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.

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.

A multiparameter approach to monitor disease activity in collagen-induced arthritis.

INTRODUCTION: Disease severity in collagen-induced arthritis (CIA) is commonly assessed by clinical scoring of paw swelling and histological examination of joints. Although this is an accurate approach, it is also labour-intensive and the application of less invasive and less time-consuming methods is of great interest. However, it is still unclear which of these methods represents the most discriminating measure of disease activity. METHODS: We undertook a comparative analysis in which different measurements of inflammation and tissue damage in CIA were studied on an individual mouse level. We compared the current gold standard methods - clinical scoring and histological examination - with alternative methods based on scoring of X-ray or micro-computed tomography (CT) images and investigated the significance of systemically expressed proteins, involved in CIA pathogenesis, that have potential as biomarkers. RESULTS: Linear regression analysis revealed a marked association of serum matrix metalloproteinase (MMP)-3 levels with all features of CIA including inflammation, cartilage destruction and bone erosions. This association was improved by combined detection of MMP-3 and anti-collagen IgG2a antibody concentrations. In addition, combined analysis of both X-ray and micro-CT images was found to be predictive for cartilage and bone damage. Most remarkably, validation analysis using an independent data set proved that variations in disease severity, induced by different therapies, could be accurately represented by predicted values based on the proposed parameters. CONCLUSIONS: Our analyses revealed that clinical scoring, combined with serum MMP-3, anti-collagen IgG2a measurement and scoring of X-ray and micro-CT images, yields a comprehensive insight into the different aspects of disease activity in CIA.

Invariant natural killer T cells are natural regulators of murine spondylarthritis.

OBJECTIVE: To investigate the role of invariant natural killer T (iNKT) cells in TNF(DeltaARE/+) mice, an animal model of spondylarthritis (SpA) with both gut and joint inflammation. METHODS: The frequency and activation of iNKT cells were analyzed on mononuclear cells from the lymph nodes and livers of mice, using flow cytometry with alpha-galactosylceramide/CD1d tetramers and quantitative polymerase chain reaction for the invariant V(alpha)14-J(alpha)18 rearrangement. Bone marrow-derived dendritic cells (DCs) were obtained by expansion of primary cells with granulocyte-macrophage colony-stimulating factor followed by coculture with iNKT cell hybridomas, and interleukin-2 release into the cocultures was then measured by enzyme-linked immunosorbent assay (ELISA). Cytokine levels were determined by ELISA or cytometric bead array analyses of freshly isolated DCs and iNKT cells in mixed cocultures. TNF(DeltaARE/+) mice were backcrossed onto J(alpha)18(-/-) and CD1d(-/-) mice, and disease onset was evaluated by clinical scoring, positron emission tomography, and histology. CD1d levels were analyzed on mononuclear cells in paired blood and synovial fluid samples from patients with SpA compared with healthy control subjects. RESULTS: In the absence of iNKT cells, symptoms of gut and joint inflammation in TNF(DeltaARE/+)mice were aggravated. Invariant NKT cells were activated during the course of the disease. This was linked to an enrichment of inflammatory DCs, characterized by high levels of CD1d, particularly at draining sites of inflammation. A similar increase in CD1d levels was observed on DCs from patients with SpA. Inflammatory DCs from TNF(DeltaARE/+) mice stimulated iNKT cells to produce immunomodulatory cytokines, in the absence of exogenous stimulation. Prolonged, continuous exposure, but not short-term exposure, to tumor necrosis factor (TNF) was found to be responsible for the enhanced DC-NKT cell crosstalk. CONCLUSION: This mode of iNKT cell activation represents a natural counterregulatory mechanism for the dampening of TNF-driven inflammation.

Cutting edge: the chemokine receptor CXCR3 retains invariant NK T cells in the thymus.

The current model used to define T cell export from the thymus suggests that emigrating lymphocytes seed the peripheral organs as functionally mature cells. This model holds true for the majority of T cells exported from the thymus with the exception of invariant NK T (iNKT) cells. iNKT cells undergo lineage expansion after positive selection and acquire NK receptor expression once fully mature; yet, the majority of mature iNKT cells are retained in the thymus by an as of yet unidentified mechanism. In this study we demonstrate that mature iNKT cells are retained in the thymus by the chemokine receptor CXCR3. We propose that the expression of CXCR3 ligands in the thymic medullary epithelium promotes the chemotactic retention of mature iNKT thymocytes and prevents leakage of iNKT cells into the peripheral circulation.