Sphingosine kinase 1 is critically involved in nitric oxide-mediated human endothelial cell migration and tube formation

Sphingosine kinases (SKs) convert sphingosine to sphingosine 1-phosphate (S1P), which is a bioactive lipid that regulates a variety of cellular processes including proliferation, differentiation and migration.

Development of replication-defective lymphocytic choriomeningitis virus vectors for the induction of potent CD8+ T cell immunity

Lymphocytic choriomeningitis virus (LCMV) exhibits natural tropism for dendritic cells and represents the prototypic infection that elicits protective CD8(+) T cell (cytotoxic T lymphocyte (CTL)) immunity. Here we have harnessed the immunobiology of this arenavirus for vaccine delivery. By using producer cells constitutively synthesizing the viral glycoprotein (GP), it was possible to replace the gene encoding LCMV GP with vaccine antigens to create replication-defective vaccine vectors. These rLCMV vaccines elicited CTL responses that were equivalent to or greater than those elicited by recombinant adenovirus 5 or recombinant vaccinia virus in their magnitude and cytokine profiles, and they exhibited more effective protection in several models. In contrast to recombinant adenovirus 5, rLCMV failed to elicit vector-specific antibody immunity, which facilitated re-administration of the same vector for booster vaccination. In addition, rLCMV elicited T helper type 1 CD4+ T cell responses and protective neutralizing antibodies to vaccine antigens. These features, together with low seroprevalence in humans, suggest that rLCMV may show utility as a vaccine platform against infectious diseases and cancer.

Inhibition of direct and indirect TLR-mediated activation of human NK cells by low molecular weight dextran sulfate

NK cells express toll-like receptors (TLR) that recognize conserved pathogen or damage associated molecular patterns and play a fundamental role in innate immunity. Low molecular weight dextran sulfate (DXS), known to inhibit the complement system, has recently been reported by us to inhibit TLR4-induced maturation of human monocyte-derived dendritic cells (MoDC). In this study, we investigated the capability of DXS to interfere with human NK cell activation triggered directly by TLR2 agonists or indirectly by supernatants of TLR4-activated MoDC. Both TLR2 agonists and supernatants of TLR4-activated MoDC activated NK cells phenotypically, as demonstrated by the analysis of NK cell activation markers (CD56, CD25, CD69, NKp30, NKp44, NKp46, DNAM-1 and NKG2D), and functionally as shown by increased NK cell degranulation (CD107a surface expression) and IFN-gamma secretion. DXS prevented the up-regulation of NK cell activation markers triggered by TLR2 ligands or supernatants of TLR4-activated MoDC and dose-dependently abrogated NK cell degranulation and IFN-gamma secretion. In summary our results suggest that DXS may be a useful reagent to inhibit the direct and indirect TLR-mediated activation of NK cells.

Estrogen Receptor α Signaling in T Lymphocytes Is Required for Estradiol-Mediated Inhibition of Th1 and Th17 Cell Differentiation and Protection against Experimental Autoimmune Encephalomyelitis

Estrogen treatment exerts a protective effect on experimental autoimmune encephalomyelitis (EAE) and is under clinical trial for multiple sclerosis therapy. Estrogens have been suspected to protect from CNS autoimmunity through their capacity to exert anti-inflammatory as well as neuroprotective effects. Despite the obvious impacts of estrogens on the pathophysiology of multiple sclerosis and EAE, the dominant cellular target that orchestrates the anti-inflammatory effect of 17β-estradiol (E2) in EAE is still ill defined. Using conditional estrogen receptor (ER) α-deficient mice and bone marrow chimera experiments, we show that expression of ERα is critical in hematopoietic cells but not in endothelial ones to mediate the E2 inhibitory effect on Th1 and Th17 cell priming, resulting in EAE protection. Furthermore, using newly created cell type-specific ERα-deficient mice, we demonstrate that ERα is required in T lymphocytes, but neither in macrophages nor dendritic cells, for E2-mediated inhibition of Th1/Th17 cell differentiation and protection from EAE. Lastly, in absence of ERα in host nonhematopoietic tissues, we further show that ERα signaling in T cells is necessary and sufficient to mediate the inhibitory effect of E2 on EAE development. These data uncover T lymphocytes as a major and nonredundant cellular target responsible for the anti-inflammatory effects of E2 in Th17 cell-driven CNS autoimmunity.

The ubiquitin ligase XIAP recruits LUBAC for NOD2 signaling in inflammation and innate immunity

Nucleotide-binding and oligomerization domain (NOD)-like receptors constitute a first line of defense against invading bacteria. X-linked Inhibitor of Apoptosis (XIAP) is implicated in the control of bacterial infections, and mutations in XIAP are causally linked to immunodeficiency in X-linked lymphoproliferative syndrome type-2 (XLP-2). Here, we demonstrate that the RING domain of XIAP is essential for NOD2 signaling and that XIAP contributes to exacerbation of inflammation-induced hepatitis in experimental mice. We find that XIAP ubiquitylates RIPK2 and recruits the linear ubiquitin chain assembly complex (LUBAC) to NOD2. We further show that LUBAC activity is required for efficient NF-κB activation and secretion of proinflammatory cytokines after NOD2 stimulation. Remarkably, XLP-2-derived XIAP variants have impaired ubiquitin ligase activity, fail to ubiquitylate RIPK2, and cannot facilitate NOD2 signaling. We conclude that XIAP and LUBAC constitute essential ubiquitin ligases in NOD2-mediated inflammatory signaling and propose that deregulation of NOD2 signaling contributes to XLP-2 pathogenesis.

The Emerging Role of TLR and Innate Immunity in Cardiovascular Disease

Cardiovascular disease is a complex disorder involving multiple pathophysiological processes, several of which involve activation of toll-like receptors (TLRs) of the innate immune system. As sentinels of innate immunity TLRs are nonclonally germline-encoded molecular pattern recognition receptors that recognize exogenous as well as tissue-derived molecular dangers signals promoting inflammation. In addition to their expression in immune cells, TLRs are found in other tissues and cell types including cardiomyocytes, endothelial and vascular smooth muscle cells. TLRs are differentially regulated in various cell types by several cardiovascular risk factors such as hypercholesterolemia, hyperlipidemia, and hyperglycemia and may represent a key mechanism linking chronic inflammation, cardiovascular disease progression, and activation of the immune system. Modulation of TLR signaling by specific TLR agonists or antagonists, alone or in combination, may be a useful therapeutic approach to treat various cardiovascular inflammatory conditions such as atherosclerosis, peripheral arterial disease, secondary microvascular complications of diabetes, autoimmune disease, and ischemia reperfusion injury. In this paper we discuss recent developments and current evidence for the role of TLR in cardiovascular disease as well as the therapeutic potential of various compounds on inhibition of TLR-mediated inflammatory responses.

Mechanisms of nanoparticle-mediated photomechanical cell damage

Laser-assisted killing of gold nanoparticle targeted macrophages was investigated. Using pressure transient detection, flash photography and transmission electron microscopy (TEM) imaging, we studied the mechanism of single cell damage by vapor bubble formation around gold nanospheres induced by nanosecond laser pulses. The influence of the number of irradiating laser pulses and of particle size and concentration on the threshold for acute cell damage was determined. While the single pulse damage threshold is independent of the particle size, the threshold decreases with increasing particle size when using trains of pulses. The dependence of the cell damage threshold on the nanoparticle concentration during incubation reveals that particle accumulation and distribution inside the cell plays a key role in tissue imaging or cell damaging.

Endothelial cell antibody-mediated rejection and successful retransplantation in a heart transplanted patient

Antibody-mediated rejection (AMR) plays a significant role in cardiac allograft dysfunction, and recently a consensus regarding the diagnosis of AMR has been published. To our knowledge, it has not previously been reported that acute graft failure related to AMR, and antiendothelial cell antibodies can successfully be diagnosed to allow the patient to receive the outlined treatment and undergo a subsequent retransplantation.

Antagonistic and synergistic effects of glucocorticoids and IL-7 on CD4+ T cell activation

Glucocorticoids (GCs) are steroidal compounds widely used to treat chronic and acute inflammatory diseases. In particular, GCs at pharmacological doses induce apoptosis of activated and naïve T cells, inhibit their proliferation and block pro-inflammatory cytokine secretion. At physiological concentrations, the effect of these steroids on T cell immunity are not yet fully understood, and various studies reported paradoxical roles exerted by GCs on T cell immunity. Here, we show that GCs surprisingly induce proliferation of activated CD4(+) T cells in the presence of IL-7, a cytokine secreted in the thymus and at mucosal sites. Increased proliferation is dependent on a GC-mediated survival of mitotic cells. Moreover, we observe a downmodulation of Th1 cytokine secretion in cells treated with GCs, an outcome which is not affected by the presence of IL-7. GCs exert thus a positive role in the presence of IL-7 by enhancing proliferation of CD4(+) T cells and simultaneously a negative role by suppressing pro-inflammatory cytokine production.

Activation of the orphan endothelial receptor Tie1 modifies Tie2-mediated intracellular signaling and cell survival

A critical role for Tie1, an orphan endothelial receptor, in blood vessel morphogenesis has emerged from mutant mouse studies. Moreover, it was recently demonstrated that certain angiopoietin (Ang) family members can activate Tie1. We report here that Ang1 induces Tie1 phosphorylation in endothelial cells. Tie1 phosphorylation was, however, Tie2 dependent because 1) Ang1 failed to induce Tie1 phosphorylation when Tie2 was down-regulated in endothelial cells; 2) Tie1 phosphorylation was induced in the absence of Ang1 by either a constitutively active form of Tie2 or a Tie2 agonistic antibody; 3) in HEK 293 cells Ang1 phosphorylated a form of Tie1 without kinase activity when coexpressed with Tie2, and Ang1 failed to phosphorylate Tie1 when coexpressed with kinase-defective Tie2. Ang1-mediated AKT and 42/44MAPK phosphorylation is predominantly Tie2 mediated, and Tie1 down-regulates this pathway. Finally, based on a battery of in vitro and in vivo data, we show that a main role for Tie1 is to modulate blood vessel morphogenesis by virtue of its ability to down-regulate Tie2-driven signaling and endothelial survival. Our new observations help to explain why Tie1 null embryos have increased capillary densities in several organ systems. The experiments also constitute a paradigm for how endothelial integrity is fine-tuned by the interplay between closely related receptors by a single growth factor.

Synergistic inhibition in cell-cell fusion mediated by the matrix and nucleocapsid protein of canine distemper virus

Canine distemper virus (CDV) causes a chronic, demyelinating, progressive or relapsing neurological disease in dogs, because CDV persists in the CNS. Persistence of virulent CDV, such as the A75/17 strain has been reproduced in cell cultures where it is associated with a non-cytolytic infection with very limited cell-cell fusion. This is in sharp contrast to attenuated CDV infection in cell cultures, such as the Onderstepoort (OP) CDV strain, which produces extensive fusion activity and cytolysis. Fusion efficiency may be determined by the structure of the viral fusion protein per se but also by its interaction with other structural proteins of CDV. This was studied by combining genes derived from persistent and non-persistent CDV strains in transient transfection experiments. It was found that fusion efficiency was markedly attenuated by the structure of the fusion protein of the neurovirulent A75/17-CDV. Moreover, we showed that the interaction of the surface glycoproteins with the M protein of the persistent strain greatly influenced fusion activity. Site directed mutagenesis showed that the c-terminus of the M protein is of particular importance in this respect. Interestingly, although the nucleocapsid protein alone did not affect F/H-induced cell-cell fusion, maximal inhibition occurred when the latter was added to combined glycoproteins with matrix protein. Thus, the present study suggests that very limited fusogenicity in virulent CDV infection, which favours persistence by limiting cell destruction involves complex interactions between all viral structural proteins.

LRH-1-mediated glucocorticoid synthesis in enterocytes protects against inflammatory bowel disease

Liver receptor homolog-1 (LRH-1) is a nuclear receptor involved in intestinal lipid homeostasis and cell proliferation. Here we show that haploinsufficiency of LRH-1 predisposes mice to the development of intestinal inflammation. Besides the increased inflammatory response, LRH-1 heterozygous mice exposed to 2,4,6-trinitrobenzene sulfonic acid show lower local corticosterone production as a result of an impaired intestinal expression of the enzymes CYP11A1 and CYP11B1, which control the local synthesis of corticosterone in the intestine. Local glucocorticoid production is strictly enterocyte-dependent because it is robustly reduced in epithelium-specific LRH-1-deficient mice. Consistent with these findings, colon biopsies of patients with Crohn's disease and ulcerative colitis show reduced expression of LRH-1 and genes involved in the production of glucocorticoids. Hence, LRH-1 regulates intestinal immunity in response to immunological stress by triggering local glucocorticoid production. These findings underscore the importance of LRH-1 in the control of intestinal inflammation and the pathogenesis of inflammatory bowel disease.