infect. immun. doi:10.1128/iai.00949-06 accepted · or a 30 minute 0.2% trypsin digest for the...

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Salmonella Pathogenesis in Caveolin-1 (-/-) Deficient Mice:

Role of Cav-1 in Innate Immunity and the Inflammatory Immune Response

Freddy A. Medina 1,2, Cecilia J. de Almeida 1,2, Elliott Dew 1,2, Jiangwei Li 1,2, Gloria

Bonnucelli 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. Lisanti 1,2,4 *

1 Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461

2 Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA

19107

3 Departments of Pathology and Medicine, Albert Einstein College of Medicine, Bronx, NY 10461

4 Muscular and Neurodegenerative Disease Unit, University of Genova and G.Gaslini Pediatric Institute,

Largo Gaslini 5, 16147 Genova, Italy

Key Words: Caveolin-1, Bacterial Pathogenesis, Macrophage Immune Function, Salmonella

Running Title: Caveolin-1 in Innate Immunity

*Corresponding Author: Dr. Michael P. Lisanti, 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: [email protected] or [email protected]

Abbreviations used in this paper: p.o., per os; i.v., intravenous

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Copyright © 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.00949-06 IAI Accepts, published online ahead of print on 18 September 2006

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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 LPS. Here, we sought to determine the role of

caveolin-1 expression in Salmonella pathogenesis. Cav-1-/- mice displayed a significant decrease

in survival when challenged with Salmonella typhimurium. Spleen and tissue burdens were

significantly higher in Cav-1-/- mice. However, infection of Cav-1-/- macrophages with

Salmonella 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 are likely to be responsible

for the marked susceptibility of caveolin-1 deficient mice to Salmonella typhimurium.ACCEPTED


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Salmonella is one of the most common causes of food poisoning in the U.S. 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 U.S. annually, although there are only over 40, 000 reported

cases each year (1). Salmonella 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 Salmonella typhi (16). Once Salmonella 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 through blood circulation 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 IL-1, IL-6, IL-12, TNF- , IFN- (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

50-100 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 and are concentrated. Although it has been mainly shown that

caveolin-1 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,

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

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caveolin-1 occurs in adipocytes, endothelial cells, muscle cells, and fibroblast, while regulated

expression has been observed in immune cells .

The existence of the 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 LPS (21). However, it still remains unknown whether

caveolin-1 plays a role in the development 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). S. typhimurium enters host cells through lipid rafts

utilizing a mechanism that is dependent on the presence of cholesterol (20).

Here, we report for the first time a role for caveolin-1 in bacterial pathogenesis. Cav-1-/-

mice displayed an increased bacterial tissue burden and susceptibility to S. 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.ACCEPTED


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Materials and Methods

Reagents

The anti-caveolin-1 (N-20) rabbit polyclonal antibody (N-20) directed against caveolin-1

residues 2-21 was obtained from Santa Cruz Biotechnology, Inc. The polyclonal antibody that

recognizes inducible nitric oxide synthase (iNOS) was purchased from BD Pharmingen.

Antibodies directed against total STAT and phospho-STAT3 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 following commercial sources: cell culture reagents

(Life Technologies, Gaithersburg, MD); Salmonella 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 (NIH) guidelines. Cav-1-/- mice were generated as we previously described (Razani

2001). Cav-1+/+

and Cav-1-/-

mice were generated through heterozygous matings and are in the

C57Bl/6 genetic background.

Salmonella typhimurium culture

Salmonella 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 media and subcultured

in high salt (0.3 M NaCl) LB media until an optical density of 0.8 at 600 nm was obtained.

Bacteria were harvested by centrifugation, washed twice in PBS and appropriately diluted.

Infection with Salmonella typhimurium

Inocula were prepared by diluting S. typhimurium cultures in phosphate-buffered saline (PBS) to

1 x 103 CFU/100 l for intravenous and 1 x 10

5 CFU/100 l for oral administration. Age-

matched mice (12-14 weeks) were infected by oral gavage or injected in the tail vein with S.

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typhimurium SB300 and monitored daily for survival. Administered colony forming units were

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 three-day infected mice and culture supernatants

from peritoneal macrophages we 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 washing, pre-mixed detection antibodies were added to each well and

incubated for 1 hour. 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 10-25 naïve mice. Macrophages were

plated at a density of 2-5x105 in 24-well plates and allowed to adhere for 2 hours after harvesting

cells with a 6 ml peritoneal lavage. Non-adherent cells were removed by washing and

macrophages were cultured in Dulbecos’ modified Eagles 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 non-opsonized bacteria at a multiplicity of infection (M.O.I) 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% FCS and 100 g/ml gentamicin to kill the remaining extracellular bacteria.

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Internalized bacteria were obtained by lysing macrophages with 0.1% Triton X-100 for 10 min.

Colony forming units were determined by plating serial dilutions in duplicate on LB plates

containing 100 g/ml streptomycin.

Histopathological Examination

Spleens and livers from three-day infected mice were collected and fixed in 10% phosphate

buffered formalin and paraffin embedded sections (5 M) 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 minute 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 IgG followed by avidin biotinylated-horseradish peroxidase

complexes (Vectastain ABC kit: Vector Laboratories). Sections were developed with DAB

(Vector laboratories) and counterstained with hematoxylin.

Western blots

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 BCA reagent (Pierce), and the volume required for 40 g of protein was

determined. Samples were then separated by SDS-PAGE (12.5% acrylamide) and transferred to

nitrocellulose. The nitrocellulose membranes were stained with Ponceau S (to visualize protein

bands), followed by immunoblot 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 (BSA) 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 (1:5000 dilution, Pierce) were used to visualize bound primary antibodies

with the Supersignal chemiluminescence substrate (Pierce).

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Statistics

Statistical analysis of data was performed using the log-rank test for survival studies and by two-

tailed student’s t-test from GraphPad Prism (GraphPad, San Diego, CA). Differences between

experimental groups were considered significant for P values of < 0.05. Data is representative of

at least three independent experiments.

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Results

Cav-1-/- mice are highly susceptible to S. typhimurium strain SB300.

Cav-1+/+ and Cav-1-/- mice were challenged i.v. with 1x 103 C.F.U. of Salmonella

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 6-8 days

(Fig. 1A). Mice were also administered S. 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 S. typhimurium.

To evaluate the memory responses of Cav-1-/- mice against S. typhimurium, mice were

vaccinated i.v. with 5 x 105 CFU of SL7207, an attenuated auxotrophic mutant. At day 45 post-

vaccination, mice were challenged with a lethal dose of S. typhimurium. There was no significant

difference in survival between the groups (See Supplemental Fig. 1).

Cav-1-/-

mice display increased bacterial burden in tissues.

Bacterial growth was assessed at various intervals in the spleens and livers of S.

typhimurium infected mice to determine whether it might play a role in the increased

susceptibility of Cav-1-/-

mice. After 24 hours 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 post-

infection. Cav-1-/-

mice had ~3-fold and ~6.5-fold increases in the spleens and livers,

respectively. These differences were greater at 5 days post-infection where 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 & B).

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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 Supplemental Fig. 2).

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- , TNF- , 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 S. 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 may 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-

, TNF- , and IL-6 at 3 days post-infection (Fig. 3A &B). Concentrations of CCL2, CCL5, IL-

1 , and IL-12 were similar between the groups. Production of the anti-inflammatory cytokine

IL-10 was reduced,although is was not statistically significant (see figure Fig. 3B).

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 S. typhimurium were nearly double than those found in Cav-1+/+

mice (Fig. 4).

Both groups had comparable low levels of serum nitric oxide in non-infected mice (data not

shown).

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 stimulated with Salmonella LPS (Fig. 5A). Furthermore, as observed in vivo,

levels of CXCL1, CXCL10, CCL5, TNF- , and IL-6 are significantly increased in Cav-1-/-

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macrophages compared to Cav-1+/+ (Fig. 5B & C). We also examined nitric oxide production

from macrophages in vitro and found the same observations 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 & B).

Reduced Stat3 signaling in Cav-1-/- macrophages.

Previous studies have shown that caveolin-1 binds to Stat3 (38). We next examined Stat3

signaling in isolated peritoneal macrophages treated with S. 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 hour after

stimulation and continued up to 24 hours (Fig.7).

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 difference 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 non-necrotic liver granulomas from Cav-1-/-

mice

showed increased neutrophil infiltration in granulomas compared to Cav-1+/+ (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 spleen sections from infected Cav-1+/+

mice revealed infiltration of white pulp by polymorphonuclear leukocytes (PMN). Surprisingly,

Cav-1-/-

mice lacked such infiltration (data not shown). Immunohistochemical staining revealed

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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.

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Discussion

Our results demonstrate that caveolin-1 plays a critical role in protection against a

virulent S. typhimurium strain. Cav-1-/-

mice infected with S. typhimurium both orally and

intravenously had a significant reduction in survival. In addition, Cav-1-/-

mice displayed a

reduced ability to resist S. 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 S. 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

(SV) 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 co-

purify 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 by pathogen

modification of lysosomes to prevent fusion, the pathogen’s inherent ability to survive the harsh

lysosomal environment and utilizing caveolae to enter cells. Another advantage of caveolar

entry is the concentration of signaling molecules and the availability of key protein mediators of

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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 S. typhimurium.

Furthermore, ablation of cholesterol renders S. typhimurium unable to penetrate host cells.

Some of the components of the type III secretion system from S. 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 (3).

The increased susceptibility of Cav-1-/-

mice demonstrated that caveolae play a role in

controlling S. typhimurium. Cav-1-/-

mice started displaying a significant increase in their

bacterial burden after three days of being infection. This data suggested that caveolin-1 could be

altering the ability of S. typhimurium to enter and infect tissues. Therefore, we examined whether

uptake of S. typhimurium was compromised in Cav-1-/-

macrophages. Our results showed that

S. typhimurium infectivity was not altered in Cav-1-/-

macrophages and that caveolin-1 signaling

is not necessary for is its internalization. Therefore, S. typhimurium entry into macrophages

involves utilization of lipid rafts but not cholesterol rich caveolae.

Our results showed that Cav-1-/-

mice displayed an exacerbated production of several

chemokines, such as IFN- , TNF- , 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 S.

typhimurium infection at a faster rate than Cav-1+/+

mice. Nitric oxide is essential for protecting

host from Salmonella as it displays cytoprotective, immunoregulatory, and antimicrobial

properties. Paradoxically, an overactive response to Salmonella LPS may cause deleterious

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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 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 S. 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 and IL-6, as well as

several chemokines (MIP-1 , MIP-1 , MIP-2 , KC) and hematopoietic growth and survival

factors (GM-CSF) (34, 47). Studies of mice treated with antibodies against specific cytokine or

chemokines and in mice where these inflammatory mediators have been knocked out show their

importance in controlling Salmonella infection and enhancing host survival (2, 9, 12). and 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 Salmonella LPS partially recapitulated our in vivo observations. This

suggests that other immune or non-immune 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.

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Caveolin-1 has been shown to co-localize 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

suggest 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 (SOCS) 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 S. 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

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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 an ~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 formally possible that the Cav-1-/- phenotypes we observe may be due to

changes in the metabolism of cholesterol in macrophages. We and others have previously shown

that caveolin-1 is involved in regulating the trafficking of cholesterol (reviewed in (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, as 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.ACCEPTED


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Acknowledgements

This work was supported by grants from the National Institutes of Health (NIH), and the

American Heart Association (AHA).

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Figure Legends

Figure 1. Cav-1-/- mice display significantly reduced survival rates upon challenge with a highly

virulent S. typhimurium strain. Cav-1+/+ (dashed line) and Cav-1-/- (solid line) mice were

administered 1 x 103 C.F.U. i.v (A) or 104 C.F.U. 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 both i.v and p.o. challenged mice (P 0.001).

Figure 2. Numbers of live S. typhimurium 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 S. typhimurium and bacterial counts from (A) spleen and (B) liver homogenates were

determined by culture on agar plates at days 1, 3, and 5 post-infection. Data are represented as

the mean number of CFU ± s.e.m. Statistically significant differences were less than P 0.05.

Figure 3. Dramatic increase of serum cytokine production in Cav-1-/- mice in response to S.

typhimurium infection. Mice were bled after 3 days of challenge with S. typhimurium and the

serum (A) chemokine and (B) cytokine levels of the proinflammatory cytokines and the anti-

inflammatory cytokines were measured. Data are represented as the mean ± s.e.m. (n 7).

Statistically significant differences were less than P 0.05.

Figure 4. Increased production of serum nitric oxide in Cav-1-/- mice in response to S.

typhimurium infection. Mice were bled after 3 days of challenge with S. typhimurium and the

serum nitric oxide levels were determined (n 7). Statistically significant differences were less

than P 0.05.

Figure 5. Increased production of inflammatory cytokines from LPS stimulated Cav-1-/-

macrophages. Macrophages from Cav-1+/+ and Cav-1-/- mice were isolated by peritoneal lavage.

Cav-1+/+ and Cav-1-/- macrophages were cultured with 1 μg/ml of S. typhimurium for 24 h. A)

Western blot analysis of caveolin-1 expression levels are shown. -actin was employed as an

equal loading control. Note the increase in caveolin-1 expression in response to LPS in Cav-1+/+

macrophages. B & C) Concentrations of chemokines and cytokines in the culture supernatants

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were measured by LINCOplex. Data are represented as the mean ± s.e.m from triplicate wells.

Statistically significant differences were less than P 0.05.

Figure 6. Increased nitric oxide production and iNOS activity in Cav-1-/- macrophages.

Macrophages from Cav-1+/+ and Cav-1-/- mice were isolated by peritoneal lavage. Cav-1+/+ and

Cav-1-/- macrophages were cultured with 1 μg/ml of S. typhimurium for 24 h and nitric oxide

levels were measured or iNOS expression was assessed from lysates. Data are represented as the

mean ± s.e.m from triplicate wells. Statistically significant differences were less than P 0.05.

Figure 7. Reduced STAT3 phosphorylation in Cav-1-/- macrophages. Cav-1+/+ and Cav-1-/-

macrophages were cultured with 1 μg/ml of S. typhimurium for 0, 0.5, 1, 3 and 24 hrs. Lysates

from stimulated macrophages were tested for pSTAT3, striped and re-probed for STAT3.

Figure 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 S.

typhimurium were counted in 25 light microscopic fields. Data are represented as the mean ±

s.e.m. Statistical analysis by 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 post-infection.

Figure 9. Neutrophil recruitment in S. typhimurium infected Cav-1+/+ and Cav-1-/- mice. (A)

Cav-1-/- mice show an increase in neutrophil recruitment in liver granulomas at 3 days post-

infection. (B) Spleen sections from S. typhimurium infected Cav-1+/+ mice show neutrophil

infiltration in the white pulp,while sections from Cav-1-/- mice lack any such infiltration.

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infect. immun. doi:10.1128/iai.00949-06 accepted · or a 30 minute 0.2% trypsin digest for the...

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