied extensively and was found to be implicated in numerous autoimmune diseases, such as rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, psoriasis, systemic lupus erythematosus, juvenile idiopathic arthritis, and diabetes. Thus, agents that can inhibit TNF-α have great potential for prevention and treatment of chronic diseases. To date, the U.S. Food and Drug Administration has approved many TNF-α blockers, such as etanercept, infliximab, adalimumab, certolizumab pegol, and golimumab. These agents can block TNF-α actions and be used to treat different diseases. However, the uses of TNF-α blockers are not without serious adverse effects. Therefore, natural TNF-α blockers are best for developing safe, efficacious, and affordable agents for prevention and treatment of chronic diseases. The current review details the TNFS, functions of TNF-α in normal and disease conditions, roles of TNF-α blockers, and advantages and disadvantages.

Targeting tumor necrosis factor receptors in ankylosing spondylitis.

Over the past two decades, considerable advances in our understanding of inflammatory and immune pathways have allowed for the growing use of targeted biologic therapy. Most notably, the introduction of tumor necrosis factor (TNF) inhibitors has dramatically changed the management of autoimmune inflammatory disorders, including ankylosing spondylitis (AS). Despite the efficacy of TNF inhibitors documented in multiple clinical trials, anti-TNF therapy in AS is far from foolproof; it is associated with serious adverse effects and limited response to therapy in some patients. Moreover, specific questions regarding the role of TNF as a mediator of AS remain unanswered. Therefore, additional efforts are needed in order to better understand the role of TNF in the pathogenesis of AS and to develop safer and more effective treatment strategies. The purpose of this review is to better the understanding of the physiologic and pathogenic roles of TNF signaling in the course of AS. Relevant TNF biology and novel approaches to TNF blockade in AS are discussed.

Understanding how combinatorial targeting of TLRs and TNFR family costimulatory members promote enhanced T cell responses.

INTRODUCTION: Due to the ability of pathogen-associated molecular patters and tumor necrosis factor receptor (TNFR) family costimulatory agonists to boost T cell responses, studies have combined Toll-like receptor (TLR) ligands with TNFR family costimulatory receptor agonists to induce impressive and long-lasting T cell responses. Although some studies have determined how these combinatorial vaccines promote enhanced T cell responses, much remains unknown about the mechanism used by these combinations to promote synergistic T cell responses - especially in settings of infectious diseases or cancer. AREAS COVERED: In this review, we look in detail at the signaling pathways induced by combinatorial targeting of TLR and TNFR family costimulatory members that help them promote synergistic T cell responses. Understanding this can greatly aid the development of novel vaccine regimens that promote cellular immune responses, which is essential for treating certain infectious diseases and cancer. EXPERT OPINION: Vaccines against some infectious diseases as well as therapeutic cancer vaccines require cellular immunity. Therefore, we evaluate here how signaling pathways induced by TLR ligand and costimulatory agonist combinations promote enhanced T cell responses during immunization with model antigens, viral pathogens, or tumor antigens. Once pathways that drive these combinatorial vaccines to boost T cell activation are identified, they can be incorporated in vaccines designed to target pathogens or cancer.

A New Venue of TNF Targeting.

The first Food and Drug Administration-(FDA)-approved drugs were small, chemically-manufactured and highly active molecules with possible off-target effects, followed by protein-based medicines such as antibodies. Conventional antibodies bind a specific protein and are becoming increasingly important in the therapeutic landscape. A very prominent class of biologicals are the anti-tumor necrosis factor (TNF) drugs that are applied in several inflammatory diseases that are characterized by dysregulated TNF levels. Marketing of TNF inhibitors revolutionized the treatment of diseases such as Crohn's disease. However, these inhibitors also have undesired effects, some of them directly associated with the inherent nature of this drug class, whereas others are linked with their mechanism of action, being pan-TNF inhibition. The effects of TNF can diverge at the level of TNF format or receptor, and we discuss the consequences of this in sepsis, autoimmunity and neurodegeneration. Recently, researchers tried to design drugs with reduced side effects. These include molecules with more specificity targeting one specific TNF format or receptor, or that neutralize TNF in specific cells. Alternatively, TNF-directed biologicals without the typical antibody structure are manufactured. Here, we review the complications related to the use of conventional TNF inhibitors, together with the anti-TNF alternatives and the benefits of selective approaches in different diseases.

Small tumor necrosis factor receptor biologics inhibit the tumor necrosis factor-p38 signalling axis and inflammation.

Despite anti-TNF therapy advancements for inflammatory diseases such as rheumatoid arthritis, the burden of diseases remains high. An 11-mer TNF peptide, TNF70-80, is known to stimulate selective functional responses compared to the parent TNF molecule. Here, we show that TNF70-80 binds to the TNF receptor, activating p38 MAP kinase through TNF receptor-associated factor 2. Using truncated TNFR mutants, we identify the sequence in TNFRI which enables p38 activation by TNF70-80. Peptides with this TNFRI sequence, such as TNFRI206-211 bind to TNF and inhibit TNF-induced p38 activation, respiratory burst, cytokine production and adhesion receptor expression but not F-Met-Leu-Phe-induced respiratory burst in neutrophils. TNFRI206-211 does not prevent TNF binding to TNFRI or TNF-induced stimulation of ERK, JNK and NF-κB. TNFRI206-211 inhibits bacterial lipopolysaccharide-induced peritonitis, carrageenan-induced and antigen-induced paw inflammation, and respiratory syncytial virus-induced lung inflammation in mice. Our findings suggest a way of targeting TNF-p38 pathway to treat chronic inflammatory disorders.

Structural principles of tumor necrosis factor superfamily signaling.

The tumor necrosis factor (TNF) ligand and receptor superfamilies play an important role in cell proliferation, survival, and death. Stimulating or inhibiting TNF superfamily signaling pathways is expected to have therapeutic benefit for patients with various diseases, including cancer, autoimmunity, and infectious diseases. We review our current understanding of the structure and geometry of TNF superfamily ligands, receptors, and their interactions. A trimeric ligand and three receptors, each binding at the interface of two ligand monomers, form the basic unit of signaling. Clustering of multiple receptor subunits is necessary for efficient signaling. Current reports suggest that the receptors are prearranged on the cell surface in a "nonsignaling," resting state in a large hexagonal structure of antiparallel dimers. Receptor activation requires ligand binding, and cross-linking antibodies can stabilize the receptors, thereby maintaining the active, signaling state. On the other hand, an antagonist antibody that locks receptor arrangement in antiparallel dimers effectively blocks signaling. This model may aid the design of more effective TNF signaling-targeted therapies.

Teriparatide rapidly improves pain-like behavior in ovariectomized mice in association with the downregulation of inflammatory cytokine expression.

Recent studies have indicated that teriparatide, an anti-osteoporosis agent, significantly improves back pain regardless of the presence of vertebral fracture in osteoporosis patients. The aims of this study were to examine whether teriparatide improves pain-like behavior in an ovariectomized (OVX) mouse model, and to evaluate changes in osteoclast marker levels and inflammatory cytokine expression levels induced by teriparatide treatment in bone tissue in association with improvements in pain-like behavior. OVX and sham operations were performed in 8-week-old mice, followed by teriparatide treatment for 2 weeks. Pain-like behavior tests (von Frey, paw flick and spontaneous pain test), and the measurement of serum tartrate-resistant acid phosphatase 5b (TRAP5b) level and inflammatory cytokine (interleukin [IL]-1ß, IL-6 and tumor necrosis factor [TNF]-α) expression levels in the bone tissue were conducted after teriparatide treatment in OVX mice. Pain-like behavior in the von Frey test was significantly improved by teriparatide treatment in OVX mice. With regard to the early phase (within the first 7 days of treatment), teriparatide significantly improved pain-like behavior in the von Frey test, the paw flick test and the spontaneous pain test. Teriparatide significantly inhibited the expression of IL-1ß, IL-6 and TNF-α in OVX mice in the early phase of the treatment, while the TRAP5b level in OVX mice was not significantly affected. We demonstrated that the teriparatide-induced rapid improvement effect on pain-like behavior in OVX mice was associated with the downregulation of inflammatory cytokine expression, including IL-1ß, IL-6 and TNF-α.

CD52 inhibits Toll-like receptor activation of NF-κB and triggers apoptosis to suppress inflammation.

Soluble CD52 is a small glycoprotein that suppresses T-cell activation, but its effect on innate immune cell function is unknown. Here we demonstrate that soluble CD52 inhibits Toll-like receptor and tumor necrosis factor receptor signaling to limit activation of NF-κB and thereby suppress the production of inflammatory cytokines by macrophages, monocytes and dendritic cells. At higher concentrations, soluble CD52 depletes the short-lived pro-survival protein MCL-1, contributing to activation of the BH3-only proteins BAX and BAK to cause intrinsic apoptotic cell death. In vivo, administration of soluble CD52 suppresses lipopolysaccharide (LPS)-induced cytokine secretion and other features of endotoxic shock, whereas genetic deletion of CD52 exacerbates LPS responses. Thus, soluble CD52 exhibits broad immune suppressive effects that signify its potential as an immunotherapeutic agent.

Etanercept is effective as monotherapy or in combination with methotrexate in rheumatoid arthritis: subanalysis of an observational study.

Approximately 30% of patients with rheumatoid arthritis receiving biological disease-modifying antirheumatic drugs (bDMARDs) take them as monotherapy. Although etanercept (ETN) monotherapy has been evaluated in clinical trials, data in the real-world setting are sparse. We compared the efficacy and safety of ETN, given alone or in combination with methotrexate (MTX), in routine clinical practice. This was a subanalysis of patients who received either ETN alone or ETN + MTX during a 52-week prospective, observational study conducted at 329 German centers. The primary endpoint was "Funktionsfragebogen Hannover" (Hannover Functional Ability Questionnaire [FFbH]; low FFbH = worse function) functional remission at week 26 and week 52. Secondary endpoints included the 28-joint count Disease Activity Score (DAS28), DAS28 remission (DAS28 < 2.6), and adverse events (AEs). Participating centers applied ETN monotherapy in 43.1% of patients and ETN + MTX in 56.9%. A smaller proportion of patients achieved FFbH functional remission with ETN vs ETN + MTX (31.9%, 95% confidence interval [CI] 29.1-34.9% vs 39.8%, 37.2-42.5%, respectively; p < 0.001) at 26 weeks and at 52 weeks (38.4%, 35.1-41.7% vs 44.3%, 41.5-47.2%, respectively; p = 0.007). After 52 weeks, the mean DAS28 (±SD) decreased from 5.5 ± 1.3 to 3.4 ± 1.4 (ETN) vs 5.3 ± 1.3 to 3.2 ± 1.3 (ETN + MTX) and DAS28 remission was achieved by 32.5% (95% CI 29.0-36.1%) of patients with ETN vs 38.8% (35.8-41.9%; p = 0.007) with ETN + MTX. Overall, 20.6 (ETN) and 19.7% (ETN + MTX) of patients reported treatment-related AEs. Patients received ETN monotherapy almost as often as ETN + MTX. ETN + MTX appeared marginally more effective than ETN monotherapy in some, but not all, outcomes measured.

An Innovative High-Throughput Screening Approach for Discovery of Small Molecules That Inhibit TNF Receptors.

Tumor necrosis factor receptor 1 (TNFR1) is a transmembrane receptor that binds tumor necrosis factor or lymphotoxin-alpha and plays a critical role in regulating the inflammatory response. Upregulation of these ligands is associated with inflammatory and autoimmune diseases. Current treatments reduce symptoms by sequestering free ligands, but this can cause adverse side effects by unintentionally inhibiting ligand binding to off-target receptors. Hence, there is a need for new small molecules that specifically target the receptors, rather than the ligands. Here, we developed a TNFR1 FRET biosensor expressed in living cells to screen compounds from the NIH Clinical Collection. We used an innovative high-throughput fluorescence lifetime screening platform that has exquisite spatial and temporal resolution to identify two small-molecule compounds, zafirlukast and triclabendazole, that inhibit the TNFR1-induced IκBα degradation and NF-κB activation. Biochemical and computational docking methods were used to show that zafirlukast disrupts the interactions between TNFR1 pre-ligand assembly domain (PLAD), whereas triclabendazole acts allosterically. Importantly, neither compound inhibits ligand binding, proving for the first time that it is possible to inhibit receptor activation by targeting TNF receptor-receptor interactions. This strategy should be generally applicable to other members of the TNFR superfamily, as well as to oligomeric receptors in general.

Death receptor 6 contributes to autoimmunity in lupus-prone mice.

Expansion of autoreactive follicular helper T (Tfh) cells is tightly restricted to prevent induction of autoantibody-dependent immunological diseases, such as systemic lupus erythematosus (SLE). Here we show expression of an orphan immune regulator, death receptor 6 (DR6/TNFRSF21), on a population of Tfh cells that are highly expanded in lupus-like disease progression in mice. Genome-wide screening reveals an interaction between syndecan-1 and DR6 resulting in immunosuppressive functions. Importantly, syndecan-1 is expressed specifically on autoreactive germinal centre (GC) B cells that are critical for maintenance of Tfh cells. Syndecan-1 expression level on GC B cells is associated with Tfh cell expansion and disease progression in lupus-prone mouse strains. In addition, Tfh cell suppression by DR6-specific monoclonal antibody delays disease progression in lupus-prone mice. These findings suggest that the DR6/syndecan-1 axis regulates aberrant GC reactions and could be a therapeutic target for autoimmune diseases such as SLE.