Caveolin-1-deficient mice show defects in innate immunity and inflammatory immune response during Salmonella enterica serovar Typhimurium infection

Abstract

A number of studies have shown an association of pathogens with caveolae. To this date, however, there are no studies showing a role for caveolin-1 in modulating immune responses against pathogens. Interestingly, expression of caveolin-1 has been shown to occur in a regulated manner in immune cells in response to lipopolysaccharide (LPS). Here, we sought to determine the role of caveolin-1 (Cav-1) expression in Salmonella pathogenesis. Cav-1(-/-) mice displayed a significant decrease in survival when challenged with Salmonella enterica serovar Typhimurium. Spleen and tissue burdens were significantly higher in Cav-1(-/-) mice. However, infection of Cav-1(-/-) macrophages with serovar Typhimurium did not result in differences in bacterial invasion. In addition, Cav-1(-/-) mice displayed increased production of inflammatory cytokines, chemokines, and nitric oxide. Regardless of this, Cav-1(-/-) mice were unable to control the systemic infection of Salmonella. The increased chemokine production in Cav-1(-/-) mice resulted in greater infiltration of neutrophils into granulomas but did not alter the number of granulomas present. This was accompanied by increased necrosis in the liver. However, Cav-1(-/-) macrophages displayed increased inflammatory responses and increased nitric oxide production in vitro in response to Salmonella LPS. These results show that caveolin-1 plays a key role in regulating anti-inflammatory responses in macrophages. Taken together, these data suggest that the increased production of toxic mediators from macrophages lacking caveolin-1 is likely to be responsible for the marked susceptibility of caveolin-1-deficient mice to S. enterica serovar Typhimurium.

FIG. 1.

Cav-1^−/− mice display significantly reduced survival rates upon challenge with a highly virulent serovar Typhimurium strain. Cav-1^+/+ (WT, wild type) and Cav-1^−/− (C1KO, Cav-1 knockout) mice were administered 1 × 10³ CFU i.v (A) or 10⁴ CFU of p.o. (B) and monitored daily for survival (n ≥ 9). Survival curves were analyzed with the log rank test and revealed statistically significant differences for mice challenged both i.v and p.o. (P ≤ 0.001).

FIG. 2.

Numbers of live serovar Typhimurium organisms in the spleens or livers of Cav-1^+/+ and Cav-1^−/− mice after bacterial challenge. Cav-1^+/+ (n = 5) and Cav-1^−/− mice (n = 5) were infected i.v. with 1 × 10³ CFU of serovar Typhimurium, and bacterial counts from spleen (A) and liver (B) homogenates were determined by culture on agar plates at days 1, 3, and 5 postinfection. Data are the mean number of CFU ± standard error of the mean. Differences were considered statistically significant at P values of ≤0.05.

FIG. 3.

Dramatic increase of serum cytokine production in Cav-1^−/− mice in response to serovar Typhimurium infection. Mice were bled after 3 days of challenge with serovar Typhimurium and the serum chemokine (A) and cytokine (B) levels of the proinflammatory cytokines and the anti-inflammatory cytokines were measured. Data are the mean ± standard error of the mean (n ≥ 7). Differences were considered statistically significant at P values of ≤0.05. WT, Cav-1^+/+ (wild type); C1KO, Cav-1^−/− (knockout).

FIG. 4.

Increased production of serum nitric oxide in Cav-1^−/− mice in response to serovar Typhimurium infection. Mice were bled after 3 days of challenge with serovar Typhimurium, and the serum nitric oxide levels were determined (n ≥ 7). Differences were considered statistically significant at P values of ≤0.05.

FIG. 5.

Increased production of inflammatory cytokines from LPS-stimulated Cav-1^−/− macrophages. Macrophages from Cav-1^+/+ (WT, wild type) and Cav-1^−/− (KO, knockout) mice were isolated by peritoneal lavage. Cav-1^+/+ and Cav-1^−/− macrophages were cultured with 1 μg/ml of serovar Typhimurium for 24 h. (A) Western blot analysis of caveolin-1 expression levels are shown. β-actin was employed as an equal loading control. Note the increase in caveolin-1 expression in response to LPS in Cav-1^+/+ macrophages. (B and C) Concentrations of chemokines and cytokines in the culture supernatants were measured by LINCOplex. Data are the mean ± standard error of the mean from triplicate wells. Differences were considered statistically significant at P values of ≤0.05.

FIG. 6.

Increased nitric oxide production and iNOS activity in Cav-1^−/− macrophages. Macrophages from Cav-1^+/+ (WT, wild type) and Cav-1^−/− (KO, knockout) mice were isolated by peritoneal lavage. Cav-1^+/+ and Cav-1^−/− macrophages were cultured with 1 μg/ml of serovar Typhimurium for 24 h, and nitric oxide levels were measured or iNOS expression was assessed from lysates. Data are the mean ± standard error of the mean from triplicate wells. Differences were considered statistically significant at P values of ≤0.05.

FIG. 7.

Reduced STAT3 phosphorylation in Cav-1^−/− macrophages. Cav-1^+/+ (WT, wild type) and Cav-1^−/− (KO, knockout) macrophages were cultured with 1 μg/ml of serovar Typhimurium for 0, 0.5, 1, 3, and 24 h. Lysates from stimulated macrophages were tested for pSTAT3, stripped, and reprobed for STAT3.

FIG. 8.

Granuloma burden and histopathological evaluation of liver section stained by H-E. (A) Liver granulomas from Cav-1^+/+ and Cav-1^−/− mice (n ≥ 5) infected with 1 × 10³ CFU serovar Typhimurium were counted in 25 light microscopic fields. Data are the mean ± standard error of the mean. Statistical analysis by a Student's t test revealed that there was no statistically significant difference. (B) Cav-1^−/− mice show an increased amount of liver necrosis at 3 days postinfection.

FIG. 9.

Neutrophil recruitment in serovar Typhimurium-infected Cav-1^+/+ and Cav-1^−/− mice. (A) Cav-1^−/− mice show an increase in neutrophil recruitment in liver granulomas at 3 days postinfection. (B) Spleen sections from serovar Typhimurium-infected Cav-1^+/+ mice show neutrophil infiltration in the white pulp, while sections from Cav-1^−/− mice lack any such infiltration.