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. 2014 Dec;82(12):4952–4958. doi: 10.1128/IAI.02150-14

Recognition of Streptococcus pneumoniae and Muramyl Dipeptide by NOD2 Results in Potent Induction of MMP-9, Which Can Be Controlled by Lipopolysaccharide Stimulation

Marloes Vissers a,b, Yvonne Hartman a,b, Laszlo Groh a,b, Dirk J de Jong c, Marien I de Jonge a,b, Gerben Ferwerda a,b,✉
Editor: A Camilli
PMCID: PMC4249303  PMID: 25183734

Abstract

Matrix metallopeptidase 9 (MMP-9) is a protease involved in the degradation of extracellular matrix collagen. Evidence suggests that MMP-9 is involved in pathogenesis during Streptococcus pneumoniae infection. However, not much is known about the induction of MMP-9 and the regulatory processes involved. We show here that the Gram-positive bacteria used in this study induced large amounts of MMP-9, in contrast to the Gram-negative bacteria that were used. An important pathogen-associated molecular pattern (PAMP) for Gram-positive bacteria is muramyl dipeptide (MDP). MDP is a very potent inducer of MMP-9 and showed a dose-dependent MMP-9 induction. Experiments using peripheral blood mononuclear cells (PBMCs) from Crohn's disease patients with nonfunctional NOD2 showed that MMP-9 induction by Streptococcus pneumoniae and MDP is NOD2 dependent. Increasing amounts of lipopolysaccharide (LPS), an important PAMP for Gram-negative bacteria, resulted in decreasing amounts of MMP-9. Moreover, the induction of MMP-9 by MDP could be counteracted by simultaneously adding LPS. The inhibition of MMP-9 expression by LPS was found to be regulated posttranscriptionally, independently of tissue inhibitor of metalloproteinase 1 (TIMP-1), an endogenous inhibitor of MMP-9. Collectively, these data show that Streptococcus pneumoniae is able to induce large amounts of MMP-9. These high MMP-9 levels are potentially involved in Streptococcus pneumoniae pathogenesis.

INTRODUCTION

Streptococcus pneumoniae is frequently found in the upper respiratory tract of healthy humans and can reside there asymptomatically for a long period. However, S. pneumoniae can become pathogenic and cause a wide range of diseases, from a relatively mild ear infection, otitis media, to sometimes fatal diseases, such as pneumonia, sepsis, and meningitis. The World Health Organization (WHO) estimates that 1.6 million people die every year from pneumococcal infections.

Matrix metallopeptidase 9 (MMP-9) belongs to a family of zinc-binding proteolytic enzymes involved in shaping the extracellular matrix. MMP-9 belongs to the gelatinases and is able to cleave type IV collagen, which is a major component of the basement membrane (1–3).

Several studies have shown the importance of MMP-9 in defense against S. pneumoniae infections (4, 5). Infection of MMP-2/MMP-9 knockout mice with S. pneumoniae led to more bacteria, a greater influx of immune cells, higher cytokine levels in the lungs, and, ultimately, a lower survival rate (4). These studies also showed that MMP-9 is crucial for phagocytosis of S. pneumoniae by neutrophils. In a pneumococcal meningitis model using MMP-9 knockout mice, no difference was found in the leukocyte count or course of disease. However, it was shown that MMP-9 is crucial for the clearance of S. pneumoniae from blood (5). Both studies suggest that MMP-9 is essential for clearance of this bacterium.

The induction of MMP-9 can also have harmful effects. Depletion of lung dendritic cells (DCs) in mice led to enhanced resistance to a challenge infection with S. pneumoniae, a delayed bacterial systemic dissemination, fewer inflammatory mediators in serum, and reduced bacterial loads (6). The study showed that S. pneumoniae induced MMP-9 in these DCs and that S. pneumoniae used the increased production of MMP-9 to disseminate to other tissues. Some serotypes of S. pneumoniae express the virulence factor zinc metalloproteinase C (ZmpC), which is able to cleave the inactive precursor of MMP-9, leading to activation of this enzyme. It was hypothesized that S. pneumoniae might use this active MMP-9 to invade the human body and cause invasive disease (7). These studies suggest that although MMP-9 is crucial for clearance of this bacterium from blood, it might also enable S. pneumoniae to enter the human body and become invasive.

Thus, although the induction of MMP-9 might have both beneficial and harmful effects, MMP-9 must be considered important in the pathogenesis of S. pneumoniae infection. At the moment, not much is known about the induction of MMP-9 and the regulatory processes involved. Therefore, the goal of this study was to determine how MMP-9 is induced and which receptors are involved.

MATERIALS AND METHODS

Bacteria.

Streptococcus pneumoniae TIGR4 was routinely culture in Todd-Hewitt broth containing 0.5% yeast extract at 37°C with agitation (200 to 250 rpm). The optical density at 620 nm (OD620) was measured, and bacteria were grown to an OD620 of 0.3. Viable bacterial counts were determined by plating serial dilutions in phosphate-buffered saline (PBS) on blood agar plates. TIGR4 was washed with PBS and heat killed at 65°C for 30 min.

Moraxella catarrhalis BBH18 was routinely cultured in brain heart infusion (BHI) broth (Becton, Dickinson) at 37°C with agitation (200 to 250 rpm). The OD620 was measured, and bacteria were grown to an OD620 of 1.0. Viable bacterial counts were determined by plating serial dilutions in PBS on supplemented BHI agar plates.

Nontypeable Haemophilus influenzae (NTHI) was grown in BHI broth (Becton, Dickinson) supplemented with 10 μg/ml hemin (Sigma) and 2 μg/ml β-NAD (Merck) at 37°C with agitation (200 to 250 rpm). The OD620 was measured, and bacteria were grown to an OD620 of 0.5. Viable bacterial counts were determined by plating serial dilutions in PBS on supplemented BHI agar plates. NTHI was washed with PBS and heat killed at 65°C for 1 h.

PBMC isolation and stimulation assays.

After obtaining informed consent, blood was drawn from the cubital vein of healthy volunteers, namely, patients with Crohn's disease who are homozygous for the 3020insC mutation in the nucleotide-binding oligomerization domain 2 (NOD2) receptor and patients with Crohn's disease and with a functional NOD2 protein (wild type [WT]). The study was approved by the Central Committee on Research involving Human Subjects of the Radboud University Medical Center. Blood was drawn into 10-ml EDTA tubes (Monoject). The peripheral blood mononuclear cell (PBMC) fraction was obtained by density gradient centrifugation using Lymphoprep (Axis-Shield), following a previously described protocol (8). In short, blood was diluted with an equal volume of PBS. The diluted blood was added on top of the Lymphoprep and centrifuged at 800 × g to separate plasma from the PBMC fraction. PBMCs were harvested, washed three times in PBS, and resuspended in culture medium (RPMI 1640 GlutaMAXI medium [Invitrogen] with 1% penicillin-streptomycin [Invitrogen]). Cells were counted using a counting chamber, and the number was adjusted to 5 × 106 cells/ml. A total of 5 × 105 cells in a 100-μl volume were added to round-bottom 96-well plates (Nunc) and incubated with an equal volume of culture medium (negative control) or various stimuli: heat-killed Streptococcus pneumoniae, heat-killed Staphylococcus aureus (Invivogen), heat-killed Moraxella catarrhalis, heat-killed Haemophilus influenzae, Pam3CSK4 (Pam3Cys; EMC Microcollections), peptidoglycan from S. aureus (PGN-SA; Invivogen), peptidoglycan from Escherichia coli (PGN-EB; Invivogen), lipoteichoic acid (LTA; Invivogen), ultrapure lipopolysaccharide from E. coli serotype O111:B4 (LPS; Invivogen), ultrapure FLA-ST (flagellin; Invivogen), CpG ODN 2336 (CpG; Invivogen), E. coli DNA (Invivogen), l-Ala-γ-d-Glu-mDAP (tri-DAP; Invivogen), MurNAc-l-Ala-γ-d-Glu-mDAP (mTriDAP; InvivoGen), MurNAc-Ala-d-iso-Gln-Lys (mTriLYS; Invivogen), muramyl dipeptide (MDP; InvivoGen), or combinations of ligands. Combinations of ligands were added simultaneously. After 24 h, the supernatants were collected and stored at −20°C for cytokine measurement. After 4 h, the cells were resuspended in 170 μl RLT (Qiagen, Germany) buffer with 1% β-mercaptoethanol and stored at −80°C for quantitative PCR.

Cytokine measurements.

MMP-9 (R&D Systems), tissue inhibitor of metalloproteinase 1 (TIMP-1; R&D Systems), and interleukin-1β (IL-1β; Sanquin, The Netherlands) concentrations were measured in cell supernatants by use of commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions. MMP-9 had a detection limit of 156 pg/ml, TIMP-1 had a detection limit of 156 pg/ml, and IL-1β had a detection limit of 20 pg/ml.

cDNA synthesis and quantitative PCR.

Total RNA was extracted using an RNeasy kit (Qiagen, Germany), genomic DNA was removed using Turbo DNase (Ambion), and cDNA was synthesized using SuperScript reverse transcriptase (Invitrogen), according to the manufacturers' instructions. Quantitative PCR measurements for MMP-9 (accession no. NM_004994.2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (accession no. NM_002046.3) were performed using commercially available TaqMan gene expression assays (Applied Biosystems). The PCR conditions were as follows: initial denaturation for 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Mean relative mRNA expression from two replicate measurements was normalized to GAPDH expression for each sample. The fold change in gene expression relative to normal (negative control) was calculated using the ΔΔCT method.

Statistical analysis.

Fold upregulation was compared to 1, and statistical significance was calculated by the one-tailed Wilcoxon signed-rank test. Cytokine data were compared between groups by using the one-tailed Mann-Whitney U test. Cytokine data were compared within groups by using the one-tailed Wilcoxon signed-rank test. Data shown are representative of two or more independent experiments performed in duplicate, with a minimum n value of 5, except for the data in Fig. S1 in the supplemental material, for which we used only three volunteers. Graph-Pad Prism 5.03 was used for statistics (GraphPad Software). P values of <0.05 were considered statistically significant. Data are given as means plus standard errors of the means (SEM).

RESULTS

MMP-9 is induced by Gram-positive bacteria.

To determine which respiratory bacteria are able to induce MMP-9, we stimulated human PBMCs with heat-killed Gram-positive bacteria (Streptococcus pneumoniae and Staphylococcus aureus) and heat-killed Gram-negative bacteria (Moraxella catarrhalis and Haemophilus influenzae).

MMP-9 was induced by both Gram-positive bacteria but not by the Gram-negative bacteria (Fig. 1A). As a control to show that both Moraxella catarrhalis and Haemophilus influenzae are able to induce cytokines, IL-1β was measured. IL-1β showed the opposite effect. Gram-negative bacteria induced large amounts of IL-1β, in contrast to the Gram-positive bacteria, which induced only small amounts of IL-1β (Fig. 1B). This shows that the Gram-positive bacteria we used are more efficient at MMP-9 induction, whereas the Gram-negative bacteria we used induce large amounts of IL-1β.

FIG 1.

FIG 1

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MMP-9 is induced by Gram-positive bacteria. PBMCs isolated from five healthy volunteers were stimulated with heat-killed Streptococcus pneumoniae (SP) (multiplicity of infection [MOI] = 1), heat-killed Staphylococcus aureus (SA) (MOI = 1), heat-killed Moraxella catarrhalis (MC) (MOI = 1), and heat-killed Haemophilus influenzae (HI) (MOI = 1). Levels of MMP-9 (A) and IL-1β (B) in culture supernatants were measured by ELISA after 24 h of stimulation at 37°C. Data are presented as means + SEM of results for pooled duplicates of all samples. The data are representative of at least two experiments. The significance of differences in cytokine production after stimulation with a ligand or with medium was determined using the Wilcoxon signed-rank test (*, P < 0.05).

MMP-9 is efficiently induced by MDP, in a dose-dependent manner.

To study in more detail which pattern recognition receptors (PRRs) are able to induce MMP-9, we stimulated PBMCs with different ligands specific for PRRs described to recognize bacteria. We used Pam3Cys (Toll-like receptors 1 and 2 [TLR1/2]), PGN-SA (TLR2), PGN-EB (TLR2), LTA (TLR2/6), LPS (TLR4), flagellin (TLR5), CpG (TLR9), E. coli DNA (TLR9), tri-DAP (NOD1), mTriDAP (NOD1/2), MDP (NOD2), and mTriLYS (NOD2). Stimulation of TLR2, TLR5, and especially NOD2 induced large amounts of MMP-9 (Fig. 2A). This pattern was specific for MMP-9, as IL-1β showed a different pattern. IL-1β was induced mainly by stimulation of TLR2 (Fig. 2B). In conclusion, NOD2 stimulation leads to potent MMP-9 production.

FIG 2.

FIG 2

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MMP-9 is efficiently induced by MDP. PBMCs isolated from at least five healthy volunteers were stimulated with Pam3Cys (10 μg/ml), PGN-SA (10 μg/ml), PGN-EB (10 μg/ml), LTA (10 μg/ml), LPS (1 ng/ml), flagellin (100 ng/ml), CpG (10 μg/ml), DNA from E. coli (10 μg/ml), tri-DAP (1 μg/ml), mTriDAP (5 μg/ml), MDP (1 μg/ml), or mTriLYS (1 μg/ml). Levels of MMP-9 (A) and IL-1β (B) in culture supernatants were measured by ELISA after 24 h of stimulation at 37°C. Data are presented as means + SEM of the results for pooled duplicates of all samples. The data are representative of at least two experiments. The significance of differences in cytokine production after stimulation with a ligand or with medium was determined using the Wilcoxon signed-rank test (*, P < 0.05).

Figure 2A shows that the most efficient ligand for MMP-9 induction was MDP. MDP is present mostly in Gram-positive bacteria. An important pathogen-associated molecular pattern (PAMP) for Gram-negative bacteria is LPS. Therefore, we performed a dose-response experiment with both MDP and LPS. MMP-9 induction by MDP showed a dose-dependent response in which larger amounts of MDP induced larger amounts of MMP-9 (see Fig. S1A in the supplemental material). LPS also showed a dose-dependent response. Interestingly, this dose-dependent response worked antagonistically, as a larger amount of LPS induced less MMP-9 (see Fig. S1B). This suggests that LPS may control MMP-9 induction by MDP.

MMP-9 induction by MDP is NOD2 dependent and can be controlled by LPS.

The receptor described to recognize MDP is NOD2 (9). To test whether the MMP-9 induction was dependent on NOD2, PBMCs from patients with Crohn's disease who have a point mutation in their NOD2 gene, rendering it nonfunctional, were used. As a control, PBMCs from healthy volunteers were used.

Stimulation with MDP resulted in high MMP-9 levels in healthy volunteers (Fig. 3A). However, Crohn's disease patients with the mutation did not induce MMP-9 in response to MDP. LPS induced small amounts of MMP-9, and this was not NOD2 dependent, as Crohn's disease patients with the mutation showed levels of MMP-9 comparable to those of the healthy volunteers. LTA also induced small amounts of MMP-9, and this was partly NOD2 dependent. Simultaneous stimulation of PBMCs from healthy volunteers with both LPS and MDP did not result in MMP-9 levels that were comparable to or higher than MMP-9 levels induced by MDP alone. In contrast, costimulation with LPS seemed to control the MMP-9 induction by MDP, leading to reduced MMP-9 levels comparable to those obtained with LPS stimulation alone.

FIG 3.

FIG 3

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MMP-9 induction by MDP is NOD2 dependent and can be controlled by LPS. PBMCs isolated from at least six healthy volunteers with wild-type NOD2 (controls) and from at least five patients with Crohn's disease who are homozygous for the 3020insC NOD2 mutation (patients; NOD2fs) were stimulated with LTA (10 μg/ml), LPS (1 ng/ml), MDP (5 µg/ml), or a combination of MDP (5 μg/ml) and LPS (1 ng/ml) (A) or stimulated with live Streptococcus pneumoniae (MOI = 0.2), bead-beaten Streptococcus pneumoniae (MOI = 0.2), heat-killed (HK) Streptococcus pneumoniae (MOI = 0.2), heat-killed Moraxella catarrhalis (MOI = 0.02), or heat-killed Haemophilus influenzae (MOI = 0.02) (B). Levels of MMP-9 in culture supernatants were measured by ELISA after 24 h of incubation at 37°C. Data are presented as means + SEM of the results for pooled duplicates of every sample and are representative of at least two experiments performed. Cytokine responses after stimulation were compared to those of PBMCs stimulated with medium by using the Wilcoxon signed-rank test. Cytokine responses compared between groups were tested using the Mann-Whitney U test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Stimulation with live, bead-beaten, and heat-killed S. pneumoniae all resulted in high MMP-9 levels in healthy volunteers, although live S. pneumoniae induced lower levels than those induced by heat-killed or bead-beaten S. pneumoniae (Fig. 3B). Crohn's disease patients showed a reduced MMP-9 response to all S. pneumoniae stimulations. This suggests that the MMP-9 induction by S. pneumoniae is largely NOD2 dependent. M. catarrhalis did not induce MMP-9 in healthy volunteers, whereas H. influenzae induced some MMP-9, in a partly NOD2-dependent manner.

As a control to see whether the effect was solely dependent on the mutation of the NOD2 gene, not on other secondary effects of Crohn's disease, PBMCs from Crohn's disease patients with a functional NOD2 gene were stimulated with MDP. These PBMCs showed results comparable to those for the healthy volunteers (see Fig. S2A in the supplemental material), proving that the results are solely attributable to the NOD2 mutation.

In conclusion, MDP- and Streptococcus pneumoniae-dependent induction of MMP-9 is largely NOD2 dependent and can be controlled by LPS.

The inhibitory effect of LPS is regulated at the posttranscriptional level.

To study whether the MMP-9 induction was regulated at the transcriptional level, mRNA levels of MMP-9 were measured. MMP-9 mRNA was upregulated by MDP in the healthy volunteers but not in the Crohn's disease patients with the mutation (Fig. 4). This suggests that NOD2-dependent MMP-9 induction is regulated at the transcriptional level.

FIG 4.

FIG 4

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The inhibitory effect of LPS is regulated at the posttranscriptional level. PBMCs isolated from five healthy volunteers with wild-type NOD2 (controls) and from five patients with Crohn's disease who are homozygous for the 3020insC NOD2 mutation (patients; NOD2fs) were stimulated with MDP (5 μg/ml), LPS (1 ng/ml), or a combination of MDP (5 μg/ml) and LPS (1 ng/ml). MMP-9 transcription was measured by quantitative PCR and expressed as the fold increase compared with unstimulated cells. Cells were lysed in RLT buffer after 4 h of incubation at 37°C. Data are presented as means + SEM of the results for pooled duplicates of every sample. The data are representative of at least two experiments. Fold upregulation was compared between groups by using the Mann-Whitney U test (*, P < 0.05).

As a control, PBMCs from Crohn's disease patients with a functional NOD2 gene were stimulated. These PBMCs showed results comparable to those for the healthy volunteers (see Fig. S2B in the supplemental material), proving that the regulation of MMP-9 mRNA levels after MDP stimulation is solely attributable to NOD2.

Stimulation with only LPS and costimulation with MDP and LPS led to upregulation of MMP-9 mRNA levels, to levels comparable to those measured after MDP stimulation (Fig. 4). This suggests that LPS controls MMP-9 production at the posttranscriptional level.

Inhibition of MMP-9 by LPS is not TIMP-1 dependent.

The conclusion that LPS inhibits MMP-9 induction in a posttranscriptional manner led to the hypothesis that LPS induces proteins that are able to inhibit MMP-9, e.g., tissue inhibitors of metalloproteinases (TIMPs). TIMP-1 is described to form a complex with MMP-9, so we hypothesized that LPS stimulation induced more TIMP-1 than MDP stimulation did and that LPS could inhibit MMP-9 induction by MDP in this manner. However, measurement of TIMP-1 showed no major differences after stimulation with MDP, LPS (Fig. 5A), or Gram-positive or Gram-negative heat-killed bacteria (Fig. 5B). This suggests that LPS does not inhibit MMP-9 induction in a TIMP-1-dependent manner.

FIG 5.

FIG 5

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Inhibition of MMP-9 by LPS is not TIMP-1 dependent. PBMCs isolated from five healthy volunteers were stimulated with MDP (5 μg/ml), LPS (1 ng/ml), a combination of MDP (5 μg/ml) and LPS (1 ng/ml), heat-killed Streptococcus pneumoniae (SP) (MOI = 1), heat-killed Staphylococcus aureus (SA) (MOI = 1), heat-killed Moraxella catarrhalis (MC) (MOI = 1), or heat-killed Haemophilus influenzae (HI) (MOI = 1). Levels of TIMP-1 in culture supernatants were measured by ELISA after 24 h of incubation at 37°C. Data are presented as means + SEM of the results for pooled duplicates of every sample and are representative of at least two experiments. Cytokine responses were compared between groups by using the Mann-Whitney U test. Cytokine responses after stimulation were compared to those of PBMCs stimulated with medium by using the Wilcoxon signed-rank test (*, P < 0.05).

DISCUSSION

In this study, we show that the two Gram-positive bacteria used in this study induce large amounts of MMP-9, in contrast to the two Gram-negative bacteria. Both S. pneumoniae and MDP are potent inducers of MMP-9, in an NOD2-dependent manner. Interestingly, the Gram-negative PAMP LPS could control the MDP-dependent MMP-9 induction. This inhibition was regulated posttranscriptionally but was not TIMP-1 dependent. Collectively, these data show that Gram-positive bacteria are able to induce larger amounts of MMP-9 than the two Gram-negative bacteria. The high MMP-9 levels induced by S. pneumoniae may be involved in S. pneumoniae pathogenesis.

The Gram-positive bacteria used are very potent in inducing MMP-9 compared to the Gram-negative bacteria. The induction of MMP-9 by Gram-positive bacteria has been described in the literature (6, 10, 11). The two Gram-negative bacteria studied here did not induce high levels of MMP-9. In contrast, others found high MMP-9 plasma levels in patients infected with Gram-negative bacteria (11, 12). A possible explanation is that the MMP-9 induction in Gram-negative infections is a secondary effect. Gram-negative bacteria are more potent inducers of IL-1β, and this cytokine has been described to induce MMP-9 (13, 14). In our experiments, we did not see this secondary effect, maybe due to the fact that we stimulated the immune cells for only 24 h. Thus, possibly, Gram-positive bacteria induce high levels of MMP-9 in a direct and fast manner, whereas Gram-negative bacteria possibly induce MMP-9 in a slower and indirect manner.

Stimulation of multiple receptors can lead to MMP-9 induction. However, the most potent one seems to be NOD2. MDP stimulation especially leads to high MMP-9 levels. These data are in accordance with the results of a study from Lappas, who showed that MDP is able to induce MMP-9 in human primary myometrial cells (15). LPS was previously found to induce MMP-9 (16), and this was also shown in our experiments. However, in our study, increasing amounts of LPS resulted in a reduced MMP-9 induction, and the high MMP-9 induction by MDP could be controlled by LPS stimulation. This effect has not been described before in the literature.

PBMCs from Crohn's disease patients homozygous for the 3020insC mutation to the NOD2 gene were used to show that both MDP and S. pneumoniae signal via NOD2. Several studies have shown that MDP signaling in these homozygous Crohn's disease patients is abrogated (9, 17). We showed that MDP-mediated MMP-9 induction is completely NOD2 dependent. However, MMP-9 induction by S. pneumoniae is largely NOD2 dependent. This is likely due to the fact that S. pneumoniae has multiple PAMPs and can therefore stimulate multiple PRRs. S. pneumoniae can also stimulate, for example, TLR2 (18–20), which can also induce MMP-9, as shown in this study. We also show that heat-killed S. pneumoniae and bead-beaten S. pneumoniae induce higher MMP-9 levels than those induced by live S. pneumoniae. This is possibly due to the fact that live S. pneumoniae might contain less soluble MDP, as heat killing and bead beating might release MDP from the bacterium.

Unfortunately, the exact mechanism by which LPS controls MMP-9 induction was not found. However, we did show that this inhibition is probably posttranscriptionally regulated. MMPs are known for their strong posttranscriptional regulation (21). Cytokines and growth factors have been described to modulate mRNA stability of several MMPs (22, 23). After translation, MMPs are secreted as latent zymogens (preproteins) that need to be cleaved to become active. Furthermore, endogenous MMP inhibitors also regulate the level of activity. The ELISA we used does not differentiate between the preprotein and active MMP-9. Therefore, we cannot draw any conclusions on active versus inactive MMP-9 in our assays.

MMPs themselves can be produced by different cell types, such as endothelial cells (24), epithelial cells (25), fibroblasts (26), inflammatory cells such as monocytes and phagocytes (16, 27, 28), and neutrophils (27), which are a major cellular source of MMP-9. Because we used a purified PBMC model, the most probable source of MMP-9 in our experiments was therefore the monocytes or DCs. The inhibitor of MMP-9 also has to be produced by one of the cell types present in the PBMC population. Proteins described to regulate MMP-9 activity are neutrophil gelatinase-associated lipocalin (NGAL) (29), α2-macroglobulin (30), IL-10 (31), and tissue inhibitors of metalloproteinases (TIMPs) (32). NGAL is produced by neutrophils (33), which are not present in our PBMC model, so NGAL is not a likely candidate. The α2-macroglobulin molecule is synthesized mainly in the liver but also locally, by macrophages or fibroblasts (34). This suggests that α2-macroglobulin is also not very likely to be the inhibitor of the MDP-dependent MMP-9 induction. A protein that can be produced by inflammatory cells present in PBMCs is IL-10 (35). However, IL-10 regulates MMP-9 expression at the mRNA level (31), and in our model, the MMP-9 expression levels were comparable after LPS and MDP stimulation. TIMPs are endogenous inhibitors of MMPs and can be produced by PBMCs (36). TIMP-1 can form a complex with MMP-9 and thereby inactivate it (31). A logical posttranscriptional manner of inhibition would be a higher level of TIMP-1 production after LPS stimulation, with the TIMP-1 then binding MMP-9 and thereby inactivating it. However, we did not find any differences in TIMP-1 induction by the different ligands or bacteria.

Certain strains of S. pneumoniae display the zinc metalloproteinase ZmpC on their surfaces. ZmpC can cleave MMP-9 into its active form. Oggioni et al. tested clinical isolates of S. pneumoniae for the presence of ZmpC (7). The ZmpC gene was not found in any of the nasal or conjunctival swabs tested. However, it was present in 1 of 13 meningitis isolates and in 6 of 11 pneumonia isolates. Although these numbers are too small to allow us to draw firm conclusions, they do suggest that the ZmpC protease might play a role in pneumococcal virulence and pathogenicity by cleaving MMP-9. Since we have shown that LPS seems to inhibit early MMP-9 induction, perhaps the presence of certain Gram-negative bacteria during S. pneumoniae colonization prevents S. pneumoniae from becoming invasive by reducing the amount of MMP-9 released.

Moreover, upregulation of MMP-9 has been correlated with multiple diseases, e.g., cardiovascular disease (37), chronic obstructive pulmonary disease (COPD) emphysema (38), and severe respiratory syncytial virus (RSV) infections (39). For severe RSV infections, a correlation between higher MMP-9 levels and severity of disease has been found (39). However, stimulation of PBMCs from infants with RSV did not lead to MMP-9 induction. In this study, we have shown that certain bacterial ligands, such as MDP, are important drivers of MMP-9 induction. It would be interesting to study the possible role of Gram-positive bacteria in these types of diseases.

In conclusion, Gram-positive bacteria and MDP strongly induce MMP-9 in an NOD2-dependent manner. S. pneumoniae can potentially use this high-level MMP-9 induction to enhance its virulence and cause invasive disease.

Supplementary Material

Supplemental material
supp_82_12_4952__index.html (1KB, html)

ACKNOWLEDGMENTS

We thank J. Langereis, S. de Vries, and M. Habets for providing us with heat-killed Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae, respectively.

M.V. and G.F. are supported by the Virgo Consortium, funded by Dutch government project FES0908, and by the Netherlands Genomics Initiative (project 050-060-452).

We declare no commercial or financial conflicts of interest.

Footnotes

Published ahead of print 2 September 2014

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.02150-14.

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