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Virus-Like Particle-Induced Protection Against MRSA Pneumonia Is Dependent on IL-13 and Enhancement of Phagocyte Function

2012; Elsevier BV; Volume: 181; Issue: 1 Linguagem: Inglês

10.1016/j.ajpath.2012.03.018

ISSN

1525-2191

Autores

Agnieszka Rynda‐Apple, Erin Dobrinen, Mark McAlpine, Amanda Read, Ann Harmsen, Laura Richert, Matthew Calverley, Kyler B. Pallister, Jovanka M. Voyich, James Wiley, Ben Johnson, Mark Young, Trevor Douglas, Allen G. Harmsen,

Tópico(s)

Viral gastroenteritis research and epidemiology

Resumo

The importance of the priming of the lung environment by past infections is being increasingly recognized. Exposure to any given antigen can either improve or worsen the outcome of subsequent lung infections, depending on the immunological history of the host. Thus, an ability to impart transient alterations in the lung environment in anticipation of future insult could provide an important novel therapy for emerging infectious diseases. In this study, we show that nasal administration of virus-like particles (VLPs) before, or immediately after, lethal challenge with methicillin-resistant Staphylococcus aureus (MRSA) of mice i) ensures complete recovery from lung infection and near absolute clearance of bacteria within 12 hours of challenge, ii) reduces host response-induced lung tissue damage, iii) promotes recruitment and efficient bacterial clearance by neutrophils and CD11c+ cells, and iv) protects macrophages from MRSA-induced necrosis. VLP-mediated protection against MRSA relied on innate immunity. Complete recovery occurred in VLP-dosed mice with severe combined immunodeficiency, but not in wild-type mice depleted of either Ly6G+ or CD11c+ cells. Early IL-13 production associated with VLP-induced CD11c+ cells was essential for VLP-induced protection. These results indicate that VLP-induced alteration of the lung environment protects the host from lethal MRSA pneumonia by enhancing phagocyte recruitment and killing and by reducing inflammation-induced tissue damage via IL-13–dependent mechanisms. The importance of the priming of the lung environment by past infections is being increasingly recognized. Exposure to any given antigen can either improve or worsen the outcome of subsequent lung infections, depending on the immunological history of the host. Thus, an ability to impart transient alterations in the lung environment in anticipation of future insult could provide an important novel therapy for emerging infectious diseases. In this study, we show that nasal administration of virus-like particles (VLPs) before, or immediately after, lethal challenge with methicillin-resistant Staphylococcus aureus (MRSA) of mice i) ensures complete recovery from lung infection and near absolute clearance of bacteria within 12 hours of challenge, ii) reduces host response-induced lung tissue damage, iii) promotes recruitment and efficient bacterial clearance by neutrophils and CD11c+ cells, and iv) protects macrophages from MRSA-induced necrosis. VLP-mediated protection against MRSA relied on innate immunity. Complete recovery occurred in VLP-dosed mice with severe combined immunodeficiency, but not in wild-type mice depleted of either Ly6G+ or CD11c+ cells. Early IL-13 production associated with VLP-induced CD11c+ cells was essential for VLP-induced protection. These results indicate that VLP-induced alteration of the lung environment protects the host from lethal MRSA pneumonia by enhancing phagocyte recruitment and killing and by reducing inflammation-induced tissue damage via IL-13–dependent mechanisms. The immune system is a plastic entity, in which the history of prior exposures modulates responses to future insults. It is well documented that the occurrence and even the exact sequence of infections can skew the immune response to a subsequent threat toward an inflammatory or anti-inflammatory phenotype,1Walzl G. Tafuro S. Moss P. Openshaw P.J. Hussell T. Influenza virus lung infection protects from respiratory syncytial virus-induced immunopathology.J Exp Med. 2000; 192: 1317-1326Crossref PubMed Scopus (114) Google Scholar, 2Folkerts G. Walzl G. Openshaw P.J. 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Colonic bacterial infection abrogates eosinophilic pulmonary disease.J Infect Dis. 2006; 193: 223-230Crossref PubMed Google Scholar and even the stage of host development.2Folkerts G. Walzl G. Openshaw P.J. Do common childhood infections "teach" the immune system not to be allergic?.Immunol Today. 2000; 21: 118-120Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar The modulatory effect of former insults can be long lasting and can influence the outcome and prognosis of an individual long after the infection has been cleared.2Folkerts G. Walzl G. Openshaw P.J. Do common childhood infections "teach" the immune system not to be allergic?.Immunol Today. 2000; 21: 118-120Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 7Wissinger E. Goulding J. Hussell T. Immune homeostasis in the respiratory tract and its impact on heterologous infection.Semin Immunol. 2009; 21: 147-155Crossref PubMed Scopus (75) Google Scholar For example, acute influenza infection can decrease immune-mediated lung damage associated with subsequent respiratory syncytial virus infection and inhibit replication of vaccinia virus, but it can also enhance replication of lymphocytic choriomeningitis virus and murine cytomegalovirus.7Wissinger E. Goulding J. Hussell T. Immune homeostasis in the respiratory tract and its impact on heterologous infection.Semin Immunol. 2009; 21: 147-155Crossref PubMed Scopus (75) Google Scholar Microbial products, such as CpG, lipopolysaccharide, or other bacterial toxins, can induce protection against a variety of subsequent respiratory pathogens.8Edwards L. Williams A.E. Krieg A.M. Rae A.J. Snelgrove R.J. Hussell T. Stimulation via Toll-like receptor 9 reduces Cryptococcus neoformans-induced pulmonary inflammation in an IL-12-dependent manner.Eur J Immunol. 2005; 35: 273-281Crossref PubMed Scopus (48) Google Scholar, 9Williams A.E. Edwards L. Humphreys I.R. Snelgrove R. Rae A. Rappuoli R. Hussell T. Innate imprinting by the modified heat-labile toxin of Escherichia coli (LTK63) provides generic protection against lung infectious disease.J Immunol. 2004; 173: 7435-7443Crossref PubMed Scopus (57) Google Scholar, 10Watkins A.C. Caputo F.J. Badami C. Barlos D. Xu da Z. Lu Q. Feketeova E. Deitch E.A. Mesenteric lymph duct ligation attenuates lung injury and neutrophil activation after intraperitoneal injection of endotoxin in rats.J Trauma. 2008; 64: 126-130Crossref PubMed Scopus (35) Google Scholar The homeostatic profile of the lung can also be altered by noninfectious substances. Morphine reduces Toll-like receptor 9–NF-κB signaling, which leads to impaired clearance of Streptococcus pneumoniae.11Wang J. Barke R.A. Charboneau R. Schwendener R. Roy S. Morphine induces defects in early response of alveolar macrophages to Streptococcus pneumoniae by modulating TLR9-NF-kappa B signaling.J Immunol. 2008; 180: 3594-3600Crossref PubMed Google Scholar Cigarette smoke down-regulates Toll-like receptor 2 expression on alveolar macrophages (AMs)12Droemann D. Goldmann T. Tiedje T. Zabel P. Dalhoff K. Schaaf B. Toll-like receptor 2 expression is decreased on alveolar macrophages in cigarette smokers and COPD patients.Respir Res. 2005; 6: 68Crossref PubMed Scopus (193) Google Scholar and inhibits expression of proinflammatory cytokines,13Chen H. Cowan M.J. Hasday J.D. Vogel S.N. Medvedev A.E. Tobacco smoking inhibits expression of proinflammatory cytokines and activation of IL-1R-associated kinase, p38, and NF-kappaB in alveolar macrophages stimulated with TLR2 and TLR4 agonists.J Immunol. 2007; 179: 6097-6106Crossref PubMed Google Scholar which may increase susceptibility to respiratory bacterial infections. Immune imprinting by the nontoxic heat-labile endotoxin (LTK63) of Escherichia coli, delivered as a mucosal adjuvant, induces a transient inflammatory response that reduces inflammation to subsequent type 1 and 2 helper T-cell pathogens (namely, influenza, respiratory syncytial virus, and Cryptococcus neoformans).9Williams A.E. Edwards L. Humphreys I.R. Snelgrove R. Rae A. Rappuoli R. Hussell T. Innate imprinting by the modified heat-labile toxin of Escherichia coli (LTK63) provides generic protection against lung infectious disease.J Immunol. 2004; 173: 7435-7443Crossref PubMed Scopus (57) Google Scholar Therefore, the ability to transiently modulate the immune environment in preparation for a future infectious challenge, without the need for the induction of harmful inflammation, would be beneficial as preventative and therapeutic treatments. We have previously reported that pulmonary delivery of a noninfectious, self-assembled protein cage nanoparticle (PCN) induced low-grade inflammation in the absence of tissue damage, yet it enhanced the protection of mice against subsequent sublethal and lethal infections by unrelated viruses, including influenza, severe acute respiratory syndrome virus, and pneumovirus.14Wiley J.A. Richert L.E. Swain S.D. Harmsen A. Barnard D.L. Randall T.D. Jutila M. Douglas T. Broomell C. Young M. Harmsen A.G. Inducible bronchus-associated lymphoid tissue elicited by a protein cage nanoparticle enhances protection in mice against diverse respiratory viruses.PLoS One. 2009; 4: e7142Crossref PubMed Scopus (105) Google Scholar This protection is associated with the formation of inducible bronchus-associated lymphoid tissue (iBALT) that modulates the local lung environment in the absence of harmful inflammation.14Wiley J.A. Richert L.E. Swain S.D. Harmsen A. Barnard D.L. Randall T.D. Jutila M. Douglas T. Broomell C. Young M. Harmsen A.G. Inducible bronchus-associated lymphoid tissue elicited by a protein cage nanoparticle enhances protection in mice against diverse respiratory viruses.PLoS One. 2009; 4: e7142Crossref PubMed Scopus (105) Google Scholar, 15Rangel-Moreno J. Hartson L. Navarro C. Gaxiola M. Selman M. Randall T.D. Inducible bronchus-associated lymphoid tissue (iBALT) in patients with pulmonary complications of rheumatoid arthritis.J Clin Invest. 2006; 116: 3183-3194Crossref PubMed Scopus (379) Google Scholar PCN is a hollow, spherical protein cage that self-assembles from 24 identical subunits of small heat shock protein, derived from Methanocaldococcus jannaschii. Although PCNs are not of viral origin, their small size (12-nm diameter), self-assembled particulate structure, immunogenicity, and lack of genetic material characterize them as virus-like particles (VLPs).14Wiley J.A. Richert L.E. Swain S.D. Harmsen A. Barnard D.L. Randall T.D. Jutila M. Douglas T. Broomell C. Young M. Harmsen A.G. Inducible bronchus-associated lymphoid tissue elicited by a protein cage nanoparticle enhances protection in mice against diverse respiratory viruses.PLoS One. 2009; 4: e7142Crossref PubMed Scopus (105) Google Scholar, 16Flenniken M.L. Willits D.A. Harmsen A.L. Liepold L.O. Harmsen A.G. Young M.J. Douglas T. Melanoma and lymphocyte cell-specific targeting incorporated into a heat shock protein cage architecture.Chem Biol. 2006; 13: 161-170Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 17Kaiser C.R. Flenniken M.L. Gillitzer E. Harmsen A.L. Harmsen A.G. Jutila M.A. Douglas T. Young M.J. Biodistribution studies of protein cage nanoparticles demonstrate broad tissue distribution and rapid clearance in vivo.Int J Nanomedicine. 2007; 2: 715-733PubMed Google Scholar Known for their potent adjuvant-like properties,18Jennings G.T. Bachmann M.F. The coming of age of virus-like particle vaccines.Biol Chem. 2008; 389: 521-536Crossref PubMed Scopus (338) Google Scholar VLPs can enhance immune responses to parental infectious agents, such as hepatitis C,19Lechmann M. Murata K. Satoi J. Vergalla J. Baumert T.F. Liang T.J. Hepatitis C virus-like particles induce virus-specific humoral and cellular immune responses in mice.Hepatology. 2001; 34: 417-423Crossref PubMed Scopus (103) Google Scholar dengue virus,20Zhang S. Liang M. Gu W. Li C. Miao F. Wang X. Jin C. Zhang L. Zhang F. Zhang Q. Jiang L. Li M. Li D. Vaccination with dengue virus-like particles induces humoral and cellular immune responses in mice.Virol J. 2011; 8: 333Crossref PubMed Scopus (48) Google Scholar Norwalk virus,21Velasquez L.S. Shira S. Berta A.N. Kilbourne J. Medi B.M. Tizard I. Ni Y. Arntzen C.J. Herbst-Kralovetz M.M. Intranasal delivery of Norwalk virus-like particles formulated in an in situ gelling, dry powder vaccine.Vaccine. 2011; 29: 5221-5231Crossref PubMed Scopus (80) Google Scholar, 22Herbst-Kralovetz M. Mason H.S. Chen Q. Norwalk virus-like particles as vaccines.Expert Rev Vaccines. 2010; 9: 299-307Crossref PubMed Scopus (100) Google Scholar or human papillomavirus-induced cervical cancer.23Barr E. Sings H.L. Prophylactic HPV vaccines: new interventions for cancer control.Vaccine. 2008; 26: 6244-6257Crossref PubMed Scopus (64) Google Scholar, 24Harper D.M. Currently approved prophylactic HPV vaccines.Expert Rev Vaccines. 2009; 8: 1663-1679Crossref PubMed Scopus (125) Google Scholar In addition, VLPs have been used as a platform for targeting cells16Flenniken M.L. Willits D.A. Harmsen A.L. Liepold L.O. Harmsen A.G. Young M.J. Douglas T. Melanoma and lymphocyte cell-specific targeting incorporated into a heat shock protein cage architecture.Chem Biol. 2006; 13: 161-170Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar and as a delivery vehicle to present the immune system with fused or conjugated heterologous epitopes,25Chackerian B. Rangel M. Hunter Z. Peabody D.S. Virus and virus-like particle-based immunogens for Alzheimer's disease induce antibody responses against amyloid-beta without concomitant T cell responses.Vaccine. 2006; 24: 6321-6331Crossref PubMed Scopus (98) Google Scholar and even to induce autoantibodies to self molecules involved in chronic diseases.25Chackerian B. Rangel M. Hunter Z. Peabody D.S. Virus and virus-like particle-based immunogens for Alzheimer's disease induce antibody responses against amyloid-beta without concomitant T cell responses.Vaccine. 2006; 24: 6321-6331Crossref PubMed Scopus (98) Google Scholar, 26Fulurija A. Lutz T.A. Sladko K. Osto M. Wielinga P.Y. Bachmann M.F. Saudan P. Vaccination against GIP for the treatment of obesity.PLoS One. 2008; 3: e3163Crossref PubMed Scopus (87) Google Scholar The work reported by our laboratory adds yet another dimension to the VLP-mediated immune modulation (namely, the ability to induce protective immunity to a variety of unrelated antigens).14Wiley J.A. Richert L.E. Swain S.D. Harmsen A. Barnard D.L. Randall T.D. Jutila M. Douglas T. Broomell C. Young M. Harmsen A.G. Inducible bronchus-associated lymphoid tissue elicited by a protein cage nanoparticle enhances protection in mice against diverse respiratory viruses.PLoS One. 2009; 4: e7142Crossref PubMed Scopus (105) Google Scholar Invasive methicillin-resistant Staphylococcus aureus (MRSA) infections result annually in more deaths (>18,000 cases) than any other single infectious agent in the United States.27Klevens R.M. Morrison M.A. Nadle J. Petit S. Gershman K. Ray S. Harrison L.H. Lynfield R. Dumyati G. Townes J.M. Craig A.S. Zell E.R. Fosheim G.E. McDougal L.K. Carey R.B. Fridkin S.K. Invasive methicillin-resistant Staphylococcus aureus infections in the United States.JAMA. 2007; 298: 1763-1771Crossref PubMed Scopus (2857) Google Scholar In fact, the mortality associated with MRSA exceeds the mortality resulting from combined, human immunodeficiency, hepatitis, and influenza viruses.28Boucher H.W. Corey G.R. Epidemiology of methicillin-resistant Staphylococcus aureus.Clin Infect Dis. 2008; 46: S344-S349Crossref PubMed Scopus (674) Google Scholar, 29Miller L.S. Cho J.S. Immunity against Staphylococcus aureus cutaneous infections.Nat Rev Immunol. 2011; 11: 505-518Crossref PubMed Scopus (322) Google Scholar The pulsed-field gel electrophoresis type USA300 is the most prevalent community-associated MRSA strain in United States. USA300 typically causes skin and soft tissue infections; however, it is also capable of causing severe staphylococcal diseases, including necrotizing pneumonia that can transpire in otherwise healthy individuals.30Graves S.F. Kobayashi S.D. DeLeo F.R. Community-associated methicillin-resistant Staphylococcus aureus immune evasion and virulence.J Mol Med (Berl). 2010; 88: 109-114Crossref PubMed Scopus (66) Google Scholar, 31Deleo F.R. Otto M. Kreiswirth B.N. Chambers H.F. Community-associated methicillin-resistant Staphylococcus aureus.Lancet. 2010; 375: 1557-1568Abstract Full Text Full Text PDF PubMed Scopus (1064) Google Scholar In the current study, PCN administration before, or shortly after, respiratory infection of mice with MRSA minimized morbidity and prevented mortality. Major reduction of bacterial burden was observed as early as 8 hours after infection in PCN-dosed mice. This protection was associated with improved recruitment of neutrophils and enhanced antibacterial activity of both neutrophils and lung CD11c+ cells. The reliance on innate immunity for PCN-mediated protection was indicated by the complete recovery of MRSA-infected mice with severe combined immunodeficiency (SCID) dosed with PCN and the significant loss of protection in PCN-dosed wild-type (WT) mice depleted of either Ly6G+ neutrophils or CD11c+ cells. Interestingly, PCN-mediated protection was not limited to the lung, because bone marrow (BM) cells from mice intranasally dosed with PCN, but not with PBS, efficiently killed MRSA in vitro. Phenotypical analyses revealed that PCN-dosed mice retained a high M1:M2 macrophage ratio throughout the infection; however, the efficient bacterial killing in PCN-dosed mice was dependent on the early presence of IL-13 and IL-4, in that mice deficient in either of these cytokines had reduced PCN-mediated protection against MRSA pneumonia. Male and female WT BALB/c, C57BL/6 (CD45.2), SCID, C-X-C chemokine receptor (CXCR) 2 knockout (KO), IL-13 KO, and CCR2 KO mice were bred at Montana State University, Bozeman. The breeding pairs of IL-13 KO mice were originally generated by Dr. Andrew N.J. McKenzie (Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK) and were kindly provided by Dr. Andrij Holian (University of Montana, Missoula). The breeding pairs of WT and remaining KO mice were obtained from The Jackson Laboratories (Bar Harbor, MI). All mice were maintained at Montana State University Animal Resources Center in individual ventilated cages under high efficiency particulate arresting-filter barrier conditions and were fed sterile food and water ad libitum. All animal care procedures were in accordance with institutional polices for animal health and well-being; 7- to 8-week-old mice were used in all experiments. PCNs were prepared as previously described.14Wiley J.A. Richert L.E. Swain S.D. Harmsen A. Barnard D.L. Randall T.D. Jutila M. Douglas T. Broomell C. Young M. Harmsen A.G. Inducible bronchus-associated lymphoid tissue elicited by a protein cage nanoparticle enhances protection in mice against diverse respiratory viruses.PLoS One. 2009; 4: e7142Crossref PubMed Scopus (105) Google Scholar, 16Flenniken M.L. Willits D.A. Harmsen A.L. Liepold L.O. Harmsen A.G. Young M.J. Douglas T. Melanoma and lymphocyte cell-specific targeting incorporated into a heat shock protein cage architecture.Chem Biol. 2006; 13: 161-170Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar Mice were lightly anesthetized with oxygen-delivered isoflurane, USP (Piramal, Bethlehem, PA), and were intranasally dosed with 50 μL of sterile PBS or PCN containing 100 μg of protein. This dosing procedure was repeated five times either daily or every couple of days for 2 weeks, as designated in the figures. The LAC strain of MRSA (pulsed-field type USA300) was a kind gift from Dr. Jovanka Voyich (Montana State University). The USA300 MRSA was used for infections because it has been described as a causative agent of severe community-onset pneumonia in healthy individuals.32Francis J.S. Doherty M.C. Lopatin U. Johnston C.P. Sinha G. Ross T. Cai M. Hansel N.N. Perl T. Ticehurst J.R. Carroll K. Thomas D.L. Nuermberger E. Bartlett J.G. Severe community-onset pneumonia in healthy adults caused by methicillin-resistant Staphylococcus aureus carrying the Panton-Valentine leukocidin genes.Clin Infect Dis. 2005; 40: 100-107Crossref PubMed Scopus (652) Google Scholar Before MRSA infection, PCN- and PBS-dosed mice were rested for 6 to 7 days to ensure removal of nanoparticles from the body.17Kaiser C.R. Flenniken M.L. Gillitzer E. Harmsen A.L. Harmsen A.G. Jutila M.A. Douglas T. Young M.J. Biodistribution studies of protein cage nanoparticles demonstrate broad tissue distribution and rapid clearance in vivo.Int J Nanomedicine. 2007; 2: 715-733PubMed Google Scholar Fresh or frozen bacteria grown to OD600 1.5 in tryptic soy broth were used for in vivo infections and in vitro cultures. Between 5 × 107 and 5 × 108 (where indicated) microorganisms were used for the infection dose. The erythromycin-sensitive MRSA strain (AH1263 USA300 community-associated MRSA ErmS Lac)33Boles B.R. Thoendel M. Roth A.J. Horswill A.R. Identification of genes involved in polysaccharide-independent Staphylococcus aureus biofilm formation.PLoS One. 2010; 5: e10146Crossref PubMed Scopus (294) Google Scholar was provided by Dr. Alex Horswill (University of Iowa, Iowa City). Mice were sacrificed at the designated time points by i.p. administration of a lethal dose of sodium pentobarbital (90 mg/kg), followed by exsanguination when the mice no longer exhibited a pedal reflex. Bronchoalveolar lavage fluid (BALF) was obtained by washing the lungs with 2 mL of 3 mmol/L EDTA in PBS in two aliquots of 1 mL each. The cellular composition of BALF was determined by hemocytometer cell counts and differential counts of cytospins after staining with Quick-Diff solution (Siemens; Medical Solutions Diagnostics, Tarrytown, NY). After centrifugation, the cell-free BALF supernatant was recovered and stored at −80°C for future use to determine levels of cytokines and lung tissue damage. Lungs used for histological analyses were instilled and fixed in 10% buffered formalin phosphate (Fisher Scientific, Fairlawn, NJ) for 24 hours. Paraffin-embedded lung sections (5 μm thick) were stained with H&E and evaluated under a microscope (Eclipse E800; Nikon Inc., Melville, NY) at ×10 and ×60 objective magnification. Mice were monitored bidaily up to 96 hours after infection. Lung bacterial burden was determined based on the counted number of colony-forming units (CFUs) cultured out of 100 μL of serially diluted whole lung homogenates (LHs) on tryptic soy agar (TSA) plates at 37°C in 5% CO2. Plated colonies were counted after an overnight incubation. Mice previously dosed with PCN or PBS were i.p. injected with anti-CD11c monoclonal antibody (mAb) [HB224/N418; for depletion of AMs and dendritic cells (DCs)] or anti-Ly6G mAb (1A8; for depletion of neutrophils), both obtained from BioXcell (West Lebanon, NH). The total volume of 500 μL (containing 250 μg of either Ab) was delivered by injection on days −3, −1, and 0 in relation to MRSA infection (day 0). Cell depletion was verified by flow cytometry at sacrifice and was determined to be at least 98% efficient for depletion of Ly6G+ and >97% efficient for depletion of CD11c+ cells in both PCN- and PBS-dosed mice. Neutrophils were isolated from the BM of naïve BALB/c mice by Percoll gradient, as previously described.34Siemen D.W. Schepetkin I.A. Kirpotina L.N. Lei B. Quinn M.T. Neutrophil Isolation from Nonhuman Species.Methods in Molecular Biology. 2007; 412: 21-34Crossref PubMed Google Scholar Briefly, BM was flushed from dissected tibias and femurs, resuspended in HBSS (Mediatech Inc., Herndon, VA), supplemented with 0.1% bovine serum albumin and 1% glucose, and filtered through a 70-μm nylon cell strainer (BD, Franklin Lakes, NJ) to remove clumps and bone particles. BM cells were centrifuged (600 × g, 10 minutes) and resuspended in 45% Percoll PLUS solution (GE Healthcare Bio-Sciences Corp, Piscataway, NJ). The Percoll gradient was prepared by layering the polypropylene tube with 81%, 62%, 55%, and 50% of Percoll solution in HBSS. The BM cells in 45% Percoll were carefully suspended on a top of the gradient. After centrifugation (1600 × g, 30 minutes), the neutrophil fraction (band between 81% and 62% Percoll gradient) was carefully removed and washed twice with HBSS. The purity of the neutrophil fraction was confirmed by microscopic examination of cells after the Diff-Quick staining procedure and was assessed to be >97% pure. Purified neutrophils were labeled with Vybrant-DiI (Molecular Probes, Eugene, OR) dye, according to the manufacturer's recommendations. Mice were dosed nasally with PCN or PBS for 5 consecutive days. One week after the last dose, mice were infected with MRSA and, 1 hour later, adoptively transferred through tail vein injection with 1 × 107 Vybrant-labeled neutrophils. At 4 hours after adoptive transfer, mice were sacrificed. The number of Vybrant+ cells in BALF was determined by fluorescence-activated cell sorting (FACS). Cytokine analyses were performed on cell-free BALF. Levels of interferon-γ, tumor necrosis factor (TNF)-α, IL-6, and monocyte chemoattractant protein (MCP)-1 were determined by Cytometric Bead Array (BD Bioscience, San Jose, CA), according to manufacturer's recommendations, and analyzed by BD LSRII Flow Cytometer (BD Bioscience) using BD Cytometric Bead Array analysis software. Levels of IL-13 and IL-4 were determined by a sandwich enzyme-linked immunosorbent assay kit (Ready-SET-Go; eBioscience, San Diego, CA). BALF samples were tested in triplicate. A single-cell suspension was obtained by centrifugation of BALF. Red blood cells were lysed with AKC Lysis Buffer (150 mmol/L NH4Cl, 1 mmol/L KHCO3, and 0.1 mmol/L Na2EDTA; pH 7.2 to 7.4) in room temperature for 5 minutes. Cells were washed with PBS twice and blocked with Fc receptor block (2.4G2 hybridoma made in house) for 20 minutes at room temperature. Cells were stained with fluorochrome-labeled antibodies to extracellular markers: CD11c (clone N418; eBioscience), CD11b (clone M1/70; eBioscience), F4/80 (clone BM8; eBioscience), major histocompatibility complex (MHC) II (clone M5/114.15.2; eBioscience), and CD206 (clone MR5D3; AbD Serotec, Raleigh, NC). After a 30-minutes incubation, cells were washed twice in FACS buffer (1% fetal bovine serum/PBS) and stained with streptavidin–allophycocyanin for 30 minutes. After staining for extracellular markers, cells were washed in FACS buffer and fixed in 4% paraformaldehyde for 20 minutes. Fixed cells were washed once in FACS buffer and in 0.2% saponin made in FACS buffer, and they were stained for 30 minutes with phosphatidylethanolamine-conjugated Abs to mouse IL-13 (clone eBio13A; eBioscience) or with rat IgG1 isotype control Ab (clone eBRG1; eBioscience). Samples were acquired on FACSCanto running FACSDiva software (both obtained from BD Bioscience). Approximately 50,000 events were routinely acquired per sample. FlowJo software (Tree Star, Inc., Ashland, OR) was used for analysis. A single-cell suspension obtained after red blood cell lysis and filtration (30 μm) of LH was run through magnetic-activated cell sorting (MACS)–positive selection columns to obtain pure Ly6G+ and Ly6G− cell populations (>97% and >98% purity, respectively). In separate experiments, LH cells were first selected for CD11c+ cells (positive MACS CD11c selection) and the CD11c− cells were subsequently depleted of Ly6G+ cells by MACS Ly6G depletion columns (>97% purity). The purity of all fractions was confirmed by FACS, according to manufacturers' recommendations. For these experiments, designated populations of cells were plated in a 1:1 ratio with normal mouse serum (NMS)–opsonized MRSA (50% NMS, 30 minutes, 37°C, 5% CO2) on NMS-coated flat-well culture plates (100% NMS, 30 minutes, 37°C, 5% CO2). The killing assay was conducted at 37°C for 1.5 and 3 hours. After this time, 0.1% saponin was added to each well to release internalized bacteria and total CFUs were determined for each experimental sample by plating the 10-fold dilutions on TSA plates. In the experiments in which two different variants of the LAC strain were used (in vivo, MT1263 erythromycin-sensitive LAC; and in vitro, WT LAC erythromycin resistant), the bacterial burden was evaluated by plating on TSA/erythromycin plates (25 μg/mL). This assay was performed as previously described.35Voyich J.M. Braughton K.R. Sturdevant D.E. Whitney A.R. Said-Salim B. Porcella S.F. Long R.D. Dorward D.W. Gardner D.J. Kreiswirth B.N. Musser J.M. DeLeo F.R. Insights into mechanisms used by Staphylococcus aureus to avoid destruction by human neutr

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