ORIGINAL RESEARCH Matrix Metalloproteinases Vary with Airway Microbiota Composition and Lung Function in Non–Cystic Fibrosis Bronchiectasis Steven L. Taylor1, Geraint B. Rogers2, Alice C.-H. Chen1, Lucy D. Burr1,3, Michael A. McGuckin1, and David J. Serisier1,3 1
Immunity, Infection, and Inflammation Program, Mater Research Institute, University of Queensland and Translational Research Institute, Woolloongabba, Australia; 2SAHMRI Infection and Immunity Theme, School of Medicine, Flinders University, Bedford Park, Australia; and 3Department of Respiratory Medicine, Mater Adult Hospital, South Brisbane, Australia
Abstract Rationale: Despite growing evidence for the roles of airway remodeling and bacterial infection in the progression of non–cystic fibrosis bronchiectasis, relationships between collagen-degrading proteases and chronic airway infection are poorly understood. Objectives: The aim of this study was to determine which matrix metalloproteinases (MMPs) are elevated in bronchiectasis, whether these MMP levels vary based on patients’ dominant infective microbe, and how these levels correlate with clinical measures of disease severity. Methods: We determined concentrations of nine MMPs and four tissue inhibitors of metalloproteinases (TIMPs) in induced sputum from 86 patients with bronchiectasis and 8 healthy control subjects by Luminex protein assay. Concentrations were then assessed in relation to lung function, inflammatory markers, and airway microbiota composition, determined by 16S rRNA gene amplicon sequencing. Airway microbiota composition was classified as Pseudomonas aeruginosa–dominated, Haemophilus influenzae–dominated, or dominated by another species. MMP-8 and MMP-9 activity levels were also measured in a subset of patients.
Measurements and Main Results: MMP-1, -3, -7, -8, and -9 and TIMP-2 and -4 levels, as well as MMP-8/TIMP-1 and MMP-9/TIMP1 ratios, were significantly higher in patients with bronchiectasis than in healthy control subjects (all: P , 0.001, except MMP-7: P , 0.05). Patients with bronchiectasis with H. influenzae–dominated airway infections demonstrated higher MMP-2 levels (P , 0.01) and MMP-8 activity (P , 0.05) than those with P. aeruginosa–dominated airway infections. Among patients with bronchiectasis, there were significant inverse correlations between FEV1 as a percentage of predicted value, MMP-8 and MMP-1 levels, and MMP-8/TIMP-1 and MMP-9/TIMP-1 ratios (P , 0.01). Conclusions: Increased MMP levels (particularly MMP-8 and MMP-1) and MMP/TIMP ratios in patients with bronchiectasis compared with healthy control subjects correlated with lower lung function and higher levels of inflammatory markers. Further, MMP profiles differed in patients with bronchiectasis according to the dominant pathogen determined by gene sequencing, raising the possibility of differential airway remodeling according to airway microbiology. Keywords: airway remodeling; Haemophilus influenzae; Pseudomonas aeruginosa; tissue inhibitor of metalloproteinase
(Received in original form November 11, 2014; accepted in final form January 31, 2015 ) Supported by the Mater Adult Respiratory Research Trust Fund. Author Contributions: S.L.T., G.B.R., A.C.-H.C., L.D.B., M.A.M., and D.J.S.: contributed to the conception, execution, analysis, and reporting of the study and approved the final version of the manuscript. D.J.S.: acts as the guarantor for the paper and takes responsibility for the integrity of the paper as a whole. Correspondence and requests for reprints should be addressed to David J. Serisier, M.B. B.S., D.M., Department of Respiratory Medicine, Level 9, Mater Adult Hospital, Raymond Terrace, South Brisbane, QLD 4101, Australia. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Ann Am Thorac Soc Vol 12, No 5, pp 701–707, May 2015 Copyright © 2015 by the American Thoracic Society DOI: 10.1513/AnnalsATS.201411-513OC Internet address: www.atsjournals.org
Non–cystic fibrosis (non-CF) bronchiectasis is characterized by a cycle of recurrent bacterial infection, inflammation, and structural remodeling of the bronchi (1). Structural remodeling ultimately leads to irreversible dilation of
the bronchi and impaired mucociliary clearance (1). The composition of the airway microbiota has been shown to differ substantially between patients with bronchiectasis and to correlate with disease
Taylor, Rogers, Chen, et al.: Proteases and Microbiota in Bronchiectasis
severity (2–5). The two bacterial species most commonly isolated by culture are Pseudomonas aeruginosa and Haemophilus influenzae (2, 3), with the majority of patients with bronchiectasis having positive cultures for one of these two species (3). 701
ORIGINAL RESEARCH Commonly, either P. aeruginosa or H. influenzae is numerically dominant in the airways, typically representing more than 90% of bacteria present (6, 7). Although these two common opportunistic pathogens share certain similarities (e.g., they are both Gram-negative, rod-shaped, facultative anaerobic Gammaproteobacteria), they are associated with very different clinical courses when dominant in bronchiectasis lung infections (3, 6). P. aeruginosa–dominated infections are associated with an accelerated decline in lung function, more frequent pulmonary exacerbations, greater sputum production, and a more frequent requirement for antibiotic therapy (6, 8–10). The basis for the difference in disease severity associated with P. aeruginosa and H. influenzae infections is not fully understood. However, our recent data raise the possibility of differential airway inflammatory pathway activation according to baseline infecting organism (11). The extracellular matrix (ECM) is a primary structural component of the lung with a highly complex and regulated system of matrix deposition and degradation (12). ECM components are degraded by a family of proteolytic enzymes known as matrix metalloproteinases (MMPs) (13, 14). In bronchiectasis, MMPs and tissue inhibitors of metalloproteinases (TIMPs) are produced by a wide range of cell types, including epithelial cells, fibroblasts, alveolar macrophages, and neutrophils, the latter of which is the most prevalent inflammatory cell type in bronchiectasis (14–16). MMPs also have a diverse role in immunity that is not directly related to ECM modulation, including activation of cytokines, facilitation of cell migration, and activation of defensins (17). The ratios of individual MMPs to TIMPs have been reported to be informative in a number of other respiratory infections and diseases (18–25); however, they have not been explored in bronchiectasis. Concentrations of some individual MMPs and TIMPs have been investigated in bronchiectasis airway secretions previously, which highlighted potential mechanisms in airway disease progression (13, 15, 26–31). Though informative, these studies were limited by the size of the patient cohorts and the limited number of MMPs and TIMPs assessed. Furthermore, no prior studies have evaluated proteases in relation to airway microbiota. Therefore, detailed analyses of the ECM degradation pathway 702
in bronchiectasis, and its relationship to both airway microbiology and disease course, would prove informative. We hypothesized that MMP levels would be elevated in patients with bronchiectasis compared with healthy control subjects. Further, given our recent data suggesting that microbiota-based subgrouping of patients is more clinically informative than standard culture, we assessed MMP levels and activity according to these groupings. Within patients with bronchiectasis, we hypothesized that MMP concentrations would be higher in patients in whom P. aeruginosa was the dominant infective bacterial species. To test these hypotheses, concentrations of a panel of MMPs and TIMPs were correlated with bacterial species predominance, respiratory clinical outcomes, and inflammatory markers. Specifically, levels of nine MMPs and four TIMPs were determined in induced sputum samples from healthy control subjects and from adult patients with non-CF bronchiectasis stratified according to dominant infective species.
Methods Study Population
Induced sputum was collected from a cohort of 86 adult patients with non-CF
bronchiectasis at the beginning of a randomized controlled clinical trial (32) and from eight healthy volunteers. Full details of inclusion and exclusion criteria for the study, the current subgroup, and the control group are provided in the online supplement. Patients with bronchiectasis were not receiving systemic corticosteroids and were macrolide-naive. No patients in the study were receiving nebulized antibiotics. Healthy control subjects had no evidence of respiratory disease, no history of use of any bronchoactive medications, and normal spirometry (FEV1, FVC, and FEV1:FVC ratio all lying within the normal range). The study was approved by the Mater Human Research Ethics Committee, Brisbane, Australia, and all subjects provided their written, informed consent to participate. The details of the healthy control subjects and patients with bronchiectasis are summarized in Table 1. Induced Sputum Collection and Processing
Induced sputum was collected as described previously (33). Briefly, sputum induction was performed with 4.5% hypertonic saline, diluted 1:4 with phosphate-buffered saline, then centrifuged at 10,000 3 g for 10 minutes. The supernatant was aliquoted,
Table 1. Clinical characteristics of healthy control subjects and patients with non–cystic fibrosis bronchiectasis
Patients (n) Female (n) Age, yr FEV1% Prebronchodilator Postbronchodilator Duration of bronchiectasis, yr Exacerbations in prior 12 mo (n) LCQ score SGRQ total score C-reactive protein, mg/L Sputum % neutrophils* Bacterial dominance (n) Haemophilus influenzae Pseudomonas aeruginosa Other†
Control
Non-CF Bronchiectasis
8 5 40.5 (11.0)
86 53 63.0 (8.8)
95.5 (12.9) 98.0 (12.0)
66.5 70.7 42.3 4.7 14.8 39.1 5.7 94.3
(17.0) (18.4) (20.9) (2.6) (3.4) (13.8) (5.5) (6.3)
31 22 33
Definition of abbreviations: CF = cystic fibrosis; FEV1% = forced expiratory volume in 1 second as a percentage of the predicted value; LCQ = Leicester Cough Questionnaire; SGRQ = St. George’s Respiratory Questionnaire. Data are mean 6 standard deviation. *Neutrophils in sputum as a percent of total nonsquamous cells. † Patients with numerically dominant bacterial species other than P. aeruginosa and H. influenzae.
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ORIGINAL RESEARCH with half added to Roche cOmplete Protease Inhibitor Cocktail (Roche Applied Science, Mannheim, Germany), and samples were immediately stored at 2808 C. Multiplex Protein Assay
Nine MMPs and four TIMPs were measured using Magnetic Luminex Performance Assay multiplex kits (R&D Systems, Minneapolis, MN) by Cardinal Bioresearch (New Farm, Australia). Sputum MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP9, MMP-10, MMP-12, and MMP-13 concentrations were measured in one assay, and TIMP-1, TIMP-2, TIMP-3 and TIMP4 concentrations were measured in a separate assay, as per the manufacturer’s instructions and as described in Data Supplement E2. In a number of samples, concentrations of MMP-8 or MMP-9 exceeded the highest standard value. In these cases, concentrations were determined by extrapolation from the standard curve, and separate MMP-8 and
A
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MMP-9 activity assays were performed for further validation. Concentrations of MMP-13 and TIMP-3 fell below the detection threshold in the majority of samples and were therefore excluded from any further analysis. MMP-8 and MMP-9 Activity Assay
MMP-8 and MMP-9 proteolytic activity was measured in induced sputum from all patients with P. aeruginosa– and H. influenzae–dominant infections. MMP-8 activity was measured using a human MMP8 activity assay (QuickZyme Biosciences, Leiden, The Netherlands). MMP-9 activity was assessed using the Fluorokine Human MMP Kit (R&D Systems).
Inflammatory Markers
Serum C-reactive protein (CRP) levels and neutrophils in sputum (as a percentage of total nonsquamous cells [neutrophil %]) were measured using established laboratory techniques as previously described (32). Neutrophil measurements in sputum from 73 of the 86 patients with bronchiectasis were recorded. Inflammatory cytokines IL-8 and IL-1b were measured by ELISA (BD Biosciences, San Jose, CA). Bacterial Dominance
Clinical Measures
Clinical measures analyzed in this study included FEV1 as a percentage of predicted value (FEV1%), Leicester Cough Questionnaire (LCQ) score, St. George’s Respiratory Questionnaire (SGRQ) score,
Control Bronchiectasis
***
*
Identification of numerically dominant bacterial species was performed by bacterial tag-encoded FLX amplicon pyrosequencing as previously described (6, 34). A full description of the methodology is provided in Data Supplement E2. Patients were
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Concentration (pg/mL)
and reported number of exacerbations in the preceding 12 months. Details of clinical data collection are provided in Data Supplement E2.
*** 106
106 105 10
105
***
***
4
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***
103 103 102
B MMP-9/TIMP-1
102 101 100 10–1
10
–2
MMP-1
MMP-2
MMP-3
***
MMP-7
102
MMP-8/TIMP-1
101
MMP-8
MMP-9 MMP-10 MMP-12
102
TIMP-1 TIMP-2
TIMP-4
***
101 100 10–1 10–2
Figure 1. Matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP) concentrations in induced sputum from healthy control subjects (n = 8) and patients with non–cystic fibrosis (non-CF) bronchiectasis (n = 86). (A) In patients with non-CF bronchiectasis, MMP-1, MMP-3, MMP-7, MMP-8, MMP-9, TIMP-2, and TIMP-4 were significantly elevated compared with levels in healthy control subjects. (B) MMP-8/TIMP-1 and MMP-9/TIMP-1 ratios were significantly increased in patients with non-CF bronchiectasis. Box plots represent interquartile range, median, and minimum and maximum values. *P , 0.05, ***P , 0.001 (Mann-Whitney U test).
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ORIGINAL RESEARCH MMP-8/TIMP-1 and MMP-9/TIMP-1 ratios were higher in patients with bronchiectasis than in healthy control subjects (P = 0.0002 and P , 0.0003, respectively) (Figure 1B). MMP and TIMP Concentrations and Activities Differ between Patients with Bronchiectasis Based on Dominant Infective Microbe
Patients with bronchiectasis were stratified on the basis of their numerically dominant
A
Statistics
Results Elevated Concentrations of MMPs, TIMPs, and MMP/TIMP Ratios in Bronchiectasis
Concentrations of MMPs and TIMPs in the induced sputum from patients with bronchiectasis and healthy control subjects are shown in Figure 1A and Table E4. MMP-1, MMP-3, MMP-7, MMP-8, MMP9, TIMP-2, and TIMP-4 were significantly higher in samples from patients with bronchiectasis than in samples from healthy control subjects (P , 0.001 for all except MMP-7, where P , 0.05). MMP-8 and MMP-9 were the most abundant MMPs, and TIMP-1 was the most abundant TIMP. However, TIMP-1 did not differ between healthy control subjects and patients with bronchiectasis (P = 0.939). 704
107
*** P. aeruginosa H. influenzae Other
**
** ***
**
Concentration (pg/mL)
*** 106
105
*** **
104
103
102
101
MMP-1
MMP-2
MMP-8
B 108 Active MMP-8 (pg/mL)
Statistical analyses were performed with the GraphPad Prism program (version 5.1; GraphPad Software, San Diego, CA). All data were tested for normal distribution by Pearson omnibus normality test with and without log transformation. Control versus bronchiectasis data and MMP-8 and MMP-9 activity data were analyzed by two-tailed Mann-Whitney U test. Analysis of variance for MMP and TIMP levels according to bacterial dominance was performed using the Kruskal-Wallis test with Dunn’s post hoc test for pairwise comparisons. Correlations were analyzed by using Spearman’s rank-order correlation. A confidence interval of 95% was chosen for all analyses except correlations, where a confidence interval of 99% was used to define statistical significance as a measure to account for multiple testing.
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infective pathogen (dominated by either P. aeruginosa or H. influenzae or a species other than these two) by gene sequencing and MMP and TIMP concentrations, and MMP-8 and MMP-9 activities were analyzed. MMP-8, MMP-9, and TIMP-2 concentrations were significantly lower in patients with infections dominated by bacterial species other than P. aeruginosa or H. influenzae, with the same trend observed for MMP-1, MMP-3, and MMP-7 concentrations (Figure 2A and Figure E5).
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MMP-9
TIMP-2
108
*
Active MMP-9 (pg/mL)
divided on the basis of dominant bacterial species as having infections dominated by P. aeruginosa (n = 22) or H. influenzae (n = 31) or by a species other than these two (n = 33). Stratifying patients in this manner has proven to be more clinically informative than culture-based methods (6). Further, previous quantification of the relative abundance of bacterial genera in this patient cohort has revealed that the airway microbiota is highly polarized in patients with H. influenzae– and P. aeruginosa– dominant infections, with dominance of either exceeding 80% abundance (5). A summary of the dominant microbes in the patient cohort is provided in Table E3.
107
106
105
104
Figure 2. Matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP) levels and activity in induced sputum from patients with bronchiectasis stratified by dominant microbe. Patients were categorized by airway microbiota as dominated by Pseudomonas aeruginosa (n = 22), Haemophilus influenzae (n = 31), or another bacterial species (n = 33). (A) Select MMP and TIMP concentrations in bronchiectasis sputum grouped as P. aeruginosa, H. influenzae, or other. (B) Patients in which H. influenzae was dominant had increased MMP-8 activity, but not MMP-9 activity, compared with those in whom P. aeruginosa was dominant. Box plots represent interquartile range, median, and minimum and maximum values. Analysis was carried out by using (A) the Kruskal-Wallis test with Dunn’s posttest and (B) the Mann-Whitney U test. *P , 0.05, **P , 0.01, ***P , 0.001.
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ORIGINAL RESEARCH Unexpectedly, no MMPs or TIMPs were significantly higher in patients with P. aeruginosa–dominated infections than in patients with H. influenzae–dominated infections. In fact, MMP-2 levels and MMP-8 activity were lower in patients with P. aeruginosa–dominated infections than in those with H. influenzae–dominated infections (P , 0.01 and P , 0.03, respectively) (Figure 2). MMP Concentrations Significantly Correlate with Lung Function and Inflammatory Markers
Correlations of select MMPs and TIMPs with lung function and inflammatory markers are shown in Table 2 and Figure E6. Significant inverse correlations were seen between FEV1% and levels of MMP-1, MMP-8, and MMP-8/TIMP-1 and MMP9/TIMP-1 ratios. As expected, MMP-1, MMP-8, and MMP-9 (the three MMPs most increased in bronchiectasis), as well as TIMP-2, positively correlated with multiple inflammatory markers (sputum neutrophil %, induced sputum IL-8 and IL-1b levels, and serum CRP level), as did MMP-8/ TIMP-1 and MMP-9/TIMP-1 ratios. MMP-2 did not correlate with any clinical measure or inflammatory marker (data not shown). Subgroup analysis based on dominant microbe was also performed to explore differences in correlations between MMPs and TIMPs with lung function and inflammatory markers. We found that, though not statistically significant, the directions of correlations between MMP-8, MMP-9, and TIMP-2 with FEV1% in patients with H. influenzae–dominated infection were positive rather than inverse
(r = 0.055, r = 0.203, and r = 0.056, respectively) (Table E7).
Discussion We found that five of the nine MMPs and two of the four TIMPs measured were significantly elevated in the induced sputum of patients with bronchiectasis compared with that of healthy control subjects. We confirmed predicted associations between MMP-1 and MMP-8 with lower lung function (as measured by FEV1 %) and with markers of local and systemic inflammation (as measured by percent of neutrophils in sputum, IL-8 and IL-1b levels for local inflammation, and serum CRP levels for systemic inflammation). We further showed that, whereas the most abundant MMPs (MMP-8 and MMP-9) were increased in bronchiectasis, their primary inhibitor (TIMP-1) was not, indicating a protease– antiprotease imbalance. Stratification of patients with bronchiectasis on the basis of the dominant bacterial species revealed that MMP and TIMP levels differed between patients with P. aeruginosa–dominated, H. influenzae– dominated, or other taxa-dominated communities in a manner that was surprising. Specifically, patients with H. influenzae–dominated infection, who tend to manifest less severe disease than patients with P. aeruginosa–dominated infection (5, 6, 32), had significantly increased MMP-2 concentrations and MMP-8 activities. Concentrations of MMP-1, MMP-8, and MMP-9, all of which were
significantly increased in bronchiectasis, have been investigated previously in this context. Hseih and colleagues reported that a polymorphism causing increased MMP-1 secretion by peripheral blood mononuclear cells was related to worse clinical measures of disease (27). Our research also shows that MMP-1 is increased in bronchiectasis and correlates with poorer lung function and increased inflammation; however, MMP-1 was at a relatively low abundance compared with other MMPs and is therefore likely to represent a minor proportion of overall protease activity. Low MMP-1 level relative to MMP-8 and MMP-9 is consistent with previous literature and is likely due to a cellular source, with MMP1 being released primarily by fibroblasts and monocytes and MMP-8 and MMP-9 being primarily neutrophil-derived (30, 35, 36). Our findings with respect to MMP-8 and MMP-9 are consistent with other research showing MMP-8 and MMP-9 to be increased in lung biopsies of patients with bronchiectasis compared with healthy control subjects, as well as localized with neutrophils (31). MMP-8 has also been detected in bronchoalveolar lavage fluid (BALF) and cultured bronchial epithelial cells, monocytes, and macrophages of patients with bronchiectasis (29, 30). We extend this knowledge by showing that these MMPs are also elevated in the induced sputum of patients with bronchiectasis, with MMP-8 correlating with poorer lung function. Contrary to our expectations, patients with P. aeruginosa–dominated airway infection did not demonstrate elevations in
Table 2. Correlations between clinical measures of disease and selected MMPs, TIMPs, and MMP/TIMP ratios
FEV1% Neutrophil %† Serum CRP level IL-8¶ IL-1b¶
MMP-1
MMP-8
MMP-9
TIMP-2
MMP-8/TIMP-1
20.338* 0.418‡ 0.445x 0.698x 0.721x
20.295* 0.501x 0.357‡ 0.745x 0.859x
20.261 0.458x 0.402‡ 0.752x 0.808x
20.265 0.459x 0.321* 0.786x 0.806x
20.338* 0.519x 0.441x 0.684x 0.849x
MMP-9/TIMP-1 20.318* 0.403‡ 0.429x 0.485x 0.667x
Definition of abbreviations: CRP = C-reactive protein; FEV1% = forced expiratory volume in 1 second as a percentage of the predicted value; MMP = matrix metalloproteinase; TIMP = tissue inhibitor of metalloproteinase. Data are Spearman’s rank-order correlation coefficients. *P , 0.01. † Neutrophil measurements in sputum as a percent of total nonsquamous cells were recorded for 73 patients. ‡ P , 0.001. x P , 0.0001. ¶ Induced sputum IL-8 and IL-1b concentrations.
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ORIGINAL RESEARCH either the levels or the activity of any MMP when compared with H. influenzae– dominated infection. Rather, MMP-8 activity and MMP-2 concentration were significantly lower in patients with P. aeruginosa–dominated infection than in those with H. influenzae–dominated infection. These differential MMP levels may be important in understanding the reasons for differences in disease severity and phenotype between subjects with infection by these two organisms and complements our recent finding of differential inflammatory pathway activation according to baseline microbiology (11). Furthermore, we have recently described significant differences in response to macrolide therapy according to baseline sputum microbiology (32, 34), and differential activation of either (or both) MMPs or inflammatory pathways may be relevant to these effects. Of particular interest was the 2.4-fold mean increase in MMP-2 in patients with H. influenzae–dominated infection compared with P. aeruginosa–dominated infection. MMP-2, along with MMP-9, is a gelatinase capable of degrading type IV collagen in basement membranes (14). MMP-2 is secreted by lymphocytes, epithelial cells, and fibroblasts, whereas MMP-9 is expressed primarily in inflammatory cells such as neutrophils (37, 38). Suga and colleagues examined differences in MMP-2 and MMP-9 activity in various interstitial lung diseases and found that MMP-9 was increased in patients with extensive structural remodeling, whereas MMP-2 was increased in patients with less severe and generally reversible architectural remodeling (38). They further showed that BALF from patients with increased MMP9 contained more neutrophils, whereas BALF from patients with increased MMP2 contained more lymphocytes, and they concluded that these matrix-degrading proteins are likely markers of disease prognosis due to predominance of different inflammatory cells. It is interesting to speculate that a similar variation in inflammatory profile exists between patients with bronchiectasis with H. influenzae–dominated versus P. aeruginosa–dominated airway infection. H. influenzae–dominated infection may subtly promote less neutrophil-specific airway inflammation and subsequently promote 706
more MMP-2 secretion from other cell types. Alternatively, as MMP-2 is secreted by epithelial cells (37), decreased MMP-2 levels in P. aeruginosa–dominated airway infection may simply be a reflection of decreased viable airway epithelium related to a more severe disease phenotype. As there is a substantial difference in clinical severity between patients colonized by P. aeruginosa compared with H. influenzae, MMP-2 levels may prove to be a marker for a milder disease phenotype, as Suga and colleagues and others have suggested (14, 38). There was also significantly higher MMP-8 activity in patients with H. influenzae–dominated infection than in those with P. aeruginosa–dominated infection. As MMP-8 inversely correlated with lung function, this appears to contradict accepted literature that patients with P. aeruginosa infection have lower lung function than those with H. influenzae infection. However, much is yet unknown about bronchiectasis and the contribution of airway infections to disease progression and lung remodeling. The magnitude of difference in MMP-8 activity may be negligible in relation to airway remodeling. Further, MMP-8 concentrations in patients with H. influenzae–dominated infection did not demonstrate inverse correlations with FEV1%, in contrast to the levels in patients with infection dominated by P. aeruginosa or another bacterial species. Further research into the relationship between H. influenzae infection and MMP-8 interactions is required to understand these findings. An imbalance between MMP-8 and MMP-9 and their primary inhibitor, TIMP1, was found to correlate with poorer lung function and increased inflammation, which has not previously been identified in bronchiectasis. This finding is consistent with research in other respiratory diseases, including CF, asthma, chronic obstructive pulmonary disease, and chronic bronchitis (19, 22–25). MMP-8, MMP-9, and TIMP-1 are synthesized by neutrophils and stored in secondary granules, as well as produced by other cell types (22, 39). Indeed, we found positive correlations between MMP-8 and MMP-9 (but not TIMP-1) levels with sputum neutrophil %, which supports literature reports that neutrophils are the primary cellular source of MMP-8 and MMP-9 in bronchiectasis (31). Jackson and
colleagues previously showed that human neutrophil elastase (HNE) can degrade TIMP-1 (19) and also activates pro-MMP-9 in CF (40). They concluded that HNE is likely a contributor to MMP/TIMP dysregulation. As part of a linked investigation (data not shown), HNE activity was assessed in this bronchiectasis patient cohort and was found to be significantly higher in patients with P. aeruginosa–dominated infection than in those without P. aeruginosa–dominated infection and also correlated with MMP-9 activity. Hence, if HNE were primarily responsible for driving MMP/TIMP imbalance, significantly higher MMP-9 activity would also be anticipated in patients with P. aeruginosa–dominated infection than in those with H. influenzae–dominated infection, which was not observed. This implies HNE is not the sole contributor to the MMP/TIMP imbalance. Our results represent cross-sectional rather than longitudinal data, which we acknowledge limits our ability to assign causality. However, this study was designed to explore the gap of knowledge surrounding the complex relationships in bronchiectasis between MMPs, clinical phenotypes, and airway microbiota composition for the purpose of designing future testable hypotheses. Furthermore, the sample size and comprehensive analyses undertaken, within a rigorous, prospective, randomized controlled trial exceeds all prior evaluations of MMPs and TIMPs in this disease. In summary, we report that, of nine MMPs, increased MMP-1 and MMP-8 concentrations alone were associated with lower lung function, with concentrations higher in patients with P. aeruginosa– and H. influenzae–dominant airway infections. We also show that MMP-2 was higher in patients with airway infection dominated by H. influenzae than in those whose airway infection was dominated by P. aeruginosa, suggesting differential MMP activation according to airway microbiology. Finally, as found in other respiratory diseases, an imbalance between the amount of MMP-8 and MMP-9 compared with their inhibitor, TIMP-1, is apparent in bronchiectasis and correlates inversely with FEV1%. n Author disclosures are available with the text of this article at www.atsjournals.org.
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ORIGINAL RESEARCH References 1 Cole PJ. Inflammation: a two-edged sword—the model of bronchiectasis. Eur J Respir Dis Suppl 1986;147:6–15. 2 Chalmers JD, Smith MP, McHugh BJ, Doherty C, Govan JR, Hill AT. Short- and long-term antibiotic treatment reduces airway and systemic inflammation in non-cystic fibrosis bronchiectasis. Am J Respir Crit Care Med 2012;186:657–665. 3 King PT, Holdsworth SR, Freezer NJ, Villanueva E, Holmes PW. Microbiologic follow-up study in adult bronchiectasis. Respir Med 2007;101:1633–1638. 4 Tunney MM, Einarsson GG, Wei L, Drain M, Klem ER, Cardwell C, Ennis M, Boucher RC, Wolfgang MC, Elborn JS. Lung microbiota and bacterial abundance in patients with bronchiectasis when clinically stable and during exacerbation. Am J Respir Crit Care Med 2013;187:1118–1126. 5 Rogers GB, van der Gast CJ, Cuthbertson L, Thomson SK, Bruce KD, Martin ML, Serisier DJ. Clinical measures of disease in adult non-CF bronchiectasis correlate with airway microbiota composition. Thorax 2013;68:731–737. 6 Rogers GB, Zain NM, Bruce KD, Burr LD, Chen AC, Rivett DW, McGuckin MA, Serisier DJ. A novel microbiota stratification system predicts future exacerbations in bronchiectasis. Ann Am Thorac Soc 2014;11:496–503. 7 Rogers GB, van der Gast CJ, Serisier DJ. Predominant pathogen competition and core microbiota divergence in chronic airway infection. ISME J 2015;9:217–225. 8 Ho PL, Chan KN, Ip MS, Lam WK, Ho CS, Yuen KY, Tsang KW. The effect of Pseudomonas aeruginosa infection on clinical parameters in steady-state bronchiectasis. Chest 1998;114:1594–1598. 9 Evans SA, Turner SM, Bosch BJ, Hardy CC, Woodhead MA. Lung function in bronchiectasis: the influence of Pseudomonas aeruginosa. Eur Respir J 1996;9:1601–1604. 10 Shoemark A, Ozerovitch L, Wilson R. Aetiology in adult patients with bronchiectasis. Respir Med 2007;101:1163–1170. 11 Chen AC, Martin ML, Lourie R, Rogers GB, Burr LD, Hasnain SZ, Bowler SD, McGuckin MA, Serisier DJ. Adult non-cystic fibrosis bronchiectasis is characterised by airway luminal th17 pathway activation. PloS One 2015;10:e0119325. 12 McKleroy W, Lee TH, Atabai K. Always cleave up your mess: targeting collagen degradation to treat tissue fibrosis. Am J Physiol Lung Cell Mol Physiol 2013;304:L709–L721. 13 Bergin DA, Hurley K, Mehta A, Cox S, Ryan D, O’Neill SJ, Reeves EP, McElvaney NG. Airway inflammatory markers in individuals with cystic fibrosis and non-cystic fibrosis bronchiectasis. J Inflamm Res 2013;6:1–11. 14 Gueders MM, Foidart JM, Noel A, Cataldo DD. Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs in the respiratory tract: potential implications in asthma and other lung diseases. Eur J Pharmacol 2006;533:133–144. 15 Maisi P, Prikk K, Sepper R, Pirila¨ E, Salo T, Hietanen J, Sorsa T. Soluble membrane-type 1 matrix metalloproteinase (MT1-MMP) and gelatinase A (MMP-2) in induced sputum and bronchoalveolar lavage fluid of human bronchial asthma and bronchiectasis. APMIS 2002;110:771–782. 16 Zheng L, Shum H, Tipoe GL, Leung R, Lam WK, Ooi GC, Tsang KW. Macrophages, neutrophils and tumour necrosis factor-alpha expression in bronchiectatic airways in vivo. Respir Med 2001;95:792–798. 17 Elkington PT, Friedland JS. Matrix metalloproteinases in destructive pulmonary pathology. Thorax 2006;61:259–266. 18 Friedland JS, Shaw TC, Price NM, Dayer JM. Differential regulation of MMP-1/9 and TIMP-1 secretion in human monocytic cells in response to Mycobacterium tuberculosis. Matrix Biol 2002;21:103–110. 19 Jackson PL, Xu X, Wilson L, Weathington NM, Clancy JP, Blalock JE, Gaggar A. Human neutrophil elastase-mediated cleavage sites of MMP-9 and TIMP-1: implications to cystic fibrosis proteolytic dysfunction. Mol Med 2010;16:159–166. 20 Lanchou J, Corbel M, Tanguy M, Germain N, Boichot E, Theret N, Clement B, Lagente V, Malledant Y. Imbalance between matrix metalloproteinases (MMP-9 and MMP-2) and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in acute respiratory distress syndrome patients. Crit Care Med 2003;31:536–542. 21 El-Solh AA, Amsterdam D, Alhajhusain A, Akinnusi ME, Saliba RG, Lynch SV, Wiener-Kronish JP. Matrix metalloproteases in bronchoalveolar lavage fluid of patients with type III Pseudomonas aeruginosa pneumonia. J Infect 2009;59:49–55.
22 Cataldo D, Munaut C, Noel ¨ A, Frankenne F, Bartsch P, Foidart JM, Louis R. Matrix metalloproteinases and TIMP-1 production by peripheral blood granulocytes from COPD patients and asthmatics. Allergy 2001;56:145–151. 23 Obase Y, Rytila¨ P, Metso T, Pelkonen AS, Tervahartiala T, Turpeinen M, Makel ¨ a¨ M, Saarialho-Kere U, Selroos O, Sorsa T, et al. Effects of inhaled corticosteroids on metalloproteinase-8 and tissue inhibitor of metalloproteinase-1 in the airways of asthmatic children. Int Arch Allergy Immunol 2010;151:247–254. 24 Vignola AM, Riccobono L, Mirabella A, Profita M, Chanez P, Bellia V, Mautino G, D’Accardi P, Bousquet J, Bonsignore G. Sputum metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio correlates with airflow obstruction in asthma and chronic bronchitis. Am J Respir Crit Care Med 1998;158:1945–1950. 25 Hoshino M, Nakamura Y, Sim J, Shimojo J, Isogai S. Bronchial subepithelial fibrosis and expression of matrix metalloproteinase-9 in asthmatic airway inflammation. J Allergy Clin Immunol 1998;102:783–788. 26 Goeminne PC, Vandooren J, Moelants EA, Decraene A, Rabaey E, Pauwels A, Seys S, Opdenakker G, Proost P, Dupont LJ. The Sputum Colour Chart as a predictor of lung inflammation, proteolysis and damage in non-cystic fibrosis bronchiectasis: a case–control analysis. Respirology 2014;19:203–210. 27 Hsieh MH, Chou PC, Chou CL, Ho SC, Joa WC, Chen LF, Sheng TF, Lin HC, Wang TY, Chang PJ, et al. Matrix metalloproteinase-1 polymorphism (21607G) and disease severity in non-cystic fibrosis bronchiectasis in Taiwan. PLoS One 2013;8:e66265. 28 Karakoc GB, Inal A, Yilmaz M, Altintas DU, Kendirli SG. Exhaled breath condensate MMP-9 levels in children with bronchiectasis. Pediatr Pulmonol 2009;44:1010–1016. 29 Prikk K, Maisi P, Pirila¨ E, Sepper R, Salo T, Wahlgren J, Sorsa T. In vivo collagenase-2 (MMP-8) expression by human bronchial epithelial cells and monocytes/macrophages in bronchiectasis. J Pathol 2001;194:232–238. 30 Sepper R, Konttinen YT, Ding Y, Takagi M, Sorsa T. Human neutrophil collagenase (MMP-8), identified in bronchiectasis BAL fluid, correlates with severity of disease. Chest 1995;107:1641–1647. 31 Zheng L, Lam WK, Tipoe GL, Shum IH, Yan C, Leung R, Sun J, Ooi GC, Tsang KW. Overexpression of matrix metalloproteinase-8 and -9 in bronchiectatic airways in vivo. Eur Respir J 2002;20:170–176. 32 Serisier DJ, Martin ML, McGuckin MA, Lourie R, Chen AC, Brain B, Biga S, Schlebusch S, Dash P, Bowler SD. Effect of long-term, low-dose erythromycin on pulmonary exacerbations among patients with non-cystic fibrosis bronchiectasis: the BLESS randomized controlled trial. JAMA 2013;309:1260–1267. 33 Rogers GB, Skelton S, Serisier DJ, van der Gast CJ, Bruce KD. Determining cystic fibrosis-affected lung microbiology: comparison of spontaneous and serially induced sputum samples by use of terminal restriction fragment length polymorphism profiling. J Clin Microbiol 2010;48:78–86. 34 Rogers GB, Bruce KD, Martin ML, Burr LD, Serisier DJ. The effect of long-term macrolide treatment on respiratory microbiota composition in non-cystic fibrosis bronchiectasis: an analysis from the randomised, double-blind, placebo-controlled BLESS trial. Lancet Respir Med 2014;2:988–996. 35 Elkington P, Shiomi T, Breen R, Nuttall RK, Ugarte-Gil CA, Walker NF, Saraiva L, Pedersen B, Mauri F, Lipman M, et al. MMP-1 drives immunopathology in human tuberculosis and transgenic mice. J Clin Invest 2011;121:1827–1833. 36 Walker NF, Clark SO, Oni T, Andreu N, Tezera L, Singh S, Saraiva L, Pedersen B, Kelly DL, Tree JA, et al. Doxycycline and HIV infection suppress tuberculosis-induced matrix metalloproteinases. Am J Respir Crit Care Med 2012;185:989–997. 37 Murphy G, Docherty AJ. The matrix metalloproteinases and their inhibitors. Am J Respir Cell Mol Biol 1992;7:120–125. 38 Suga M, Iyonaga K, Okamoto T, Gushima Y, Miyakawa H, Akaike T, Ando M. Characteristic elevation of matrix metalloproteinase activity in idiopathic interstitial pneumonias. Am J Respir Crit Care Med 2000;162:1949–1956. 39 Cockeran R, Mitchell TJ, Feldman C, Anderson R. Pneumolysin induces release of matrix metalloproteinase-8 and -9 from human neutrophils. Eur Respir J 2009;34:1167–1170. 40 Gaggar A, Li Y, Weathington N, Winkler M, Kong M, Jackson P, Blalock JE, Clancy JP. Matrix metalloprotease-9 dysregulation in lower airway secretions of cystic fibrosis patients. Am J Physiol Lung Cell Mol Physiol 2007;293:L96–L104.
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Immunity, Infection, and Inflammation Program, Mater Research Institute, University of Queensland and Translational Research Institute, Woolloongabba, Australia; 2SAHMRI Infection and Immunity Theme, School of Medicine, Flinders University, Bedford Park, Australia; and 3Department of Respiratory Medicine, Mater Adult Hospital, South Brisbane, Australia
Abstract Rationale: Despite growing evidence for the roles of airway remodeling and bacterial infection in the progression of non–cystic fibrosis bronchiectasis, relationships between collagen-degrading proteases and chronic airway infection are poorly understood. Objectives: The aim of this study was to determine which matrix metalloproteinases (MMPs) are elevated in bronchiectasis, whether these MMP levels vary based on patients’ dominant infective microbe, and how these levels correlate with clinical measures of disease severity. Methods: We determined concentrations of nine MMPs and four tissue inhibitors of metalloproteinases (TIMPs) in induced sputum from 86 patients with bronchiectasis and 8 healthy control subjects by Luminex protein assay. Concentrations were then assessed in relation to lung function, inflammatory markers, and airway microbiota composition, determined by 16S rRNA gene amplicon sequencing. Airway microbiota composition was classified as Pseudomonas aeruginosa–dominated, Haemophilus influenzae–dominated, or dominated by another species. MMP-8 and MMP-9 activity levels were also measured in a subset of patients.
Measurements and Main Results: MMP-1, -3, -7, -8, and -9 and TIMP-2 and -4 levels, as well as MMP-8/TIMP-1 and MMP-9/TIMP1 ratios, were significantly higher in patients with bronchiectasis than in healthy control subjects (all: P , 0.001, except MMP-7: P , 0.05). Patients with bronchiectasis with H. influenzae–dominated airway infections demonstrated higher MMP-2 levels (P , 0.01) and MMP-8 activity (P , 0.05) than those with P. aeruginosa–dominated airway infections. Among patients with bronchiectasis, there were significant inverse correlations between FEV1 as a percentage of predicted value, MMP-8 and MMP-1 levels, and MMP-8/TIMP-1 and MMP-9/TIMP-1 ratios (P , 0.01). Conclusions: Increased MMP levels (particularly MMP-8 and MMP-1) and MMP/TIMP ratios in patients with bronchiectasis compared with healthy control subjects correlated with lower lung function and higher levels of inflammatory markers. Further, MMP profiles differed in patients with bronchiectasis according to the dominant pathogen determined by gene sequencing, raising the possibility of differential airway remodeling according to airway microbiology. Keywords: airway remodeling; Haemophilus influenzae; Pseudomonas aeruginosa; tissue inhibitor of metalloproteinase
(Received in original form November 11, 2014; accepted in final form January 31, 2015 ) Supported by the Mater Adult Respiratory Research Trust Fund. Author Contributions: S.L.T., G.B.R., A.C.-H.C., L.D.B., M.A.M., and D.J.S.: contributed to the conception, execution, analysis, and reporting of the study and approved the final version of the manuscript. D.J.S.: acts as the guarantor for the paper and takes responsibility for the integrity of the paper as a whole. Correspondence and requests for reprints should be addressed to David J. Serisier, M.B. B.S., D.M., Department of Respiratory Medicine, Level 9, Mater Adult Hospital, Raymond Terrace, South Brisbane, QLD 4101, Australia. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Ann Am Thorac Soc Vol 12, No 5, pp 701–707, May 2015 Copyright © 2015 by the American Thoracic Society DOI: 10.1513/AnnalsATS.201411-513OC Internet address: www.atsjournals.org
Non–cystic fibrosis (non-CF) bronchiectasis is characterized by a cycle of recurrent bacterial infection, inflammation, and structural remodeling of the bronchi (1). Structural remodeling ultimately leads to irreversible dilation of
the bronchi and impaired mucociliary clearance (1). The composition of the airway microbiota has been shown to differ substantially between patients with bronchiectasis and to correlate with disease
Taylor, Rogers, Chen, et al.: Proteases and Microbiota in Bronchiectasis
severity (2–5). The two bacterial species most commonly isolated by culture are Pseudomonas aeruginosa and Haemophilus influenzae (2, 3), with the majority of patients with bronchiectasis having positive cultures for one of these two species (3). 701
ORIGINAL RESEARCH Commonly, either P. aeruginosa or H. influenzae is numerically dominant in the airways, typically representing more than 90% of bacteria present (6, 7). Although these two common opportunistic pathogens share certain similarities (e.g., they are both Gram-negative, rod-shaped, facultative anaerobic Gammaproteobacteria), they are associated with very different clinical courses when dominant in bronchiectasis lung infections (3, 6). P. aeruginosa–dominated infections are associated with an accelerated decline in lung function, more frequent pulmonary exacerbations, greater sputum production, and a more frequent requirement for antibiotic therapy (6, 8–10). The basis for the difference in disease severity associated with P. aeruginosa and H. influenzae infections is not fully understood. However, our recent data raise the possibility of differential airway inflammatory pathway activation according to baseline infecting organism (11). The extracellular matrix (ECM) is a primary structural component of the lung with a highly complex and regulated system of matrix deposition and degradation (12). ECM components are degraded by a family of proteolytic enzymes known as matrix metalloproteinases (MMPs) (13, 14). In bronchiectasis, MMPs and tissue inhibitors of metalloproteinases (TIMPs) are produced by a wide range of cell types, including epithelial cells, fibroblasts, alveolar macrophages, and neutrophils, the latter of which is the most prevalent inflammatory cell type in bronchiectasis (14–16). MMPs also have a diverse role in immunity that is not directly related to ECM modulation, including activation of cytokines, facilitation of cell migration, and activation of defensins (17). The ratios of individual MMPs to TIMPs have been reported to be informative in a number of other respiratory infections and diseases (18–25); however, they have not been explored in bronchiectasis. Concentrations of some individual MMPs and TIMPs have been investigated in bronchiectasis airway secretions previously, which highlighted potential mechanisms in airway disease progression (13, 15, 26–31). Though informative, these studies were limited by the size of the patient cohorts and the limited number of MMPs and TIMPs assessed. Furthermore, no prior studies have evaluated proteases in relation to airway microbiota. Therefore, detailed analyses of the ECM degradation pathway 702
in bronchiectasis, and its relationship to both airway microbiology and disease course, would prove informative. We hypothesized that MMP levels would be elevated in patients with bronchiectasis compared with healthy control subjects. Further, given our recent data suggesting that microbiota-based subgrouping of patients is more clinically informative than standard culture, we assessed MMP levels and activity according to these groupings. Within patients with bronchiectasis, we hypothesized that MMP concentrations would be higher in patients in whom P. aeruginosa was the dominant infective bacterial species. To test these hypotheses, concentrations of a panel of MMPs and TIMPs were correlated with bacterial species predominance, respiratory clinical outcomes, and inflammatory markers. Specifically, levels of nine MMPs and four TIMPs were determined in induced sputum samples from healthy control subjects and from adult patients with non-CF bronchiectasis stratified according to dominant infective species.
Methods Study Population
Induced sputum was collected from a cohort of 86 adult patients with non-CF
bronchiectasis at the beginning of a randomized controlled clinical trial (32) and from eight healthy volunteers. Full details of inclusion and exclusion criteria for the study, the current subgroup, and the control group are provided in the online supplement. Patients with bronchiectasis were not receiving systemic corticosteroids and were macrolide-naive. No patients in the study were receiving nebulized antibiotics. Healthy control subjects had no evidence of respiratory disease, no history of use of any bronchoactive medications, and normal spirometry (FEV1, FVC, and FEV1:FVC ratio all lying within the normal range). The study was approved by the Mater Human Research Ethics Committee, Brisbane, Australia, and all subjects provided their written, informed consent to participate. The details of the healthy control subjects and patients with bronchiectasis are summarized in Table 1. Induced Sputum Collection and Processing
Induced sputum was collected as described previously (33). Briefly, sputum induction was performed with 4.5% hypertonic saline, diluted 1:4 with phosphate-buffered saline, then centrifuged at 10,000 3 g for 10 minutes. The supernatant was aliquoted,
Table 1. Clinical characteristics of healthy control subjects and patients with non–cystic fibrosis bronchiectasis
Patients (n) Female (n) Age, yr FEV1% Prebronchodilator Postbronchodilator Duration of bronchiectasis, yr Exacerbations in prior 12 mo (n) LCQ score SGRQ total score C-reactive protein, mg/L Sputum % neutrophils* Bacterial dominance (n) Haemophilus influenzae Pseudomonas aeruginosa Other†
Control
Non-CF Bronchiectasis
8 5 40.5 (11.0)
86 53 63.0 (8.8)
95.5 (12.9) 98.0 (12.0)
66.5 70.7 42.3 4.7 14.8 39.1 5.7 94.3
(17.0) (18.4) (20.9) (2.6) (3.4) (13.8) (5.5) (6.3)
31 22 33
Definition of abbreviations: CF = cystic fibrosis; FEV1% = forced expiratory volume in 1 second as a percentage of the predicted value; LCQ = Leicester Cough Questionnaire; SGRQ = St. George’s Respiratory Questionnaire. Data are mean 6 standard deviation. *Neutrophils in sputum as a percent of total nonsquamous cells. † Patients with numerically dominant bacterial species other than P. aeruginosa and H. influenzae.
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ORIGINAL RESEARCH with half added to Roche cOmplete Protease Inhibitor Cocktail (Roche Applied Science, Mannheim, Germany), and samples were immediately stored at 2808 C. Multiplex Protein Assay
Nine MMPs and four TIMPs were measured using Magnetic Luminex Performance Assay multiplex kits (R&D Systems, Minneapolis, MN) by Cardinal Bioresearch (New Farm, Australia). Sputum MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP9, MMP-10, MMP-12, and MMP-13 concentrations were measured in one assay, and TIMP-1, TIMP-2, TIMP-3 and TIMP4 concentrations were measured in a separate assay, as per the manufacturer’s instructions and as described in Data Supplement E2. In a number of samples, concentrations of MMP-8 or MMP-9 exceeded the highest standard value. In these cases, concentrations were determined by extrapolation from the standard curve, and separate MMP-8 and
A
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MMP-9 activity assays were performed for further validation. Concentrations of MMP-13 and TIMP-3 fell below the detection threshold in the majority of samples and were therefore excluded from any further analysis. MMP-8 and MMP-9 Activity Assay
MMP-8 and MMP-9 proteolytic activity was measured in induced sputum from all patients with P. aeruginosa– and H. influenzae–dominant infections. MMP-8 activity was measured using a human MMP8 activity assay (QuickZyme Biosciences, Leiden, The Netherlands). MMP-9 activity was assessed using the Fluorokine Human MMP Kit (R&D Systems).
Inflammatory Markers
Serum C-reactive protein (CRP) levels and neutrophils in sputum (as a percentage of total nonsquamous cells [neutrophil %]) were measured using established laboratory techniques as previously described (32). Neutrophil measurements in sputum from 73 of the 86 patients with bronchiectasis were recorded. Inflammatory cytokines IL-8 and IL-1b were measured by ELISA (BD Biosciences, San Jose, CA). Bacterial Dominance
Clinical Measures
Clinical measures analyzed in this study included FEV1 as a percentage of predicted value (FEV1%), Leicester Cough Questionnaire (LCQ) score, St. George’s Respiratory Questionnaire (SGRQ) score,
Control Bronchiectasis
***
*
Identification of numerically dominant bacterial species was performed by bacterial tag-encoded FLX amplicon pyrosequencing as previously described (6, 34). A full description of the methodology is provided in Data Supplement E2. Patients were
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***
107
Concentration (pg/mL)
and reported number of exacerbations in the preceding 12 months. Details of clinical data collection are provided in Data Supplement E2.
*** 106
106 105 10
105
***
***
4
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***
103 103 102
B MMP-9/TIMP-1
102 101 100 10–1
10
–2
MMP-1
MMP-2
MMP-3
***
MMP-7
102
MMP-8/TIMP-1
101
MMP-8
MMP-9 MMP-10 MMP-12
102
TIMP-1 TIMP-2
TIMP-4
***
101 100 10–1 10–2
Figure 1. Matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP) concentrations in induced sputum from healthy control subjects (n = 8) and patients with non–cystic fibrosis (non-CF) bronchiectasis (n = 86). (A) In patients with non-CF bronchiectasis, MMP-1, MMP-3, MMP-7, MMP-8, MMP-9, TIMP-2, and TIMP-4 were significantly elevated compared with levels in healthy control subjects. (B) MMP-8/TIMP-1 and MMP-9/TIMP-1 ratios were significantly increased in patients with non-CF bronchiectasis. Box plots represent interquartile range, median, and minimum and maximum values. *P , 0.05, ***P , 0.001 (Mann-Whitney U test).
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703
ORIGINAL RESEARCH MMP-8/TIMP-1 and MMP-9/TIMP-1 ratios were higher in patients with bronchiectasis than in healthy control subjects (P = 0.0002 and P , 0.0003, respectively) (Figure 1B). MMP and TIMP Concentrations and Activities Differ between Patients with Bronchiectasis Based on Dominant Infective Microbe
Patients with bronchiectasis were stratified on the basis of their numerically dominant
A
Statistics
Results Elevated Concentrations of MMPs, TIMPs, and MMP/TIMP Ratios in Bronchiectasis
Concentrations of MMPs and TIMPs in the induced sputum from patients with bronchiectasis and healthy control subjects are shown in Figure 1A and Table E4. MMP-1, MMP-3, MMP-7, MMP-8, MMP9, TIMP-2, and TIMP-4 were significantly higher in samples from patients with bronchiectasis than in samples from healthy control subjects (P , 0.001 for all except MMP-7, where P , 0.05). MMP-8 and MMP-9 were the most abundant MMPs, and TIMP-1 was the most abundant TIMP. However, TIMP-1 did not differ between healthy control subjects and patients with bronchiectasis (P = 0.939). 704
107
*** P. aeruginosa H. influenzae Other
**
** ***
**
Concentration (pg/mL)
*** 106
105
*** **
104
103
102
101
MMP-1
MMP-2
MMP-8
B 108 Active MMP-8 (pg/mL)
Statistical analyses were performed with the GraphPad Prism program (version 5.1; GraphPad Software, San Diego, CA). All data were tested for normal distribution by Pearson omnibus normality test with and without log transformation. Control versus bronchiectasis data and MMP-8 and MMP-9 activity data were analyzed by two-tailed Mann-Whitney U test. Analysis of variance for MMP and TIMP levels according to bacterial dominance was performed using the Kruskal-Wallis test with Dunn’s post hoc test for pairwise comparisons. Correlations were analyzed by using Spearman’s rank-order correlation. A confidence interval of 95% was chosen for all analyses except correlations, where a confidence interval of 99% was used to define statistical significance as a measure to account for multiple testing.
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infective pathogen (dominated by either P. aeruginosa or H. influenzae or a species other than these two) by gene sequencing and MMP and TIMP concentrations, and MMP-8 and MMP-9 activities were analyzed. MMP-8, MMP-9, and TIMP-2 concentrations were significantly lower in patients with infections dominated by bacterial species other than P. aeruginosa or H. influenzae, with the same trend observed for MMP-1, MMP-3, and MMP-7 concentrations (Figure 2A and Figure E5).
107
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MMP-9
TIMP-2
108
*
Active MMP-9 (pg/mL)
divided on the basis of dominant bacterial species as having infections dominated by P. aeruginosa (n = 22) or H. influenzae (n = 31) or by a species other than these two (n = 33). Stratifying patients in this manner has proven to be more clinically informative than culture-based methods (6). Further, previous quantification of the relative abundance of bacterial genera in this patient cohort has revealed that the airway microbiota is highly polarized in patients with H. influenzae– and P. aeruginosa– dominant infections, with dominance of either exceeding 80% abundance (5). A summary of the dominant microbes in the patient cohort is provided in Table E3.
107
106
105
104
Figure 2. Matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP) levels and activity in induced sputum from patients with bronchiectasis stratified by dominant microbe. Patients were categorized by airway microbiota as dominated by Pseudomonas aeruginosa (n = 22), Haemophilus influenzae (n = 31), or another bacterial species (n = 33). (A) Select MMP and TIMP concentrations in bronchiectasis sputum grouped as P. aeruginosa, H. influenzae, or other. (B) Patients in which H. influenzae was dominant had increased MMP-8 activity, but not MMP-9 activity, compared with those in whom P. aeruginosa was dominant. Box plots represent interquartile range, median, and minimum and maximum values. Analysis was carried out by using (A) the Kruskal-Wallis test with Dunn’s posttest and (B) the Mann-Whitney U test. *P , 0.05, **P , 0.01, ***P , 0.001.
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ORIGINAL RESEARCH Unexpectedly, no MMPs or TIMPs were significantly higher in patients with P. aeruginosa–dominated infections than in patients with H. influenzae–dominated infections. In fact, MMP-2 levels and MMP-8 activity were lower in patients with P. aeruginosa–dominated infections than in those with H. influenzae–dominated infections (P , 0.01 and P , 0.03, respectively) (Figure 2). MMP Concentrations Significantly Correlate with Lung Function and Inflammatory Markers
Correlations of select MMPs and TIMPs with lung function and inflammatory markers are shown in Table 2 and Figure E6. Significant inverse correlations were seen between FEV1% and levels of MMP-1, MMP-8, and MMP-8/TIMP-1 and MMP9/TIMP-1 ratios. As expected, MMP-1, MMP-8, and MMP-9 (the three MMPs most increased in bronchiectasis), as well as TIMP-2, positively correlated with multiple inflammatory markers (sputum neutrophil %, induced sputum IL-8 and IL-1b levels, and serum CRP level), as did MMP-8/ TIMP-1 and MMP-9/TIMP-1 ratios. MMP-2 did not correlate with any clinical measure or inflammatory marker (data not shown). Subgroup analysis based on dominant microbe was also performed to explore differences in correlations between MMPs and TIMPs with lung function and inflammatory markers. We found that, though not statistically significant, the directions of correlations between MMP-8, MMP-9, and TIMP-2 with FEV1% in patients with H. influenzae–dominated infection were positive rather than inverse
(r = 0.055, r = 0.203, and r = 0.056, respectively) (Table E7).
Discussion We found that five of the nine MMPs and two of the four TIMPs measured were significantly elevated in the induced sputum of patients with bronchiectasis compared with that of healthy control subjects. We confirmed predicted associations between MMP-1 and MMP-8 with lower lung function (as measured by FEV1 %) and with markers of local and systemic inflammation (as measured by percent of neutrophils in sputum, IL-8 and IL-1b levels for local inflammation, and serum CRP levels for systemic inflammation). We further showed that, whereas the most abundant MMPs (MMP-8 and MMP-9) were increased in bronchiectasis, their primary inhibitor (TIMP-1) was not, indicating a protease– antiprotease imbalance. Stratification of patients with bronchiectasis on the basis of the dominant bacterial species revealed that MMP and TIMP levels differed between patients with P. aeruginosa–dominated, H. influenzae– dominated, or other taxa-dominated communities in a manner that was surprising. Specifically, patients with H. influenzae–dominated infection, who tend to manifest less severe disease than patients with P. aeruginosa–dominated infection (5, 6, 32), had significantly increased MMP-2 concentrations and MMP-8 activities. Concentrations of MMP-1, MMP-8, and MMP-9, all of which were
significantly increased in bronchiectasis, have been investigated previously in this context. Hseih and colleagues reported that a polymorphism causing increased MMP-1 secretion by peripheral blood mononuclear cells was related to worse clinical measures of disease (27). Our research also shows that MMP-1 is increased in bronchiectasis and correlates with poorer lung function and increased inflammation; however, MMP-1 was at a relatively low abundance compared with other MMPs and is therefore likely to represent a minor proportion of overall protease activity. Low MMP-1 level relative to MMP-8 and MMP-9 is consistent with previous literature and is likely due to a cellular source, with MMP1 being released primarily by fibroblasts and monocytes and MMP-8 and MMP-9 being primarily neutrophil-derived (30, 35, 36). Our findings with respect to MMP-8 and MMP-9 are consistent with other research showing MMP-8 and MMP-9 to be increased in lung biopsies of patients with bronchiectasis compared with healthy control subjects, as well as localized with neutrophils (31). MMP-8 has also been detected in bronchoalveolar lavage fluid (BALF) and cultured bronchial epithelial cells, monocytes, and macrophages of patients with bronchiectasis (29, 30). We extend this knowledge by showing that these MMPs are also elevated in the induced sputum of patients with bronchiectasis, with MMP-8 correlating with poorer lung function. Contrary to our expectations, patients with P. aeruginosa–dominated airway infection did not demonstrate elevations in
Table 2. Correlations between clinical measures of disease and selected MMPs, TIMPs, and MMP/TIMP ratios
FEV1% Neutrophil %† Serum CRP level IL-8¶ IL-1b¶
MMP-1
MMP-8
MMP-9
TIMP-2
MMP-8/TIMP-1
20.338* 0.418‡ 0.445x 0.698x 0.721x
20.295* 0.501x 0.357‡ 0.745x 0.859x
20.261 0.458x 0.402‡ 0.752x 0.808x
20.265 0.459x 0.321* 0.786x 0.806x
20.338* 0.519x 0.441x 0.684x 0.849x
MMP-9/TIMP-1 20.318* 0.403‡ 0.429x 0.485x 0.667x
Definition of abbreviations: CRP = C-reactive protein; FEV1% = forced expiratory volume in 1 second as a percentage of the predicted value; MMP = matrix metalloproteinase; TIMP = tissue inhibitor of metalloproteinase. Data are Spearman’s rank-order correlation coefficients. *P , 0.01. † Neutrophil measurements in sputum as a percent of total nonsquamous cells were recorded for 73 patients. ‡ P , 0.001. x P , 0.0001. ¶ Induced sputum IL-8 and IL-1b concentrations.
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ORIGINAL RESEARCH either the levels or the activity of any MMP when compared with H. influenzae– dominated infection. Rather, MMP-8 activity and MMP-2 concentration were significantly lower in patients with P. aeruginosa–dominated infection than in those with H. influenzae–dominated infection. These differential MMP levels may be important in understanding the reasons for differences in disease severity and phenotype between subjects with infection by these two organisms and complements our recent finding of differential inflammatory pathway activation according to baseline microbiology (11). Furthermore, we have recently described significant differences in response to macrolide therapy according to baseline sputum microbiology (32, 34), and differential activation of either (or both) MMPs or inflammatory pathways may be relevant to these effects. Of particular interest was the 2.4-fold mean increase in MMP-2 in patients with H. influenzae–dominated infection compared with P. aeruginosa–dominated infection. MMP-2, along with MMP-9, is a gelatinase capable of degrading type IV collagen in basement membranes (14). MMP-2 is secreted by lymphocytes, epithelial cells, and fibroblasts, whereas MMP-9 is expressed primarily in inflammatory cells such as neutrophils (37, 38). Suga and colleagues examined differences in MMP-2 and MMP-9 activity in various interstitial lung diseases and found that MMP-9 was increased in patients with extensive structural remodeling, whereas MMP-2 was increased in patients with less severe and generally reversible architectural remodeling (38). They further showed that BALF from patients with increased MMP9 contained more neutrophils, whereas BALF from patients with increased MMP2 contained more lymphocytes, and they concluded that these matrix-degrading proteins are likely markers of disease prognosis due to predominance of different inflammatory cells. It is interesting to speculate that a similar variation in inflammatory profile exists between patients with bronchiectasis with H. influenzae–dominated versus P. aeruginosa–dominated airway infection. H. influenzae–dominated infection may subtly promote less neutrophil-specific airway inflammation and subsequently promote 706
more MMP-2 secretion from other cell types. Alternatively, as MMP-2 is secreted by epithelial cells (37), decreased MMP-2 levels in P. aeruginosa–dominated airway infection may simply be a reflection of decreased viable airway epithelium related to a more severe disease phenotype. As there is a substantial difference in clinical severity between patients colonized by P. aeruginosa compared with H. influenzae, MMP-2 levels may prove to be a marker for a milder disease phenotype, as Suga and colleagues and others have suggested (14, 38). There was also significantly higher MMP-8 activity in patients with H. influenzae–dominated infection than in those with P. aeruginosa–dominated infection. As MMP-8 inversely correlated with lung function, this appears to contradict accepted literature that patients with P. aeruginosa infection have lower lung function than those with H. influenzae infection. However, much is yet unknown about bronchiectasis and the contribution of airway infections to disease progression and lung remodeling. The magnitude of difference in MMP-8 activity may be negligible in relation to airway remodeling. Further, MMP-8 concentrations in patients with H. influenzae–dominated infection did not demonstrate inverse correlations with FEV1%, in contrast to the levels in patients with infection dominated by P. aeruginosa or another bacterial species. Further research into the relationship between H. influenzae infection and MMP-8 interactions is required to understand these findings. An imbalance between MMP-8 and MMP-9 and their primary inhibitor, TIMP1, was found to correlate with poorer lung function and increased inflammation, which has not previously been identified in bronchiectasis. This finding is consistent with research in other respiratory diseases, including CF, asthma, chronic obstructive pulmonary disease, and chronic bronchitis (19, 22–25). MMP-8, MMP-9, and TIMP-1 are synthesized by neutrophils and stored in secondary granules, as well as produced by other cell types (22, 39). Indeed, we found positive correlations between MMP-8 and MMP-9 (but not TIMP-1) levels with sputum neutrophil %, which supports literature reports that neutrophils are the primary cellular source of MMP-8 and MMP-9 in bronchiectasis (31). Jackson and
colleagues previously showed that human neutrophil elastase (HNE) can degrade TIMP-1 (19) and also activates pro-MMP-9 in CF (40). They concluded that HNE is likely a contributor to MMP/TIMP dysregulation. As part of a linked investigation (data not shown), HNE activity was assessed in this bronchiectasis patient cohort and was found to be significantly higher in patients with P. aeruginosa–dominated infection than in those without P. aeruginosa–dominated infection and also correlated with MMP-9 activity. Hence, if HNE were primarily responsible for driving MMP/TIMP imbalance, significantly higher MMP-9 activity would also be anticipated in patients with P. aeruginosa–dominated infection than in those with H. influenzae–dominated infection, which was not observed. This implies HNE is not the sole contributor to the MMP/TIMP imbalance. Our results represent cross-sectional rather than longitudinal data, which we acknowledge limits our ability to assign causality. However, this study was designed to explore the gap of knowledge surrounding the complex relationships in bronchiectasis between MMPs, clinical phenotypes, and airway microbiota composition for the purpose of designing future testable hypotheses. Furthermore, the sample size and comprehensive analyses undertaken, within a rigorous, prospective, randomized controlled trial exceeds all prior evaluations of MMPs and TIMPs in this disease. In summary, we report that, of nine MMPs, increased MMP-1 and MMP-8 concentrations alone were associated with lower lung function, with concentrations higher in patients with P. aeruginosa– and H. influenzae–dominant airway infections. We also show that MMP-2 was higher in patients with airway infection dominated by H. influenzae than in those whose airway infection was dominated by P. aeruginosa, suggesting differential MMP activation according to airway microbiology. Finally, as found in other respiratory diseases, an imbalance between the amount of MMP-8 and MMP-9 compared with their inhibitor, TIMP-1, is apparent in bronchiectasis and correlates inversely with FEV1%. n Author disclosures are available with the text of this article at www.atsjournals.org.
AnnalsATS Volume 12 Number 5 | May 2015
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