Elevated inflammatory response in caveolin-1 deficient mice with P.

aeruginosa infection
is mediated by STAT3 and NF-B

Kefei Yuan*, Canhua Huang*¶, John Fox, Madeleine Gaid, Andrew Weaver, Guoping Li,
Brij B. Singh, Hongwei Gao# and Min Wu¶.

*The State Key Laboratory for Biotherapy, West China Hospital, Sichuan University,
Chengdu 610041, China; #Center for Experimental Therapeutics and Reperfusion Injury,
Department of Anesthesiology, Perioperative & Pain Medicine, Brigham and Women's
Hospital, Harvard Medical School; and Department of Biochemistry and Molecular Biology,
University of North Dakota, Grand Forks, ND58203-9037.
Running head: Cav-1 deficiency impacts P. aeruginosa infection
¶
Address correspondence to: min.wu@med.und.edu, Tel: 701 777-4875, Fax: 701 777-2382;
or hcanhua@hotmail.com, Tel: +86-13258370346, Fax: 86-28-85164060

negative strategy demonstrated that

Caveolin-1 (Cav-1), an important
composition protein within the
flask-shaped membrane invaginations
termed caveolae, may play a role in host
defense against infections. However, the
phenotype in P. aeruginosa-infected cav1
knockout (KO) mice is still unresolved
and the mechanism involved is almost
entirely unknown. Using a respiratory
infection model, we confirmed a crucial
role played by Cav-1 in host defense
against this pathogen because Cav-1 KO
mice showed increased mortality, severe
lung injury, and systemic dissemination
as compared to wild-type (WT)
littermates. In addition, cav1 KO mice
exhibited elevated inflammatory
cytokines (IL-6, TNF- and IL-12a),
decreased phagocytic ability of
macrophages, and increased superoxide
release in the lung, liver, and kidney. We
further studied relevant cellular signaling
processes and found that STAT3 and
NF-B are markedly activated. Our data
revealed that the Cav-1-STAT3-NF-κB
axis is responsible for dysregulated
cytokine response, which is attributable
to increased mortality and disease
progression. Moreover, down-regulating
Cav-1 in cell culture with a dominant
  1
STAT3 activation was essential for the
translocation of NF-B into the nucleus,
confirming the observations from cav1
KO mice. Collectively, our studies
indicate that Cav-1 is critical for
inflammatory responses regulating the
STAT3-NF-B pathway and thereby
impacting P. aeruginosa infection.
P. aeruginosa accounts for 25% of
Gram-negative bacteria isolated from
hospitals associated with high morbidity
and mortality (1). P. aeruginosa frequently
infects immunocompromised individuals,
such as those affected by
ventilator-associated infection, severe burns,
and cancer (2). More than 80% of cystic
fibrosis patients suffer severe P. aeruginosa
infection (3). This bacterium becomes
increasingly resistant to various antibiotics.
Further understanding the mechanism of
host-pathogen interaction may result in
effective approaches to preventing this
infection. Thus, P. aeruginosa has been a
focus of airway infectious diseases (1,4-6).
Recent studies suggest that P.
aeruginosa also invades the lung epithelial
cells through lipid raft-mediated
endocytosis (7-9), which is a possible
reason why this bacterium develops such
formidable resistance to antibiotics (10).
The invasion process of P. aeruginosa may
involve certain lipid raft-associated proteins,
including the caveolin family of proteins (9).
Using caveolin 1 (cav1) KO mice, two
recent studies investigated the role of Cav-1
during P. aeruginosa infection (11,12);
however, their observations were very
different. Thus, the role of Cav-1 in this
infection needs to be clearly characterized.
Caveolins are a family of integral
membrane proteins involved in caveolae
formation and receptor-dependant
endocytosis (13-15). Cav-1 and -2 are
co-expressed in various cells, such as
endothelial cells, airway epithelial cells, and
type I pneumocytes. Cav-1 is the major
component of caveolae, flask-shaped
plasma membrane invaginations. In the
absence of Cav-1, Cav-2 cannot reach the
plasma membrane and is degraded (16).
Through the hetero-oligomeric complex
formed between Cav-1 and Cav-2, Cav-2
can translocate to lipid rafts of the plasma
membrane. The lipid rafts, also known as
membrane microdomains, contain
glycosphingolipids, cholesterol, and
signaling/receptor proteins (17-19), and
play a crucial role in many cellular
activities including airway bacterial
invasion.
Signal transducers and activators of
transcription (STATs) are SH2 domain
containing transcription factors involved in
inflammatory response in carcinogenesis
and host defense (20-23). STATs are
activated through receptors for cytokines
and hormones, and these receptors do not
contain any intrinsic enzymatic activity,
thus they depend on tyrosine kinases that
can interact with the intracellular domain of
the receptor. STAT signaling is initiated by
phosphorylating and activating Janus
  2
kinases (JAKs), which in turn lead to
phosphorylation of tyrosine residues on
receptors (24). These receptors serve as
docking sites for STATs. Suppressors of
cytokine signaling (SOCSs) function as
negative regulators of STATs and operate by
binding and inhibiting JAKs or competing
with STATs for phosphotyrosine binding
sites on cytokine receptors (25). Recently,
STAT3 was found to interact with Cav-1
and heat shock protein 90 in plasma
membrane rafts during E. coli infection (26).
Cav-1 is also related to a JAK2/STAT5
pathway since Cav-1 is homologous to the
pseudosubstrate for SOCS. Thus, Cav-1
may down-regulate JAK/STAT pathways,
modulating pro-inflammatory response.
Since Cav-1 was found to be associated
with STAT3 in lipid rafts by a
co-localization study, it is also possible that
a Cav-1 cascade may impact the PI3K/Akt
pathway through molecular interactions,
which may serve as a regulator for STAT3
(27). Thus, Cav-1 may help maintain the
balance between host response and tissue
damage that may be caused by
overproduction of inflammatory cytokines.
To better characterize the role of Cav-1
in P. aeruginosa infection, we sought to
determine the pathogenic mechanism in
cav1 KO mice. We found that cav1 KO
mice manifested more severe infection,
including increased bacterial burdens,
inflammation, oxidative stress, and
susceptibility to infection. Our studies also
showed that the STAT3-NF-κB pathway is
responsible for the dysregulated response
during P. aeruginosa infection.
Experimental procedures
Mice-cav1 KO and WT control mice
(B6129SF2/J) were obtained from The
Jackson Laboratory (Bar Harbor, ME) (28).
Mice were housed and bred in the animal
facility at University of North Dakota
(UND), and the animal experiments were
performed in accordance to the institutional
animal care and use committee guidelines.
We anesthetized mice with 45 mg/kg
ketamine, and intranasally instilled 0.5×107
(PAK, 6 mice/group) to 1×107 (PAO1)
colony-forming units (CFUs) of P.
aeruginosa and mice were killed when they
were moribund (29). After
bronchoalveolar lavage (BAL), the trachea
and lung were excised for homogenization
or fixed in 10% formalin.
Cells-Mouse AM cells were isolated by
BAL as described (30). AM cells were
grown in RPMI 1640 medium
supplemented with 10% newborn calf
serum and penicillin/streptomycin
antibiotics in 5% CO2 incubator. MLE-12
cells were obtained from ATCC and
maintained following the manufacturer’s
instructions.
Bacteria strains-P. aeruginosa strain PAO1
wild-type (WT) was a gift from S. Lory
(Harvard Medical School, Boston, MA).
PAK and PAO1-GFP were obtained from G.
Pier (Channing Laboratory, Harvard
Medical School, Boston, MA) (31).
Infection experiments-Bacteria were grown
overnight in Luria-Bertani (LB) broth at
37°C with vigorous shaking. The next day,
the bacteria were pelleted by centrifugation
at 5,000 × g and resuspended in 10 ml of
fresh LB broth and allowed to grow until
the mid-logarithmic phase. OD 600 nm was
measured, density was adjusted to ～0.25
OD (0.1 OD = 1 × 108 cells/ml). Cells were
washed once with PBS after overnight
culture in serum containing medium and
changed to serum-free and antibiotic-free
medium immediately before infection (29).
Except dose-determination assays, cells
were infected by P. aeruginosa in
multiplicity of infection (MOI) of 10:1
  3
bacteria-cell ratio for indicated time points.
Cell transfection-MLE-12 cells were
transfected with yellow fluorescent protein
(YFP)-Cav-1, YFP-Cav-1Δ51-169
dominant negative (DN) plasmids using
LipofectAmine 2000 reagent (Invitrogen
Life Technologies, Carlsbad, CA) in
serum-free RPMI 1640 medium following
the manufacturer’s instruction. The cells
were lysed after 24 h transient expression
(32).
Inflammatory cytokine profiling-Cytokine
concentrations were measured by ELISA kit
(eBioscience company, San Diego, CA) in
samples of cell culture medium, BAL fluid
and lung homogenates collected at indicated
times after infection. The MLE-12 cells
were treated as described above. Culture
medium was collected after infection. For
BAL fluid, the trachea was surgically
exposed and cannulated, lungs were
lavaged 5 times with 1.0 ml volume of
lavage fluid, the lavageates were pooled,
and cells were removed by centrifugation.
For lung homogenates, excised lungs were
ground in 500 μl PBS. 96-well plates
(Corning Costar 9018) were coated with
100 μl/well of capture antibody in coating
buffer and incubated overnight at 4°C (33).
100ul aliquots of serum samples were
added to the coated microtiter wells. The
cytokine concentrations were determined
with corresponding detection
HRP-conjugated antibodies. The values
were read at 450 nm and analyzed.
Western blotting-Mouse monoclonal Abs
against Cav-1, phospho-STAT3, IL-6,
NF-κB, and rabbit polyclonal Ab against
phospho-NF-κB and goat polyclonal Abs
against TNF-α, SOCS3 were from Santa
Cruz Biotechnology. Rabbit monoclonal Ab
against glyceraldehydes-3-phosphate
dehydrogenase (GAPDH) was from Cell
Signaling Technology. The samples derived
from cells and lung homogenates were grown either on coverslips in a 24-well
lysed and quantitated. The lysates were plate or in glass-bottom dishes (MatTek,
boiled for 5 min, and protease inhibitor Ashland, MA). For immunostaining, the
cocktail added. The supernatants were cells were fixed in 3.7% paraformaldehyde,
collected, and an equal amount of each permeabilized with 0.2% Triton X-100 in
sample was loaded onto 10% PBS, and non-specific binding site was
SDS-polyacrylamide mini-gels and blocked with blocking buffer for 30 min.
electrophoresed to resolve proteins. The Cells were incubated with primary Abs at
proteins were then transferred to 1/500 dilution in blocking buffer for 1 h and
polyvinylidine difluoride membranes washed three times with wash buffer. After
(Pierce Biotechnology, Rockford, IL) and incubation with appropriate
blocked 2 h at room temperature using 5% fluorophore-conjugated secondary Abs, the
non-fat milk blocking buffer. Membranes coverslips were mounted on slides with
were incubated overnight at 4°C with Vecta-shield mounting medium (36). DAPI
appropriate first antibodies diluted at (Sigma-Aldrich, St. Louis, MO) was used to
1:1,000 in 5% bovine serum albumin (BSA) stain the nucleus. The images were captured
western antibody buffer. After washing by LSM 510 Meta confocal microscope
three times with washing solution, the (Carl Zeiss MicroImaging, Thornwood,
antigen-antibody complexes were incubated NY), and processed using the software
for 45 min at room-temperature with provided by the manufacturer.
horseradish peroxidase-conjugated Luciferase Assay-Transient transfections
secondary antibody (Rockland were performed with 2×105 cells plated in
Immunochemicals, Gilbertsville, PA) 6-well plates by using 2 μg of DNA and 3
diluted 1:2,000 (34). Signals were μl of Lipofect-Amine 2000 reagent
visualized using enhanced (Invitrogen Life Technologies, Carlsbad,
chemiluminescence detection kit CA) in serum-free RPMI 1640 medium
(SuperSignal West Pico; Pierce). following the manufacturer’s instruction. 24
RT-PCR analysis-RNA was extracted from h after transfection, the cells were infected
Lung homogenates and cells with Trizol with PAK at 10:1 MOI for 1 h (37). Cell
(Invitrogen Life Technologies, Carlsbad, lysates were subjected to luciferase activity
CA) according to the manufacturer’s analysis by using the Dual-Luciferase
instructions. For detected genes, reverse Reporter Assay System (Promega, Madison,
transcription (RT) was performed using 1.5 WI).
g of RNA, RNase ribonuclease inhibitor, 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltet
Oligo dT and cloned AMV reverse razolium bromide (MTT) assay-This assay
transcriptase (Invitrogen Life Technologies, measures color change of MTT upon
Carlsbad, CA). PCR products were reduction by enzymes to assess the viability
separated by 1.0% agarose gel of cells. BAL fluid was centrifuged and
electrophoresis containing ethidium cells were cultured in a 96 well plate. After
bromide and visualized under UV light. The incubation with P. aeruginosa, MTT dye
results for each gene were normalized in was added at final concentration of 1 μg/ml
comparison with GAPDH expression (35). per well. The cells were incubated at 37 °C
Confocal microscopy and indirect for 60 min or until color change occurred.
immunofluorescence staining-Cells were The dye is yellow in color and upon

  4
reduction by enzymes, which gave a blue
formazan product. The reaction was stopped
by adding 100 μl of stop solution (10%
DMSO; 10% SDS in 50 mM HEPES
buffer). The plate was left at room
temperature overnight for complete
dissolution of formazan crystals. Next day,
the plate was read at 560 nm absorbance
using a plate reader to quantify the dye
conversion (38). Duplicates were done for
each sample and control. Background
correction was done with blanks containing
dye alone.
NBT assay-This assay is based on color
change of NBT dye upon reduction by
released superoxide. Cells were treated as
above and same amount of dye added as
above. The dye is yellow in color and upon
reduction by superoxide, which gave a blue
formazan product (39).
Dihydrodichlorofluorescein diacetate
(H2DCF-DA or H2DCF) assay-H2DCF dye
(Molecular Probes, Carlsbad, CA) does not
normally fluoresce and emits green
fluorescence upon reaction with superoxide
inside cells. Cells were treated as above and
equal amount(s) of dye added. After 10 min
incubation, fluorescence was measured
using a fluorimeter (BioTek, Winooski, VT)
(39,40).
Lipid peroxidation assay-Malondialdehyde
(MDA) is an end product of lipid
peroxidation process, and was measured in
a colorimetric assay (Calbiochem, San
Diego, CA) according to the manufacturer’s
instructions. Homogenized lung tissue in
62.5 mM Tris-HCl (pH=6.8) supplemented
with Complete-Mini Protease Inhibitor
(Roche Diagnostics) in equal protein
amounts were used in the assay. Duplicates
were done for each sample and control (41).
Phagocytosis assay-AM cells obtained from
BAL fluid were plated in 96 well plates and
grown overnight. The cells were treated
  5
with the serum-free medium for 1 h. Then
GFP-PAO1 was used to infect the cells at
10:1 MOI. After 1 h incubation at 37 °C, the
wells were washed and treated with
polymyxin B 100 μg/ml for 1 h to kill any
remaining extracellular bacteria (29). The
number of phagocytosed bacteria was
counted using Synergy HT fluorimeter
(BioTek, Winooski, VT) with 485 ± 20 nm
excitation and 528 ± 20 nm emission filters.
Background correction was done for
autofluorescence. Duplicates were done for
each sample and control. Alternatively,
classical colony forming unit (CFU) was
performed to quantitate phagocytosis as
described previously.
Myeloperoxidase (MPO) assay-MPO assay
was performed as described. Samples were
homogenized in 50 mM
hexadecyltrimethylammonium bromide
(HTAB) 50 mM KH2PO4, pH 6.0, and 0.5
mM EDTA, 1 ml/100 mg tissue and
centrifuged or 15 min at 12,000 rpm at 4°C.
Supernatants were decanted and 100 ml of
reaction buffer (0.167 mg/ml O-dianisidine,
50 mM KH2PO4, pH 6.0 and 0.0005% mM
H2O2) was added to 100 ml sample.
Absorbance was read at 460 nm at 2 min
intervals. Duplicates were done for each
sample and control (41).
Bacteria burden assay-AM cells from BAL
fluid and ground lung, spleen, liver, kidney
tissues were homogenized with PBS and
were spread on LB plates to enumerate
bacteria levels. The plates were cultured in
37°C incubator overnight and colonies were
counted. Duplicates were done for each
sample and control.
Histopathology analysis-Lung tissues were
fixed in 10% formalin then embedded in
paraffin using a routine histologic
procedure. Four-micrometer sections were
cut and stained by standard H&E and
examined for differences in morphology
post-infection (42).
Statistical analysis-All experiments were
performed in triplicate and repeated at least
three times. Data were presented as percent
changes compared with the controls ± SD
from the three independent experiments.
Group means were compared by One-way
ANOVA [Turkey’s post-hoc], using SPSS
software, and difference was accepted at p <
0.05 (39). Survival test was represented by
Kaplan-Meier survival curves, using SPSS
software.
Results
Decreased survival in cav1 KO mice-To
determine the role of Cav-1 in P.
aeruginosa infection, we intranasally
instilled PAK (0.5×107 CFU/mouse) to
cav1 KO and WT mice (with otherwise
similar genetic backgrounds). As shown
in Fig. 1, cav1 KO mice exhibited increased
lethality (50% cav1 KO mice died within 24
h post infection). At 60 h, all KO mice died
while 50% of the wt control mice remained
alive. The result is represented by
Kaplan-Meier survival curves (p = 0.017,
Log-rank test). These findings suggest that
Cav-1 is required for host defense in P.
aeruginosa infection in acute pneumonia
models.
Increased bacterial burdens and oxidation
in cav1 KO lungs-To fully analyze the cause
of the lethality, moribund mice were killed
and organs were aseptically removed for
various assays. Lung homogenates were
used to measure bacterial burdens. Cav-1
KO mice showed significantly increased
CFUs of PAK in the lung tissue and AM
cells when compared with WT mice (Fig.
2A; p ＜ 0.001, one-way ANOVA),
indicating severe lung injury and
pneumonia. To quantitatively determine
polymorphonuclear neutrophil (PMN)
infiltration, BAL fluid and serum were
  6
analyzed for the percentage of PMN cells.
PMN penetration to the lung was increased
in the BAL fluid and serum of cav1 KO
mice when compared to WT mice (Fig.
2B-C). 
P. aeruginosa infection was previously
shown to induce the release of reactive
oxygen species (ROS) in lung, which may
accumulate and eventually cause lung
injury (43). To measure this oxidative stress,
cav1 KO and WT mice were infected by
PAK and AM cells were obtained.
Compared to WT mice, AM cells of cav1
KO mice showed increased oxidative stress
18 h post infection as determined by NBT
assays (Fig. 2D). These results were further
confirmed using the H2DCF assay (Fig. 2E),
a sensitive fluorescence method for
quantifying superoxide. Moreover,
decreased mitochondrial membrane
potential was also observed in cav1 KO
mice compared to WT mice using JC-1
fluorescence assays (Fig. 2F), indicating
that increased oxidation resulted in
apoptotic cell death. These data collectively
suggest that increased ROS may hamper
cell survival by increasing apoptosis and
may significantly damage the lung and
other organs. These results are consistent
with the increased PMN penetration in the
lung and serum in cav1 KO mice (Fig.
2B-C).
Under P. aeruginosa infection, PMN are
expected to infiltrate into the lung to clear
bacteria through ROS and proteases.
Although bacterial clearance is dependent
in part on the production of ROS, excessive
ROS accumulation may cause lung injury.
As a direct indicator of lung injury, lung
histology was examined 18 h post PAK
infection. Although both cav1 KO mice and
WT control mice showed signs of
pneumonia, significant histological
alterations and increased PMN infiltration
were only observed in the lungs of cav1 KO
mice, indicating more severe lung injury in
these animals (Fig. 2G). The insets
demonstrate the typical regions with serious
tissue damage or inflammatory response.
Infection dissemination in cav1 KO mice-A
logical question is whether or not the
intranasal inoculation only caused a local
infection within the lung. Using tissue
homogenates, we assessed bacterial burdens
in several other organs. CFUs of PAK were
also increased in the spleen, liver, and
kidneys, indicating that bacteria were able
to spread from the original inoculation site
(lungs) (Fig. 3A-C). The spleen, which is
the most sensitive indicator for bacterial
spread, displayed marked increase in
bacterial burdens (Fig. 3A). Consistent with
the PMN penetration into the serum (Fig.
2C), these results also suggest that the
severe lung injury and bacterial spread into
other organs are the possible cause of
mortality in these mice.
To confirm the dissemination results, we
next determined myeloperoxidase (MPO)
activity in the lung and other organs to
again probe neutrophil penetration, since
MPO is a recognized influx for oxidation in
tissue. As expected, similar MPO activity
was observed in the lung, liver, and kidney
(Fig. 3D-F). Increased MPO in organs other
than the lung (e.g., liver and kidney)
suggest that the superoxide release may
result from systemic spread of the invading
PAK bacteria.
As increased oxidation can also cause
tissue injury by oxidative degradation of
lipids, we used a thiobarbituric acid reactive
substances (TBARS) assay to detect lipid
peroxidation in the lung, liver, and kidney
tissue. Our results show that lipid
peroxidation was significantly increased in
all of the PAK infected organs of cav1 KO
mice as compared to WT mice (Fig. 3G-I).
  7
The lung showed a marked increase in lipid
peroxidation compared to the liver and
kidney, suggesting that the lung was the
main target. These data were consistent with
the severity of lung injury determined using
MPO assay and superoxide detection,
suggesting that the increased lipid
peroxidation in KO mice is relevant to the
progression of lung injury by oxidation.
Altered AM function in cav1 KO mice-AM
cells play a crucial role in bacterial
clearance by phagocytosis (43). To explore
this relationship, we also measured the
ingested PAK bacteria in AM cells.
Increased bacterial burdens were found in
the AM cells of cav1 KO mice compared to
those of WT mice as assessed by CFU (Fig.
4A). However, this result still cannot
address whether the bacteria were dealt
with through active phagocytosis or passive
uptake. To determine the function of AM
cells, equal amounts of PAO1-GFP, a strain
of P. aeruginosa with green fluorescence
(31,39), were added into cultured AM cells
isolated from cav1 KO mice or WT mice
for 2 h incubation. This allowed us to
conveniently quantify phagocytosis by
measuring fluorescence intensity using a
fluorescent plate reader (BioTek) (32,44).
Phagocytosis of cav1 KO AM cells was
significantly lower than that of WT AM,
indicating that Cav-1 may play a role in
phagocytosis by AM cells (Fig. 4B). These
results also indicate that increased
pre-ingestion of the pathogen resulted in
reduced phagocytic capacity in AM
following infection.
Survival of AM cells post infection is
also an essential factor for maintaining their
bacterial clearance function. Thus, to
determine the AM viability, AM cells were
infected with PAK for 1 h at MOI 10:1.
After adding polymyxin B (to remove the
surface bacteria, Sigma-Aldrich), survival
levels of AM were measured, which again
showed a decrease in the survival of AM
cells in cav1 KO mice after PAK infection
(Fig. 4C). These results indicate that Cav-1
may play a critical role in AM-mediated
host defense against PAK infection.
Altered inflammatory response in cav1 KO
mice-Cav-1 has been implicated in
bacteria-induced inflammatory responses.
Cytokine concentrations of BAL fluid were
measured 18 h post PAK infection to probe
various cytokines for reflecting
inflammatory response. The BAL fluid of
cav1 KO mice showed no significant
difference in inflammatory response
compared to that of WT mice. However, in
cav1 mouse lungs, IL-2 and IL-10 levels
were significantly elevated compared with
WT mice as assayed by ELISA (p < 0.05).
Furthermore, TNF- and IL-6 levels were
increased by approximately 4-fold in cav1
KO mice (p < 0.01) (Fig. 5A-D). This
indicates that the lung epithelium may be
the main source of increased cytokines. To
validate the above data, mRNA levels of the
above-quantified cytokines were also
measured by semi-quantitative RT-PCR.
mRNA expressions of TNF-α, IL-6, IFN-,
and IL-12a were all up-regulated by
approximately 2 to 4 fold as determined by
densitometry (Fig. 5E). Similarly, levels of
TNF-, IFN-, IL-1, IL-12a, and IL-6
were increased in cav1 KO lung tissues as
assessed by western blotting with
densitometrical analysis, particularly IL-12a
and IL-6 (Fig. 5F). Collectively, these data
indicate that cav1 KO mice showed more
intense pro-inflammatory response (TNF-,
IL-1 and IL-6) following PAK infection
as compared to WT mice.
Activation of NF-B pathway and STAT3 by
PAK infection in cav1 KO mice-To illustrate
the mechanism that causes the dysregulated
response to PAK infection in cav1 KO mice,
  8
we assessed several signaling proteins in
lung tissues by western blotting (Fig. 5G).
We found that NF-B was highly activated
in protein expression (2-fold) and
phosphorylation (1.4-fold). To further
dissect the pathway of cytokine response,
we also analyzed STAT activation. STAT3,
a transcription factor associated with
NF-B activation in various cellular
processes, showed significant expression in
cav1 KO mouse lungs (4-fold) and
phospho-STAT3 was drastically increased
(24-fold, Fig. 5G). Since STAT5 was not
significantly increased (data not shown),
our data suggest that STAT3 might play a
specific regulatory role in this infection (20).
We then evaluated the immediate up-stream
regulator of STAT3, and found that
phospho-JAK2 was dramatically increased
in cav1 KO mice (34-fold) (Fig. 5G). We
also analyzed roles of SOCS3, a negative
regulator of STAT3. A significant increase
in SOCS3 was seen in cav1 KO mice as
compared to WT mice by western blotting
analysis (data not shown), suggesting that
SOCS3 was responsive to the
infection-induced inflammatory response.
These results strongly indicate the
activation of STAT3 in cav1 KO mice as
compared to WT mice.
PAO1 infection displays similar phenotypes
in cav1 KO mice-To determine whether the
infection response and its mechanism are
similar across different bacterial strains, we
studied the immune response to another
well-studied P. aeruginosa strain - PAO1 in
cav1 KO mice. A similar infection condition
was used, except for a higher CFU count
because PAO1 is less cytotoxic than PAK.
Consistent with observations in PAK
infection, cav1 KO mice also showed
elevated bacterial burdens in lungs and in
other major organs (the liver, spleen, and
kidney) after PAO1 infection (Fig. 6A-D).
Our findings demonstrate that lung tissues
were severely injured in cav1 KO mice as
compared to WT mice. In addition, sepsis
was also observed in cav1 KO mice versus
WT mice by PAO1 infection. Furthermore,
inflammatory response indicated by
cytokines and superoxide was also
increased in cav1 KO mice following PAO1
infection (data not shown). We then
investigated the NF-B pathway and
STAT3 activation using western blotting
analysis. Similar to PAK strain, PAO1 strain
also induced significant inflammatory
responses (pNF-B 6.4-fold, pSTAT3
5.4-fold, and pJAK2 5.8-fold) in cav1 KO
mice as compared to WT mice. These
results indicate that PAO1 infection also
activated the STAT3 pathway along with
NF-B activation (Fig. 6E). In addition,
levels of inflammatory cytokines, IL-6 (7.3
fold) and TNF- (5.6 fold), were also
increased in cav1 KO mice as compared to
WT mice. Interestingly, the SOCS3 level
was much higher in cav1 KO mice than WT
mice (data not shown), which was also
more significantly activated than in the
PAK-infected mice (Fig. 5G). These data
indicate that PAO1 infection with cav1 KO
mice exhibited a similar phenotype to the
PAK strain and that a more rigorous
inflammatory response was induced by the
lower-cytotoxicity strain PAO1, as
compared to PAK.
NF-B pathway is altered by mutating
Cav-1 in MLE-12 cells and by STAT3
inhibitor-To better understand the role of
STAT3 in the NF-B pathway, we
transfected murine alveolar epithelial
MLE-12 cells with either WT cav1 or a
dominant negative (DN) cav1 expressing
plasmid (45). MLE-12 cells have been
widely used as a model for analyzing
murine lung epithelial function (39,41,46).
Transfected cells were then infected with
  9
PAK for 1 h at MOI 10:1 and lysed for CFU
assay. As expected, increased bacterial
burdens were observed in cav1 DN
MLE-12 cells (Fig. 7A). Next, we assessed
the mRNA levels for NF-B, JAK2, and
STAT3, which were not significantly
activated at an early phase (1 h post
infection). However, mRNA levels of IL-6,
and IL-12a significantly increased. To
ascertain the role of STAT3, WP1066 (a
STAT3 inhibitor, Sigma-Aldrich) was used
to pre-treat the cav1 DN cells. WP1066 has
been demonstrated to inhibit the
phosphorylation of STAT3 at low M range,
thereby effectively blocking STAT3
signaling (47). After treatment with the
STAT3 inhibitor, activation of IL-6 and
TNF- was abolished, while the total
mRNA levels were not altered (Fig. 7B).
These data support the view that STAT3
plays a crucial role in cytokine production.
Furthermore, we determined the protein
levels of the aforementioned cytokines and
their regulators. Consistent with the mRNA
expression, the protein levels of IL-6 and
TNF- were increased in cav1 DN
transfected cells when compared to WT
controls by semi-quantitative RT-PCR (Fig.
7C), which showed that cav1 DN
transfection resulted in a similar profile as
observed in cav1 KO mice. By contrast,
protein levels of IL-6 and TNF-α were
suppressed by WP1066 pretreatment.
Importantly, phosphorylation of NF-B,
JAK2, and STAT3 was all significantly
increased in cav1 DN transfected cells
versus the control. Similarly, WP1066
pre-treatment abolished the phosphorylation
of NF-B, JAK2, and STAT3 in cav1 DN
cells (Fig. 7C). These results confirm our
findings observed in cav1 KO mice,
indicating that the typical phenotypes may
be attributable to a dysregulated
pro-inflammatory cytokine response
through the Cav-1-STAT3-NF-B axis.
Inactive (resting) NF-B and STAT3
are normally retained in the cytosol of
MLE-12 cells (48). We hypothesized that
these factors can be activated by infection,
which may induce their translocation to the
nucleus (49). We examined the localization
of NF-B by confocal microscopy, and
found that both NF-B and STAT3 were
activated and translocated into the nucleus
in cav1 DN MLE-12 cells, but not in
vector-treated MLE-12 cells (Fig. 8A-B).
To further probe the activation of NF-B,
we transfected MLE-12 cells with luciferase
expression of NF-B. Consistent with the
imaging data, luciferase promoter assay
defined the activation of NF-B (Fig. 8C).
Importantly, we reaffirmed that the
activation of NF-B was also associated
with the STAT3 pathway, because the
elevation in luciferase activity was
significantly inhibited by WP1066. A
simplified model represents the pathway
implicated in cav1 KO mice and cav1 DN
cells (Fig. 8D).
Discussion
In this study, we investigated the
phenotype of P. aeruginosa infection in
cav1 KO mice. Severe disease phenotypes
in cav1 KO mice were observed in our
experiments including decreased survival,
increased inflammatory response, and more
severe lung injury compared to WT mice.
We reasoned that dysregulated cytokine
response and increased superoxide release
contribute to this exaggerated pathogenesis.
Importantly, we have identified a novel
mechanism involved in the phenotype, in
which the STAT3 pathway is responsible for
the dysregulated inflammatory response.
Our results are consistent with the
hypothesis that Cav-1 plays an essential
role in the clearance of P. aeruginosa,
  10
because both phagocytosis of the bacteria
by alveolar macrophages and cytokine
production by alveolar epithelial cells were
Cav-1-dependent (50). Similar phenotypes
have been observed with other pathogens
such as S. Typhimurium (51), showing
increased susceptibility, increased
production of cytokines, and elevated
bacterial burdens in cav1 KO mice. Thus,
Cav-1 may render critical immune defense
against a wide range of microorganisms.
Previous studies have demonstrated
that caveolins are widely expressed in lung
epithelial cells (52,53). Because caveolins
are associated with lipid rafts (54), they are
thought to be an important part of host
response to P. aeruginosa infection in lung
epithelial cells (7). The majority of previous
studies have focused on illustrating the role
of caveolins in regulating the cell responses
to pathogen virulence factors like LPS
(28,55-57). However, limited reports have
actually investigated the role of Cav-1 in
the innate immunity of the lung against P.
aeruginosa infection. Two previous reports
showed different phenotypes in cav1 KO
mice with a single pathogen P. aeruginosa
(11,12). This discrepancy must be addressed
in order to understand the real role of this
critical protein. Our timely studies reveal
that Cav-1 is indispensable in host defense
against P. aeruginosa infection, consistent
with the observation made by Gadjeva et al.
Furthermore, our studies reveal
mechanistically that Cav-1 is a powerful
negative regulator in pro-inflammatory
cytokines during this infection.
We have demonstrated that ROS levels
were significantly increased and that
mitochondrial potential was decreased in
AM cells of cav1 KO mice as compared to
that found in WT mice. These data showed
an elevated oxidative stress in cav1 KO
mice, which might cause lung injury and
systemic bacterial infection.
Over-production of superoxide can impair
innate immunity, including the phagocytosis
function of AM, the cells that form the front
line of innate defense in the lung. Indeed,
AM cells have been shown to be
dysfunctional in cav1 KO mice, exhibiting
reduced phagocytosis and increased
susceptibility to cell death after P.
aeruginosa infection. Although similar
methods were utilized in cross-pathogen
comparison study by Lisanti et al., no
difference in bacterial ingestion was found
between cav1 KO mice and WT mice with
S. Typhimurium infection (51). This
discrepancy may be due to inherent
differences in the two pathways, or the
distinct experimental settings. In addition,
PMN cells, another important cell type
responsible for bacterial clearance, show
lowered phagocytosis against P. aeruginosa
under Cav-1 deficiency (12). Together,
these observations demonstrate that the
bacterial clearance mechanism is impaired
in Cav-1 deficient mice, at least in the case
of P. aeruginosa infection. Furthermore, the
sustained recruitment of PMN cells into the
lung necessary for bacterial clearance may
release excess superoxide and proteases,
resulting in more severe lung injury and
systemic bacterial infection.
Another major contributing factor to
lung injury and mortality is inflammatory
response. We found that the lungs of cav1
KO mice exhibit increases in
pro-inflammatory cytokines, but a similar
increase was not found in the BAL fluid of
these mice. Thus, lung epithelial cells might
also contribute to the production of
cytokines (41,58,59), while macrophages
and neutrophils are traditionally regarded as
the main players in inflammatory responses
(60). To further explore the dysregulation of
inflammatory response in KO mice,
  11
modifications of the cell signaling pathway
in the lungs were examined. In particular,
we found NF-B, a major transcription
factor for cytokine production in alveolar
epithelial cells, to be highly activated (61).
To investigate the molecular events
associated with this activation, we studied
the JAK/STAT pathway. Interestingly,
STAT3, but not STAT5 was found to be
highly activated in cav1 KO mice following
infection. The interaction between NF-B
and STAT3 has been discussed extensively
in various experimental models, particularly
in cancer research, whereas the
STAT3-NF-B axis is still relatively
undefined in Gram-negative infections.
Among the cytokines whose expression was
altered, TNF-α and IL-6 were most
significantly up-regulated. Considering that
TNF-α and IL-6 are able to induce the
activation of NF-B and STAT3,
respectively, these significant changes
might be partially attributable to positive
feedback (22,62). Interestingly, we also
observed significantly increased expression
and phosphorylation of Akt and
GSK3data not shown, which may be the
other pathways responding to or resulting
from the dysregulated cytokine production,
and may interact with the JAK2/STAT3
pathway in Cav-1 deficient animals.
Consistent with our observations, previous
reports have showed the relevance of the
PI3K/Akt axis (27) and GSK3to a
JAK/STAT pathway (63). These indicate
that in cav1 KO mice, infection by various
bacteria may activate several different
pathways to compensate for the loss of this
critical protein. Indeed, there is little doubt
that Cav-1 is important for regulating lipid
raft-mediated endocytosis and various other
cellular events (32,64,65). Despite such
activation in various pathways, loss of
Cav-1 cannot be completely rescued to
combat infections and usually leads to interesting question for future studies.
lethal consequences, because crucial In conclusion, we have demonstrated a
cellular signaling systems may have been typical phenotype of P. aeruginosa infection
ill-regulated. For example, Cav-1 has been in cav1 KO mice, and our data indicate an
previously reported to interact with STAT3 important role for Cav-1 in innate immunity
directly through molecular interactions (51). in mice. Cav-1 deficiency impaired the
Deficiency in Cav-1 may impact STAT5 phagocytic ability and other immune
activity (66), while the ultimate effect on defense mechanisms, resulting in sustained
STAT5 activity is cell-dependent (51). infiltration of PMN cells into the lung and
Furthermore, no previous studies have intense inflammatory response. Along with
linked Cav-1 with the STAT3 pathway in the bacterial infection-induced oxidative
response to a respiratory pathogen. Thus, stress, the accumulation of PMN cells and
linking Cav-1 with STAT3-NF-B is a failure of bacterial clearance may cause
novel finding for this host response. Finally, severe lung injury and systemic bacterial
the role of the STAT3 pathway in AM is dissemination, which finally results in host
currently unclear and is worth studying. mortality. In addition, we revealed a novel
Thus, we hypothesized that STAT3 Cav-1-STAT-3-NF-B axis directly
might play a vital role in regulating NF-B contributing to a dysregulated cytokine
activation. We used MLE-12 cells to profile in cav1 KO mice. More importantly,
establish a Cav-1 deficient lung epithelial we confirmed this Cav-1-STAT-3-NF-B
culture model. Our data indicate that pathway in cav1 knockdown MLE-12 cells
transfection of a cav1 DN plasmid in during P. aeruginosa infection. Taken
MLE-12 cells showed a similar together, these observations have provided
inflammatory response and cell signaling an insight into the role of Cav-1 in innate
activation as seen in cav1 KO mice. STAT3 immunity against P. aeruginosa and might
inhibitor WP1066 was then used to confirm indicate novel targets for clinical therapy.
the role of STAT3 in MLE-12 cells. As
expected, STAT3 inhibition decreased
NF-B activation and reduced the
inflammatory response, indicating that
STAT3 activation is essential for NF-B
activation and down-stream inflammatory
response modifications. Taken together, our
data indicate that Cav-1 initiates a negative
regulation in the STAT3-NF-B pathway.
Although SOCS3, a chief negative regulator
of SOCS3, was also increased in cav1 KO
mice (data not shown), the over-activation
of STAT3 and associated inflammatory
response was still not contained. Thus, our
study suggests that Cav-1 may potentiate
the negative regulatory effects of SOCS3 on
inflammatory response, while loss of Cav-1
significantly dampens this role, an

  12
References:

1.  Driscoll, J. A., S. L. Brody, and M. H. Kollef. (2007) Drugs 67, 351‐368 
2.  Lyczak, J. B., Cannon, C. L., and Pier, G. B. (2000) Microbes Infect 2, 1051‐1060 
3.  Heijerman, H. (2005) J Cyst Fibros 4 Suppl 2, 3‐5 
4.  Fagon, J. Y., J. Chastre, Y. Domart, J.L. Trouillet, and C. Gibert. (1996) Clin. Infect. Dis. 23, 538‐542 
5.  Dunn, M., and R. G. Wunderink. (1995) Clin. Chest Med. 16, 95‐109 
6.  Crouch, B. S., R. G. Wunderink, C. B. Jones, and K. V., Jr. Leeper. (1996) Chest 109, 1019‐1029 
7.  Zaas, D. W., M. J. Duncan, G. Li, J. R. Wright, and S. N. Abraham. (2005) J. Biol. Chem. 280, 4864‐4872 
8.  Grassme, H., V. Jendrossek, A. Riehle, G. VonKurthy, J. Berger, H. Schwarz, M. Weller, R.    Kolesnick, 
and E. Gulbins. (2003) Nat. Med. 9, 322‐330 
9.  Garcia‐Medina, R., W. M. Dunne, P. K. Singh, and S. L. Brody. (2005) Infect. Immun. 73, 8298‐8305 
10.  Chastre, J., and Fagon, J. Y. (2002) Am J Respir Crit Care Med 165, 867‐903 
11.  Zaas, D. W., Z. D. Swan, B. J. Brown, G. Li, S. H. Randell, S. Degan, M. E. Sunday, J. R. Wright, and S. N. 
Abraham. (2009) J. Biol. Chem. 284, 9955‐9964 
12.  Gadjeva, M., C. Paradis‐Bleau, G. P. Priebe, R. Fichorova, and G. B. Pier. (2010) J. Immunol. 184, 
296‐302 
13.  Scherer, P. E., T. Okamoto, M. Chun, I. Nishimoto, H. F. Lodish, and M. P. Lisanti. (1996) Proc. Natl. 
Acad. Sci. U.S.A. 93, 131‐135 
14.  Tang, Z., P. E. Scherer, T. Okamoto, K. Song, C. Chu, D. S. Kohtz, I. Nishimoto, H. F. Lodish, and M. P. 
Lisanti. (1996) J. Biol. Chem. 271, 2255‐2261 
15.  Williams, T. M., and M. P. Lisanti. (2004) Genome Biol. 5, 214 
16.  Mora, R., V. L. Bonilha, A. Marmorstein, P. E. Scherer, D. Brown, M. P. Lisanti, and E. Rodriguez‐Boulan. 
(1999) J. Biol. Chem. 274, 25708‐25717 
17.  Thomas, S., A. P. Pais, S. Casares, and T. D. Brumeanu. (2004) Molecular Immunology 41, 399‐409 
18.  Thomas, S., R. S. Kumar, and T. D. Brumeanu. (2004) AITE 52, 215‐224 
19.  Korade, Z. (2008) Neuropharmacology 55, 1265‐1273 
20.  Grivennikov, S. I. a. M. K. (2010) Cytokine & Growth Factor Reviews 21, 11‐19 
21.  Ivashkiv, L. B. (2010) Cell Host Microbe 8, 132‐135 
22.  Takeda, K., T. Kaisho, N. Yoshida, J. Takeda, T. Kishimoto, and S. Akira. (1998) J. Immunol. 161, 
4652‐4660 
23.  Jones, M. R., Quinton, L. J., Simms, B. T., Lupa, M. M., Kogan, M. S., and Mizgerd, J. P. (2006) J Infect 
Dis 193, 360‐369 
24.  Wang, L., Kurosaki, T., and Corey, S. J. (2007) Oncogene 26, 2851‐2859 
25.  Norkina, O., Dolganiuc, A., Catalano, D., Kodys, K., Mandrekar, P., Syed, A., Efros, M., and Szabo, G. 
(2008) Alcohol Clin Exp Res 32, 1565‐1573 
26.  Maruvada, R., Argon, Y., and Prasadarao, N. V. (2008) Cell Microbiol 10, 2326‐2338 
27.  Shen‐Tu, G., Schauer, D. B., Jones, N. L., and Sherman, P. M. (2010) Lab Invest 90, 266‐281 
28.  Drab, M., P. Verkade, M. Elger, M. Kasper, M. Lohn, B. Lauterbach, J. Menne, C. Lindschau, F. Mende, 
and F. C. Luft. (2001) Science 293, 2449‐2452 
29.  Kannan, S., Audet, A., Huang, H., Chen, L. J., and Wu, M. (2008) J Immunol 180, 2396‐2408 
30.  Wisniowski, P. E., Spech, R. W., Wu, M., Doyle, N. A., Pasula, R., and Martin II, W. J. (2000) Am J Respir 

  13
 Crit Care Med 162, 733‐739 
31.  Priebe, G. P., Brinig, M. M., Hatano, K., Grout, M., Coleman, F. T., Pier, G. B., and Goldberg, J. B. (2002) 
Infect Immun 70, 1507‐1517 
32.  Kannan, S., Audet, A., Knittel, J., Mullegama, S., Gao, G. F., and Wu, M. (2006) European J Immunol 36, 
1739‐1752 
33.  Kannan, K., Huang, H., Seeger, D., Audet, A., Chen, Y., Huang, C., Gao, H., Li, S., and Wu, M. (2009) 
PLoS One 4, e4891 
34.  Wu, M., Stockley, P. G., and Martin II, W. J. (2002) Electrophoresis 23, 2373‐2376 
35.  Wu, M., Harvey, K. A., Ruzmetov, N., Welch, Z. R., Sech, L., Jackson, K., Stillwell, W., Zaloga, G. P., and 
Siddiqui, R. A. (2005) Int J Cancer 117, 340‐348 
36.  Kannan, S., Pang, H., Foster, D., Rao, Z., and Wu, M. (2006) Cell Death Differ 13, 311‐323 
37.  Yan, C., Cao, J., Wu, M., Jiang, T., Yoshimura, A., and Gao, H. (2010) J Biol Chem 285, 37227‐37239 
38.  Wu, M., Brown, W. L., and Stockley, P. G. (1995) Bioconjugate Chem 6, 587‐595 
39.  Wu, M., Huang, H., Zhang, W., Kannan, S., Weaver, A., Mckibben, M., Herington, D., Zeng, H., and Gao, 
H. (2011) Infect Immun 79, 75‐87 
40.  Wu, M., Audet, A., Cusic, J., Seeger, D., Cochran, R., and Ghribi, O. (2009) J Neurochem 111, 
1011‐1021 
41.  Kannan, S., H. Huang, D. Seeger, A. Audet, Y. Chen, C. Huang, H. Gao, S. Li, and M. Wu. (2009) Plos 
One 4, e4891 
42.  Wu, M., Hussain, S., He, H. Y., Pasula, R., Smith, P. A., and Martin II, W. J. (2001) Proc Natl Acad Sci U S 
A 98, 14589‐14594 
43.  Suntres, Z. E., O. Abdelwahab, and P. N. Shek. (2002) Microbial Pathogenesis 32, 27‐34 
44.  Kannan, S., A. Audet, H. Huang, L. Chen, and M.    Wu. (2008) J. Immunol. 180, 2396‐2408 
45.  Brazer, S. C., Singh, B. B., Liu, X., Swaim, W., and Ambudkar, I. S. (2003) J Biol Chem 278, 27208‐27215 
46.  Renier, M., Tamanini, A., Nicolis, E., Rolfini, R., Imler, J. L., Pavirani, A., and Cabrini, G. (1995) Hum 
Gene Ther 6, 1275‐1283 
47.  Kenneth, R. F., A. H. Charles, and L. C. Timothy. (2005) Clin. Cancer Res. 11, A277 
48.  Sugahara, K., A. Mizutani and H. Yamamoto. (2010) Am. J. Respir. Crit. Care Med. 181, A3613 
49.  Arita, Y., T. Ito, T. Oono, K. Kawabe, T. Hisano, and R. Takayanagi. (2008) World J. Gastroenterol. 14, 
4473‐4479 
50.  Kowalski, M. P., A. Dubouix‐Bourandy, M. Bajmoczi, D. E. Golan, T. Zaidi, Y. S. Coutinho‐Sledge, M. P. 
Gygi, S. P. Gygi, E. A. Wiemer, and G. B. Pier. (2007) Science 317, 130‐132 
51.  Medina, F. A., C. J. de Almeida, E. Dew, J. Li, G. Bonuccelli, T. M. Williams, A. W. Cohen, R. G. Pestell, P. 
G. Frank, H. B. Tanowitz, and M. P. Lisanti. (2006) Infect. Immun. 74, 6665‐6674 
52.  Barth, K., K. Weinhold, A. Guenther, M. T. Young, H. Schnittler, and M. Kasper. (2007) FEBS Journal 274, 
3021‐3033 
53.  Barar, J., L. Campbell, A. J. Hollins, N. P. B. Thomas, M. W. Smith, C. J. Morris, and M. Gumbleton. 
(2007) Biochemical and Biophysical Research Communications 359, 360‐366 
54.  Jacobson, K., O. G. Mouritsen and R. G. Anderson. (2007) Nat. Cell Biol. 9, 7‐14 
55.  Shaykhiev, R., J. Sierigk, C. Herr, G. Krasteva, W. Kummer, and R. Bals. (2010) The FASEB Journal 24, 
4756‐4766 
56.  Garrean, S., X. P. Gao, V. Brovkovych, J. Shimizu, Y. Y. Zhao, S. M. Vogel, and A. B. Malik. (2006) J. 
Immunol. 177, 4853‐4860 
57.  Cohen, A. W., R. Hnasko, W. Schubert, and M. P. Lisanti. (2004) Physiol. Rev. 84, 1341‐1379 

  14
58.  Amano, H., K. Morimoto, M. Senba, H. Wang, and Y. Ishida. (2004) J. Immunol. 172, 398‐409 
59.  Thorley, A. J., P. Goldstraw, A. Young, and T. D. Tetley. (2005) Am. J. Respir. Cell Mol. Biol. 32, 262‐267 
60.  Ozaki, T., Maeda, M., Hayashi, H., Nakamura, Y., Moriguchi, H., Kamei, T., Yasuoka, S., and Ogura, T. 
(1989) Am Rev Respir Dis 140, 1595‐1601 
61.  Moodie, F. M., Marwick, J. A., Anderson, C. S., Szulakowski, P., Biswas, S. K., Bauter, M. R., Kilty, L., and 
Rahman, I. (2004) FASEB J 10, 1897‐1899 
62.  Wang, C., M. M. Mayo, and A. S. Baldwin. (1996) Science 274, 784‐787 
63.  Nakayama, M., Hisatsune, J., Yamasaki, E., Isomoto, H., Kurazono, H., Hatakeyama, M., Azuma, T., 
Yamaoka, Y., Yahiro, K., Moss, J., and Hirayama, T. (2009) J Biol Chem 284, 1612‐1619 
64.  Anderson, R. G. (1998) Annu. Rev. Biochem. 67, 199‐225. 
65.  Anderson, R. G., and K. Jacobson. (2002) Science 296, 1821‐1825 
66.  Park, D. S., Lee, H., Frank, P. G., Razani, B., Nguyen, A. V., Parlow, A. F., Russell, R. G., Hulit, J., Pestell, R. 
G., and Lisanti, M. P. (2002) Mol Biol Cell 13, 3416‐3430 

FOOTNOTES
This project was supported by NIH ES014690 and American Heart Association Scientist
Development Grant (MW); and by NIH 5R01HL092905-04 and 3R01HL092905-02S1 (HG).
We thank S. Rolling of UND imaging core for help with confocal imaging.

The abbreviations used are: AM, alveolar macrophage; BAL, bronchoalveolar lavage; Cav-1,
caveolin-1; DN, dominant negative; MOI, multiplicity of infection; MPO,
myeloperoxidase; YFP, yellow fluorescent protein; PA, Pseudomonas Aeruginosa; JAK,
Janus kinase; SOCS, suppressors of cytokine signaling; H2DCF, Dihydrodichlorofluorescein
diacetate; TBARS, thiobarbituric acid reactive substances.
Competing interests’ statement: The authors declare that they have no competing financial
interests.

  15
 FIGURE LEGENDS
Figure 1. cav1 KO mice displayed increased susceptibility to P. aeruginosa infection.
cav1 KO mice and WT mice were intranasally challenged with 0.5×107 CFU/mouse PAK.
Then, the mice were maintained up to 72 h. Survival test is represented by Kaplan-Meier
survival curves (p = 0.017, 95% confidence interval: 18.4–44.5, log-rank test).

Figure 2. Increased bacterial burdens, PMN, and oxidation injury in the lung of cav1
KO mice. A, The lungs showed significantly increased bacterial burdens after infection with
PAK in cav1 KO mice compared to WT mice. cav1 KO mice and WT mice were infected
with 0.5×107 CFU/mouse PAK. Frozen tissues were homogenized in PBS. The same
amounts of tissue were evaluated for bacterial colonies for CFUs/gram tissue. B-C, Increased
PMN infiltration and acute inflammatory response were observed in the lung and serum of
cav1 KO mice compared to WT mice. After HEMA-3 staining (Fisher Scientific), PMN cell
percentages were calculated versus total nuclear cells. D, Superoxide production of AM cells
was significantly increased in cav1 KO mice compared to WT mice using NBT assay (1
g/ml). The same amounts of AM cells were infected with PAK at MOI 10:1 for 1 h. The
optical density for NBT was quantified at 560 nm. E, Oxidative stress was increased in cav1
KO mice determined by H2DCF assay (5 M). The fluorescence was quantified at 488 nm.
F, cav1 KO mice showed significantly lower mitochondrial potential than WT mice assessed
by JC-1 fluorescent assay (0.1 g/ml). The fluorescence was quantified at 532 nm. G.
Increased lung injury and inflammatory assessed by histology. cav1 KO mice and WT mice
were infected with 0.5×107 CFU/mouse of PAK for 24 h. Mice were dissected and lungs
were embedded in formalin. Sections were analyzed by H&E staining. The data are
representative of three experiments (One-way ANOVA [Turkey’s post-hoc], * p < 0.05, ** p
< 0.01).

Figure 3. Increased dissemination and oxidation in cav1 KO mice. A-C. The spleen, liver,
and kidneys showed significantly increased bacterial burdens in cav1 KO mice compared
with WT mice after PAK infection. D-F, Increased MPO activity in cav1 KO mouse lung,
liver, and kidney tissue compared to WT mice. G-I, Increased levels of lipid peroxidation
were observed in the lung, liver, and kidney tissues of cav1 KO mice compared to WT mice
by TBARS assay. The data are representative of three experiments (One-way ANOVA
[Turkey’s post-hoc], * p < 0.05, ** p < 0.01).

Figure 4. AM cell function is altered in cav1 KO mice. A. Increased bacterial burdens in

AM cells after infection with PAK in cav1 KO mice compared with WT mice. B, cav1 KO
mice and WT control mice were infected with 0.5×107 CFU/mouse of PAK for 18 h. The
same amounts of AM cells were infected with PAO1-GFP at MOI 10:1 for 1 h. Fluorescence
intensity showed decreased phagocytosis in cav1 KO mice. C, Survival percentages of AM
cells were measured by MTT assay. The data are representative of three experiments
(One-way ANOVA [Turkey’s post-hoc], * p < 0.05, ** p < 0.01).

Figure 5. PAK infection induced intense inflammatory response in cav1 KO mice. A-D,
Increased inflammatory cytokines in BALF of cav1 KO mice compared to WT mice

  16
assessed by ELISA. Mice (6 mice/group) were infected with 0.5×107 CFU/mouse of PAK for
18 h. BAL fluid was collected and cytokines measured. E-F, Increased inflammatory
cytokines in BAL fluid of cav1 KO mice compared to WT mice assessed by RT-PCR (E) and
western blotting (F). Frozen lung tissue of cav1 KO mice and WT mice at 18 h
post-infection was lysed for protein and mRNA assays. G, PAK infection activated the
NF-B pathway and STAT3 in cav1 KO mice. cav1 KO mice and WT mice were infected
with 0.5×107 CFU/mouse of PAK for 18 h. Gel data were quantified using densitometry with
Quantity one, and data are representative of three experiments (One-way ANOVA [Turkey’s
post-hoc], * p < 0.05, ** p < 0.01).

Figure 6. PAO1 infection caused similar phenotypes in cav1 KO mice as seen in PAK
infection. A-D, Increased bacterial burdens were observed in cav1 KO lung, spleen, liver,
and kidney tissues after PAO1 infection. E, Lung tissues of PAO1 infected cav1 KO mice
and WT mice (5 mice/group) were lysed for measuring cytokines and cell signaling protein
levels by western blotting (One-way ANOVA [Turkey’s post-hoc], ** p < 0.01). Gel data
were quantified using densitometry with Quantity one, and data are representative of three
experiments.

Figure 7. cav1 mutation in MLE-12 cells activated the STAT3-NF-κB pathway by PAK
infection. A, MLE-12 cells were transfected with a cav1 DN or cav1 WT plasmid. After 24 h,
cells were infected with PAK at MOI 10:1 for 1 h. The STAT3-NF-B pathway was
determined by western blotting. Cells were treated by STAT3 inhibitor WP1066 (2 M) for
0.5 h before infection. B, mRNA expression levels of IL-6 and TNF- were measured with
RT-PCR. C, After PAK infection, cav1 DN MLE-12 or WT MLE-12 cells were lysed to
determine expression and phosphorylation of cytokines and cell signaling proteins by
western blotting (three samples/each treatment). Gel data were quantified using densitometry
with Quantity one, and data are representative of three experiments in triplicate (One-way
ANOVA [Turkey’s post-hoc], * p < 0.05).

Figure 8. Elevated nuclear translocation of NF-B and STAT3 induced in cav1 DN

MLE-12 cells. A-B, WT and cav1 DN MLE-12 cells were infected with PAK for 1 h.
Localization of NF-B and STAT3 were visualized by indirect immunofluorescence staining.
Nuclear translocation of NF-B and STAT3 were detected in both WT and cav1 DN
MLE-12 cells (arrows showing the nuclear translocation). WP1066 (2 M) was used to
pre-treat the cells for 0.5 h before infection. C, Activation of NF-B was detected in both
WT and cav1 DN MLE-12 cells using Luciferase promoter assay. Transient transfections
were performed with 2×105 cells plated in 6-well plates using 2 g of DNA and 3 l of
LipofectAmine 2000 reagent for 24 h. After 1 h PAK infection at 10:1 MOI, cell lysates were
subjected to luciferase activity analysis using the Dual-Luciferase Reporter Assay System.
The data are representative of three experiments in triplicate (One-way ANOVA [Turkey’s
post-hoc], * p < 0.05). D, Diagram delineates a pathway involved in Cav-1 KO cells against
P. aeruginosa infection. Cav-1 deficiency impacts the phosphorylation of both NF-B and
STAT3 in cytoplasm by P. aeruginosa infection. The translocation of both factors to the
nucleus continuously induces cytokine production and aggravates disease progression.

  17
 100%
Cav1 KO
WT

80%
Survival

60%

40%

20%

Time post infection (hours)

Figure 1
 Lung BAL 800 Serum
A B 350 ＊＊ C ＊
3 ＊＊ 700

106/g tissue
300 μ

l
μ 600

l
250

PMN cells/
500
2

PMN cells/
200 400
150 300
× 1 100 200
CFU

50 100
0 0 0
WT Cav1 WT Cav1 WT Cav1

D 0.35 ＊ E 70 0 ＊ F 180

Mitochondrial Potential
160
0.3 60 0

H2DCF Assay (RFU)
NBT Assay (RLU)

140
0.25 50 0 120
0.2 40 0 100 ＊
0.15 30 0 80
60
0.1 20 0
40
0.05 10 0
20
0 0 0
WT Cav1 WT Ca v1 WT Cav1
G WT Cav1
Normal                                 PAK Normal                                 PAK

50 μ m Figure 2
A Spleen
B Liver C Kidney
＊＊
＊ ＊
2 2 2

106/g tissue
106/g tissue

106/g tissue
1 1 1
× × ×

CFU
CFU

CFU
0 0 0
WT Cav1 WT Cav1 WT Cav1

D Lung
E Liver
F Kidney
0.05

MPO Assay (RLU)
＊＊ ＊
＊

MPO Assay (RLU)
MPO Assay (RLU)

0.04 0.03
0.04
0.03
0.02 0.03
0.02 0.02
0.01 0.01
0.01
0 0 0
WT Cav1 WT Cav1 WT Cav1

G Lung H I
Liver Kidney
Lipid peroxidation (RLU)

Lipid peroxidation (RLU)
Lipid peroxidation (RLU)

0.5 0.25 ＊
＊＊ 0.2
＊
0.4 0.2
0.15
0.3 0.15
0.1
0.2 0.1

0.1 0.05 0.05

0 0 0
WT Cav1 WT Cav1 WT Cav1
Figure 3
 4000
A B C

Phagocytosis (RFU)
140%
3500
8 120%
＊＊ 3000 ＊
2500 100%
CFU/cell

6 ＊

Survival 
2000 80%
4 1500 60%
1000 40%
2
500 20%
0 0 0%
WT Cav1 WT Cav1 WT Cav1 WT+PAK Cav1+PAK

Figure 4
 A  B  C  6 ＊＊
D
2.5 6 12 ＊＊

TNF‐ (ng/ml)
5

IL‐10   (ng/ml)

IL‐6 (ng/ml)
IL‐2  (ng/ml)
2 5 10
4
4 8
1.5 3
3 6
1 2 2 4
0.5 1
1 2
0
0 0 0
WT         Cav1 WT           Cav1 WT          Cav1 WT          Cav1

Control PAK
E Control PAK
F  Control PAK G 
WT Cav1 WT Cav1
WT Cav1 WT Cav1 WT Cav1 WT Cav1
Cav‐1
IFN‐ Cav‐1
pNF‐B
IL‐1 IFN‐ NF‐B
IL‐6 IL‐1
pJAK2
IL‐12a IL‐6 JAK2
TNF‐ IL‐12a pSTAT3
GAPDH TNF‐ STAT3
GAPDH GAPDH
4.5 ＊＊
4 IFN -γ 12 30
p N F-κB ＊＊
Relative Density

Relative Density
Relative Density

IL -1 β IFN -γ ＊＊ ＊＊
3.5 10 25 N F-κB
IL -6 IL -1 β
3 ＊ 8 IL -6 p JAK2
IL -1 2 a 20
2.5 TN F-α ＊ ＊ IL -1 2 a p STAT3
2 6 TN F-α ＊ 15 STAT3
1.5 4 ＊ 10
1 ＊
2 ＊ ＊
5 ＊
0.5
0 0 0
(-) PAK (-) PAK (-) PAK
Figure 5
A B C D
Lung Spleen Liver Kidney
106/g tissue

106/g tissue

106/g tissue
106/g tissue
55 ＊＊
＊＊ 4 3
＊＊ ＊＊ 2 ＊＊
44 3
33 2
2 1
× 22 × × ×
1
11 1
CFU

CFU

CFU
00 CFU 0 0 0
WTWT Cav1
Cav1 WT Cav1 WT Cav1 WT Cav1

E WT                  Cav1 WT                      Cav1
10 ＊＊
Cav‐1 pJAK2

Relative Density
p N F-κB ＊
8 p STAT3 ＊
pNF‐B JAK2 ＊ ＊
p JAK2
6 IL -6
NF‐B IL‐6 4 TN F-α

pSTAT3 TNF‐ 2

STAT3 GAPDH 0
WT                      Cav1

Figure 6
 A B Control PAK
120 (—)       Cav1      DN   WP1066 (—)      Cav1       DN  WP1066
＊
100 NF‐B
CFU/104 cells

80 JAK2

60 STAT3

40 IL‐6
TNF‐
20
GAPDH
0
(-) Cav1 DN WP1066 (-) Cav1 DN WP1066

Relative Density
IL -6 ＊
2
TN F-α
Control PAK
1.5
＊
C 1
Control PAK
0.5
(—)       Cav1      DN   WP1066 (—)      Cav1      DN   WP1066
Cav1‐YFP 0
△Cav1‐YFP (-) DN WP1066
pNF‐B
NF‐B
5 ＊＊
pJAK2 Relative Density
p N F-κB
JAK2 4 p JAK2 ＊
p STAT3
pSTAT3 ＊
3 IL -6 ＊
STAT3 TN F-α ＊
2
IL‐6
TNF‐ 1

GAPDH 0
Figure 7 (-) DN WP1066
A NF‐B DAPI           Merge with DIC  B STAT3                    DAPI            Merge with DIC 

(—) (—)

PAK PAK

Cav1 DN Cav1 DN
+ PAK + PAK

WP1066 WP1066
+ PAK + PAK
Relative Luciferase Activity 

C 3000 ＊
D
2500

2000

1500

1000

500

0
(-) Cav1 DN WP1066 (-) Cav1 DN WP1066

Control       PAK
Figure 8