Ceramide in Bacterial Infections and Cystic Fibrosis

Article in press - uncorrected proof

Biol. Chem., Vol. 389, pp. 1371–1379, November 2008 • Copyright by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2008.162

Review

Ceramide in bacterial infections and cystic fibrosis

Heike Grassme´ , Katrin Anne Becker, ordered phase and thus form distinct domains in the cell Yang Zhang and Erich Gulbins* membrane (Simons and Ikonen, 1997; Brown and Lon- don, 1998). Results from these studies indicate that Department of Molecular Biology, University of sphingolipids and cholesterol associate via hydrophilic Duisburg-Essen, Hufelandstrasse 55, D-45122 Essen, and hydrophobic interactions, resulting in an organized Germany membrane domain with a higher melting point than other * Corresponding author phospholipids in model membranes investigated in bio- e-mail: [email protected] physical experiments (Simons and Ikonen, 1997; Brown and London, 1998). In particular, the head groups of (gly- co)sphingolipids and the hydroxyl group of cholesterol Abstract seem to stabilize these domains via hydrophilic interactions, whereas the acyl chains of sphingolipids and the Ceramide is formed by the activity of sphingomyelinases, sterol ring form hydrophobic van der Waals’ interactions by degradation of complex sphingolipids, reverse cera- (for a review see Kolesnick et al., 2000). In addition, cho- midase activity or de novo synthesized. The formation of lesterol seems to stabilize these domains by filling the ceramide within biological membranes results in the for- void spaces between bulky sphingolipids, and thus mation of large ceramide-enriched membrane domains. extraction of cholesterol results in destruction of these These domains serve the spatial and temporal organi- small membrane domains (Xu et al., 2001; Megha et al., zation of receptors and signaling molecules. The acid 2006). It was suggested that this lateral organization of sphingomyelinase-ceramide system plays an important the cell membrane resulted in the formation of sphingo- role in the infection of mammalian host cells with lipid- and cholesterol-enriched membrane domains, also bacterial pathogens such as Neisseria gonorrhoeae, named rafts (Simons and Ikonen, 1997). However, at Escherichia coli, Staphylococcus aureus, Listeria mono- present only indirect evidence exists to support the pres- cytogenes, Salmonella typhimurium and Pseudomonas ence of rafts in cells under in vivo conditions, for instance aeruginosa. Ceramide and ceramide-enriched membrane at physiological temperature, and the concept of rafts is platforms are also involved in the induction of apoptosis still somewhat controversial. in infected cells, such as in epithelial and endothelial cells after infection with Pseudomonas aeruginosa and Staphy- Sphingomyelinases and ceramide-enriched lococcus aureus, respectively. Finally, ceramide-enriched membrane domains membrane platforms are critical regulators of the release Ceramide-enriched membrane platforms are membrane of pro-inflammatory cytokines upon infection. The domains that are either generated from rafts or inde- diverse functions of ceramide in bacterial infections pendent of rafts (Kolesnick et al., 2000; Gulbins and suggest that ceramide and ceramide-enriched membrane Kolesnick, 2003). Here we focus on the generation of domains are key players in host responses to many path- ceramide by the activity of sphingomyelinases that ogens and thus are potential novel targets to treat hydrolyze sphingomyelin, the most prevalent sphingolipid infections. in the cell membrane (Quintern et al., 1989; Calderon and Keywords: acid sphingomyelinase; bacteria; ceramide; DeVries, 1997). Sphingomyelin is an amide ester of a cystic fibrosis; virus. hydrophobic ceramide moiety and a hydrophilic phos- phorylcholine headgroup. Ceramide is composed of a D-erythro-sphingosine and a fatty acid containing 2–32 Ceramide, microdomains and ceramide- carbon atoms in the acyl chain (Hakomori, 1983; Rabio- enriched membrane platforms net et al., 2008). Sphingomyelinases are characterized by their pH optimum, with acid, neutral and alkaline sphin- Biophysical aspects of membrane domains gomyelinase identified (Quintern et al., 1989; Chatterjee et al., 1999; Duan et al., 2003). Since sphingomyelin is In 1972, Singer and Nicolson suggested a random dis- predominantly present in the outer leaflet of the cell tribution of lipids and proteins in the cell membrane membrane (Calderon and DeVries, 1997), the hydrolysis named the fluid mosaic model of the cell membrane of sphingomyelin results in ceramide-enriched mem- (Singer and Nicolson, 1972), although they did not rule brane domains that are primarily in the outer leaflet of the out some ordering of the membrane. This model also cell membrane, or in general in anti-cytoplasmic leaflets suggested that the membrane exists in a fluid-disordered of cellular membranes. It remains to be determined status. However, biophysical studies in the last 15 years whether a relevant exchange of ceramide between the revised this model and suggested the formation of small outer and inner leaflets of the cell membrane occurs domains in the cell membrane that exist in a liquid- under in vivo conditions, but Johnston and Johnston

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(2008) demonstrated that hydrolysis of sphingomyelin to resulted in the rapid formation of large ceramide-enriched ceramide in a bilayer promotes flip-flop of ceramide, membrane platforms that were visualized by fluores- which has also been shown to occur in erythrocytes cence microscopy of giant vesicles (Holopainen et al., (Lopez-Montero et al., 2005). Several studies demon- 1998; Nurminen et al., 2002). These platforms sponta- strated that ceramide in the outer leaflet of the cell neously formed small vesicles that budded into giant membrane is formed by hydrolysis of sphingomyelin by vesicles. It should be noted that the formation of these the activity of acid sphingomyelinase, which is present in ceramide-enriched membrane platforms did not require vesicles, mostly lysosomes and secretory lysosomes, in any staining with antibodies and, most importantly, did mammalian cells (Schissel et al., 1996, 1998; Grassme not require any other cellular components such as the et al., 2001a). The sorting of acid sphingomyelinase cytoskeleton. These studies suggest that ceramide- between different compartments seems to be controlled enriched membrane platforms spontaneously form in cell by its glycosylation pattern, which determines transport membranes owing to the biophysical properties of cera- of mannose-6-phosphate-modified acid sphingomyeli- mide, although these data certainly do not exclude the nase into lysosomes, whereas complex-glycosylated possibility that in vivo formation of ceramide-enriched sphingomyelinase is secreted upon stimulation, for membrane domains also involves the cytoskeleton and/ instance via interleukin (IL)-1 receptors (Schissel et al., or other components of the cell. In vitro studies also indi- 1996, 1998). Several studies demonstrated that stimuli cate that even small amounts of ceramide incorporated such as CD95, DR5 and CD40 or infection with some into rafts transfer fluid phospholipid layers into a gel-like pathogenic bacteria and viruses mobilize intracellular phase (Veiga et al., 1999). Atomic force microscopy vesicles, a process that results in exposure of acid sphin- studies confirmed the phase separation in artificial gomyelinase on the outer leaflet of the cell membrane C16-ceramide-enriched glycerol-phospholipid/cholesterol (Grassme et al., 2001b, 2002, 2003a,b, 2005; Cremesti membranes (ten Grotenhuis et al., 1996). et al., 2001; Dumitru and Gulbins, 2006). The activity of A recent study (Johnston and Johnston, 2008) used a the acid sphingomyelinase results in hydrolysis of sphin- combination of atomic force microscopy and total inter- gomyelin and generation of ceramide. Ceramide exhibits nal reflection fluorescence to directly visualize clustering special biophysical properties that mediate association of of small membrane domains into larger domains in arti- ceramide molecules and the formation of small cera- ficial membranes composed of 1,2-dioleoyl-sn-glycero- mide-enriched microdomains (Holopainen et al., 1998; 3-phosphocholine/sphingomyelin/cholesterol mixtures Kolesnick et al., 2000; Nurminen et al., 2002). These upon treatment with Bacillus cereus sphingomyelinase to microdomains spontaneously fuse to larger domains and generate ceramide or incubation with C -ceramide. finally large ceramide-enriched membrane domains, also 16 Atomic force microscopy permits investigation of the named platforms, that can reach a width of up to 5 mm nanoscale organization of a membrane, whereas total (Gulbins and Kolesnick, 2003). Accumulation of ceramide internal reflection fluorescence permits improved analysis within rafts results in a change in the biophysical prop- of the dynamics of distinct membrane domains. The erties of these domains. Thus, the formation of ceramide studies revealed that enzymatic hydrolysis of sphingo- increases the hydrophobicity and changes the fluidity of myelin to ceramide in model membranes resulted in very membrane domains, since ceramide molecules are tightly packed and form highly ordered microdomains. Fur- rapid reorganization of the membrane, clustering of thermore, ceramide accumulation may change the domains and the formation of larger distinct domains that membrane height and very likely results in a change in presumably correspond to ceramide-enriched membrane raft composition, since ceramide molecules may com- domains. The addition of C16-ceramide also resulted in pete with cholesterol within rafts (Megha and London, the formation of larger domains in these bilayers, albeit 2004). with different kinetics and less impact on membrane organization. Although it is impossible to directly com- Visualization of ceramide-enriched membrane pare ceramide-enriched membrane domains in cellular domains membranes with the coalescence of small to larger domains observed in these experiments, the studies Changes in the plasma membrane upon generation of suggest many similarities between these membrane ceramide might be critical for the signaling function of domains. Furthermore, the studies on isolated mem- ceramide. Ceramide-enriched membrane platforms were branes imply that the amount of ceramide generated in demonstrated by several methods in vivo and in vitro. a membrane critically determines the reorganization of First, several groups demonstrated the formation of cera- membranes into different types of distinct domains. mide-enriched membrane platforms upon stimulation with CD95, DR5 and CD40 and infection with P. aeruginosa or rhinoviruses in lymphocytes, epithelial cells or Functions of ceramide-enriched membrane fibroblasts (Grassme et al., 2001b, 2002, 2003a,b, 2005; platforms Cremesti et al., 2001; Dumitru and Gulbins, 2006). These studies used fluorescence and electron microscopy to Ceramide-enriched membrane domains seem to function visualize the domains and translocation of acid sphin- as a sorting unit in the cell that permits the spatial and gomyelinase onto the extracellular leaflet of the plasma temporal organization of receptors and signaling mole- membrane. Studies on unilamellar vesicles composed of cules. Thus, it was shown that several receptors phosphatidylcholine/sphingomyelin demonstrated that and signaling molecules aggregate and cluster in cera- treatment with bead-immobilized sphingomyelinase mide-enriched membrane platforms upon stimulation or Article in press - uncorrected proof

Ceramide in bacterial infections and cystic fibrosis 1373 infection with pathogens, respectively. The receptors Ceramide and bacterial infections include CD95 (Grassme et al., 2001b; Cremesti et al., 2001), CD40 (Grassme et al., 2002), DR5 (Dumitru and Acid sphingomyelinase and ceramide were shown to play Gulbins, 2006), FcgRII (Shakor et al., 2004), CD14 (Pfeif- a central role in infection with several pathogens and in fer et al., 2001), CFTR (Kowalski and Pier, 2004) and sig- several aspects of host-pathogen interactions. naling molecules such as NADPH-oxidase (Zhang et al., 2001a, 2003), caspase 8 (Eramo et al., 2004) and Kv1.3 Neisseria gonorrhoeae (Bock et al., 2003). In particular, stress stimuli such as g- irradiation (Santana et al., 1996), UV light (Zhang et al., Initial studies on the role of ceramide in bacterial infection 2001b; Rotolo et al., 2005; Kashkar et al., 2005), platelet- investigated the role of acid sphingomyelinase and cera- activating factor (Go¨ ggel et al., 2004), cisplatin (Lacour mide in N. gonorrhoeae infection of human epithelial and et al., 2004; Rebillard et al., 2007) and Cu2q treatment myeloid cells (Grassme et al., 1997; Hauck et al., 2000). (Lang et al., 2007) also trigger the formation of large cera- These studies revealed activation of acid sphingomyeli- mide-enriched membrane platforms, but it is unknown nase and the formation of ceramide on infection of which molecules in these platforms mediate their cellular human cells with N. gonorrhoeae (Figure 1A). The signi- effects. ficance of these findings was indicated by inhibition of The exact molecular mechanisms that mediate clustering of receptors and signaling molecules within or at ceramide-enriched membrane platforms are unknown. The generation of ceramide in rafts dramatically alters the composition and decreases the fluidity of rafts that may trap activated receptors, associated signaling molecules and/or farnesylated/geranylated intracellular molecules preferentially interacting with ceramide-enriched domains. Other molecules may be excluded from these domains owing to biophysical and energetic reasons. In addition, the generation of ceramide may alter the height of the plasma membrane, resulting in the trapping of molecules that fit into these domains better than in other domains of the plasma membrane (Bock and Gul- bins, 2002). A change in membrane height on ceramide generation was recently shown in vitro in studies on artificial membranes treated with recombinant sphingomyelinase (Johnston and Johnston, 2008), supporting the notion that a change in receptor conformation on ligand binding may promote preferential interaction with ceramide-enriched membrane domains. Further studies (Chiantia et al., 2008) demonstrated that proteins and lipids such as placental alkaline phosphatase and the ganglioside GM1 are enriched in domains with high affinity to the liquid-ordered phase, whereas proteins and lipids with low affinity to the liquid-ordered phase are excluded. Ceramide generation in membranes dramatically decreased the planar diffusion of these proteins, which resulted in trapping and finally clustering of a protein in ceramide-enriched domains. It should be noted that the spatial distribution of ceramide generated within cells, for instance by neutral sphingomyelinase or intra-vesicular acid sphingomyelinase, requires definition. Ceramide generated by these enzymes might also reorganize intracellular membranes, but it may also interact with and regulate enzymes such as cathepsin D (Heinrich et al., 1999), phospholipase A2 (Huwiler et al., 2001), kinase suppressor of Ras (KSR) (Zhang et al., 1997), ceramide-activated protein serine- Figure 1 Functions of acid sphingomyelinase and ceramide threonine phosphatases (CAPP) (Dobrowsky and Han- during bacterial infections. nun, 1993), protein kinase C isoforms (Muller et al., 1995) Acid sphingomyelinase functions in the outer leaflet of the cell and c-Raf-1 (Yao et al., 1995). Cathepsin D is activated membrane to mediate internalization of pathogens (A) and in in lysosomes by the activation of acid sphingomyelinase lysosomes to mediate fusion of phagosomes with lysosomes (B). Acid sphingomyelinase also seems to be involved in matu- upon treatment of cells with tumor necrosis factor. Acti- ration of phagolysosomes (B). Finally, surface and intracellular vation and/or interaction with ceramide results in trans- ceramide generated by acid sphingomyelinase activity is impor- location of the protein into the cytoplasm and induction tant for induction of cell death upon infection with some patho- of cell death (Heinrich et al., 2004). gens (C). Article in press - uncorrected proof

1374 H. Grassme´ et al.

N. gonorrhoeae uptake by either genetic deficiency or 2001). However, the molecular mechanisms by which pharmacological inhibition of acid sphingomyelinase. ceramide and acid sphingomyelinase are involved in S. Subsequent studies demonstrated that CEACAM pro- aureus infections are still unknown. teins are the receptors for internalization of N. gonorrhoeae (Bos et al., 1997). Further studies demonstrated Salmonella typhimurium that at least some CEACAM receptors associate with and cluster in rafts upon infection with N. gonorrhoeae Infection of macrophages with S. typhimurium results in (Muenzner et al., 2008). Studies with mutant CEACAM uptake and intracellular killing of the bacteria (McCollister receptors revealed that the transmembrane domain of et al., 2007). Intracellular killing of the pathogen was CEACAM 1 is responsible for its association with lipid defective in acid sphingomyelinase-deficient macro- rafts (Muenzner et al., 2008), reminiscent of the findings phages, although this did not correlate with acid sphin- for CD40 discussed above. gomyelinase activation and was rather dependent on constitutive expression of the enzyme (Figure 1B). On the other hand, macrophages responded by secreting acid Listeria monocytogenes sphingomyelinase and an attractive hypothesis is that the Studies with L. monocytogenes demonstrated that acid secreted sphingomyelinase is internalized together with sphingomyelinase-deficient mice are approximately 100- the pathogen to mediate endosome-lysosome fusion, fold more sensitive to infection with this bacterium than which seems to be impaired in acid sphingomyelinase- wild-type mice (Utermo¨ hlen et al., 2003). Acid sphingo- deficient macrophages. myelinase-deficient macrophages internalized L. monocytogenes, in contrast to findings for N. gonorrhoeae. Escherichia coli/LPS Thus, the role of ceramide in bacterial internalization is Infection of immature dendritic cells with E. coli was very likely cell-type-specific, with different mechanisms shown to activate acid sphingomyelinase, trigger cera- for bacterial uptake in macrophages and epithelial cells. mide production and induce apoptosis (Figure 1C), Acid sphingomyelinase-deficient mice displayed interfer- effects that were all mimicked by lipopolysaccharide on g serum levels similar to those in wild-type mice. Like- (LPS) (Falcone et al., 2004). It was previously shown that wise, secretion of IL-1 and -6 from macrophages on LPS triggers endothelial cell death dependent on acid infection with L. monocytogenes was not dependent on sphingomyelinase expression (Haimovitz-Friedman et al., expression of acid sphingomyelinase. Although NO pro- 1997). Apoptosis of immature dendritic cells was pre- duction was also normal in acid sphingomyelinase-defi- vented by functional inhibition of acid sphingomyelinase cient macrophages on infection, macrophages lacking using imipramine, which induces acid sphingomyelinase acid sphingomyelinase were unable to kill the bacteria degradation in lysosomes, probably by interfering with and thus restrict growth of the bacteria caused by binding of the enzyme to the lysosomal membrane and delayed maturation of phagosomes into lysosomes subsequent proteolysis (Hurwitz et al., 1994). (Figure 1B) (Schramm et al., 2008). In wild-type macrophages, the phagosome rapidly fused with the lysosome Mycobacteria to form a phago-lysosome and to kill and digest bacteria. In contrast, phagosomes in acid sphingomyelinase-defi- Recent studies (Utermo¨ hlen et al., 2008) demonstrated cient macrophages were still positive for late endosomal that acid sphingomyelinase-deficient mice were resistant markers, even 2 h after infection, and lacked typical lyso- to Mycobacterium avium and survive infection, whereas somal proteins. Furthermore, transfer of the lysosomal wild-type mice died after approximately 80 days. Acid matrix into the lysosome and thus full fusion of the two sphingomyelinase-deficient mice were able to control the vesicles seemed to be inhibited by deficiency of acid bacteria within small granulomas, whereas M. avium sphingomyelinase in L. monocytogenes-infected macro- formed large infiltrates with hypertrophic macrophages phages. Once fused, phagolysosomes in acid sphingo- containing masses of bacteria in wild-type mice. Giant myelinase-deficient macrophages contained lower cells typical of mycobacterial infection in wild-type mice activities of bacteriocidal proteases. The impaired phago- were absent in acid sphingomyelinase-deficient mice. At lysosome maturation in acid sphingomyelinase-deficient present, the exact mechanisms that mediate the high macrophages correlated with development of sepsis and resistance of acid sphingomyelinase-deficient mice to greatly increased mortality of acid sphingomyelinase- M. avium infections are unknown. deficient mice. These data suggest a novel function of acid sphingomyelinase in infectious biology, i.e., control Pseudomonas aeruginosa of the maturation and fusion of intracellular phagosomes with lysosomes. Further studies revealed a critical role of acid sphingomyelinase and ceramide-enriched membrane platforms Staphylococcus aureus in the infection of epithelial cells with P. aeruginosa. P. aeruginosa infections are very important in patients with Whereas acid sphingomyelinase and ceramide mediate systemic bacterial infections, ventilator-associated pneu- internalization of N. gonorrhoeae into epithelial cells and monia and patients with cystic fibrosis. The clinical sig- fusion of intracellular vesicles in L. monocytogenes- nificance is highlighted by the fact that 40% of deaths in infected macrophages, the enzyme was shown to be patients with ventilator-associated pneumonia are involved in the induction of cell death on infection of caused by P. aeruginosa (Crouch Brewer et al., 1996). endothelial cells with S. aureus (Figure 1C) (Esen et al., Infection of mammalian cells with this pathogen triggers Article in press - uncorrected proof

Ceramide in bacterial infections and cystic fibrosis 1375 rapid activation of acid sphingomyelinase that correlates The role of ceramide-enriched membrane platforms in with translocation of the enzyme onto the extracellular the control of cytokine release (Grassme et al., 2003b) is leaflet of the cell membrane to the bacterial infection site much less defined and it is unknown whether these (Grassme et al., 2003b). The activity of surface sphin- domains directly regulate IL-1 synthesis, for instance, via gomyelinase results in ceramide hydrolysis and the for- activation of caspase 1, or whether ceramide-enriched mation of ceramide-enriched membrane platforms that membrane platforms indirectly regulate cytokine release, seem to be generated from rafts, since they are also pos- for instance, by induction of cell death or internalization. itive for GM1, a typical raft marker. Ceramide-enriched Experiments using cells or mice genetically deficient membranes were shown to be critical for the internali- for acid sphingomyelinase or treated with drugs that dis- zation of P. aeruginosa into epithelial cells and fibro- rupt rafts by interference with the cholesterol metabo- blasts, the induction of death of infected cells, and lism, i.e., b-cyclodextrin, nystatin or filipin, confirmed the controlled release of cytokines (Grassme et al., 2003b). critical role of ceramide-enriched membrane platforms Ceramide-enriched membrane platforms may regulate and rafts in bacterial elimination in vitro and in vivo on internalization by clustering the cystic fibrosis conduc- pulmonary infection with P. aeruginosa (Grassme et al., tance regulator (Cftr) or ganglioside asialo-GM1, which 2003b; Kowalski and Pier, 2004). The inability of acid were shown to function as receptors for P. aeruginosa sphingomyelinase-deficient mice to eliminate bacteria involved in internalization of the bacteria (Saiman and from the lung might be caused by a defect in bacteria Prince, 1993; Pier et al., 1996). The notion that ceramide- internalization, whereas overwhelming lung inflammation enriched membrane platforms cluster Cftr is consistent in these animals on P. aeruginosa infection might be with findings that Cftr moves into the raft fraction after linked to a defect in apoptosis in epithelial cells, since infection and that destruction of rafts prevents internali- apoptosis was shown to down-regulate and inhibit zation of P. aeruginosa into pulmonary epithelial cells inflammation (Henson et al., 2001). (Kowalski and Pier, 2004). Raft proteins that seem to be Most of the events described above in internalization critical for internalization and/or induction of cell death and induction of cell death require expression of the type on P. aeruginosa infection include Src-like tyrosine III secretion system in P. aeruginosa (Galan and Collmer, kinases and the major vault protein (MVP) (Kannan et al., 1999). P. aeruginosa mutants that lack this secretion 2006, 2008; Kowalski et al., 2007; Dudez et al., 2008). system fail to trigger death and to invade epithelial cells Another Src-like kinase, Lyn, associates with rafts and (Jendrossek et al., 2003). Although it is unknown whether mediates activation of phosphatidyl-inositol-3-kinase ceramide-enriched membrane platforms play a role in the and Akt, as well as a respiratory burst, on P. aeruginosa transfer of bacterial proteins into mammalian host cells infection (Kannan et al., 2006, 2008). Destruction of rafts via the type III secretion system, it is an interesting prevented these events. Rafts and Lyn are critical for the speculation that the specific environment in ceramide- internalization of P. aeruginosa, and induction of apop- enriched membrane platforms permits adhesion to and/ tosis and inflammatory cytokines, as observed in Lyn- or penetration of the multi-protein complex comprising deficient mast cells (Kannan et al., 2006), highlighting the this bacterial secretion system. role of rafts in P. aeruginosa infections. MVP is another The role of rafts in P. aeruginosa internalization is not protein recruited into rafts upon infection with P. aerugi- restricted to the lung, but also applies to other tissues. nosa (Kowalski et al., 2007). Vault proteins may have a Thus, raft destruction also prevented infection of corneal function in nucleo-cytoplasmic transport, whereas a epithelial cells with P. aeruginosa, which is commonly function in infectious biology was previously unknown. involved in contact lens-mediated infections of the eye MVP formed a cluster at the site of the bacterial infection (Yamamoto et al., 2005; Zaidi et al., 2008). These rafts very similar to ceramide-enriched membrane platforms. also seem to contain Cftr that might function as a recep- Infection of MVP-deficient mice revealed a defect in inter- tor for P. aeruginosa in the cornea (Zaidi et al., 2008). nalization of the pathogens into lung epithelial cells and Although very likely, it is still unknown whether ceramide an increase in mortality. The exact function of MVP in the and acid sphingomyelinase are involved in the infection infection process is unknown, but it might be a scaffold of corneal epithelial cells with P. aeruginosa in vivo. protein contributing to the organization of raft domains at the infection site. Receptors such as Cftr may also activate infected cells via clustered rafts and ceramide-enriched membrane Ceramide in cystic fibrosis platforms. Thus, it was shown that destruction of rafts prevented NF-kB nuclear translocation in respiratory Cystic fibrosis is the most common autosomal recessive epithelial cells on P. aeruginosa infection (Kowalski and disorder, at least in Western countries, with an incidence Pier, 2004). of 1 in 2500 births. It is caused by mutations in Cftr Ceramide-enriched membrane platforms cluster CD95, (Riordan et al., 1989) and characterized by chronic lung which was previously shown to be critically involved in inflammation, and frequent and chronic infections of the induction of cell death on infection of epithelial cells with lung with P. aeruginosa, as well as S. aureus, Burkhol- P. aeruginosa (Grassme et al., 2000). Consistent with the deria cepacia and Haemophilus influenzae. Although notion of a role of ceramide-enriched membrane plat- patients also suffer from severe gastrointestinal and forms in apoptosis is the observation that raft destruction hepatic symptoms, pulmonary inflammation and infec- prevents cellular apoptosis in vitro and in vivo (Grassme tion determine the course of the disease and the low life et al., 2003b; Kowalski and Pier, 2004). expectancy of patients. Article in press - uncorrected proof

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At present it is unknown how Cftr deficiency results in ceptibility of Cftr-deficient mice, and normalization of increased pulmonary inflammation and the high suscep- ceramide prevented both DNA deposition and infection tibility of patients to bacterial infections of the lung. It was susceptibility in airways of Cftr-deficient mice. shown that even non-infected Cftr-deficient mice exhibit Ceramide might also be critically involved in chronic increased IL-8 concentrations in the respiratory tract inflammation of the lung, a second highlight of cystic (Zahm et al., 1997; Joseph et al., 2005), and that aborted fibrosis. Thus, even non-infected Cftr-deficient mice embryos with cystic fibrosis already display signs of showed increased numbers of neutrophils and macro- inflammation in the lung (Verhaeghe et al., 2007). The phages in the lung, as well as accumulation of the pro- chronic inflammation in cystic fibrosis patients might be inflammatory mediators IL-1 and -8. These changes that involved in their susceptibility to P. aeruginosa infections, indicate chronic inflammation were corrected upon nor- although this hypothesis is not proven. malization of pulmonary ceramide concentrations. At We recently proposed a novel patho-physiological present, it is unknown how ceramide triggers chronic concept in cystic fibrosis (Teichgra¨ ber et al., 2008). Cftr inflammation in the lung. Furthermore, it needs to be deficiency results in an increase in pH in vesicles that defined whether chronic inflammation and/or chronic contain acid sphingomyelinase and acid ceramidase. It bacterial infection causes pulmonary fibrosis in cystic should be noted that the role of Cftr in regulation of fibrosis and whether ceramide plays a role in this vesicular pH is controversial, but different findings might process. be explained by the use of different cells and, more On first view it might be confusing that acid sphingo- importantly, the analysis of different vesicle populations myelinase activation and subsequent ceramide formation that express different sets of ion channels and trans- are required for defense against P. aeruginosa in wild- porters to regulate pH (Barasch et al., 1991; Dunn et al., type mice, whereas a constitutive increase in ceramide 1994; Di et al., 2006). However, alkalization of acid sphin- in the lung of cystic fibrosis mice and patients sensitizes gomyelinase- and acid ceramidase-positive vesicles in to P. aeruginosa infection. However, it is obvious that epithelial cells results in an imbalance in the enzymes: increased activity in a system normally used for infection whereas an increase in pH to 6.0 results in almost inac- defense results in biological changes that finally sensitize tive acid ceramidase (in fact at this pH the enzyme has the lung to infection, for instance, by DNA deposition in reverse activity, producing ceramide from sphingosine), bronchi, although these changes are primarily unrelated acid sphingomyelinase activity is only decreased by to any infection. In cystic fibrosis patients, P. aeruginosa 30–40% (He et al., 2003; Teichgra¨ ber et al., 2008). The infection may even result in a vicious cycle by further imbalance in the two enzymes results in ceramide accu- increasing the activity of acid sphingomyelinase on top mulation in Cftr-deficient cells. Age-dependent ceramide of an already increased ceramide concentration, whereas accumulation was detected in respiratory epithelial cells transient release of ceramide in healthy individuals seems and in submucosal glands of two different strains of unin- to be important for elimination of the bacteria. This situa- fected Cftr-deficient mice and in lung specimens and tion is very reminiscent of other highly organized biolog- nasal epithelial cells from patients with cystic fibrosis. ical systems such as the coagulation system, in which The latter indicates that relevant ceramide accumulation normal levels prevent bleeding, whereas activities that also occurs in humans. Inhibition of acid sphingomyeli- are increased or too low lead to spontaneous coagulation nase in heterozygous Cftr-/-/Smpd1q/- mice or by phar- or bleeding, respectively. macological inhibition with amitriptyline (Hurwitz et al., 1994) decreased ceramide concentrations in the lungs of Cftr-deficient mice and normalized pulmonary ceramide Perspectives concentrations in these animals (Teichgra¨ ber et al., 2008). Amitriptyline does not directly inhibit acid sphingomyeli- The studies described in the present review suggest that nase, but induces degradation of the enzyme in acidic ceramide and acid sphingomyelinase are potential tar- compartments, as described above for imipramine gets for the treatment of some bacterial infections and (Hurwitz et al., 1994). Amitriptyline also decreases acid diseases that are critically associated with chronic infec- ceramidase expression (Elojeimy et al., 2006), an effect tions, such as cystic fibrosis. However, since ceramide that is, however, almost irrelevant in a scenario with very seems to act via changes in biological membranes result- low acid ceramidase activity. ing in temporal and spatial organization of receptors and Changes in cholesterol metabolism in Cftr-deficient signaling molecules, there is no simple approach to tar- mice affect ceramide concentrations in lungs of these get ceramide in infections. Targeting different pools of mice (Teichgra¨ ber et al., 2008). Severely increased cho- acid sphingomyelinase, such as surface acid sphingo- lesterol concentrations were previously shown to inhibit myelinase or lysosomal acid sphingomyelinase, might acid sphingomyelinase (Bhuvaneswaran et al., 1985), open a possibility to achieve specific modification of the similar to the phenotype of Niemann-Pick type C. acid sphingomyelinase/ceramide system during different Although the molecular details still require definition, bacterial infections. A different scenario applies for the studies further demonstrated that accumulated cera- P. aeruginosa infections: a sufficient response to P. aeru- mide triggers death of respiratory epithelial cells in Cftr- ginosa requires expression of acid sphingomyelinase and deficient bronchi, resulting in the release of dead cells acute formation of ceramide, whereas constitutively into the bronchial lumen, DNA release, and finally the increased production of ceramide such as in cystic fibro- deposition of DNA clumps and plugs in small bronchi. sis results in increased susceptibility to bacterial infec- DNA in the airways is critical for the high infection sus- tions. Thus, treatment of cystic fibrosis using blockers of Article in press - uncorrected proof

Ceramide in bacterial infections and cystic fibrosis 1377 acid sphingomyelinase must be personalized and titrated Duan, R.D., Bergman, T., Xu, N., Wu, J., Cheng, Y., Duan, J., to retain sufficient acid sphingomyelinase activity. This is Nelander, S., Palmberg, C., and Nilsson, A. (2003). Identifi- reminiscent of the administration of many highly efficient cation of human intestinal alkaline sphingomyelinase as a novel ecto-enzyme related to the nucleotide phosphodies- drugs, such as drugs that control blood coagulation, terase family. J. Biol. Chem. 278, 38528–28536. which must also be tightly controlled to balance between Dudez, T., Borot, F., Huang, S., Kwak, B.R., Bacchetta, M., coagulation and spontaneous bleeding. However, since Oliero, M., Stanton, B.A., and Chanson, M. (2008). CFTR in inhibitors of acid sphingomyelinase usually do not com- lipid raft-TNFR1 complex modulates gap junctional intercel- pletely block the enzyme, it might be possible to treat lular communication and IL-8 secretion. Biochim. Biophys. cystic fibrosis patients with inhibitors of acid sphingo- Acta 1783, 779–788. myelinase. First clinical studies are already under way to Dumitru, C.A., and Gulbins, E. (2006). TRAIL activates acid sphingomyelinase via a redox mechanism and releases cera- test this novel concept in cystic fibrosis patients. mide to trigger apoptosis. Oncogene 25, 5612–5625. Dunn, K.W., Park, J., Semrad, C.E., Gelman, D.L., Shevell, T., and McGraw, T.E. (1994). Regulation of endocytic trafficking Acknowledgments and acidification are independent of the cystic fibrosis transmembrane regulator. J. Biol. Chem. 269, 5336–5345. 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