Tumor necrosis factor α receptor 1A transduces the inhibitory effect on axon regeneration triggered by IgG anti-ganglioside GD1a antibodies.

Anti-ganglioside antibodies (anti-Gg Abs) have been linked to delayed/poor clinical recovery in both axonal and demyelinating forms of Guillain-Barrè Syndrome (GBS). In many instances, the incomplete recovery is attributed to the peripheral nervous system's failure to regenerate. The cross-linking of cell surface gangliosides by anti-Gg Abs triggers inhibition of nerve repair in both in vitro and in vivo axon regeneration paradigms. This mechanism involves the activation of the small GTPase RhoA, which negatively modulates the growth cone cytoskeleton. At present, the identity/es of the receptor/s responsible for transducing the signal that ultimately leads to RhoA activation remains poorly understood. The aim of this work was to identify the transducer molecule responsible for the inhibitory effect of anti-Gg Abs on nerve repair. Putative candidate molecules were identified through proteomic mass spectrometry of ganglioside affinity-captured proteins from rat cerebellar granule neurons (Prendergast et al., 2014). These candidates were evaluated using an in vitro model of neurite outgrowth with primary cultured dorsal root ganglion neurons (DRGn) and an in vivo model of axon regeneration. Using an shRNA-strategy to silence putative candidates on DRGn, we identified tumor necrosis factor receptor 1A protein (TNFR1A) as a transducer molecule for the inhibitory effect on neurite outgrowth from rat/mouse DRGn cultures of a well characterized mAb targeting the related gangliosides GD1a and GT1b. Interestingly, lack of TNFr1A expression on DRGn abolished the inhibitory effect on neurite outgrowth caused by anti-GD1a but not anti-GT1b specific mAbs, suggesting specificity of GD1a/transducer signaling. Similar results were obtained using primary DRGn cultures from TNFR1a-null mice, which did not activate RhoA after exposure to anti-GD1a mAbs. Generation of single point mutants at the stalk region of TNFR1A identified a critical amino acid for transducing GD1a signaling, suggesting a direct interaction. Finally, passive immunization with an anti-GD1a/GT1b mAb in an in vivo model of axon regeneration exhibited reduced inhibitory activity in TNFR1a-null mice compared to wild type mice. In conclusion, these findings identify TNFR1A as a novel transducer receptor for the inhibitory effect exerted by anti-GD1a Abs on nerve repair, representing a significant step forward toward understanding the factors contributing to poor clinical recovery in GBS associated with anti-Gg Abs.

Distinct Role of TNFR1 and TNFR2 in Protective Immunity Against Orientia tsutsugamushi Infection in Mice.

Infection with Orientia tsutsugamushi, an obligate intracellular bacterium, can cause mild or severe scrub typhus. Some patients develop acute lung injury, multi-organ failure, and fatal infection; however, little is known regarding key immune mediators that mediate infection control or disease pathogenesis. Using murine models of scrub typhus, we demonstrated in this study the requirement of TNF-TNFR signaling in protective immunity against this infection. Mice lacking both TNF receptors (TNFR1 and TNFR2) were highly susceptible to O. tsutsugamushi infection, displaying significantly increased tissue bacterial burdens and succumbing to infection by day 9, while most wild-type mice survived through day 20. This increased susceptibility correlated with poor activation of cellular immunity in inflamed tissues. Flow cytometry of lung- and spleen-derived cells revealed profound deficiencies in total numbers and activation status of NK cells, neutrophils, and macrophages, as well as CD4 and CD8 T cells. To define the role of individual receptors in O. tsutsugamushi infection, we used mice lacking either TNFR1 or TNFR2. While deficiency in either receptor alone was sufficient to increase host susceptibility to the infection, TNFR1 and TNFR2 played a distinct role in cellular responses. TNF signaling through TNFR1 promoted inflammatory responses and effector T cell expansion, while TNFR2 signaling was associated with anti-inflammatory action and tissue homeostasis. Moreover, TNFRs played an intrinsic role in CD8+ T cell activation, revealing an indispensable role of TNF in protective immunity against O. tsutsugamushi infection.

XIAP restrains TNF-driven intestinal inflammation and dysbiosis by promoting innate immune responses of Paneth and dendritic cells.

Deficiency in X-linked inhibitor of apoptosis protein (XIAP) is the cause for X-linked lymphoproliferative syndrome 2 (XLP2). About one-third of these patients suffer from severe and therapy-refractory inflammatory bowel disease (IBD), but the exact cause of this pathogenesis remains undefined. Here, we used XIAP-deficient mice to characterize the mechanisms underlying intestinal inflammation. In Xiap−/− mice, we observed spontaneous terminal ileitis and microbial dysbiosis characterized by a reduction of Clostridia species. We showed that in inflamed mice, both TNF receptor 1 and 2 (TNFR1/2) cooperated in promoting ileitis by targeting TLR5-expressing Paneth cells (PCs) or dendritic cells (DCs). Using intestinal organoids and in vivo modeling, we demonstrated that TLR5 signaling triggered TNF production, which induced PC dysfunction mediated by TNFR1. TNFR2 acted upon lamina propria immune cells. scRNA-seq identified a DC population expressing TLR5, in which Tnfr2 expression was also elevated. Thus, the combined activity of TLR5 and TNFR2 signaling may be responsible for DC loss in lamina propria of Xiap−/− mice. Consequently, both Tnfr1−/−Xiap−/− and Tnfr2−/−Xiap−/− mice were rescued from dysbiosis and intestinal inflammation. Furthermore, RNA-seq of ileal crypts revealed that in inflamed Xiap−/− mice, TLR5 signaling was abrogated, linking aberrant TNF responses with the development of a dysbiosis. Evidence for TNFR2 signaling driving intestinal inflammation was detected in XLP2 patient samples. Together, these data point toward a key role of XIAP in mediating resilience of TLR5-expressing PCs and intestinal DCs, allowing them to maintain tissue integrity and microbiota homeostasis.

Differential role of TNFR1 and TNFR2 in the development of imiquimod-induced mouse psoriasis.

Tumor necrosis factor alpha (TNF) has been implicated in the pathogenesis of psoriasis and anti-TNF therapeutics are used in the treatment of psoriasis in the clinic. However, considerable proportion of patients fail to respond to anti-TNF treatment. Furthermore, anti-TNF therapy induces de novo development of psoriasis in some patients with other type of autoimmune disorders. Therefore, further understanding of the role of TNF-TNFR signaling in pathogenesis of psoriasis remains a critical to devise safer and more effective treatment. In this study, it is shown that in imiquimod-induced mouse psoriasis model, TNF receptor type 1 (TNFR1) deficiency inhibited the development of skin diseases. In sharp contrast, TNF receptor type 2 (TNFR2) deficiency led to more severe psoriasis that was associated with increased Th1 and Th17 responses and reduced number of CD4+ Foxp3+ regulatory T cells (Tregs). Importantly, adoptive transfer of WT Tregs was able to attenuate inflammatory responses in imiquimod-treated TNFR2-/- mice, suggestive of a role of malfunctioned Tregs in mice deficient in TNFR2. RNA sequencing data revealed that Tregs deficient in TNFR2 exhibited down-regulation of different biological processes linked to proliferative expansion. Taken together, our study clearly indicated that TNFR1 was pathogenic in mouse psoriasis. In contrast, through boosting the proliferative expansion of Tregs, TNFR2 was protective in this model. The data thus suggest that TNFR1-specific antagonist or TNFR2-specific agonist may be useful in the treatment of patients with psoriasis.

Changes in Cerebrospinal Fluid Balance of TNF and TNF Receptors in Naïve Multiple Sclerosis Patients: Early Involvement in Compartmentalised Intrathecal Inflammation.

An imbalance of TNF signalling in the inflammatory milieu generated by meningeal immune cell infiltrates in the subarachnoid space in multiple sclerosis (MS), and its animal model may lead to increased cortical pathology. In order to explore whether this feature may be present from the early stages of MS and may be associated with the clinical outcome, the protein levels of TNF, sTNF-R1 and sTNF-R2 were assayed in CSF collected from 122 treatment-naïve MS patients and 36 subjects with other neurological conditions at diagnosis. Potential correlations with other CSF cytokines/chemokines and with clinical and imaging parameters at diagnosis (T0) and after 2 years of follow-up (T24) were evaluated. Significantly increased levels of TNF (fold change: 7.739; p < 0.001), sTNF-R1 (fold change: 1.693; p < 0.001) and sTNF-R2 (fold change: 2.189; p < 0.001) were detected in CSF of MS patients compared to the control group at T0. Increased TNF levels in CSF were significantly (p < 0.01) associated with increased EDSS change (r = 0.43), relapses (r = 0.48) and the appearance of white matter lesions (r = 0.49). CSF levels of TNFR1 were associated with cortical lesion volume (r = 0.41) at T0, as well as with new cortical lesions (r = 0.56), whilst no correlation could be found between TNFR2 levels in CSF and clinical or MRI features. Combined correlation and pathway analysis (ingenuity) of the CSF protein pattern associated with TNF expression (encompassing elevated levels of BAFF, IFN-γ, IL-1ß, IL-10, IL-8, IL-16, CCL21, haptoglobin and fibrinogen) showed a particular relationship to the interaction between innate and adaptive immune response. The CSF sTNF-R1-associated pattern (encompassing high levels of CXCL13, TWEAK, LIGHT, IL-35, osteopontin, pentraxin-3, sCD163 and chitinase-3-L1) was mainly related to altered T cell and B cell signalling. Finally, the CSF TNFR2-associated pattern (encompassing high CSF levels of IFN-ß, IFN-λ2, sIL-6Rα) was linked to Th cell differentiation and regulatory cytokine signalling. In conclusion, dysregulation of TNF and TNF-R1/2 pathways associates with specific clinical/MRI profiles and can be identified at a very early stage in MS patients, at the time of diagnosis, contributing to the prediction of the disease outcome.

The TNFR1 Antagonist Atrosimab Is Therapeutic in Mouse Models of Acute and Chronic Inflammation.

Therapeutics that block tumor necrosis factor (TNF), and thus activation of TNF receptor 1 (TNFR1) and TNFR2, are clinically used to treat inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease and psoriasis. However, TNFR1 and TNFR2 work antithetically to balance immune responses involved in inflammatory diseases. In particular, TNFR1 promotes inflammation and tissue degeneration, whereas TNFR2 contributes to immune modulation and tissue regeneration. We, therefore, have developed the monovalent antagonistic anti-TNFR1 antibody derivative Atrosimab to selectively block TNFR1 signaling, while leaving TNFR2 signaling unaffected. Here, we describe that Atrosimab is highly stable at different storage temperatures and demonstrate its therapeutic efficacy in mouse models of acute and chronic inflammation, including experimental arthritis, non-alcoholic steatohepatitis (NASH) and experimental autoimmune encephalomyelitis (EAE). Our data support the hypothesis that it is sufficient to block TNFR1 signaling, while leaving immune modulatory and regenerative responses via TNFR2 intact, to induce therapeutic effects. Collectively, we demonstrate the therapeutic potential of the human TNFR1 antagonist Atrosimab for treatment of chronic inflammatory diseases.

Contributing role of TNF, IL-10, sTNFR1 and TNF gene polymorphisms in disease severity of leptospirosis.

The immune response is hypothesized as an important factor in the disease outcome of leptospirosis. Exaggerated immune response may promote tissue damage that lead to severe disease outcome. In this study TNF, IL-10, sTNFR1 levels were measured among sixty-two hospitalized leptospirosis confirmed patients in Sri Lanka. Thirty-one serum samples from healthy individuals were obtained as controls. PCR-RFLP method was used to identify TNF gene polymorphisms and to determine their association with TNF expression and disease severity in leptospirosis. TNF (p = 0.0022) and IL-10 (p < 0.0001) were found to be significantly elevated in leptospirosis patients, while sTNFR1 (p < 0.0001) was significantly suppressed. TNF was not significantly elevated in patients with complications while the anti-inflammatory cytokine IL-10 was significantly elevated among patients with complications (p = 0.0011) and with mortality (p = 0.0088). The ratio of IL-10 to TNF was higher among patients with complications (p = 0.0008) and in fatal cases (p = 0.0179). No association between TNF gene polymorphisms and TNF expression was detected due to the low frequency of heterozygous and mutated genes present in this study population. Thus the findings of the study show that elevated levels of IL-10 in the acute phase of disease could lead to severe outcomes and a high IL-10/TNF ratio is observed in patients with complications due to leptospirosis.

Mediterranean fever gene variants modify clinical phenotypes of idiopathic multi-centric Castleman disease.

Four cases of idiopathic multi-centric Castleman disease (iMCD) reportedly have variants in hereditary autoinflammatory disease-related genes; however, the frequency and role of these variants in iMCD is still unknown. We therefore investigated such gene variants among patients with iMCD and aimed to reveal the relationship between iMCD and autoinflammatory disease-related genes. We reviewed 14 Japanese iMCD patients who were recruited between January 2015 and September 2019. All patients met both the Japanese tentative diagnostic criteria for Castleman disease and the international consensus diagnostic criteria for iMCD. We performed genetic analyses for 31 autoinflammatory disease-related genes by targeted next-generation sequencing. The MEFV gene variants were observed in 10 of 14 patients with iMCD. Although iMCD had a high percentage of exons 2 or 3 variants of MEFV, comparison of data from healthy Japanese subjects indicated that there was no significant difference in the percentage between healthy Japanese subjects and patients with iMCD. Variants of uncertain significance (VUS) in the TNFRSF1A and CECR1 genes were observed in two of the patients, respectively. We divided patients into two groups-those with MEFV variants (excluding E148Q variants) and those without MEFV variants-and compared the clinical characteristics between these two groups. Patients with MEFV variants, excluding E148Q variants, exhibited a significantly higher likelihood of fever and significantly lower levels of hemoglobin than those lacking MEFV variants. Our results indicated that patients with iMCD tended to have a high frequency of MEFV gene variants and the presence of such variants can affect iMCD clinical phenotypes.

Higher-order assemblies in immune signaling: supramolecular complexes and phase separation.

Signaling pathways in innate and adaptive immunity play vital roles in pathogen recognition and the functions of immune cells. Higher-order assemblies have recently emerged as a central principle that governs immune signaling and, by extension, cellular communication in general. There are mainly two types of higher-order assemblies: 1) ordered, solid-like large supramolecular complexes formed by stable and rigid protein-protein interactions, and 2) liquid-like phase-separated condensates formed by weaker and more dynamic intermolecular interactions. This review covers key examples of both types of higher-order assemblies in major immune pathways. By placing emphasis on the molecular structures of the examples provided, we discuss how their structural organization enables elegant mechanisms of signaling regulation.

TNF-TNFR1 Signaling Enhances the Protection Against Neospora caninum Infection.

Neospora caninum is a protozoan associated with abortions in ruminants and neuromuscular disease in dogs. Classically, the immune response against apicomplexan parasites is characterized by the production of proinflammatory cytokines, such as IL-12, IFN-γ and TNF. TNF is mainly produced during the acute phases of the infections and binds to TNF receptor 1 (CD120a, p55, TNFR1) activating a variety of cells, hence playing an important role in the induction of the inflammatory process against diverse pathogens. Thus, in this study, we aimed to evaluate the role of TNF in cellular and humoral immune responses during N. caninum infection. For this purpose, we used a mouse model of infection based on wildtype (WT) and genetically deficient C57BL/6 mice in TNFR1 (Tnfr1-/-). We observed that Tnfr1-/- mice presented higher mortality associated with inflammatory lesions and increased parasite burden in the brain after the infection with N. caninum tachyzoites. Moreover, Tnfr1-/- mice showed a reduction in nitric oxide (NO) levels in vivo. We also observed that Tnfr1-/- mice showed enhanced serum concentration of antigen-specific IgG2 subclass, while IgG1 production was significantly reduced compared to WT mice, suggesting that TNFR1 is required for regular IgG subclass production and antigen recognition. Based on our results, we conclude that the TNF-TNFR1 complex is crucial for mediating host resistance during the infection by N. caninum.

TNFRp75-dependent immune regulation of alveolar macrophages and neutrophils during early Mycobacterium tuberculosis and Mycobacterium bovis BCG infection.

TNF signalling through TNFRp55 and TNFRp75, and receptor shedding is important for immune activation and regulation. TNFRp75 deficiency leads to improved control of Mycobacterium tuberculosis (M. tuberculosis) infection, but the effects of early innate immune events in this process are unclear. We investigated the role of TNFRp75 on cell activation and apoptosis of alveolar macrophages and neutrophils during M. tuberculosis and M. bovis BCG infection. We found increased microbicidal activity against M. tuberculosis occurred independently of IFNy and NO generation, and displayed an inverse correlation with alveolar macrophages (AMs) apoptosis. Both M. tuberculosis and M. bovis BCG induced higher expression of MHC-II in TNFRp75-/- AMs; however, M bovis BCG infection did not alter AM apoptosis in the absence of TNFRp75. Pulmonary concentrations of CCL2, CCL3 and IL-1ß were increased in TNFRp75-/- mice during M, bovis BCG infection, but had no effect on neutrophil responses. Thus, TNFRp75-dependent regulation of mycobacterial replication is virulence dependent and occurs independently of early alveolar macrophage apoptosis and neutrophil responses.

Vaccine targeting TNF epitope 1-14 do not suppress host defense against Mycobacterium bovis Bacillus Calmette-Guérin infection.

Anti-TNF inhibitors are efficacious in the treatment of chronic inflammatory diseases such as rheumatoid arthritis (RA), Crohn's disease (CD), juvenile idiopathic arthritis (JIA), and ankylosing spondylitis (AS). However, more and more clinical case reports revealed that anti-TNF inhibitors could increase the risk of viral, fungal, and bacterial (especially intracellular) infection. In this study, based on Immune Epitope Database (IEDB) online B cell epitope prediction and the knowledge of TNF three dimensional (3D) structure we developed a novel vaccine (DTNF114-TNF114) that targeting TNF epitope 1-14, which produced antibodies only partially binding to trans-membrane TNF (tmTNF), therefore partially sparing tmTNF-TNFR1/2 interaction. Immunization with DTNF114-TNF114 significantly protected and prolonged the survival rate of mice challenged with lipopolysaccharide (LPS); and in the mCherry expressing Mycobacterium bovis Bacillus Calmette-Guérin (mCherry-BCG) infection model, DTNF114-TNF114 immunization significantly decreased soluble TNF (solTNF) level in serum, meanwhile did not suppress host immunity against infection. Thus, this novel and infection concern-free vaccine provides a potential alternative or supplement to currently clinically used anti-TNF inhibitors.

Tumor Necrosis Factor Receptors: Pleiotropic Signaling Complexes and Their Differential Effects.

Since its discovery in 1975, TNFα has been a subject of intense study as it plays significant roles in both immunity and cancer. Such attention is well deserved as TNFα is unique in its engagement of pleiotropic signaling via its two receptors: TNFR1 and TNFR2. Extensive research has yielded mechanistic insights into how a single cytokine can provoke a disparate range of cellular responses, from proliferation and survival to apoptosis and necrosis. Understanding the intracellular signaling pathways induced by this single cytokine via its two receptors is key to further revelation of its exact functions in the many disease states and immune responses in which it plays a role. In this review, we describe the signaling complexes formed by TNFR1 and TNFR2 that lead to each potential cellular response, namely, canonical and non-canonical NF-κB activation, apoptosis and necrosis. This is followed by a discussion of data from in vivo mouse and human studies to examine the differential impacts of TNFR1 versus TNFR2 signaling.

Selective Blockade of TNFR1 Improves Clinical Disease and Bronchoconstriction in Experimental RSV Infection.

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis in infants and young children. Although some clinical studies have speculated that tumor necrosis factor (TNF)-α is a major contributor of RSV-mediated airway disease, experimental evidence remains unclear or conflicting. TNF-α initiates inflammation and cell death through two distinct receptors: TNF-receptor (TNFR)1 and TNFR2. Here we delineate the function of TNF-α by short-lasting blockade of either receptor in an experimental BALB/c mouse model of RSV infection. We demonstrate that antibody-mediated blockade of TNFR1, but not TNFR2, results in significantly improved clinical disease and bronchoconstriction as well as significant reductions of several inflammatory cytokines and chemokines, including IL-1α, IL-1ß, IL-6, Ccl3, Ccl4, and Ccl5. Additionally, TNFR1 blockade was found to significantly reduce neutrophil number and activation status, consistent with the concomitant reduction of pro-neutrophilic chemokines Cxcl1 and Cxcl2. Similar protective activity was also observed when a single-dose of TNFR1 blockade was administered to mice following RSV inoculation, although this treatment resulted in improved alveolar macrophage survival rather than reduced neutrophil activation. Importantly, short-lasting blockade of TNFR1 did not affect RSV peak replication in the lung. This study suggests a potential therapeutic approach for RSV bronchiolitis based on selective blockade of TNFR1.

Autoantibodies from SLE patients induce programmed cell death in murine fibroblast cells through interaction with TNFR1 receptor.

Various pathological processes are known to be associated with the production of IgG autoantibodies, which have high affinity for self-antigens and often cause tissue injury and the development of autoimmune diseases. However, the mechanism of their cytotoxic activity is not clearly understood yet. Here, we have shown that the action of these autoantibodies on cells expressing TNFR1 (the cell surface receptor for TNFα) can cause both caspase-dependent apoptosis and necroptosis of these cells, with suppression of apoptosis resulting in switching to RIP1-dependent necroptosis. Analysis of necroptotic mechanisms has shown that a critical point of necroptosis is phosphorylation of RIP1 and RIP3 kinases, which is followed by the involvement of lysosomes and mitochondria in this process. The induction of cytotoxicity is initiated by the interaction of autoantibodies with TNFR1, and autoantibodies can therefore be regarded as a new functional ligand for this receptor. The innate immunity protein Tag7 (PGLYRP1) described in our recent studies is also a ligand for TNFR1 and competes with autoantibodies for binding with it. Supposedly, the cytotoxic effect of autoantibodies is one of the factors responsible for autoimmune diseases that lead to tissue injury.

Characterization of the Inflammatory Response to Severe COVID-19 Illness.

Rationale: Coronavirus disease (COVID-19) is a global threat to health. Its inflammatory characteristics are incompletely understood.Objectives: To define the cytokine profile of COVID-19 and to identify evidence of immunometabolic alterations in those with severe illness.Methods: Levels of IL-1ß, IL-6, IL-8, IL-10, and sTNFR1 (soluble tumor necrosis factor receptor 1) were assessed in plasma from healthy volunteers, hospitalized but stable patients with COVID-19 (COVIDstable patients), patients with COVID-19 requiring ICU admission (COVIDICU patients), and patients with severe community-acquired pneumonia requiring ICU support (CAPICU patients). Immunometabolic markers were measured in circulating neutrophils from patients with severe COVID-19. The acute phase response of AAT (alpha-1 antitrypsin) to COVID-19 was also evaluated.Measurements and Main Results: IL-1ß, IL-6, IL-8, and sTNFR1 were all increased in patients with COVID-19. COVIDICU patients could be clearly differentiated from COVIDstable patients, and demonstrated higher levels of IL-1ß, IL-6, and sTNFR1 but lower IL-10 than CAPICU patients. COVID-19 neutrophils displayed altered immunometabolism, with increased cytosolic PKM2 (pyruvate kinase M2), phosphorylated PKM2, HIF-1α (hypoxia-inducible factor-1α), and lactate. The production and sialylation of AAT increased in COVID-19, but this antiinflammatory response was overwhelmed in severe illness, with the IL-6:AAT ratio markedly higher in patients requiring ICU admission (P < 0.0001). In critically unwell patients with COVID-19, increases in IL-6:AAT predicted prolonged ICU stay and mortality, whereas improvement in IL-6:AAT was associated with clinical resolution (P < 0.0001).Conclusions: The COVID-19 cytokinemia is distinct from that of other types of pneumonia, leading to organ failure and ICU need. Neutrophils undergo immunometabolic reprogramming in severe COVID-19 illness. Cytokine ratios may predict outcomes in this population.

Structural optimization of a TNFR1-selective antagonistic TNFα mutant to create new-modality TNF-regulating biologics.

Excessive activation of the proinflammatory cytokine tumor necrosis factor-α (TNFα) is a major cause of autoimmune diseases, including rheumatoid arthritis. TNFα induces immune responses via TNF receptor 1 (TNFR1) and TNFR2. Signaling via TNFR1 induces proinflammatory responses, whereas TNFR2 signaling is suggested to suppress the pathophysiology of inflammatory diseases. Therefore, selective inhibition of TNFR1 signaling and preservation of TNFR2 signaling activities may be beneficial for managing autoimmune diseases. To this end, we developed a TNFR1-selective, antagonistic TNFα mutant (R1antTNF). Here, we developed an R1antTNF derivative, scR1antTNF-Fc, which represents a single-chain form of trimeric R1antTNF with a human IgG-Fc domain. scR1antTNF-Fc had properties similar to those of R1antTNF, including TNFR1-selective binding avidity, TNFR1 antagonistic activity, and thermal stability, and had a significantly extended plasma t1/2in vivo In a murine rheumatoid arthritis model, scR1antTNF-Fc and 40-kDa PEG-scR1antTNF (a previously reported PEGylated form) delayed the onset of collagen-induced arthritis, suppressed arthritis progression in mice, and required a reduced frequency of administration. Interestingly, with these biologic treatments, we observed an increased ratio of regulatory T cells to conventional T cells in lymph nodes compared with etanercept, a commonly used TNF inhibitor. Therefore, scR1antTNF-Fc and 40-kDa PEG-scR1antTNF indirectly induced immunosuppression. These results suggest that selective TNFR1 inhibition benefits the management of autoimmune diseases and that R1antTNF derivatives hold promise as new-modality TNF-regulating biologics.

TNF-α pathway and T-cell immunity are activated early during the development of diabetic nephropathy in Type II Diabetes Mellitus.

Aim of the present study was to investigate the possible involvement of TNF-α signaling pathway and T-lymphocyte activation in DN. Eighty-two diabetic patients [39 male, age 69.5(56-78)years] were divided into three groups, according to Albumin/Creatinine ratio (ACR) levels, Group I (ACR < 30 µg/mg), Group II (ACR 30-300 µg/mg), Group III (ACR > 300 µg/mg). Urinary Tumor Necrosis Factor-α (TNF-α), and serum TNF-α, ΤNF-receptor 1 (TNFR1), TNFR2, B7-1, CD28, Cytoxic T-Lymphocyte-Associated protein-4 (CTLA4), were estimated. There were significant differences between Groups I, II, III regarding the concentration of urinary TNF-α (p < .001), serum TNFR1 (p < .001), serum TNFR2(p < .001), CTLA4 (p < .001) and CD28(p = .034). In multivariate analysis, independent parameters correlated with ACR were serum TNFR1 (p = .003), TNFR2 (p = .012) and urinary TNF-α (p = .015) levels. There was a significant correlation between markers of T-cell activation and TNF-α signaling pathway activation. Activation of TNF-α signaling pathway and T-lymphocytes seem to synergize and participate in the development of DN in type II DM.

Dendritic Cells of Mesenteric and Regional Lymph Nodes Contribute to Yersinia enterocolitica O:3-Induced Reactive Arthritis in TNFRp55-/- Mice.

Dendritic cells (DCs) participate in the pathogenesis of several diseases. We investigated DCs and the connection between mucosa and joints in a murine model of Yersinia enterocolitica O:3-induced reactive arthritis (ReA) in TNFRp55-/- mice. DCs of mesenteric lymph nodes (MLN) and joint regional lymph nodes (RLN) were analyzed in TNFRp55-/- and wild-type mice. On day 14 after Y. enterocolitica infection (arthritis onset), we found that under TNFRp55 deficiency, migratory (MHChighCD11c+) DCs increased significantly in RLN. Within these RLN, resident (MHCintCD11c+) DCs increased on days 14 and 21. Similar changes in both migratory and resident DCs were also detected on day 14 in MLN of TNFRp55-/- mice. In vitro, LPS-stimulated migratory TNFRp55-/- DCs of MLN increased IL-12/23p40 compared with wild-type mice. In addition, TNFRp55-/- bone marrow-derived DCs in a TNFRp55-/- MLN microenvironment exhibited higher expression of CCR7 after Y. enterocolitica infection. The major intestinal DC subsets (CD103+CD11b-, CD103-CD11b+, and CD103+CD11b+) were found in the RLN of Y. enterocolitica-infected TNFRp55-/- mice. Fingolimod (FTY720) treatment of Y. enterocolitica-infected mice reduced the CD11b- subset of migratory DCs in RLN of TNFRp55-/- mice and significantly suppressed the severity of ReA in these mice. This result was associated with decreased articular IL-12/23p40 and IFN-γ levels. In vitro FTY720 treatment downregulated CCR7 on Y. enterocolitica-infected bone marrow-derived DCs and purified MLN DCs, which may explain the mechanism underlying the impairment of DCs in RLN induced by FTY720. Taken together, data indicate the migration of intestinal DCs to RLN and the contribution of these cells in the immunopathogenesis of ReA, which may provide evidence for controlling this disease.

Sustained hyperammonemia induces TNF-a IN Purkinje neurons by activating the TNFR1-NF-κB pathway.

BACKGROUND: Patients with liver cirrhosis may develop hepatic encephalopathy. Rats with chronic hyperammonemia exhibit neurological alterations mediated by peripheral inflammation and neuroinflammation. Motor incoordination is due to increased TNF-a levels and activation of its receptor TNFR1 in the cerebellum. The aims were to assess (a) whether peripheral inflammation is responsible for TNF-a induction in hyperammonemic rats, (b) the cell type(s) in which TNF-a is increased, (c) whether this increase is associated with increased nuclear NF-κB and TNFR1 activation, (d) the time course of TNF-a induction, and (e) if TNF-a is induced in the Purkinje neurons of patients who die with liver cirrhosis. METHODS: We analyzed the level of TNF-a mRNA and NF-κB in microglia, astrocytes, and Purkinje neurons in the cerebellum after 1, 2, and 4 weeks of hyperammonemia. We assessed whether preventing peripheral inflammation by administering an anti-TNF-a antibody prevents TNF-a induction. We tested whether TNF-a induction is reversed by R7050, which inhibits the TNFR1-NF-κB pathway, in ex vivo cerebellar slices. RESULTS: Hyperammonemia induced microglial and astrocyte activation at 1 week. This was followed by TNF-a induction in both glial cell types at 2 weeks and in Purkinje neurons at 4 weeks. The level of TNF-a mRNA increased in parallel with the TNF-a protein level, indicating that TNF-a was synthesized in Purkinje cells. This increase was associated with increased NF-κB nuclear translocation. The nuclear translocation of NF-κB and the increase in TNF-a were reversed by R7050, indicating that they were mediated by the activation of TNFR1. Preventing peripheral inflammation with an anti-TNF-a antibody prevents TNF-a induction. CONCLUSION: Sustained (4 weeks) but not short-term hyperammonemia induces TNF-a in Purkinje neurons in rats. This is mediated by peripheral inflammation. TNF-a is also increased in the Purkinje neurons of patients who die with liver cirrhosis. The results suggest that hyperammonemia induces TNF-a in glial cells and that TNF-a released by glial cells activates TNFR1 in Purkinje neurons, leading to NF-κB nuclear translocation and the induction of TNF-a expression, which may contribute to the neurological alterations observed in hyperammonemia and hepatic encephalopathy.