The American Journal of Pathology, Vol. 184, No. 8, August 2014

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IMMUNOPATHOLOGY AND INFECTIOUS DISEASES

Mast Cells Protect against Pseudomonas aeruginosae Induced Lung Injury Robert D. Junkins,*y Svetlana O. Carrigan,* Zhengli Wu,*y Andrew W. Stadnyk,*y Elizabeth Cowley,z Thomas Issekutz,*y Jason Berman,*yx and Tong-Jun Lin*yx From the Departments of Pediatrics,* Microbiology and Immunology,y Physiology and Biophysics,z and Pathology,x Dalhousie University, Halifax, Nova Scotia, Canada Accepted for publication May 12, 2014. Address correspondence to Tong-Jun Lin, Ph.D., IWK Health Centre, 5850 University Ave, Halifax, NS, Canada B3K 6R8. E-mail: tong-jun.lin@dal. ca.

Pseudomonas aeruginosa, an opportunistic pathogen, is the leading cause of morbidity and mortality in immune-compromised individuals. Maintaining the integrity of the respiratory epithelium is critical for an effective host response to P. aeruginosa. Given the close spatial relationship between mast cells and the respiratory epithelium, and the importance of tightly regulated epithelial permeability during lung infections, we examined whether mast cells influence airway epithelial integrity during P. aeruginosa lung infection in a mouse model. We found that mast celledeficient KitW-sh/KitW-sh mice displayed greatly increased epithelial permeability, bacterial dissemination, and neutrophil accumulation compared with wild-type animals after P. aeruginosa infection; these defects were corrected on reconstitution with mast cells. An in vitro Transwell co-culture model further demonstrated that a secreted mast cell factor decreased epithelial cell apoptosis and tumor necrosis factor production after P. aeruginosa infection. Together, our data demonstrate a previously unrecognized role for mast cells in the maintenance of epithelial integrity during P. aeruginosa infection, through a mechanism that likely involves prevention of epithelial apoptosis and tumor necrosis factor production. Our understanding of mechanisms of the host response to P. aeruginosa will open new avenues for the development of successful preventative and treatment strategies. (Am J Pathol 2014, 184: 2310e2321; http:// dx.doi.org/10.1016/j.ajpath.2014.05.009)

The respiratory epithelium acts as the first line of defense against invading pathogens within the lungs by acting as a physical barrier to infection, and promoting clearance of pathogens from the lungs.1,2 Disruption of epithelial integrity is a hallmark of lung injury.3,4 During bacterial infection, lung injury is further characterized by accumulation of neutrophils within the lungs, increased dissemination of bacteria from the site of infection, and lung epithelial apoptosis.5,6 Many bacterial and viral pathogens have evolved mechanisms to disrupt epithelial integrity, resulting in enhanced pathogenicity.7e9 Pseudomonas aeruginosa is a Gramnegative opportunistic bacterial pathogen resistant to many commonly used antibiotics. Chronic lung infection with P. aeruginosa is a major concern among immunocompromised patients and represents the leading cause of morbidity and mortality in cystic fibrosis.2,7,8,10 Furthermore, acute P. aeruginosa infection is the second most common cause Copyright ª 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2014.05.009

of ventilator-associated pneumonia, leading to poor clinical outcomes.11 Given the damaging effects of P. aeruginosa infection and the emergence of multieantibiotic-resistant strains, understanding mechanisms involved in host defense against the bacteria will assist in developing preventative and therapeutic strategies for treating patients susceptible to P. aeruginosa infection.12,13 During P. aeruginosa infection, epithelial barrier function plays an important role as intact epithelial layers increase resistance to bacterial internalization and infiltration.14e16 Apoptosis occurs in the early phase of P. aeruginosa pneumonia and is recognized as a feature of P. aeruginosaeinduced lung injury.6 Blockade of P. aeruginosaeinduced apoptosis improves permeability and lung fluid balance.6 Supported by Cystic Fibrosis Canada (T.-J.L.) and Izaak Walton Killam Health Centre (R.D.J. and S.O.C.) studentships. R.D.J. and S.O.C. contributed equally to this work. Disclosures: None declared.

Mast Cells Prevent Lung Injury P. aeruginosa expresses a plethora of virulence factors that disrupt host defense mechanisms and promote bacterial invasion and survival.17 Exotoxins secreted through the type III secretion system,18 quorum-sensing molecules,19 rhamnolipids,20 exotoxin A,21 and elastase21,22 have all been implicated in increasing epithelial permeability during P. aeruginosa infection. However, host factors involved in maintaining airway epithelial integrity during P. aeruginosa infection remain incompletely understood. Mast cells are long-lived bone marrowederived cells resident in tissues throughout the body. Within the lung, they are abundant, representing up to 2% of the alveolar wall and protruding into the airspace of the lung, ideally placing them as sentinel cells of the immune system and first responders to respiratory tract infections.23 On encountering microbes, mast cells play critical roles in initiation and coordination of the innate and acquired immune responses.24,25 Depending on the stimulation encountered, mast cells are capable of producing numerous inflammatory and immunomodulatory factors, which include preformed granules containing histamine and proteases as well as de novo synthesized cytokines and arachidonic acid metabolites. Furthermore, mast cells can influence surrounding cells through both the production of soluble mediators and cell-to-cell contact, mediating both innate and adaptive immune responses.26e28 Although mast cells are classically considered as a proinflammatory cell type, mounting evidence also suggests that these cells have immunomodulatory functions.29,30 The role of mast cells during bacterial infection in vivo has been studied in a variety of models using mast celledeficient mice or mast cell stabilizers.31e37 In some models, mast cell mediators have been implicated in neutrophil recruitment and function as well as bacterial clearance.16,17 However, because of the diversity of immunomodulatory mediators produced by mast cells and the arsenal of Fc and pattern recognition receptors expressed on their surface, the contribution of mast cells to host immune responses is highly variable and often pathogen specific.38e40 In vivo, whether mast cells modulate the host response to P. aeruginosa lung infection has not been reported previously. KitW-sh/KitW-sh mice have a spontaneous mutation in the upstream regulatory elements of the c-kit gene,41 which renders them almost completely devoid of mature mast cells at many anatomical sites, including the lungs,42 while avoiding additional phenotypic abnormalities, such as anemia and infertility, observed in other mast celledeficient mice harboring mutations in the coding region of the c-kit gene.43 This feature, combined with the ability to reconstitute these mice with bone marrowederived mast cells (BMMCs),42 makes them a valuable tool for studying mast cell biological features. Given the close spatial relationship between mast cells and the respiratory epithelium, and the importance of tightly regulated epithelial permeability during lung infections, we set out to examine whether mast cells influence airway epithelial integrity during P. aeruginosa lung infection.

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P. aeruginosa lung infection was induced in wild-type and mast celledeficient KitW-sh/KitW-sh mice. Increased epithelial permeability, bacterial dissemination, and an accumulation of neutrophil were observed within the lungs of mast celledeficient animals. Reconstitution of KitW-sh/KitW-sh mice with mast cells reduced the P. aeruginosaeinduced increase of epithelial permeability and bacterial dissemination, suggesting a protective role for mast cells during P. aeruginosaeinduced acute lung damage. By using an in vitro Transwell model, we confirmed that mast cells and mast cellederived factors promote epithelial integrity. We further showed that mast cells reduced P. aeruginosae mediated epithelial cell apoptosis and epithelial-derived tumor necrosis factor (TNF) production. Thus, mast cells contribute to maintaining epithelial integrity in the airways during P. aeruginosa lung infection.

Materials and Methods Animals Mast celledeficient KitW-sh/KitW-sh mice and C57BL/6 mice were purchased from Charles River Laboratories (Saint Constant, QC, Canada). Mice received food and water ad libitum and were housed in pathogen-free facilities at the IWK Health Centre (Halifax, NS, Canada) animal facility. KitW-sh/KitW-sh mice were matched with C57BL/6 mice for age and sex. The protocols were approved by the University Committee on Laboratory Animals, Dalhousie University (Halifax), in accordance with the guidelines of the Canadian Council on Animal Care.

Antibodies Rabbit antiecaspase-3 (number 9661) was purchased from New England Biolabs (Whitby, ON, Canada). Rabbit antie zonula occludens protein 1 (ZO-1; 402200), rabbit antioccludin (404700), and Alexa 594econjugated goat anti-rabbit IgG (A11072) were purchased from Life Technologies (Burlington, ON, Canada). Goat anti-actin (sc1616), horseradish peroxidaseeconjugated goat anti-rabbit IgG (sc-2004), and horseradish peroxidaseeconjugated rabbit anti-goat IgG (sc-2768) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Bacterial Preparation and Infection of Mice P. aeruginosa mucoid strain 8821 was a gift from Dr. Ananda Chakrabarty (University of Illinois, Chicago, IL). P. aeruginosa samples were cultured as described previously.44 Briefly, suspension cultures were grown until reaching the early stationary phase. Bacteria were washed in phosphate buffer and resuspended in saline. Mice were anesthetized and intranasally infected with P. aeruginosa strain 8821 [1  109 colony-forming units (CFUs) per mouse] in 20 mL of total inoculum volumes, followed by 10 mL saline.

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Junkins et al Mice were sacrificed at various time points after infection, and bronchoalveolar lavage fluid (BALF) was obtained by lavaging the lung with 3 1 mL phosphate buffer solution containing 100 mg/mL soybean trypsin inhibitor (Sigma Aldrich, St. Louis, MO). BALF recovery of 800  100 mL was consistently achieved. Lung tissue was also collected and homogenized for detection of myeloperoxidase (MPO) activity and histological features, as described previously.45,46

Cytokine and Chemokine Production

MPO Assay

Immunofluorescence Microscopy

The MPO assay was used to determine the infiltration of neutrophils into the lungs of the mice, as previously described.45,46 Briefly, samples in duplicate (75 mL) were mixed with an equal volume of the substrate [3,30 ,5,50 -tetramethyl-benzidine dihydrochloride (3 mmol/L), Resorcinol (120mmol/L), and H2O2 (2.2 mmol/L)] for 2 minutes. The reaction was stopped by adding 150 mL of 2 mol/L H2SO4. The optical density was measured at 450 nm.

Immunofluorescence of 16HBE14o- cells grown on transwell filters was performed, as described previously, with a few modifications.48 Briefly, cells were grown on transwell filters for 6 to 8 days at a density of 150,000 cells per 0.33 cm2 filter in Dulbecco’s modified Eagle’s medium containing 10% FBS. Confluence of the monolayers was confirmed by measurement of TEER. HMC-1 cells were added at a density of 5  105 cells/mL to the basolateral (bottom) chamber of half of the transwells in a total volume of 800 mL. The other half of the wells received 800 mL of IMDM containing 10% FBS as a mast cell free control. The indicated number of P. aeruginosa bacteria was added to the apical (top) chambers of the transwells in 200 mL of IMDM containing 10% FBS. Twenty-four hours later, cells were fixed and permeabilized using BD Cytofix/Cytoperm, according to the manufacturer’s directions (BD Biosciences). Cells were blocked in 5% goat serum (Santa Cruz Biotechnology), and then incubated with the indicated primary antibodies, followed by Alexa 594econjugated goat anti-rabbit IgG (Life Technologies). Images were captured using fluorescence microscopy (Nikon Eclipse E600; Nikon, Tokyo, Japan) or confocal laser-scanning microscopy (Zeiss LSM510; Zeiss, Toronto, ON, Canada).

Histological Characteristics Lungs were perfused with neutral-buffered formalin for Harris H&E staining or with Carnoy’s fixative for Alcian blue staining. Mice lungs were fixed in formalin or Carnoy’s fixative overnight, then in 100% ethanol for paraffin embedding, and divided into sections. Slides were deparaffinized with solvent/clearing agent (Citrisolv; Fisher Scientific, Ottawa, ON, Canada) and rehydrated through decreasing concentrations of ethanol. Slides were subjected to H&E staining to illustrate lung histological features or Alcian blue staining to illustrate mast cells.

morphological features and the presence of metachromatic granules, at which time they were used for this study.

The concentration of TNF in culture supernatants was determined by ELISA, as previously described, using antibody pairs from R&D Systems (Minneapolis, MN).45,46

Cell Culture Image Analysis The human epithelial 16HBE14o- cells were maintained on flasks coated with 10 mg/mL human fibronectin (BD Biosciences, Mississauga, ON, Canada), 100 mg/mL bovine serum albumin (Sigma Aldrich), and 30 mg/mL type I bovine collagen (BD Biosciences). For transwell experiments, cells were grown on 0.4-mm transwell filters (Fisher Scientific), coated as described above, for 6 to 8 days at a density of 150,000 cells per 0.33 cm2 filter in minimal essential medium containing 10% fetal bovine serum (FBS) and 50 U/mL of both penicillin and streptomycin. Confluence of the monolayers was confirmed by measurement of transepithelial electrical resistance (TEER). The human mast cell line HMC-1 was cultured in Iscove’s modified Dulbecco’s medium containing 10% FBS and 50 U/mL of both penicillin and streptomycin. Highly purified cord bloodederived mast cells (CBMCs; >95% purity) were obtained by long-term culture of cord blood progenitor cells, as previously described.47 The percentage of mast cells in the cultures was determined by toluidine blue staining of cytocentrifuged samples. Mature mast cells after >8 weeks in culture were identified by their

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Area analysis was performed from tagged image file format images of monolayers stained for caspase-3, as described above. Unedited images captured using identical microscope settings were analyzed using Photoshop (Adobe, San Jose, CA).

In Vivo Epithelial Permeability Measurement Vascular and epithelial permeability in lungs of mice infected with P. aeruginosa was determined using Evans Blue Dye. Mice were intranasally infected with P. aeruginosa strain 8821, as described above. At 18 hours before sacrifice, mice were injected i.p. with 400 mL of 0.5% Evans Blue Dye (Sigma Aldrich) in phosphate buffer solution. Twenty-four hours after infection, mice were sacrificed, and serum, the first 1 mL wash of BALF, was collected, as described above. Serum was diluted 1:20 in phosphate buffer solution in all samples and used to calculate relative permeability indexes in individual mice.

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Mast Cells Prevent Lung Injury BALF was centrifuged for 10 minutes at 18,000  g to remove cells and bacteria. Permeability changes were calculated as a ratio of optical density at 620 nm of BALF to diluted serum.

and secondary antibodies, as indicated, and detected by an enhanced chemiluminescence detection system (Western Lightning Plus-ECL; PerkinElmer, Waltham, MA). Scanning densitometry analysis was performed using Scion Image version 4.0.3.2 (Scion Corporation, Frederick, MD).

In Vitro Epithelial Permeability Measurement The 16HBE14o- cells were grown on 0.4-mm transwell filters (Fisher Scientific) for 6 to 8 days at a density of 150,000 cells per 0.33-mm filter in Dulbecco’s modified Eagle’s medium containing 10% FBS. Monolayers were considered confluent when TEER exceeded 600 U*cm2, at which point transwells were used for experiments. HMC-1 cells were added at a density of 5  105 cells/mL to the lower, basolateral chamber of half of the transwells in a 24-well plate in a total volume of 800 mL. The other half of the wells received 800 mL of IMDM containing 10% FBS as a mast cell free control. The indicated number of P. aeruginosa bacteria was added to the apical (top) chambers of the transwells in 200 mL of IMDM containing 10% FBS. TEER was measured 24 hours later using an EVOM Epithelial Voltohmeter, according to the manufacturer’s instructions (World Precision Instruments, Sarasota, FL). For the determination of monolayer permeability, cells were grown on transwell filters and treated as described above. Twenty-four hours after bacterial inoculation, 5 mL of 4-kDa dextran conjugated to fluorescein isothiocyanate (FITCedextran 4000; Sigma Aldrich) was added to the apical (top) chamber to achieve a final concentration of 5 mg/mL, and the chamber was incubated for 3 hours at  37 C and 5% CO2. After incubation, media from the bottom chambers were collected and fluorescence measured at 518 nm using a fluorometer (Fluoroscan Ascent F; Thermo Scientific, Waltham, MA). Transepithelial FITCedextran 4000 diffusion was calculated using bare filter diffusion as 100%.

Mast Cell Reconstitution Mast celledeficient KitW-sh/KitW-sh mice were reconstituted with BMMCs cultured from C57BL/6 mice, as described previously.49 Briefly, mice were reconstituted with 5  106 BMMCs in 200 mL RPMI 1640 medium delivered i.v. Animals were used for experiments 8 weeks after reconstitution. Reconstitution of mast celledeficient KitW-sh/ KitW-sh mice with BMMCs was confirmed by Alcian blue staining of paraffin-embedded lung sections, as described previously.49

Western Blot Analysis and Scanning Densitometry Western blot analysis and scanning densitometry were performed as described previously.50 Briefly, cell lysates (10 to 20 mg) were subjected to electrophoresis in 10% or 12% SDSepolyacrylamide gels. Gels were transferred to a polyvinylidene difluoride membrane, blotted with primary

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Statistical Analysis Data are presented as means  SEM of the indicated number of experiments. Statistical significance between multiple treatments was determined by one-way analysis of variance and post hoc Tukey’s honest significance test. Alternatively, when two independent variables were analyzed, a two-way analysis of variance and a Bonferroni multiple-comparison test were used. Statistical analysis was performed using GraphPad Prism software version 5.04 (GraphPad Software Inc., La Jolla, CA).

Results Mast CelleDeficient Mice Display Increased Epithelial Permeability after P. aeruginosa Lung Infection Increased epithelial permeability is a hallmark of acute lung injury. To examine the biological contribution of mast cells to epithelial integrity in vivo, an acute P. aeruginosa lung infection model was used in wild-type C57BL/6, KitW-sh/ KitW-sh mast celledeficient, and KitW-sh/KitW-sh mice reconstituted with mast cells derived from the bone marrow of C57BL/6 mice. Animals were infected intranasally with P. aeruginosa 8821, a mucoid strain isolated from the lungs of a patient with cystic fibrosis.51 At 8 hours later, mice were injected i.p. with Evans Blue Dye. Twenty-four hours after infection, mice were sacrificed and leakage of the dye into the BALF was measured and expressed as a ratio to serum concentration to examine epithelial permeability (Figure 1A). Mast celledeficient mice displayed greatly increased Evans Blue Dye leakage in the BALF, implicating mast cells in maintaining epithelial integrity in vivo after P. aeruginosa infection. Reconstitution of mast celle deficient mice with wild-type C57BL/6 BMMCs repopulated the lungs with mast cells (Supplemental Figure S1), and was able to significantly decrease P. aeruginosae induced changes in epithelial permeability. Intact respiratory epithelium plays an essential barrier function in the lung and prevents bacterial dissemination into the blood. To examine the biological impact of mast cells on epithelial barrier function, wild-type and mast celledeficient mice were infected intranasally with P. aeruginosa strain 8821. Dissemination of the bacteria into the blood stream was monitored 4, 24, 48, and 72 hours after infection (Figure 1B). Few bacteria were isolated from the blood of wild-type animals after P. aeruginosa lung infection, suggesting the infection was effectively contained in the lung. In contrast, significant dissemination of the bacteria into the blood of mast celledeficient mice was observed starting 24 hours after

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Mast celledeficient mice display increased lung epithelial permeability and bacterial dissemination after P. aeruginosa lung infection. C57BL/6 (black bars), KitW-sh/KitW-sh reconstituted with C57BL/6 bone marrow-derived mast cells (BMMCs) (Wsh þ BMMC; gray bars) and KitW-sh/ KitW-sh (Wsh; white bars) mice were left uninfected (NT, n Z 3) or infected intranasally with 1  109 CFU P. aeruginosa strain 8821 for 24 hours (24 hours, n Z 8 to 18). At 18 hours before sacrifice, mice received i.p. injections of Evans Blue Dye. A: Dye leakage into the BALF was assessed and is presented as the OD620 nm ratio of BALF to a 1:20 dilution of serum (permeability index). B: C57BL/6 and Wsh mice were infected intranasally with 1  109 CFU P. aeruginosa strain 8821, then serum was collected and cultured 4, 24, 48, and 72 hours after infection to monitor bacterial dissemination. C: Bacterial dissemination into the blood from overnight cultured serum (n Z 10  SEM). *P < 0.05, **P < 0.01, and ***P < 0.001.

infection (P < 0.001 compared with C57BL/6 control). Despite enhanced bacterial dissemination in mast celle deficient animals, the numbers of bacteria in the blood eventually began to decrease 72 hours after infection, and no mortality was observed in wild-type or mast celledeficient mice.

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To ensure this phenotype was due to the absence of mast cells in KitW-sh/KitW-sh mast celledeficient mice, dissemination of the bacteria into the blood 24 hours after infection was monitored in wild-type C57BL/6, KitW-sh/KitW-sh mast celledeficient, and KitW-sh/KitW-sh mast celledeficient mice reconstituted with wild-type BMMCs (Figure 1C). Bacterial dissemination was significantly reduced after reconstitution of KitW-sh/KitW-sh mast celledeficient mice with BMMCs, demonstrating that impaired epithelial barrier function is due to the absence of mast cells. Increased epithelial permeability also contributes to the aberrant accumulation of neutrophils in the lung.5 To examine the impact of mast cell deficiency on neutrophil infiltration into the lungs, wild-type and mast celledeficient mice were infected intranasally with P. aeruginosa strain 8821. Mice were sacrificed 4, 24, 48, or 72 hours later, and lung tissue was collected and assayed for activity of the neutrophil-specific MPO (Figure 2A) or fixed for H&E staining (Figure 2C). MPO activity was increased in the lungs of mast celledeficient mice after infection compared with wild-type animals (P < 0.001), and histological samples displayed increased numbers of infiltrating leukocytes. Furthermore, as with epithelial permeability and bacterial dissemination, increased neutrophil infiltration into the lungs was restored to near wild-type levels in mast celledeficient mice after reconstitution with wild-type mast cells (Figure 2B). Together with observed effects on epithelial permeability and bacterial dissemination, these data suggest that mast cells play a protective role in maintaining epithelial integrity after P. aeruginosa infection in vivo.

To further explore a role of mast cells on epithelial barrier function, we established an in vitro model in which 16HBE14o- human bronchial epithelial cells were grown on 0.4-mm pore size transwell filters. The basolateral chambers of each well received either HMC-1 human mast cells or media control. Monolayers were then infected apically with increasing doses of P. aeruginosa bacteria for 24 hours, after which epithelial permeability and barrier function were measured by TEER (Figure 3A) and FITCdextran diffusion (Figure 3B), respectively. Monolayers displayed a dose-dependent decrease in TEER (P < 0.001) and a dose-dependent increase in FITC-dextran diffusion (P < 0.001), indicating a loss of epithelial integrity and increased permeability. More important, changes in TEER (P < 0.001) and FITC-dextran diffusion (P < 0.001) were significantly decreased by the presence of basolateral mast cells, implicating mast cells in maintaining epithelial integrity after P. aeruginosa infection. Similar results were obtained using primary human CBMCs from two separate donors, demonstrating that this phenomenon is not restricted to the HMC-1 mast cell line (Supplemental Figure S2, A and B).

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Mast Cells Prevent Lung Injury cells alone, with or without P. aeruginosa stimulation for 24 hours. These cell supernatants contained secreted factors from each culture condition and were added to the basolateral chamber of 16HBE14o- transwells, which were then infected apically with P. aeruginosa bacteria. The impact of each treatment on TEER (Figure 4A) and FITC-dextran diffusion (Figure 4B) were measured 24 hours later. All supernatants from samples containing HMC-1 cells were able to improve epithelial integrity to levels observed when mast cells were present. These results suggest that mast cellederived factors are involved in maintaining epithelial integrity, and that stimulation with P. aeruginosa and epithelial co-culture are not required for the production of the mast cell product. Similar results were obtained with cell-free supernatants from CBMC cells and CBMC-16HBE14o- transwell co-culture supernatants prepared as described above, again demonstrating that this phenomenon is not restricted to the HMC-1 mast cell line (Supplemental Figure S3).

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Mast celledeficient mice display enhanced neutrophil recruitment in their lungs after P. aeruginosa lung infection. C57BL/6 (white bars) and KitW-sh/KitW-sh (Wsh; black bars) mice were intranasally infected with P. aeruginosa (strain 8821, 1  109 CFU per mouse) or left untreated (NT). A: After 4, 24, 48, or 72 hours, mice were sacrificed and neutrophil accumulation in the right lung was measured using an MPO assay. B: C57BL/6, KitW-sh/KitW-sh reconstituted with C57BL/6 bone marrowderived mast cells (BMMCs) (Wsh þ BMMC; gray bars), and Wsh were left NT or infected as described above for 24 hours, after which neutrophil infiltration into the lungs was assessed using an MPO assay. C: The upper lobe of the left lung was also collected at each time point for H&E staining, and greater leukocyte infiltration in lungs of KitW-sh/KitW-sh mice is apparent (n Z 8  SEM). *P < 0.05, ***P < 0.001. Original magnification, 100 (C).

Enhanced Epithelial Integrity after P. aeruginosa Infection Is Mediated by Mast CelleDerived Factors To further confirm a role of mast cells in promoting epithelial integrity, cell-free supernatants were prepared from the basolateral chamber of 16HBE14o- transwells, which had been cultured in the presence or absence of HMC-1 mast cells, with or without P. aeruginosa stimulation for 24 hours. Additional cell-free supernatants were prepared from HMC-1

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Figure 3 Mast cells (MCs) prevent bronchial epithelium permeability changes induced by P. aeruginosa. Human mast HMC-1 cells were resuspended in antibiotic-free IMDM/10% FBS at 5  105 cells/mL and added to half of the wells in a 24-well plate in 800 mL total volume (basolateral, bottom chamber). IMDM was added to the other chambers as a mast cell free control. P. aeruginosa in a range of infective doses was added to the apical (top) chambers of transwells in 200 mL of IMDM containing 10% FBS. A: Transepithelial electrical resistance was measured 24 hours later. Twenty-four hours after bacterial inoculation, 5 mL of FITC-dextran 4000 was added to all top chambers and incubated for 3 hours. B: A sample of fixed volume from the bottom chambers was collected, and fluorescence was measured. Data are represented as a percentage of FITC-dextran 4000 diffusion across bare filter (n Z 5  SEM). *P < 0.05, **P < 0.01, and ***P < 0.001. NT, untreated.

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Enhanced epithelial integrity after P. aeruginosa (Ps.a) infection is mediated by secreted mast cell factors. Human epithelial 16HBE14o- cells were grown to confluence on Transwells for 6 days. Epithelial integrity was confirmed using TEER. Basolateral wells received 800 mL of IMDM, human mast HMC-1 cells at a density of 0.5  106 cells/mL, or the indicated cell-free supernatants. Cell-free supernatants were prepared from the basolateral chambers of 16HBE14o- transwells in the presence or absence of HMC-1 cells, with or without apical infection with 1  106 CFU P. aeruginosa strain 8821 for 24 hours. Alternatively, supernatants were collected from HMC-1 cells in the absence of 16HBE-14oco-culture, with or without infection with P. aeruginosa strain 8821 at a multiplicity of infection of 1:10 for 24 hours. Supernatants were cleared of cells by centrifugation. Monolayers were left uninfected, or infected with 1  106 CFU P. aeruginosa strain 8821 in the apical chamber. Twenty hours later, 5 mL of FITC-dextran was added apically. Twenty-four hours after infection, TEER was measured (A), then basolateral supernatants were collected and assayed for FITC-dextran diffusion (B) (n Z 4  SEM). *P < 0.05, ***P < 0.001.

Mast Cells Do Not Significantly Impact Tight Junction Integrity after P. aeruginosa Infection Mast cells have previously been implicated as key regulators of epithelial integrity in the intestine,52e54 where tight junction proteins have a prominent role in maintaining epithelial integrity. To determine whether mast cells affect tight junction organization in vitro during P. aeruginosa

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infection, 16HBE14o- monolayers were grown on transwell filters until TEER was established. Half of the monolayers received HMC-1 mast cells in the basolateral chamber, whereas the other half received media control. Cells were left untreated or infected apically with increasing doses of P. aeruginosa bacteria for 24 hours, after which monolayers were fixed and stained for ZO-1 (Figure 5A) or occludin (Figure 5B) by immunofluorescence. A loss of tight junction integrity was observed after P. aeruginosa infection, as indicated by the decreased localization of occludin and ZO-1 staining at cell interfaces. The presence of mast cells in the basolateral chamber had no significant impact on the localization of either tight junction protein. Lysates were also collected from transwell monolayers treated with increasing doses of P. aeruginosa in the presence or absence of mast cells, and were subjected to Western blot analysis for ZO-1 and occludin (Figure 5C). Consistent with previous reports, P. aeruginosa resulted in a dose-dependent decrease in the levels of ZO-1 and occludin.21,22 However, the presence of basolateral mast cells had little impact on the expression of either tight junction protein. Together, these data demonstrate that mast cells do not affect tight junction protein localization or expression during P. aeruginosa infection.

Mast Cells Decrease P. aeruginosaeInduced Epithelial Cell Apoptosis P. aeruginosa has previously been shown to induce significant epithelial cell apoptosis via caspase-3edependent mechanisms.6,55 Caspase-3 activation has been used as an apoptosis marker. We examined the impact of mast cell coculture on P. aeruginosaeinduced epithelial cell apoptosis. The 16HBE14o- human bronchial epithelial cells were grown on transwell filters until significant TEER was established. Basolateral chambers received either HMC-1 human mast cells or media control. Monolayers were left untreated or were infected apically with increasing doses of P. aeruginosa bacteria. Twenty-four hours later, monolayers were fixed and stained for active caspase-3 (Figure 6A). Area analysis was used to quantify the percentage of apoptotic cells (Figure 6B). Caspase-3 activation was further monitored by Western blot analysis in cell lysates collected from transwell monolayers treated with increasing doses of P. aeruginosa in the presence or absence of basolateral mast cells (Figure 6, C and D). A dose-dependent activation of caspase-3 was observed after P. aeruginosa infection (P < 0.001) and was significantly reduced by co-culture with mast cells (P < 0.001). These results suggest that mast cells reduce caspase-3 activation after P. aeruginosa infection. TNF is a key mediator of both acute lung injury56,57 and apoptosis of bronchial epithelial cells in the lungs.58 To determine the impact of mast cell co-culture on TNF production after P. aeruginosa infection, apical supernatants were collected from 16HBE14o- cells grown on transwells and infected with increasing doses of P. aeruginiosa

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Figure 5

Mast cells do not significantly affect tight junction integrity after P. aeruginosa (Ps.a.) infection. HBE cells were grown to confluence on transwells for 6 days. Epithelial integrity was confirmed using TEER. Basolateral wells received 800 mL of IMDM or HMC-1 cells at a density of 0.5  106 cells/mL. Monolayers were left uninfected (NT) or infected with increasing infectious doses of P. aeruginosa strain 8821 in the apical chamber. Twenty-four hours later, cells were fixed and strained for cleaved ZO-1 (A) or occludin (B) and imaged by confocal microscopy. C: Alternatively, cells were grown and treated as described above, then lysates were prepared and subjected to Western blot analysis for ZO-1, occluding, and actin. Representative images of two to three independent experiments are shown.

bacteria in the presence or absence of basolateral mast cells. ELISA was used to assay apical secretion of TNF (Figure 6E). Apical TNF levels increased in a dosedependent manner after P. aeruginosa infection (P < 0.001). However, co-culture with basolateral mast cells was able to abrogate P. aeruginosaeinduced TNF production (P < 0.001). Similar results were obtained in vitro using primary human CBMCs (Supplemental Figure S2C). Together, these results suggest that mast cells reduce epithelial cell apoptosis through a mechanism that likely involves suppressing TNF production and caspase-3 activation after P. aeruginosa lung infection.

Discussion The epithelium is the first cellular barrier encountered by invading bacteria. In the lung, a healthy epithelium with complex junctional structures allows for the selective passage of molecules and maintenance of well-defined polarized functions.59 Dysfunction in epithelial permeability affects bacterial dissemination and contributes to lung injury.60,61 P. aeruginosa is the leading cause of morbidity and mortality among patients with cystic fibrosis2,7,8,10 and uses a range of strategies to disrupt host lung epithelial permeability.17e22 How host factors counteract these pathogen-induced lung epithelial permeability changes is not entirely understood. In this study, mast celledeficient KitW-sh/KitW-sh mice were used to demonstrate that mast cell deficiency leads to increased lung epithelial permeability, P. aeruginosa dissemination into the blood stream, and

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neutrophil infiltration into the lung. By using an in vitro transwell co-culture model, we found that mast cellederived factors reduced P. aeruginosaeinduced increases in epithelial permeability and that mast cells decreased P. aeruginosaeinduced epithelial cell apoptosis and epithelial-derived TNF production. Together, these findings demonstrate a previously unrecognized role for mast cells in maintaining the integrity of the lung epithelium and preventing lung injury during P. aeruginosa lung infection. The role of mast cells in acute lung injury has previously been addressed in several different models, which yielded conflicting results. In models of ischemia-reperfusion injury, mast cellederived tryptase and mast cell protease 7 have been shown as key mediators of acute lung injury,62,63 and pharmacological inhibition of mast cell function has been shown to reduce lung injury after liver transplantation.64 A similar role for mast cells has also been proposed during H5N1 influenza infection, where mast cell stabilizers reduced lung injury, leading to reduced mortality in vivo.65 These studies suggest that the contribution of mast cells to lung injury likely involves mast cellederived proteinases. In contrast, mast cells have recently been shown to reduce acute lung injury in a model of pulmonary lipopolysaccharide (LPS) challenge.66 In this model, local production of transforming growth factor-b1 stimulated production of IL-6 from mast cells, which induced neutrophil apoptosis and decreased acute lung injury.66 Similarly, we found that mast cells reduce P. aeruginosae induced lung injury in vitro and in vivo. However, our findings point toward an alternative mechanism of action, because we did not see any differences in the levels of TGF-b1 or IL-6 in the lungs of the mast celledeficient animals when compared

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Figure 6 Mast cell co-culture decreases P. aeruginosa (Ps.a.)einduced caspase-3 (Casp 3) activity and TNF production in bronchial epithelial cells: Human epithelial 16HBE14o- cells were grown to confluence on transwells for 6 days. Epithelial integrity was confirmed using TEER. Basolateral wells received 800 mL of IMDM or HMC-1 cells at a density of 0.5  106 cells/mL. Monolayers were left uninfected (NT) or infected with increasing doses of P. aeruginosa strain 8821 in the apical chamber. A: Twenty-four hours later, cells were fixed and stained for active caspase-3. B: Area analysis was used to determine the percentage of caspase-3epositive cells. C: Alternatively, cells were grown and treated as above, then cell lysates were prepared and subjected to Western blot analysis for active caspase-3. D: Results were quantified using scanning densitometry. E: Apical supernatants were collected and analyzed for tumor necrosis factor (TNF) by ELISA (n Z 3 to 5  SEM). *P < 0.05, **P < 0.01, and ***P < 0.001.

with wild-type animals after P. aeruginosa lung infection (data not shown). Furthermore, we were able to replicate mast cellemediated improvements in epithelial integrity using an in vitro transwell model in which no neutrophils were present. Hence, our findings demonstrate, for the first time to our knowledge, that mast cells directly counter acute lung injury during P. aeruginosa lung infection through maintaining epithelial integrity. In addition, our study highlights the divergent biological roles of mast cells at different mucosal surfaces throughout the body. Mast cells arrive at mucosal sites as immature precursor cells, and only complete their differentiation once recruited to the tissue in which they become resident.67 As a result, mast cell phenotypes are extremely plastic, and are largely determined by the biochemical nature of the tissue in which they reside.68e71 In the current study, we demonstrated a role for mast cells in preventing changes in epithelial permeability in the lung. These findings stand in contrast to extensive research demonstrating that mast cell proteases are responsible for the increasing epithelial permeability in the intestines in response to diverse stimuli.12,19,20,25,53,72,73 Mast cells do not secrete proteases in response to stimulation with bacterial products.74,75 In addition, we and others76 found that, unlike in the intestines, mast cells do not affect tight junction integrity in the lungs.

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Thus, our results highlight a unique and protective role of mast cells in regulating epithelial permeability in the lung. The localization of mast cells to mucosal interfaces ideally positions them to be sentinel cells of the immune system, recognizing and coordinating immune responses against invading pathogens. To effectively respond to diverse infectious agents, mast cells possess a wide array of pattern recognition and Fc receptors, and are capable of rapidly producing a plethora of bactericidal, proinflammatory, and immunomodulatory mediators.77 As a result, the role of mast cells during infections is highly pathogen specific. For example, during Klebsiella pneumoniae lung infection, mast cells are required for neutrophil recruitment and bacterial clearance.32 In contrast, during Mycoplasma pulmonis lung infection, mast celledeficient mice showed a significant and persistent accumulation of neutrophils, suggesting that mast cells dampen neutrophil recruitment in the lung.29 Recently, Chan et al78 described a broad immunosuppressive role for mast cells in the bladder during Escherichia coli infection. Taken together with the observed role for mast cells in protecting against P. aeruginosaeinduced acute lung damage, these findings demonstrate the ability of mast cells to respond to lung infections with a pathogen-specific and biologically relevant immune response.

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Mast Cells Prevent Lung Injury Our results appear to be in contrast with other groups who used the mast cell stabilizer cromolyn to inhibit mast cell function in rats treated with LPS from P. aeruginosa.79 Administration of cromolyn reduced LPS-induced neutrophil accumulation in the rat lung, suggesting that mast cells promote LPS-induced lung inflammation.79 These differences can be explained by the fact that cromolyn has selective effects on some aspects of mast cell functions, such as degranulation, but has limited effects on others, such as cytokine and chemokine production.80 Furthermore, in addition to its partial effects on mast cells, cromolyn also has non-specific effects on other cell responses and has been reported to directly affect neutrophil activity.81 In addition, because of the complex interplay of the arsenal of virulence factors expressed by P. aeruginosa, LPS alone may display different effects in the lungs than those seen during infection with whole bacteria. These differences in experimental models, and the fact that herein we used live infection, may explain the discrepancies observed. These studies provide interesting insights into the divergent roles of mast cells in response to various pathogenic stimuli, and highlight the complexity of the role of mast cells during P. aeruginosa lung infection. The current study demonstrates a previously unappreciated and biologically relevant role for mast cells in preventing bacteria-induced acute lung injury during P. aeruginosa lung infection. The absence of mast cells resulted in increased epithelial permeability, an increased accumulation of neutrophils within the parenchyma of the lung, and significant dissemination of bacteria from the site of infection. In cystic fibrosis, a decrease in total mast cell numbers was observed in small airways and pulmonary vessels.82 Cystic fibrosis is often associated with P. aeruginosa infection and chronic lung damage. It is tempting to speculate that a decrease of mast cell number may contribute to the lung damage in cystic fibrosis and activation of mast cells may serve as a new approach for the management of cystic fibrosis. However, we caution that the specific role of mast cells in cystic fibrosis remains to be determined and the popular way of mast cell activation in vivo in animal models by compound 48/80 has not been tested in humans. The next step toward therapeutic intervention would be the identification of the specific mast cellederived factor(s) responsible for the observed protective effects. Our initial studies have excluded cytokines known to influence acute lung injury, such as IL-6, IL-10, and TGF-b1 in the in vitro and in vivo models (data not shown). Future studies should examine additional mast cell mediators, such as IL-4 and IL-13. These cytokines are produced by mast cells, and both IL-4e and IL-13edeficient mice display greatly increased lung damage in response to various stimuli.83e86 The administration of both recombinant IL-4 and IL-13 has been shown to reduce immune complex-induced lung injury and TNF production in vivo.87 Mast cells are able to produce a plethora of biologically important mediators. Characterization of mast cellederived mediator(s)

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responsible for maintaining epithelial integrity remains to be a top priority.

Acknowledgment P. aeruginosa mucoid strain 8821 was a gift from Dr. Ananda Chakrabarty (University of Illinois, Chicago, IL).

Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2014.05.009.

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Mast cells protect against Pseudomonas aeruginosa-induced lung injury.

Pseudomonas aeruginosa, an opportunistic pathogen, is the leading cause of morbidity and mortality in immune-compromised individuals. Maintaining the ...
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