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Infection and Immunity, December 2006, p. 6665-6674, Vol. 74, No. 12
0019-9567/06/$08.00+0     doi:10.1128/IAI.00949-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Caveolin-1-Deficient Mice Show Defects in Innate Immunity and Inflammatory Immune Response during Salmonella enterica Serovar Typhimurium Infection{triangledown} ,{dagger}

Freddy A. Medina,1,2 Cecilia J. de Almeida,1,2 Elliott Dew,1,2 Jiangwei Li,1,2 Gloria Bonuccelli,1,2 Terence M. Williams,1,2 Alex W. Cohen,1,2 Richard G. Pestell,2 Philippe G. Frank,1,2 Herbert B. Tanowitz,3 and Michael P. Lisanti1,2,4*

Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461,1 Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107,2 Departments of Pathology and Medicine, Albert Einstein College of Medicine, Bronx, New York 10461,3 Muscular and Neurodegenerative Disease Unit, University of Genova and G. Gaslini Pediatric Institute, Largo Gaslini 5, 16147 Genova, Italy4

Received 14 June 2006/ Returned for modification 23 July 2006/ Accepted 2 September 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Salmonella is one of the most common causes of food poisoning in the United States. This gram-negative bacterium most commonly causes gastroenteritis in infected individuals but occasionally can result in a life-threatening systemic infection. It is estimated that 2 to 4 million cases of salmonellosis occur in the United States annually, although there are only over 40,000 reported cases each year (3). Salmonella enterica serovar Typhimurium-infected mice do not display typical symptoms from individuals affected with this pathogen but, rather, develop systemic disease resembling human typhoid fever. This infection model has provided insight into Salmonella pathogenesis since mice are resistant to S. enterica serovar Typhi (16). Once Salmonella organisms are ingested, they travel through the gastrointestinal tract until they encounter M cells of the Peyer's patches, which facilitate their entry into the underlying tissue. The organisms invade epithelial cells, where they multiply intracellularly and subsequently spread hematogenously to other organs. Growth mainly occurs in the liver and the spleen resulting in hepato-spleenomegaly. Within these organs bacteria are phagocytosed by macrophages that compose the reticuloendothelial system and spread of the organism to other organs is controlled (11). Control of Salmonella infection is also mediated by the host immune system, which releases a number of proinflammatory cytokines such as interleukin-1 (IL-1), IL-6, IL-12, tumor necrosis factor alpha (TNF-{alpha}), and gamma interferon (IFN-{gamma}) (11). However, these cytokines are also responsible for causing organ damage.

Caveolae are a subset of lipid rafts that are rich in glycosphingolipids and cholesterol. Expression of caveolin-1 drives the formation of this lipid raft subset to create a characteristic 50- to 100-nm flask-shaped membrane invagination (27, 33, 46). Our previous studies have shown that mice deficient in caveolin-1 expression are also devoid of caveolae (31). Caveolin-1 organizes and modulates the function of molecules involved in signal transduction by acting as a scaffold to which they bind. Although research has shown that caveolin-1 mainly negatively regulates proteins that it interacts with, it also can function to enhance signaling. Previous studies have shown that signaling molecules such as heteromeric G proteins, inducible nitric oxide synthase (iNOS), and Src family tyrosine kinases accumulate in caveolae. Therefore, it is not surprising that caveolae have been implicated in a variety of cellular processes such as endocytosis, apoptosis, cholesterol trafficking, proliferation, and signaling. Constitutive expression of caveolin-1 occurs in adipocytes, endothelial cells, muscle cells, and fibroblasts, while regulated expression has been observed in immune cells.

The existence of caveolar lipid rafts in immune cells was once a controversial topic. There is now an increasing amount of evidence showing the presence of caveolin-1 in a variety of immune cells (13, 18, 19). Interestingly, expression of caveolin-1 has been shown to occur in a regulated manner in response to lipopolysaccharide (LPS) (21). However, it still remains unknown whether caveolin-1 plays a role in the development of more complex immune responses that occur against pathogens.

A number of studies have shown the association of pathogens with caveolae. In many cases, particularly for bacterial pathogens and their endotoxins, such interactions might have evolved to facilitate entry of the pathogen into host cells, avoiding routes that would lead normally to pathogen destruction (7). Serovar Typhimurium enters host cells through lipid rafts by utilizing a mechanism that is dependent on the presence of cholesterol (20).

Here, we report for the first time a role for caveolin-1 (Cav-1) in bacterial pathogenesis. Cav-1–/– mice displayed an increased bacterial tissue burden and susceptibility to serovar Typhimurium. This was combined with a significant increase in nitric oxide, cytokine, and chemokine production. Moreover, macrophages were shown to be responsible for this defect.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents. The anti-caveolin-1 (N-20) rabbit polyclonal antibody directed against caveolin-1 residues 2 to 21 was obtained from Santa Cruz Biotechnology, Inc. The polyclonal antibody that recognizes iNOS was purchased from BD Pharmingen. Antibodies directed against total STAT3 and phospho-STAT3 (pSTAT3) were obtained from BD Pharmingen (San Jose, CA). The antibody directed against murine neutrophils was purchased from Serotec, Inc. (Raleigh, N.C.). Anti-F4/80 was purchased from R&D Systems (Minneapolis, MN). Other reagents were obtained from the indicated commercial sources: cell culture reagents (Life Technologies, Gaithersburg, MD) and S. enterica serovar Typhimurium lipopolysaccharide (Sigma, St. Louis, MO).

Animals. All animals were housed and maintained in a pathogen-free environment/barrier facility at the Institute for Animal Studies at the Albert Einstein College of Medicine under National Institutes of Health guidelines. Cav-1–/– mice were generated as we previously described (31). Cav-1+/+ and Cav-1–/– mice were generated through heterozygous matings and are in the C57BL/6 genetic background.

S. enterica serovar Typhimurium culture. The serovar Typhimurium strains used were SB300 (mouse passaged wild-type SL1344; rpsL hisG) and the auxotrophic mutant SL7207, which was used as a live attenuated vaccine. Overnight cultures of bacteria were grown in low-salt (0.09 M NaCl) LB medium and subcultured in high-salt (0.3 M NaCl) LB medium until an optical density of 0.8 at 600 nm was obtained. Bacteria were harvested by centrifugation, washed twice in phosphate-buffered saline (PBS), and appropriately diluted.

Infection with S. enterica serovar Typhimurium. Inocula were prepared by diluting serovar Typhimurium cultures in PBS to 1 x 103 CFU/100 µl for intravenous (i.v.) and 1 x 105 CFU/100 µl for oral (or per os [p.o.]) administration. Age-matched mice (12 to 14 weeks) were infected by oral gavage or were injected in the tail vein with serovar Typhimurium SB300 and monitored daily for survival. The number of administered CFU was confirmed by plating serial dilutions of the resuspended inoculum. Livers and spleens from Salmonella-infected mice were removed at various times after infection and homogenized in PBS with a Polytron homogenizer. Serial dilutions of liver and spleen homogenates were prepared and plated in duplicate in LB agar plates containing 50 µg/ml streptomycin. Colonies were counted after an overnight incubation at 37°C.

Determination of nitric oxide, chemokine, and cytokine levels. Serum and cell culture supernatant nitric oxide levels were measured using a nitric oxide colorimetric assay kit (Stressgen). Serum from 3-day-infected mice and culture supernatants from peritoneal macrophages were analyzed for cytokine levels using LINCOplex technology. Samples were read using a Luminex reader (Linco Research, St. Charles, MO). Briefly, Luminex beads were incubated with 25 µl of filtered serum in a blocked 96-well plate overnight at room temperature. After a washing step, premixed detection antibodies were added to each well and incubated for 1 h. Streptavidin-phycoerythrin was added, and samples were incubated for an additional 30 min. Samples were washed and read in a Luminex 100 machine to determine cytokine and chemokine concentrations.

Mouse peritoneal macrophages. Mouse peritoneal macrophages were harvested from 10 to 25 naïve mice. Macrophages were plated at a density of 2 x 105 to 5 x 105 in 24-well plates and allowed to adhere for 2 h after cells were harvested with a 6-ml peritoneal lavage. Nonadherent cells were removed by washing, and macrophages were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum.

Salmonella intracellular survival. Salmonella cultures were incubated for 20 min in DMEM containing 10% normal mouse serum to allow opsonization. Infection of resident peritoneal macrophages was performed using opsonized or nonopsonized bacteria at a multiplicity of infection of 10 for 30 min at 37°C. Cells were washed three times with PBS and incubated for 90 min at 37°C in DMEM containing 10% fetal calf serum and 100 µg/ml gentamicin to kill the remaining extracellular bacteria. Internalized bacteria were obtained by lysing macrophages with 0.1% Triton X-100 for 10 min. The number of CFU was determined by plating serial dilutions in duplicate on LB plates containing 100 µg/ml streptomycin.

Histopathological examination. Spleens and livers from 3-day-infected mice were collected and fixed in 10% phosphate-buffered formalin, and paraffin-embedded sections (5 µM) were prepared. Sections were stained by hematoxylin and eosin (H-E) or for the presence of neutrophils and macrophages to examine granuloma formation. Fixed sections were deparaffinized and subjected to antigen retrieval by microwave irradiation in 0.01 M, pH 6, trisodium citrate buffer for the anti-neutrophil antibody or a 30-min 0.2% trypsin digest for the anti-F4/80 antibody. Endogenous peroxidase was quenched using 0.3% hydrogen peroxide in methanol. Sections were blocked with normal rabbit serum and incubated with primary antibodies. Primary antibodies were detected using biotinylated rabbit anti-rat immunoglobulin G, followed by avidin biotinylated-horseradish peroxidase complexes (Vectastain ABC kit; Vector Laboratories). Sections were developed with diaminobenzidine (Vector laboratories) and counterstained with hematoxylin.

Western blotting. Macrophages were stimulated for various times with LPS. Subsequently, they were washed with PBS and treated with lysis buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 1% Triton X-100, 60 mM octyl glucoside) containing protease inhibitors (Boehringer Mannheim). Cell lysates were then centrifuged at 12,000 x g for 10 min to remove insoluble debris. Protein concentrations were quantified using the bicinchoninic acid reagent (Pierce), and the volume required for 40 µg of protein was determined. Samples were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12.5% acrylamide) and transferred to nitrocellulose. The nitrocellulose membranes were stained with Ponceau S (to visualize protein bands), followed by immunoblotting analysis. All subsequent wash buffers contained 10 mM Tris, pH 8.0, 150 mM NaCl, and 0.05% Tween-20, which was supplemented with 1% bovine serum albumin and 2% nonfat dry milk (Carnation) for the blocking solution and the antibody diluent. Primary antibodies were used at a 1:500 dilution. Horseradish peroxidase-conjugated secondary antibodies (1:5,000 dilution; Pierce) were used to visualize bound primary antibodies with the Supersignal chemiluminescence substrate (Pierce).

Statistics. Statistical analysis of data was performed using the log rank test for survival studies and by a two-tailed Student's t test from GraphPad Prism (San Diego, CA). Differences between experimental groups were considered significant for P values of <0.05. Data are representative of at least three independent experiments.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cav-1–/– mice are highly susceptible to S. enterica serovar Typhimurium strain SB300. Cav-1+/+ and Cav-1–/– mice were challenged i.v. with 1 x 103 CFU of serovar Typhimurium in order to determine if caveolin-1 had any role in the host response against this pathogen. The median survival for Cav-1+/+ mice was 13 days, and mice were able to survive up to 23 days after infection. In contrast, Cav-1–/– mice displayed a lower survival rate, with a median survival of 7 days. All Cav-1–/– mice had succumbed to the infection within 6 to 8 days (Fig. 1A). Mice were also administered serovar Typhimurium orally, since this is the natural route utilized by the bacteria for infection. Results mirrored those obtained with i.v. challenge. Cav-1–/– mice showed a median survival of 9 days, which was significantly less than the median survival of 14 days for Cav-1+/+ mice (Fig. 1B). These results demonstrate that caveolin-1 exerts a protective effect against serovar Typhimurium.


Figure 1
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  		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 x 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).

 
To evaluate the memory responses of Cav-1–/– mice against serovar Typhimurium, mice were vaccinated i.v. with 5 x 105 CFU of strain SL7207, an attenuated auxotrophic mutant. At day 45 postvaccination, mice were challenged with a lethal dose of serovar Typhimurium. There was no significant difference in survival between the groups (see Fig. S1 in the supplemental material).

Cav-1–/– mice display increased bacterial burden in tissues. Bacterial growth was assessed at various intervals in the spleens and livers of S. enterica serovar Typhimurium-infected mice to determine whether it might play a role in the increased susceptibility of Cav-1–/– mice. After 24 h of infection, Cav-1–/– mice exhibited a slightly higher but not significant bacterial load in both the liver and spleen. The number of bacteria found in the spleens and livers of infected animals was significantly higher at 3 days postinfection. Cav-1–/– mice had ~3-fold and ~6.5-fold increases in the spleens and livers, respectively. These differences were greater at 5 days postinfection, when the bacterial load in the spleen of Cav-1–/– mice exhibited a ~37-fold increase and a ~95-fold increase in the liver (Fig. 2A and B).


Figure 2
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  		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 x 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.

 
Salmonella targets the reticuloendothelial system, which is mainly composed of phagocytic cells such as macrophages, in order to replicate and survive. Therefore, we isolated resident peritoneal macrophages from Cav-1+/+ and Cav-1–/– mice to examine their ability to ingest opsonized Salmonella. We did not observe any difference in the uptake of opsonized bacteria regardless of the presence or absence of caveolin-1 (see Fig. S2 in the supplemental material).

Increased chemokine, cytokine, and nitric oxide production in Cav-1–/– mice. Infection with Salmonella results in the production of a number of chemokines and cytokines, such as IFN-{gamma}, TNF-{alpha}, and IL-6, which have been shown to have a role in the control of Salmonella infection in mice. Once mice are unable to control serovar Typhimurium and the infection becomes overwhelming and systemic, the production of cytokines and nitric oxide becomes deleterious. Therefore, we examined whether the susceptibility Cav-1 –/– mice might be a result of altered chemokine and cytokine expression. Results show that Cav-1 –/– mice have exaggerated production of the chemokines CCL3, CXCL1, and CXCL10 and the cytokines IFN-{gamma}, TNF-{alpha}, and IL-6 at 3 days postinfection (Fig. 3A and B). Concentrations of CCL2, CCL5, IL-1{alpha}, and IL-12 were similar between the groups. Production of the anti-inflammatory cytokine IL-10 was reduced, although this was not statistically significant (Fig. 3B).


Figure 3
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  		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).

 
We next investigated if nitric oxide, which plays a role in containing microbial proliferation, could also be up-regulated. Nitric oxide levels in the sera of Cav-1–/– mice challenged with serovar Typhimurium were nearly double the levels found in Cav-1+/+ mice (Fig. 4). Both groups had comparable low levels of serum nitric oxide in noninfected mice (data not shown).


Figure 4
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  		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.

 
Cav-1–/– macrophages also have increased chemokine, cytokine, and nitric oxide production. Several of the key chemokines and cytokines expressed in response to Salmonella LPS are produced by macrophages. Our results show that caveolin-1 is induced in peritoneal macrophages when they are stimulated with Salmonella LPS (Fig. 5A). Furthermore, as observed in vivo, levels of CXCL1, CXCL10, CCL5, TNF-{alpha}, and IL-6 are significantly increased in Cav-1–/– macrophages compared to Cav-1+/+ (Fig. 5B and C). We also examined nitric oxide production from macrophages in vitro with the same results as during infection in vivo. Cav-1–/– macrophages had a higher expression level of iNOS that resulted in an increased production of nitric oxide compared to wild type (Fig. 6A and B).


Figure 5
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  		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. ß-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.

 

Figure 6
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  		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.

 
Reduced Stat3 signaling in Cav-1–/– macrophages. Previous studies demonstrated that caveolin-1 binds to Stat3 (38). We next examined Stat3 signaling in isolated peritoneal macrophages treated with serovar Typhimurium LPS since mice deficient in this gene exhibit exacerbated cytokine production (15, 22, 23). Macrophage lysates were examined at several time points for pSTAT3 levels, followed by normalization to total STAT3. Total levels of STAT3 remained unchanged after LPS stimulation. However, differences in pSTAT3 were detected. Decreased pSTAT3 levels were detected 1 h after stimulation and continued up to 24 h (Fig. 7).


Figure 7
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  		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.

 
Histopathology of Cav-1–/– mice infected with Salmonella. In order to understand the pathological changes occurring in Cav-1–/– mice during infection, we examined histological sections of spleens and livers since these are the major organs where Salmonella replication occurs. Examination of liver sections revealed a slight increase in the number of granulomas in infected Cav-1–/– mice, but the differences between the groups were not statistically significant (Fig. 8A). No size differences between the groups were observed (data not shown). Liver sections from Cav-1–/– mice showed an increased prevalence of necrotic tissue (Fig. 8B). Interestingly, the nonnecrotic liver granulomas from Cav-1–/– mice showed increased neutrophil infiltration in granulomas compared to Cav-1+/+ mice (Fig. 9A). The microabscesses contained mostly neutrophils with some macrophages. We did not observe any differences in macrophage infiltration. They were found mostly scattered in sections from both mouse genotypes (data not shown). Examination of H-E-stained spleen sections from infected Cav-1+/+ mice revealed infiltration of white pulp by polymorphonuclear leukocytes. Surprisingly, Cav-1–/– mice lacked such infiltration (data not shown). Immunohistochemical staining revealed that the infiltration was mostly neutrophilic in nature. As seen in Fig. 9B, spleens of Cav-1–/– mice had a pronounced deficiency in neutrophil of accumulation.


Figure 8
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  		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 x 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.

 

Figure 9
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  		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.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results demonstrate that caveolin-1 plays a critical role in protection against a virulent S. enterica serovar Typhimurium strain. Cav-1–/– mice infected with S. enterica serovar Typhimurium both orally and intravenously had a significant reduction in survival. In addition, Cav-1–/– mice displayed a reduced ability to resist serovar Typhimurium growth. This resulted in an increased bacterial burden in their livers and spleen. These in vivo studies suggest that host susceptibility to infection is, at least in part, determined at the very early stages of infection, before the development of adaptive immunity. Therefore, the rapid susceptibility of Cav-1–/– mice suggests that caveolin-1 contributes to the host's innate response against serovar Typhimurium. To our knowledge, this is the first report showing a role for caveolin-1 in the host-mediated response against a bacterial pathogen.

A number of pathogens have been shown to enter cells through lipid rafts. For example, cholesterol depletion, which disrupts all classes of lipid rafts, including caveolae, inhibits entry by several Chlamydia trachomatis serovars (24, 41). Likewise, the expression of dominant negative Cav-1 mutants, interrupting caveolar endocytosis, also limits the uptake of simian virus 40, a well-described virus which selectively enters cells via a caveolae-mediated process (35). Furthermore, signal transduction events initialized by bacteria and viruses seeking cellular entry are often localized to molecules residing within caveolae and even involve the tyrosine phosphorylation of Cav-1 (5, 24, 25, 29, 30). Several studies have shown that inhibition of these caveolae-dependent signaling events results in the ablation of specific bacteria and virus entry into cells (30, 42). In addition, it has been shown that the receptors for bacteria and viruses copurify with Cav-1 and localize to caveolae, thus indicating that these structures may provide an abundant means by which pathogenic agents gain entry into host cells (4, 24, 39, 40). Pathogen entry via caveolae is an attractive means of access into host cells. Bacteria utilize many mechanisms to avoid the host immune system. For example, pathogens can avoid being killed by evading the lysosomal or endosomal fusion pathways. This can occur as a result of the pathogen modification of lysosomes to prevent fusion, the pathogen's inherent ability to survive the harsh lysosomal environment, and the use of caveolae to enter cells. Another advantage of caveolar entry is the concentration of signaling molecules and the availability of key protein mediators of vesicle formation, docking, and fusion (36, 45). Therefore, it is not surprising that a number of pathogens exploit caveolae to enter cells.

Salmonella entry into host cells is a complex and active, rather than passive, process. This microbe utilizes a bacterial type III secretory system to induce its uptake into host cells by exporting proteins across its cell wall and the host cell vacuolar membrane and into the cytosol (26). In addition, this secretory system is essential for replication in macrophages (6, 14). Previous studies have shown that cholesterol accumulates at the entry site of serovar Typhimurium. Furthermore, ablation of cholesterol renders serovar Typhimurium unable to penetrate host cells. Some of the components of the type III secretion system from serovar Typhimurium appear to associate with lipid rafts and may play a role in inducing its reorganization to initiate bacterial uptake. Once inside the cell, Salmonella resides in a cholesterol-rich vacuolar compartment that it actively modifies to avoid fusion with lysosomes (2).

The increased susceptibility of Cav-1–/– mice demonstrated that caveolae play a role in controlling serovar Typhimurium. Cav-1–/– mice started displaying a significant increase in their bacterial burden after 3 days of being infected. These data suggested that caveolin-1 could be altering the ability of serovar Typhimurium to enter and infect tissues. Therefore, we examined whether uptake of serovar Typhimurium was compromised in Cav-1–/– macrophages. Our results showed that serovar Typhimurium infectivity was not altered in Cav-1–/– macrophages and that caveolin-1 signaling is not necessary for is its internalization. Therefore, serovar Typhimurium entry into macrophages involves utilization of lipid rafts but not cholesterol-rich caveolae.

Our results demonstrate that Cav-1–/– mice displayed an exacerbated production of several chemokines, such as IFN-{gamma}, TNF-{alpha}, and IL-6, and chemokines. In addition, serum nitric oxide levels were nearly double of those observed in Cav-1+/+ mice. Interestingly, despite the enhanced production of these inflammatory mediators and nitric oxide, Cav-1–/– mice succumbed to serovar Typhimurium infection at a faster rate than Cav-1+/+ mice. Nitric oxide is essential for protecting the host from Salmonella as it displays cytoprotective, immunoregulatory, and antimicrobial properties. Paradoxically, an overactive response to Salmonella LPS may cause deleterious effects to the host, resulting in toxic shock that is associated with tissue damage and eventual death.

Macrophages are one of the main targets during Salmonella infection. Not only do they serve as a site for bacterial replication, but they also alert the immune system of an imminent infection. Our experiments demonstrated that macrophages were responsible for the exaggerated immune response observed in Cav-1+/+ mice. The control of Salmonella by the immune system requires production of chemokines and cytokines (8). LPS is the major constituent of the outer membranes of gram-negative bacteria, such as serovar Typhimurium, and is known to be responsible in the induction of chemokine and cytokine production. Previous studies have shown that Salmonella induces secretion of the proinflammatory cytokines IL-1, TNF-{alpha} and IL-6, as well as several chemokines (macrophage inflammatory protein 1{alpha} [MIP-1{alpha}], MIP-1ß, MIP-2{alpha}, and KC) and hematopoietic growth and survival factors (granulocyte-macrophage colony-stimulating factor) (34, 47). Studies of mice treated with antibodies against specific cytokine or chemokines and of mice where these inflammatory mediators have been knocked out show their importance in controlling Salmonella infection and enhancing host survival (1, 9, 12) and indicate that they have the capacity to initiate a protective systemic inflammatory response in the host that is aimed at elimination of gram-negative bacteria. Previous studies and our observations show that caveolin-1 expression occurs in a regulated manner in response to LPS. These results suggest that caveolin-1 may play a role in immune regulation in macrophages. Resident peritoneal macrophages stimulated with Salmonella LPS partially recapitulated our in vivo observations. This suggests that other immune or nonimmune cells may be involved in the increased chemokine and cytokine production. LPS also stimulates production of reactive nitrogen intermediates that lead to bacterial killing. Supernatants from Cav-1–/– macrophages stimulated with Salmonella LPS displayed elevated levels of nitric oxide. This correlates with the increased expression of iNOS observed in Cav-1–/– macrophages. Previous studies have shown that caveolin-1 can bind directly to iNOS. Furthermore, expression of caveolin-1 in a human colon carcinoma cell line that expresses very low levels of caveolin-1 results in decreased iNOS expression and activity. Other studies have observed an increase in iNOS expression in the hearts of Cav-1–/– mice. These results show that caveolin-1 plays a key immunoregulatory role in macrophages.

Caveolin-1 has been shown to colocalize with STAT3 (37). Additional studies showed that caveolin-1 binds directly to STAT3 and pSTAT3 (38). We explored whether caveolin-1 played a role in STAT3 signaling in Cav-1–/– macrophages because mice deficient in STAT3 also exhibit exaggerated immune responses to LPS similar to those observed in Cav-1–/– mice (15, 22, 23, 43). Our results showed that the reduced phosphorylation of STAT3 in Cav-1–/– macrophages suggests that caveolin-1 may play a role in controlling cytokine production through STAT3. It is also possible that caveolin-1 may also regulate cytokine signaling through a series of conserved residues it contains in its scaffolding domain that it shares with the suppressor of cytokine signaling pseudosubstrate domain (28).

The production of chemokines in response to Salmonella in vivo results in recruitment of leukocytes to the site of infection. Salmonella preferentially targets the reticuloendothelial system found in the liver and spleen of animals. Therefore, we conducted experiments to determine whether the augmented production of cytokines and chemokines in Cav-1–/– mice was associated with increased leukocyte trafficking and organ damage. Sections of livers and spleens were stained and evaluated for histopathological changes at 3 days after challenge. Despite the increased chemokine production, the number of granulomas formed in the liver of Cav-1–/– mice was not higher. However, the enhanced expression of chemokines from Cav-1–/– mice is likely responsible for the increased neutrophil infiltrate in granulomas. Neutrophils are one of the earliest immune cells recruited during serovar Typhimurium infection, while changes in macrophage numbers occur in the subsequent days of infection (17). This may explain why we did not observe any differences in the number of macrophages found in granulomas. Interestingly, while neutrophils were readily observed in the white pulp of the spleen of Cav-1+/+ mice, we did not observe any neutrophil infiltrate in Cav-1–/– mice. The importance of neutrophils in Salmonella immunity has been previously established in studies where antibody depletion of neutrophils using an antibody rendered mice more susceptible to infection (44). In addition to these findings, we observed an increased amount of liver damage and necrosis in Cav-1–/– mice.

In summary, our current findings are important because they demonstrate conclusively for the first time that caveolin-1 plays a role in immunity against bacterial pathogens. Although caveolae did not appear to be necessary for Salmonella invasion, we were able to show that caveolin-1 is expressed in macrophages, it plays a role in innate immune defense, and it regulates macrophage cytokine production and signaling.

One limitation of the current study is that Cav-1–/– mice also show a ~95% down-regulation of caveolin-2 expression (31). Thus, caveolin-2 may also play a role in the phenotypes that we describe here. However, previous studies using Cav-1–/– mice have shown that virtually all of the Cav-1–/– mouse phenotypes are due to loss of caveolin-1, and not caveolin-2, expression (32). Nevertheless, future studies using Cav-2 –/– mice will be needed to definitively clarify this issue. In addition, further studies are clearly warranted to determine if the Cav-1–/– phenotypes that we observe here are also generally applicable to other gram-negative, as well as gram-positive, bacterial pathogens.

Finally, it is possible that the Cav-1–/– phenotypes we observe may be due to changes in the metabolism of cholesterol in macrophages. We along with others have previously shown that caveolin-1 is involved in regulating the trafficking of cholesterol (reviewed in reference 33). Most recently, we evaluated the status of cholesterol homeostasis in Cav-1–/– macrophages (10). Interestingly, we showed that Cav-1–/– macrophages, upon cholesterol loading, are significantly enriched in esterified cholesterol but depleted of free cholesterol, compared with their wild-type counterparts (10). Thus, these changes in free cholesterol content may also contribute to alterations in lipid raft-mediated signaling in macrophages.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the National Institutes of Health and the American Heart Association. Cecilia J. de Almeida was supported by a Fogarty International Training Grant (1D43TW007129). This project was funded, in part, under a grant from the Pennsylvania Department of Health.

The Pennsylvania Department of Health specifically disclaims responsibility for any analyses, interpretations or conclusions.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Cancer Biology, Kimmel Cancer Center, Bluemle Life Sciences Building, Room 933, 233 S. 10th Street, Philadelphia, PA 19107. Phone: (215) 503-9295. Fax: (215) 923-1098. E-mail: michael.lisanti{at}jefferson.edu. Back

{triangledown} Published ahead of print on 18 September 2006. Back

Editor: W. A. Petri, Jr.

{dagger} Supplemental material for this article can be found at http://iai.asm.org/. Back


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 Introduction
 Materials and Methods
 Results
 Discussion
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Infection and Immunity, December 2006, p. 6665-6674, Vol. 74, No. 12
0019-9567/06/$08.00+0     doi:10.1128/IAI.00949-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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