Association between biomechanical alterations and migratory ability of semaphorin-3A-treated thymocytes.

BACKGROUND: Class 3 semaphorins are soluble proteins involved in cell adhesion and migration. Semaphorin-3A (Sema3A) was initially shown to be involved in neuronal guidance, and it has also been reported to be associated with immune disorders. Both Sema3A and its receptors are expressed by most immune cells, including monocytes, macrophages, and lymphocytes, and these proteins regulate cell function. Here, we studied the correlation between Sema3A-induced changes in biophysical parameters of thymocytes, and the subsequent repercussions on cell function. METHODS: Thymocytes from mice were treated in vitro with Sema3A for 30min. Scanning electron microscopy was performed to assess cell morphology. Atomic force microscopy was performed to further evaluate cell morphology, membrane roughness, and elasticity. Flow cytometry and/or fluorescence microscopy were performed to assess the F-actin cytoskeleton and ROCK2. Cell adhesion to a bovine serum albumin substrate and transwell migration assays were used to assess cell migration. RESULTS: Sema3A induced filopodia formation in thymocytes, increased membrane stiffness and roughness, and caused a cortical distribution of the cytoskeleton without changes in F-actin levels. Sema3A-treated thymocytes showed reduced substrate adhesion and migratory ability, without changes in cell viability. In addition, Sema3A was able to down-regulate ROCK2. CONCLUSIONS: Sema3A promotes cytoskeletal rearrangement, leading to membrane modifications, including increased stiffness and roughness. This effect in turn affects the adhesion and migration of thymocytes, possibly due to a reduction in ROCK2 expression. GENERAL SIGNIFICANCE: Sema3A treatment impairs thymocyte migration due to biomechanical alterations in cell membranes.

Biomechanical and functional properties of trophoblast cells exposed to Group B Streptococcus in vitro and the beneficial effects of uvaol treatment.

BACKGROUND: Group B streptococcus (GBS) is the main bacteria that infects pregnant women and can cause abortion and chorioamnionitis. The impact of GBS effects on human trophoblast cells remains largely elusive, and actions toward anti-inflammatory strategies in pregnancy are needed. A potent anti-inflammatory molecule, uvaol is a triterpene from olive oil and its functions in trophoblasts are unknown. We aimed to analyze biomechanical and functional effects of inactivated GBS in trophoblast cells, with the addition of uvaol to test potential benefits. METHODS: HTR-8/SVneo cells were treated with uvaol and incubated with inactivated GBS. Cell viability and death were analyzed. Cellular elasticity and topography were accessed by atomic force microscopy. Nitrite production was evaluated by Griess reaction. Nuclear translocation of NFkB p65 was detected by immunofluorescence and Th1/Th2 cytokines by bead-based multiplex assay. RESULTS: GBS at 108 CFU increased cell death, which was partially prevented by uvaol. Cell stiffness, cytoskeleton organization and morphology were changed by GBS, and uvaol partially restored these alterations. Nuclear translocation of NFkB p65 began 15 min after GBS incubation and uvaol inhibited this process. GBS decreased IL-4 secretion and increased IL-1ß, IFN-γ and IL-2, whereas uvaol reverted this. CONCLUSIONS: The increased inflammation and cell death caused by GBS correlated with biomechanical and cytoskeleton changes found in trophoblast cells, while uvaol was effective its protective role. GENERAL SIGNIFICANCE: Uvaol is a natural anti-inflammatory product efficient against GBS-induced inflammation and it has potential to be acquired through diet in order to prevent GBS deleterious effects in pregnancy.