Accelerated Clearance and Degradation of Cell-Free HIV by Neutralizing Antibodies Occurs via FcγRIIb on Liver Sinusoidal Endothelial Cells by Endocytosis.

Neutralizing Abs suppress HIV infection by accelerating viral clearance from blood circulation in addition to neutralization. The elimination mechanism is largely unknown. We determined that human liver sinusoidal endothelial cells (LSEC) express FcγRIIb as the lone Fcγ receptor, and using humanized FcγRIIb mouse, we found that Ab-opsonized HIV pseudoviruses were cleared considerably faster from circulation than HIV by LSEC FcγRIIb. Compared with humanized FcγRIIb-expressing mice, HIV clearance was significantly slower in FcγRIIb knockout mice. Interestingly, a pentamix of neutralizing Abs cleared HIV faster compared with hyperimmune anti-HIV Ig (HIVIG), although the HIV Ab/Ag ratio was higher in immune complexes made of HIVIG and HIV than pentamix and HIV. The effector mechanism of LSEC FcγRIIb was identified to be endocytosis. Once endocytosed, both Ab-opsonized HIV pseudoviruses and HIV localized to lysosomes. This suggests that clearance of HIV, endocytosis, and lysosomal trafficking within LSEC occur sequentially and that the clearance rate may influence downstream events. Most importantly, we have identified LSEC FcγRIIb-mediated endocytosis to be the Fc effector mechanism to eliminate cell-free HIV by Abs, which could inform development of HIV vaccine and Ab therapy.

Oligodendroglial TNFR2 Mediates Membrane TNF-Dependent Repair in Experimental Autoimmune Encephalomyelitis by Promoting Oligodendrocyte Differentiation and Remyelination.

UNLABELLED: Tumor necrosis factor (TNF) is associated with the pathophysiology of various neurological disorders, including multiple sclerosis. It exists as a transmembrane form tmTNF, signaling via TNF receptor 2 (TNFR2) and TNFR1, and a soluble form, solTNF, signaling via TNFR1. Multiple sclerosis is associated with the detrimental effects of solTNF acting through TNFR1, while tmTNF promotes repair and remyelination. Here we demonstrate that oligodendroglial TNFR2 is a key mediator of tmTNF-dependent protection in experimental autoimmune encephalomyelitis (EAE). CNP-cre:TNFR2(fl/fl) mice with TNFR2 ablation in oligodendrocytes show exacerbation of the disease with increased axon and myelin pathology, reduced remyelination, and increased loss of oligodendrocyte precursor cells and mature oligodendrocytes. The clinical course of EAE is not improved by the solTNF inhibitor XPro1595 in CNP-cre:TNFR2(fl/fl) mice, indicating that for tmTNF to promote recovery TNFR2 in oligodendrocytes is required. We show that TNFR2 drives differentiation of oligodendrocyte precursor cells, but not proliferation or survival. TNFR2 ablation leads to dysregulated expression of microRNAs, among which are regulators of oligodendrocyte differentiation and inflammation, including miR-7a. Our data provide the first direct in vivo evidence that TNFR2 in oligodendrocytes is important for oligodendrocyte differentiation, thereby sustaining tmTNF-dependent repair in neuroimmune disease. Our studies identify TNFR2 in the CNS as a molecular target for the development of remyelinating agents, addressing the most pressing need in multiple sclerosis therapy nowadays. SIGNIFICANCE STATEMENT: Our study, using novel TNF receptor 2 (TNFR2) conditional KO mice with selective TNFR2 ablation in oligodendrocytes, provides the first direct evidence that TNFR2 is an important signal for oligodendrocyte differentiation. Following activation by transmembrane TNF, TNFR2 initiates pathways that drive oligodendrocytes into a reparative mode contributing to remyelination following disease. This identifies TNFR2 as a new molecular target for the development of therapeutic agents in multiple sclerosis.

Transmembrane TNF-α is sufficient for articular inflammation and hypernociception in a mouse model of gout.

Gout manifests as recurrent episodes of acute joint inflammation and pain due to the deposition of monosodium urate (MSU) crystals within the affected tissue in a process dependent on NLRP3 inflammasome activation. The synthesis, activation, and release of IL-1ß are crucial for MSU-induced inflammation. The current study evaluated the mechanism by which TNF-α contributed to MSU-induced inflammation. Male C57BL/6J or transgenic mice were used in this study and inflammation was induced by the injection of MSU crystals into the joint. TNF-α was markedly increased in the joint after the injection of MSU. There was inhibition in the infiltration of neutrophils, production of CXCL1 and IL-1ß, and decreased hypernociception in mice deficient for TNF-α or its receptors. Pharmacological blockade of TNF-α with Etanercept or pentoxyfylline produced similar results. Mechanistically, TNF-α blockade resulted in lower amounts of IL-1ß protein and pro-IL-1ß mRNA transcripts in joints. Gene-modified mice that express only transmembrane TNF-α had an inflammatory response similar to that of WT mice and blockade of soluble TNF-α (XPro™1595) did not decrease MSU-induced inflammation. In conclusion, TNF-α drives expression of pro-IL-1ß mRNA and IL-1ß protein in experimental gout and that its transmembrane form is sufficient to trigger MSU-induced inflammation in mice.

Altered expression of oligodendrocyte and neuronal marker genes predicts the clinical onset of autoimmune encephalomyelitis and indicates the effectiveness of multiple sclerosis-directed therapeutics.

Experimental autoimmune encephalomyelitis (EAE) is a valuable model for studying immunopathology in multiple sclerosis (MS) and for exploring the interface between autoimmune responses and CNS tissue that ultimately leads to lesion development. In this study, we measured gene expression in mouse spinal cord during myelin oligodendrocyte gp35-55 peptide-induced EAE, using quantitative RT-PCR, to identify gene markers that monitor individual hallmark pathological processes. We defined a small panel of genes whose longitudinal expression patterns provided insight into the timing, interrelationships, and mechanisms of individual disease processes and the efficacy of therapeutics for the treatment of MS. Earliest transcriptional changes were upregulation of Il17a and sharp downregulation of neuronal and oligodendrocyte marker genes preceding clinical disease onset, whereas neuroinflammatory markers progressively increased as symptoms and tissue lesions developed. EAE-induced gene-expression changes were not altered in mice deficient in IKKß in cells of the myeloid lineage compared with controls, but the administration of a selective inhibitor of soluble TNF to mice from the day of immunization delayed changes in the expression of innate inflammation, myelin, and neuron markers from the presymptomatic phase. Proof of principle that the gene panel shows drug screening potential was obtained using a well-established MS therapeutic, glatiramer acetate. Prophylactic treatment of mice with glatiramer acetate normalized gene marker expression, and this correlated with the level of therapeutic success. These results show that neurons and oligodendrocytes are highly sensitive to CNS-directed autoimmunity before the development of clinical symptoms and immunopathology and reveal a role for soluble TNF in mediating the earliest changes in gene expression.

Central but not systemic administration of XPro1595 is therapeutic following moderate spinal cord injury in mice.

BACKGROUND: Glial cell activation and overproduction of inflammatory mediators in the central nervous system (CNS) have been implicated in acute traumatic injuries to the CNS, including spinal cord injury (SCI). Elevated levels of the proinflammatory cytokine tumor necrosis factor (TNF), which exists in both a soluble (sol) and a transmembrane (tm) form, have been found in the lesioned cord early after injury. The contribution of solTNF versus tmTNF to the development of the lesion is, however, still unclear. METHODS: We tested the effect of systemically or centrally blocking solTNF alone, using XPro1595, versus using the drug etanercept to block both solTNF and tmTNF compared to a placebo vehicle following moderate SCI in mice. Functional outcomes were evaluated using the Basso Mouse Scale, rung walk test, and thermal hyperalgesia analysis. The inflammatory response in the lesioned cord was investigated using immunohistochemistry and western blotting analyses. RESULTS: We found that peripheral administration of anti-TNF therapies had no discernable effect on locomotor performances after SCI. In contrast, central administration of XPro1595 resulted in improved locomotor function, decreased anxiety-related behavior, and reduced damage to the lesioned spinal cord, whereas central administration of etanercept had no therapeutic effects. Improvements in XPro1595-treated mice were accompanied by increases in Toll-like receptor 4 and TNF receptor 2 (TNFR2) protein levels and changes in Iba1 protein expression in microglia/macrophages 7 and 28 days after SCI. CONCLUSIONS: These studies suggest that, by selectively blocking solTNF, XPro1595 is neuroprotective when applied directly to the lesioned cord. This protection may be mediated via alteration of the inflammatory environment without suppression of the neuroprotective effects of tmTNF signaling through TNFR2.

Systemically administered anti-TNF therapy ameliorates functional outcomes after focal cerebral ischemia.

BACKGROUND: The innate immune system contributes to the outcome after stroke, where neuroinflammation and post-stroke systemic immune depression are central features. Tumor necrosis factor (TNF), which exists in both a transmembrane (tm) and soluble (sol) form, is known to sustain complex inflammatory responses associated with stroke. We tested the effect of systemically blocking only solTNF versus blocking both tmTNF and solTNF on infarct volume, functional outcome and inflammation in focal cerebral ischemia. METHODS: We used XPro1595 (a dominant-negative inhibitor of solTNF) and etanercept (which blocks both solTNF and tmTNF) to test the effect of systemic administration on infarct volume, functional recovery and inflammation after focal cerebral ischemia in mice. Functional recovery was evaluated after one, three and five days, and infarct volumes at six hours, 24 hours and five days after ischemia. Brain inflammation, liver acute phase response (APR), spleen and blood leukocyte profiles, along with plasma microvesicle analysis, were evaluated. RESULTS: We found that both XPro1595 and etanercept significantly improved functional outcomes, altered microglial responses, and modified APR, spleen T cell and microvesicle numbers, but without affecting infarct volumes. CONCLUSIONS: Our data suggest that XPro1595 and etanercept improve functional outcome after focal cerebral ischemia by altering the peripheral immune response, changing blood and spleen cell populations and decreasing granulocyte infiltration into the brain. Blocking solTNF, using XPro1595, was just as efficient as blocking both solTNF and tmTNF using etanercept. Our findings may have implications for future treatments with anti-TNF drugs in TNF-dependent diseases.

Antibody-mediated coengagement of FcγRIIb and B cell receptor complex suppresses humoral immunity in systemic lupus erythematosus.

Engagement of the low-affinity Ab receptor FcγRIIb downregulates B cell activation, and its dysfunction is associated with autoimmunity in mice and humans. We engineered the Fc domain of an anti-human CD19 Ab to bind FcγRIIb with high affinity, promoting the coengagement of FcγRIIb with the BCR complex. This Ab (XmAb5871) stimulated phosphorylation of the ITIM of FcγRIIb and suppressed BCR-induced calcium mobilization, proliferation, and costimulatory molecule expression of human B cells from healthy volunteers and systemic lupus erythematosus (SLE) patients, as well as B cell proliferation induced by LPS, IL-4, or BAFF. XmAb5871 suppressed humoral immunity against tetanus toxoid and reduced serum IgM, IgG, and IgE levels in SCID mice engrafted with SLE or healthy human PBMC. XmAb5871 treatment also increased survival of mice engrafted with PBMC from a unique SLE patient. Unlike anti-CD20 Ab, coengagement of FcγRIIb and BCR complex did not promote B cell depletion in human PBMC cultures or in mice. Thus, amplification of the FcγRIIb inhibitory pathway in activated B cells may represent a novel B cell-targeted immunosuppressive therapeutic approach for SLE and other autoimmune diseases that should avoid the complications associated with B cell depletion.

Reduction of total IgE by targeted coengagement of IgE B-cell receptor and FcγRIIb with Fc-engineered antibody.

BACKGROUND: Sequestration of IgE to prevent its binding to high-affinity IgE receptor FcεRI on basophils and mast cells is an effective therapy for allergic asthma. IgE production requires differentiation of activated IgE(+) B cells into plasma cells upon allergen sensitization. B-cell receptor signaling is suppressed by the inhibitory IgG Fc receptor FcγRIIb; therefore, we reasoned that a therapeutic antibody that coengages FcγRIIb and IgE B-cell receptor would not only sequester IgE but also suppress its production by blocking IgE(+) B-cell activation and differentiation to IgE-secreting plasma cells. OBJECTIVE: To explore the effects of IgE sequestration versus IgE suppression by comparing omalizumab to FcγRIIb-optimized anti-IgE antibodies in humanized mouse models of immunoglobulin production. METHODS: By using a murine anti-IgE antibody as a template, we humanized, increased IgE binding, and modified its Fc domain to increase affinity for FcγRIIb. We next compared effects of this antibody (XmAb7195) versus omalizumab on the secretion of IgE and other isotypes in human PBMC cultures and in PBMC-engrafted severe combined immunodeficiency mice. RESULTS: Relative to omalizumab, XmAb7195 has a 5-fold higher affinity for human IgE and more than 400-fold higher affinity for FcγRIIb. In addition to sequestering soluble IgE, XmAb7195 inhibited plasma cell differentiation and consequent human IgE production through coengagement of IgE B-cell receptor with FcγRIIb. In PBMC-engrafted mice, XmAb7195 reduced total human IgE (but not IgG or IgM) levels by up to 40-fold relative to omalizumab. CONCLUSION: XmAb7195 acts by IgE sequestration coupled with an FcγRIIb-mediated inhibitory mechanism to suppress the formation of IgE-secreting plasma cells and reduce both free and total IgE levels.

Virally infected and matured human dendritic cells activate natural killer cells via cooperative activity of plasma membrane-bound TNF and IL-15.

Recombinant adenovirus-engineered dendritic cells (Ad.DCs) are potent immunologic adjuvants of antiviral and anticancer vaccines. The effectiveness of Ad.DC-based vaccines may depend on the ability of Ad.DCs to crosstalk with natural killer (NK) cells and to activate, polarize, and bridge innate and adaptive immunity. We investigated, for the first time, whether and how human Ad.DCs activate NK cells, and compared the Ad.DC function with that of immature DCs and matured DCs (mDCs). We found that adenovirus transduction and lipopolysaccharide/interferon-gamma-induced maturation increased expression of transmembrane tumor necrosis factor (TNF) and trans-presented (trans) interleukin-15 (IL-15) on DCs, leading to enhanced NK cell activation without enhancing DC susceptibility to NK cell-mediated killing. This crosstalk enhanced NK cell CD69 expression, interferon-gamma secretion, proliferation, and antitumor activities, with Ad.DCs being significantly more effective than immature DCs, but less effective than mDCs. The Ad.DC and mDC crosstalk with NK cells was largely prevented by physical separation of DCs and NK cells, and neutralization of total TNF and IL-15, but not by selective sequestration of soluble TNF. These findings demonstrate that both Ad.DCs and mDCs can efficiently promote innate immune functions by activation of NK cells through the cooperative activities of tmTNF and trans-IL-15 mediated by cell-to-cell contact.

Transmembrane tumour necrosis factor is neuroprotective and regulates experimental autoimmune encephalomyelitis via neuronal nuclear factor-kappaB.

Tumour necrosis factor mediates chronic inflammatory pathologies including those affecting the central nervous system, but non-selective tumour necrosis factor inhibitors exacerbate multiple sclerosis. In addition, TNF receptor SF1A, which encodes one of the tumour necrosis factor receptors, has recently been identified as a multiple sclerosis susceptibility locus in genome-wide association studies in large patient cohorts. These clinical data have emphasized the need for a better understanding of the beneficial effects of tumour necrosis factor during central nervous system inflammation. In this study, we present evidence that the soluble and transmembrane forms of tumour necrosis factor exert opposing deleterious and beneficial effects, respectively, in a multiple sclerosis model. We compared the effects, in experimental autoimmune encephalomyelitis, of selectively inhibiting soluble tumour necrosis factor, and of both soluble and transmembrane tumour necrosis factor. Blocking the action of soluble tumour necrosis factor, but not of soluble tumour necrosis factor and transmembrane tumour necrosis factor, protected mice against the clinical symptoms of experimental autoimmune encephalomyelitis. Therapeutic benefit was independent of changes in antigen-specific immune responses and focal inflammatory spinal cord lesions, but was associated with reduced overall central nervous system immunoreactivity, increased expression of neuroprotective molecules, and was dependent upon the activity of neuronal nuclear factor-κB, a downstream mediator of neuroprotective tumour necrosis factor/tumour necrosis factor receptor signalling, because mice lacking IκB kinase ß in glutamatergic neurons were not protected by soluble tumour necrosis factor blockade. Furthermore, blocking the action of soluble tumour necrosis factor, but not of soluble tumour necrosis factor and transmembrane tumour necrosis factor, protected neurons in astrocyte-neuron co-cultures against glucose deprivation, an in vitro neurodegeneration model relevant for multiple sclerosis, and this was dependent upon contact between the two cell types. Our results show that soluble tumour necrosis factor promotes central nervous system inflammation, while transmembrane tumour necrosis factor is neuroprotective, and suggest that selective inhibition of soluble tumour necrosis factor may provide a new way forward for the treatment of multiple sclerosis and possibly other inflammatory central nervous system disorders.

Inhibition of soluble tumour necrosis factor is therapeutic in experimental autoimmune encephalomyelitis and promotes axon preservation and remyelination.

Tumour necrosis factor is linked to the pathophysiology of various neurodegenerative disorders including multiple sclerosis. Tumour necrosis factor exists in two biologically active forms, soluble and transmembrane. Here we show that selective inhibition of soluble tumour necrosis factor is therapeutic in experimental autoimmune encephalomyelitis. Treatment with XPro1595, a selective soluble tumour necrosis factor blocker, improves the clinical outcome, whereas non-selective inhibition of both forms of tumour necrosis factor with etanercept does not result in protection. The therapeutic effect of XPro1595 is associated with axon preservation and improved myelin compaction, paralleled by increased expression of axon-specific molecules (e.g. neurofilament-H) and reduced expression of non-phosphorylated neurofilament-H which is associated with axon damage. XPro1595-treated mice show significant remyelination accompanied by elevated expression of myelin-specific genes and increased numbers of oligodendrocyte precursors. Immunohistochemical characterization of tumour necrosis factor receptors in the spinal cord following experimental autoimmune encephalomyelitis shows tumour necrosis factor receptor 1 expression in neurons, oligodendrocytes and astrocytes, while tumour necrosis factor receptor 2 is localized in oligodendrocytes, oligodendrocyte precursors, astrocytes and macrophages/microglia. Importantly, a similar pattern of expression is found in post-mortem spinal cord of patients affected by progressive multiple sclerosis, suggesting that pharmacological modulation of tumour necrosis factor receptor signalling may represent an important target in affecting not only the course of mouse experimental autoimmune encephalomyelitis but human multiple sclerosis as well. Collectively, our data demonstrate that selective inhibition of soluble tumour necrosis factor improves recovery following experimental autoimmune encephalomyelitis, and that signalling mediated by transmembrane tumour necrosis factor is essential for axon and myelin preservation as well as remyelination, opening the possibility of a new avenue of treatment for multiple sclerosis.

Allergic lung inflammation is mediated by soluble tumor necrosis factor (TNF) and attenuated by dominant-negative TNF biologics.

Tumor Necrosis Factor (TNF) is a pleiotropic cytokine consisting of soluble and transmembrane forms, with distinct roles in inflammation and immunity. TNF is an important factor in allergic airway inflammation. However, the disparate functions of soluble (sol) and transmembrane (tm) TNF in lung pathology are not well understood. Our aim was to assess the activities of solTNF and tmTNF in murine models of allergic airway disease, and to evaluate the efficacy of solTNF-selective inhibition. We used ovalbumin sensitization and challenge of TNF knockout, tmTNF knockin, and wild-type C57BL/6 mice to distinguish differences in airway inflammation and hyperreactivity mediated by solTNF and tmTNF. Functions of solTNF and tmTNF in hyperresponsive, wild-type Balb/c mice were assessed by comparing dominant-negative anti-TNF biologics, which antagonize solTNF yet spare tmTNF, to etanercept, a nonselective inhibitor of both TNF forms. Responses in transgenic C57BL/6 mice demonstrated that solTNF, and not tmTNF, is necessary to drive airway inflammation. In Balb/c mice, dominant-negative TNF biologics administered during immunization decreased the recruitment of eosinophils and lymphocytes into the bronchoalveolar space and lung parenchyma, reduced specific serum IgE, goblet-cell hyperplasia, and eosinophilic inflammation, and suppressed methacholine-induced airway hyperreactivity. Concentrations of IL-5, CCL5/RANTES, CCL11/eotaxin, and CCL17/TARC were also reduced in bronchoalveolar lavage. Dominant-negative TNFs reduced lung eosinophilia, even when given only during antigen challenge. The selective inhibition of soluble TNF suppresses inflammation, hyperreactivity, and remodeling in transgenic and wild-type murine models of allergic airway disease, and may offer safety advantages in therapies that preserve the immunoprotective functions of transmembrane TNF.

Inhibition of B cell activation following in vivo co-engagement of B cell antigen receptor and Fcγ receptor IIb in non-autoimmune-prone and SLE-prone mice.

Engagement of Fcγ receptor IIb (FcγRIIb) suppresses B cell activation and represents a promising target for therapy in autoimmunity. Obexelimab is a non-depleting anti-human CD19 mAb with an Fc region engineered to have high affinity for human FcγRIIb, thereby co-engaging BCR and FcγRIIb. To assess its ability to suppress B cell activation in vivo, we generated non-autoimmune-prone C57BL/6 (B6) and SLE-prone NZM 2328 (NZM) mice in which the human FcγRIIb extracellular domain was knocked into the mouse Fcgr2b locus (B6.hRIIb and NZM.hRIIb mice, respectively, the latter retaining features of SLE). XENP8206, a mAb which bears the same FcγRIIb-enhanced human Fc domain as does obexelimab but which recognizes murine CD19 rather than human CD19, inhibited in vitro BCR-triggered activation of B cells from both B6.hRIIb and NZM.hRIIb mice. Following administration of XENP8206 to B6.hRIIb or NZM.hRIIb mice, B cell numbers in the spleen and lymph nodes remained stable but became hyporesponsive to BCR-triggered activation for at least 14 days. These findings demonstrate proof-of-principle that pharmacologic co-engagement of BCR and human FcγRIIb inhibits B cell activation in non-autoimmune and SLE-prone hosts while preserving B cell numbers. These observations lay a strong foundation for clinical trials in human SLE with agents that co-engage BCR and FcγRIIb. Moreover, B6.hRIIb and NZM.hRIIb should serve as powerful in vivo models in the elucidation of the cellular and molecular underpinnings of the changes induced by BCR/FcγRIIb co-engagement.

Soluble TNF, but not membrane TNF, is critical in LPS-induced hepatitis.

BACKGROUND & AIMS: : Bacillus Calmette-Guérin (BCG) infection causes hepatic injury following granuloma formation and secretion of cytokines which renders mice highly sensitive to endotoxin-mediated hepatotoxicity. Tumor necrosis factor (TNF) is required for granuloma formation and is one of the most important cytokines in liver injury. TNF inhibitors are effective therapies for inflammatory diseases. However, clinical use of non-selective TNF inhibitors is associated with an increased risk of infections. This work investigates the differential roles of soluble TNF (solTNF) and membrane TNF (memTNF) in BCG infection, BCG/LPS- and D-GALN/LPS-induced liver injury. METHODS: We have used both genetic and pharmacologic approaches and analyzed liver injury, TLR4, cytokine and iNOS activation induced by BCG, BCG/LPS and D-GALN/LPS. RESULTS: BCG infection-induced liver injury is seen in wild-type mice but not in TNF(-/-), memTNF knock-in (KI), and sTNFR1-Fc transgenic mice. Severity of BCG-induced liver injury is correlated with BCG-granuloma number and hepatic expression of TLR4 and iNOS. In addition, protection from liver damage caused by BCG/LPS or D-GALN/LPS administration was observed in TNF(-/-), memTNF KI and sTNFR1-Fc transgenic mice. To extend the genetic findings, we then evaluated whether selective pharmacological inhibition of solTNF by dominant-negative (DN)-TNF neutralization and non-selective inhibition of solTNF and memTNF by anti-TNF antibodies and etanercept (TNFR2-IgG1) can protect the mice from liver injury. Both selective and non-selective inhibition of solTNF protected mice from BCG/LPS and D-GALN/LPS-induced liver damage. CONCLUSIONS: These data suggest that memTNF is not mediating liver injury and that selective inhibition of solTNF sparing memTNF may represent a new therapeutic strategy to treat immune-mediated inflammatory liver diseases.

Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies.

The humoral immune response requires antigen-specific B cell activation and subsequent terminal differentiation into plasma cells. Engagement of B cell antigen receptor (BCR) on mature B cells activates an intracellular signaling cascade, including calcium mobilization, which leads to cell proliferation and differentiation. Coengagement by immune complex of BCR with the inhibitory Fc receptor FcgammaRIIb, the only IgG receptor expressed on B cells, inhibits B cell activation signals through a negative feedback loop. We now describe antibodies that mimic the inhibitory effects of immune complex by high-affinity coengagement of FcgammaRIIb and the BCR coreceptor complex on human B cells. We engineered the Fc domain of an anti-CD19 antibody to generate variants with up to approximately 430-fold greater affinity to FcgammaRIIb. Relative to native IgG1, the FcgammaRIIb binding-enhanced (IIbE) variants strongly inhibited BCR-induced calcium mobilization and viability in primary human B cells. Inhibitory effects involved phosphorylation of SH2-containing inositol polyphosphate 5-phosphatase (SHIP), which is known to be involved in FcgammaRIIb-induced negative feedback of B cell activation by immune complex. Coengagement of BCR and FcgammaRIIb by IIbE variants also overcame the anti-apoptotic effects of BCR activation. The use of a single antibody to suppress B cell functions by coengagement of BCR and FcgammaRIIb may represent a novel approach in the treatment of B cell-mediated autoimmune diseases.

Topical Administration of a Soluble TNF Inhibitor Reduces Infarct Volume After Focal Cerebral Ischemia in Mice.

BACKGROUND: Tumor necrosis factor, which exists both as a soluble (solTNF) and a transmembrane (tmTNF) protein, plays an important role in post-stroke inflammation. The objective of the present study was to test the effect of topical versus intracerebroventricular administration of XPro1595 (a solTNF inhibitor) and etanercept (a solTNF and tmTNF inhibitor) compared to saline on output measures such as infarct volume and post-stroke inflammation in mice. METHODS: Adult male C57BL/6 mice were treated topically (2.5 mg/ml/1µl/h for 3 consecutive days) or intracerebroventricularly (1.25 mg/kg/0.5 ml, once) with saline, XPro1595, or etanercept immediately after permanent middle cerebral artery occlusion (pMCAO). Mice were allowed to survive 1 or 3 days. Infarct volume, microglial and leukocyte profiles, and inflammatory markers were evaluated. RESULTS: We found that topical, and not intracerebroventricular, administration of XPro1595 reduced infarct volume at both 1 and 3 days after pMCAO. Etanercept showed no effect. We observed no changes in microglial or leukocyte populations. XPro1595 increased gene expression of P2ry12 at 1 day and Trem2 at 1 and 3 days, while decreasing Cx3cr1 expression at 1 and 3 days after pMCAO, suggesting a change in microglial activation toward a phagocytic phenotype. CONCLUSION: Our data demonstrate that topical administration of XPro1595 for 3 consecutive days decreases infarct volumes after ischemic stroke, while modifying microglial activation and the inflammatory response post-stroke. This suggests that inhibitors of solTNF hold great promise for future neuroprotective treatment in ischemic stroke.

Blocking soluble tumor necrosis factor signaling with dominant-negative tumor necrosis factor inhibitor attenuates loss of dopaminergic neurons in models of Parkinson's disease.

The mechanisms that trigger or contribute to loss of dopaminergic (DA) neurons in Parkinson's disease (PD) remain unclear and controversial. Elevated levels of tumor necrosis factor (TNF) in CSF and postmortem brains of PD patients and animal models of PD implicate this proinflammatory cytokine in the pathophysiology of the disease; but a role for TNF in mediating loss of DA neurons in PD has not been clearly demonstrated. Here, we report that neutralization of soluble TNF (solTNF) in vivo with the engineered dominant-negative TNF compound XENP345 (a PEGylated version of the TNF variant A145R/I97T) reduced by 50% the retrograde nigral degeneration induced by a striatal injection of the oxidative neurotoxin 6-hydroxydopamine (6-OHDA). XENP345 was neuroprotective only when infused into the nigra, not the striatum. XENP345/6-OHDA rats displayed attenuated amphetamine-induced rotational behavior, indicating preservation of striatal dopamine levels. Similar protective effects were observed with chronic in vivo coinfusion of XENP345 with bacterial lipopolysaccharide (LPS) into the substantia nigra, confirming a role for solTNF-dependent neuroinflammation in nigral degeneration. In embryonic rat midbrain neuron/glia cell cultures exposed to LPS, even delayed administration of XENP345 prevented selective degeneration of DA neurons despite sustained microglia activation and secretion of solTNF. XENP345 also attenuated 6-OHDA-induced DA neuron toxicity in vitro. Collectively, our data demonstrate a role for TNF in vitro and in vivo in two models of PD, and raise the possibility that delaying the progressive degeneration of the nigrostriatal pathway in humans is therapeutically feasible with agents capable of blocking solTNF in early stages of PD.

Trypanosoma brucei growth control by TNF in mammalian host is independent of the soluble form of the cytokine.

Infection of C57Bl/6 mice by pleomorphic African trypanosomes Trypanosoma brucei and T. congolense is characterized by parasitemia waves coupled with the production of systemic levels of TNF. This cytokine is known to control T. brucei growth, but also to contribute to tissue damage, shortening the survival time of infected mice. Using a dominant-negative version of TNF to discriminate between the effects of the membrane-form versus the soluble form of TNF, we show that the second form is involved in neither parasite control nor induction of liver injury. Therefore, soluble TNF is likely not a major contributor to disease outcome. We propose that membrane-bound TNF is responsible for both T. brucei control and host pathology.

Inhibition of TNF reduces mechanical orofacial hyperalgesia induced by Complete Freund's Adjuvant by a TRPV1-dependent mechanism in mice.

BACKGROUND: Inflammation in the orofacial region results in pain and is associated with many pathological states, including migraine, neuralgias and temporomandibular disorder. Although extensively studied, the mechanisms responsible for these conditions are not known and effective treatments are lacking. We reported earlier that the proinflammatory cytokine tumor necrosis factor (TNF) plays an important role in regulation of trigeminal ganglion (TG) neuron function in vitro. In the present study we investigated the role of TNF in mechanical hypersensitivity in mice. METHODS: We employed the Complete Freund's Adjuvant (CFA)-induced model of orofacial pain and evaluated the effect of blocking of soluble TNF activity by peripheral administration of the novel dominant negative TNF biologic, XPro1595. RESULTS: We show that CFA administration into the lower lip causes hyperalgesia and an increase in both expression of transient receptor potential vanilloid subfamily member 1 (TRPV1) mRNA and in the average intensity of TRPV1 protein immunoreactivity in TG neurons. We also show that intraperitoneal administration of XPro1595 prevents both CFA-induced mechanical hypersensitivity and, as shown in immunohistochemical staining - upregulation of TRPV1 protein expression in TG neurons. CONCLUSIONS: We conclude that one of the possible regulatory mechanisms of TNF in pain involves upregulation of the nociceptor TRPV1, and that peripheral treatment with a selective anti-soluble TNF biologic can prevent hyperalgesia caused by inflammation in the orofacial region. Therefore, these new findings suggest that XPro1595 may serve as a novel treatment for orofacial pain disorders.

Therapeutic inhibition of soluble brain TNF promotes remyelination by increasing myelin phagocytosis by microglia.

Multiple sclerosis (MS) is an inflammatory CNS demyelinating disease in which remyelination largely fails. Transmembrane TNF (tmTNF) and TNF receptor 2 are important for remyelination in experimental MS models, but it is unknown whether soluble TNF (solTNF), a major proinflammatory factor, is involved in regeneration processes. Here, we investigated the specific contribution of solTNF to demyelination and remyelination in the cuprizone model. Treatment with XPro1595, a selective inhibitor of solTNF that crosses the intact blood-brain barrier (BBB), in cuprizone-fed mice did not prevent toxin-induced oligodendrocyte loss and demyelination, but it permitted profound early remyelination due to improved phagocytosis of myelin debris by CNS macrophages and prevented disease-associated decline in motor performance. The beneficial effects of XPro1595 were absent in TNF-deficient mice and replicated in tmTNF-knockin mice, showing that tmTNF is sufficient for the maintenance of myelin and neuroprotection. These findings demonstrate that solTNF inhibits remyelination and repair in a cuprizone demyelination model and suggest that local production of solTNF in the CNS might be one reason why remyelination fails in MS. These findings also suggest that disinhibition of remyelination by selective inhibitors of solTNF that cross the BBB might represent a promising approach for treatment in progressive MS.