The inositol phosphatase SHIP controls Salmonella enterica serovar Typhimurium infection in vivo

Abstract

The SH2 domain-containing inositol 5'-phosphatase, SHIP, negatively regulates various hematopoietic cell functions and is critical for maintaining immune homeostasis. However, whether SHIP plays a role in controlling bacterial infections in vivo remains unknown. Salmonella enterica causes human salmonellosis, a disease that ranges in severity from mild gastroenteritis to severe systemic illness, resulting in significant morbidity and mortality worldwide. The susceptibility of ship(+/+) and ship(-/-) mice and bone marrow-derived macrophages to S. enterica serovar Typhimurium infection was compared. ship(-/-) mice displayed an increased susceptibility to both oral and intraperitoneal serovar Typhimurium infection and had significantly higher bacterial loads in intestinal and systemic sites than ship(+/+) mice, indicating a role for SHIP in the gut-associated and systemic pathogenesis of serovar Typhimurium in vivo. Cytokine analysis of serum from orally infected mice showed that ship(-/-) mice produce lower levels of Th1 cytokines than do ship(+/+) animals at 2 days postinfection, and in vitro analysis of supernatants taken from infected bone marrow-derived macrophages derived to mimic the in vivo ship(-/-) alternatively activated (M2) macrophage phenotype correlated with these data. M2 macrophages were the predominant population in vivo in both oral and intraperitoneal infections, since tissue macrophages within the small intestine and peritoneal macrophages from ship(-/-) mice showed elevated levels of the M2 macrophage markers Ym1 and Arginase 1 compared to ship(+/+) cells. Based on these data, we propose that M2 macrophage skewing in ship(-/-) mice contributes to ineffective clearance of Salmonella in vivo.

FIG. 1.

SHIP controls susceptibility to Salmonella in vivo. (A) ship^+/+and ship^−/− mice were infected orally with 10⁶ live or 10⁶ and 10⁸ heat-killed (HK) serovar Typhimurium SL13344, and the time of death was assessed over a 3-week period. (B) ship^+/+and ship^−/− mice were infected i.p. with 10² live or 10² and 10⁸ heat-killed (HK) serovar Typhimurium SL13344, and the time of death was assessed over a 3-week period. For both panels, three independent experiments were performed with a total of 12 animals for both ship^+/+and ship^−/− mice.

FIG. 2.

SHIP is required to control Salmonella but not Citrobacter replication in vivo. (A) ship^+/+and ship^−/− mice were infected orally with 10⁶ serovar Typhimurium SL13344 and sacrificed at 2 days postinfection. Colons, small intestines (SI), livers, MLN, and spleens were harvested from the mice, homogenized, and plated to enumerate the bacterial load. (B) ship^+/+and ship^−/− mice were infected i.p. with 10² serovar Typhimurium SL13344 and sacrificed at 5 days postinfection. Colons, small intestines, livers, MLN, and spleens were harvested from the mice, homogenized, and plated to enumerate bacterial load. (C) ship^+/+and ship^−/− mice were infected orally with 10⁸ C. rodentium DBS100 and sacrificed at 7 days postinfection. Colons were harvested from the mice, homogenized, and plated to enumerate the bacterial load. For all figures, three independent experiments were performed with a total of 12 animals for both ship^+/+and ship^−/− mice.

FIG. 3.

SHIP deficiency leads to altered levels of inflammatory cytokine production after Salmonella infection in vivo. ship^+/+and ship^−/− mice were infected orally with 10⁶ serovar Typhimurium SL1344 (A), i.p. with 10² serovar Typhimurium SL1344 (B), or left uninfected (C) and sacrificed at 2 days postinfection or 5 days postinfection, respectively. Blood samples were obtained via cardiac puncture, and serum was separated for cytokine analysis. Cytokines were analyzed by using the flow cytometry-based CBA mouse inflammation kit. For panels A and B, three independent experiments were performed with a total of 12 animals for both ship^+/+and ship^−/− mice.

FIG. 4.

M2 macrophage markers are found in the guts and peritoneal cavities of ship^−/− mice. (A and B) Sections of small intestine were taken from uninfected (ship^+/+ and ship^−/−) and orally infected ship^+/+and ship^−/− mice (ship^+/+ SL1344 and ship^−/− SL1344) at day 2 postinfection and stained for the M2 macrophage markers YM1 or Arg1 (green) or and F4/80 (red) as a macrophage control. (C) Sections of small intestine were taken from uninfected ship^+/+ and ship^−/− mice (−SL1344) and orally infected ship^+/+and ship^−/− mice (+SL1344) at day 2 postinfection and stained with hematoxylin and eosin to show the pathology. All photographs were taken at ×40 magnification. (D) Peritoneal macrophages were obtained from uninfected (−SL1344) or i.p.-infected (+SL1344) ship^+/+and ship^−/− mice at 2 days postinfection. Cells were washed, counted, and lysed directly into Western sample buffer for protein analysis. Both bands are specific for Arg1 (35). For panels A to D, the results of representative experiments are shown.

FIG. 5.

Fold replication of serovar Typhimurium in M1 versus M2 macrophages. (A) BMDMs were obtained from ship^+/+ and ship^−/− mice and derived in the presence of FBS alone (M1) or FBS plus 2% mouse serum (M2) and infected with serovar Typhimurium SL1344. (B) RAW 264.7 macrophages were grown in either DMEM plus 10% FBS (M1) or skewed to an M2 phenotype by the addition of 100 ng of IL-4/ml (M2) 24 h prior to infection. For both panels, the cells were seeded and infected with serovar Typhimurium SL1344 at an MOI of 10 in a gentamicin protection assay, and the bacteria were enumerated at 2 and 24 h postinfection. The graphs represent the fold replication of intracellular serovar Typhimurium by dividing the CFU values obtained at 24 h by those obtained at 2 h. For both panels, three independent experiments were performed, with each treatment being performed in triplicate, for a total nine wells for each treatment.

FIG. 6.

Salmonella-infected BMDMs from ship^−/− mice derived under M2 inducing conditions show decreased levels of inflammatory cytokines compared to ship^+/+ cells. (A to D) BMDMs were obtained from ship^+/+and ship^−/− mice and derived in the presence of FBS plus 2% mouse serum for 10 days. Cells were seeded and either left untreated (−), infected with serovar Typhimurium SL1344 (SL1344) or heat-killed serovar Typhimurium SL1344 (HK) at an MOI of 10, or treated with 100 ng of serovar Typhimurium LPS (LPS)/ml for 8 h, and supernatants were collected. Cytokine analysis was performed by using ELISAs. For all four panels, three independent experiments were performed with each treatment being performed in triplicate for a total nine wells for each treatment.

FIG. 7.

ship^−/− BMDMs are not more susceptible to death in vitro upon infection with Salmonella. BMDMs were obtained from ship^+/+and ship^−/− mice and derived in the presence of FBS plus 2% mouse serum for 10 days. Cells were seeded and either left untreated (−), infected with serovar Typhimurium SL1344 (SL1344) or heat-killed serovar Typhimurium SL1344 (HK) at an MOI of 10, or treated with 100 ng of serovar Typhimurium LPS/ml (LPS) for 8 h (A) or 24 h (B). Cells were collected and stained with the cell death marker 7AAD and analyzed via flow cytometry. Three independent experiments were performed, with each treatment being performed in triplicate for a total nine wells for each treatment.