Trans-sialidases are key enzymes in the life cycle of African trypanosomes in both, mammalian host and insect vector and have been associated with the disease trypanosomiasis, namely sleeping sickness and nagana. Besides the previously reported TconTS1, we have identified three additional active trans-sialidases, TconTS2, TconTS3 and TconTS4, and three trans-sialidase like genes in Trypanosoma congolense. At least TconTS1, TconTS2 and TconTS4 are found in the bloodstream of infected animals. We have characterised the enzymatic properties of recombinant proteins expressed in eukaryotic fibroblasts using fetuin as model blood glycoprotein donor substrate. One of the recombinant trans-sialidases, TconTS2, had the highest specific activity reported thus far with very low sialidase activity. The active trans-sialidases share all the amino acids critical for the catalytic reaction with few variations in the predicted binding site for the leaving or acceptor glycan. However, these differences cannot explain the orders of magnitudes between their transfer activities, which must be due to other unidentified structural features of the proteins or substrates selectivity. Interestingly, the phylogenetic relationships between the lectin domains correlate with their specific trans-sialylation activities. This raises the question whether and how the lectin domains regulate the trans-sialidase reaction. The identification and enzymatic characterisation of the trans-sialidase family in T. congolense will contribute significantly towards the understanding of the roles of these enzymes in the pathogenesis of Animal African Trypanosomiasis.
Correction to: Tie2 Activation Promotes Protection and Reconstitution of the Endothelial Glycocalyx in Human SepsisThromb Haemost 2019; 119(11): 1827-1838DOI: 10.1055/s-0039-1695768
Sphingosine 1-phosphate (S1P) is a lysosphingolipid with anti-atherogenic properties, but mechanisms underlying its effects remain unclear. We here investigated atherosclerosis development in cholesterol-rich diet-fed LDL receptor-deficient mice with high or low overexpression levels of S1P receptor type 1 (S1P1) in macrophages. S1P1-overexpressing macrophages showed increased activity of transcription factors PU.1, IRF8, and LXR and were skewed towards a M2-distinct phenotype characterized by enhanced production of IL-10, IL-1RA, and IL-5, increased ATP-binding cassette transporter A1- and G1-dependent cholesterol efflux, increased expression of MerTK and efferocytosis, and reduced apoptosis due to elevated Bcl6 and MafB. A similar macrophage phenotype was observed in mice administered S1P1-selective agonist KRP203. Mechanistically, the enhanced PU.1, IRF8, and LXR activity in S1P1-overexpressing macrophages led to down-regulation of the cAMP-dependent protein kinase A and activation of the signaling cascade encompassing protein kinases Akt and mTOR complex 1 (mTORC1) as well as the late endosomal/lysosomal adaptor MAPK and mTOR activator 1 (Lamtor-1). Atherosclerotic lesions in aortic roots and brachiocephalic arteries were profoundly or moderately reduced in mice with high and low S1P1 overexpression in macrophages, respectively. We conclude that S1P1 signaling polarizes macrophages towards an anti-atherogenic functional phenotype and countervails the development of atherosclerosis in mice.
Soluble fms-like tyrosine kinase-1 (sFlt-1) is a secreted protein that binds heparan sulfate expressed on the endothelial glycocalyx (eGC). In this paper we analyze how excess sFlt-1 causes conformational changes in the eGC, leading to monocyte adhesion, a key event triggering vascular dysfunction. In vitro exposure of primary human umbilical vein endothelial cells to excess sFlt-1 decreased eGC height and increased stiffness as determined by atomic force microscopy (AFM). Yet, structural loss of the eGC components was not observed, as indicated by Ulex europaeus agglutinin I and wheat germ agglutinin staining. Moreover, the conformation observed under excess sFlt-1, a collapsed eGC, is flat and stiff with unchanged coverage and sustained content. Functionally, this conformation increased the endothelial adhesiveness to THP-1 monocytes by about 35%. Heparin blocked all these effects, but the vascular endothelial growth factor did not. In vivo administration of sFlt-1 in mice also resulted in the collapse of the eGC in isolated aorta analyzed ex vivo by AFM. Our findings show that excess sFlt-1 causes the collapse of the eGC and favors leukocyte adhesion. This study provides an additional mechanism of action by which sFlt-1 may cause endothelial dysfunction and injury.
Bacterial challenge of keratinocytes with the abundant skin commensal Staphylococcus epidermidis induces distinct innate immune responses, but the underlying molecular mechanisms are still emerging. We report that the aryl hydrocarbon receptor (AhR) was activated in human primary keratinocytes infected with S. epidermidis, leading to induction of the AhR-responsive gene cytochrome P450 1A1 (CYP1A1). In addition, functional AhR was required for S. epidermidis-mediated induction of IL-1β expression in keratinocytes. AhR-dependent gene induction of IL-1β and CYP1A1 was mediated by factor(s) < 2 kDa secreted by S. epidermidis. Blockade of the AhR in a 3D organotypic skin equivalent infected with S. epidermidis attenuated the S. epidermidis-induced CYP1A1 and IL-1β expression. Moreover, S. epidermidis also induced expression of IL-1α and of the antimicrobial peptide human β-defensin-3 in an AhR-dependent manner in a 3D skin equivalent. An increased outgrowth of S. epidermidis on the surface of skin explants treated with a specific AhR inhibitor further indicate a pivotal role of the AhR in mediating an epidermal defense response. Taken together, our data expand the role of the AhR in innate immunity and support a previously unappreciated contribution for the AhR in cutaneous defense.
The endothelial glycocalyx (eGC), a carbohydrate-rich layer lining the luminal surface of the endothelium, provides a first vasoprotective barrier against vascular leakage and adhesion in sepsis and vessel inflammation. Angiopoietin-2 (Angpt-2), an antagonist of the endothelium-stabilizing receptor Tie2 secreted by endothelial cells, promotes vascular permeability through cellular contraction and junctional disintegration. We hypothesized that Angpt-2 might also mediate the breakdown of the eGC. Using confocal and atomic force microscopy, we show that exogenous Angpt-2 induces a rapid loss of the eGC in endothelial cells in vitro. Glycocalyx deterioration involves the specific loss of its main constituent heparan sulphate, paralleled by the secretion of the heparan sulphate-specific heparanase from late endosomal/lysosomal stores. Corresponding in vivo experiments revealed that exogenous Angpt-2 leads to heparanase-dependent eGC breakdown, which contributes to plasma leakage and leukocyte recruitment in vivo. Our data indicate that eGC breakdown is mediated by Angpt-2 in a non-redundant manner.
Endothelial cells are a critical target of the soluble Fms-like tyrosine kinase-1 (sFlt-1), a soluble factor increased in different diseases with varying degrees of renal impairment and endothelial dysfunction, including chronic kidney disease (CKD). Although the mechanisms underlying endothelial dysfunction are multifactorial and complex, herein, we investigated the damaging effects of sFlt-1 on structural and functional changes in endothelial cells. Our results evidenced that sera from patients with CKD stiffen the endothelial cell cortex in vitro, an effect correlated with sFlt-1 levels and prevented by sFlt-1 neutralization. Besides, we could show that recombinant sFlt-1 leads to endothelial stiffening in vitro and in vivo. This was accompanied by cytoskeleton reorganization and changes in the endothelial barrier function, as observed by increased actin polymerization and endothelial cell permeability, respectively. These results depended on the activation of the p38 MAPK and were blocked by the specific inhibitor SB203580. However, sFlt-1 only minimally affected the expression of stiffness-sensitive genes. These findings bring new insight into the mechanism of action of sFlt-1 and its biological effects that cannot be exclusively ascribed to the regulation of angiogenesis.
