Amyloid Beta Peptide Is an Endogenous Negative Allosteric Modulator of Leptin Receptor.

INTRODUCTION: Metabolic dysfunction is now recognized as a pivotal component of Alzheimer's disease (AD), the most common dementia worldwide. However, the precise molecular mechanisms linking metabolic dysfunction to AD remain elusive. OBJECTIVE: Here, we investigated the direct impact of soluble oligomeric amyloid beta (Aß) peptides, the main molecular hallmark of AD, on the leptin system, a major component of central energy metabolism regulation. METHODS: We developed a new time-resolved fluorescence resonance energy transfer-based Aß binding assay for the leptin receptor (LepR) and studied the effect of Aß on LepR function in several in vitro assays. The in vivo effect of Aß on LepR function was studied in an Aß-specific AD mouse model and in pro-opiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus. RESULTS: We revealed specific and high-affinity (Ki = 0.1 nM) binding of Aß to LepR. Pharmacological characterization of this interaction showed that Aß binds allosterically to the extracellular domain of LepR and negatively affects receptor function. Negative allosteric modulation of LepR by Aß was detected at the level of signaling pathways (STAT-3, AKT, and ERK) in vitroand in vivo. Importantly, the leptin-induced response of POMC neurons, key players in the regulation of metabolic function, was completely abolished in the presence of Aß. CONCLUSION: Our data indicate that Aß is a negative allosteric modulator of LepR, resulting in impaired leptin action, and qualify LepR as a new and direct target of Aß oligomers. Preventing the interaction of Aß with LepR might improve both the metabolic and cognitive dysfunctions in AD condition.

A novel leptin receptor antagonist uncouples leptin's metabolic and immune functions.

Leptin links body energy stores to high energy demanding processes like reproduction and immunity. Based on leptin's role in autoimmune diseases and cancer, several leptin and leptin receptor (LR) antagonists have been developed, but these intrinsically lead to unwanted weight gain. Here, we report on the uncoupling of leptin's metabolic and immune functions based on the cross talk with the epidermal growth factor receptor (EGFR). We show that both receptors spontaneously interact and, remarkably, that this complex can partially overrule the lack of LR activation by a leptin antagonistic mutein. Moreover, this leptin mutant induces EGFR phosphorylation comparable to wild-type leptin. Exploiting this non-canonical leptin signalling pathway, we identified a camelid single-domain antibody that selectively inhibits this LR-EGFR cross talk without interfering with homotypic LR signalling. Administration in vivo showed that this single-domain antibody did not interfere with leptin's metabolic functions, but could reverse the leptin-driven protection against starvation-induced thymic and splenic atrophy. These findings offer new opportunities for the design and clinical application of selective leptin and LR antagonists that avoid unwanted metabolic side effects.

Leptin's metabolic and immune functions can be uncoupled at the ligand/receptor interaction level.

The adipocyte-derived cytokine leptin acts as a metabolic switch, connecting the body's metabolism to high-energy consuming processes such as reproduction and immune responses. We here provide genetic and biochemical evidence that the metabolic and immune functions of leptin can be uncoupled at the receptor level. First, homozygous mutant fatt/fatt mice carry a spontaneous splice mutation causing deletion of the leptin receptor (LR) immunoglobulin-like domain (IGD) in all LR isoforms. These mice are hyperphagic and morbidly obese, but display only minimal changes in size and cellularity of the thymus, and cellular immune responses are unaffected. These animals also displayed liver damage in response to concavalin A comparable to wild-type and heterozygous littermates. Second, treatment of healthy mice with a neutralizing nanobody targeting IGD induced weight gain and hyperinsulinaemia, but completely failed to block development of experimentally induced autoimmune diseases. These data indicate that leptin receptor deficiency or antagonism profoundly affects metabolism, with little concomitant effects on immune functions.

A bidirectional crosstalk between iNKT cells and adipocytes mediated by leptin modulates susceptibility for T cell mediated hepatitis.

BACKGROUND & AIMS: Immunometabolism is an emerging field of clinical investigation due to the obesity epidemic worldwide. A reciprocal involvement of immune mediators in the body energy metabolism has been recognized for years, but is only partially understood. We hypothesized that the adipokine leptin could provide an important modulator of iNKT cells. METHODS: The expression of leptin receptor (LR) on resting and activated iNKT cells was measured by flow cytometry. FACS-sorted hepatic iNKT cells were stimulated with anti-CD3/CD28Ab coated beads in the absence or presence of a neutralizing anti-leptin Ab. Furthermore, we evaluated the outcome of LR blocking nanobody treatment in ConA induced hepatitis and towards metabolic parameters in WT and iNKT cell deficient mice. RESULTS: The LR is expressed on iNKT cells and leptin suppresses iNKT cell proliferation and cytokine production in vitro. LR deficient iNKT cells are hyper-responsive further enforcing the role of leptin as an important inhibitor of iNKT cell function. Consistently, in vivo blockade of LR signaling exacerbated ConA hepatitis in wild-type but not in iNKT cell deficient mice, through both Janus kinase (JAK)2 and mitogen-activated protein kinase (MAPK) dependent mechanisms. Moreover, LR inhibition altered fat pad features and was accompanied by insulin resistance, only in wild-type mice. Curiously, this interaction was strictly dependent on MAPK mediated LR signaling in iNKT cells and uncoupled from the more central effects of leptin. CONCLUSIONS: Our data support a new concept of immune regulation by which leptin protects towards T cell mediated hepatitis via modulation of iNKT cells.

Antagonizing leptin: current status and future directions.

The adipocyte-derived hormone/cytokine leptin acts as a metabolic switch, connecting the body's nutritional status to high energy consuming processes such as reproduction and immune responses. Inappropriate leptin responses can promote autoimmune diseases and tumorigenesis. In this review we discuss the current strategies to modulate leptin signaling and the possibilities for their use in research and therapy.

The role of leptin in the development of pulmonary neutrophilia in infection and acute lung injury.

OBJECTIVES: One of the hallmarks of severe pneumonia and associated acute lung injury is neutrophil recruitment to the lung. Leptin is thought to be up-regulated in the lung following injury and to exert diverse effects on leukocytes, influencing both chemotaxis and survival. We hypothesized that pulmonary leptin contributes directly to the development of pulmonary neutrophilia during pneumonia and acute lung injury. DESIGN: Controlled human and murine in vivo and ex vivo experimental studies. SETTING: Research laboratory of a university hospital. SUBJECTS: Healthy human volunteers and subjects hospitalized with bacterial and H1N1 pneumonia. C57Bl/6 and db/db mice were also used. INTERVENTIONS: Lung samples from patients and mice with either bacterial or H1N1 pneumonia and associated acute lung injury were immunostained for leptin. Human bronchoalveolar lavage samples obtained after lipopolysaccharide-induced lung injury were assayed for leptin. C57Bl/6 mice were examined after oropharyngeal aspiration of recombinant leptin alone or in combination with Escherichia coli- or Klebsiella pneumoniae-induced pneumonia. Leptin-resistant (db/db) mice were also examined using the E. coli model. Bronchoalveolar lavage neutrophilia and cytokine levels were measured. Leptin-induced chemotaxis was examined in human blood- and murine marrow-derived neutrophils in vitro. MEASUREMENTS AND MAIN RESULTS: Injured human and murine lung tissue showed leptin induction compared to normal lung, as did human bronchoalveolar lavage following lipopolysaccharide instillation. Bronchoalveolar lavage neutrophilia in uninjured and infected mice was increased and lung bacterial load decreased by airway leptin administration, whereas bronchoalveolar lavage neutrophilia in infected leptin-resistant mice was decreased. In sterile lung injury by lipopolysaccharide, leptin also appeared to decrease airspace neutrophil apoptosis. Both human and murine neutrophils migrated toward leptin in vitro, and this required intact signaling through the Janus Kinase 2/phosphatidylinositol-4,5-bisphosphate 3-kinase pathway. CONCLUSIONS: We demonstrate that pulmonary leptin is induced in injured human and murine lungs and that this cytokine is effective in driving alveolar airspace neutrophilia. This action appears to be caused by direct effects of leptin on neutrophils.

Inhibition of the connexin 43 elevation may be involved in the neuroprotective activity of leptin against brain ischemic injury.

Leptin is a multifunctional hormone produced by the ob gene and is secreted by adipocytes that regulate food intake and energy metabolism. Numerous studies demonstrated that leptin is a novel neuroprotective effector, however, the mechanisms are largely unknown. Herein, we demonstrate the protective activities of leptin after ischemic stroke and provide the first evidence for the involvement of the connexin 43 (Cx43) in leptin-mediated neuroprotection. We found that leptin treatment reduces the infarct volume, improves animal behavioral parameters, and inhibits the elevation of Cx43 expression in vivo. In vitro, leptin reverses ischemia-induced SY5Y and U87 cells Cx43 elevation, secreted glutamate levels in medium and SY5Y cell death, these roles could be abolished by leptin receptor blocker. Additionally, leptin administration upregulated the extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation. Moreover, ERK1/2 inhibitors pretreatment reversed the effects of leptin on Cx43 expression, glutamate levels and cell apoptosis. In conclusion, the present study demonstrated that leptin can reduce the Cx43 expression and cell death both in vivo and in vitro via ERK1/2 signaling pathway. This result provides a novel regulatory signaling pathway of the neuroprotective effects of leptin and may contribute to ischemic brain injury prevention and therapy.

High efficacy of huCD20-targeted AcTaferon in humanized patient derived xenograft models of aggressive B cell lymphoma.

Type I interferon (IFN) is a potent antitumoral drug, with an important history in the treatment of hematologic malignancies. However, its pleiotropic nature leads to severe dose-limiting toxicities that blunt its therapeutic potential. To achieve selective targeting of specific immune or tumor cells, AcTakines (Activity-on-Target Cytokines), i.e., immunocytokines utilizing attenuated cytokines, and clinically optimized A-Kines™ were developed. In syngeneic murine models, the CD20-targeted murine IFNα2-based AcTaferons (AFNs) have demonstrated clear antitumoral effects, with excellent tolerability. The current study explores the antitumoral potential of the humanized huCD20-Fc-AFN in 5 different humanized patient derived xenograft (PDX) models of huCD20+ aggressive B non-Hodgkin lymphomas (B-NHLs). The huCD20-Fc-AFN consists of a huCD20-specific single-domain antibody (VHH) linked through a heterodimeric 'knob-in-hole' human IgG1 Fc molecule to an attenuated huIFNα2 sequence. An in vitro targeting efficacy of up to 1.000-fold could be obtained, without detectable in vivo toxicities, except for selective (on-target) and reversible B cell depletion. Treatment with huCD20-Fc-AFN significantly increased the median overall survival (mOS) in both non-humanized (mOS 31 to 45 days; HR = 0.26; p = 0.001), and humanized NSG/NOG mice (mOS 34 to 80 days; HR = 0.37; p < 0.0001). In humanized mice, there was a trend for increased survival when compared to equimolar rituximab (mOS 49 to 80 days; HR = 0.73; p = 0.09). The antitumoral effects of huCD20-Fc-AFN were partly due to direct effects of type I IFN on the tumor cells, but additional effects via the human immune system are essential to obtain long-term remissions. To conclude, huCD20-Fc-AFN could provide a novel therapeutic strategy for huCD20-expressing aggressive B-NHLs.

Selection of non-competitive leptin antagonists using a random nanobody-based approach.

The adipocyte-derived cytokine leptin acts as a metabolic switch, connecting the body's metabolism to high-energy consuming processes such as reproduction and immune responses. Accumulating evidence suggests that leptin plays a role in human pathologies, such as autoimmune diseases and cancer, thus providing a rationale for the development of leptin antagonists. In the present study, we generated and evaluated a panel of neutralizing nanobodies targeting the LR (leptin receptor). A nanobody comprises the variable domain of the naturally occurring single-chain antibodies found in members of the Camelidae family. We identified three classes of neutralizing nanobodies targeting different LR subdomains: i.e. the CRH2 (cytokine receptor homology 2), Ig-like and FNIII (fibronectin type III) domains. Only nanobodies directed against the CRH2 domain inhibited leptin binding. We could show that a nanobody that targets the Ig-like domain potently interfered with leptin-dependent regulation of hypothalamic NPY (neuropeptide Y) expression. As a consequence, daily intraperitoneal injection increased body weight, body fat content, food intake, liver size and serum insulin levels. All of these characteristics resemble the phenotype of leptin and LR-deficient animals. The results of the present study support proposed models of the activated LR complex, and demonstrate that it is possible to block LR signalling without affecting ligand binding. These nanobodies form new tools to study the mechanisms of BBB (blood-brain barrier) leptin transport and the effect of LR inhibition in disease models.

10th antibody industrial symposium: new developments in antibody and adoptive cell therapies.

The annual "Antibody Industrial Symposium", co-organized by LabEx MAbImprove and MabDesign, held its 10th anniversary edition in Montpellier, France, on June 28-29, 2022. The meeting focused on new results and concepts in antibody engineering (naked, mono- or multi-specific, conjugated to drugs or radioelements) and also on new cell-based therapies, such as chimeric antigenic receptor (CAR)-T cells. The symposium, which brought together scientists from academia and industry, also addressed issues concerning the production of these molecules and cells, and the necessary steps to ensure a strong intellectual property protection of these new molecules and approaches. These two days of exchanges allowed a rich discussion among the various actors in the field of therapeutic antibodies.

Leptin modulates innate and adaptive immune cell recruitment after cigarette smoke exposure in mice.

Leptin, a pleiotropic type I cytokine, was recently demonstrated to be expressed by resident lung cells in chronic obstructive pulmonary disease patients and asymptomatic smokers. To elucidate the functional role of leptin in the onset of chronic obstructive pulmonary disease, we tested leptin-deficient ob/ob mice (C57BL/6), leptin receptor-deficient db/db mice (C57BKS), and littermates in a model of cigarette smoke (CS)-induced pulmonary inflammation. Wild-type (WT) C57BL/6 mice were exposed for 4 or 24 wk to control air or CS. Pulmonary leptin expression was analyzed by immunohistochemistry and real-time PCR. Pulmonary inflammation upon 4 wk CS exposure was evaluated in bronchoalveolar lavage fluid (BALF) and lung tissue of WT, ob/ob, and db/db mice. Immunohistochemical analysis revealed leptin expression in bronchial epithelial cells, pneumocytes, alveolar macrophages, and bronchial/vascular smooth muscle cells. The 4 and 24 wk CS exposure increased leptin expression in bronchial epithelial cells and pneumocytes versus air-exposed WT mice (p<0.05). The 4 wk CS exposure resulted in increased accumulation of neutrophils, dendritic cells, macrophages, and lymphocytes in BALF and lung tissue of WT, ob/ob, and db/db mice. CS-exposed ob/ob and db/db mice showed in general higher numbers of neutrophils and lower numbers of CD4+, CD8+, and dendritic cells versus CS-exposed WT mice. Consistently, CXCL1 levels were enhanced in BALF of CS-exposed ob/ob and db/db mice versus WT mice (p<0.05). Exogenous leptin administration completely restored the skewed inflammatory profile in ob/ob mice. These data reveal an important role of leptin in modulating innate and adaptive immunity after CS inhalation in mice.

Safe eradication of large established tumors using neovasculature-targeted tumor necrosis factor-based therapies.

Systemic toxicities have severely limited the clinical application of tumor necrosis factor (TNF) as an anticancer agent. Activity-on-Target cytokines (AcTakines) are a novel class of immunocytokines with improved therapeutic index. A TNF-based AcTakine targeted to CD13 enables selective activation of the tumor neovasculature without any detectable toxicity in vivo. Upregulation of adhesion markers supports enhanced T-cell infiltration leading to control or elimination of solid tumors by, respectively, CAR T cells or a combination therapy with CD8-targeted type I interferon AcTakine. Co-treatment with a CD13-targeted type II interferon AcTakine leads to very rapid destruction of the tumor neovasculature and complete regression of large, established tumors. As no tumor markers are needed, safe and efficacious elimination of a broad range of tumor types becomes feasible.

Techniques: new pharmacological perspectives for the leptin receptor.

The function of leptin, initially confined to its role in energy homeostasis and obesity, has now expanded to the regulation of reproduction, glucose homeostasis, bone formation, wound healing and the immune system. Both stimulation and inhibition of the molecular target of leptin, the leptin receptor (LR), might find applications in disease treatment. Recent advances in the understanding of LR activation mechanisms have led to the design of LR antagonists. Several assays have been developed for the screening and evaluation of LR ligands. Both the extracellular and the intracellular domains of the LR are potential drug targets. The bioluminescence resonance energy transfer technique can be used to screen for compounds that target the extracellular part of the LR, and we propose that the novel reverse mammalian protein-protein interaction trap technique can be used to screen compounds that affect intracellular aspects of LR signalling. These assays can be easily adapted to other pharmacologically relevant receptors.

Leptin induces inflammation-related genes in RINm5F insulinoma cells.

BACKGROUND: Leptin acts not only on hypothalamic centers to control food intake but has additional functions in peripheral tissues, e.g. inhibition of insulin secretion from pancreatic islets. The leptin receptor (LEPRb) is a class I cytokine receptor that mediates activation of STAT transcription factors. In this study, we characterise the regulation of inflammation-related genes by leptin in insulinoma cells and compare the effect of transcriptional regulation by leptin with that of other cytokines. RESULTS: We have used RINm5F insulinoma cells as a model system for a peripheral target cell of leptin. Six transcripts encoding inflammation-related proteins were found to be upregulated by activation of LEPRb, namely lipocalin-2, pancreatitis-associated protein, preprotachykinin-1, fibrinogen-beta, tissue-type plasminogen activator (tPA) and manganese-dependent superoxide dismutase (MnSOD). Four of these transcripts (fibrinogen-beta, lipocalin-2, tPA, MnSOD) were also induced by the proinflammatory cytokine interleukin-1beta (IL-1beta). Interferon-gamma alone had no effect on the leptin-induced transcripts but enhanced the upregulation by IL-1beta of lipocalin-2, tPA and MnSOD mRNA levels. Experiments with LEPRb point mutants revealed that the upregulation of the inflammation-related genes depended on the presence of tyrosine-1138 which mediates the activation of the transcription factors STAT1 and STAT3. Reporter gene assays showed that leptin induced the expression of preprotachykinin-1 and lipocalin-2 on the level of promoter regulation. Finally, leptin treatment increased caspase 3-like proteolytic activity in RINm5F cells. CONCLUSION: The present data show that leptin induces a cytokine-like transcriptional response in RINm5F cells, consistent with the proposed function of leptin as a modulator of immune and inflammatory responses.

The Leptin Receptor Complex: Heavier Than Expected?

Under normal physiological conditions, leptin and the leptin receptor (ObR) regulate the body weight by balancing food intake and energy expenditure. However, this adipocyte-derived hormone also directs peripheral processes, including immunity, reproduction, and bone metabolism. Leptin, therefore, can act as a metabolic switch connecting the body's nutritional status to high energy consuming processes. We provide an extensive overview of current structural insights on the leptin-ObR interface and ObR activation, coupling to signaling pathways and their negative regulation, and leptin functioning under normal and pathophysiological conditions (obesity, autoimmunity, cancer, … ). We also discuss possible cross-talk with other receptor systems on the receptor (extracellular) and signaling cascade (intracellular) levels.

Heteromeric MAPPIT: a novel strategy to study modification-dependent protein-protein interactions in mammalian cells.

We recently reported a two-hybrid trap for detecting protein-protein interactions in intact mammalian cells (MAPPIT). The bait protein was fused to a STAT recruitment-deficient, homodimeric cytokine receptor and the prey protein to functional STAT recruitment sites. In such a configuration, STAT-dependent responses can be used to monitor a given bait-prey interaction. Using this system, we were able to demonstrate both modification-independent and tyrosine phosphorylation- dependent interactions. Protein modification in this approach is, however, strictly dependent on the receptor-associated JAK tyrosine kinases. We have now extended this concept by using extracellular domains of the heteromeric granulocyte/macrophage colony-stimulating factor receptor (GM-CSFR). Herein, the bait was fused to the (beta)c chain and its modifying enzyme to the GM-CSFRalpha chain (or vice versa). We demonstrate several serine phosphorylation-dependent interactions in the TGFbeta/Smad pathway using the catalytic domains of the ALK4 or ALK6 serine/threonine kinase receptors. In all cases tested, STAT-dependent signaling was completely abolished when mutant baits were used wherein critical serine residues were replaced by alanines. This approach operates both in transient and stable expression systems and may not be limited to serine phosphorylation but has the potential for studying various different types of protein modification-dependent interactions in intact cells.

Hyperleptinemia is associated with impaired pulmonary host defense.

We have previously reported that obesity attenuates pulmonary inflammation in both patients with acute respiratory distress syndrome (ARDS) and in mouse models of the disease. We hypothesized that obesity-associated hyperleptinemia, and not body mass per se, drives attenuation of the pulmonary inflammatory response and that this e_ect could also impair the host response to pneumonia. We examined the correlation between circulating leptin levels and risk, severity, and outcome of pneumonia in 2 patient cohorts (NHANES III and ARDSNet-ALVEOLI) and in mouse models of diet-induced obesity and lean hyperleptinemia. Plasma leptin levels in ambulatory subjects (NHANES) correlated positively with annual risk of respiratory infection independent of BMI. In patients with severe pneumonia resulting in ARDS (ARDSNet-ALVEOLI), plasma leptin levels were found to correlate positively with subsequent mortality. In obese mice with pneumonia, plasma leptin levels were associated with pneumonia severity, and in obese mice with sterile lung injury, leptin levels were inversely related to bronchoalveolar lavage neutrophilia, as well as to plasma IL-6 and G-CSF levels. These results were recapitulated in lean mice with experimentally induced hyperleptinemia. Our findings suggest that the association between obesity and elevated risk of pulmonary infection may be driven by hyperleptinemia.

Functional analysis of leptin receptor activation using a Janus kinase/signal transducer and activator of transcription complementation assay.

The leptin receptor (LR), a member of the class I cytokine receptor family, is composed of a single subunit. Its extracellular domain consists of two so-called cytokine receptor homology domains, separated by an Ig-like domain, and two additional fibronectin type III modules. Requirements for LR activation were examined using a complementation strategy. Two LR mutants, LR-FFY-Deltabox 1 and LR-F3, deficient in Janus kinase or signal transducer and activator of transcription (STAT) activation, respectively, were only able to generate a STAT3-dependent signal when coexpressed. Based on the requirements for Janus kinase/STAT signaling, and on the lack of complementation with similar receptor constructs, but containing the extracellular domain of the homodimeric erythropoietin receptor, this observation can be explained only by higher order LR clustering. Using a panel of deletion mutants we were able to define a role for the cytokine receptor homology 1 and Ig-like domains in leptin signaling. Moreover, we demonstrate a nonredundant function for the individual receptor chains within the homomeric LR complex. Based on these data, we propose a possible model for LR clustering.

Leptin: From structural insights to the design of antagonists.

After its discovery in 1994, it soon became clear that leptin acts as an adipocyte-derived hormone with a central role in the control of body weight and energy homeostasis. However, a growing body of evidence has revealed that leptin is a pleiotropic cytokine with activities on many peripheral cell types. Inappropriate leptin signaling can promote autoimmunity, certain cardiovascular diseases, elevated blood pressure and cancer, which makes leptin and the leptin receptor interesting targets for antagonism. Profound insights in the leptin receptor (LR) activation mechanisms are a prerequisite for the rational design of these antagonists. In this review, we focus on the molecular mechanisms underlying leptin receptor activation and signaling. We also discuss the current strategies to interfere with leptin signaling and their therapeutic potential.

An abnormally glycosylated isoform of erythropoietin in hemangioblastoma is associated with polycythemia.

BACKGROUND: Hemangioblastomas express erythropoietin and the patients often present with polycythemia. METHODS: Serum erythropoietin was measured using a commercial immunoassay, a functional erythropoietin assay and iso-electric focusing. RESULTS: Despite the polycythemia, serum erythropoietin remained low, while a functional erythropoietin-assay showed a 4-5 higher activity in serum compared to the immunoassay. Iso-electric focusing of serum erythropoietin indicated overrepresentation of highly sialylated erythropoietin isoforms produced by the tumor. As a result, altered affinity of the monoclonal antibody used in the immunoassay for the hypersialylated isoforms was suggested. CONCLUSION: Analysis of erythropoietin isoforms may be helpful in distinguishing the ectopic erythropoietin isoforms from normally glycosylated erythropoietin.