Newborn Screening for Acid Sphingomyelinase Deficiency: Prevalence and Genotypic Findings in Italy
Vincenza GragnanielloChiara CazzorlaDaniela GueraldiChristian LoroElena PorcùLeonardo SalviatiAlessandro P. BurlinaAlberto Burlina
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Acid sphingomyelinase deficiency (ASMD) is a rare lysosomal storage disorder with a broad clinical spectrum. Early diagnosis and initiation of treatment are crucial for improving outcomes, yet the disease often goes undiagnosed due to its rarity and phenotypic heterogeneity. This study aims to evaluate the feasibility and disease incidence of newborn screening (NBS) for ASMD in Italy. Dried blood spot samples from 275,011 newborns were collected between 2015 and 2024 at the Regional Center for Expanded NBS in Padua. Acid sphingomyelinase activity was assayed using tandem mass spectrometry. Deidentified samples with reduced enzyme activity underwent second-tier testing with LysoSM quantification and SMPD1 gene analysis. Two samples were identified with reduced sphingomyelinase activity and elevated LysoSM levels. Both carried two SMPD1 variants, suggesting a diagnosis of ASMD. Molecular findings included novel and previously reported variants, some of uncertain significance. The overall incidence was 1 in 137,506 newborns and the PPV was 100%. This study demonstrates the feasibility of NBS for ASMD in Italy and provides evidence of a higher disease incidence than clinically reported, suggesting ASMD is an underdiagnosed condition. Optimized screening algorithms and second-tier biomarker testing can enhance the accuracy of NBS for ASMD. The long-term follow-up of identified cases is necessary for genotype–phenotype correlation and improving patient management.Keywords:
Acid sphingomyelinase
In this review, we summarize implications of the acid sphingomyelinase/ ceramide system in ischemic stroke. Acid sphingomyelinase catalyzes the formation of the bioactive sphingolipid ceramide which coalesces into membrane platforms and has a pivotal role in inflammation, cell signaling and death. Cerebral ischemia increases acid sphingomyelinase activity and elevates brain ceramide levels, which has been associated with the exacerbation of ischemic injury and deterioration of stroke outcome. In view of the fact that lowering acid sphingomyelinase activity and ceramide level was shown to protect against ischemic injury and ameliorate neurological deficits, the acid sphingomyelinase/ ceramide system might represent a promising target for stroke therapies.
Acid sphingomyelinase
Sphingolipid
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Phosphorylcholine
Acid sphingomyelinase
Sphingolipid
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Niemann–Pick disease
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Sphingomyelin located in the plasma membrane is hydrolyzed by enzyme-neutral, Mg++-dependent sphingomyelinase, whereas sphingomyelin located in endosomes and lysosomes is hydrolyzed by acid, Zn++-independent sphingomyelinase. In both cases, ceramide and phosphocholine are the products of sphingomyelin hydrolysis. Ceramide is recognized to be the second messenger on the sphingomyelin signaling pathway [1]. Different cell types, such as human and murine macrophages, human skin fibroblasts and human monocytes, have been shown to secrete Zn++-dependent acid sphingomyelinase, and the enzyme has been named secretory sphingomyelinase [4]. Secretory sphingomyelinase is also the product of the acid sphingomyelinase gene [4]. It has also been shown that human vascular endothelial cells are a very rich source of the enzyme. They secrete the enzyme both apically and basolaterally, thus contributing to serum and arterial wall sphingomyelinase. The acid sphingomyelinase secreted by the endothelial cells is partially Zn++-independent [2]. Two forms of acid sphingomyelinase have been shown to be present in the serum: Zn++-dependent and Zn++-independent [6] [7]. The secretory sphingomyelinase is claimed to play an important role in the development of atherosclerosis. Though the optimum pH for the enzyme is around 5, it hydrolyzes sphingomyelin present in atherogenic low density lipoproteins (LDL) at a neutral pH. Hydrolysis of sphingomyelin in this lipoprotein fraction results in its subendothelial aggregation [3] [5]. Aggregated LDL in turn, induce macrophage cell formation [8]. In the light of this role of the secretory sphingomyelinase, it seems important to recognize factors regulating the enzyme's activity in the arterial bed. Inflammatory cytokines such as interleukin-1β and interferon-γ have been shown to increase secretion of the enzyme in vitro [2]. Serum enzyme activity increases considerably during acute systemic inflammation in mice [9]. The activities of the two enzymes have been shown to remain stable in serum of patients with metabolic bone disease [6]. In patients with hemophagocytic lymphohistiocytosis, the activity of Zn++-dependent sphingomyelinase was elevated 10-20-fold whereas the activity of Zn++-independent form of the enzyme was elevated only by around 2-fold [10]. Diabetes is a disease that accelerates atherosclerosis development. The aim of the present study was to examine the activity of secretory sphingomyelinase in the serum of patients with type 2 diabetes. Activity of the Zn++-dependent form of the enzyme was found to be markedly elevated in the patient group compared to control subjects.
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Dear Editor, Acid sphingomyelinase deficiency (ASMD), more commonly classified under the subtypes Niemann-Pick disease type A (NPD A) and Niemann-Pick disease type B (NPD B), is a primary lysosomal hydrolase defect that develops due to the deficiency of acid sphingomyelinase (ASM). When there is a lack of this enzyme, sphingomyelin builds up in the cells inducing damage to the tissues1. In a study conducted by McGovern et al2, 18 out of 103 patients with Niemann-Pick disease (NPD) died due to complications such as pneumonia, liver failure, and hemorrhage. Despite the alarming morbidity, supportive measures and lifestyle changes were the main conduits to managing this disease until recently. ASMD is caused by the deficiency of the enzyme ASM, which is encoded by the gene sphingomyelin phosphodiesterase 1. ASM-deficient NPD is passed down as an autosomal recessive trait, but several reports are present where it was present in heterozygous individuals as well1. ASM, found in lysosomes, takes part in membrane degradation. It primarily acts on sphingomyelin, which is a major constituent of the cell membrane and breaks it into ceramide. Because of ASMD, there is a build-up of sphingomyelin, procuring huge lipid-laden foam cells which results in the impairment of the proper functioning of organs. NPD A is characterized by a severe deficit of ASM. The accumulation of sphingomyelin in neurons causes neurodegeneration and typically death by 3 years of age. The organs most commonly affected are the liver, spleen, and lungs, culminating in hepatosplenomegaly and respiratory crisis. NPD B is generally considered the milder form of NPD A. By comparison, patients with the type B variant have organomegaly but no neurological manifestations1. Patients with NPD are recommended lifestyle interventions to suppress further exacerbation of the disease. Some of the current guidelines for the treatment of NPD are caution in contact sports to reduce the chances of spleen rupture, regular hepatic assessments, complete blood counts, chest radiographs for proper lung function, periodic testing for peripheral neuropathy and neuropsychology, ECGs, and lipid profiles to avoid heart complications3. As of date, no disease-specific drugs have been developed to cure NPD. However, enzyme replacement therapy with the recombinant human ASM, Olipudase Alfa-rpcp, under the brand name XENPOZYME, has been approved to treat ASMD. Xenpozyme is a recombinant protein that is administered intravenously. It works by replacing the defective enzyme ASM through enzyme replacement therapy and performing its function alternatively. Thus, it lessens the build-up of sphingomyelin just as the original enzyme would. The data obtained from the ASCEND and ASCEND-PEDS clinical trials, where people from different age groups having NPD A or NPD B were evaluated, indicates the promising results of the drug. In the ASCEND clinical trials, 36 patients were randomized to receive either Xenpozyme or a placebo for 52 weeks. Patients treated with Xenpozyme had a 22% improvement in lung’s diffusing capacity for carbon monoxide (DLco) and had a reduction in spleen size by 39.5%, compared with a 3% DLco improvement and 0.5% increase in spleen size for the patients in the placebo group4. In the ASCEND-Peds trial, 20 pediatric patients received Xenpozyme. After 52 weeks, there was a 33% increase in the DLco and a decrease in spleen size by 49% compared with baseline4. In another study, five adult patients with chronic ASMD were given escalating doses of olipudase alfa intravenously for 26 weeks. The follow-up data that were analyzed 30 months after the trials stated that there was a 47.3% and a 35.6% decrease in the spleen and liver size as compared with the baseline. The DLco also improved to 67.1% from 53.2%5. Headaches, nasopharyngitis, URTIs, coughs, urticaria, and anaphylactic reactions were the noted side-effects seen in the patients present in ASCENDS Trial whereas, in the ASCEND-PEDS trial, pyrexia, cough, vomiting, nasopharyngitis, diarrhea, headache, upper respiratory tract infections, contusion, abdominal pain, nasal congestion, rash, urticaria, and epistaxis were the reported adverse effects4. Xenpozyme is currently the only approved drug for ASMD. Although more data is needed, the approval of this novel drug has raised optimism and hope to provide better treatment for patients suffering from ASMD. Ethical approval Not applicable. Sources of funding None. Authors’ contribution W.S. : conceptualization, writing – original draft, final approval and agreeing to the accuracy of the work. T.S.: final approval and agreeing to accuracy of work. Conflicts of interest disclosure The authors declare that they have no financial conflict of interest with regard to the content of this report. Research registration unique identifying number (UIN) None. Guarantor Wania Sultan and Tasmiyah Siddiqui.
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Abstract: Niemann‐Pick disease types A and B are two clinical forms of an inherited lysosomal storage disorder characterized by accumulation of sphingomyelin due to deficient activity of the lysosomal enzyme, acid sphingomyelinase. Patients with both types have hepatosplenomegaly, but only those with type A have nervous system involvement leading to death in early infancy. The residual activities of lysosomal sphingomyelinase in types A and B have never been well characterized because of limitations in both in vitro enzymatic assays and loading tests on intact cells. To evaluate the effective level of sphingomyelinase activity, intact, living cultured Epstein‐Barr virus‐transformed lymphoid cells were incubated with a radiolabeled sphingomyelin that was first associated to human low‐density lipoproteins. This lipoprotein‐associated sphingomyelin was targeted to lysosomes, thereby permitting selective hydrolysis by the lysosomal sphingomyelinase. Short‐term pulse‐chase experiments allowed the determination of the initial rates of degradation; in normal cells, the half‐time of sphingomyelin degradation averaged 4.5 h. Whereas cells from the severe neuronopathic type A form of Niemann‐Pick disease exhibited about 0.15% residual sphingomyelinase activity, cells from patients with the visceral type B form exhibited about 4%, i.e., 27 times more. Cells from heterozygous Niemann‐Pick subjects showed about 70% residual activity. These results provide the first approach to measuring the effective activity of a lysosomal enzyme and represent an accurate method for the differential diagnosis of Niemann‐Pick disease types A and B. They also support the hypothesis of relationships among the effective in situ residual enzyme activity, the amount of stored substrate, and the severity of the ensuing lysosomal storage disorder.
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Despite of the importance of the acid sphingomyelinase for sphingomyelin homeostasis and sphingolipid signalling, potent and selective inhibitors for this enzyme are rare. An increasing set of data on the inhibition of acid sphingomyelinase in different disease models using indirect inhibitors has been generated and strongly implies acid sphingomyelinase as an emerging drug target. Very recently, some new and promising inhibitors from different substance classes have been developed. In this review, previous and current developments in the field are summarized.
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Niemann–Pick disease
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Plasma secretion of acid sphingomyelinase is a hallmark of cellular stress response resulting in the formation of membrane embedded ceramide-enriched lipid rafts and the reorganization of receptor complexes. Consistently, decompartmentalization of ceramide formation from inert sphingomyelin has been associated with signaling events and regulation of the cellular phenotype. Herein, we addressed the question of whether the secretion of acid sphingomyelinase is involved in host response during sepsis. We found an exaggerated clinical course in mice genetically deficient in acid sphingomyelinase characterized by an increased bacterial burden, an increased phagocytotic activity, and a more pronounced cytokine storm. Moreover, on a functional level, leukocyte-endothelial interaction was found diminished in sphingomyelinase-deficient animals corresponding to a distinct leukocytes' phenotype with respect to rolling and sticking as well as expression of cellular surface proteins. We conclude that hydrolysis of membrane-embedded sphingomyelin, triggered by circulating sphingomyelinase, plays a pivotal role in the first line of defense against invading microorganisms. This function might be essential during the early phase of infection leading to an adaptive response of remote cells and tissues. Plasma secretion of acid sphingomyelinase is a hallmark of cellular stress response resulting in the formation of membrane embedded ceramide-enriched lipid rafts and the reorganization of receptor complexes. Consistently, decompartmentalization of ceramide formation from inert sphingomyelin has been associated with signaling events and regulation of the cellular phenotype. Herein, we addressed the question of whether the secretion of acid sphingomyelinase is involved in host response during sepsis. We found an exaggerated clinical course in mice genetically deficient in acid sphingomyelinase characterized by an increased bacterial burden, an increased phagocytotic activity, and a more pronounced cytokine storm. Moreover, on a functional level, leukocyte-endothelial interaction was found diminished in sphingomyelinase-deficient animals corresponding to a distinct leukocytes' phenotype with respect to rolling and sticking as well as expression of cellular surface proteins. We conclude that hydrolysis of membrane-embedded sphingomyelin, triggered by circulating sphingomyelinase, plays a pivotal role in the first line of defense against invading microorganisms. This function might be essential during the early phase of infection leading to an adaptive response of remote cells and tissues. Sepsis is defined as a syndrome with a complex continuum of host responses to invading microorganisms. These pathophysiological changes affect more than 1.5 million patients in Europe alone. Similarly, 20% of patients admitted to intensive care units (ICUs) in the United States suffer from severe sepsis, which is the leading cause of mortality in noncardiac ICUs. Despite the development of early intervention and intensive care, mortality resulting from sepsis is unacceptably high, reaching 30 to 50% in hospitals worldwide (1Martin G.S. Mannino D.M. Eaton S. Moss M. The epidemiology of sepsis in the United States from 1979 through 2000.N. Engl. J. Med. 2003; 348: 1546-1554Crossref PubMed Scopus (4818) Google Scholar, 2Levy M.M. Dellinger R.P. Townsend S.R. Linde-Zwirble W.T. Marshall J.C. Bion J. Schorr C. Artigas A. Ramsay G. Beale R. et al.The Surviving Sepsis Campaign: results of an international guideline-based performance improvement program targeting severe sepsis.Intensive Care Med. 2010; 36: 222-231Crossref PubMed Scopus (620) Google Scholar, 3Levy M.M. Artigas A. Phillips G.S. Rhodes A. Beale R. Osborn T. Vincent J.L. Townsend S. Lemeshow S. Dellinger R.P. Outcomes of the Surviving Sepsis Campaign in intensive care units in the USA and Europe: a prospective cohort study.Lancet Infect. Dis. 2012; 12: 919-924Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). The host response to infection is mediated by pathogen components (e.g., lipopolysaccharides or zymosan) and by host-activated enzymes, mediators (such as proinflammatory cytokines), and cells changing their phenotype. The resulting remote organ failure is associated with a poor outcome. Generation of the lipid mediator ceramide has been suggested as one major route of cellular response to stress (4Hannun Y.A. Obeid L.M. The Ceramide-centric universe of lipid-mediated cell regulation: stress encounters of the lipid kind.J. Biol. Chem. 2002; 277: 25847-25850Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar). Activation of ceramide-generating enzymes that hydrolyze cell membrane embedded sphingomyelin is implicated in mediating and regulating diverse cellular processes, such as proliferation, differentiation, apoptosis, and inflammation (5van Blitterswijk W.J. van der Luit A.H. Veldman R.J. Verheij M. Borst J. Ceramide: second messenger or modulator of membrane structure and dynamics?.Biochem. J. 2003; 369: 199-211Crossref PubMed Scopus (387) Google Scholar, 6Gulbins E. Li P.L. Physiological and pathophysiological aspects of ceramide.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2006; 290: R11-R26Crossref PubMed Scopus (185) Google Scholar). Moreover, ceramide formation is involved in the pathogenesis of numerous diseases, such as cancer, atherosclerosis, pulmonary edema, and cardiovascular disease (7Jenkins R.W. Canals D. Hannun Y.A. Roles and regulation of secretory and lysosomal acid sphingomyelinase.Cell. Signal. 2009; 21: 836-846Crossref PubMed Scopus (224) Google Scholar, 8Smith E.L. Schuchman E.H. The unexpected role of acid sphingomyelinase in cell death and the pathophysiology of common diseases.FASEB J. 2008; 22: 3419-3431Crossref PubMed Scopus (179) Google Scholar, 9Gulbins E. Regulation of death receptor signaling and apoptosis by ceramide.Pharmacol. Res. 2003; 47: 393-399Crossref PubMed Scopus (134) Google Scholar, 10Goggel R. Winoto-Morbach S. Vielhaber G. Imai Y. Lindner K. Brade L. Brade H. Ehlers S. Slutsky A.S. Schutze S. et al.PAF-mediated pulmonary edema: a new role for acid sphingomyelinase and ceramide.Nat. Med. 2004; 10: 155-160Crossref PubMed Scopus (265) Google Scholar). Five isoforms of specific, sphingomyelin hydrolyzing phosphodiesterases or sphingomyelinases have been identified (11Goni F.M. Alonso A. Sphingomyelinases: enzymology and membrane activity.FEBS Lett. 2002; 531: 38-46Crossref PubMed Scopus (292) Google Scholar, 12Claus R.A. Dorer M.J. Bunck A.C. Deigner H.P. Inhibition of sphingomyelin hydrolysis: targeting the lipid mediator ceramide as a key regulator of cellular fate.Curr. Med. Chem. 2009; 16: 1978-2000Crossref PubMed Scopus (53) Google Scholar). The secreted acid sphingomyelinase (aSMase) is the only isoform associated with extracellular hydrolysis of sphingomyelin and has been found to be secreted by macrophages, human skin fibroblasts, and human vascular endothelial cells (13Marathe S. Kuriakose G. Williams K.J. Tabas I. Sphingomyelinase, an enzyme implicated in atherogenesis, is present in atherosclerotic lesions and binds to specific components of the subendothelial extracellular matrix.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2648-2658Crossref PubMed Scopus (105) Google Scholar, 14Marathe S. Schissel S.L. Yellin M.J. Beatini N. Mintzer R. Williams K.J. Tabas I. Human vascular endothelial cells are a rich and regulatable source of secretory sphingomyelinase. Implications for early atherogenesis and ceramide-mediated cell signaling.J. Biol. Chem. 1998; 273: 4081-4088Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Apical secretion by endothelial cells was stimulated by a variety of proinflammatory mediators and cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, IFN-β, or IL-1β, but also by membrane constituents of gram-negative bacteria (14Marathe S. Schissel S.L. Yellin M.J. Beatini N. Mintzer R. Williams K.J. Tabas I. Human vascular endothelial cells are a rich and regulatable source of secretory sphingomyelinase. Implications for early atherogenesis and ceramide-mediated cell signaling.J. Biol. Chem. 1998; 273: 4081-4088Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). It was previously shown that patients with chronic or acute systemic inflammation or infection such as sepsis exhibited an enhanced sphingolytic activity in comparison to controls (15Claus R.A. Bunck A.C. Bockmeyer C.L. Brunkhorst F.M. Losche W. Kinscherf R. Deigner H.P. Role of increased sphingomyelinase activity in apoptosis and organ failure of patients with severe sepsis.FASEB J. 2005; 19: 1719-1721Crossref PubMed Scopus (99) Google Scholar, 16Doehner W. Bunck A.C. Rauchhaus M. von Haehling S. Brunkhorst F.M. Cicoira M. Tschope C. Ponikowski P. Claus R.A. Anker S.D. Secretory sphingomyelinase is upregulated in chronic heart failure: a second messenger system of immune activation relates to body composition, muscular functional capacity, and peripheral blood flow.Eur. Heart J. 2007; 28: 821-828Crossref PubMed Scopus (73) Google Scholar). During progression of the disease, a further increase in the activity of secreted aSMase parallel to severity of illness predicts a fatal outcome (15Claus R.A. Bunck A.C. Bockmeyer C.L. Brunkhorst F.M. Losche W. Kinscherf R. Deigner H.P. Role of increased sphingomyelinase activity in apoptosis and organ failure of patients with severe sepsis.FASEB J. 2005; 19: 1719-1721Crossref PubMed Scopus (99) Google Scholar). Furthermore, in mononuclear cells of septic patients, the concentration of ceramide was increased and correlated positively with plasma TNF-α levels and was higher among patients who developed dysfunction of remote organs (17Drobnik W. Liebisch G. Audebert F.X. Frohlich D. Gluck T. Vogel P. Rothe G. Schmitz G. Plasma ceramide and lysophosphatidylcholine inversely correlate with mortality in sepsis patients.J. Lipid Res. 2003; 44: 754-761Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 18Delogu G. Famularo G. Amati F. Signore L. Antonucci A. Trinchieri V. Di Marzio L. Cifone M.G. Ceramide concentrations in septic patients: a possible marker of multiple organ dysfunction syndrome.Crit. Care Med. 1999; 27: 2413-2417Crossref PubMed Scopus (55) Google Scholar). A 2- to 3-fold rise in plasma sphingolytic activity was also observed in animal models after application of endotoxin or proinflammatory cytokines (15Claus R.A. Bunck A.C. Bockmeyer C.L. Brunkhorst F.M. Losche W. Kinscherf R. Deigner H.P. Role of increased sphingomyelinase activity in apoptosis and organ failure of patients with severe sepsis.FASEB J. 2005; 19: 1719-1721Crossref PubMed Scopus (99) Google Scholar, 19Wong M.L. Xie B. Beatini N. Phu P. Marathe S. Johns A. Gold P.W. Hirsch E. Williams K.J. Licinio J. et al.Acute systemic inflammation up-regulates secretory sphingomyelinase in vivo: a possible link between inflammatory cytokines and atherogenesis.Proc. Natl. Acad. Sci. USA. 2000; 97: 8681-8686Crossref PubMed Scopus (136) Google Scholar). As the inert membrane constituent, sphingomyelin is preferentially distributed in the outer leaflet of cellular membranes. After inflammation-triggered secretion, aSMase gains unlimited access to its substrate and produces huge amounts of ceramide, affecting membrane organization (20Jenkins R.W. Canals D. Idkowiak-Baldys J. Simbari F. Roddy P. Perry D.M. Kitatani K. Luberto C. Hannun Y.A. Regulated secretion of acid sphingomyelinase: implications for selectivity of ceramide formation.J. Biol. Chem. 2010; 285: 35706-35718Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 21Nikolova-Karakashian M.N. Rozenova K.A. Ceramide in stress response.Adv. Exp. Med. Biol. 2010; 688: 86-108Crossref PubMed Scopus (101) Google Scholar). In an instantaneous manner, ceramide induced the release of reactive oxygen species in endothelial cells, decreasing endothelium-dependent vasorelaxation (22Zhang A.Y. Yi F. Zhang G. Gulbins E. Li P.L. Lipid raft clustering and redox signaling platform formation in coronary arterial endothelial cells.Hypertension. 2006; 47: 74-80Crossref PubMed Scopus (177) Google Scholar, 23Zhang D.X. Yi F.X. Zou A.P. Li P.L. Role of ceramide in TNF-alpha-induced impairment of endothelium-dependent vasorelaxation in coronary arteries.Am. J. Physiol. Heart Circ. Physiol. 2002; 283: H1785-H1794Crossref PubMed Scopus (61) Google Scholar). These observations support the concept that secreted aSMase is a key player in cytokine secretion during host response. This sequence of cellular response to inflammatory stress also put forward the hypothesis that an unfavorable outcome of sepsis-associated remote organ failure might be linked to regulating the secretion and activation of aSMase. Transgenic mice with a deficiency in aSMase function (knockout [KO]) (aged 8–10 weeks) and their wild-type (WT) littermates (aged 8–12 weeks) were used in this study (24Horinouchi K. Erlich S. Perl D.P. Ferlinz K. Bisgaier C.L. Sandhoff K. Desnick R.J. Stewart C.L. Schuchman E.H. Acid sphingomyelinase deficient mice: a model of types A and B Niemann-Pick disease.Nat. Genet. 1995; 10: 288-293Crossref PubMed Scopus (411) Google Scholar). Animals were randomly selected for each experiment, were maintained under artificial day-night conditions at room temperature, and received a standard diet and water ad libitum. All experiments were performed in accordance with the German legislation on protection of animals and with permission of the regional animal welfare committee. Mice were anesthetized with a combination of ketamine and xylazin injected intraperitoneally before any surgical procedure. During the surgical procedures, body temperature was maintained at 37°C. Sepsis was induced using the peritoneal contamination and infection (PCI) model as previously described (25Gonnert F.A. Kunisch E. Gajda M. Lambeck S. Weber M. Claus R.A. Bauer M. Kinne R.W. Hepatic Fibrosis in a Long-term Murine Model of Sepsis.Shock. 2012; 37: 399-407Crossref PubMed Scopus (23) Google Scholar). In the present study, peritonitis was induced by the injection of a 1:4 diluted stool suspension (5 µl/g body weight [BW]) intraperitoneally. Two postinsult time points were selected to represent early sepsis (6 h) and late sepsis (18–24 h). For survival analysis, aSMase WT and KO animals (n =≥15 per genotype) were monitored every 3 h after peritonitis induction over 72 h. During this experiment, animals received volume resuscitation (balanced saline solution) of 25 µl/g BW twice per day subcutaneously over the period of the survival study. Log-rank statistics were used for data analysis. Blood and organs were collected from deeply anesthetized WT and KO mice (n =≥4 per genotype per time point) 6 h after PCI. Sodium citrate was used as an anticoagulant. After blood was harvested, whole liver and lungs were collected in a sterile manner. To avoid bacterial contamination, collected organs were briefly washed in 70% ethanol. The organs were later homogenized in sterile 0.9% saline solution. Enriched brain-heart infusion (2 ml) was added to each sample, and dilutions up to five orders of magnitude were prepared. These dilutions were plated on Schädler's and blood agars and incubated under aerobic and anaerobic conditions for 48 h. The number of bacterial colonies was counted. Data were evaluated with consideration of blood volume or normalized to wet weight of organs. Whole blood was collected by heart puncture from sham-treated and septic animals (n =≥15 per genotype per time point) 6 h after peritonitis induction. Leukocyte and platelet counts were determined for aSMase WT and KO animals using the automated veterinary hematology analyzer Poch-100iv-Diff (Sysmex, Leipzig, Germany). Leukocyte subpopulations were also analyzed in whole blood collected from both genotypes. Blood smears were prepared and stained using Giemsa. The slides were then processed for analysis using the automated analyzer Cellavision DM 96 (Sysmex, Leipzig, Germany). Serum was collected from aSMase WT and KO animals at three time points (n =≥8 per genotype per time point) for analysis of laboratory markers of organ dysfunction using the clinical chemistry analyzer Fuji Dri-Chem 3500i (Sysmex, Leipzig, Germany) according to the manufacturer's instructions. Phagocytotic activity of granulocytes and monocytes was determined by flow cytometry after incubating whole blood samples with FITC-labeled and opsonized Escherichia coli (Phagotest; Becton Dickinson, Franklin Lakes, NJ). Briefly, 6 h after PCI, blood was collected from sham and septic WT and KO animals (n =≥4 per genotype per time point) using Li-heparin as an anticoagulant. Diluted blood was incubated with labeled E. coli (1.7 × 109/ml) for 20 min at 37°C followed by washing, lysis, and staining. The samples were measured by flow cytometry according to the manufacturer's instructions (software: CellQuest Pro, version 5.1.1). After "gating" FITC-positive monocytes and granulocytes, groups were compared with each other. Cytokines (IL-6, IL-10, monocyte chemotactic protein [MCP]-1, and TNF-α) were quantified in EDTA plasma samples collected from sham and septic WT and KO animals at three time points (n =≥6 per genotype per time point) using cytometric bead assay (Becton Dickinson) according to the manufacturer's instructions. Livers were collected from WT and KO animals (n =≥4 per genotype per time point) at three time points. Transmigration of leukocytes into liver tissue was evaluated by specific staining of granulocytes in liver sections. These sections (3 µm) were deparaffinized with decreasing ethanol series. The LEUCOGNOST® NASDCL kit (Merck Millipore, Darmstadt, Germany) was used for staining transmigrated granulocytes following the user's manual. At 400× magnification, stained cells were counted in at least 20 visual fields by two independent and experienced scientists blinded to treatment or genotype. In vivo imaging of the liver was performed with an epifluorescence microscope as described in detail previously (26Recknagel P. Claus R.A. Neugebauer U. Bauer M. Gonnert F.A. In vivo imaging of hepatic excretory function in the rat by fluorescence microscopy.J Biophotonics. 2012; 5: 571-581Crossref PubMed Scopus (17) Google Scholar) to evaluate leukocyte endothelium interactions 6 h after insult in sham-treated and in septic aSMase WT and KO animals (n = 5 per genotype per time point). Leukocytes were labeled in vivo with carboxyfluorescein diacetate succinimidyl ester (1 mg/kg BW) administered via a central line 10 min before surgical instrumentation. At least five regions of interest per mouse of either postsinusoidal venules (40× objective lens) or sinusoids (10× objective lens) were recorded over 30 s and 5 s, respectively (GFP filterset, 470–495 nm excitation and 525–550 nm emission band pass filters). Leukocytes were considered as "rollers" when they rolled across the endothelium or stuck to the endothelium for a few seconds and then detached. Leukocytes were considered as "stickers" when they remained stuck to the endothelium throughout the observation period. The total number of leukocytes in the postsinusoidal venules was calculated per mm2 endothelial surface (length of observed vessel segment × diameter ×π). Rollers and stickers in postsinusoidal venules were calculated per 100 counted leukocytes. Stickers in sinusoids as well as transmigrated leukocytes were calculated per mm2 liver surface (27Gonnert F.A. Recknagel P. Seidel M. Jbeily N. Dahlke K. Bockmeyer C.L. Winning J. Losche W. Claus R.A. Bauer M. Characteristics of clinical sepsis reflected in a reliable and reproducible rodent sepsis model.J. Surg. Res. 2011; 170: e123-e134Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Expression of surface proteins CD49d, CD62L, and CD11b was analyzed using flow cytometry according to the manufacturer's instructions (software: CellQuest Pro, version 5.1.1). Briefly, whole blood was collected from aSMase WT and KO animals (n = 9 per genotype per time point). Samples of whole blood were separately incubated with FITC-labeled CD49d, CD62L, and CD11b antibodies. The geo-mean of fluorescence intensity (MFI) of all gated leukocytes was documented. Whole blood was collected from aSMase WT and KO animals at baseline and 6 h after sepsis induction (n =≥12 per genotype per time point). The ceramide pattern was fixed by the addition of an unspecific, broad-spectrum inhibitor of hydrolases (diisopropylfluorophosphate) during the lysis of red blood cells. Washed and pelleted leukocytes were pooled (to obtain 1–2 million cells per sample; six samples per group) and resuspended in methanol. After addition of 20 pmol C17-ceramide, chloroform, methanol. and distilled water to the cells followed by intense vortexing and centrifugation, the lower organic phase was obtained for further analysis. Lipids were then resolved in methanol after evaporation of the organic phase via the SpeedVac SC201 ARC vacuum system (Thermo Fisher Scientific GmbH, Dreieich, Germany). Analysis was performed using a quadrupole/time-of-flight mass spectrometer (Agilent, Waldbronn, Germany) connected to a rapid-resolution liquid chromatograph. High-purity nitrogen for the mass spectrometer was produced by a nitrogen generator (Parker Balston, Maidstone, UK). Chromatographic separations were obtained using a ZORBAX Eclipse XDB-C18 as described previously (Agilent Technologies, Waldbronn, Germany) (28Luth A. Neuber C. Kleuser B. Novel methods for the quantification of (2E)-hexadecenal by liquid chromatography with detection by either ESI QTOF tandem mass spectrometry or fluorescence measurement.Anal. Chim. Acta. 2012; 722: 70-79Crossref PubMed Scopus (13) Google Scholar). The injection volume per sample was 10 µl. Best results were obtained with an isocratic elution (acetonitrile/2-propanol 3:2 with 1% formic acid) at a flow rate of 1 ml/min for 15 min. For mass spectrometric measurements, we used the following ion source conditions and gas settings for positive LC-MS/MS: sheath gas temperature: 400°C, sheath gas flow: 9 l/min, nebulizer pressure = 30 psig, drying gas temperature = 350°C, drying gas flow = 8 l/min, capillary voltage = 2,000 V, fragmentor voltage = 355 V, nozzle voltage = 2,000 V. All ceramides gave the same fragment ion of m/z 264.27 at different retention times depending on their chain length. Quantification was performed using Mass Hunter software (Agilent). Calibration curves of reference ceramide values were performed from 1 to 100 pmol and were constructed by linear fitting using the least squares linear regression calculation. The resulting slope of the calibration curve was used for calculating the concentration of the respective analyte in the unknowns. Citrated plasma was collected from aSMase WT and KO animals (n =≥6 per genotype per time point) 6 h after the insult. aSMase activity was determined by the hydrolysis of fluorescently labeled sphingomyelin (NBD-SM; Molecular Probes, Eugene, OR) as a substrate, chromatographic product separation, and image analysis as described previously (15Claus R.A. Bunck A.C. Bockmeyer C.L. Brunkhorst F.M. Losche W. Kinscherf R. Deigner H.P. Role of increased sphingomyelinase activity in apoptosis and organ failure of patients with severe sepsis.FASEB J. 2005; 19: 1719-1721Crossref PubMed Scopus (99) Google Scholar, 29Loidl A. Claus R. Deigner H.P. Hermetter A. High-precision fluorescence assay for sphingomyelinase activity of isolated enzymes and cell lysates.J. Lipid Res. 2002; 43: 815-823Abstract Full Text Full Text PDF PubMed Google Scholar). Plasma samples were diluted 1:10 with the incubation buffer (sodium acetate, pH 5.0) before analysis. The final composition of the reaction mixture was 12 μl plasma, and the extraction was carried out using the SpeedVac concentrator Plus (Eppendorf, Hamburg, Germany). Whole blood was collected from aSMase WT and KO animals at baseline and 6 h after sepsis induction (n = 6 per genotype per time point). Granulocytes were labeled with a PE-labeled Ly-6G antibody (eBioscience Inc., San Diego, CA). For the detection of reactive oxygen species (ROS), we used the FagoFlowEx kit according to the manufacturer's instructions (EXBIO, Vestec, Czech Republic). Briefly, blood samples were incubated with E. coli and dihydrorhodamine-123. After red blood cell lysis, the resuspended leukocytes were analyzed for detection of ROS formation using flow cytometry according to the manufacturer's instructions (software: CellQuest Pro, version 5.1.1). The fluorescence signal was expressed as MFI. We measured MFI for sham samples and those incubated with E. coli. Results are represented as a stimulation index, which was calculated by normalization of the E. coli mix against the control. Whole blood and tissue samples (liver and lung) were collected from aSMase KO mice and their WT littermates 6 and 24 h after sepsis (n = 2 per genotype per time point). Organs were shock frozen for later isolation of total RNA. Samples were analyzed by a pangenomic microarray mouseWG-6 v1.1 expression bead chips using an iScan platform (Illumina, San Diego, CA) measuring the variation of expression rate of >42.000 transcripts. Subsequent to biostatistical analysis, validation of expression data of representative genes was performed by quantitative real-time PCR. Data were normalized to unchanged reference transcripts (Actb, Gapdh, Hprt), and relative changes were plotted against sham-treated WT littermates. Data were obtained from randomly selected animals for each time point. Analyses were generally performed in an independent setting with respect to the measure. Thus, each animal reflects a discrete set of data. For ethical reasons, when possible and appropriate different tissues, organs, and whole blood samples from one animal were used for several measurements (i.e., samples for determination of leukocyte counts, leukocyte subpopulations, and platelet counts in whole blood were obtained from the same animals). The same applied for the analyses of bacterial burden from blood and organs and granulocyte migration studies. All data were examined for normal distribution, and appropriate tests were applied. Statistical differences between groups were analyzed using ANOVA followed by Student-Newman-Keuls post hoc test or ANOVA (Kruskal-Wallis test) followed by pairwise multiple comparison procedures (Dunn's method) or Mann-Whitney rank-sum test. Data are given as boxplots, which show the median and the first and third quartiles and whiskers (10th and 90th percentiles). P values below 0.05 were considered statistically significant. Discriminating values of ceramide content in circulating leukocytes highlighted the differences between the two genotypes. Despite the loss of sphingomyelinase, cell-bound ceramide was found significantly increased at baseline in KO animals compared with the WT littermates. Reflecting the biological significance of ceramide generation, levels of ceramide in leukocytes significantly increased only in WT animals 6 h after the septic insult (Fig. 1). For characterization of the bacterial load, solid organs and blood samples collected from WT and KO mice were investigated for their content of aerobic and anaerobic bacteria. The two genotypes exhibited differences in bacterial load subsequent to sepsis. In KO animals, the number of colony-forming units (CFUs) of aerobes and anaerobes in liver as well as the aerobes in blood were significantly higher compared with WT animals. Blood anaerobes were similarly high in both WT and KO groups. Surprisingly, the number of CFU in lungs of KO was lower compared with WT animals. CFUs of anaerobes did not significantly increase in the KO animals 6 h after sepsis induction (Fig. 2). As a negative control, vehicle-treated control mice (sham) exhibited no CFU in blood or in liver and lung. The levels of proinflammatory mediators reflect important markers of disease progression and are linked to the activation status of the innate immune system. After PCI, cytokine levels increased over time (baseline, 6 h, and 24 h) independent of the genotype (Fig. 3). In WT and KO animals, 6 h after sepsis induction, cytokine levels of IL-6 and MCP significantly increased in both genotypes. However, we measured a significantly higher increase in the levels of the prototypical anti- and proinflammatory cytokines TNF-α and IL-10 in KO animals 6 h after sepsis induction compared with WT animals. At 24 h, a further increase in cytokine levels of TNF-α and MCP could be observed in KO animals compared with WT littermates without reaching significance. To evaluate the distinct cytokine profiles as a cause or consequence of sphingomyelinase activity and ceramide formation, we further investigated the inflammatory response in a systems biology approach using a tissue-specific transcriptomal analysis. Across all tissues (circulating leukocytes, liver, and lung), more than 26,000 transcripts were found expressed. After normalization and hierarchical clustering analysis, only seven annotated transcripts exhibited a different expression compared with untreated animals of each genotype (e.g., Gpnmb, Hexa, CD83). In aSMase KO animals with peritonitis, transcripts were found differentially regulated when compared with WT littermates at both time points (n = 258 at 6 h; n = 315 at 24 h) (Fig. 4A). The transcripts for Tnfa, Nfkbia, Casp4, Ccl4, Ccl7, Mapkapk2, Csf3, Stat3, and Il1rn were found to be differentially regulated (supplementary Table I). These transcripts included those crucially involved in inflammation, immune regulation, and inflammatory response against invading pathogens. qPCR confirmed a sharp increase of selected transcripts relevant to regulation of inflammatory response, such as cytokines and cytokine-regulated transcripts (e.g., Tnf, Tnfaip3) in KO animals (Fig. 4
Acid sphingomyelinase
Sphingomyelin phosphodiesterase
Sphingolipid
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Acid sphingomyelinase
Niemann–Pick disease
Dried blood
Sphingolipid
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Effective treatment strategies for severe coronavirus disease (COVID-19) remain scarce. Hydrolysis of membrane-embedded, inert sphingomyelin by stress responsive sphingomyelinases is a hallmark of adaptive responses and cellular repair. As demonstrated in experimental and observational clinical studies, the transient and stress-triggered release of a sphingomyelinase, SMPD1, into circulation and subsequent ceramide generation provides a promising target for FDA-approved drugs. Here, we report the activation of sphingomyelinase-ceramide pathway in 23 intensive care patients with severe COVID-19. We observed an increase of circulating activity of sphingomyelinase with subsequent derangement of sphingolipids in serum lipoproteins and from red blood cells (RBC). Consistent with increased ceramide levels derived from the inert membrane constituent sphingomyelin, increased activity of acid sphingomyelinase (ASM) accurately distinguished the patient cohort undergoing intensive care from healthy controls. Positive correlational analyses with biomarkers of severe clinical phenotype support the concept of an essential pathophysiological role of ASM in the course of SARS-CoV-2 infection as well as of a promising role for functional inhibition with anti-inflammatory agents in SARS-CoV-2 infection as also proposed in independent observational studies. We conclude that large-sized multicenter, interventional trials are now needed to evaluate the potential benefit of functional inhibition of this sphingomyelinase in critically ill patients with COVID-19.
Acid sphingomyelinase
Sphingomyelin phosphodiesterase
Sphingolipid
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Citations (21)