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• W2022635059 abstract "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 Pseudomonas aeruginosa-infected cav1 knock-out (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 with wild-type (WT) littermates. In addition, cav1 KO mice exhibited elevated inflammatory cytokines (IL-6, TNF-α, 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-κB are markedly activated. Our data revealed that the Cav-1/STAT3/NF-κB axis is responsible for a dysregulated cytokine response, which contributes to increased mortality and disease progression. Moreover, down-regulating Cav-1 in cell culture with a dominant negative strategy demonstrated that STAT3 activation was essential for the translocation of NF-κB 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-κB pathway and thereby impacting P. aeruginosa infection. 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 Pseudomonas aeruginosa-infected cav1 knock-out (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 with wild-type (WT) littermates. In addition, cav1 KO mice exhibited elevated inflammatory cytokines (IL-6, TNF-α, 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-κB are markedly activated. Our data revealed that the Cav-1/STAT3/NF-κB axis is responsible for a dysregulated cytokine response, which contributes to increased mortality and disease progression. Moreover, down-regulating Cav-1 in cell culture with a dominant negative strategy demonstrated that STAT3 activation was essential for the translocation of NF-κB 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-κB pathway and thereby impacting P. aeruginosa infection. Pseudomonas aeruginosa accounts for 25% of Gram-negative bacteria isolated from hospitals and is associated with high morbidity and mortality (1Driscoll J.A. Brody S.L. Kollef M.H. Drugs. 2007; 67: 351-368Crossref PubMed Scopus (576) Google Scholar). P. aeruginosa frequently infects immunocompromised individuals, such as those affected by ventilator-associated infection, severe burns, and cancer (2Lyczak J.B. Cannon C.L. Pier G.B. Microbes Infect. 2000; 2: 1051-1060Crossref PubMed Scopus (959) Google Scholar). More than 80% of cystic fibrosis patients suffer severe P. aeruginosa infection (3Heijerman H. J. Cyst. Fibros. 2005; 4: 3-5Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). This bacterium becomes increasingly resistant to various antibiotics. Further understanding the mechanism of the host-pathogen interaction may result in effective approaches to preventing this infection. Thus, P. aeruginosa has been a focus of airway infectious diseases (1Driscoll J.A. Brody S.L. Kollef M.H. Drugs. 2007; 67: 351-368Crossref PubMed Scopus (576) Google Scholar, 4Fagon J.Y. Chastre J. Domart Y. Trouillet J.L. Gibert C. Clin. Infect. Dis. 1996; 23: 538-542Crossref PubMed Scopus (159) Google Scholar, 5Dunn M. Wunderink R.G. Clin. Chest Med. 1995; 16: 95-109PubMed Google Scholar, 6Crouch Brewer S. Wunderink R.G. Jones C.B. Leeper Jr., K.V. Chest. 1996; 109: 1019-1029Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). Recent studies suggest that P. aeruginosa also invades the lung epithelial cells through lipid raft-mediated endocytosis (7Zaas D.W. Duncan M.J. Li G. Wright J.R. Abraham S.N. J. Biol. Chem. 2005; 280: 4864-4872Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 8Grassmé H. Jendrossek V. Riehle A. von Kürthy G. Berger J. Schwarz H. Weller M. Kolesnick R. Gulbins E. Nat. Med. 2003; 9: 322-330Crossref PubMed Scopus (457) Google Scholar, 9Garcia-Medina R. Dunne W.M. Singh P.K. Brody S.L. Infect. Immun. 2005; 73: 8298-8305Crossref PubMed Scopus (93) Google Scholar), which is a possible reason why this bacterium develops such formidable resistance to antibiotics (10Chastre J. Fagon J.Y. Am. J. Respir. Crit. Care Med. 2002; 165: 867-903Crossref PubMed Scopus (2133) Google Scholar). The invasion process of P. aeruginosa may involve certain lipid raft-associated proteins, including the caveolin family of proteins (9Garcia-Medina R. Dunne W.M. Singh P.K. Brody S.L. Infect. Immun. 2005; 73: 8298-8305Crossref PubMed Scopus (93) Google Scholar). Using caveolin-1 (cav1) 3The abbreviations used are: CavcaveolinAMalveolar macrophageBALbronchoalveolar lavageDNdominant negativem.o.i.multiplicity of infectionMPOmyeloperoxidaseSOCSsuppressors of cytokine signalingAbantibodyNBTnitro blue tetrazoliumANOVAanalysis of variancePMNpolymorphonuclear neutrophilROSreactive oxygen species. KO mice, two recent studies investigated the role of Cav-1 during P. aeruginosa infection (11Zaas D.W. Swan Z.D. Brown B.J. Li G. Randell S.H. Degan S. Sunday M.E. Wright J.R. Abraham S.N. J. Biol. Chem. 2009; 284: 9955-9964Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 12Gadjeva M. Paradis-Bleau C. Priebe G.P. Fichorova R. Pier G.B. J. Immunol. 2010; 184: 296-302Crossref PubMed Scopus (38) Google Scholar); however, their observations were very different. Thus, the role of Cav-1 in this infection needs to be clearly characterized. caveolin alveolar macrophage bronchoalveolar lavage dominant negative multiplicity of infection myeloperoxidase suppressors of cytokine signaling antibody nitro blue tetrazolium analysis of variance polymorphonuclear neutrophil reactive oxygen species. Caveolins are a family of integral membrane proteins involved in caveola formation and receptor-dependent endocytosis (13Scherer P.E. Okamoto T. Chun M. Nishimoto I. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 131-135Crossref PubMed Scopus (486) Google Scholar, 14Tang Z. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar, 15Williams T.M. Lisanti M.P. Genome Biol. 2004; 5: 214Crossref PubMed Scopus (351) Google Scholar). 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 (16Mora R. Bonilha V.L. Marmorstein A. Scherer P.E. Brown D. Lisanti M.P. Rodriguez-Boulan E. J. Biol. Chem. 1999; 274: 25708-25717Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). 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 (17Thomas S. Preda-Pais A. Casares S. Brumeanu T.D. Mol. Immunol. 2004; 41: 399-409Crossref PubMed Scopus (49) Google Scholar, 18Thomas S. Kumar R.S. Brumeanu T.D. Arch. Immunol. Ther. Exp. 2004; 52: 215-224PubMed Google Scholar, 19Korade Z. Kenworthy A.K. Neuropharmacology. 2008; 55: 1265-1273Crossref PubMed Scopus (211) Google Scholar) 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 the inflammatory response in carcinogenesis and host defense (20Grivennikov S.I. Karin M.A. Cytokine Growth Factor Rev. 2010; 21: 11-19Crossref PubMed Scopus (798) Google Scholar, 21Ivashkiv L.B. Cell Host Microbe. 2010; 8: 132-135Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar, 22Takeda K. Kaisho T. Yoshida N. Takeda J. Kishimoto T. Akira S. J. Immunol. 1998; 161: 4652-4660PubMed Google Scholar, 23Jones M.R. Quinton L.J. Simms B.T. Lupa M.M. Kogan M.S. Mizgerd J.P. J. Infect. Dis. 2006; 193: 360-369Crossref PubMed Scopus (82) Google Scholar). 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 kinases (JAKs), which in turn lead to phosphorylation of tyrosine residues on receptors (24Wang L. Kurosaki T. Corey S.J. Oncogene. 2007; 26: 2851-2859Crossref PubMed Scopus (41) Google Scholar). 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 (25Norkina O. Dolganiuc A. Catalano D. Kodys K. Mandrekar P. Syed A. Efros M. Szabo G. Alcohol Clin. Exp. Res. 2008; 32: 1565-1573Crossref PubMed Scopus (51) Google Scholar). Recently, STAT3 was found to interact with Cav-1 and heat shock protein 90 in plasma membrane rafts during Escherichia coli infection (26Maruvada R. Argon Y. Prasadarao N.V. Cell. Microbiol. 2008; 10: 2326-2338Crossref PubMed Scopus (22) Google Scholar). Cav-1 is also related to a JAK2/STAT5 pathway because Cav-1 is homologous to the pseudosubstrate for SOCS. Thus, Cav-1 may down-regulate JAK/STAT pathways, modulating the proinflammatory response. Because 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 (27Shen-Tu G. Schauer D.B. Jones N.L. Sherman P.M. Lab. Invest. 2010; 90: 266-281Crossref PubMed Scopus (8) Google Scholar). 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. KO and wild-type (WT) control mice (B6129SF2/J) were obtained from The Jackson Laboratory (Bar Harbor, ME) (28Drab M. Verkade P. Elger M. Kasper M. Lohn M. Lauterbach B. Menne J. Lindschau C. Mende F. Luft F.C. Schedl A. Haller H. Kurzchalia T.V. Science. 2001; 293: 2449-2452Crossref PubMed Scopus (1292) Google Scholar). Mice were housed and bred in the animal facility at the University of North Dakota, and the animal experiments were performed in accordance with the institutional animal care and use committee guidelines. We anesthetized mice with 45 mg/kg ketamine and intranasally instilled 0.5 × 107 (PAK; six mice/group) to 1 × 107 (PAO1) colony-forming units (cfu) of P. aeruginosa, and mice were killed when they were moribund (29Kannan S. Audet A. Huang H. Chen L.J. Wu M. J. Immunol. 2008; 180: 2396-2408Crossref PubMed Scopus (69) Google Scholar). After bronchoalveolar lavage (BAL), the trachea and lung were excised for homogenization or fixed in 10% formalin. Mouse AM cells were isolated by BAL as described (30Wisniowski P.E. Spech R.W. Wu M. Doyle N.A. Pasula R. Martin 2nd, W.J. Am. J. Respir. Crit. Care Med. 2000; 162: 733-739Crossref PubMed Scopus (18) Google Scholar). AM cells were grown in RPMI 1640 medium supplemented with 10% newborn calf serum and penicillin/streptomycin antibiotics in a 5% CO2 incubator. MLE-12 cells were obtained from ATCC and maintained following the manufacturer's instructions. P. aeruginosa strain PAO1 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) (31Priebe G.P. Brinig M.M. Hatano K. Grout M. Coleman F.T. Pier G.B. Goldberg J.B. Infect. Immun. 2002; 70: 1507-1517Crossref PubMed Scopus (57) Google Scholar). 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, resuspended in 10 ml of fresh LB broth, and allowed to grow until the midlogarithmic phase. Optical density (OD) at 600 nm was measured, and density was adjusted to 653,740.25 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 (29Kannan S. Audet A. Huang H. Chen L.J. Wu M. J. Immunol. 2008; 180: 2396-2408Crossref PubMed Scopus (69) Google Scholar). Except for dose determination assays, cells were infected by P. aeruginosa in a multiplicity of infection (m.o.i.) of 10:1 bacteria-cell ratio for the indicated time points. MLE-12 cells were transfected with yellow fluorescent protein (YFP)-Cav-1 and YFP-Cav-1Δ51–169 dominant negative (DN) plasmids using Lipofectamine 2000 reagent (Invitrogen) in serum-free RPMI 1640 medium following the manufacturer's instruction. The cells were lysed after 24 h of transient expression (32Kannan S. Audet A. Knittel J. Mullegama S. Gao G.F. Wu M. Eur. J. Immunol. 2006; 36: 1739-1752Crossref PubMed Scopus (49) Google Scholar). Cytokine concentrations were measured by an ELISA kit (eBioscience Co., San Diego, CA) in samples of cell culture medium, BAL fluid, and lung homogenates collected at the 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 five 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 of PBS. 96-well plates (Corning Costar 9018) were coated with 100 μl/well capture antibody in coating buffer and incubated overnight at 4 °C (33Kannan S. Huang H. Seeger D. Audet A. Chen Y. Huang C. Gao H. Li S. Wu M. PLoS One. 2009; 4: e4891Crossref PubMed Scopus (59) Google Scholar). 100-μl 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. Mouse monoclonal Abs against Cav-1, phospho-STAT3, IL-6, and NF-κB; rabbit polyclonal Ab against phospho-NF-κB; and goat polyclonal Abs against TNF-α and SOCS3 were from Santa Cruz Biotechnology, Inc. Rabbit monoclonal Ab against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Cell Signaling Technology. The samples derived from cells and lung homogenates were lysed and quantitated. The lysates were boiled for 5 min, and protease inhibitor mixture was added. The supernatants were collected, and an equal amount of each sample was loaded onto 10% SDS-polyacrylamide minigels and electrophoresed to resolve proteins. The proteins were then transferred to polyvinylidene difluoride membranes (Pierce) and blocked for 2 h at room temperature using 5% nonfat milk blocking buffer. Membranes were incubated overnight at 4 °C with appropriate first antibodies diluted at 1:1,000 in 5% bovine serum albumin (BSA) Western antibody buffer. After washing three times with washing solution, the antigen-antibody complexes were incubated for 45 min at room temperature with horseradish peroxidase-conjugated secondary antibody (Rockland Immunochemicals, Gilbertsville, PA) diluted 1:2,000 (34Wu M. Stockley P.G. Martin 2nd, W.J. Electrophoresis. 2002; 23: 2373-2376Crossref PubMed Scopus (57) Google Scholar). Signals were visualized using an enhanced chemiluminescence detection kit (SuperSignal West Pico, Pierce). RNA was extracted from lung homogenates and cells with TRIzol (Invitrogen) according to the manufacturer's instructions. For detected genes, RT was performed using 1.5 μg of RNA, RNase ribonuclease inhibitor, oligo(dT), and cloned avian myeloblastosis virus reverse transcriptase (Invitrogen). PCR products were separated by 1.0% agarose gel electrophoresis containing ethidium bromide and visualized under UV light. The results for each gene were normalized in comparison with GAPDH expression (35Wu M. Harvey K.A. Ruzmetov N. Welch Z.R. Sech L. Jackson K. Stillwell W. Zaloga G.P. Siddiqui R.A. Int. J. Cancer. 2005; 117: 340-348Crossref PubMed Scopus (105) Google Scholar). Cells were grown either on coverslips in a 24-well plate or in glass bottom dishes (MatTek, Ashland, MA). For immunostaining, the cells were fixed in 3.7% paraformaldehyde and permeabilized with 0.2% Triton X-100 in PBS, and the nonspecific binding site was blocked with blocking buffer for 30 min. Cells were incubated with primary Abs at a 1:500 dilution in blocking buffer for 1 h and washed three times with wash buffer. After incubation with appropriate fluorophore-conjugated secondary Abs, the coverslips were mounted on slides with VECTASHIELD mounting medium (36Kannan S. Pang H. Foster D.C. Rao Z. Wu M. Cell Death Differ. 2006; 13: 311-323Crossref PubMed Scopus (43) Google Scholar). DAPI (Sigma-Aldrich) was used to stain the nucleus. The images were captured by an LSM 510 Meta confocal microscope (Carl Zeiss MicroImaging, Thornwood, NY) and processed using the software provided by the manufacturer. Transient transfections were performed with 2 × 105 cells plated in 6-well plates by using 2 μg of DNA and 3 μl of Lipofectamine 2000 reagent (Invitrogen) in serum-free RPMI 1640 medium following the manufacturer's instruction. 24 h after transfection, the cells were infected with PAK at 10:1 m.o.i. for 1 h (37Yan C. Cao J. Wu M. Zhang W. Jiang T. Yoshimura A. Gao H. J. Biol. Chem. 2010; 285: 37227-37239Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Cell lysates were subjected to luciferase activity analysis by using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). This assay measures the color change of 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide upon reduction by enzymes to assess the viability of cells. BAL fluid was centrifuged, and cells were cultured in a 96-well plate. After incubation with P. aeruginosa, 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide dye was added at a final concentration of 1 μg/ml per well. The cells were incubated at 37 °C for 60 min or until the color change occurred. The dye is yellow in color and upon reduction by enzymes forms 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. The next day, the plate was read at 560-nm absorbance using a plate reader to quantify the dye conversion (38Wu M. Brown W.L. Stockley P.G. Bioconjug. Chem. 1995; 6: 587-595Crossref PubMed Scopus (78) Google Scholar). Duplicates were done for each sample and control. Background correction was done with blanks containing dye alone. This assay is based on the color change of NBT dye upon reduction by released superoxide. Cells were treated as above, and same amount of dye as used above was added. The dye is yellow in color and upon reduction by superoxide forms a blue formazan product (39Wu M. Huang H. Zhang W. Kannan S. Weaver A. McKibben M. Herington D. Zeng H. Gao H. Infect. Immun. 2011; 79: 75-87Crossref PubMed Scopus (43) Google Scholar). Dihydrodichlorofluorescein diacetate dye (Molecular Probes, Carlsbad, CA) does not normally fluoresce but emits green fluorescence upon reaction with superoxide inside cells. Cells were treated as above, and an equal amount(s) of dye was added. After a 10-min incubation, fluorescence was measured using a fluorometer (BioTek, Winooski, VT) (39Wu M. Huang H. Zhang W. Kannan S. Weaver A. McKibben M. Herington D. Zeng H. Gao H. Infect. Immun. 2011; 79: 75-87Crossref PubMed Scopus (43) Google Scholar, 40Wu M. Audet A. Cusic J. Seeger D. Cochran R. Ghribi O. J. Neurochem. 2009; 111: 1011-1021Crossref PubMed Scopus (24) Google Scholar). Malondialdehyde is an end product of the lipid peroxidation process and was measured in a colorimetric assay (Calbiochem) 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 was used in the assay. Duplicates were done for each sample and control (41Kannan S. Huang H. Seeger D. Audet A. Chen Y. Huang C. Gao H. Li S. Wu M. PLoS One. 2009; 4: e4891Crossref PubMed Scopus (67) Google Scholar). AM cells obtained from BAL fluid were plated in 96-well plates and grown overnight. The cells were treated with the serum-free medium for 1 h. Then GFP-PAO1 was used to infect the cells at 10:1 m.o.i. After a 1-h incubation at 37 °C, the wells were washed and treated with 100 μg/ml polymyxin B for 1 h to kill any remaining extracellular bacteria (29Kannan S. Audet A. Huang H. Chen L.J. Wu M. J. Immunol. 2008; 180: 2396-2408Crossref PubMed Scopus (69) Google Scholar). The number of phagocytosed bacteria was counted using a Synergy HT fluorometer (BioTek) 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 units were counted to quantitate phagocytosis as described previously. The MPO assay was performed as follows. Samples were homogenized in 50 mm hexadecyltrimethylammonium bromide, 50 mm KH2PO4, pH 6.0, 0.5 mm EDTA at 1 ml/100 mg of tissue and centrifuged for 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, 0.0005% mm H2O2) were added to 100 ml of sample. Absorbance was read at 460 nm at 2-min intervals. Duplicates were done for each sample and control (41Kannan S. Huang H. Seeger D. Audet A. Chen Y. Huang C. Gao H. Li S. Wu M. PLoS One. 2009; 4: e4891Crossref PubMed Scopus (67) Google Scholar). AM cells from BAL fluid and ground lung, spleen, liver, and kidney tissues were homogenized with PBS and spread on LB plates to enumerate levels of bacteria. The plates were cultured in a 37 °C incubator overnight, and colonies were counted. Duplicates were done for each sample and control. Lung tissues were fixed in 10% formalin and then embedded in paraffin using a routine histologic procedure. Four-micrometer sections were cut, stained by standard H&E, and examined for differences in morphology postinfection (42Wu M. Hussain S. He Y.H. Pasula R. Smith P.A. Martin 2nd, W.J. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 14589-14594Crossref PubMed Scopus (51) Google Scholar). All experiments were performed in triplicate and repeated at least three times. Data are presented as percent changes compared with the controls ±S.D. from the three independent experiments. Group means were compared by one-way ANOVA (Tukey's post hoc) using SPSS software, and a difference was accepted at p < 0.05 (39Wu M. Huang H. Zhang W. Kannan S. Weaver A. McKibben M. Herington D. Zeng H. Gao H. Infect. Immun. 2011; 79: 75-87Crossref PubMed Scopus (43) Google Scholar). The survival test was represented by Kaplan-Meier survival curves using SPSS software. 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 postinfection). At 60 h, all KO mice died, whereas 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. 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 colony-forming units 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 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 with WT mice (Fig. 2, B and 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 (43Suntres Z.E. Omri A. Shek P.N. Microb. Pathog. 2002; 32: 27-34Crossref PubMed Scopus (46) Google Scholar). To measure this oxidative stress, cav1 KO and WT mice were infected by PAK, and AM cells were obtained. Compared with WT mice, AM cells of cav1 KO mice showed increased oxidative stress 18 h postinfection as determined by NBT assays (Fig. 2D). These results were further confirmed using the dihydrodichlorofluorescein diacetate assay (Fig. 2E), a sensitive fluorescence method for quantifying superoxide. Moreover, a decreased mitochondrial membrane potential was also observed in cav1 KO mice as compared with 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. 2, B and C). Under P. aeruginosa infection, PMNs 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. 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. Colony-forming units 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. 3, A–C). The spleen, which is the most sensitive indicator for bacterial spread, displayed a 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 MPO activity in the lung and other organs to again probe neutrophil penetration because MPO is a recognized influx for oxidation in tissue. As expected, similar MPO activity was observed in the lung, liver, and kidney (Fig. 3, D–F). Increased MPO in organs other than the lung (e.g. liver and kidney) suggests 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 substance 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 with WT mice (Fig. 3, G–I). The lung showed a marked increase in lipid peroxidation compared with the liver and kidney, suggesting that the lung was the main target. These data were consistent with the severity of lung injury determined using the MPO assay and superoxide detection, suggesting that the increased lipid peroxidation in KO mice is relevant to the progression of lung injury by oxidation. AM cells play a crucial role in bacterial clearance by phagocytosis (43Suntres Z.E. Omri A. Shek P.N. Microb. Pathog. 2002; 32: 27-34Crossref PubMed Scopus (46) Google Scholar). 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 with those of WT mice as assessed by colony-forming units (Fig. 4A). However, this re" @default.
• W2022635059 created "2016-06-24" @default.
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• W2022635059 date "2011-06-01" @default.
• W2022635059 modified "2023-10-15" @default.
• W2022635059 title "Elevated Inflammatory Response in Caveolin-1-deficient Mice with Pseudomonas aeruginosa Infection Is Mediated by STAT3 Protein and Nuclear Factor κB (NF-κB)" @default.
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