Mycobacterial Infection is Promoted by Neutral Sphingomyelinase 2 Regulating a Signaling Cascade Leading to Activation of β1-Integrin

Background/Aims: Mycobacteria-induced diseases, especially tuberculosis, cause more than 1 million deaths each year, which is higher than any other single bacterial pathogen. Neutral sphingomyelinase 2 (Nsm2) has been implied in many physiological processes and diseases, but the role of Nsm2 in pathogen-host interactions and mycobacterial infections has barely been studied. Methods: We investigated the role of the Nsm2/ceramide system in systemic infection of mice and murine macrophages with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) as a model for mycobacterial infection. For in vitro assays we isolated bone marrow-derived macrophages from Wildtype mice or Nsm2-heterozygous and investigated the role of Nsm2 for macrophage migration/clustering as well as the involvement of p38 mitogen-activated protein kinases (p38K), c-Jun N-terminal kinase (JNK), β1-integrin and Rac1 activity by Western blot and microscopic studies. For in vivo assays we injected mice intravenously with BCG and analyzed infected tissues for the role of Nsm2-mediated activation of β1-integrin in granuloma formation and bacterial burden. Results: Our results reveal that BCG infection of macrophages results in rapid stimulation of Nsm2. Genetic and pharmacological studies demonstrate that Nsm2 stimulates a signaling cascade via p38K and JNK to an activation of surface β1-integrin and Rac1 that leads to the formation of granuloma-like macrophages clusters in vitro and granuloma in vivo. Heterozygosity of Nsm2 in macrophages or antibody-mediated neutralization of active b1-integrin reduced macrophage clusters in vitro and granuloma formation in vivo. Most importantly, Nsm2 heterozygosity or treatment with neutralizing antibodies against β1-integrin protected mice from systemic BCG infections and chronic infections of the liver and spleen. Conclusion: The findings indicate that the Nsm2/ ceramide system plays an important role in systemic infection of mice with mycobacteria by regulating a signaling cascade via p38K, JNK, b1-integrin and Rac1

CD95 Rapidly Clusters in Cells of Diverse Origins

We have shown that CD95-mediated cell death requires a clustering of the receptor in distinct sphingolipid-rich domains of the cell membrane (Grassmé et al., 2000, Cremesti et al., 2000). These domains form in response to acid sphingomyelinase (ASM)-induced ceramide generation. However, recent studies challenged the finding of early CD95 clustering (Algeciras-Schimnich et al., 2002). Here, six independent groups tested clustering of CD95 in diverse cell type including primary cells ex vivo and established cell lines. The studies show clustering of CD95 within seconds to minutes in all cell types tested by the different groups. In addition, clustering of CD95 was detected after stimulation of cells using three agonistic anti-CD95 antibodies (CH11, APO-1-3 and JO2), CD95 ligand and stimuli that induce an upregulation and activation of the endogenous CD95/CD95 ligand system. The data confirm our previous studies and suggest rapid, i.e., within seconds to minutes, CD95 clustering as a general phenomenon occurring in many cell types

Neutral Sphingomyelinase in Physiological and Measles Virus Induced T Cell Suppression

T cell paralysis is a main feature of measles virus (MV) induced immunosuppression. MV contact mediated activation of sphingomyelinases was found to contribute to MV interference with T cell actin reorganization. The role of these enzymes in MV-induced inhibition of T cell activation remained equally undefined as their general role in regulating immune synapse (IS) activity which relies on spatiotemporal membrane patterning. Our study for the first time reveals that transient activation of the neutral sphingomyelinase 2 (NSM2) occurs in physiological co-stimulation of primary T cells where ceramide accumulation is confined to the lamellum (where also NSM2 can be detected) and excluded from IS areas of high actin turnover. Genetic ablation of the enzyme is associated with T cell hyper-responsiveness as revealed by actin dynamics, tyrosine phosphorylation, Ca2+-mobilization and expansion indicating that NSM2 acts to suppress overshooting T cell responses. In line with its suppressive activity, exaggerated, prolonged NSM2 activation as occurring in co-stimulated T cells following MV exposure was associated with aberrant compartmentalization of ceramides, loss of spreading responses, interference with accumulation of tyrosine phosphorylated protein species and expansion. Altogether, this study for the first time reveals a role of NSM2 in physiological T cell stimulation which is dampening and can be abused by a virus, which promotes enhanced and prolonged NSM2 activation to cause pathological T cell suppression

Regulation of the Inflammasome by Ceramide in Cystic Fibrosis Lungs

Background: Cystic fibrosis (CF), the most common autosomal recessive disorder in Western countries, is characterized by chronic pulmonary inflammation, reduced mucociliary clearance, and increased susceptibility to infection. Our studies using Cftr-deficient mice and human CF specimens showed that ceramide accumulates in CF lungs and mediates increased cell death, susceptibility to infections, and inflammation. Methods: We used Cftr-deficient and syngenic wildtype mice as well as Cftr-deficient mice heterozygous for the acid sphingomyelinase. We determined activation and topology of inflammasome components as well as expression of tight junction proteins by confocal microscopy, western blotting and ELISA. Results: We demonstrate an upregulation and membrane recruitment of the adapter protein apoptosis-associated speck-like protein (Asc), a major component of the inflammasome, and caspase 1, an activation of Jun N-terminal kinase as well as an altered distribution and a degradation of the tight junction proteins ZO-1, ZO-2 and Occludin in lungs of CF mice. All of these events are abrogated in CF mice that are heterozygous for the acid sphingomyelinase and, therefore, show normal levels of ceramide in their lungs. These alterations indicate an activation of the inflammasome by ceramide in the lungs of CF mice. Consistent with this notion, we observe a normalization of the increased levels of the cytokines IL-1β and KC/IL-8 in lungs of CF mice upon treatment with caspase 1 inhibitors. Conclusion: Our data suggest a signaling cascade from ceramide via the inflammasome to caspase 1, the release of cytokines and an alteration of tight junction proteins in CF epithelia

Ceramide and NSM2 accumulate within the lamellum in co-stimulated T cells.

<p>A. Ceramide and f-actin were co-detected in T cells 15 min after seeding onto co-stimulatory slides (left panels, with intensities indicated by false colour representation, bottom panels). Size bar: 5 µm. Subcellular distribution of actin (in red) and ceramide (in green) within the lamellum (LM) or the lamellipodium (LP) are representatively shown as profiles (middle panel, intensity profile plane indicated by the arrow) or blow ups imaged at lower or higher z planes (distance approximately 600 nm)(right panels, enlargements of boxed area middle panel). B. NSM2 and f-actin were co-detected 24 h following nucleofection of p-NSM2-GFP (NSM2: green, f-actin: red). Subcellular distribution of NSM2-GFP is shown after deconvolution and 3D reconstruction (right panel). Size bar: 5 µm.</p

Acid sphingomyelinase is involved in CEACAM receptor-mediated phagocytosis of Neisseria gonorrhoeae

The interaction with human phagocytes is a hallmark of symptomatic Neisseria gonorrhoeae infections. Gonococcal outer membrane proteins of the Opa family induce the opsoninindependent uptake of the bacteria that relies on CEACAM receptors and an active signaling machinery of the phagocyte. Here, we show that CEACAM receptor-mediated phagocytosis of Opa52-expressing N. gonorrhoeae into human cells results in a rapid activation of the acid sphingomyelinase. Inhibition of this enzyme by imipramine or SR33557 abolishes opsonin-independent internalization without affecting bacterial adherence. Reconstitution of ceramide, the product of acid sphingomyelinase activity, in imipramine- or SR33557-treated cells restores internalization of the bacteria. Furthermore, we demonstrate that CEACAM receptor-initiated stimulation of other signalling molecules, in particular Src-like tyrosine kinases and Jun Nterminal kinases, requires acid sphingomyelinase. These studies provide evidence for a crucial role of the acid sphingomyelinase for CEACAM receptor-initiated signalling events and internalization of Opa52-expressing N. gonorrhoeae into human neutrophils