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CORD-19:12c408d995ef90da15a01ba61777b04b3aa3bb11 JSONTXT 8 Projects

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Id Subject Object Predicate Lexical cue
T1 0-96 Sentence denotes Stimulated Innate Resistance of Lung Epithelium Protects Mice Broadly against Bacteria and Fungi
T2 98-106 Sentence denotes Abstract
T3 107-148 Sentence denotes Pneumonia is a serious problem worldwide.
T4 149-269 Sentence denotes We recently demonstrated that innate defense mechanisms of the lung are highly inducible against pneumococcal pneumonia.
T5 270-565 Sentence denotes To determine the breadth of protection conferred by stimulation of lung mucosal innate immunity, and to identify cells and signaling pathways activated by this treatment, mice were treated with an aerosolized bacterial lysate, then challenged with lethal doses of bacterial and fungal pathogens.
T6 566-738 Sentence denotes Mice were highly protected against a broad array of Gram-positive, Gram-negative, and class A bioterror bacterial pathogens, and the fungal pathogen, Aspergillus fumigatus.
T7 739-895 Sentence denotes Protection was associated with rapid pathogen killing within the lungs, and this effect was recapitulated in vitro using a respiratory epithelial cell line.
T8 896-1032 Sentence denotes Gene expression analysis of lung tissue showed marked activation of NF-kB, type I and II IFN, and antifungal Card9-Bcl10-Malt1 pathways.
T9 1033-1158 Sentence denotes Cytokines were the most strongly induced genes, but the inflammatory cytokines TNF and IL-6 were not required for protection.
T10 1159-1227 Sentence denotes Lung-expressed antimicrobial peptides were also highly up-regulated.
T11 1228-1496 Sentence denotes Taken together, stimulated innate resistance appears to occur through the activation of multiple host defense signaling pathways in lung epithelial cells, inducing rapid pathogen killing, and conferring broad protection against virulent bacterial and fungal pathogens.
T12 1497-1797 Sentence denotes Augmentation of innate antimicrobial defenses of the lungs might have therapeutic value for protection of patients with neutropenia or impaired adaptive immunity against opportunistic pneumonia, and for defense of immunocompetent subjects against a bioterror threat or epidemic respiratory infection.
T13 1799-1840 Sentence denotes Pneumonia is a serious problem worldwide.
T14 1841-1961 Sentence denotes We recently demonstrated that innate defense mechanisms of the lung are highly inducible against pneumococcal pneumonia.
T15 1962-2257 Sentence denotes To determine the breadth of protection conferred by stimulation of lung mucosal innate immunity, and to identify cells and signaling pathways activated by this treatment, mice were treated with an aerosolized bacterial lysate, then challenged with lethal doses of bacterial and fungal pathogens.
T16 2258-2430 Sentence denotes Mice were highly protected against a broad array of Gram-positive, Gram-negative, and class A bioterror bacterial pathogens, and the fungal pathogen, Aspergillus fumigatus.
T17 2431-2587 Sentence denotes Protection was associated with rapid pathogen killing within the lungs, and this effect was recapitulated in vitro using a respiratory epithelial cell line.
T18 2588-2724 Sentence denotes Gene expression analysis of lung tissue showed marked activation of NF-kB, type I and II IFN, and antifungal Card9-Bcl10-Malt1 pathways.
T19 2725-2850 Sentence denotes Cytokines were the most strongly induced genes, but the inflammatory cytokines TNF and IL-6 were not required for protection.
T20 2851-2919 Sentence denotes Lung-expressed antimicrobial peptides were also highly up-regulated.
T21 2920-3188 Sentence denotes Taken together, stimulated innate resistance appears to occur through the activation of multiple host defense signaling pathways in lung epithelial cells, inducing rapid pathogen killing, and conferring broad protection against virulent bacterial and fungal pathogens.
T22 3189-3489 Sentence denotes Augmentation of innate antimicrobial defenses of the lungs might have therapeutic value for protection of patients with neutropenia or impaired adaptive immunity against opportunistic pneumonia, and for defense of immunocompetent subjects against a bioterror threat or epidemic respiratory infection.
T23 3490-3692 Sentence denotes Keywords: innate immunity; pneumonia; immunocompromised host; lung epithelium Among both healthy and immunosuppressed people, pneumonia is the leading cause of premature death in the world (1) (2) (3) .
T24 3693-3938 Sentence denotes Nosocomial pneumonias result in more deaths than any other hospital infection (4) , and impose a particular burden on immunocompromised patients, such as those with hematologic malignancies and others undergoing cytotoxic therapies (5) (6) (7) .
T25 3939-4240 Sentence denotes The lower respiratory tract is also a frequent locus of emerging infections, such as severe acute respiratory syndrome (8) or epidemic influenza (9) , and of difficult to manage pathogens, such as multiple drug resistant Pseudomonas aeruginosa or extensively drug resistant Mycobacterium tuberculosis.
T26 4241-4464 Sentence denotes Furthermore, the respiratory route is the most likely portal of entry for bioterror agents, and the only means by which such pathogens have been delivered successfully in the modern era (i.e., inhalation anthrax) (10, 11) .
T27 4465-4610 Sentence denotes Although the need for gas exchange leaves the lungs structurally vulnerable to infection, they successfully contain and eliminate most pathogens.
T28 4611-4825 Sentence denotes The airway and alveolar epithelia, in particular, are critical to the prevention of infection through their capacity to recruit leukocytes and to initiate antimicrobial innate immune responses (12) (13) (14) (15) .
T29 4826-5053 Sentence denotes We have recently found that stimulation of lung innate immune mechanisms with an aerosolized bacterial lysate confers a high level of protection against challenge with otherwise lethal inocula of Streptococcus pneumoniae (16) .
T30 5054-5169 Sentence denotes Pretreatment with the lysate induces complete protection against challenge if given 4 to 24 hours before challenge.
T31 5170-5334 Sentence denotes Lesser levels of protection occur at shorter and longer intervals between treatment and challenge, and some protection occurs even when treatment follows challenge.
T32 5335-5485 Sentence denotes Protection does not depend on recruited neutrophils or resident mast cells and alveolar macrophages, and is specific to the airway route of infection.
T33 5486-5695 Sentence denotes The survival benefit correlates in magnitude and time with rapid bacterial killing within the lungs, and is associated with increased concentrations of numerous antimicrobial polypeptides in lung lining fluid.
T34 5696-5774 Sentence denotes We have termed the protective phenomenon, stimulated innate resistance (StIR).
T35 5775-6001 Sentence denotes In contrast to the highly refined epitope sensing of adaptive immune responses, innate immunity relies on rapid recognition of pathogen-associated molecular patterns (PAMPs) shared across many pathogen species (17) (18) (19) .
T36 6002-6183 Sentence denotes Suppression of adaptive immunity and hematopoiesis, as observed in hematologic malignancy and/or cytotoxic chemotherapy, does not obviate the lung's innate immune response to PAMPs.
T37 6184-6310 Sentence denotes Rather, profoundly immunosuppressed patients generate remarkable cytokine responses to pneumonia (20) , and we have shown that
T38 6311-6364 Sentence denotes Pneumonia results in significant worldwide mortality.
T39 6365-6578 Sentence denotes Stimulating antimicrobial signaling to induce broad protection against respiratory pathogens has novel scientific and clinical implications for protection against opportunistic, bioterror, and epidemic infections.
T40 6579-6746 Sentence denotes (Received in original form July 10, 2008 and in final form March 9, 2009) innate lung defenses are highly inducible in mice receiving myeloablative chemotherapy (16) .
T41 6747-7047 Sentence denotes Because of the characteristic breadth of innate immunity, and the complexity of the lysate used to stimulate protection against S. pneumoniae, the survival effect induced by the aerosolized bacterial lysate might be expected to extend to other bacterial pathogens, or even to other microbial classes.
T42 7048-7281 Sentence denotes Here, we tested this hypothesis by treating mice with the lysate before challenge with numerous respiratory pathogens, including nosocomial pathogens and National Institute of Allergy and Infectious Diseases class A bioterror agents.
T43 7282-7481 Sentence denotes We find dramatic protection against diverse bacterial and fungal pathogens, and that this effect appears to arise from the simultaneous induction of multiple antimicrobial pathways in the epithelium.
T44 7482-7707 Sentence denotes These findings could provide a basis for novel clinical strategies to protect immunosuppressed patients from fatal infectious complications, and to safeguard broader populations from emerging infections or bioterror assaults.
T45 7708-7817 Sentence denotes Nontypeable Haemophilus influenzae (NTHi) was stored, grown, and harvested as previously described (16, 21) .
T46 7818-7921 Sentence denotes In the current work, the cell pellet was washed and resuspended in 20 ml 0.9% sodium chloride solution.
T47 7922-8211 Sentence denotes This suspension was passed three times through an Emulsi-Flex C5 cell disruptor (Avestin, Ottawa, ON, Canada) at greater then 10,000 psi, then diluted to 4-5 mg/ml in 0.9% sodium chloride solution by bicinchoninic assay (Pierce, Rockford, IL), and centrifuged at 15,000 3 g for 10 minutes.
T48 8212-8398 Sentence denotes The supernatant was collected, the protein concentration was adjusted to 2.5 mg/ml, and the lysate was sterilized by passage through a 0.2-mm filter and frozen in 8 ml aliquots at 2808C.
T49 8399-8485 Sentence denotes Treatment of mice with aerosolized lysate was performed as previously described (16) .
T50 8486-8602 Sentence denotes Experiments with S. pneumoniae, P. aeruginosa, Staphylococcus aureus, and Aspergillus fumigatus were conducted at M.
T51 8603-8605 Sentence denotes D.
T52 8606-8900 Sentence denotes Anderson Cancer Center using specific pathogen-free, 5-to 9-week-old female BALB/c mice (Harlan, Indianapolis, IN), except for the experiments with IL-6-deficient C57BL/6 il6 2/2 and littermate C57BL/6 il6 1/1 mice of either sex that were bred within our colony and used at 5 to 8 weeks of age.
T53 8901-9123 Sentence denotes Experiments with Bacillus anthracis, Yersinia pestis, and Francisella tularensis were conducted at the University of Texas Medical Branch in Galveston using 6-to 10-week-old female Swiss-Webster mice (Taconic, Hudson, NY).
T54 9124-9261 Sentence denotes Mice were handled in accordance with the institutional animal care and use committees of each institution, and were killed if distressed.
T55 9262-9378 Sentence denotes For the clinical pathogens, the challenges were targeted to inocula that induced 80-100% mortality (LD 80 -LD 100 ).
T56 9379-9597 Sentence denotes S. pneumoniae serotype 4, P. aeruginosa strain PA103, and Klebsiella pneumoniae serotype 2 were suspended in PBS and delivered by aerosol using an AeroMist nebulizer driven by 10 L/min of 5% CO 2 in air for 60 minutes.
T57 9598-9832 Sentence denotes This was similar to the NTHi lysate exposure, except that an additional 5 ml of the bacterial suspensions were added to the nebulizer after 30 minutes, in addition to the 10 ml placed at the beginning, with a total of 10 ml delivered.
T58 9833-10076 Sentence denotes Panton-Valentine leukocidin (PVL) toxinproducing methicillin-resistant S. aureus USA300 was suspended in PBS, and 20 ml was instilled into the left naris of mice anesthetized with an intraperitoneal injection of 2.5% tribromoethanol (16 ml/g).
T59 10077-10142 Sentence denotes For the bioterror pathogens, the dose was targeted to 5 3 LD 50 .
T60 10143-10264 Sentence denotes Ames strain B. anthracis spores, Y. pestis CO92, and F. tularensis Schu 4 were delivered by nasal instillation (22, 23) .
T61 10265-10522 Sentence denotes Mice were anesthetized by intraperitoneal injection of ketamine (48 mg/kg) and xylazine (9.6 mg/kg), then suspended vertically by their upper incisors, and 20 ml of pathogen suspension was instilled into each naris, followed by 10 ml of PBS into each naris.
T62 10523-10597 Sentence denotes All survival studies were performed using groups of 10 mice per condition.
T63 10598-10701 Sentence denotes Conidia of strain Af293 were stored as frozen stock (1 3 10 9 conidia/ml) in 20% glycerol in PBS (24) .
T64 10702-10863 Sentence denotes A 1-ml aliquot of stock was plated on yeast extract agar plates at 378C in 5% CO 2 for 3 days, then harvested by gentle scraping in PBS containing 0.1% Tween-20.
T65 10864-11031 Sentence denotes The suspension was filtered through 40-mm filters, centrifuged at 2,500 3 g for 10 minutes, washed, resuspended in PBS, and aerosolized as for S. pneumoniae challenge.
T66 11032-11113 Sentence denotes Conidia counts in the nebulizer suspension were determined using a hemacytometer.
T67 11114-11293 Sentence denotes Mice were immunosuppressed by intraperitoneal injection of cyclophosphamide (75 mg/gm) and cortisone acetate (300 mg/kg) 4 days and 1 day before challenge with A. fumigatus (24) .
T68 11294-11574 Sentence denotes For assessment of lung fungal burden 24 hours after challenge, real-time quantitative PCR for A. fumigatus 18S rRNA was performed as previously described (24) , and Aspergillus galactomannan was measured by a single-incubation sandwich ELISA, as detailed in the online supplement.
T69 11575-11675 Sentence denotes C57BL/6 mice were treated, or not, with the aerosolized NTHi lysate, then killed after 2 or 4 hours.
T70 11676-11765 Sentence denotes To reduce leukocytes, the lung vasculature was perfused and the airways lavaged with PBS.
T71 11766-11945 Sentence denotes The lungs were homogenized, total RNA was isolated, and cRNA was synthesized, amplified, labeled, and hybridized on Sentrix Mouse-6 Expression BeadChips (Illumina, San Diego, CA).
T72 11946-12433 Sentence denotes Primary microarray data were deposited at the National Center for Biotechnology Information Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih. gov/geo/, accession number GSE13685) consistent with minimum information about a microarray experiment standards, and were analyzed using an ANOVA-based scheme after quantile normalization between samples and conditions, followed by pathway analysis using multiple strategies described in the MATERIALS AND METHODS in the online supplement.
T73 12434-12558 Sentence denotes Lung lavage, cell counts, and bronchoalveolar lavage (BAL) cytokine ELISAs were performed as previously described (16, 21) .
T74 12559-12849 Sentence denotes For histological analysis, the aortas of anesthetized mice were transected, and the lungs perfused in situ with PBS via the right cardiac ventricle, then fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.0) infused through a tracheal cannula at 10-15 cm H 2 O pressure at 218C.
T75 12850-13085 Sentence denotes The lungs were removed from the thoracic cavity and further fixed overnight at 48C, then serially dehydrated and embedded in paraffin, sectioned, and stained for light microscopy with hematoxylin and eosin or Gomori methanamine silver.
T76 13086-13223 Sentence denotes For all pathogens, bacterial concentrations in nebulizers, nasal instillate, and lung tissue were determined by plating serial dilutions.
T77 13224-13440 Sentence denotes For experiments involving anti-TNF antibody, mice were injected with 1 mg of rat/mouse chimeric monoclonal antibody cV1q (Centocor, Horsham, PA) in 100 ml PBS 5 or 24 hours before NTHi lysate treatment, as indicated.
T78 13441-13644 Sentence denotes TNF blocking efficacy was confirmed by prevention of TNF-induced neutrophil infiltration after intranasal instillation of recombinant mouse TNF (PeproTech, Rocky Hill, NJ), as previously described (21) .
T79 13645-13799 Sentence denotes MLE-15 murine lung epithelial cells were cultured in six-well plates (z3 3 10 6 cells/well) in RPMI supplemented with 10% FCS and penicillin/streptomycin.
T80 13800-14077 Sentence denotes When grown to confluence, media were changed to serum-and antibiotic-free RPMI, and the cells were treated (or not) with NTHi lysate at a concentration of 100 mg/ml for 4 or 24 hours before inoculation with z100 CFU S. pneumoniae serotype 4 or 1,200 B. anthracis Sterne spores.
T81 14078-14171 Sentence denotes Pathogen burden was assessed 4 hours after infection by serial dilution culture of the media.
T82 14172-14336 Sentence denotes Alternate wells without MLE-15 cells were infected with or without addition of NTHi lysate to demonstrate the absence of intrinsic antibiotic effects of the lysate.
T83 14337-14462 Sentence denotes To further characterize the response to treatment, we investigated differential gene expression in isolated epithelial cells.
T84 14463-14642 Sentence denotes The analysis was performed identically to the whole-lung studies, harvesting total RNA from MLE-15 cells 2 hours after treatment with 100 mg/ml NTHi lysate or equal volume of PBS.
T85 14643-14792 Sentence denotes As with the whole-lung studies, the primary data were deposited at the National Center for Biotechnology Information GEO (accession number GSE13740).
T86 14793-14885 Sentence denotes Summary statistics for bacterial counts in lung tissue were compared using Student's t test.
T87 14886-15072 Sentence denotes Proportions of mice surviving pathogen challenges were compared using Fisher's exact text, and log-rank comparisons of survival distribution were performed using Kaplan-Meier estimation.
T88 15073-15236 Sentence denotes All data shown are representative of at least two independent experiments, and were not combined for analysis, because of differences in microbial challenge doses.
T89 15237-15302 Sentence denotes Analyses were performed using SAS/STAT (SAS Institute, Cary, NC).
T90 15303-15552 Sentence denotes Given the striking protection conferred against S. pneumoniae by NTHi lysate, and the characteristic breadth of innate immune responses, we tested whether stimulation with the aerosolized NTHi lysate protected against additional bacterial pathogens.
T91 15553-15893 Sentence denotes The gram-negative bacilli, P. aeruginosa and K. pneumoniae, are important causes of pneumonia in hospitalized and immunocompromised patients (3), and gram-positive methicillin-resistant S. aureus expressing Panton-Valentine leukocidin is a highly virulent bacterium that causes severe pneumonia in both the community and the hospital (25) .
T92 15894-16134 Sentence denotes Aerosolization of the NTHi lysate 18 to 24 hours before bacterial challenge provided nearly complete protection against lethality from aerosolized P. aeruginosa and K. pneumoniae, or intranasally instilled S. aureus ( Figures 1A-1C , left).
T93 16135-16322 Sentence denotes Pretreatment with the NTHi lysate resulted in rapid killing of all three bacterial species within the lungs ( Figures 1A-1C , right), similar to our prior observations with S. pneumoniae.
T94 16323-16596 Sentence denotes NTHi lysate-induced reductions in lung pathogen burden immediately after infection were associated with clearance of pathogens from the lungs and survival, whereas sham-treated mice experienced progressive lung infections and sepsis (see Table E1 in the online supplement).
T95 16597-16769 Sentence denotes Our observation of protection against highly pathogenic grampositive and gram-negative clinical organisms suggests that protection might also extend to agents of bioterror.
T96 16770-16973 Sentence denotes B. anthracis is the most likely agent to be used in a bioterror attack, because it is highly virulent, available from infected soil worldwide, and its spores are stable and readily aerosolized (22, 23) .
T97 16974-17179 Sentence denotes Intranasal instillation of 5 3 LD 50 B. anthracis Ames strain spores caused 100% mortality in mice that did not receive NTHi lysate pretreatment, but ,20% mortality in pretreated mice ( Figure 1D , left).
T98 17180-17402 Sentence denotes Protection was associated with a significant reduction in lung bacterial CFUs, even though B. anthracis was delivered as a spore; however, the degree of reduction was less than with the other bacteria ( Figure 1D , right).
T99 17403-17569 Sentence denotes Pretreatment with the aerosolized NTHi lysate similarly reduced mortality and lung bacterial burden from intranasal instillation of 5 3 LD 50 Y. pestis ( Figure 1E ).
T100 17570-17769 Sentence denotes NTHi lysate pretreatment also increased the time to median mortality, and reduced the lung bacterial burden (F), where pathogen burden was measured 24 hours after challenge due to very small inocula.
T101 17770-17879 Sentence denotes All mice were observed for at least 3 days beyond the period shown, and no additional mortality was observed.
T102 17880-18093 Sentence denotes In all experiments, survival was significantly better for NTHi lysatetreated mice on the last day of observed mortality (P , 0.001; n 5 10 mice per group for survival studies), except with F. tularensis (P 5 0.1).
T103 18094-18263 Sentence denotes NTHi lysate-treated mice in all experiments, including F. tularensis, showed greater Kaplan-Meier-estimated median survival (*P , 0.001 compared with sham-treated mice).
T104 18264-18387 Sentence denotes Lung pathogen burden is reported as the mean (6SEM) ( † P , 0.01, ‡ P , 0.05; n 5 4-5 mice per group for bacterial counts).
T105 18388-18552 Sentence denotes after intranasal instillation of 5 3 LD 50 F. tularensis, although it did not significantly reduce overall mortality from this intracellular pathogen ( Figure 1F ).
T106 18553-18751 Sentence denotes Depending on the length of potential exposure to an infectious threat, the time course of bacterial protection conferred by StIR may necessitate repetitive treatments of an at-risk population (16) .
T107 18752-18968 Sentence denotes Consistent with our recently reported findings of stimulated resistance to influenza virus after multiple treatments (26) , no tachyphylaxis of the bacterial-protective effect was observed with repetitive treatments.
T108 18969-19137 Sentence denotes Whether mice were exposed to multiple treatments in 1 week, or to weekly treatments for 8 weeks, we observed robust protection against bacterial pathogens ( Figure 2 ).
T109 19138-19232 Sentence denotes We next tested whether NTHi lysate-induced protection would extend to a nonbacterial pathogen.
T110 19233-19252 Sentence denotes Pneumonia due to A.
T111 19253-19378 Sentence denotes fumigatus is an important cause of mortality in immunocompromised patients (3), so protection against this fungus was tested.
T112 19379-19625 Sentence denotes Mice that were not immunosuppressed experienced no mortality from an aerosolized inoculum of 3 3 10 8 conidia/ml (data not shown), but mice immunosuppressed with cyclophosphamide and cortisol experienced approximately 70% mortality ( Figure 3A ).
T113 19626-19709 Sentence denotes Pretreatment with the NTHi lysate reduced mortality to less than 10% ( Figure 3A ).
T114 19710-19930 Sentence denotes Using a quantitative galactomannan assay ( Figure 3B ) and a quantitative RT-PCR assay for Aspergillus 18S rRNA ( Figure 3C ), the lung pathogen burden was found to be reduced approximately 10-fold 1 day after infection.
T115 19931-20210 Sentence denotes Histopathologic evaluation of unpretreated immunosuppressed mice demonstrated abundant hyphal elements with extensive tissue invasion by 24 hours after challenge, in contrast to NTHi lysate-pretreated mice that demonstrated few fungal forms and little inflammation ( Figure 3D ).
T116 20211-20443 Sentence denotes This suggests that, similar to protection against bacteria, NTHi lysate-stimulated protection against this fungus arises primarily from the induction of a microbicidal environment rather than the generation of cellular inflammation.
T117 20444-20867 Sentence denotes Before challenge with P. aeruginosa (3 3 10 10 CFU/ml), Swiss-Webster mice were treated with NTHi lysate 7, 4, and 1 day before the infection, or only once 1 day before infection, or with PBS alone. (B) C57BL/6 mice were treated with eight weekly NTHi lysate treatments, only one treatment, or PBS before challenge with P. aeruginosa (3.3 3 10 10 CFU/ml) (n 5 10 mice/group; *P , 0.001, † P 5 0.0007 compared with no NTHi).
T118 20869-21008 Sentence denotes To gain insight into the mechanism of protection confered by StIR, we compared the gene expression profiles of treated and untreated lungs.
T119 21009-21160 Sentence denotes Maximal protection against S. pneumoniae was achieved by 4 hours after treatment (16) , so gene expression patterns were examined within this interval.
T120 21161-21254 Sentence denotes Mice were exposed to the aerosolized NTHi lysate for 20 minutes, then killed at 2 or 4 hours.
T121 21255-21457 Sentence denotes Because we have previously shown that alveolar macrophages and recruited neutrophils are dispensable for StIR, we sought to maximize our ability to detect gene expression changes in the lung parenchyma.
T122 21458-21650 Sentence denotes As such, the lungs were lavaged to remove alveolar macrophages and other leukocytes in the airway lumen, and the pulmonary circulation was perfused with PBS to remove intravascular leukocytes.
T123 21651-21733 Sentence denotes Transcripts isolated from the lungs were then analyzed by whole-genome microarray.
T124 21734-21960 Sentence denotes We identified 3,011 differentially expressed genes at 2 hours after treatment using a false discovery rate (FDR) of less than 0.05 (not shown), and 164 genes when applying an FDR of less than 0.0001 ( Figure 4A and Table E2 ).
T125 21961-22168 Sentence denotes Most differentially expressed genes were up-regulated relative to baseline, most remained differentially expressed at 4 hours, and very few genes were differentially expressed at 4 hours, but not at 2 hours.
T126 22169-22534 Sentence denotes The differentially expressed genes encode a broad array of proteins involved in host defense, including cytokines and chemokines, pattern recognition receptors, intracellular signaling proteins, antimicrobial polypeptides, reactive oxygen species generators, and numerous other modifiers of the local host environment ( Figure 4B , Table E2 , and the GEO database).
T127 22535-22713 Sentence denotes Pathway analysis demonstrated NF-kB signaling to be the most enriched process, followed closely by IFN-a/b and -g, IL-10, IL-6, and Toll-like receptor signaling (Figures 4C-4D ).
T128 22714-23094 Sentence denotes Other engaged networks included the Card9-Bcl10-Malt1 pathway that is important for antifungal defense (27) , phosphatidylinositol 3-kinase (PI3K)/Akt, Janus kinase (JAK)/signal transducers and activators of transcription (STAT) (JAK2, SOCS3, STAT2, STAT3, STAT5a) (also reported in the IFN network), p38 mitogen-activated protein kinase and phosphatase and tensin homolog (PTEN).
T129 23095-23216 Sentence denotes Together, these results indicate rapid and broad activation of antimicrobial and antiapoptotic pathways within the lungs.
T130 23217-23370 Sentence denotes Analysis showed cytokine-encoding genes to be the most highly up-regulated individual transcripts after NTHi lysate treatment ( Figure 4B and Table E2 ).
T131 23371-23560 Sentence denotes Protein-level inflammatory cytokine concentrations also increased in BAL fluid after treatment, with increases of 1,569-fold for TNF and 186-fold for IL-6 (Figures 5A and 5D) after 4 hours.
T132 23561-23772 Sentence denotes As protective responses to PAMPs have been shown to be IL-6-and TNF-dependent in other settings (28) , we sought to determine whether NTHi lysate-induced resistance was dependent on these inflammatory cytokines.
T133 23773-24058 Sentence denotes Using an anti-TNF antibody that we have previously shown sufficient to block inflammatory cell infiltration after intratracheal TNF instillation (21), we found no difference in the protection induced by NTHi lysate treatment, nor in induced rapid pathogen killing (Figures 5B and 5C ).
T134 24059-24314 Sentence denotes Although IL-6 2/2 mice have an increased baseline susceptibility to bacterial infections relative to wild-type C57BL/6 mice (29), we were still able to induce protection and rapid pathogen killing with NTHi lysate in the mutant mice ( Figures 5E and 5F ).
T135 24315-24420 Sentence denotes Therefore, although inflammatory cytokines rise dramatically after stimulation, they may not be required.
T136 24421-24558 Sentence denotes These results are consistent with our prior findings that neutrophil influx parallels induction of protection, but is not required (16) .
T137 24559-24770 Sentence denotes We previously postulated the respiratory epithelium to be the most likely effector of StIR in the lung (16), so we investigated whether lung-expressed antimicrobial genes were induced by exposure to NTHi lysate.
T138 24771-24968 Sentence denotes Table 1 lists 30 transcripts with products known to have antimicrobial activity that are highly up-regulated after NTHi treatment, and that have previously been shown to be expressed by lung cells.
T139 24969-25146 Sentence denotes As shown, these include a broad array of products with diverse antimicrobial mechanisms, ranging from direct pathogen killing to pathogen opsonization to nutrient sequestration.
T140 25147-25284 Sentence denotes Many of the product proteins were previously noted to be up-regulated on our proteomic analyses of BAL fluid after NTHi lysate treatment.
T141 25285-25490 Sentence denotes As inducible protection is consistently associated with rapid, substantial reductions in pathogen burden, we have inferred that StIR is achieved primarily through promotion of an antimicrobial environment.
T142 25491-25749 Sentence denotes Epithelial cells are the most likely mediators of this effect, based on the large epithelial surface area of the lungs, the proximity of the epithelium to pathogens, and the induction of epithelium-expressed antimicrobial genes (Table 1 ) and proteins (16) .
T143 25750-26014 Sentence denotes Figure 6 shows that pretreatment of MLE-15 respiratory epithelial cells with NTHi lysate for 24 hours substantially reduces the pathogen burden in the culture media when inoculated with S. pneumoniae, B. anthracis, or P. aeruginosa ( Figures 6A-6C, respectively) .
T144 26015-26067 Sentence denotes This observation was not restricted to MLE-15 cells.
T145 26068-26205 Sentence denotes Rather, we consistently observed inducible bacterial killing by a variety of murine and human respiratory epithelial cells ( Figure E1 ).
T146 26206-26405 Sentence denotes Additional media-only wells (i.e., no MLE-15 cells) confirmed that the antibacterial effect results from stimulation of the epithelial cells, rather than an intrinsic antibiotic effect of the lysate.
T147 26406-26514 Sentence denotes In fact, the lysate appeared to promote pathogen growth in the absence of stimulated cells (Figures 6D-6F) .
T148 26515-26722 Sentence denotes In contrast to our findings with respiratory epithelial cells, we did not observe similar inducible bacterial killing after treating dendritic cell or macrophage cell lines with NTHi lysate (Figures 6G-6I) .
T149 26723-26932 Sentence denotes Having found that the bacterial killing effect of StIR could be recapitulated by isolated respiratory epithelial cells, we assessed whether this occurred in association with similar changes in gene expression.
T150 26933-27191 Sentence denotes After treatment of MLE-15 cells with NTHi lysate or PBS (sham), we again found induction of broad gene expression changes in the NTHi lysate-treated cells, including many of the same inflammatory cytokines induced in the wholelung model (Figures 7A and 7B) .
T151 27192-27307 Sentence denotes The induction of inflammatory cytokines by treatment was also confirmed at the protein level ( Figures 7C and 7D) .
T152 27308-27408 Sentence denotes Using an FDR of 0.01, we found 207 differentially expressed transcripts after NTHi lysate treatment.
T153 27409-27574 Sentence denotes In the interval between the in vivo and in vitro analyses, the commercially available Illumina Sentrix Mouse-6 BeadChip was changed to a new version (v1.0b to v1.1).
T154 27575-27772 Sentence denotes To compare the whole-lung gene expression patterns to those of the in vitro studies, while accounting for minor changes in the probe sets, we decided to compare only the best-described transcripts.
T155 27773-27890 Sentence denotes As such, we compared the RefSeq-annotated transcripts induced by NTHi lysate treatment with an FDR of less than 0.05.
T156 27891-28063 Sentence denotes This yielded 379 common differentially expressed transcripts, of which 318 (83.9%) changed in a similar manner (i.e., were either both up-or down-regulated) in both models.
T157 28064-28198 Sentence denotes Considering the potential effectors of the bacterial killing effect, we looked specifically at the antimicrobial peptides in Table 1 .
T158 28199-28382 Sentence denotes A total of 18 of the 30 (60.0%) antimicrobial peptide transcripts identified in whole-lung model were also increased when the epithelial cells were stimulated in isolation (Table 2) .
T159 28383-28666 Sentence denotes Of these, five transcripts were identified to be increased in all three analyses performed to date (proteomic analysis of mouse BAL fluid after treatment [16] , gene expression analysis of whole mouse lungs after treatment, and gene expression analysis of epithelial cells in vitro).
T160 28667-28753 Sentence denotes These were chitinase 3-like 1, lipocalin 2, hemopexin, haptoglobin, and calgranulin B.
T161 28754-28905 Sentence denotes We show here that stimulation of the lungs with a complex mixture of bacterial products results in rapid activation of multiple antimicrobial pathways.
T162 28906-29075 Sentence denotes This activation is associated with broad and powerful protection against respiratory challenge by gram-negative bacterial, gram-positive bacterial, and fungal pathogens.
T163 29076-29230 Sentence denotes Mechanistically, we support the hypothesis that the principal means of protection is through elaboration of antimicrobial products by the lung epithelium.
T164 29231-29470 Sentence denotes The activation of multiple pattern recognition receptors, which our gene expression data suggest occurs after NTHi lysate treatment, can result in synergizing, tolerizing, or priming interactions at the cellular level (28, (30) (31) (32) .
T165 29471-29710 Sentence denotes However, for the whole organism, survival of a microbial challenge relies on only two host strategies: tolerance (the ability to limit the damage caused by the pathogen); and resistance (the ability to limit the pathogen burden) (33, 34) .
T166 29711-30014 Sentence denotes Although previous studies, including our own studies with endotoxin, have shown limited resistance stimulated with single Toll-like receptor ligands (16, (35) (36) (37) , treatment with NTHi lysate results in profound protection from otherwise lethal pneumonias caused by disparate classes of organisms.
T167 30015-30129 Sentence denotes For all investigated pathogens, protection is associated with significant and rapid reductions in pathogen burden.
T168 30130-30186 Sentence denotes We have applied to this phenomenon the descriptor, StIR.
T169 30187-30340 Sentence denotes Pneumonia with gram-negative, gram-positive, and fungal pathogens is a common and often fatal complication among immunocompromised patients (3, 38, 39) .
T170 30341-30471 Sentence denotes The use of prophylactic antibiotics to prevent infection cultivates microbial antibiotic resistance and causes host drug toxicity.
T171 30472-30630 Sentence denotes We have previously demonstrated that protection against bacterial pneumonia by stimulation of lung innate immunity does not depend upon recruited neutrophils.
T172 30631-30797 Sentence denotes Here, we similarly find that the aerosolized lysate protects against fungal pneumonia, despite neutrophil suppression with cyclophosphamide and cortisol ( Figure 3) .
T173 30798-31067 Sentence denotes Therefore, patients undergoing myeloablative chemotherapy as part of cancer treatment, or in preparation for hematopoietic stem cell transplantation, might benefit from stimulation of lung innate immunity during vulnerable periods of transient, severe myelosuppression.
T174 31068-31330 Sentence denotes Because the augmentation of innate immune defenses is independent of adaptive immune function, the protective response can be induced even in the setting of T-cell suppression by fludarabine, or immunosuppression with calcineurin inhibitors or alkylating agents.
T175 31331-31557 Sentence denotes Nontypeable Haemophilus influenzae (NTHi) lysate up-regulated antimicrobial genes were checked against expression sequence-tagged National Center for Biotechnology Information databases to identify those expressed in the lung.
T176 31558-31690 Sentence denotes The top 30 lung-expressed antimicrobial genes are shown, ranked by fold-change over baseline at 2 hours after NTHi lysate treatment.
T177 31691-31771 Sentence denotes * Protein products previously shown to be induced by NTHi lysate exposure (16) .
T178 31772-31905 Sentence denotes The 2001 anthrax attacks in the United States highlighted the challenges of defending populations against bioterror agents (40, 41) .
T179 31906-32018 Sentence denotes Presently, only military personnel and first responders have access to anthrax vaccination in the United States.
T180 32019-32211 Sentence denotes Even if an adequate vaccine stockpile could be maintained, as antianthrax acquired immunity requires months to develop, such vaccination would not protect the general population acutely (40) .
T181 32212-32293 Sentence denotes Furthermore, vaccines are not available for all potential bioterror agents (42) .
T182 32294-32488 Sentence denotes Perhaps most important, early in an attack, the identity of a pathogen may not be immediately evident, delaying the determination of appropriate preventative and/or postexposure therapies (40) .
T183 32489-32650 Sentence denotes Therefore, the broad protection conferred by stimulation of innate immunity is attractive for management of large populations in the event of a bioterror attack.
T184 32651-32754 Sentence denotes We have demonstrated increased mean survival for every bioterror pathogen against which we have tested.
T185 32755-32868 Sentence denotes The current studies provide our first insight into the cellular effectors of the resistant phenotype in the lung.
T186 32869-33113 Sentence denotes Whereas neutrophils, mast cells, and alveolar macrophages are expendable in NTHi lysate-induced protection, we show here in vitro that respiratory epithelial cells are sufficient to sense the treatment and to generate an antimicrobial response.
T187 33114-33347 Sentence denotes Having direct contact with the treatment, close apposition to deposited pathogens, and established capacity to generate antimicrobial products, the respiratory epithelium seems well suited to serve the StIR effector function in vivo.
T188 33348-33527 Sentence denotes Although the 11 most strongly up-regulated genes after treatment encode cytokines, common mediators of inflammation, such as TNF and IL-6, are not required for induced resistance.
T189 33528-33633 Sentence denotes This is consistent with our previous finding that neutrophil infiltration is not required for protection.
T190 33634-33825 Sentence denotes On the other hand, our proteomic (16) and genomic ( Table 1 ) analyses indicate that the respiratory epithelium expresses numerous antimicrobial products in response to NTHi lysate treatment.
T191 33826-33910 Sentence denotes Our gene expression analyses identified several intriguing StIR candidate mediators.
T192 33911-34091 Sentence denotes The three known mouse acute-phase serum amyloid A (SAA) genes (43) comprise three of the four most highly up-regulated lung antimicrobial genes after in vivo NTHi lysate treatment.
T193 34092-34300 Sentence denotes These enigmatic apolipoproteins promote host defense through widely varied mechanisms, including cathelicidin-like direct killing, pathogen opsonization, and induction of leukocyte chemotaxis (44) (45) (46) .
T194 34301-34573 Sentence denotes Although only Saa3 was significantly up-regulated on the in vitro array, it is notable that the other two transcripts were higher at the 4-hour than the 2-hour time point in the in vivo studies, so it is possible that we had not observed the maximal expression at 2 hours.
T195 34574-34758 Sentence denotes Also upregulated are the lipocalins, orosomucoid 1, orosomucoid 2, and lipocalin 2, that have recently been shown critical in host defenses against bacterial pathogens (47) (48) (49) .
T196 34759-34943 Sentence denotes Their protective functions seem to result mainly from iron sequestration (48, 49) , although lipocalin 2 also appears key to IL-22-induced defenses in the respiratory epithelium (47) .
T197 34944-35151 Sentence denotes Lipocalin 2 is one of five antimicrobial effectors that have been found to be up-regulated on all of our proteomic and gene expression screens after NTHi lysate treatment, making it an interesting candidate.
T198 35152-35287 Sentence denotes However, we have not seen similarly broad or robust protection in previous studies addressing the antimicrobial effects of lipocalin 2.
T199 35288-35431 Sentence denotes Therefore, we suspect that, if lipocalin 2 does participate in NTHi lysate-induced StIR, it is probably one of several antimicrobial effectors.
T200 35432-35654 Sentence denotes The SAA proteins, the lipocalins, and the other antimicrobial products that we identified on these analyses will each be carefully explored as potential effectors, as they may play different roles with different pathogens.
T201 35655-35841 Sentence denotes Investigations to identify novel NTHi-induced antimicrobial effectors are also underway, as well as exploration of other possible effector mechanisms, such as augmented barrier function.
T202 35843-35879 Sentence denotes Serum amyloid A 2 (NM_011314.1) 1.10
T203 35880-36069 Sentence denotes The antimicrobial transcripts up-regulated in Table 1 were evaluated in MLE-15 murine lung epithelial cells 2 hours after treatment with 100 mg/ml nontypeable Haemophilus influenzae lysate.
T204 36070-36112 Sentence denotes The 18 up-regulated transcripts are shown.
T205 36113-36256 Sentence denotes * Candidate effectors identified on proteomic screens of BAL fluid, whole-lung gene expression analysis, and in vitro gene expression analysis.
T206 36257-36267 Sentence denotes Figure 7 .
T207 36268-36363 Sentence denotes Broad induction of gene expression changes in NTHi lysate-treated respiratory epithelial cells.
T208 36364-36506 Sentence denotes MLE-15 cells were treated with 100 mg/ml NTHi lysate for 2 hours, then RNA was harvested to analyze treatment-induced gene expression changes.
T209 36507-36579 Sentence denotes A total of 207 differentially expressed transcripts were identified (A).
T210 36580-36645 Sentence denotes Red, increased gene expression; green, decreased gene expression.
T211 36646-36754 Sentence denotes These included many of the same inflammatory cytokines found to be up-regulated in the whole-lung model (B).
T212 36755-36811 Sentence denotes These changes were confirmed at the protein level (C-E).
T213 36812-36882 Sentence denotes Cytokine levels are reported as mean (6SEM) (*P , 0.0001, † P , 0.01).
T214 36883-36961 Sentence denotes Whether StIR is a purely epithelial phenomenon in the lung remains to be seen.
T215 36962-37252 Sentence denotes We found that epithelial cells in isolation can be stimulated to kill bacteria, and that this is associated with gene expression patterns and cytokine elaboration that is very similar to that noted in the whole lung system, supporting our hypothesis that the epithelium is critical to StIR.
T216 37253-37499 Sentence denotes In striking contrast, macrophages and dendritic cells could not be stimulated by the lysate to directly kill extracellular bacteria in vitro (Figure 6 ), consistent with our prior findings that macrophages are not required for in vivo StIR (16) .
T217 37500-37682 Sentence denotes Nonetheless, dendritic cells may sense the stimulus in vivo and reinforce the epithelial effector response through local signaling via IL-22/STAT-3dependent mechanisms (47, 50, 51) .
T218 37683-37829 Sentence denotes In summary, we have demonstrated that stimulation of lung innate immunity provides protection of remarkable breadth against respiratory pathogens.
T219 37830-38093 Sentence denotes Therapeutic manipulation of StIR may be possible to reduce the burden of respiratory infections in immunosuppressed patients, and to reduce the risk of infection of the general public during a bioterror attack with a pathogen introduced via the respiratory route.
T220 38094-38253 Sentence denotes Conflict of Interest Statement: M.H. is a cofounder of Pulmotect, Inc., owns shares in the company, and has received a $50,000 research grant from the company.
T221 38254-38469 Sentence denotes D.K. has been reimbursed by Merck, Pfizer, Enzon, and Astellas (2008-2009, ,$10,000 total) for participating as a speaker and/or lecturer in various meeting, symposiums, and conferences, and advisory board meetings.
T222 38470-38557 Sentence denotes He has also received industry grants from Astellas and Merck in excess of $20,000 each.
T223 38558-38791 Sentence denotes C.G.C., M.J.T., and B.F.D. are inventors of the subject matter disclosed in the patent application ''Compositions and Methods for Stimulation of Lung Innate Immunity,'' filed by the Board of Regents of the University of Texas system.
T224 38792-38927 Sentence denotes None of the other authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.