Constitutive acid sphingomyelinase enhances early and late macrophage killing of Salmonella enterica serovar Typhimurium

Abstract

We have identified acid sphingomyelinase (ASM) as an important player in the early and late anti-Salmonella activity of macrophages. A functional ASM participated in the killing activity of macrophages against wild-type Salmonella enterica serovar Typhimurium. The role of ASM in early macrophage killing of Salmonella appears to be linked to an active NADPH phagocyte oxidase enzymatic complex, since the flavoprotein inhibitor diphenyleneiodonium not only blocked a productive respiratory burst but also abrogated the survival advantage of Salmonella in macrophages lacking ASM. Lack of ASM activity also increased the intracellular survival of an isogenic DeltaspiC::FRT Salmonella strain deficient in a translocator and effector of the Salmonella pathogenicity island 2 (SPI2) type III secretion system, suggesting that the antimicrobial activity associated with ASM is manifested regardless of the SPI2 status of the bacteria. Constitutively expressed ASM is responsible for the role that this lipid-metabolizing hydrolase plays in the innate host defense of macrophages against Salmonella. Accordingly, the ASM activity and intracellular concentration and composition of ceramide, gangliosides, and neutral sphingolipids did not increase upon Salmonella infection. Salmonella triggered, nonetheless, a significant increase in the secreted fraction of ASM. Collectively, these findings have elucidated a novel role for constitutive ASM in the anti-Salmonella activity of murine macrophages.

FIG. 1.

ASM enzymatic activity of murine macrophages. (A) Peritoneal macrophages isolated from ASM^−/− and ASM^+/+ mice were assessed for ASM activity by incubating cellular lysates with NBD-sphingomyelin (NBD-SM) for 60 min at 37°C. Enzymatic production of NBD-ceramide (NBD-cer) was visualized following high-performance TLC of lipid extracts. NBD-ceramide (1 pmol) was loaded as a control (lane 1). (B) The amount of ceramide produced by intracellular ASM was quantified by regression analysis using an NBD-ceramide standard curve. The results are expressed as fmol ceramide/10⁵ macrophages (mφ)/h (mean ± standard deviation) (n = 3). *, P < 0.05, compared to ASM^−/− control by unpaired, two-way t test.

FIG. 2.

Intracellular survival of Salmonella is enhanced in macrophages lacking ASM. The antimicrobial activities of macrophages from ASM^+/+ and ASM^−/− mice against wild-type (wt) (A) and spiC-deficient (B) Salmonella bacteria were monitored over time. Percent survival is expressed as the mean ± standard deviation (n = 6 to 10). Logarithmically transformed data were analyzed by two-way ANOVA. *, P < 0.05 for comparison of ASM^+/+ and ASM^−/− antimicrobial activities as determined by a Bonferroni posttest. (C) Internalization of bacteria by ASM^+/+ and ASM^−/− macrophages was estimated by recording the number of bacteria capable of forming a colony following 35 min of infection. Data are expressed as mean numbers of CFU ± SEM (n = 16). (D) Phagocytic indices were calculated for ASM^+/+ and ASM^−/− macrophages by enumerating the number of intracellular FITC-labeled Salmonella bacteria per macrophage by fluorescence microscopy. A total of 50 to 60 macrophages were analyzed from six separate slides per group. The numbers of bacteria per macrophage are represented in a box-and-whisker plot as median, intraquartile, and total ranges.

FIG. 3.

Generation of ROS and RNS by Salmonella-infected macrophages is independent of ASM. (A) The generation of RNS by Salmonella-infected macrophages from ASM^+/+ and ASM^−/− mice was determined by measuring the accumulation of nitrite in supernatants at the indicated times. Data are expressed as μM nitrite/10⁵ macrophages (mφ) (mean ± SEM). (B) The oxidative burst of ASM-proficient or -deficient macrophages was measured as lucigenin-dependent chemiluminescence after Salmonella challenge. Selected samples were treated with 10 μM DPI at the time of the infection. Results are expressed as mean relative luminescence units (RLU) ± SEM. The data are from three to six independent observations.

FIG. 4.

ASM-mediated anti-Salmonella activity of macrophages depends on a functional NADPH oxidase. (A) The effect that the flavoprotein inhibitor DPI has on the survival of wild-type (wt) and spiC-deficient Salmonella was studied in macrophages from ASM^+/+ and ASM^−/− mice. Data are expressed as mean percent survival ± standard deviation. (B) The effect of the NOS inhibitor MMLA on the survival of Salmonella in ASM^+/+ and ASM^−/− macrophages was determined after 20 h of infection. DPI and MMLA were added to the macrophages at the time of the infection. Data are expressed as mean percent survival ± SEM. (C) Mean ASM activity ± SEM was determined for untreated or DPI-treated ASM^+/+ macrophages (mφ) prior to (0 min) or 30 min following challenge with Salmonella. The data are from four to seven independent observations.

FIG. 5.

Salmonella infection decreases constitutive ASM activity of macrophages. (A) Intracellular ASM activity was assessed at the indicated times following challenge of periodate-elicited macrophages (mφ) with wild-type (wt) or spiC-deficient (n = 4 to 6) Salmonella. (B) Comparison of the ASM activities of resident and periodate-elicited Salmonella-infected ASM^+/+ macrophages (n = 4 to 8). ASM activities are expressed as mean fmol ceramide/10⁵ macrophages/h ± standard deviation. p.i., postinfection. (C) Total sphingolipids extracted from control (lanes C) or Salmonella-infected (lanes I) ASM^+/+ or ASM^−/− macrophages were visualized following separation by high-performance TLC. Mixed gangliosides GM1 (6), GD1a (7), and GD1b (8) (lane MG); neutral glycosphingolipids cerebroside (1), lactosyl ceramide (2), ceramide trihexoside (4), and globoside (5) (lane S1); and sphingolipid mix of cerebrosides (1), sulfatides (3), and sphingomyelin (5) (lane S2) were used as standards.

FIG. 6.

Infection of macrophages with Salmonella stimulates extracellular ASM secretion. (A) Secreted ASM activity of ASM^−/− or ASM^+/+ macrophages was assessed in cellular supernatants by TLC as hydrolysis of NBD-sphingomyelin (NBD-SM) to NBD-ceramide (NBD-cer). (B) Secreted ASM (S-ASM) from uninfected controls and E. coli- or Salmonella-challenged ASM^+/+ macrophages (mφ) was quantified in supernatants after 4 h of accumulation. The data are expressed as the mean ± standard deviation (n = 4). Statistical analysis of untransformed data was performed with a two-way ANOVA. *, P < 0.05, compared to uninfected controls as determined by a Bonferroni posttest.