Abstract
Pulmonary damage by Pseudomonas aeruginosa during cystic fibrosis lung infection and ventilator-associated pneumonia is mediated both by pathogen virulence factors and host inflammation. Impaired immune function due to tissue damage and inflammation, coupled with pathogen multidrug resistance, complicates management of these deep-seated infections. Therefore, preservation of lung function and effective immune clearance may be enhanced by selectively controlling inflammation. Pathological inflammation during P. aeruginosa pneumonia is driven by interleukin-1β (IL-1β). This proinflammatory cytokine is canonically regulated by caspase-family inflammasome proteases, but we report that plasticity in IL-1β proteolytic activation allows for its direct maturation by the pseudomonal protease LasB. LasB promotes IL-1β activation, neutrophilic inflammation, and destruction of lung architecture characteristic of severe P. aeruginosa pulmonary infection. Discovery of this IL-1β regulatory mechanism provides a distinct target for anti-inflammatory therapeutics, such that matrix metalloprotease inhibitors blocking LasB limit inflammation and pathology during P. aeruginosa pulmonary infections.
Highlights
IL-1β drives pathology during pulmonary infection by Pseudomonas aeruginosa.
The Pseudomonas protease LasB cleaves and activates IL-1β independent of canonical and noncanonical inflammasomes
Metalloprotease inhibitors active against LasB limit inflammation and bacterial growth
Research in Context Inflammation is highly damaging during lung infections by the opportunistic pathogen Pseudomonas aeruginosa. Sun et al. demonstrate that the Pseudomonas LasB protease directly activates IL-1β in an inflammasome-independent manner. Inhibition of IL-1β conversion by LasB protects against neutrophilic inflammation and destruction of the lung. Adjunctive therapeutics that limit pathological inflammation induced by infection would be beneficial for the treatment of pulmonary infections when used with conventional antibiotics.
Introduction
Pseudomonas aeruginosa is a prominent cause of severe opportunistic pulmonary infections associated with mechanical ventilation and the genetic disease cystic fibrosis (CF). P. aeruginosa infection is often refractory to antibiotic therapy due to multidrug resistance, making it a World Health Organization and U.S. Centers for Disease Control priority pathogen for therapeutic development. P. aeruginosa infection destroys lung architecture and function due to inflammatory- and neutrophil-mediated degradation of mucin layers and structural proteins of the pulmonary connective tissue 1,2. Neutrophil cytokines such as IL-1β 3,4 and IL-8 5, the latter itself regulated by IL-1β 6, initiate and maintain this inflammatory cycle. Anti-inflammatory agents can mitigate tissue destruction to preserve pulmonary function during P. aeruginosa pneumonia 7 and CF 8,9.
Newly synthesized IL-1β (pro-IL-1β) is inactive and requires proteolytic processing into a mature active form. Canonically, this is carried out by the inflammasome, a macromolecular complex of intracellular pattern recognition receptors and the proteases caspase-1 or caspase-11 10. During infection, inflammasomes are formed upon detection of pathogen-associated molecular patterns (PAMPs), including many present in P. aeruginosa such as flagellin (FliC), the type III secretion basal body rod (PscI), the type IV pilin (PilA), RhsT, exolysin (ExlA), exotoxin A (ExoA), cyclic 3′-5′ diguanylate (c-di-GMP), and lipopolysaccharide (LPS), which are varyingly detected by NLRC4, NLRP3, or caspase-11 11-19. Some pathogens limit inflammation by targeting the inflammasome 20, and P. aeruginosa dampens inflammasome activation via the effector ExoU 18. Despite the multitude of inflammasome-activating signals that P. aeruginosa express, caspases, NLRP3, and NLRC4 are not essential for pro-IL-1β maturation in macrophages, epithelial cells, or neutrophils infected with P. aeruginosa 21,22. Correspondingly, P. aeruginosa-infected caspase-1-/- and caspase-1/11-/- mice succumb to a destructive neutrophilic pulmonary inflammation against which IL-1 receptor (IL-1R1-/-) mice are protected 23. These observations highlight the contribution of IL-1β to P. aeruginosa infection but suggest there are mechanisms for its maturation other than the inflammasome.
The pathological cascade of protease dysregulation and activation seen during severe P. aeruginosa lung infections provide a possibility for IL-1β maturation by alternative mechanisms. Caspase-8 24-26, and the neutrophil granular proteases elastase (NE) and proteinase 3 (PR3) 3,4,27, cleave pro-IL-1β, but this does not always result in active cytokine 28. Bronchial secretions, however, also possess abundant protease activity from microbial sources 2. Here we find that IL-1β is not exclusively matured by host proteases, and that P. aeruginosa protease LasB also drives this inflammatory pathway. Targeting this bacterial protease may, therefore, provide supportive therapy to limit inflammatory pathology in pulmonary infection.
Results
IL-1β drives neutrophilic inflammation during P. aeruginosa lung infection
Inflammation drives poor clinical outcomes during P. aeruginosa lung infection 29. C57Bl/6 mice infected intratracheally with P. aeruginosa had markedly disrupted airway architecture within 24 h, concurrent with neutrophil infiltration into the lung tissue and bronchoalveolar lavage fluid (BAL) (Figure 1A). We examined the contribution of pro-inflammatory cytokines to this process using the FDA-approved IL-1 receptor (IL-1R1) antagonist anakinra, which directly inhibits both IL-1β and IL-1α, but not other critical proinflammatory cytokines such as KC/CXCL1, IL-6, or TNFα (Figure 1B). As observed during human infections, P. aeruginosa persisted in the BAL (Figure 1C) and lung tissue (Figure 1D) despite significant neutrophil infiltration that was partially IL-1-dependent (Figure 1E).
IL-1β is typically released by secretion or cell lysis and requires additional maturation, activities which are all mediated by the inflammasome proteases caspases -1 or -11 10. CFU and release of IL-1α was unaltered in P. aeruginosa-infected caspase-1/11-/- C57Bl/6 mice, but surprisingly, IL-1β release was also only modestly attenuated (Figure 1F). This pool of extracellular IL-1β has the potential to mediate proinflammatory signaling as an IL-1R1 agonist when the inhibitory pro-domain has been removed. Neutrophil granular proteases may provide such activation 3,4,15,21, however, since neutrophil recruitment is itself IL-1β-dependent (Figure 1A, 1E), and these neutrophils themselves may later inactivate IL-1β 28, we reasoned that additional proteases initiate the process.
P. aeruginosa induces IL-1β maturation independent of the inflammasome
To more specifically measure only IL-1β that is active, we made use of transgenic reporter cells expressing luciferase under the control of the IL-1R (Figure 2A) similar to previously 30. Consistent with our in vivo observations, caspase-1/11-/- bone-marrow-derived macrophages (BMM) still released cytokines that activated IL-1R1 reporter cells upon infection with P. aeruginosa PAO1 (Figure 2B). This activity was conserved across numerous P. aeruginosa isolates. In contrast, an ionophore that activates the NLRP3 inflammasome, nigericin, was completely dependent on caspases for the activation of IL-1 signaling. Furthermore, P. aeruginosa infection of human cell lines relevant to lung infection (macrophages, THP-1; neutrophils, HL60; type II alveolar epithelial cells, A549) still stimulated IL-1 signaling in the presence of the caspase-1/11-specific inhibitor YVAD-cmk (Figure 2C). Monoclonal antibodies specific to IL-1R1 or IL-1β, but not IL-1α, inhibited IL-1 signal from caspase-1/11-/- BMM (Figure 2D). The absolute quantity of each cytokine measured by enzyme-linked immunosorbent assay (pro- and mature-forms) remained unchanged (Figure 2D). Together, these results indicate that P. aeruginosa stimulates IL-1 signaling through a pool of extracellular IL-1β that is active and matured independently of caspase-1/11.
IL-1β is activated by the P. aeruginosa LasB protease
Proteases contributing to IL-1β activation were evaluated using small molecule inhibitors specific to each protease class. Inhibition of metalloproteases, and not cysteine proteases (e.g. caspases-1, 11, and 8) or serine proteases (e.g. NE and PR3), abrogated IL-1β signaling in P. aeruginosa-infected caspase-1/11-/- BMM (Figure 3A). P. aeruginosa encodes several secreted metalloproteases, and by examining mutants of each (ΔlasA, ΔlasB, ΔaprA), we found LasB to be the most active protease overall as measured by hydrolysis of casein during agar plate growth (Figure 3B), and was the major contributor to caspase-1/11-independent IL-1β signaling (Figure 3C). Complementation with the LasB coding sequence under its native promoter restored the ability of ΔlasB P. aeruginosa to induce IL-1β signaling in infected caspase-1/11-/- BMM (Figure 3D). Furthermore, activation was independent of il1b expression (Figure 2E). These data show that LasB induces IL-1 signaling independently of caspase-1/11.
LasB-activated IL-1β is active
Incubation with recombinant LasB was sufficient to convert recombinant human pro-IL-1β into an active form (Figure 4A). Further examination of pro-IL-1β cleavage by LasB, again using recombinant forms of each protein, showed several intermediate cleavage products which accumulate as a stable product that is degraded no further (Figure 4B), similar to what occurs upon IL-1β maturation by caspase-1 31. Analysis of these fragments by Edman sequencing identified cleavage sites that were all in the N-terminus of pro-IL-1β. Examination of N-terminal truncated IL-1β by in vitro transcription/translation showed a defined region flanking the caspase-1 cleavage site (N-term fragment 117) is sufficient to generate active cytokine (Figure 4C). We further examined proteolysis in this ∼ 20 amino acid region with a series of internally quenched fluorescent peptides and found that LasB preferentially cleaved within the sequence HDAPVRSLN of pro-IL-1β (Figure 4D). Mass spectroscopy confirmed that LasB cleaved between Ser-121 and Leu-122 (Figure 4D, Figure S1), at a site conserved between mice and humans that matches the smallest IL-1β form we observed during SDS-PAGE (Figure 4B). This site also matches the substrate specificity profile for LasB (Figure 4E), which shows a distinct preference for cleaving peptide bonds when Ser or Thr are in the P1 position (amino-terminal side of bond) and hydrophobic amino acids such as Phe, Leu, Nle, Tyr, Trp and Ile in the P1′ position (Supplementary Spreadsheets 1-3), generated using a mass spectrometry-based substrate profiling assay previously validated with other microbial proteases 32,33. During infection, the signature of IL-1β-targeted proteolysis (Figure 4F) is consistent with a significant role for LasB-mediated maturation (hydrolysis of HDAPVRSLN) compared to caspase-1 (hydrolysis of EAYVHDAPV) 30. These data support the model that the pro-domain of IL-1β is promiscuous to protease activation and that the location of specific cleavages can dictate subsequent signaling activity.
Metalloprotease inhibitors of LasB prevent IL-1β -mediated pathological inflammation
Since IL-1β inhibition protects against lung damage (Figure 1A, 1B), and because LasB drives IL-1β maturation (Figure 3C, 4D), we examined whether protease inhibitors active against LasB limit lung injury. Two investigational hydroxamate-based anti-neoplastic metalloprotease inhibitors, marimastat and ilomastat, inhibited LasB cleavage of the IL-1β-derived substrate (Figure 5A) and P. aeruginosa activation of IL-1β (Figure 5B) at sub-antimicrobial concentrations (Figure S2A). During murine pulmonary infection, marimastat and ilomastat each showed therapeutic effects to reduce IL-1β (Figure 5C), neutrophil recruitment (Figure 5D), pulmonary pathology (Figure 5E), and invasion (Figure S2B). Together this data suggests that inhibiting metalloproteases, including LasB, can reduce inflammation during infections by P. aeruginosa.
Discussion
Opportunistic P. aeruginosa lung infections can destroy tissue structure and impair organ function. Our findings reveal a mechanism by which a bacterial protease, LasB, contributes to pathological inflammation by directly activating IL-1β. LasB is one of the most abundant virulence factors in the lung microenvironment during P. aeruginosa infection and can cleave numerous host factors 34, even exerting broadly anti-inflammatory influences through destructive proteolysis of PAMPs such as flagellin 35, and various cytokines including IFN, IL-6, IL-8, MCP-1, TNF, trappin-2 and RANTES 36-39. Consequently, LasB-deficient bacteria may preferentially induce a KC, IL-6, and IL-8 dominant inflammatory response 36, whereas we find wild-type P. aeruginosa induce a strong IL-1β response.
LasB activates IL-1β through direct proteolytic removal of its inhibitory amino-terminal pro-domain, bypassing the necessity for host caspases. The LasB and caspase-1 mechanisms for generating mature IL-1β are distinguishable by substrate specificity (a hydrophobic P1’ vs aspartic acid P1 site), enzyme class (metalloprotease vs cysteine protease), and cellular source (microbial vs host). LasB activation of pro-IL-1β in both the intra- and extracellular milieu is entirely feasible, given the abundance of intracellular proteins released by pyroptosis and necrosis during infections 10,40 and the abundance of LasB 41. We recently hypothesized that IL-1β evolved as a sensor of diverse proteases 30, a model further supported by the present discovery of a P. aeruginosa protease with this activity.
In lung infection, LasB activation of IL-1β augments neutrophil recruitment and promotes destruction of the pulmonary tissue. IL-1β inhibition protects against this pathology, however, clinical interventions to date have utilized expensive biologics (e.g. IL-1R1 antagonists) associated with increased risk for severe infections 30,42. The proteolytic activation of IL-1β may be a more tractable pharmacological target, made possible by disambiguation of the molecular networks involved and, perhaps amenable to the repurposing existing proteases inhibitors. Alpha-1-antitrypsin suppresses NE-mediated degradation of the CF lung 43,44, potentially also limiting pro-IL-1β maturation by NE 27. This strategy may also act against pro-IL-1β maturation by LasB, which is also inhibited by alpha-1-antitrypsin 45. Metalloprotease inhibitors such as marimastat and ilomastat may also be beneficial in treating CF 46 not only for inhibiting matrix metalloproteases, but also by cross-inhibiting LasB (Figure 5).
MATERIALS AND METHODS
Bacterial strains and plasmids
All bacterial strains, plasmids, and primers used in this study are listed in Table 1. lasB and the upstream 260 bp regulatory region in PAO1 were cloned into pUC18T-mini-Tn7T-hph 47 using Polymerase Incomplete Primer Extension (PIPE) cloning 48 with primers lasB-F, lasB-R, Tn7-F, and Tn7-R. Transformants into Top10 cells were selected on LB agar plates containing 100 µg/mL Hygromycin B (Life Technologies). Stable complementation into PAO1 ΔlasB was performed as previously described 47, and transformants selected with 400 µg/mL Hygromycin B. pET-LasB with a C-terminal His tag was constructed by sequential PIPE cloning with the primers LasB-A, LasB-B, LasB-C, and LasB-D, and proteins were expressed and purified by conventional methods as previously described 30. pET-pro-IL-1β and the purification of pro-IL-1β have been previously described 30. Constructs for the expression of IL-1β mutants were generated by PIPE cloning from pET-pro-IL-1β 30 with the corresponding primers sets in Table 1, and proteins were expressed and purified in the same manner as for pro-IL-1β previously 30. Bacteria were routinely propagated in Luria broth (LB) medium at 37 °C. For infections, bacterial cultures were grown to late exponential phase (OD600 1.2) then washed and diluted in PBS.
Animal Experiments
The UCSD or Emory University Institutional Animal Care and Use Committees approved all animal use. Eight-to-ten week old male or female C57Bl/6 and isogenic caspase-1/-11-/- mice were anesthetized with ketamine/xylazine intraperitoneally, then 107 CFU PAO1 inoculated intratracheally in 30 µl of 1x PBS, 25 µg/kg Ilomastat, and 25 µg/kg Marimastat. Mice were euthanized by CO2 asphyxiation, and bronchiolar lavage fluid or lung homogenate were dilution plated onto LB agar plates for CFU enumeration, or quantification of cytokines or proteolysis. Bronchiolar lavage fluid cells were counted on a hemocytometer with cytologic examination on cytospin preparations fixed and stained using Hema 3 (Fisher HealthCare™). Histologic sections were prepared from formalin-fixed and paraffin-embedded lungs, stained with hematoxylin and eosin (H&E). Cytospin and histology slides were imaged on a Hamamatsu Nanozoomer 2.0Ht Slide Scanner.
In vitro infection models
Macrophages were generated from femur exudates of wild-type C57Bl/6 (Jackson Laboratories) or caspase-1/11-/- (kindly provided by R. Flavell) mice using M-CSF containing L929 cell supernatants as previously 30. THP-1, HL60, and A549 cells were propagated by standard protocols detailed previously 49. One hour before infection, the media was replaced with RPMI lacking phenol red, fetal bovine serum, and antibiotics. Inhibitor treatments were added 1 h before infection and include: 20 µg/mL Anakinra (Amgen), 100 ng/mL rIL-1β (R&D Systems), 5 µM caspase inhibitors zVAD-fmk, YVAD-fmk, DEVD-fmk, and IETD-fmk (R&D Systems), 10 µg/mL complete protease inhibitor cocktail (Roche), 1x protease inhibitors AEBSF, Antipain, Aprotinin, Bestatin, EDTA, E-64, Phosphoramidon, Pepstatin, and PMSF (G-Biosciences). Except when noted, cells were routinely infected by co-incubation with P. aeruginosa at a multiplicity of infection of 10, spun into contact for 3 min at 300 g, and cells or supernatants were harvested for analysis after 2 h.
Cytokine measurements
Relative IL-1 signaling by cells was measured in 50 µl of supernatant from infected or treated cells, then incubated with 1 µM okadaic acid 30 min before transfer onto transgenic IL-1R reporter cells (Invivogen). After 18 h, reporter cell supernatants were analyzed for secreted alkaline phosphatase activity using HEK-Blue Detection reagent (Invivogen). Cytokines were quantified by enzyme-linked immunosorbent assay following the manufacturer’s protocol (R&D Systems). Expression was examined in cells lysed with RIPA (Millipore). RNA was isolated (Qiagen), cDNA synthesized with SuperScript III and Oligo(dT)20 primers (Invitrogen), and qPCR performed with KAPA SYBR Fast (Kapa Biosystems) with primers for il1b and relative expression normalized to gapdh and compared by ΔΔCt as previously 50. In vitro transcription/translation was performed with the corresponding primers in Table 1 using pET-pro-IL-1β as a template and following the manufacturer’s recommendations in 10 µl reaction volumes (TNT Coupled Reticulocyte Lysate; Promega). Loading for IL-1R reporter assays was normalized by total IL-1β product measured by enzyme-linked immunosorbent assay (R&D Systems).
Substrate specificity profiling
10 nM LasB was incubated in triplicate with a mixture of 228 synthetic tetradecapeptides (0.5 µM each) in PBS, 2mM DTT as described previously 51. After 15, 60, 240 and 1200 min, aliquots were removed, quenched with 6.4 M GuHCl, immediately frozen at -80°C. Controls were performed with LasB treated with GuHCl prior to peptide exposure. Samples were acidified to pH<3.0 with 1% formic acid, desalted with C18 LTS tips (Rainin), and injected into a Q-Exactive Mass Spectrometer (Thermo) equipped with an Ultimate 3000 HPLC. Peptides separated by reverse phase chromatography on a C18 column (1.7 µm bead size, 75 µm x 20 cm, 65°C) at a flow rate of 400 nl/min using a linear gradient from 5% to 30% B, with solvent A: 0.1% formic acid in water and solvent B: 0.1% formic acid in acetonitrile. Survey scans were recorded over a 150–2000 m/z range (70000 resolutions at 200 m/z, AGC target 1×106, 75 ms maximum). MS/MS was performed in data-dependent acquisition mode with HCD fragmentation (30 normalized collision energy) on the 10 most intense precursor ions (17500 resolutions at 200 m/z, AGC target 5×104, 120 ms maximum, dynamic exclusion 15 s).
Peak integration and data analysis were performed using Peaks software (Bioinformatics Solutions Inc.). MS2 data were searched against the tetradecapeptide library sequences and a decoy search was conducted with sequences in reverse order with no protease digestion specified. Data were filtered to 1% peptide and protein level false discovery rates with the target-decoy strategy. Peptides were quantified with label free quantification and data normalized by LOWESS and filtered by 0.3 peptide quality. Missing and zero values are imputed with random normally distributed numbers in the range of the average of smallest 5% of the data±SD. Enzymatic progress curves of each unique peptide were obtained by performing nonlinear least-squares regression on their peak areas in the MS precursor scans using the first-order enzymatic kinetics model: Y = (plateau-Y0)×(1-exp(–t×kcat/KM×[E0]))+Y0, where E0 is the total enzyme concentration. Nonlinear regression was performed on cleavage products only if the following criteria were met: Peptides were detected in at least 2 of the 3 replicates and the peak intensity of peptides increased by >50,000 and >5-fold over the course of the assay. Proteolytic efficiency was solved from the progress curves by estimating total enzyme concentration and is reported as kcat/KM and clustered into 8 groups by Jenks optimization method. IceLogo software was used for visualization of amino-acid frequency using cleavage sequences in the top 3 clusters (118 most efficiently cleaved peptides). Mass spectrometry deposited:ftp://massive.ucsd.edu/MSV000081623.
Protease Measurements
Internally-quenched peptides 7-Methoxycoumarin- (Mca) labeled on the amino terminus and 2, 4-dinitrophenyl (Dnp) on the carboxy terminus were synthesized with the sequences of IFFDTWDNE, TWDNEAYVH, EAYVHDAPV, and HDAPVRSLN, corresponding to amino acids 103-111, 107-115, 111-119, and 115-123 of the reference human pro-IL-1β sequence (UniProt: P01584; CPC Scientific). In triplicate, 10 µM peptides were incubated in PBS, 1 mM CaCl2, 0.01% Tween-20, with 5 nM human caspase-1 (Enzo) or LasB (Elastin Products Co.). The reaction was continuously monitored using an EnSpire plate reader (PerkinElmer) with 323nm fluorophore excitation and 398nm emission and the maximum kinetic velocity calculated as previously 30. The cleavage site was determined by incubating 10 nM of LasB with 10 µM of HDAPVRSLN. At 20, 40 and 60 min intervals each reaction was quenched with 6.4 M GuHCl and the cleavage products desalted and analyzed by mass spectrometry as described above, except using a 20-min linear gradient from 5% to 50% B and only selecting top 5 peptides for MS/MS.
Statistical analysis
Statistical significance was calculated by unpaired Student t test (*, P < 0.05; **, P < 0.005) using GraphPad Prism unless otherwise indicated. Data are representative of at least three independent experiments. For iceLogo plots only amino acids with significantly (P < 0.05) increased or decreased frequency are shown.
Author contributions
J.S., A.J.O., V.N., and C.N.L. designed experiments and interpreted the data. J.S., D.L., J.K., J.O., Z.J., E.A.S., A.J.O., and C.N.L conducted the studies. J.S., V.N., and C.N.L. wrote the manuscript with the assistance of all of the authors.
Acknowledgments
We thank Christopher Lietz (UCSD) for assistance with mass spectrometry and data analysis, the UCSD Histopathology Core facility, the UCSD Neuroscience Microscopy Shared Facility (P30 NS047101), Jason Munguia (UCSD) for technical assistance, Colin Manoil (UW) for Pseudomonas transposon mutants (P30 DK089507) and Joanna Goldberg (Emory) for helpful advice and discussions. J.S. received support from NIH/NIGMS T32 GM007752, E.A.S. from NIH/NCI T32 CA121938, J.M.K. from a UC President’s Postdoctoral Fellowship, Z.J. from the UC San Diego Chancellor’s Research Excellence Scholarship, A.J.O. from NIH/NIBIB AI1333393, V.N. from NIH/NICHD grant U54 HD090259 and NIH/NHLBI R01 HL125352, and C.L. the A.P. Giannini Foundation and NIH/NIAID K22 AI130223. C.N.L. has a research agreement with Antabio examining inhibitors of LasB. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Footnotes
All authors declare no conflicts of interest