The TNF-like weak inducer of the apoptosis/fibroblast growth factor-inducible molecule 14 axis mediates histamine and platelet-activating factor-induced subcutaneous vascular leakage and anaphylactic shock.

BACKGROUND: Anaphylaxis includes mast cell (MC) activation, but less is known about downstream mechanisms (ie, vascular permeability controlled by endothelial cells [ECs]). The TNF-like weak inducer of apoptosis (TWEAK) and its sole receptor, fibroblast growth factor-inducible molecule 14 (Fn14), belong to the TNF superfamily and are involved in proinflammatory responses. OBJECTIVE: We sought to investigate the role of TWEAK/Fn14 axis in anaphylaxis. METHODS: In vivo vascular permeability and mouse models of passive systemic anaphylaxis (PSA) and active systemic anaphylaxis were applied to wild-type (WT), TWEAK- and Fn14-deficient mice (TWEAK-/- and Fn14-/-, respectively). Primary bone marrow-derived mast cells (BMMCs) and ECs from WT and Fn14-/- or TWEAK-/- mice were studied. The TWEAK/Fn14 axis was also investigated in human samples. RESULTS: Mice with PSA and active systemic anaphylaxis had increased Fn14 and TWEAK expression in lung tissues and increased serum soluble TWEAK concentrations. TWEAK and Fn14 deficiencies prevent PSA-related symptoms, resulting in resistance to decreased body temperature, less severe reactions, and maintained physical activity. Numbers of MCs after PSA are similar between genotypes in different tissue regions, such as ear skin and the trachea, tongue, peritoneum, lungs, and bone marrow. Moreover, in vitro studies revealed no differences in degranulation or mediator release between WT and Fn14-/- BMMCs after IgE-FcεRI stimulation. In vivo and in vitro histamine and platelet-activating factor administration increases Fn14 receptor expression in lungs and ECs. Moreover, Fn14 deficiency in ECs maintained in vitro impermeability when stimulated by mediators or activated BMMCs but not by TWEAK-/- BMMCs, indicating that Fn14 is crucial for endothelial barrier function. TWEAK/Fn14 deletion or TWEAK-blocking antibody prevented histamine/platelet-activating factor-induced vascular subcutaneous permeability. Circulating soluble TWEAK levels were increased in patients with anaphylaxis, and plasma from those patients increased Fn14 expression in ECs. CONCLUSION: The TWEAK/Fn14 axis participates in anaphylactic reactions. Inhibition of TWEAK/Fn14 interaction could be efficacious in anaphylaxis therapy.

Experimental atopic dermatitis is dependent on the TWEAK/Fn14 signaling pathway.

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) acts through its receptor fibroblast growth factor inducible 14 (Fn14), and participates in skin inflammation. Both TWEAK and Fn14 are highly expressed in skin lesions of patients with atopic dermatitis. The purpose of this study was to further explore the effect of Fn14 inhibition on experimental atopic dermatitis. Experimental atopic dermatitis was induced in the wild-type and Fn14 knock-out BALB/c mice. The effect of TWEAK/Fn14 interaction on keratinocytes was studied in an in-vitro model of atopic dermatitis. Fn14 deficiency ameliorates skin lesions in the mice model, accompanied by less infiltration of inflammatory cells and lower local levels of proinflammatory cytokines, including TWEAK, TNF-α and interleukin (IL)-17. Fn14 deficiency also attenuates the up-regulation of TNFR1 in skin lesions of atopic dermatitis. Moreover, topical TWEAK exacerbates skin lesion in the wild-type but not in the Fn14 knock-out mice. In vitro, TWEAK enhances the expressions of IL-17, IL-18 and IFN-γ in keratinocytes under atopic dermatitis-like inflammation. These results suggest that Fn14 deficiency protects mice from experimental atopic dermatitis, involving the attenuation of inflammatory responses and keratinocyte apoptosis. In the context of atopic dermatitis-like inflammation, TWEAK modulates keratinocytes via a TNFR1-mediated pathway.

CD1d is a novel cell-surface marker for human monocytic myeloid-derived suppressor cells with T cell suppression activity in peripheral blood after allogeneic hematopoietic stem cell transplantation.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that regulate immune responses in cancer and various pathological conditions. However, the phenotypic and functional heterogeneity of human MDSCs represents a major hurdle for the development of therapeutic strategies targeting or regulating MDSCs in tumor progression, inflammation, and graft-versus-host disease (GVHD). We previously shown that circulating HLA-DR-CD14+ monocytic MDSCs are a major contributor to clinical outcomes after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In this study, we identified, using high-throughput screening, a set of surface markers that are strongly expressed in HLA-DR-CD14+ monocytic MDSCs isolated from the peripheral blood (PB) of patients receiving allo-HSCT. Subsequent experiments showed the consistent dominant expression of CD1d in monocytic MDSCs of allo-HSCT PB in comparison with granulocytic MDSCs. In addition, CD1d-expressing cells isolated from PB of allo-HSCT patients showed the suppressive activity of T cell proliferation and higher expression of MyD88 and IDO compared with CD1d- cells. Our results suggest that CD1d could be a valuable marker for further therapeutic evaluation of human monocytic MDSCs for immune-related diseases, including GVHD.

Biomeasures and mechanistic modeling highlight PK/PD risks for a monoclonal antibody targeting Fn14 in kidney disease.

Discovery of the upregulation of fibroblast growth factor-inducible-14 (Fn14) receptor following tissue injury has prompted investigation into biotherapeutic targeting of the Fn14 receptor for the treatment of conditions such as chronic kidney diseases. In the development of monoclonal antibody (mAb) therapeutics, there is an increasing trend to use biomeasures combined with mechanistic pharmacokinetic/pharmacodynamic (PK/PD) modeling to enable decision making in early discovery. With the aim of guiding preclinical efforts on designing an antibody with optimized properties, we developed a mechanistic site-of-action (SoA) PK/PD model for human application. This model incorporates experimental biomeasures, including concentration of soluble Fn14 (sFn14) in human plasma and membrane Fn14 (mFn14) in human kidney tissue, and turnover rate of human sFn14. Pulse-chase studies using stable isotope-labeled amino acids and mass spectrometry indicated the sFn14 half-life to be approximately 5 hours in healthy volunteers. The biomeasures (concentration, turnover) of sFn14 in plasma reveals a significant hurdle in designing an antibody against Fn14 with desired characteristics. The projected dose (>1 mg/kg/wk for 90% target coverage) derived from the human PK/PD model revealed potential high and frequent dosing requirements under certain conditions. The PK/PD model suggested a unique bell-shaped relationship between target coverage and antibody affinity for anti-Fn14 mAb, which could be applied to direct the antibody engineering towards an optimized affinity. This investigation highlighted potential applications, including assessment of PK/PD risks during early target validation, human dose prediction and drug candidate optimization.

Lipopolysaccharide Upregulated Intestinal Epithelial Cell Expression of Fn14 and Activation of Fn14 Signaling Amplify Intestinal TLR4-Mediated Inflammation.

TLR4 in intestinal epithelial cells has been shown both inflammatory and homeostatic roles following binding of its cognate ligand lipopolysaccharide (LPS). TWEAK-Fn14 axis plays an important role in pathologies caused by excessive or abnormal inflammatory responses. This study aimed to evaluate potential cross-talk between TLR4 and TWEAK/Fn14 system in porcine small intestinal epithelial cells. Our in vivo results showed that, compared with the age-matched normal control piglets, increased expression of Fn14 in epithelium and decreased TWEAK expression in lamina propria were detected in the small intestinal of piglets stimulated with LPS. Consistent with this finding, treatment with LPS increased the expression of Fn14 and TLR4 while decreased TWEAK expression in porcine small intestinal epithelial cell lines SIEC02. Interestingly, modulating Fn14 activation using agonistic anti-Fn14 decreased TLR4-mediated TNF-α production by SIEC02. In addition, pretreatment of LPS-stimulated SIEC02 with recombinant TWEAK protein suppresses the expression of Fn14 and TNF-α and inhibits the negative impact of LPS on the tight junctional protein occludin expression. In conclusion, this study demonstrates that the TWEAK-independent Fn14 activation augments TLR4-mediated inflammatory responses in the intestine of piglets. Furthermore, the TWEAK-dependent suppression of Fn14 signaling may play a role in intestinal homeostasis.

Quantitative analysis of cell surface antigen-antibody interaction using Gaussia princeps luciferase antibody fusion proteins.

Cell surface antigen-specific antibodies are of substantial diagnostic and therapeutic importance. The binding properties of such antibodies are usually evaluated by cell-free assays, in particular surface plasmon resonance (SPR) analysis, or flow cytometry. SPR analyses allow the detailed quantitative and dynamic evaluation of the binding properties of antibodies, but need purified, typically recombinantly produced antigens. It can, however, be difficult to produce the required antigen. Furthermore, cellular factors influencing the antigen-antibody interaction are not considered by this method. Flow cytometry-based analyses do not have these limitations, but require elaborated calibration controls for absolute quantification of bound molecules. To overcome the limitations of SRP and flow cytometry in the characterization of cell surface antigen-specific antibodies, we developed Fn14-specific antibody 18D1 as an example of an antibody fusion protein format that includes the luciferase of Gaussia princeps (GpL), which enables very simple and highly sensitive cellular binding studies. We found that GpL-tagging of the C-terminus of the antibody light chain does not affect the interaction of 18D1-IgG1 with its antigen and Fc-gamma receptors (FcγRs). In accordance with this, the GpL(LC-CT)-18D1-IgG1 antibody fusion protein showed basically the same FcγR-dependent agonistic properties as the parental 18D1 antibody. Similar results were obtained with isotype switch variants of 18D1 and antibodies specific for CD95, LTßR and CD40. In sum, we demonstrate that antibody GpL fusion proteins are easily manageable and versatile tools for the characterization of cell surface antigen-antibody interactions that have the potential to considerably extend the instrumentarium for the evaluation of antibodies.

Immunoprotective mechanisms in swine within the "grey zone" in antibody response after immunization with foot-and-mouth disease vaccine.

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals caused by the FMD virus (FMDV). Vaccination represents one approach for limiting the effects of FMD. The level of protection in vaccinated animals after challenge with foot and mouth disease virus (FMDV) is closely related to the antibody titer, which can be classified into three zones: a "white zone", a "grey zone", and a "black zone". The aim of the present study was to clarify the immunoprotective mechanisms operating in the grey zone, in which vaccinated animals have intermediate antibody titers, making it difficult to predict the level of protection. Thirty-three pigs were used to analyze the distribution of lymphocyte subpopulations in whole blood and the expression levels of 40 cytokines before vaccination and challenge. The antibody titer in pigs in the grey zone ranged from 1:6-1:45. Cytotoxic T lymphocyte subpopulations, expression levels of Th1 cytokines such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-12, IL-15, IL-18, and monocyte interferon gamma inducing factor (MIG), and of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1α, transforming growth factor-α (TGF-α), and TWEAK R varied between protected and unprotected animals. The results of this study suggest that the cellular immune response is the key factor responsible for immunoprotection in vaccinated animals with antibody titers within the grey zone.