No abstract

Background: Inflammasomes are cytosolic multiprotein complexes which, upon assembly, activate the maturation and secretion of the inflammatory cytokines IL-1β and IL-18. However, participation of the NLRP3 inflammasome in ischaemic stroke remains controversial. Our aims were to determine the role of NLRP3 in brain ischaemia, and explore the mechanism involved in the potential protective effect of the neurovascular unit.Methods: WT and NLRP3 knock-out mice were subjected to ischaemia by middle cerebral artery occlusion (60 min) with or without treatment with MCC950 at different time points post-stroke. Brain injury was measured histologically with 2,3,5-triphenyltetrazolium chloride (TTC) staining. Results:We identified a time-dependent dual effect of NLRP3. While neither the pre-treatment with MCC950 nor the genetic approach (NLRP3 KO) proved to be neuroprotective, post-reperfusion treatment with MCC950 significantly reduced the infarct volume in a dose-dependent manner. Importantly, MCC950 improved the neuro-motor function and reduced the expression of different pro-inflammatory cytokines (IL-1β and TNF-α), NLRP3 inflammasome components (NLRP3 and procaspase-1), protease expression (MMP9), and endothelial adhesion molecules (ICAM and VCAM). We observed a marked protection of the blood-brain barrier (BBB), which was also reflected in the recovery of the tight junction proteins (ZO-1 and Claudin-5). Additionally, MCC950 produced a reduction of the CCL2 chemokine in blood serum and in brain tissue, which lead to a reduction in the immune cell infiltration.

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Accumulating evidence on the role of Thrombospondin-1 (TSP-1) in the immune response has emerged during the last years. In spite of the importance of TSP-1 not only as anti-angiogenic factor but also as an immunomodulatory molecule, studies on the role of TSP-1 in psoriasis have been neglected. TSP-1 and CD47 expression were analyzed in skin samples from psoriasis patients and control subjects using RT-PCR and immunofluorescence. Expression of these molecules was also evaluated in peripheral blood CD4+ T cells, moDCs, and circulating primary DCs. The functional role of TSP-1/CD47 signaling axis in psoriasis was assessed in Th17 and Treg differentiation assays. Additionally, small interfering RNA assays specific to TSP-1 were performed in CD4+ T cells and monocyte derived DC to specifically evaluate the function of this protein. Lesional skin of psoriasis patients expressed lower TSP-1 and CD47 mRNA levels compared to non-lesional skin or skin from controls. Immunofluorescence staining revealed decreased expression of CD47 in CD45+ dermal cells from psoriasis samples compared to control subjects. Peripheral CD4+ T cells and circulating primary DCs from psoriasis also expressed lower levels of CD47 compared to controls. Although no significant differences were detected in TSP-1 expression in CD4+ T cells and moDCs between patients and controls, TSP-1 expression in psoriasis patients inversely correlated with disease activity evaluated by the Psoriasis Area and Index Activity. Furthermore, exogenous TSP-1 inhibited Th17 differentiation and stimulated the differentiation of CD4+ T cells toward Treg cells. Furthermore, RNA interference specific for TSP-1 confirmed the role of this molecule as a negative regulator of T cell activation. Because of the impact of TSP-1/CD47 signaling axis in Th17 and Treg differentiation, a dysregulated expression of these molecules in the immune cells from psoriasis patients may favor the exacerbated inflammatory response in this disease.

similarly decreased in normoxia or hypoxia in both, the pVHL L188V and Y112H mutants to levels resembling those in pVHL negative cells (Fig. 4c). Taken together these data demonstrate that TSP-1 decrease in ccRCC pVHL negative cell lines is independent of HIF.

Chronic obstructive pulmonary disease (COPD) is a widespread disease, with no curative therapies nowadays. Exposure to cigarette smoking is considered the chief leading cause of COPD. Current drugs therapies improve patient quality of life, however they do not revert the progression of the disease. Therefore, a deeper study of the cellular and molecular mechanisms that underlie this pathology is required to be able to carry out targeted and effective treatments. Although the effects of cigarette smoke in the progressive deterioration of the airway have been extensively studied in COPD patients, its effects on pulmonary vasculature have been unexplored, due to the classic conception that vascular damage is a consequence of alveolar hypoxia and loss of capillary bed. In this paper, we aimed to study the effects of cigarette smoke extract (CSE) in regulating pulmonary arterial cells phenotypic modulation, in particular the effects in fibroblasts (hPAFib) and smooth muscle cells (hPASMC), and in murine pulmonary arteries. Our results demonstrated that CSE exposure had direct effects on hPAFib and hPASMC, promoting a senescent phenotype that in turn contributed, through the secretion of inflammatory molecules, to increase the proliferative potential of non-exposed cells. CSE also increased total ROS levels in hPAFib and hPASMC, and upregulated NADPH oxidase subunits NOX1 and p22 phox . Most importantly, CSE affected cell contractility and dysregulated the expression and activity of voltage-gated K + channel Kv7.4. This contributed to limit vascular responses impairing vasoconstriction and endotheliumdependent and independent relaxation.

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Background: Post-ischemic inflammation contributes to worsening of ischemic brain injury and in this process, the inflammasomes play a key role. Inflammasomes are cytosolic multiprotein complexes which upon assembly activate the maturation and secretion of the inflammatory cytokines IL-1β and IL-18. However, participation of the NLRP3 inflammasome in ischemic stroke remains controversial. Our aims were to determine the role of NLRP3 in ischemia and to explore the mechanism involved in the potential protective effect of the neurovascular unit. Methods: WT and NLRP3 knock-out mice were subjected to ischemia by middle cerebral artery occlusion (60 minutes) with or without treatment with MCC950 at different time points post-stroke. Brain injury was measured histologically with 2,3,5-triphenyltetrazolium chloride (TTC) staining. Results: We identified a time-dependent dual effect of NLRP3. While neither the pre-treatment with MCC950 nor the genetic approach (NLRP3 KO) proved to be neuroprotective, post-reperfusion treatment with MCC950 significantly reduced the infarct volume in a dose-dependent manner. Importantly, MCC950 improved the neuro-motor function and reduced the expression of different pro-inflammatory cytokines (IL-1β, TNF-α), NLRP3 inflammasome components (NLRP3, pro-caspase-1), protease expression (MMP9) and endothelial adhesion molecules (ICAM, VCAM). We observed a marked protection of the blood-brain barrier (BBB), which was also reflected in the recovery of the tight junctions proteins (ZO-1, Claudin-5). Additionally, MCC950 produced a reduction of the CCL2 chemokine in blood serum and in brain tissue, which lead to a reduction in the immune cell infiltration. Conclusions: These findings suggest that post-reperfusion NLRP3 inhibition may be an effective acute therapy for protecting the blood-brain barrier in cerebral ischemia with potential clinical translation.