The endolysosomal cysteine cathepsins L and K ... - The FASEB Journal

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The endolysosomal cysteine cathepsins L and K are involved in macrophage-mediated clearance of Staphylococcus aureus and the concomitant cytokine induction Sabrina Müller,*,‡,¶ Anja Faulhaber,* Carolin Sieber,* Dietmar Pfeifer,§ Tanja Hochberg,* Martina Gansz,*,¶ Sachin D. Deshmukh,‡ Stephanie Dauth,# Klaudia Brix,# Paul Saftig,** Christoph Peters,*,† Philipp Henneke,‡,储 and Thomas Reinheckel*,‡,†,1 *Institute of Molecular Medicine and Cell Research and †BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany; ‡Center of Chronic Immunodeficiency, §Core Facility Genomics, Department of Internal Medicine I, and 储Division of Pediatric Infectious Diseases and Rheumatology, Centre of Paediatrics and Adolescent Medicine, University Medical Center Freiburg, Freiburg, Germany; ¶Faculty of Biology, University of Freiburg, Freiburg, Germany; #School of Engineering and Science, Research Center Molecular Life Science, Jacobs University Bremen, Bremen, Germany; and **Institute of Biochemistry, Christian-AlbrechtsUniversität zu Kiel, Kiel, Germany Cysteine cathepsins are endolysosomal cysteine proteases highly expressed in macrophages; however, their individual contributions to the elimination of bacteria and bacteria-induced cytokine production by macrophages are unknown. We assessed the contribution of cysteine cathepsins to macrophage defense pathways against Staphylococcus aureus by using chemical inhibitors and by infecting primary bone marrow– derived macrophages deficient in 1 of 7 major macrophage-expressed endolysosomal cysteine proteases. We show that cysteine cathepsins are involved in the phagocytosis and killing of S. aureus. Cathepsin L was identified as an executor of nonoxidative killing. Moreover, microarray data revealed cysteine cathepsins to be important for the maximal induction of certain proinflammatory genes, such as IL6, in response to S. aureus. Cysteine cathepsin’s contribution to IL6 production was dependent on phagocytosis, and cathepsin K was identified to be a critical protease in this process. Analysis of macrophages with impaired trafficking of endolysosomal Toll-like receptors (TLRs) to the acidic

ABSTRACT

Abbreviations: AMC, 7-amino-4-methylcoumarin; BH, Benjamini-Hochberg; CpG cytosine-guanine-phosphodiester; Ctsb, cathepsin B; Ctsc, cathepsin C; Ctsh, cathepsin H; Ctsk, cathepsin K; Ctsl, cathepsin L; Ctss, cathepsin S; Ctsz, cathepsin Z; CytD, cytochalasin D; E64d, l-trans-epoxysuccinyl-leu-3methylbutylamide-ethyl ester; EEA1, early endosome antigen 1; GO, gene ontology; iNOS, inducible NO synthase; Lgmn, legumain; LAMP, lysosomal-associated membrane protein; LPS, lipopolysaccharide; MØ, macrophage; MOI, multiplicity of infection; MyD88, myeloid differentiation primary response gene 88; p.i., postinfection; PGN, peptidoglycan; RAB, Ras-related in brain; TLR, Toll-like receptor; UNC93B1, uncoordinated 93 homolog B1; WT, wild type 162

compartment revealed that they were not involved in cathepsin-dependent IL6 induction. Because IL6 production was completely dependent on the TLR-adaptor protein myeloid differentiation primary response gene 88 (MyD88), it appears that other TLRs are involved. In summary, lysosomal cysteine proteases are functionally linked to the complex bactericidal and inflammatory activities of macrophages.— Müller, S., Faulhaber, A., Sieber, C., Pfeifer, D., Hochberg, T., Gansz, M., Deshmukh, S. D., Dauth, S., Brix, K., Saftig, P., Peters, C., Henneke, P., Reinheckel, T. The endolysosomal cysteine cathepsins L and K are involved in macrophage-mediated clearance of Staphylococcus aureus and the concomitant cytokine induction. FASEB J. 28, 162–175 (2014). www.fasebj.org Key Words: proteases 䡠 lysosome 䡠 innate immune system 䡠 phagocyte 䡠 nonoxidative killing Macrophages (MØs) are professional phagocytes of the innate immune system. A major function of MØs is to take up pathogens, which are subsequently killed in the acidic environment of phagolysosomes by oxidative and nonoxidative mechanisms (1). A well-known example of nonoxidative pathogen elimination involves the peptidoglycan (PGN)-degrading glycoside hydrolase lysozyme. This enzyme cleaves the linkage of N-acetylmuramic acid and N-acetylglucosamine of PGN, a major 1 Correspondence: Institute of Molecular Medicine and Cell Research, Stefan-Meier Str. 17, 79104 Freiburg, Germany. E-mail: [email protected] doi: 10.1096/fj.13-232272 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

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component of bacterial cell walls (2). It has been shown recently that Staphylococcus aureus, a gram-positive, facultative pathogenic bacterium that causes a wide range of diseases in humans, from mild to life-threatening, is resistant to lysozyme-based degradation of PGN, thereby attenuating the host-mediated inflammatory response (3). However, inside the acidic cellular compartment, S. aureus faces more than lysozyme. In fact many degrading enzymes, especially endolysosomal proteases, reside in this microbicidal compartment (4, 5). It has been shown that mice deficient in the serine protease cathepsin G are much more prone to dying after intravenous injection of S. aureus than are wild-type (WT) mice (6). Furthermore, mice deficient in matrix metalloproteinase 12 (also termed MØ elastase) are highly susceptible to S. aureus infections, due to enhanced intracellular survival of bacteria in MØs (7). These studies indicate that proteolytic enzymes are important components for efficient killing of S. aureus. Another type of MØ protease is the C1 family of papain-like endolysosomal cysteine proteases. These so-called cysteine cathepsins have broad substrate specificity and terminally degrade proteins in the endolysosomal compartment (8). Multiple members of this protease family are highly expressed in MØs, such as cathepsin S (Ctss), as well as cathepsin B and L (Ctsb and Ctsl) (9, 10). It is well established that cysteine cathepsins contribute to the presentation of antigens by peripheral APCs, which bridge the innate and the adaptive immune system (11, 12). However, until now, whether members of this potent protease family contribute to the nonoxidative killing of S. aureus in MØs has been unknown. Recently, it has been reported that cysteine cathepsins catalyze the activating cleavage of endolysosomal pattern recognition receptors (PRRs) and Toll-like receptors (TLRs) 9, 7, and 3 (13–15), which induce a potent inflammatory response on activation (16). In addition, it has been shown that degradation of pathogens by lysozyme and other hydrolytic enzymes inside the phagolysosome is essential for an intact cytokine response (17, 18). Of interest, bacterial susceptibility to phagolysosomal degradation positively correlates with cytokine induction (19). Therefore, the uptake of pathogens and the acidification of the phagolysosome is a prerequisite for an effective cell-autonomous inflammatory response (17, 20). As endolysosomal cysteine cathepsins are known to be acidic proteases that exhibit an optimal substrate turnover at low pH and are abundantly expressed in MØs, we hypothesized that cysteine cathepsins contribute to the degradation of S. aureus in the phagolysosome and hence to the onset of inflammation. As the incidence of antibiotic-resistant S. aureus strains has dramatically increased over the past decade, it is important to understand host-mediated antibacterial defense mechanisms in detail (21). Thus, we aimed to uncover the contribution of cysteine cathepsins to the MØ response to S. aureus with regard to elimination of bacteria and cytokine induction. Potent chemical protease inhibitors and primary MØs derived from protease-deficient mice were both used in this study, to CATHEPSIN-MEDIATED ANTIBACTERIAL DEFENSE

clarify the role of endolysosomal cysteine proteases in the MØ-mediated defense against S. aureus.

MATERIALS AND METHODS Mice Mice deficient in Ctsb (22), cathepsin H (Ctsh; ref. 23), cathepsin K (Ctsk; ref. 24), Ctsl (25), Ctss (26), cathepsin Z (Ctsz; ref. 27) or legumain [Lgmn; a synonym for asparaginyl endopeptidase (AEP)] (28) were backcrossed to C57BL/6N mice. TLR9-deficient mice and TLR9-WT control mice were on a C57BL/10 background. The gp91phox⫺/⫺ mouse strain on a C57BL/6J background was purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were euthanized between 7 and 12 wk of age. Cell culture Murine bone marrow cells from hind legs were collected and pelleted (all centrifugation steps were performed at 300 g at 6°C for 5 min). Erythrocytes were lysed in 2 ml of a hypotonic lysis buffer (16.58 g NH4Cl, 2.0 g KHCO3, and 74.0 mg disodium EDTA, dissolved in 2 L ddH2O and sterile filtered). The cells were washed twice with 1⫻ DPBS and cultured in RPMI 1640 medium supplemented with 10% FCS, 1% HEPES, sodium pyruvate, MEM nonessential amino acids, 0.5% ciprofloxacin (stock concentration, 2 mg/ml) (referred to as culture medium), and 1.6% of GM-CSF-enriched, cellconditioned medium. The cells differentiated for 8 d. The medium was changed every second day. Precursor cells in the supernatant were collected and pelleted and either recultured in the same cell culture flask or cultured in a new one for expansion. The cells were cultured in a humidified atmosphere at 37°C and 5% CO2. For the experiments, MØs were plated in an appropriate number in culture medium on the prior day. Before the experiments, the MØs were starved for 3 or 14 h in RPMI 1640 medium supplemented with 1% HEPES, sodium pyruvate, and MEM nonessential amino acids (referred to as starvation medium). To irreversibly inhibit cysteine cathepsin activity, we applied 10 ␮M E64d (l-trans-epoxysuccinyl-leu-3-methylbutylamide-ethyl ester) and 10 ␮M JPM-OEt or equal volumes of DMSO as the control to the cells for 1 h. The MØs were washed with 1⫻ DPBS before the respective experiments began. Culturing of bacteria S. aureus bacteria Newman strain from a saturated overnight culture were grown in LB-medium to the midexponential growth phase (OD600⬇0.4 – 0.5) at 37°C and 180 rpm. The bacteria (500 ␮l) were mixed with an equal volume of 30% glycerol and stored at ⫺80°C. The bacterial concentration was determined by plating serial dilutions on blood agar plates. For experiments, the bacteria were thawed and washed twice with 1⫻ DPBS before being resuspended in 500 ␮l starvation medium. Phagocytosis and killing assay MØs (2.5⫻105/well) plated in 24-well plates were infected with S. aureus [multiplicity of infection (MOI) 10]. The plates were centrifuged for 2 min at 300 g and returned to the cell incubator for 10, 20, and 30 min (phagocytosis assay) or 45 min (killing assay), before the extracellular bacteria were 163

killed by addition of gentamicin sulfate (final concentration, 100 ng/␮l). The cells were lysed by addition of 300 ␮l of 0.5% saponin (diluted in 1⫻ DPBS) for 15 min at 37°C. For the killing assays, infected MØs were lysed 5 min after addition of antibiotic; this time point was referred to as 0 h and was equal to 100% in the analyses. The remaining MØs were cultivated for 1 and 3 h and lysed afterward. The lysates were serially diluted 1:10 in 1⫻ DPBS and plated on blood agar plates incubated overnight at 37°C. In addition, the supernatants were plated on blood agar plates to control efficient killing of extracellular bacteria by gentamicin sulfate. Grown colonies were correlated with intracellular bacterial survival. The experiments were performed in triplicate. Infection and stimulation of MØs MØs (8⫻104/well) plated in 96-well plates were either infected with S. aureus (MOI 5 and 10) or noninfected. To inhibit actin polymerization-dependent phagocytosis, we treated the cells with 10 ␮M cytochalasin D (CytD) 30 min before infection with MOI 10. The MØs were also stimulated with 62.5 and 250 nM cytosine-guanine-phosphodiester (CpG) oligonucleotides (ODN 1826, sequence: 5=-TCCATGACGTTCCTGACGTT-3=). The plates were centrifuged for 2 min at 300 g. After 45 min, gentamicin sulfate was added at a final concentration of 100 ng/␮l. The supernatants were collected after 9 h and stored at ⫺80°C. To compare MØs from different genotypes, we performed an MTT assay (Sigma-Aldrich, Hamburg, Germany) 6 h after treatment, according to the manufacturer’s protocol. Cytokine determination IL6 in supernatants of infected or stimulated MØs was measured by using either the IL6 Ready-Set-Go! ELISA Kit (eBioscience, Frankfurt, Germany) or the IL6 ␣LISA Kit (Perkin Elmer, Rodgau, Germany), according to the manufacturers’ protocols. ROS measurement Lucigenin (100 ␮l; stock concentration, 1 mg/ml), diluted in starvation medium, was added to 8 ⫻ 104 MØs/well of a white 96-well plate (Perkin Elmer) at a final concentration of 0 ␮g/ml and incubated at room temperature for 10 min. S. aureus (50 ␮l) diluted in starvation medium (MOI 10 and 20) was added, and the plate was centrifuged for 2 min at 300 g. Luminescence was measured for 2 h at 37°C with the Infinite200 Pro reader (Tecan, Maennedorf, Switzerland). Data are presented as relative light units (RLU) per second. Microscopy MØs were plated on glass coverslips placed in a 24-well plate (2.5⫻105 cells/well) the day before. The cells were starved for 3 h and treated for 1 h with cysteine cathepsin inhibitors or DMSO. They were washed with 1⫻ DPBS and cultured in starvation medium for an additional 3 h. The supernatants were removed, and MØs were incubated for 15 min with a 1:50,000 dilution of acridine orange (stock concentration, 10 mg/ml) in starvation medium. MØs from different genotypes were plated as described above. The cells were infected with S. aureus (MOI 10) for 1 h before they were fixed with 4% PFA at 37°C for 30 min and permeabilized with 0.2% Triton-X 100 (diluted in PBS) at room temperature for 7 min and with 164

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acetone at ⫺20°C for 4 min. A blocking step was performed with 2.5% BSA and 1.5% goat serum for 30 min at room temperature. Incubation with the primary antibodies rabbit anti-mouse Ras-related in brain 5 (Rab5), rabbit anti-mouse early endosome antigen 1 (EEA1), and rabbit anti-mRab7 (all Cell Signaling Technology, Danvers, MA, USA); rat antimouse lysosomal-associated membrane protein (LAMP)-1 (Abcam, Cambridge, UK); and rabbit anti-S. aureus (Thermo Scientific, Waltham, MA, USA) was performed at 4°C overnight, and incubation with the secondary antibodies goat anti-rabbit Cy3 (Abcam), goat anti-rabbit FITC (Abcam), and goat anti-rat Alexa Fluor 594 (Life Technologies, Paisley, UK) was performed at room temperature for 1 h. Nucleic acids were stained with Hoechst (2 ␮g/ml in PBS; Sigma-Aldrich) at room temperature for 5 min, and coverslips were mounted with Permafluor (Thermo Scientific). Microscopy images (⫻1000; ⫻100 PlnN 1.3 oil objective) were taken with an Observer.Z1 fluorescence microscope (Zeiss, Oberkochen, Germany) at 475/530 nm (␭ex/␭em) for green fluorescence, 530 –585/615 nm for red fluorescence, and 365/445– 450 nm for blue fluorescence. To obtain optical sections, we used the ApoTome.2 (Zeiss). Images were obtained with an AxioCam MRm and processed with Axio Vision software (Zeiss). Microarray analysis After pretreatment with cysteine cathepsin inhibitors, MØs plated in a 6-well plate were infected with S. aureus (MOI 5) or left untreated. Gentamicin sulfate (final concentration 100 ng/␮l) was added after 45 min. After 3 h, the MØs were washed twice with 1⫻ DPBS and lysed by 350 ␮l of RLT buffer (RNeasy Mini Kit; Qiagen, Hamburg, Germany). RNA was isolated according to the manufacturer’s protocol. RNA integrity was analyzed by capillary electrophoresis with a 2100 Bioanalyzer (Agilent Technologies, Boeblingen, Germany). Samples with an RNA integrity number ⬎9.60 were processed further. RNA was amplified with the Ambion WT Expression Kit (Life Technologies) and labeled with the aid of the GeneChip WT Terminal Labeling Kit (Affymetrix, Santa Clara, CA, USA), as described by the manufacturers’ protocols. Labeled fragments were hybridized to GeneChip Mouse Gene ST 1.0, with the GeneChip Hybridization Oven 640 (both from Affymetrix) for 16 h at 45°C and 60 rpm. After the chips were washed and stained, they were scanned with the GeneChip Scanner 3000 7G (Affymetrix). CEL files were produced from raw data with GeneChip Command Console 3.0 software (Affymetrix). Genedata Expressionist software was used for further data analysis. CEL files were imported into the Refiner Module of the Expressionist software where GeneChip (GC) background subtraction (Affymetrix) was performed with antigenomic background probes. Subsequently, quantile normalization and probe summarization were performed with the Bioconductor robust multiarray average (RMA) condensing algorithm, as implemented in the Refiner (29). To identify differentially expressed genes between the analyzed groups, the paired Bayes t test (CyberT; ref. 30) with the Bayes confidence estimate value set to 10, and a window size of 101 genes and 100% valid values in each group was used (Analyst 7.0.5a module). The false discovery rate was determined by calculating the Benjamini-Hochberg (BH) q value (31). To calculate the paired effect-size score between the experimentally analyzed groups, the Effect Size function of Analyst was used. Only genes from the categories “main” and “unmapped” (see Affymetrix transcript annotation NA32) were included in the analysis.

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Enzyme activity assay At 24 h after pretreatment with cysteine cathepsin inhibitors or DMSO, MØs were lysed on ice in sodium acetate buffer (100 mM sodium acetate, 1 mM EDTA, 0.05% Brij 35 solution, and 1 mM DTT; pH 5.5) by mechanical disruption with a Dounce homogenizer. Assay buffer (85 ␮l; 1.5 mM DTT in sodium acetate buffer) was added to the appropriate wells of a black 96-well plate. Lysate (10 ␮l) was added, and the plate was incubated at 37°C for 15 min. Substrate [5 ␮l of 500 ␮M; Z-Phe-Arg-AMC (7-amino-4-methylcoumarin); Bachem, Bubendorf, Switzerland] diluted in ddH2O was quickly added, and fluorescence intensity was measured at 460 nm at 1 min intervals for 1 h. Quantitative RT-PCR MØs were infected with different MOIs of S. aureus (as described above) and incubated for 3 h (to analyze the expression of IL6, IL12␤, and TNF-␣); 12 h [to analyze expression of inducible NO synthase (iNOS)]; and 1, 3, and 6 h (to analyze cathepsin expression) in the cell incubator. The supernatants were removed, and the cells were washed with 1⫻ DPBS before they were lysed in 350 ␮l of RLT buffer (RNeasy Mini Kit; Qiagen). RNA was then isolated and reverse transcribed into cDNA (iScript cDNA Synthesis Kit; Bio-Rad, Munich, Germany), according to the manufacturer’s protocol. Quantitative RT-PCR was performed with Platinum SYBR Green qPCR SuperMixUDG (Life Technologies), and PCR was run on the CFX96 real-time PCR system (Bio-Rad). Expression levels were normalized to ␤-actin. The following primers were used: ␤-actin forward, 5=-ACCCAGGCATTGCTGACAGG-3=, and reverse, 5=GGACAGTGAGGCCAGGATGG-3=; Ctsb forward, 5=-CCTGGGCTGGGGAGTAGAGAATGGAG-3=, and reverse, 5=-TGGAAAAAGCCCCTAAGGACTGGACAAT-3=; Ctsk forward, 5=GCCAGGATGAAAGTTGTATG-3=, and reverse, 5=-CAGGCGTTGTTCTTATTCC-3=; Ctsl forward, 5=-GCACGGCTTTTCCATGGA-3=, and reverse, 5=-CCACCTGCCTGAATTCCTCA-3=; Ctss forward, 5=-AGCTGAAAACGGGGAAGCTG-3=, and reverse, 5=GACGCACCGTGGCTTTGTAG-3=; Ctsz forward, 5=-TATGCCAGCGTCACCAGGAAC-3=, and reverse, 5=-CCTCTTGATGTTGATTCGGTCTGC-3=; IL6 forward, 5=-AACCACGGCCTTCCCTACTTC-3=, and reverse, 5=-GCCATTGCACAACTCTTTTCTCAT-3=; IL12␤ forward, 5=-GACACGCCTGAAGAAGAT GAC-3=, and reverse, 5=-TAGTCCCTTTGGTCCAGTGT-3=; iNOS forward, 5=-GCTTCACTTCCAATGCAACA-3=, and reverse, 5=-GGCTGGACTTTTCACTCTGC-3=; and TNFA forward, 5=-TCGTAGCAAACCACCAAGTG-3=, and reverse, 5=CCTTGTCCCTTGAAGAGAACC-3=. Western blot analysis MØ lysates were loaded on a 15% acrylamide gel. The proteins were separated for 2 h at 130 V and transferred onto a PVDF membrane for 1.5 h at 100 mA by semidry blotting. The membrane was blocked in 3% low-fat milk, and incubation with primary goat anti-mCtsc antibody (1:250; R&D Systems, Minneapolis, MN, USA) was performed at 4°C overnight. Secondary rabbit anti-goat IgG antibody (1:5000; R&D Systems) incubation was performed at room temperature for 1 h. Chemiluminescence was induced by the SuperSignal kit (Thermo Scientific) and detected by the Fusion SL Detection System (Peqlab, Erlangen, Germany). Statistical analysis All data are shown as means ⫾ se in independent experiments. For statistical analysis, we used the 2-tailed Student’s t CATHEPSIN-MEDIATED ANTIBACTERIAL DEFENSE

test or the 2-tailed nonparametric Mann-Whitney test, as indicated in the figure legends. For evaluation of microarray data, see Microarray Analysis above.

RESULTS Cysteine cathepsin activity was essential for efficient phagocytosis and killing of S. aureus To investigate a possible involvement of cysteine cathepsin activity in the antibacterial MØ response, we treated MØs with a combination of 2 irreversible cysteine cathepsin inhibitors (10 ␮M E64d and 10 ␮M JPM-OEt; referred to as the inhibitors). To prevent a direct interaction of the inhibitors with S. aureus, we pretreated the MØs for 1 h, and the inhibitors were removed by cell culture medium exchange before the respective experiments began (Fig. 1A). However, because we could not exclude a possible intracellular accumulation of the inhibitors and therefore contact between S. aureus and the inhibitors inside the MØs, we checked bacterial growth in the presence of the inhibitors and found no growth impairment of bacteria under these conditions (Fig. 1A, right). Cleavage of the fluorogenic cysteine cathepsin substrate Z-Phe-ArgAMC was not detectable in inhibitor-pretreated MØs, even at 24 h after inhibitor removal, indicating an efficient inhibition of target proteases over the time course of the subsequent experiments (Fig. 1A, left). As the integrity of the acidic compartment itself is a prerequisite for proper bacterial killing and for cytokine induction (1, 17), we confirmed lysosomal stability and integrity in inhibitor-pretreated MØs by acridine orange staining. Figure 1B shows rounded, green endosomes and acidic, red lysosomes indicating the unperturbed integrity of the acidic compartment in MØs after the use of the cysteine cathepsin inhibitors. Phagocytosis is the first step of the bactericidal activity of MØs. After 10, 20, and 30 min of phagocytosis, the number of MØ lysate-derived S. aureus colonies was determined (Fig. 1C). Inhibition of cysteine cathepsin activity led to an approximate 30% reduction in phagocytosis of S. aureus, as compared with the solvent-treated controls (Fig. 1C), indicating that E64d/JPM-OEt-sensitive protease activity is necessary for efficient phagocytosis of S. aureus by MØs. Next, we tested the ability of cysteine cathepsin inhibitor–pretreated MØs to kill phagocytosed S. aureus. Intracellular bacterial survival was monitored over 3 h. We found that MØs killed ingested bacteria rapidly, with only 20% survival after 3 h (Fig. 1D). In contrast, the MØs pretreated with cysteine cathepsin inhibitors showed significantly less bactericidal activity than did the solvent-treated controls. After 1 h, only 10%, and after 3 h, only 55% of intracellular bacteria were killed in the inhibitor-pretreated MØs (Fig. 1D). This result was not caused by an impaired oxidative burst due to the inhibitor treatment (Fig. 1E). However, an oxidative burst was clearly necessary for killing 165

Fig. 1. Cysteine cathepsins were essential for efficient phagocytosis and killing of S. aureus (S.a.). A) Top panel: experimental workflow. Bottom panels: cysteine cathepsin activity was assessed in lysates of MØs 24 h after pretreatment with E64d (10 ␮M) plus JPM-OEt (10 ␮M) or equal volumes of DMSO as a solvent control (representative experiment out of 3, left panel). Bacterial growth was monitored in the presence or absence of cysteine cathepsin inhibitors (n⫽2; representative experiment, right panel). B) Acridine orange staining of MØs 3 h after pretreatment with cysteine cathepsin inhibitors. Scale bar ⫽ 10 ␮m. Representative images are shown. C) Top panel: experimental workflow. Bottom panel: phagocytosis was analyzed by infecting inhibitor- and solvent-pretreated MØs with S. aureus (MOI 10) for the indicated times (nⱖ9). Statistical analysis: 1-sample t test, **P ⱕ 0.01, ***P ⱕ 0.001. D) Top panel: experimental workflow. Bottom panel: intracellular survival of S. aureus (MOI 10) in inhibitor- or solvent-pretreated MØs was monitored over 3 h (n⫽6). Statistical analysis: Mann-Whitney test. **P ⱕ 0.01. E) ROS production after infection of inhibitor- or solvent-pretreated MØs with S. aureus was analyzed by using Lucigenin (13 ng/␮l; n⫽3). F) Intracellular survival of S. aureus in gp91phox-WT and gp91phox-deficient MØs, pretreated with cysteine cathepsin inhibitors or solvent, was monitored for 3 h (n⫽3).

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the S. aureus Newman strain used in the present study, as MØs deficient in the NADPH-oxidase subunit gp91phox did not kill internalized bacteria at all within 3 h (Fig. 1F). Naturally, inhibitor pretreatment of the gp91phox-deficient MØs did not affect the already strongly impaired killing of S. aureus by these MØs, in contrast to the gp91phox-WT MØs (Fig. 1F). Ctsl was a major component of nonoxidative killing in MØs In further experiments elucidating the role of individual proteases in efficient killing of S. aureus, the Ctsl⫺/⫺ MØs showed enhanced intracellular bacterial survival compared to the WT MØs, at both 1 and 3 h postinfection (p.i.) (Fig. 2A). Notably, the Ctsl-deficient MØs showed no reduction in phagocytosis of S. aureus (Supplemental Fig. S1A). The Ctsb-, Ctsh-, Ctsk-, and Ctszdeficient MØs did not exhibit impaired bacterial killing of S. aureus, compared to WT MØs (Supplemental Fig. S1C). We also analyzed the killing capacity of MØs deficient in the E64d/JPM-OEt-insensitive cysteine protease Lgmn, without showing a difference from that of the WT MØs (Supplemental Fig. S1C). The Ctss⫺/⫺ MØs showed weakly reduced killing of S. aureus compared to the WT MØs (Fig. 2B). To address the question of whether Ctss and Ctsl synergistically contribute to bacterial killing, we bred Ctsl/Ctss doubledeficient mice. The Ctsl/Ctss double-deficient MØs killed less bacteria compared to the WT control; however, the additional deficiency of Ctss did not further decrease the already impaired bacterial killing shown for the Ctsl single-deficient MØs (compare Fig. 2A–C). In line with this finding, the pretreatment of the Ctsl-deficient MØs with cysteine cathepsin inhibitors did not further reduce bacterial killing (Fig. 2D). These data point to a specific function of Ctsl in the nonoxidative bactericidal activity of MØs. Because an altered intracellular localization of S. aureus in gp91phoxdeficient and Ctsl-deficient MØs would be a possible explanation for the observed impairment in bacterial killing, we examined the occurrence of early and late

endosomal markers, as well as the lysosomal marker LAMP-1 in these MØs after infection with S. aureus (Fig. 3). There were comparable amounts of Rab5- and EEA1-positive early endosomes, Rab7-positive late endosomes, and Lamp-1-positive, bacteria-enclosing vesicles in the WT, gp91phox-deficient, and Ctsl-deficient MØs. These vesicles were absent in noninfected MØs (Fig. 3). Cysteine cathepsin activity was essential for phagocytosisdependent cytokine response of infected MØs Knowing that degradation of bacteria inside the phagolysosome is essential for the induction of maximal cytokine levels (17, 19), we wondered whether active endolysosomal cysteine cathepsins are necessary for a potent inflammatory response. To address this question, we performed whole-transcriptome profiling by high-density microarray analysis. This approach revealed ⬃2500 genes to be altered on infection of MØs with S. aureus. However, a rather small number of the 163 genes were at least 71% or less transcribed in the S. aureus-infected, inhibitor-pretreated MØs, as compared to the controls without cathepsin inhibition (Supplemental Table S1). These genes were grouped according to their gene ontology (GO) annotation. Of note, the 2 most significantly affected GO categories by the inhibitor pretreatment were immune response and inflammatory response (Fig. 4A). Genes listed in these 2 categories are shown in Table 1. Among them are those coding for classic proinflammatory cytokines, such as IL6 and IL12␤, as well as the antimicrobial enzyme iNOS. However, the expression of other proinflammatory genes, such as the primary response genes TNF and Cxcl2, was not altered by inhibition of cysteine cathepsin activity (see Discussion). The microarray expression data were confirmed by qRT-PCR for IL6, IL12␤, TNF-␣, and iNOS (Fig. 4B–E). Of note, although endolysosomal cysteine proteases are highly expressed in the MØs, their expression was not induced by infection (Fig. 4F), which was again validated by qRT-PCR (Supplemental Fig. S2A–F). For further experiments, IL6 was used as a readout

Figure 2. Ctsl contributed to the nonoxidative killing of S. aureus (S.a.) in MØs. Intracellular survival of S. aureus (MOI 10) in MØs was monitored over 3 h. A) Ctsl-deficient MØs (n⫽4) compared to WT MØs (n⫽6). B) Ctss-deficient MØs (n⫽4) compared to WT MØs (n⫽6). C) Ctss/Ctsl double-deficient MØs (n⫽4) compared to WT MØs (n⫽3). D) Ctsl-deficient MØs pretreated with cysteine cathepsin inhibitors or solvent (n⫽4). Statistical analysis: Mann-Whitney test. *P ⱕ 0.05. CATHEPSIN-MEDIATED ANTIBACTERIAL DEFENSE

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Figure 3. Formation of bacteria-enclosed vesicles after S. aureus (S.a.) infection was comparable between genotypes. WT, gp91phox⫺/⫺, Ctsl⫺/⫺, and Ctsk⫺/⫺ MØs were infected with S. aureus (MOI 10) for 1 h. Nucleic acids derived from MØs and bacteria are stained blue. Left panels show staining patterns of noninfected WT MØs. A) Early endosomes were stained for the presence of Rab5 and EEA1 (top and middle panels, respectively), and late endosomes were stained for Rab7 (bottom panels). B) MØs were stained for the lysosomal marker LAMP-1 (red), and bacteria were stained with an S. aureus antibody (green). Bottom panels show higher magnification of images in top panels. Unless otherwise indicated, scale bars ⫽ 10 ␮m. Representative images are shown.

for proinflammatory cytokine induction. IL6 protein levels were significantly reduced in cysteine cathepsin inhibitor-pretreated MØs after S. aureus exposure, as compared to the controls (Fig. 5A). Blocking phagocytosis by the mycotoxin CytD resulted in a significant drop in levels of secreted IL6 (Fig. 5B), confirming the previously reported dependence of cytokine formation on the uptake of bacteria (17, 20). Notably, when phagocytosis was blocked, which was confirmed by FACS analysis (Supplemental Fig. S3A), the negative effect of cysteine cathepsin inhibition by IL6 production was abrogated (Fig. 5B). This indicates a phagocytosis-dependent contribution of cysteine cathepsins to the IL6 response in acidic phagolysosomes of the infected MØs. In line with this effect, IL6 production after cell surface stimulation of the MØs by lipopolysac168

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charide (LPS) via TLR4 was not affected by cysteine cathepsin inhibition (Fig. 5C). To determine the time frame during which cysteine cathepsins are needed for an intact IL6 response, we inhibited the cathepsins in S. aureus-infected MØs at several time points before and after infection (Fig. 5D). Pretreatment of MØs with cysteine cathepsin inhibitors, referred to as ⫺1 h in Fig. 5D, led to a significant reduction in IL6 levels in the supernatants of infected MØs 9 h p.i., as is shown in Fig. 5A. Adding inhibitors 1 h p.i. still resulted in a significantly reduced secretion of IL6, whereas at 2 h, only a trend toward reduced IL6 levels was measured as compared to that in the controls. Cysteine cathepsin inhibition at later time points did not affect IL6 levels in the MØ supernatants. This result reveals a cysteine cathepsin contribution to IL6 induction at early time points of infection.


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Figure 4. Transcriptome profiling of S. aureus-infected (S.a.), inhibitor-pretreated MØs. A) GO annotation of microarray data. Only categories that have P ⱕ 0.05 and ⬎5 annotated genes in our list are included. Gene list was derived using the following thresholds: BH q ⱕ 0.05 (paired Bayes t test) and ratio ⬍ 0.71 of infected inhibitor-pretreated MØs/infected DMSO-pretreated MØs. Affymetrix Mouse Gene ST array 1.0 was used as the background universe. P values reflect results obtained by GO Fisher’s exact test, as implemented in Genedata Analyst software (n⫽3). B–E) MØs pretreated with cysteine cathepsin inhibitors or solvent were infected with S. aureus (S.a.; MOI 10). Gene expression was analyzed by qRT-PCR. IL6 (n⫽5; B), IL12␤ (n⫽5; C), and D) TNF-␣ (n⫽3; D) expression was measured 3 h p.i. E) iNOS expression was measured 12 h p.i. (n⫽3) Statistical analysis: 1-sample t test. *P ⱕ 0.05, **P ⱕ 0.01. F) Median expression of cysteine cathepsins and Lgmn in S. aureus-infected and noninfected MØs measured by microarray analysis. ␤-actin is shown as the reference gene (n⫽3).

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TABLE 1. GO-annotated genes displaying reduced transcription in inhibitor-pretreated, S. aureus-infected MØs Gene symbol

Tnfsf10 Nos2 Ccl4 Gm12185: Tgtp1: Tgtp2 Cxcl10 Il12b Mx2 Il6 Cxcl9 Oas1b Ifih1 Tnfsf15 Ccl22 Mx1 Cd40 Ccl7 Cx3cl1 Il20rb Il23r Cxcl11 Tlr11 Tnf Cxcl2

Gene description

Inhibitor/DMSO

FDR

Tumor necrosis factor (ligand) superfamily, member 10 Nitric oxide synthase 2, inducible Chemokine (C-C motif) ligand 4 Predicted gene 12185: T-cell specific GTPase 1: T-cell specific GTPase 2 Chemokine (C-X-C motif) ligand 10 Interleukin 12b Myxovirus (influenza virus) resistance 2 Interleukin 6 Chemokine (C-X-C motif) ligand 9 2=–5= Oligoadenylate synthetase 1B Interferon induced with helicase C domain 1 Tumor necrosis factor (ligand) superfamily, member 15 Chemokine (C-C motif) ligand 22 Myxovirus (influenza virus) resistance 1 CD40 antigen Chemokine (C-C motif) ligand 7 Chemokine (C-X3-C motif) ligand 1 Interleukin 20 receptor beta Interleukin 23 receptor Chemokine (C-X-C motif) ligand 11 Toll-like receptor 11 Tumor necrosis factor Chemokine (C-X3-C motif) ligand 2

0.49 0.55 0.64 0.58

7.80E-06 1.51E-05 4.09E-05 4.37E-05

0.68 0.64 0.59 0.68 0.49 0.69 0.71 0.65 0.56 0.68 0.71 0.67 0.71 0.69 0.7 0.7 0.66 1.0 1.14

6.40E-05 9.19E-05 1.27E-04 2.60E-04 2.92E-04 3.16E-04 3.16E-04 4.20E-04 4.22E-04 5.58E-04 8.54E-04 0.0026 0.0028 0.0083 0.0092 0.0104 0.0143 0.9547 0.0037

MØs were pretreated with either cysteine cathepsin inhibitors or DMSO as the control before infection with S. aureus (MOI 5). Extracellular bacteria were killed by addition of gentamicin sulfate (final concentration, 100 ng/␮l) 45 min later. After an additional 3 h, cells were lysed, and RNA was isolated and transcribed to cDNA. Transcriptome profiling was performed by microarray analysis. To identify genes that were transcribed the least in infected inhibitor-pretreated MØs, we filtered according to the criteria: BH q value ⱕ 0.05 (paired Bayes t test) and ratio ⬍ 0.71 of infected inhibitor-pretreated MØs/infected DMSO-pretreated MØs. In total, 163 genes satisfied these criteria (n ⫽ 3; Supplemental Table S1). Genes depicted in the table fall into one of the two most significant GO-annotated categories: immune response and inflammatory response (Fig. 3A). Tnf and Cxcl2 are shown as examples of nonaltered gene expression after inhibition of cysteine cathepsins in infected MØs.

Active cysteine cathepsins participated in the myeloid differentiation primary response gene 88 (MyD88)dependent IL6 induction of S. aureus-infected MØs TLRs that transmit their signals via the adaptor protein MyD88 are essential for activation of MØs on infection (32). The data shown in Fig. 5E confirm that, in our experimental system, MyD88-deficient MØs produced very little IL6 after infection, whereas the WT MØs strongly induced IL6 on exposure to S. aureus, in a concentration-dependent manner. This result, together with the finding that the contribution of cysteine cathepsins to IL6 induction was phagocytosis dependent, led us to investigate the function of endolysosomal TLRs in MØs during S. aureus infection under conditions of cysteine cathepsin inhibition. Thus, MØs were pretreated with cysteine cathepsin inhibitors and stimulated with the synthetic TLR9 agonist CpG (Fig. 5F). There was no difference in IL6 levels in the supernatants of the inhibitor-pretreated MØs compared to those of the solvent-treated controls, indicating that in our system, the inhibition of cysteine cathepsins did not affect TLR9-mediated IL6 induction per se. Next, uncoordinated 93 homolog B1 (UNC93B1)-deficient MØs, which lack trafficking of TLR3, TLR7, and TLR9 to the endolysosomal compartment (33), were treated with cysteine cathepsin inhibitors before infection (Fig. 5G). Although the UNC93B1-deficient MØs 170

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secreted less than half of the IL6 produced by the WT MØs on S. aureus infection, IL6 levels in supernatants of the UNC93B1-deficient MØs were significantly reduced by inhibition of cysteine cathepsin activity (Fig. 5G). This result shows that MyD88-dependent receptors other than endolysosomal TLR3, TLR7, and TLR9 acted dependently on cysteine cathepsin activity. Ctsk was necessary for maximal IL6 induction in infected MØs To identify particular cysteine cathepsins essential for the induction of normal IL6 levels, we compared the MØs deficient in Ctsb, Ctsh, Ctsk, Ctsl, Ctss, or Ctsz in their IL6 response after infection with S. aureus to the response in the WT MØs. The MØs deficient in Ctsb, Ctsh, Ctsl, or Ctss showed no significant difference in IL6 levels compared to the WT MØs (Supplemental Fig. S1D–G). As viable intracellular bacteria also strongly induce the production of cytokines (34 –36), it is not surprising that the Ctsl-deficient MØs secreted normal amounts of IL6 (Supplemental Fig. S1F). MØs deficient in the NADPH-oxidase subunit gp91phox, which are severely impaired in their ability to kill intracellular S. aureus (Fig. 1F), secreted even more IL6 on infection than did the gp91phox-WT MØs (Supplemental Fig. S3C). In line with this result, the inhibition of cysteine cathepsins in the gp91phox-deficient MØs


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Figure 5. Cysteine cathepsin activity was necessary for the induction of normal IL6 levels. IL6 was measured in the supernatants of S. aureus-infected (S.a.), CpG-stimulated, or LPS-stimulated MØs. A, B) Inhibitor- or solvent-pretreated MØs were infected with S. aureus for 9 h. B) Phagocytosis was blocked by CytD (10 ␮M; n⫽6, normalized to control MOI 10 sample). Statistical analysis: 1-sample and paired-sample t test; n.s., not significant. *P ⱕ 0.05, **P ⱕ 0.01. C) Inhibitor- or solvent-pretreated MØs were stimulated with LPS (1 ␮g/ml) for 6 h (n⫽2). D) Cysteine cathepsin inhibitors E64d (10 ␮M) plus JPM-OEt (10 ␮M) or solvent was added to S. aureus-infected MØs (MOI 10) at different time points p.i. IL6 levels were measured after 9 h (n⫽3, normalized to the ⫺1 h control sample). Statistical analysis: 1-sample and paired-sample t test. *P ⱕ 0.05. E) WT and MyD88-deficient MØs were infected with S. aureus for 9 h (n⫽5). F) Inhibitor- and solvent-pretreated MØs were stimulated with CpG for 9 h (n⫽3). G) WT and UNC93B1-deficient MØs, pretreated with inhibitors or solvent, were infected with S. aureus (MOI 10) for 9 h (n⫽8, normalized to WT control MOI 10 sample). Statistical analysis: 2-sample t test. **P ⱕ 0.01, ***P ⱕ 0.001.

had only a weak effect on IL6 induction when the cells were infected with live bacteria, which were hardly killed in these cells. However, cysteine cathepsins were essential for maximal IL6 induction when heat-inactivated bacteria were given to the gp91phox-deficient MØs, which had to be degraded inside the endolysosomal compartment to induce maximal cytokine levels (Supplemental Fig. S3D). Consistently, in the WT MØs, the dependency of IL6 levels on cysteine cathepsin activity was stronger when heat-inactivated bacteria were added to MØs, in comparison to live S. aureus (Supplemental Fig. S3E). Strikingly, the Ctsk-deficient MØs secreted significantly less IL6 than the WT MØs after infection with S. aureus (Fig. 6A). This result was not due to a reduced uptake of bacteria, as the Ctsk-deficient MØs phagocytosed S. aureus equally to the WT MØs (Supplemental Fig. S1B). However, reduction in IL6 levels in supernatants of the Ctsk-deficient MØs was phagocytosis-dependent and in line with what had already been shown for cysteine cathepsin inhibitors (Fig. 6B). Ctsk deficiency did not alter TLR9-dependent signaling in response to CpG oligonucleotides (Fig. 6B). On the transcriptional level, S. aureus infection induced less IL6 in the Ctsk⫺/⫺ MØs than in the WT MØs (Fig. 6C) as well as less IL12␤ CATHEPSIN-MEDIATED ANTIBACTERIAL DEFENSE

and iNOS (Supplemental Fig. S1H, I). Again this reduction in IL6 mRNA levels was dependent on phagocytosis, as blocking the uptake of bacteria by CytD abrogated the difference between the Ctsk-deficient and WT MØs (Fig. 6C). The abundance of S. aureus-containing vesicles in the Ctsk-deficient MØs was comparable to that in the WT MØs (Fig. 3A, B). However, pretreatment of the Ctsk-deficient MØs with cysteine cathepsin inhibitors resulted in further reduction of IL6 levels (Fig. 6D), revealing that Ctsk is not the only E64d/JPM-OEt-sensitive protease involved in IL6 induction on infection of MØs with S. aureus. Accordingly, the supernatants of infected Ctsz-deficient MØs showed a moderate, phagocytosis-dependent reduction in IL6 levels than did the WT MØs, indicating that Ctsz may also be involved in the induction of IL6 (Fig. 6E). As more than 1 cysteine cathepsin seems to influence the induction of IL6, it is not surprising that E64d/JPM-OEt pretreatment of Ctsz-deficient MØs, as well as the MØs deficient in the Ctsb, Ctsh, Ctsk, Ctsl, or Ctss before S. aureus infection further reduced IL6 levels in the supernatants of these cells (Fig. 6F). In this context, endolysosomal cysteine cathepsins may function in a redundant fashion, whereas Ctsk and Ctsz 171

C

25

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n.s.

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control Inhib.

IL L6 levels [% of controll]

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100 75 50 25 0

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--

**

*

*

75 50 25 0

MOI 5 MOI 10 CytD +MOI 10

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S.a. MOI 10

150

100

50

0

WT Ctsb-/-Ctsk-/- Ctsl-/- Ctss-/-Ctsz-/- Lgmn-/-

CpG [62.5 nM]

Figure 6. Ctsk-deficient MØs produced less IL6 on infection with S. aureus (S.a.) than did WT MØs. A, B) IL6 was measured in the supernatants of S. aureus-infected (MOI 5 and 10) or CpG-stimulated (62.5 nM) WT and Ctsk-deficient MØs after 9 h. B) Phagocytosis was blocked by CytD (10 ␮M; n⫽3, normalized to WT MOI 10 sample). Statistical analysis: 1-sample and paired sample t test; n.s., not significant. *P ⱕ 0.05, **P ⱕ 0.01. C) IL6 expression was assessed by qRT-PCR in WT and Ctsk-deficient MØs 4 h after infection with S. aureus. Phagocytosis was blocked by CytD (10 ␮M; n⫽3, normalized to WT MOI 10 sample). Statistical analysis: 1-sample t test. *P ⱕ 0.05. D) After 9 h, IL6 was measured in the supernatants of S. aureus-infected (MOI 5 and 10) Ctsk-deficient MØs pretreated with inhibitors or solvent (n⫽3, normalized to Ctsk-KO control MOI 10 sample). Statistical analysis: paired sample t test. **P ⱕ 0.01. E) IL6 was measured after 9 h in the supernatants of S. aureus-infected (MOI 5 and 10) WT and Ctsz-deficient MØs. Phagocytosis was blocked by CytD (10 ␮M; n⫽3, normalized to WT MOI 10 sample). F) IL6 was measured in the supernatants of MØs from different genotypes pretreated with inhibitors or solvent 6 h p.i. with S. aureus (MOI 10; n⫽3–7). Statistical analysis: 1-sample t test. *P ⱕ 0.05, **P ⱕ 0.01. G) MØs of different genotypes were stimulated with CpG. IL6 was measured after 9 h (n⫽2–3).

appear to be the main players in the cysteine cathepsin– dependent IL6 response. In summary, these data indicate that cysteine cathepsin activity, and most notably, that of Ctsk, is necessary for maximal induction of IL6. Involvement of cysteine cathepsins is phagocytosis dependent and occurs within early time points of infection of MØs with S. aureus. The IL6 response is nearly completely dependent on MyD88, which is a crucial adaptor for TLR signal transduction. TLR9 is a prominent phagolysosomal TLR; however, its ability to signal is not altered by the inhibition of cysteine cathepsin activity or cathepsin knockout in primary MØs (Fig. 6G). Instead, cysteine cathepsins may be critical for efficient phagolysosomal degradation of bacteria, which is a prerequisite for the release of bacterial pathogen–associated molecular pattern as ligands for cophagocytosed plasma membrane–derived TLRs, such as TLR2 (37–39).

DISCUSSION It has been proposed that proteases are an important component of the nonoxidative killing of bacteria172

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infected phagocytes (6, 7, 40). Although endolysosomal cysteine proteases (i.e., the cysteine cathepsins), are generally considered to be part of the nonoxidative bactericidal machinery of phagocytes (1, 41), virtually no data on the actual functions of cysteine cathepsins in bacteria-infected MØs have been reported. To better understand how an S. aureus infection is controlled by the host, we investigated the contribution of cysteine cathepsin functions to 3 major components of the MØ response to S. aureus: phagocytosis, bactericidal activity, and cytokine induction. Second, by infecting primary bone marrow– derived MØs of mice deficient in 1 of 7 major MØ-expressed endolysosomal cysteine proteases, we functionally linked the MØ response to distinct cathepsins, with Ctsl being important for the efficient killing of S. aureus and Ctsk for maximal IL6 induction. Cysteine cathepsin activities were involved in the elimination of S. aureus in terms of phagocytosis and bacterial killing. We identified the endoprotease Ctsl as the cysteine cathepsin contributing to the nonoxidative killing in MØs. Of interest, the oxidative burst is a prerequisite for antimicrobial activity, as intracellular


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killing of S. aureus was not observed in gp91phox⫺/⫺ MØs (Fig. 1F). The proteolytic activity of a cysteine protease depends on the reduced SH-moiety of the cysteine at the catalytic site of the enzyme (42, 43). Indeed, reactive oxygen species (ROS) can reduce the proteolytic activity of cysteine cathepsins in MØs by 50% by diminishing the reductive capacity of the phagosome (44). However, given that cathepsins are highly expressed in MØs, the remaining activity may well be sufficient for phagolysosomal substrate cleavage under oxidative burst conditions. Furthermore, assembly of NADPH-oxidase rapidly starts after internalization of particles at the membrane of the nascent phagosome (45), and the termination of ROS production occurs as soon as 25 to 30 min into phagocytic uptake (46). As the phagosome is a highly dynamic organelle, it matures continuously by fusion with transGolgi network– derived vesicles, thereby steadily replenishing the phagolysosomal proteolytic machinery (1, 47). Thus, it is conceivable that Ctsl further degrades oxidatively damaged S. aureus in the acidic environment of the phagolysosome. This possibility is in line with the well-known increased proteolytic susceptibility of moderately oxidized proteins and the significant role of Ctsl in the degradation of oxidatively modified substrates in the lysosomal compartment (48). Alternatively, Ctsl may indirectly contribute to bacterial killing by generation of bactericidal peptides or proteins or by the activation of other antimicrobial enzymes inside the phagolysosome, as it was shown for Ctsc in S. aureusinfected neutrophils (41). However, direct evidence supporting this hypothesis is not available. By Western blot analysis, we excluded that the impaired bacterial killing in Ctsl-deficient MØs resulted from nonprocessed Ctsc, which requires cleavage by endoproteases, such as Ctsl (49), for activation (Supplemental Fig. S2G). In addition, the level of Ctsc was not affected by infection of Ctsl-deficient MØs with S. aureus (Supplemental Fig. S2H). An interference of protease inhibition with the oxidative system itself can be excluded, as ROS production was not affected by pretreatment of MØs with cysteine cathepsin inhibitors (Fig. 1E), and the Ctsl⫺/⫺ MØs showed normal iNOS levels compared with the WT MØs (Supplemental Fig. S3B). Taken together, cathepsin L may well act in concert with other degrading enzymes, such as the glycoside hydrolase lysozyme (3), to eliminate bacteria downstream of the oxidative burst. Cysteine cathepsins are not only important for the efficient clearance of S. aureus, as expression profiling revealed that the proteolytic activity of the cysteine cathepsin protease family is critical for an intact induction of several cytokines. Focusing on the proinflammatory cytokine IL6, we found that involvement of cysteine cathepsin activity in the cytokine response of infected MØs was dependent on phagocytosis and was essential during the initial phase of infection. Analysis of various cathepsin knockout MØs revealed a prominent role for cathepsin K in the IL6 response to S. aureus. It is known that the magnitude of cytokine levels CATHEPSIN-MEDIATED ANTIBACTERIAL DEFENSE

correlates with the number of internalized bacteria (17). However, although we showed that active cysteine cathepsins were required for efficient phagocytosis of S. aureus up to 30 min p.i., this finding is unlikely to explain the differences in gene expression between the inhibitor-pretreated MØs and the controls at 4 h p.i. TNF-␣ expression, which was shown to be negatively affected by reduced phagocytosis of S. aureus (17), was not altered by the inhibition of cysteine cathepsins (Table 1). Conversely, phagocytosis of S. aureus was normal in Ctsk-deficient MØs (Supplemental Fig. S1B), which produced less IL6 on infection. It appears that cysteine cathepsin activity is involved in the induction of secondary-response genes, such as IL6, IL12␤, and iNOS, but is dispensable for the induction of primary response genes, such as TNF-␣ and Cxcl2 (50 –52). There are two principal options for how cysteine cathepsins may influence the inflammatory response of infected MØs. First, phagosomal acidification has been reported to be essential for maximal enzymatic activity of acidic hydrolases, thereby contributing to the liberation of cryptic bacterial ligands, which was shown to influence the IL6 secretion of peritoneal MØs stimulated with heat-inactivated S. aureus bacteria (17). Second, it has been shown that endolysosomal TLR9 and TLR7 must be proteolytically activated by cysteine proteases before signaling to their agonists in dendritic cells and in MØs (14, 15, 53). Systematic analysis of cathepsin-deficient MØs revealed Ctsk to contribute to the IL6 response to S. aureus (Fig. 6A). However, IL6 production in response to the selective TLR9 agonist CpG was not altered in MØs deficient in Ctsb, Ctsk, Ctsl, Ctss, Ctsz, or Lgmn (Fig. 6G), in line with findings showing TLR9 response to CpG in MØs to be independent of Lgmn (53), as well as of Ctsb, Ctsk, Ctsl, and Ctss (54). Chemical inhibition of cysteine cathepsin activity significantly reduced IL6 response to S. aureus in a phagocytosis-dependent manner (Fig. 5B). However, MØs pretreated with cysteine cathepsin inhibitors showed no reduction in IL6 levels on stimulation with CpG (Fig. 5F). These data are seemingly in conflict with published results (15). However, the differences between the studies might arise from the fact that we used 5-fold lower inhibitor concentrations for a shorter time, which were still sufficient to irreversibly inhibit cysteine cathepsin activity (Fig. 1A), but may not suffice to abrogate TLR9 processing inside the phagolysosome. Recently, it was shown that in resting MØs, TLR9 translocates from the endoplasmic reticulum to the endolysosomal compartment, which was previously thought to presume an activation of cells (33, 55, 56). This finding suggests that processed TLR9 is already present in the phagolysosomes, explaining a normal IL6 response to CpG in MØs only briefly pretreated with cysteine cathepsin inhibitors. Thus, our data are in line with the initial hypothesis that degradation of bacteria in the acidic environment of the phagolysosome leads to the liberation of otherwise cryptic ligands, thereby eliciting a MyD88-dependent inflammatory response (17, 19). In this setting, Ctsk is essential; 173

however, it is not the only E64d/JPM-OEt-sensitive protease that contributes to IL6 induction of S. aureusinfected MØs (Fig. 6D). The cell-surface receptor TLR2, which is known to be important for recognition of S. aureus, is co-internalized during phagocytosis (38, 57). In this setting internalization of both the receptor and S. aureus is important for a full-blown TLR2-mediated cytokine response (19, 38). It is conceivable that cysteine cathepsin– dependent degradation of S. aureus enhances TLR2-dependent cytokine induction, thereby contributing to maximal IL6 induction. In summary, our data assign distinct roles to individual cysteine cathepsins in various processes of the MØ response to S. aureus. Ctsl contributed to the nonoxidative killing of S. aureus in MØs. Ctsk was critical for IL6 induction, probably by degrading S. aureus inside the phagolysosome of MØs. Thereby, Ctsk and other E64d/ JPM-OEt-sensitive proteases would contribute to the liberation of bacterial ligands that are subsequently recognized by MyD88-dependent receptors. Accordingly, the cysteine cathepsin family is at least bifunctional in the MØ response to S. aureus. Individual members promote bacterial elimination, whereas others induce the inflammatory cytokine response. In future, clinical investigations should address the association of cathepsin expression and susceptibility to S. aureus infections. The authors thank H. A. Chapman (University of California, San Francisco, CA, USA) and J. Joyce (Memorial Sloan-Kettering Cancer Center, New York, NY, USA) for kindly providing cathepsin S-deficient mice, M. A. Freudenberg (Max-Planck Institute for Immunobiology and Epigenetics, Freiburg, Germany) for kindly providing TLR9- and Unc93B1-deficient mice, J. Potempa (Jagiellonian University, Krakow, Poland) for the S. aureus Newman strain, M. Bogyo (Stanford University, Stanford, CA, USA) for the JPMOEt protease inhibitor, and Susanne Dollwet-Mack, Lars Ellenrieder, and Ulrike Reif (Albert Ludwigs University, Freiburg, Germany) and Bernhard Kremer (University Medical Center, Freiburg, Germany) for outstanding technical assistance. This study was supported by the Excellence Initiative of the German Federal and State Governments (EXC 294), Inflammation at Interfaces, CAU Kiel, the Deutsche Forschungsgemeinschaft SFB 850 Project B7, SFB 877, and SPP 1580, and the Centre of Chronic Immunodeficiency (CCI) Freiburg grant TP8.

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