cathepsin g and neutrophil elastase play critical and … · immunity against focal...

INFECTION AND IMMUNITY, Dec. 2011, p. 4893–4901 Vol. 79, No. 120019-9567/11/$12.00 doi:10.1128/IAI.05593-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Cathepsin G and Neutrophil Elastase Play Critical and NonredundantRoles in Lung-Protective Immunity against

Streptococcus pneumoniae in Mice�

Ines Hahn,1 Anna Klaus,1 Ann-Kathrin Janze,1 Kathrin Steinwede,1 Nadine Ding,1 Jennifer Bohling,1Christina Brumshagen,1 Helene Serrano,2 Francis Gauthier,2 James C. Paton,3

Tobias Welte,4 and Ulrich A. Maus1*Department of Experimental Pneumology1 and Clinic for Pneumology,4 Hannover School of Medicine, Hannover, Germany;

INSERM U618, Proteases et Vectorisation Pulmonaires, Universite Francois Rabelais de Tours, Tours, France2; andResearch Centre for Infectious Diseases, School of Molecular and Biomedical Science,

University of Adelaide, Adelaide, Australia3

Received 29 June 2011/Returned for modification 27 July 2011/Accepted 2 September 2011

Neutrophil serine proteases cathepsin G (CG), neutrophil elastase (NE), and proteinase 3 (PR3) have recentlybeen shown to contribute to killing of Streptococcus pneumoniae in vitro. However, their relevance in lung-protectiveimmunity against different serotypes of S. pneumoniae in vivo has not been determined so far. Here, we examined theeffect of CG and CG/NE deficiency on the lung host defense against S. pneumoniae in mice. Despite similarneutrophil recruitment, both CG knockout (KO) mice and CG/NE double-KO mice infected with focal pneumonia-inducing serotype 19 S. pneumoniae demonstrated a severely impaired bacterial clearance, which was accompaniedby lack of CG and NE but not PR3 proteolytic activity in recruited neutrophils, as determined using fluorescenceresonance energy transfer (FRET) substrates. Moreover, both CG and CG/NE KO mice but not wild-type miceresponded with increased lung permeability to infection with S. pneumoniae, resulting in severe respiratory distressand progressive mortality. Both neutrophil depletion and ablation of hematopoietic CG/NE in bone marrowchimeras abolished intra-alveolar CG and NE immunoreactivity and led to bacterial outgrowth in the lungs of mice,thereby identifying recruited neutrophils as the primary cellular source of intra-alveolar CG and NE. This is thefirst study showing a contribution of neutrophil-derived neutral serine proteases CG and NE to lung-protectiveimmunity against focal pneumonia-inducing serotype 19 S. pneumoniae in mice. These data may be important forthe development of novel intervention strategies to improve lung-protective immune mechanisms in critically illpatients suffering from severe pneumococcal pneumonia.

Streptococcus pneumoniae is the most prevalent pathogencausing community-acquired pneumonia in humans, which fre-quently progresses toward invasive pneumococcal disease as-sociated with significant morbidity and mortality worldwide(29). Alveolar macrophages play an important role in innateimmune surveillance of the lung against inhaled bacterialpathogens, which they sense via specific pattern recognitionreceptors (6, 12). Downstream activation of these lung sentinelcells characteristically triggers chemokine receptor CXCR2-mediated recruitment of neutrophilic granulocytes into thelung parenchyma and bronchoalveolar compartment to sup-port macrophage antibacterial responses (9, 23). We recentlyfor the first time showed that even a partially (10 to 25%)reduced neutrophil recruitment severely perturbed the lunghost defense against S. pneumoniae in mice, demonstratingthat indeed maximal neutrophil recruitment is essential forimmediate early control of lung bacterial infections (9). Al-though these data demonstrate that neutrophils are indispens-able for lung innate immunity against major pneumotropicpathogens such as S. pneumoniae, the molecular mechanism by

which neutrophils kill engulfed pneumococci in vivo is onlypartially defined. Basically, at least two central modes of ac-tion, i.e., reactive oxygen species (ROS)-dependent oxidativeand nonoxidative pathways primarily mediated by antimicro-bial peptides and proteases appear to contribute to neutrophilantibacterial responses (28). Recently, neutral serine proteasescathepsin G (CG), neutrophil elastase (NE), and proteinase 3(PR3) were recognized to contribute to human neutrophil kill-ing of S. pneumoniae in vitro (26). However, the role of neutralserine proteases in lung-protective immunity against S. pneu-moniae has not been determined in infection models in vivo.Moreover, it is not clear whether neutral serine protease re-dundancies observed in vitro are also active in vivo.

In the current study, we examined the role of neutral serineproteases cathepsin G and neutrophil elastase in the lung hostdefense against S. pneumoniae in vivo. We found a critical andnonredundant role for CG and NE to mediate neutrophil-depen-dent killing of S. pneumoniae in mice. This knowledge may lead tonovel therapeutic interventions for the treatment of critically illpatients suffering from severe pneumococcal pneumonia.

MATERIALS AND METHODS

Animals. Wild-type (WT) mice (129 S2/SvPasCrl) were purchased fromCharles River Laboratories. CG-deficient mice [129-CtsgGtm1.1Roes/(H)] andCG/NE double mutant mice [129-Ela2tm1(cre)RoesCtsGtm1.1Roes/(H)] generated asreported previously (28) were obtained from the European Mouse Mutant Ar-

* Corresponding author. Mailing address: Department of Experi-mental Pneumology, Hannover School of Medicine, Feodor-Lynen-Straße 21, Hannover 30625, Germany. Phone: 49-511-532-9617. Fax:49-511-532-9616. E-mail: [email protected].

� Published ahead of print on 12 September 2011.

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chive (EMMA), housed under specific-pathogen-free conditions, and used forexperiments at the age of 8 to 16 weeks. All animal experiments were approvedby our local government authorities.

Generation of chimeric mice. Chimeric mice were generated as recently de-scribed (7, 9, 19, 27). Briefly, bone marrow cells were isolated from tibias andfemurs of donor mice (wild-type mice, CG knockout [KO] mice, and CG/NE KOmice) under sterile conditions. Single-cell suspensions prepared from bone mar-row isolates were filtered through cell strainers to remove aggregates and werethen washed in Leibovitz’s L15 medium (Gibco, Invitrogen, Karlsruhe, Ger-many). Recipient wild-type mice were irradiated with a single dose of 8 Gy at adose rate of 2.5 Gy/min delivered by a linear accelerator (Siemens MD 2;Hannover, Germany) operating in a 6-MV high-energy photon delivery mode.Within 24 h after irradiation, single-cell suspensions (1 � 107 cells per mouse) ofdonor bone marrow cells were intravenously (i.v.) injected into recipients vialateral tail veins. Chimeric mice were housed under specific-pathogen-free con-ditions, with free access to autoclaved food and water. At 5 weeks after irradi-ation/bone marrow transplantation (BMT), chimeric mice were used for infec-tion experiments.

Neutrophil depletion experiments. In selected experiments, mice were de-pleted of neutrophils by i.v. administration of Ly6G-specific antibody 1A8 (250�g/mouse applied at 24 h and 12 h prior to infection) (BioXCell, West Lebanon,NH). Control mice received i.v. administration of isotype control antibody 2A8(BioXCell).

Culture and quantification of Streptococcus pneumoniae. For infection exper-iments, we used a capsular group 19 Streptococcus pneumoniae strain (EF3030),which is characterized by a relatively low virulence and which is known toprimarily cause focal pneumonia in mice (4, 8, 30), and a highly virulent capsulargroup 2 S. pneumoniae strain (D39), which is known to cause invasive pneumo-coccal disease in mice (5, 8, 17). The bacteria were grown in Todd-Hewitt broth(THB) (Oxoid, Wesel, Germany) supplemented with 20% fetal calf serum (FCS)to mid-log phase, and aliquots were snap-frozen in liquid nitrogen and stored at�80°C until use (17, 27). Pneumococci were quantified by plating serial dilutionson sheep blood agar plates (BD Biosciences, Heidelberg, Germany), followed byincubation of the plates at 37°C and 5% CO2 for 18 h and subsequent determi-nation of CFU.

Infection of mice with Streptococcus pneumoniae. Mice were infected eitherwith serotype 19 S. pneumoniae (EF3030) adjusted to 1.5 � 107 CFU/50 �lTHB/mouse or with serotype 2 S. pneumoniae (D39) adjusted to 2 � 106 CFU/50�l THB/mouse according to recently published protocols (9, 27). Chimeric miceand mice treated with neutrophil-depleting antibody 1A8 were infected with 5 �106 CFU of serotype 19 S. pneumoniae. Intratracheal instillation of S. pneu-moniae into the lungs of mice was performed by orotracheal intubation of micewith a 29-gauge Abbocath catheter (Abbott, Wiesbaden, Germany) under visualcontrol with transillumination of the neck region, followed by slow aspiration ofS. pneumoniae into the lungs of mice (8, 9). During the entire observation period,infected mice were kept in individually ventilated cages (IVC) and were moni-tored twice daily for disease symptoms and survival.

Determination of bacterial loads in BALF and lung tissue. Bacterial loadswithin the lungs of S. pneumoniae-infected mice were determined at differenttime points postinfection essentially as described recently (9, 27). Briefly, micewere euthanized with an overdose of isoflurane (Baxter, Unterschleissheim,Germany) and bronchoalveolar lavage (BAL) was performed by repeated intra-tracheal instillation of 300-�l aliquots of cold phosphate-buffered saline (PBS)supplemented with EDTA (Versen; Biochrom, Berlin, Germany) into the lungsand careful aspiration until a BAL fluid (BALF) volume of 1.5 ml was collectedas described elsewhere (9, 27). Subsequently, BAL was continued until an addi-tional volume of 4.5 ml was collected. CFU in the respective BALF aliquots werequantified by plating serial dilutions on sheep blood agar plates (BD Biosciences,Heidelberg, Germany), followed by incubation of the plates at 37°C and 5% CO2

for 18 h. Whole-lung washes were centrifuged (1,400 rpm, 4°C, 9 min), the cellpellets were pooled and resuspended in RPMI-10% FCS, and total numbers ofBALF leukocytes were determined. In addition, cell-free BALF supernatants ofthe initial 1.5-ml BALF aliquots were also subjected to enzyme-linked immu-nosorbent assay (ELISA) for determination of cytokines. For quantification ofBALF leukocytes, differential cell counts of Pappenheim-stained cytocentrifugepreparations were performed followed by multiplication of the respective valueswith total BALF cell numbers. Neutrophils were identified according to theirmorphology and typical shape of nuclei. Subsequent to BAL, individual lunglobes were removed, dissected, and homogenized in 2 ml Hanks’ balanced saltsolution (HBSS; PAA, Coelbe, Germany) without supplements using a tissuehomogenizer (IKA, Staufen, Germany). Subsequently, CFU were determined byplating serial dilutions on sheep blood agar plates followed by incubation of theplates at 37°C and 5% CO2 for 18 h.

Analysis of lung permeability. In selected experiments, lung barrier dysfunc-tion was analyzed in mice infected with S. pneumoniae essentially as describedrecently (16, 30). Briefly, 1.5 mg fluorescein isothiocyanate-labeled human albu-min (Sigma, Deisenhofen, Germany) dissolved in 150 �l 0.01 M Tris-0.9% salinewas injected intravenously. One hour later, mice were euthanized, and serum wasisolated from whole blood after centrifugation at 3,500 rpm for 15 min. Subse-quently, cell-free, undiluted BALF samples and serum samples (diluted 1/100 insaline) were placed in a 96-well microtiter plate, and fluorescence intensitieswere measured with a fluorescence spectrometer (Bio-Tek Flx800; BadFriedrichshall, Germany) operating at an absorbance wavelength of 488 nm andan emission wavelength of 525 � 20 nm. The lung permeability index is definedas the ratio of the fluorescence signals of undiluted BALF samples relative tofluorescence signals of 1/100-diluted serum samples.

Determination of arterial O2 saturation in mice. To determine the functionalconsequences of increased lung permeability in S. pneumoniae-infected miceover time, the arterial O2 saturation was measured in mice of the respectivetreatment groups longitudinally in individual mice using noninvasive, real-timepulse oximetry (MouseOx; Starr Life Sciences). Briefly, anesthetized mice in-fected with serotype 19 S. pneumoniae were prepared for real-time pulse oxim-etry by shaving of the neck region. Starting by day 1 postinfection, a collar clipwas fixed to the shaved neck region of mice and was then connected to theMouseOx system, according to the manufacturer’s instructions. After a fewminutes of equilibration, for which mice were transferred into a plastic box in thedark, arterial O2 saturation was measured in nonanesthetized mice once dailyusing the MouseOx software package. Uninfected control mice (day 0) of eithergroup were briefly anesthetized followed by shaving of their neck region andsubsequent measurement of arterial O2 saturation after a few minutes of equil-ibration of the system.

Measurement of neutrophil serine protease activity using fluorescence reso-nance energy transfer (FRET) substrates. CG, NE, and PR3 activities in bron-choalveolar recruited neutrophils of mice of the various treatment groups wereanalyzed essentially as described recently (13). Briefly, 24 h after infection withserotype 19 S. pneumoniae, mice were subjected to BAL for collection of alveolarrecruited neutrophils. Lysis of red blood cells was done with hypotonic lysissolution followed by centrifugation at 1,400 rpm for 9 min at 4°C. Subsequently,BALF cell pellets consisting of �90% neutrophils were resuspended in 200 �lice-cold PBS supplemented with 0.15% Brij 35 (Sigma-Aldrich, Steinheim, Ger-many), followed by two freeze-thaw cycles. Following the first cycle, cell prepa-rations were sonicated for 3 rounds of 10 s each with a 1-min break on ice (UW2200; Bandelin Electronics, Berlin, Germany). Cell lysates were centrifuged aftereach cycle (13,000 rpm, 25 min, 4°C), and supernatants were collected anddiluted in 50 mM phosphate-buffered saline, pH 7.4, to adjust Brij 35 concen-trations to a final 0.05%, followed by kinetic measurement of peptidase activities(13, 14). Briefly, 30-�l aliquots of lysate supernatants were diluted in 118 �lactivity buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, supplemented with 0.05%[vol/vol] Igepal CA-630) in polypropylene microplate wells at 37°C. The reac-tion was started by adding 2 �l (5 �M final) of Abz-TPFSGQ-EDDnp, Abz-QPMAVVQSVPQ-EDDnp, or Abz-VARCADYQ-EDDnp to measure CG,NE, and PR3, respectively (9). The fluorescence was recorded at �ex � 320 nmand �em � 420 nm using a microplate fluorescence reader (Spectra Max Gemini;Molecular Devices) under continuous stirring. Results were expressed per mil-lion cells as a percentage of wild-type activity.

Western blot analysis of CG/NE protein levels in BALF cells. In selectedexperiments, CG/NE protein levels were analyzed in lysed bronchoalveolar re-cruited neutrophils of mice of the various treatment groups by Western blotanalysis as recently described (25). Briefly, 24 h after infection with serotype 19S. pneumoniae, chimeric mice and neutrophil-depleted mice were subjected toBAL. Lysis of BALF cells was done with lysis buffer for 30 min on ice followedby centrifugation at 13,000 rpm for 25 min at 4°C. Subsequently, the respectivesupernatants were collected and stored at �80°C until determination of proteinconcentrations was performed using the BCA Pierce protein assay kit (ThermoScientific, Waltham, MA) according to the manufacturer’s instructions. ForSDS-polyacrylamide gel electrophoresis, protein samples were diluted 1:1 withLaemmli buffer (Bio-Rad, Munich, Germany) and heated to 95°C for 5 min and,subsequently, protein (15 to 20 �g) was loaded onto a 12.5% SDS-polyacryl-amide gel, followed by transfer of separated proteins onto a polyvinylidenedifluoride (PVDF) membrane (Millipore, Billerica, MA) using semidry-blottingtechnology. Detection of CG/NE immunoreactivity was done using I-19 antibodywith specificity for CG and cross-reacting with NE (unpublished observation)(I-19; Santa Cruz Biotechnology, Heidelberg, Germany). Beta-actin (AC-15;Sigma-Aldrich, Steinheim, Germany) was used as a housekeeping control. Pro-tein levels of CG/NE and �-actin were determined by evaluation of enhancedchemiluminescence signal (ECLplus; GE Healthcare, Buckinghamshire, United

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Kingdom) using a Vilber/Lourmat Chemismart 5000 analyzer and Bio1D soft-ware (Vilber/Lourmat, Eberhardzell, Germany).

Analysis of burst induction in bone marrow-derived neutrophils. In anotherset of experiments, bone marrow-derived neutrophils from wild-type and CG/NEKO mice were analyzed for their oxidative burst capacity. Single-cell suspensionsfrom bone marrow isolates of wild-type and CG/NE KO mice were filteredthrough a 40-�m cell strainer to remove aggregates, washed with HBSS (PAA,Pasching, Austria), and then resuspended in 2 ml HBSS and loaded onto athree-layer gradient of 81%, 67%, and 52% Percoll (GE Healthcare, Bucking-hamshire, United Kingdom) according to the manufacturer’s instructions. Aftercentrifugation at 1,500 � g for 45 min at 4°C, neutrophils were harvested fromthe 81%/67% interphase. After one wash in MACS buffer (1� PBS, 0.5% FCS),neutrophils were further enriched using the anti-Ly6G MircoBead Kit (MiltenyiBiotech, Bergisch Gladbach, Germany). Briefly, cells were incubated with 50 �lanti-Ly6G-biotin for 10 min followed by 15 min of incubation with antibiotinbeads on ice. Subsequently, cell suspensions were passed through magneticseparation (MS) columns and then eluted with MACS buffer. Highly enrichedneutrophils (95%) were then plated onto 96-well plates (2 � 105 cells/well) andwere allowed to rest at 37°C for 1 h before adding 1 mM luminol (31). Oxidativeburst was induced by addition of serotype 19 S. pneumoniae applied at a multi-plicity of infection (MOI) of 5. Relative light units (RLU) were determined usingan Flx800 fluorescence/luminescence reader and KC4 software (Bio-Tek Instru-ments, Bad Friedrichshall, Germany).

ELISA. Analysis of cytokines (interleukin-6 [IL-6], tumor necrosis factor alpha[TNF-], KC, MIP-2) in BALFs of S. pneumoniae-infected or control mice wasperformed using commercially available enzyme-linked immunosorbent assays(ELISAs), according to the manufacturer’s instructions (R&D Systems, Wies-baden, Germany).

Statistics. Significant differences between wild-type (WT) and CG KO andCG/NE KO mice were analyzed by one-way analysis of variance (ANOVA)followed by post hoc Bonferroni adjustment using SPSS for Windows software.Repeated measurements of oxygen saturation in mice of the various treatmentgroups were analyzed by ANOVA with repeated measurements and subsequentpost hoc tests with Bonferroni adjustment. Survival curves between groups wereanalyzed by log rank test. Significant differences between groups were assumedwhen P values were 0.05.

RESULTS

CG KO and CG/NE KO mice are highly susceptible toinfection with S. pneumoniae. In initial experiments, we as-sessed the role of CG and NE in lung-protective immunity topneumococcal infection, using two different serotypes, i.e., lessvirulent serotype 19 and highly invasive serotype 2 S. pneu-moniae. As shown in Fig. 1A, wild-type mice infected withserotype 19 S. pneumoniae showed an overall survival of �80%accompanied by a weak bacteremia developing at day 8 postin-fection (Fig. 1B). In contrast, CG KO and, even more so,CG/NE double-KO mice showed a progressive mortality start-ing by day 3 postinfection and resulting in overall survival of30% in CG KO mice and 10% in CG/NE KO mice, respec-tively. While S. pneumoniae-infected CG KO mice showed50% mortality on day 10 postinfection, the CG/NE KO miceshowed 50% mortality even by day 5 postinfection, althoughduring this time, no bacteremia was detectable in the doublemutant mice (Fig. 1B). We next examined the role of neutralserine proteases in lung-protective immunity against highlyinvasive serotype 2 S. pneumoniae (5). As shown in Fig. 1C andD, infection of both wild-type mice and CG/NE KO mice withserotype 2 S. pneumoniae rapidly caused bacteremia and deathin mice, with only minor, nonsignificant differences betweenexperimental groups. These data demonstrate that intrapulmo-nary CG and NE bioavailability is not sufficient to preventsepsis-induced death in mice challenged with highly invasiveserotype 2 S. pneumoniae.

Effect of serine protease CG/NE deficiency on lung bacterialloads and bronchoalveolar recruitment of neutrophils in miceinfected with S. pneumoniae. Our observation of increasedmortalities noted in CG and particularly CG/NE KO mice ledus to determine whether there are differences in bacterial loadsin the lungs of these mice. CG-deficient mice challenged withserotype 19 S. pneumoniae showed significantly increased bac-terial loads in their BALFs and lung tissue compared to wild-type mice, particularly on days 1 and 3 postinfection (Fig. 2Aand B). CG/NE KO mice demonstrated an even more dramaticoutgrowth of S. pneumoniae in their lungs, resulting in approx-imately 10-fold-increased bacterial loads compared to CG KOmice and 40- to 50-fold-increased bacterial loads in bothBALFs and lung tissue relative to wild-type mice for all inves-tigated time points (Fig. 2A and B). These data clearly supportthe assumption that both CG and NE are required for lung-protective immunity against focal pneumonia-inducing sero-type 19 S. pneumoniae and further imply that PR3 is not ableto compensate for the lack of CG and NE in this experimentalsystem.

Since infection of mice with S. pneumoniae significantly im-paired the bacterial clearance and reduced the survival of CGKO and CG/NE KO mice, we further asked whether this is dueto defective lung leukocyte migration toward the site of infec-tion. As shown in Fig. 3A, CG KO mice demonstrated asubstantial neutrophil recruitment into their lungs on day 1postinfection, while CG/NE KO mice recruited slightly butsignificantly reduced numbers of neutrophils into the lungs atthis early time point. However, on day 3 postinfection, CG/NEKO mice recruited significantly more neutrophils into theirlungs (Fig. 3A) than did controls, though these mice were stillnot able to control bacterial outgrowth in lung distal airspaces(Fig. 2). At all examined time points, numbers of alveolarmacrophages and lymphocytes were similar between groups(data not shown).

Peptidase activity of serine proteases of BALF neutrophilsfrom mice infected with S. pneumoniae. In the next set ofexperiments, we determined whether the observed stepwiseincrease of bacterial loads observed in CG/NE KO mice versusCG KO mice versus wild-type mice is related to respectivedecreases in peptidase activities in alveolar recruited neutro-phils of mice of the three treatment groups. Therefore, wedetermined the proteolytic activities of CG, NE, and PR3 inneutrophil lysates of CG KO and CG/NE KO mice relative towild-type mice at 24 h after infection with S. pneumoniae. Asshown in Fig. 3B, neutrophils collected from the lungs of S.pneumoniae-infected wild-type mice were observed to cleaverespective FRET substrates for CG, NE, and PR3, whereasneutrophil lysates of CG KO mice almost completely lackedCG proteolytic activities but exhibited similar proteolytic ac-tivities of NE and PR3. As expected, cell lysates of neutrophilscollected from the lungs of S. pneumoniae-infected CG/NE KOmice did not exhibit proteolytic activities for CG and NE andat the same time demonstrated a slightly reduced proteolyticactivity of PR3. Moreover, we measured S. pneumoniae-in-duced oxidative burst-induced ROS formation in bone mar-row-derived neutrophils isolated from CG/NE KO mice andwild-type mice (Fig. 3C). As expected, we found that bothCG/NE KO and wild-type neutrophils responded with similarROS formation to challenge with S. pneumoniae in vitro,

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thereby confirming that the dramatic increase of bacterialloads observed in CG KO and CG/NE KO mice relative towild-type mice in vivo was not due to defects in ROS formationbut rather due to lack of serine protease activities in the re-cruited neutrophils.

Effect of CG and NE deletion on lung permeability andarterial oxygen saturation in mice infected with S. pneumoniae.We next examined whether the defective bacterial clearanceobserved in CG and CG/NE KO mice would trigger increasedlung barrier dysfunction and respiratory distress in mice. In-deed, CG KO mice as well as CG/NE KO mice showed signif-icantly increased lung permeability in response to infection

with S. pneumoniae relative to wild-type mice (Fig. 4A). More-over, analysis of arterial oxygen saturation in mice of the var-ious treatment groups revealed �95% O2 saturation in allgroups on day 1 postinfection, which remained at �90% O2

saturation in wild-type mice, whereas CG KO mice demon-strated reduced arterial O2 saturation of 90% on day 4 witha further decline to 80% on day 5, resulting in increasedmortality in this group. Importantly, in CG/NE KO mice, thearterial O2 saturation declined to 90% by day 3 postinfectionand further strongly decreased to �70% by day 5 postinfection,which at the same time was accompanied by heavily increasedmortality in this experimental group (Fig. 4B). Collectively,

FIG. 1. Survival and bacteremia in wild-type mice, CG KO mice, and CG/NE KO mice after infection with S. pneumoniae. Wild-type mice, CGKO mice, and CG/NE KO mice were infected with either serotype 19 S. pneumoniae (A and B) or serotype 2 S. pneumoniae (C and D), and survival(A and C) and bacteremia (B and D) were determined during an observation period of 14 days, as indicated.



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these data and the data in Fig. 3, showing that the neutrophilinfluxes are similar in all groups, strongly suggest that thegradually increased lung permeability in cohort with the grad-ually decreased arterial oxygen saturation observed in CG KOand CG/NE KO mice contributed to the gradually increasedmortality observed in mice of the respective treatment groups.

Effect of CG and CG/NE deletion on inflammatory cytokinerelease in S. pneumoniae-infected mice. Furthermore, we mea-sured proinflammatory cytokines in BALFs of wild-type, CGKO, and CG/NE KO mice in response to S. pneumoniae in-fection. We found that particularly CG/NE KO mice re-sponded with strongly increased BALF cytokine levels ofTNF-, KC, and MIP-2 to infection with S. pneumoniae rela-tive to wild-type mice (Fig. 5). Particularly, TNF- was signif-icantly elevated at 6 h postinfection (Fig. 5A), when lung CFUcounts were similar between groups, thus suggesting a regula-tory role of CG/NE in this early cytokine response to infection.

Effect of hematopoietic CG and CG/NE deficiency on bac-terial clearance and CG/NE protein levels in chimeric miceinfected with S. pneumoniae. To evaluate the relevance of he-matopoietic CG and NE bioactivity to lung-protective immu-nity against S. pneumoniae, we irradiated wild-type mice andreconstituted them with bone marrow cells from wild-typemice, CG KO mice, or CG/NE double KO mice, followed byinfection of the resulting chimeric mice with S. pneumoniae(Fig. 6A to D). Chimeric mice lacking CG or CG/NE in theirhematopoietic system showed strongly increased lung bacterialloads in response to infection with serotype 19 S. pneumoniaerelative to wild-type irradiation control mice (Fig. 6A and B).At the same time, recruitment of neutrophils into the bron-choalveolar space was not affected by hematopoietic CG orCG/NE deficiency (Fig. 6C). However, as shown in Fig. 6D,neutrophils collected from the lungs of S. pneumoniae-infectedchimeric mice either lacking CG (CG KO BMT onto WT, Fig.6D) or both CG and NE (CG/NE KO BMT onto WT, Fig. 6D)in their hematopoietic system progressively lacked CG/NE im-munoreactivity in neutrophils recovered from their lungs bybronchoalveolar lavage, relative to the CG/NE immunoreac-

tivity observed in BALF neutrophils from irradiation controlmice (WT BMT onto WT, Fig. 6D). Collectively, these datademonstrate the critical importance of alveolar recruited neu-trophils to serve as cellular shuttles for CG/NE antibacterialactivity in the alveolar space of infected mice.

Effect of neutrophil depletion on CG/NE-dependent antibac-terial defense of the lung. To further assess the role of neu-trophils as cellular shuttles transporting CG and NE antibac-terial activity from the blood compartment into the alveolarspace, we employed an irradiation-independent experimentalapproach by specifically depleting peripheral blood neutrophilsin wild-type mice using anti-Ly6G specific antibody 1A8. Fig-ure 7A shows that administration of anti-Ly6G antibody al-most completely abolished alveolar neutrophil recruitment inmice challenged with serotype 19 S. pneumoniae. As expected,neutrophil-depleted mice showed significantly increased bac-terial loads in their BALFs and lung tissue upon S. pneumoniaechallenge relative to wild-type controls receiving control IgG(Fig. 7B). Importantly, however, such increased bacterial loads

FIG. 2. Bacterial loads in BALF and lung parenchyma of wild-typemice, CG KO mice, and CG/NE KO mice infected with S. pneumoniae.Wild-type mice (white bars), CG KO mice (hatched bars), and CG/NEKO mice (black bars) were infected with serotype 19 S. pneumoniae. Atthe indicated time points, mice were euthanized and bacterial loads inBALFs (A) and lung tissues (B) were determined, as indicated. Valuesare shown as means � standard errors of the means of n � 5 to 10mice/treatment group and time point. ** (***), P 0.01 (P 0.001)compared to wild-type mice. �� (��), P 0.01 (P 0.001) com-pared to CG KO mice.

FIG. 3. Neutrophil recruitment profiles and neutral serine proteaseand ROS activities in neutrophil lysates of S. pneumoniae-infectedwild-type, CG KO, and CG/NE KO mice. (A) Wild-type mice, CG KOmice, and CG/NE KO mice were infected with serotype 19 S. pneu-moniae. At the indicated time points, mice were euthanized and sub-jected to bronchoalveolar lavage for quantification of recruited neu-trophils. (B) Kinetic analysis using FRET substrates of neutral serineprotease activities in cell lysates of alveolar recruited neutrophils col-lected by bronchoalveolar lavage from the lungs of S. pneumoniae-infected wild-type mice (white bars), CG KO mice (hatched bars), orCG/NE KO mice (black bars), as indicated. (C) S. pneumoniae-in-duced ROS formation in highly purified neutrophils in vitro. Neutralserine protease activities and ROS formation are representative of twoindependent experiments. Values are presented as means � standarderrors of the means of n � 5 to 10 mice/treatment group and timepoint, except for n � 4 mice per treatment group (B). *, P 0.05; **,P 0.01; ***, P 0.001, compared to wild-type mice. �� (��),P 0.01 (P 0.001) compared to CG KO mice. §, P 0.05 comparedto controls.



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were accompanied by nearly complete lack of CG/NE immu-noreactivity in BALF cells of neutrophil-depleted mice (Fig.7C), whereas CG/NE protein was easily detectable in BALFcells of S. pneumoniae-infected mice pretreated with controlantibody. These data provide further evidence that neutrophilsare the primary cellular shuttle for CG/NE and illustrate thatspecific depletion of neutrophils is accompanied by completeloss of alveolar CG/NE bioavailability in mice challenged withS. pneumoniae, which ultimately results in uncontrolled out-growth of bacteria in distal air spaces.

DISCUSSION

The main goal of the current study was to evaluate the roleof cathepsin G and neutrophil elastase in the lung host defenseagainst S. pneumoniae infection in mice. Using two serotypesof S. pneumoniae exhibiting different pathogenicity profiles, wefound evidence for a critical and nonredundant contribution ofboth CG and NE to lung innate immunity particularly againstfocal pneumonia-inducing, noninvasive serotype 19 S. pneu-moniae, whereas CG and NE were not sufficient to preventinduction of septicemia in mice challenged with highly virulentserotype 2 S. pneumoniae. The defective bacterial clearanceobserved in CG KO and particularly CG/NE KO mice was notdue to impaired alveolar neutrophil recruitment or defectiveROS formation but rather due to impaired serine proteaseactivity in recruited neutrophils, overall resulting in severelyaggravated lung permeability, respiratory distress, and death.Moreover, our data show that neutrophils serve as the primary

cellular shuttle transporting CG and NE proteolytic activityinto the bronchoalveolar space. This study demonstrates acritical and nonredundant role for neutral serine proteases CGand NE in lung-protective immunity against inhaled S. pneu-moniae.

In the current study, we present several novel aspects ad-dressing the role of CG and NE in acute lung infection notreported so far in the literature. First, we demonstrate thatCG/NE were highly efficacious in preventing mortality in miceinfected with less virulent, focal pneumonia-inducing serotype19 S. pneumoniae, whereas intra-alveolar availability of CG/NEdid not protect against highly virulent, sepsis-causing serotype2 S. pneumoniae. The observed lack of effect of CG/NE intype 2 pneumococcus-infected mice is most likely due to dif-ferent kinetics by which the host is able to mount sufficientCG/NE bioavailability in the lungs, relative to the very rapidkinetics by which serotype 2 S. pneumoniae escapes local de-fense to cause sepsis in mice. Second, we illustrate that defec-tive pneumococcal killing observed in mutant mice was neitherdue to impaired alveolar neutrophil recruitment nor due todefective ROS formation in mutant neutrophils but rather dueto lack of CG/NE proteolytic activities in alveolar recruitedneutrophils, as assessed by kinetic analysis. These data providedirect evidence that proteolytic activities of neutral serine pro-teases within the infected lung are absolutely crucial for aneffective host defense against S. pneumoniae. Third, makinguse of chimeric mice and neutrophil depletion experiments, weidentified neutrophils as the primary cellular shuttle transport-

FIG. 4. Effect of CG and CG/NE deficiency on lung permeability and respiratory failure in mice infected with S. pneumoniae. Wild-type mice(white bars), CG KO mice (hatched bars), and CG/NE KO mice (black bars) were either left untreated (0-day time points in panels A and B) orinfected with serotype 19 S. pneumoniae. (A) At the indicated time points, lung permeability was determined as outlined in Materials and Methods.(B) Longitudinal analysis of arterial O2 saturation in wild-type mice (left panel), CG KO mice (middle panel), or CG/NE KO mice (right panel)left untreated (0-day time points) or infected with serotype 19 S. pneumoniae, as indicated. The data are shown as means � standard errors of themeans (n � 3 to 5 mice for control groups in panels A and B; n � 5 to 10 mice/treatment group and time point). **, P 0.01 compared to wild-typemice. ��, P 0.01 compared to CG KO mice. §, P 0.05 compared to controls (0-day values).



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ing CG and NE protein from the blood into the bronchoalveo-lar space of S. pneumoniae-infected mice. Consequently, fail-ure of single (CG KO) or double (CG/NE KO) mutant mice tomount sufficient CG or CG/NE bioactivity in their lungs uponchallenge with S. pneumoniae resulted in an acute respiratorydistress syndrome (ARDS)-like phenotype and increased mor-tality.

In this study, we explored the role of CG and CG/NE in thelung host defense against two different serotypes of S. pneu-moniae. Notably, pathogenicity profiles of S. pneumoniae differstrongly between and among serotypes (5, 15), resulting inessentially different clinical disease courses. Here, we observedthat CG KO mice and, even more so, CG/NE KO mice suc-cumbed to infection with low-virulence type 19 pneumococci,which was accompanied by weak bacteremia but severely im-paired arterial oxygenation, reflective of respiratory failure dueto acute lung injury in these mice. These observations supportour concept that particularly respiratory failure rather thanbacteremia caused the increased mortality noted in CG KOand CG/NE KO mice challenged with type 19 pneumococci.Such data strongly suggest that transient intrapulmonary bio-availability of neutrophil-derived CG/NE attenuates lung epi-thelial injury and subsequently developing respiratory distress

due to bacterial challenge. Given the increased susceptibility ofthe CG/NE KO mice to infection with S. pneumoniae com-pared to CG KO mice, it is tempting to speculate that NE KOmice would demonstrate a similar phenotype upon infectionwith S. pneumoniae, as observed in the CG KO mice, althoughNE KO mice have not been examined in the current study.

Earlier studies reported a direct role for CG and NE inbacterial and fungal pathogen elimination in vivo. CG has beenimplicated in antimicrobial defense against Staphylococcus au-reus and, together with NE, also against Aspergillus fumigatusinfection in mice (22, 28). Furthermore, neutrophil elastase isimportant for the elimination of Klebsiella pneumoniae andEscherichia coli as well as Candida albicans and Pseudomonasaeruginosa (2, 3, 10, 22). Additionally, a recent publication byStandish and Weiser (26) showed that purified human CG andNE efficiently killed S. pneumoniae in vitro, although no in vivodata on the role of CG and NE in pneumococcal killing werereported in that study. Our data close this gap by showing thatboth CG and NE play crucial and nonredundant roles in lungantibacterial responses against S. pneumoniae.

In the present study, we observed gradually increasing bac-terial loads in the lungs of CG/NE KO mice and CG KO mice,which correlated with lack of neutral serine protease activities

FIG. 5. Proinflammatory cytokine responses in the lungs of S. pneumoniae-infected wild-type mice, CG KO mice, and CG/NE KO mice.Wild-type mice (white bars), CG KO mice (hatched bars), and CG/NE KO mice (black bars) were infected with serotype 19 S. pneumoniae. Atthe indicated time points, mice were subjected to bronchoalveolar lavage for determination of BALF cytokine profiles of TNF- (A), IL-6 (B), KC(CXCL3) (C), and MIP-2 (CXCL2) (D), as indicated. Values are shown as means � standard errors of the means of n � 5 to 10 mice/treatmentgroup and time point. ** (***), P 0.01 (P 0.001) compared to wild-type mice. � (��), P 0.05 (P 0.001) compared to CG KO mice.



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in these mice, supporting a role for CG and NE in lung resis-tance against S. pneumoniae in vivo. In this regard, neutrophilsare well known to play prominent roles in antimicrobial re-sponses by at least two principal modes of action, i.e., therelease of reactive oxygen species and the production of neu-tral serine proteases and/or antimicrobial peptides (21). Ourdata are in line with a report from Reeves and colleagues, whopreviously linked these two mechanisms by showing that micedeficient in neutrophil-granule proteases were unable to resiststaphylococcal and candidal infections, despite normal super-oxide production (22). An increased ROS concentration inphagocytic vacuoles was found to be essential for cationic gran-ule protein release of CG and NE from the anionic sulfatedproteoglycan matrix, rendering neutrophil-derived neutral ser-ine proteases but not neutrophil-derived ROS induction pri-marily responsible for the bacterial killing process (20, 22). Asmentioned above, we also found that highly purified CG/NE-deficient neutrophils were able to mount normal ROS forma-tion in response to both S. pneumoniae and zymosan (data notshown) challenge in vitro.

Neutrophils have also been recognized to be important me-diators of tissue destruction in response to inflammation andinfection (11, 24). However, we believe that two points argueagainst a major role for neutrophils to have caused increasedlung permeability in the single- and double-deficient mice inthe current study. First, numbers of recruited neutrophils werequite similar in all three treatment groups, despite strong dif-ferences in lung permeability noted between groups. Second,we recently showed that neutrophils did not contribute to lungpermeability in mice challenged with the major pneumococcalcytotoxic virulence factor pneumolysin (1, 18). Given that CGKO and CG/NE KO mice had strongly increased bacterial

FIG. 6. Bacterial loads, neutrophil recruitment profiles, and CG/NE protein expression in chimeric wild-type mice, CG KO mice, or CG/NEKO mice infected with S. pneumoniae. (A to C) Chimeric wild-type mice (WT BMT onto WT, white bars), CG KO chimeric mice (CG KO BMTonto WT, hatched bars), and CG/NE KO chimeric mice (CG/NE KO BMT onto WT, black bars) were infected with serotype 19 S. pneumoniae.At the indicated time points, mice were euthanized and bacterial loads in BALFs (A) and lung tissue (B) as well as numbers of neutrophils in BALF(C) were determined, as indicated. Values are shown as means � standard errors of the means of n � 4 to 5 mice/treatment group and time point.*, P 0.05 compared to chimeric wild-type mice. §, P 0.05 compared to uninfected chimeric wild-type mice (CL). (D) Western blot analysisof CG/NE immunoreactivity in BALF cells (containing �95% neutrophils) of wild-type, CG KO, or CG/NE KO chimeric mice on day 1 afterinfection with S. pneumoniae. The given Western blot is representative of two independent experiments. BMT, bone marrow transplantation.

FIG. 7. Bacterial loads, leukocyte counts, and CG/NE protein ex-pression in neutrophil-depleted and control mice challenged with S.pneumoniae. (A and B) Wild-type mice were treated with Ly6G-spe-cific, neutrophil-depleting antibody 1A8 (black bars) or control IgG(white bars) and were then infected with serotype 19 S. pneumoniae.On day 1 postinfection, mice were euthanized and leukocyte countswere determined in BALFs (A) and bacterial loads were determined inBALFs and lung tissue (B). Values are shown as means � standarderrors of the means of n � 5 to 10 mice/treatment group and timepoint. * (**, ***), P 0.05 (P 0.01, P 0.001) compared to controlmice. (C) Western blot analysis of CG/NE immunoreactivity in BALFcells of neutrophil-depleted and control mice on day 1 after infectionwith S. pneumoniae. The Western blot shown is representative of twoindependent experiments. PMN, polymorphonuclear leukocyte.



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loads in their lungs, it appears very likely that in the absence ofneutral serine proteases, primarily pneumococcal virulencefactors such as pneumolysin contributed to the increased lungpermeability and concomitantly developing respiratory distressin mutant mice, rather than neutral serine protease-deficient,alveolar recruited neutrophils. Nevertheless, an as-yet-unex-plained issue relates to the identification of the pneumococcalsubstrate for neutrophil-derived neutral serine protease activ-ities to mediate their antibacterial activities in vivo.

In summary, the current study is the first to provide evidencethat neutrophil-derived neutral serine proteases CG and NEare absolutely crucial for effective host defense against focalpneumonia-inducing S. pneumoniae infections in vivo. Wefound neutrophil-derived CG and NE protease activities tocritically contribute to prevention of lung epithelial injury andrespiratory distress but not bacteremia in mice. These findingsmay provide important novel insights for the development ofadjunct treatment interventions to ameliorate respiratory failurein patients suffering from severe pneumococcal pneumonia.

ACKNOWLEDGMENT

The current study has been supported by the German ResearchFoundation, grant SFB 587 to U.A.M. and T.W.

REFERENCES

1. Ackerstaff, R. G., et al. 2004. Carotid endarterectomy, postoperative hyper-perfusion, and intracerebral haemorrhage. Eur. J. Vasc. Endovasc. Surg.28:679–680.

2. Belaaouaj, A., K. S. Kim, and S. D. Shapiro. 2000. Degradation of outermembrane protein A in Escherichia coli killing by neutrophil elastase. Sci-ence 289:1185–1188.

3. Belaaouaj, A., et al. 1998. Mice lacking neutrophil elastase reveal impairedhost defense against gram negative bacterial sepsis. Nat. Med. 4:615–618.

4. Briles, D. E., M. J. Crain, B. M. Gray, C. Forman, and J. Yother. 1992.Strong association between capsular type and virulence for mice amonghuman isolates of Streptococcus pneumoniae. Infect. Immun. 60:111–116.

5. Briles, D. E., et al. 2003. Immunizations with pneumococcal surface proteinA and pneumolysin are protective against pneumonia in a murine model ofpulmonary infection with Streptococcus pneumoniae. J. Infect. Dis. 188:339–348.

6. Calbo, E., and J. Garau. 2010. Of mice and men: innate immunity in pneu-mococcal pneumonia. Int. J. Antimicrob. Agents 35:107–113.

7. Hahn, I., et al. 2011. Dendritic cell depletion and repopulation in the lungafter irradiation and bone marrow transplantation in mice. Am. J. Respir.Cell Mol. Biol. 45:534–541.

8. Henken, S., et al. 2010. Efficacy profiles of daptomycin for treatment ofinvasive and noninvasive pulmonary infections with Streptococcus pneu-moniae. Antimicrob. Agents Chemother. 54:707–717.

9. Herbold, W., et al. 2010. Importance of CXC chemokine receptor 2 inalveolar neutrophil and exudate macrophage recruitment in response topneumococcal lung infection. Infect. Immun. 78:2620–2630.

10. Hirche, T. O., et al. 2008. Neutrophil elastase mediates innate host protec-tion against Pseudomonas aeruginosa. J. Immunol. 181:4945–4954.

11. Ioachimescu, O. C., and J. K. Stoller. 2005. A review of alpha-1 antitrypsindeficiency. COPD 2:263–275.

12. Kadioglu, A., and P. W. Andrew. 2004. The innate immune response topneumococcal lung infection: the untold story. Trends Immunol. 25:143–149.

13. Kalupov, T., et al. 2009. Structural characterization of mouse neutrophilserine proteases and identification of their substrate specificities: relevanceto mouse models of human inflammatory diseases. J. Biol. Chem. 284:34084–34091.

14. Korkmaz, B., et al. 2008. Measuring elastase, proteinase 3 and cathepsin Gactivities at the surface of human neutrophils with fluorescence resonanceenergy transfer substrates. Nat. Protoc. 3:991–1000.

15. Mahdi, L. K., A. D. Ogunniyi, K. S. LeMessurier, and J. C. Paton. 2008.Pneumococcal virulence gene expression and host cytokine profiles duringpathogenesis of invasive disease. Infect. Immun. 76:646–657.

16. Maus, U., et al. 2002. The role of CC chemokine receptor 2 in alveolarmonocyte and neutrophil immigration in intact mice. Am. J. Respir. Crit.Care Med. 166:268–273.

17. Maus, U. A., et al. 2007. Importance of phosphoinositide 3-kinase gamma inthe host defense against pneumococcal infection. Am. J. Respir. Crit. CareMed. 175:958–966.

18. Maus, U. A., et al. 2004. Pneumolysin-induced lung injury is independent ofleukocyte trafficking into the alveolar space. J. Immunol. 173:1307–1312.

19. Maus, U. A., et al. 2003. Monocytes are potent facilitators of alveolar neu-trophil emigration during lung inflammation: role of the CCL2-CCR2 axis.J. Immunol. 170:3273–3278.

20. Messina, C. G., E. P. Reeves, J. Roes, and A. W. Segal. 2002. Catalasenegative Staphylococcus aureus retain virulence in mouse model of chronicgranulomatous disease. FEBS Lett. 518:107–110.

21. Pham, C. T. 2006. Neutrophil serine proteases: specific regulators of inflam-mation. Nat. Rev. Immunol. 6:541–550.

22. Reeves, E. P., et al. 2002. Killing activity of neutrophils is mediated throughactivation of proteases by K� flux. Nature 416:291–297.

23. Reutershan, J., et al. 2006. Critical role of endothelial CXCR2 in LPS-induced neutrophil migration into the lung. J. Clin. Invest. 116:695–702.

24. Sandhaus, R. A. 2010. Alpha-1 antitrypsin deficiency: whom to test, whom totreat? Semin. Respir. Crit. Care Med. 31:343–347.

25. Srivastava, M., et al. 2008. Overexpression of cathepsin K in mice decreasescollagen deposition and lung resistance in response to bleomycin-inducedpulmonary fibrosis. Respir. Res. 9:54.

26. Standish, A. J., and J. N. Weiser. 2009. Human neutrophils kill Streptococ-cus pneumoniae via serine proteases. J. Immunol. 183:2602–2609.

27. Taut, K., et al. 2008. Macrophage turnover kinetics in the lungs of miceinfected with Streptococcus pneumoniae. Am. J. Respir. Cell Mol. Biol.38:105–113.

28. Tkalcevic, J., et al. 2000. Impaired immunity and enhanced resistance toendotoxin in the absence of neutrophil elastase and cathepsin G. Immunity12:201–210.

29. Welte, T., A. Torres, and D. Nathwani. 20 August 2010. Clinical and eco-nomic burden of community-acquired pneumonia among adults in Europe.Thorax [Epub ahead of print.] doi:10.1136/thx.2009.129502.

30. Winter, C., et al. 2007. Lung-specific overexpression of CC chemokine ligand(CCL) 2 enhances the host defense to Streptococcus pneumoniae infectionin mice: role of the CCL2-CCR2 axis. J. Immunol. 178:5828–5838.

31. Wu, H., T. Suzuki, B. Carey, B. C. Trapnell, and F. X. McCormack. 2011.Keratinocyte growth factor augments pulmonary innate immunity throughepithelium-driven, GM-CSF-dependent paracrine activation of alveolarmacrophages. J. Biol. Chem. 286:14932–14940.

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