RESUMEN
The V-set and transmembrane domain-containing protein (VSTM) family is a newly discovered immunoglobulin (Ig) superfamily that shares structural similarities with the B7-like transmembrane proteins. Although most VSTM5 members have been reported to exert immune-related functions, VSTM5 has been described as a regulator of neuronal morphogenesis and migration in the brain. Based on its close phylogenetic relationship with two immune checkpoints, VISTA and TIGIT, we investigated the potential role of VSTM5 in T-cell immune responses. VSTM5.Ig inhibits T-cell proliferation and cytokine production, induces T-cell apoptosis, and promotes the generation of regulatory T cells (Tregs) in in vitro T-cell assays. VSTM5 also contributes to the maintenance of T-cell anergy in vitro. Similarly, serum VSTM5.Ig produced using a recombinant plasmid in ovalbumin (OVA)-immunized mice inhibits both naive and effector T-cell immune responses. In addition, VSTM5.Ig enhances oral tolerance of cell-mediated and antibody responses in OVA-fed mice by inducing Tregs and T-cell clonal deletion. Consequently, our findings suggest that VSTM5 is a novel immune checkpoint that could be used to improve the therapeutic efficacy of tolerance-based therapies for autoimmune diseases.
Asunto(s)
Formación de Anticuerpos , Tolerancia Inmunológica , Proteínas de la Membrana , Animales , Ratones , Inmunoglobulinas , Proteínas de la Membrana/metabolismo , Ovalbúmina , Filogenia , Linfocitos T ReguladoresRESUMEN
BACKGROUND: Vascular barrier breakdown in sepsis represents a key component of the maladaptive host response to infection and the release of endothelial Angiopoietin-2 (Angpt-2) is a mechanistic driver of endothelial hyperpermeability. Angpt-2 is associated with morbidity and mortality but a targeted therapeutic approach is not available. We screened for U.S. Food and Drug Administration (FDA) approved drugs that might have off-target effects decreasing Angpt-2 and therefore, ameliorating capillary leakage. METHODS: Endothelial cells were isolated from human umbilical veins (HUVECs) and used for in vitro studies at baseline and after stimulation (FDA-library screening, RT-PCR, ELISA, immunocytochemistry, MTT assay). On the functional level, we assessed real-time transendothelial electrical resistance (TER) using an electric cell-substrate impedance sensing device. RESULTS: We found that the anti-fungal Bifonazole (BIFO) reduces spontaneous Angpt-2 release in a time- and dose-dependent manner after 8, 12, and 24âh (24âh: veh: 15.6â±â0.7 vs. BIFO: 8.6â±â0.8âng/mL, Pâ<â0.0001). Furthermore, we observed a reduction in its intra-cellular content by 33% (Pâ<â0.001). Stimulation with tumor necrosis factor α induced a strong release of Angpt-2 that could analogously be blocked by additional treatment with BIFO (veh: 1.58â±â0.2 vs. BIFO: 1.02â±â0.1, Pâ<â0.0001). Quantification of endothelial permeability by TER revealed that BIFO was sufficient to reduce Thrombin-induced barrier breakdown (veh: 0.82â±â0.1 vs. BIFO: 1.01â±â0.02, Pâ<â0.05). CONCLUSION: The antifungal BIFO reduces both release and biosynthesis of the endothelial-destabilizing factor Angpt-2 in vitro thereby improving vascular barrier function. Additional studies are needed to further investigate the underlying mechanism and to translate these findings to in vivo models.