Matrix Metalloproteinases as Drug Targets in Infections ...generally referred to as systemic...

CLINICAL MICROBIOLOGY REVIEWS, Apr. 2009, p. 224–239 Vol. 22, No. 20893-8512/09/$08.00�0 doi:10.1128/CMR.00047-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Matrix Metalloproteinases as Drug Targets in Infections Caused byGram-Negative Bacteria and in Septic Shock

Ineke Vanlaere and Claude Libert*Department for Molecular Biomedical Research, VIB, B-9052 Ghent, Belgium, and Department of Molecular Biology,

Ghent University, B-9052 Ghent, Belgium

INTRODUCTION .......................................................................................................................................................224FROM INFECTION TO SEPSIS, SEVERE SEPSIS, AND SEPTIC SHOCK..................................................224MOLECULAR MECHANISMS OF THE INFLAMMATORY RESPONSE ......................................................225MOUSE MODELS FOR STUDYING INFECTION, SEPSIS, AND SEPTIC SHOCK....................................227EXPRESSION OF MMPs DURING INFECTION, SEPSIS, AND RELATED CONDITIONS......................227

MMP Expression Is Induced by Gram-Negative Bacteria and Chlamydia .....................................................229Chlamydia .............................................................................................................................................................229Helicobacter...........................................................................................................................................................229Pseudomonas.........................................................................................................................................................229Salmonella and Escherichia ................................................................................................................................229

Expression of MMPs during Sepsis and Endotoxemia .....................................................................................229EFFECTS OF MMP INHIBITION IN SEPTIC SHOCK AND IN INFECTION MODELS...........................230STUDIES USING MMP-DEFICIENT MICE.........................................................................................................231

Phenotypes of MMP-Deficient Mice in Infection Models .................................................................................231Phenotypes of MMP-Deficient Mice in Septic Shock Models ..........................................................................233

MMPs ARE INVOLVED IN TLR4 TRIGGERING ...............................................................................................233MMPs MODULATE LPS-INDUCED INFLAMMATORY MEDIATORS..........................................................234CONCLUSIONS .........................................................................................................................................................235ACKNOWLEDGMENTS ...........................................................................................................................................235REFERENCES ............................................................................................................................................................235

INTRODUCTION

The matrix metalloproteinases (MMPs) constitute a familyof at least 25 structurally and functionally related Ca2�-con-taining, Zn2�-dependent endopeptidases (Table 1) (119).MMPs, as indicated by their name, can cleave most if not allstructural extracellular matrix (ECM) proteins, and research-ers previously focused mainly on these matrix-remodelingproperties. More recent research prompted expansion of thisclassical view. In addition to their functions as tissue-remod-eling enzymes, MMPs also act as processing enzymes thatselectively cleave a long and growing list of substrates. Theirnonmatrix targets include cell surface receptors, cytokines,chemokines, cell-cell adhesion molecules, clotting factors, andother proteinases (141). Characterization of these newly dis-covered MMP substrates and generation of MMP mutantmouse strains have demonstrated the relevance of these en-zymes in multiple processes. MMPs participate in fundamentalprocesses, such as cell proliferation, differentiation, adhesion,migration, angiogenesis, apoptosis, and inflammation (69).The remarkable diversity of MMPs in both substrates andfunctions demands tight control over these enzymes in order toavoid undesired cleavage. This control is established by theneed of MMPs for induction, secretion, and activation toachieve full activity. Compartmentalization and inhibition, for

instance by their natural inhibitors tissue inhibitors of MMPs(TIMPs) or the acute-phase reactant �2-macroglobulin, formother levels of control (119). Loss of control leads to an im-balance in the expression or activities of MMPs, and this hasbeen implicated in many disease processes. More details aboutMMPs and their structure, regulation, and function in healthand disease can be found in other, recently published reviews(31, 69, 112, 119). This review will shed light on the role ofMMPs in infection, sepsis, and septic shock.

FROM INFECTION TO SEPSIS, SEVERE SEPSIS, ANDSEPTIC SHOCK

Billions of individual microorganisms, collectively referredto as the normal flora, grow on or in the host, having developedintimate, beneficial, and sometimes essential relationships.Only a subset of microorganisms, called pathogens, can causeinfection, defined as illness caused by microbial invasion. Theoutcome of an infection depends on the pathogenicity or vir-ulence of the pathogen and on the susceptibility or resistanceof the host to that pathogen. Neither the virulence nor theresistance of the host is a constant factor. Virulence is influ-enced by factors such as nutrients, temperature, and pH,whereas resistance of the host depends on factors such as diet,age, gender, the presence of other pathogens, and underlyingdiseases and their treatment (e.g., immunosuppressive drugsafter organ transplantation or chemotherapy for cancer pa-tients), as well as on genetic factors. The toxins produced bysome pathogenic microorganisms are important virulence fac-tors. These toxins act on specific host cells or molecules, re-

* Corresponding author. Mailing address: DMBR, VIB & GhentUniversity, Technologiepark 927, B-9052 Ghent (Zwijnaarde), Bel-gium. Phone: 32-9-3313700. Fax: 32-9-3313609. E-mail: [email protected].

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sulting in specific impairment of a major host cell function(77). Bacteria are often classified as gram positive or gramnegative, based on how they stain with Gram stain. (56). Gram-positive and gram-negative bacteria cause considerably differ-ent types of infections (21). This review will focus on gram-negative infections. Gram-negative bacteria are characterizedby the presence of a unique outer membrane rich in lipopoly-saccharides (LPS). Peptidoglycans and lipoproteins, as well asflagellin of flagellated bacteria, are other molecules that formpart of the gram-negative cell wall. These molecules are allrecognized by the immune system and hence contribute to thehost response. These molecules are called pathogen-associatedmolecular patterns (PAMPs) (52).

The host’s first response to infection is local inflammation,characterized by the famous words of Celsus: calor (heat),rubor (redness), dolor (pain), and tumor (swelling). A fifthcardinal sign was added by Galen, namely, functio laesio (dis-turbance of function) (122). The innate immune system acts asthe immediate line of defense against pathogens. It consists ofsurface barriers, inflammatory cells, cytokines, chemokines,proteases, and various other components. In most cases, thesecomponents together provide effective defenses against patho-gens. However, if the immune system fails to remove thesepathogens from the local invasion site, an overwhelming infec-tion and immune response can develop, causing severe life-threatening symptoms. The archetypical example of an infec-tion that becomes systemic is entry of bacteria into thebloodstream, a condition called bacteremia. The subsequentsystemic inflammatory response is called sepsis, and if it orig-

inates from a gram-negative infection, it is known as gram-negative sepsis. The disease evolves into a severe sepsis whensigns of organ dysfunction are apparent and into septic shockif hypotension persists despite adequate fluid resuscitation(101). It is estimated that 750,000 cases of severe sepsis occurannually in the United States, with a mean mortality rate of28.6% (3). Of all sepsis cases, 38% are caused by gram-nega-tive infections. Isolates of Pseudomonas and Escherichia coliare the most common in sepsis patients (154). Trauma andburns can also trigger an exaggerated inflammatory responseand shock. When no infection is involved, the situation is moregenerally referred to as systemic inflammatory response syn-drome (SIRS). In the clinic, SIRS is diagnosed when the patientsuffers from more than one of the following clinical findings:fever, tachycardia, tachypnea, or leukocytosis. In SIRS, the signalsinitiating the inflammatory response originate from the host andare called alarmins. ATP, high-mobility group box 1, and DNAare well-studied examples of alarmins (10). Together, the endog-enous alarmins and the exogenous PAMPs constitute the largerfamily of damage-associated molecular patterns (DAMPs).

Morbidity and mortality due to severe sepsis and severeSIRS are caused by the uncontrolled inflammatory response,not by the bacteria or other insults themselves as was previ-ously thought (20). To study sepsis, animal models are used.One model is the administration of LPS. When this endotoxininduces an exaggerated inflammatory response, researchersspeak more specifically of endotoxemia and endotoxic shock.The different definitions developed at the international sepsisforum conferences are summarized in Fig. 1 (15).

Despite extensive research, there is still a lack of good ther-apies. The current treatment for sepsis, severe sepsis, andseptic shock is control of infection and support of the failingorgans. Control of infection is not only meant to eliminate theongoing infection, e.g., by use of antibiotics, but also to preventnew infections. Support of impaired organ function consists offluid resuscitation and use of vasopressors to normalize bloodpressure, mechanical ventilation for respiratory insufficiency,and kidney dialysis for kidney failure (135). Organ dysfunctionand septic shock are consequences of a dysregulated immuneresponse, and therefore extensive research on modulation ofthe immune response is being carried out. After more than 30phase III randomized trials with patients with severe sepsis,only one treatment was shown to be beneficial, namely, thetreatment with drotrecogin-alfa (activated), i.e., human recom-binant activated protein C (153). However, even this treatmentremains controversial. Therefore (and this does not happenfrequently), a new phase III trial is planned. The lack of aneffective treatment, the high prevalence, the high mortalityrate, the rapidity with which resistance to antibiotics develops,the proportional increase in the ageing population, and theassociated high economic costs all underscore the need forfurther extensive studies: only by acquiring a better under-standing of the fundamental processes involved in sepsis willwe be able to define novel targets for new therapies.

MOLECULAR MECHANISMS OF THEINFLAMMATORY RESPONSE

Inflammation can be induced by many triggers, such as al-lergens, chemicals, and cytokines, as well as DAMPs, which are

TABLE 1. Mammalian MMPs

MMPa Common name Other name(s)

MMP-1 Collagenase 1 (Mcol-A,Mcol-Bb)

Fibroblast collagenase,interstitial collagenase

MMP-2 Gelatinase A 72-kDa gelatinase, 72-kDatype IV collagenase

MMP-3 Stromelysin 1 Transin 1MMP-7 Matrilysin PUMPMMP-8 Collagenase 2 Neutrophil collagenaseMMP-9 Gelatinase B 92-kDa gelatinase, 92-kDa

type IV collagenaseMMP-10 Stromelysin 2 Transin 2MMP-11 Stromelysin 3MMP-12 MetalloelastaseMMP-13 Collagenase 3 Rat collagenaseMMP-14 MT1-MMP Membrane-type MMPMMP-15 MT2-MMPMMP-16 MT3-MMPMMP-17 MT4-MMPMMP-19 RASI-1MMP-20 EnamelysinMMP-21MMP-22MMP-23 CA-MMPMMP-24 MT5-MMPMMP-25 Leukolysin MT6-MMPMMP-26 Endometase Matrilysin 2MMP-27MMP-28 Epilysin

a MMP-4, -5, and -6 were found to be either MMP-2 or MMP-3, so they arenot unique MMPs. MMP-18 (collagenase-4) has been cloned only from Xenopus;a mammalian homologue has not been found.

b Mcol-A and -B are probably the murine homologues of MMP-1.

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recognized by pattern recognition receptors (PRRs) (52).Three major protein families have been identified as DAMPsensors: Toll-like receptors (TLRs), Nod-like receptors, andRIG-like helicases. TLRs, which are localized at the cell mem-brane, and Nod-like receptors, which are cytosolic receptors,are involved in the detection of bacterial PAMPs (6). One ofthe best studied PAMPs of gram-negative bacteria is LPS, alsocalled endotoxin. LPS is very potent, and minute amounts aresufficient to trigger the innate immune system (9). It is recog-nized by TLR4 and its coreceptors: LPS-binding protein,CD14, and myeloid differentiation 2 (147). Ligand binding

induces signaling cascades leading to the activation of tran-scription factors, such as activator protein 1, nuclear factor �B(NF-�B), and interferon regulatory factor 3 (5, 111). Thesetranscription factors induce de novo expression of multipleproinflammatory genes, leading to the release of inflammatorymediators, including cytokines, chemokines, adhesion mole-cules, and clotting factors (111). This proinflammatory re-sponse leads to endothelial alterations, recruitment and acti-vation of inflammatory cells, and a hypercoagulation state (Fig.2). Neutrophils and macrophages eliminate the pathogen bythe release of toxic products (e.g., hydrogen peroxide, lyso-

FIG. 1. General definitions. Inflammation is the host’s response to infection or other insults. Normally, a local inflammatory response leads to resolution ofthe infection or injury. However, if the inflammation becomes dysregulated, systemic activation of the innate immune system can occur. The complex clinicalfindings associated with this systemic activation are known as SIRS. SIRS is triggered by sterile inflammatory processes, e.g., pancreatitis, trauma, and burns. SIRSis considered to be present when more than one of the following clinical findings exists: fever, tachycardia, tachypnea, or leukocytosis. Sepsis is defined as asuspected or proven infection plus a SIRS. The infection is caused by bacteria, viruses, or fungi, and if the pathogen enters the circulation, the condition is knownas bacteremia, viremia, or fungemia, respectively. SIRS evolves to severe SIRS and sepsis to severe sepsis when there is organ dysfunction (e.g., hypotension,oliguria, and thrombocytopenia). Severe sepsis is called septic shock when it is complicated by serious hypotension despite fluid resuscitation. Analogously, severeSIRS can lead to shock. At each stage of the disease, recovery is possible. However, the patient’s survival chances decrease substantially in the later stages of thedisease.

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zyme, and MMPs) and phagocytosis. All these mediators andinflammatory cells are essential for a “normal” immune re-sponse but are very detrimental when their release is excessiveand uncontrolled. Simultaneously, anti-inflammatory pathwaysare also activated, leading to the release of anti-inflammatorycytokines that dampen and terminate the inflammatory re-sponse (111). During septic and endotoxic shock, homeostasisis completely lost, inflammation dominates over anti-inflam-matory pathways, and coagulation dominates over fibrinolysis.The result is tissue injury, organ failure, and very often alsodeath (Fig. 2).

MOUSE MODELS FOR STUDYING INFECTION, SEPSIS,AND SEPTIC SHOCK

Mice are susceptible to different gram-negative bacteria, andinfection can be easily established, for example, in their respi-ratory, gastrointestinal, or urinary tract. These routes of infec-tion do not always lead to sepsis. To generate sepsis experi-mentally, bacteria are administered intravenously (i.v.) orintraperitoneally (i.p.) (91). A drawback of these models is thatit is very hard to always reproduce the dose in different exper-iments. Also, very large loads of bacteria induce endotoxicrather than septic shock, because the outcome is determinedmore by the amount of LPS than by the growth and spread ofthe bacteria.

This brings us to another model mimicking sepsis: the i.v. ori.p. injection of LPS, which on its own reproduces many of thefeatures of gram-negative sepsis (19, 120). This model inducesendotoxemia, is very acute, enables precise dosing, and ishighly reproducible, but no infection is involved, which makesthe model somewhat less relevant to sepsis in humans. Never-theless, this model is still frequently used. It is noteworthy thatLPS preparations isolated from different gram-negative bacte-ria might differ in their biological effects, for example, withrespect to release of cytokines (100). Furthermore, most com-mercial LPS preparations are not pure but contain a mixture ofligands that could activate cells through TLRs other thanTLR4. Therefore, every study should clearly specify the sourceof LPS and its purity.

A third type of sepsis model relies on infection by endoge-nous flora and requires surgical intervention (20). The surgicalmodels frequently used are cecal ligation and puncture (CLP)and colon ascendens stent peritonitis. Both models generate anacute inflammatory reaction caused by a continuous influx ofenteric bacteria into the peritoneal cavity (78). These modelsare not easy to perform, and they are even more complex thanthe infection models because the septic focus generated con-sists of a mixture of different bacteria. This, however, can alsobe seen as an advantage, because it better represents the realsituation in peritonitis.

Another frequently used approach is the simultaneous in-jection of animals with LPS and D-galactosamine, a hepato-toxic drug (30). This treatment increases the sensitivity of miceto the lethal effects of LPS. However, this model is irrelevantto sepsis, since the most prominent pathological feature ishepatic necrosis, which is rarely observed during sepsis in hu-mans (88). Also, this model is mediated exclusively by tumornecrosis factor (TNF) (55), which is not the case in sepsis (18).

LPS can also be applied topically. This approach does notmirror the systemic effects of sepsis but is useful for studyingthe direct, local effects of LPS on specific organs. The mostcommonly used routes of injection are the lungs (leading toacute lung injury) and the brain (23, 159).

One must realize that there are considerable differencesbetween different models. The effects of LPS injection arethought to be solely TLR4 dependent, whereas the bacterialinfusion models also trigger other PRRs. Lipoproteins of thecell wall, flagellin, and bacterial DNA and RNA are recognizedby TLR2, TLR5, TLR9, and TLR7, respectively (95). Anotherimportant difference is the time of exposure to the challenge.Bolus LPS is sudden and lasts only briefly, whereas the stim-ulus during infections develops gradually and persists overhours or days. These and other differences have been discussedin several reviews (13, 26, 123, 131). We cannot claim that aparticular model is superior to the other models. Each modelreplicates some of the features of the disease process but failsto reproduce the whole complexity of human sepsis. In order toreach a meaningful conclusion about the role of a particularmolecule in sepsis, we should collect all the data generatedfrom the different models (83). The goal of this review is to dojust that for the MMPs.

EXPRESSION OF MMPs DURING INFECTION, SEPSIS,AND RELATED CONDITIONS

MMPs were initially believed to play a role exclusively incancer. However, we now know that many pathological condi-tions are characterized by overexpression of MMPs, suggestingthat tight regulation of MMP genes is critical for normal ho-meostasis. Unraveling the molecular mechanisms controllingMMP gene expression might identify new therapeutic targets.However, MMPs are also regulated posttranscriptionally. Forinstance, cytokines and growth factors can modulate themRNA stability of several MMPs (27, 109). After translation,the majority of MMPs are secreted as latent zymogens thatneed to be activated, either in the pericellular milieu or at thecell membrane, before they can exert their function. Excep-tions are the MMPs containing a furin-like enzyme recognitionmotif (MMP-11, MMP-28, and the membrane-type MMPs[MT-MMPs]). These MMPs can be processed into active en-zymes intracellularly (73, 116, 117). However, the extracellu-larly activated MMPs might also have intracellular activities, ashas been reported for MMP-2 (63) and suggested for MMP-7(157). The amount of active MMP is further influenced byfactors such as MMP catabolism, MMP clearance, and endog-enous inhibitors. Hence, the expression of an MMP evaluatedby measuring its mRNA does not necessarily mean that theenzyme is also active. In any case, it remains an indication thatthe protein might be involved in some way. Several expressionanalyses have shown that MMPs are indeed upregulated dur-ing infection with different gram-negative bacteria. As an ex-ample, Affymetrix gene chip analysis detected increased mmptranscripts in Peyer’s patches after infection with Salmonellaand Yersinia (40). Also, sepsis and septic shock were associatedwith higher MMP expression.



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MMP Expression Is Induced by Gram-Negative Bacteriaand Chlamydia

Chlamydia. Ramsey’s group investigated the expression ofMMPs, and particularly MMP-9, in urogenital Chlamydiamuridarum infection. They concluded that mice differing intheir susceptibility to the development of chronic chlamydialdisease also differ in the relative expression and activity ofMMPs (121). Single-nucleotide polymorphism analysis re-vealed that the human Q279R mutation, located in exon 6 ofthe mmp-9 gene, reduces the risk for severe disease followingeye infection with Chlamydia trachomatis (97).

HSP60, produced in large amounts by chlamydiae duringinfection, induced several MMPs in a concentration- and time-dependent manner. This effect was LPS independent, becauseheat treatment abolished MMP production (61).

Helicobacter. Helicobacter strains cause gastric injury by in-ducing several MMPs. Here again, it was shown that geneticvariants of MMP genes are associated with the development ofgastric ulcer in Helicobacter infection. Carriage of allele G ofthe MMP-7 promoter confers a 1.6-fold-increased risk of gas-tric ulcer in humans. Carriage of allele A of a coding single-nucleotide polymorphism in exon 6 of mmp-9 confers a 2.4-fold-increased risk. A Helicobacter pylori constituent thataugments disease risk is the pathogenicity island (PAI), whichencodes a secretion system that translocates bacterial effectormolecules into host cells. This virulence factor selectively leadsto the induction of some MMPs, such as MMP-7 (102), whileother MMPs, such as MMP-9 and MMP-2, are also upregu-lated in strains lacking this PAI (62). Levels of MMP-9 specif-ically originating from macrophages are also increased inhuman and mouse Helicobacter-associated gastritis (7). Helico-bacter infection also upregulates TIMP-1 and TIMP-3 in glan-dular epithelium and stroma (11). As already mentioned, in-duction of MMP-7 is dependent on an intact PAI and alsoseems to be regulated by p120 catenin, a component of adhe-rens junctions, and by Kaiso, a transcription repressor (102,158). Aberrant nuclear translocation of p120 in response toPAI-positive Helicobacter strains relieves the Kaiso-mediatedtranscriptional repression of MMP-7. The induction of MMP-7plays a role in stimulating migration of gastric epithelial cells(158) and hyperproliferation of gastric epithelial cells. Thishyperproliferation relies on the cleavage of the insulin growthfactor-binding protein 5 by MMP-7, which contributes to thebioavailability of insulin growth factor II (86).

Pseudomonas. In patients with cystic fibrosis, Pseudomonasaeruginosa is the most common pathogen (36) and MMP-7 ismarkedly upregulated in the lungs (76). Pulmonary infectionwith Pseudomonas aeruginosa induced the expression of bothMMP-7 and MMP-10 (57). Flagellin, not LPS, was identified asthe inductive factor released by Pseudomonas aeruginosa that

regulated MMP-7 expression (76). MMP-9 was sixfold upregu-lated in response to corneal Pseudomonas aeruginosa infection(87). Elastase, an enzyme produced by Pseudomonas aerugi-nosa, also could strongly activate pro-MMP-1, -8, and -9 underexperimental conditions (104). Investigation of the role of thiselastase in the repair of human airway epithelial cells in cultureshowed that this bacterial protease impedes closure of theairway epithelial wound by altering cell motility and causing animbalance between the pro form of MMP-2 and its activated form(25). These reports exemplify the above-mentioned complexity ofbacterial infections, in which many PAMPs (LPS, flagellin, andelastase) are present to trigger the immune system.

Salmonella and Escherichia. Gastrointestinal infection withSalmonella enterica serovar Typhimurium and Escherichia colifurther demonstrated the importance of MMP-7 in host de-fense. In the mouse, MMP-7 is coexpressed with the �-de-fensins in the Paneth cells of the small intestinal crypts, and itwas found that MMP-7 activates these defensins, enablingthem to kill bacteria (157). Generally, exposure to bacteriaseems to be the trigger for MMP-7 induction in epithelial cells.This hypothesis is further supported by the observation thatMMP-7 was not expressed by germfree mice but was inducedafter colonization with Bacteroides thetaiotaomicron, a com-mensal bacterium in the intestine (75). Serine proteinases de-rived from E. coli are specific activators of pro-MMP-2, be-cause phenylmethylsulfonyl fluoride, a serine proteaseinhibitor, completely interfered with the LPS-mediated activa-tion of pro-MMP-2 (143).

The above-mentioned reports demonstrate that MMPs, forinstance, MMP-7, might be beneficial (activation of defensins)or detrimental (gastric injury), depending on the stimulus andthe organ. This duality should be taken into considerationwhen developing new therapies.

Expression of MMPs during Sepsis and Endotoxemia

LPS induces transcription of several MMP genes, and sev-eral groups have investigated the signal transduction pathwaysinducing their expression. The induction of some MMP genesin cell cultures seemed to depend on the activation of NF-�B(58, 125) and/or mitogen-activated protein kinases p38 (64,127) and ERK1/2 (64). The increased expression of MMPsafter an LPS challenge suggests that these proteases may in-fluence the pathogenesis of endotoxemia. MMP-9 is releasedafter infusion of bacterial LPS in healthy human volunteers(2). Accordingly, increased levels of pro-MMP-9 and pro-MMP-2, as well as activated forms of MMP-9, were found inthe plasma of two patients with gram-negative sepsis. Thelevels of these MMPs were related to the severity of sepsis(118). In a clinical study of patients with septic shock, Naka-

FIG. 2. The major pathway and inflammatory mediators of sepsis and its related conditions. LPS and other microbial components (PAMPs)trigger PRRs, such as TLR4. However, TLR4 may also be triggered in the absence of infection, for instance by the enzymatic release of ECMproteins (alarmins). PAMPs and alarmins belong to the larger group of DAMPs. Systemic TLR4 triggering leads to overproduction of inflam-matory mediators, which contribute to the massive activation and recruitment of different cell types, as well as to the activation of the coagulationpathways. Neutrophils release large amounts of enzymes (e.g., MMPs) and reactive oxygen species (ROS), and the excessive activation ofcoagulation factors leads to disseminated intravascular coagulation (DIC). This, together with NO-mediated cardiovascular anomalies, willinevitably cause tissue damage and subsequent organ failure and death.



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mura et al. found that MMP-9 levels in nonsurvivors of severesepsis were higher than those in survivors and healthy controls(96). Elevated levels of MMP-9 in critically ill patients werealso observed in a study by Yassen et al. (161). A more recentstudy again confirms that patients with severe sepsis havehigher levels of MMP-9, as well as TIMP-1 and TIMP-2. Pa-tients with TIMP-1 levels of �3,200 ng/ml were 4.5 times morelikely to die than those with lower levels. The researchersconcluded that TIMP-1 might serve as a useful laboratorymarker for predicting the clinical outcome for patients withsevere sepsis (44). High levels of neutrophil MMP-8 also werefound in the peritoneal fluid of critically ill patients with sec-ondary peritonitis (43).

By analyzing the expression of some MMP and TIMP genesin livers, kidneys, spleens, and brains of mice at various timepoints after LPS injection, Pagenstecher’s group demonstratedorgan-specific and time-specific upregulation of several MMPgenes and the TIMP-1 gene (113). This LPS-induced expres-sion was dose dependent: a lethal dose of LPS induced moreMMPs for longer periods of time than a nonlethal dose (113).Endotoxemia also induces rapid changes in MMP activity inthe aortae, myocardia, and sera of LPS-injected rats (65, 66).Other studies demonstrate that the expression of MMPs fol-lowing LPS challenge can be enhanced by catecholamines(140) or ethanol abuse (74).

Different cell types can produce MMPs in response to LPS,such as endothelial cells (58), fibroblasts (156), epithelial cells(156), inflammatory cells (monocytes and phagocytes) (14, 64,125, 140), microglial cells (37), mast cells (144), and neutro-phils (118).

MMPs can also be induced indirectly by LPS or bacteria.MMPs are induced or repressed by various signals, includingcytokines, growth factors, hormones, and cell-ECM interac-tions. Many of these signals are generated during the LPSresponse or during infection. For instance, interleukin-1� (IL-1�) and TNF, both of which are important cytokines in endo-toxemia and sepsis, can induce MMP genes (60, 129). In turn,MMPs can activate these cytokines and/or release them byshedding, generating a positive feedback loop. Since MMPsalso destroy cytokines by proteolysis (51), a negative feedbackloop can be created as well. Other molecules, such as glucocor-ticoids, retinoids, and progesterone, repress expression ofMMPs. Interestingly, glucocorticoid treatment is one of thefew available therapies that show some clinical efficacy (4).Repression of MMPs by these nuclear receptors is by directaction on the promoters of their genes to suppress trans-acti-vation, as well as indirectly by inducing the transcription of theTIMPs or transforming growth factor �, which in turn suppressMMPs, such as MMP-7 (130). The production of MMP-8 andMMP-9 by neutrophils is also of interest in the context ofinflammation. MMPs are normally secreted in the extracellularenvironment following their production. Neutrophil MMP-8and MMP-9, however, are stored in the secondary and tertiarygranules of neutrophils, respectively. Upon activation, neutro-phils release the contents of their granules, rapidly leading tohigh levels of MMP-8 and MMP-9 without any need for denovo protein synthesis (118). Neutrophil MMP-8 plays a role inthe pathogenesis and progress of LPS-induced acute lung in-jury. Lung MMP-8 levels not only were elevated but also cor-related with pathological scores, the lung wet/dry weight ratio,

and the number of neutrophils (160). MMP-3 and MMP-9were also upregulated during LPS-induced neuroinflammation(93).

EFFECTS OF MMP INHIBITION IN SEPTIC SHOCKAND IN INFECTION MODELS

An obvious way to investigate whether MMPs are implicatedin endotoxic shock is by inhibiting them. MMPs can be inhib-ited by synthetic or natural compounds, as well as by endog-enously produced molecules, such as �2-macroglobulin (32),RECK (reversion-inducing cysteine-rich protein with Kazalmotifs) (103), and the TIMPs (68). TIMPs have high affinitiesfor MMPs, but their lack of selectivity and their possession ofunique, MMP-independent biological activities disfavors theiruse as inhibitors because of the potential side effects (68).

The first generation of synthetic, broad-spectrum MMP in-hibitors used hydroxamate as their zinc-binding group. Thebest known examples of this class are batimastat and marimas-tat. However, most hydroxamate inhibitors lacked specificityand also inhibited non-MMP zinc-based enzymes; conse-quently, new drugs that make use of alternative zinc-bindinggroups were developed (48).

Some antibiotics, such as the tetracyclines, also inhibitMMPs, not only by chelating the zinc and calcium ions but alsoby affecting the induction of the MMP genes. The chemicallymodified tetracyclines (CMT), which lack antibacterial activity,are most commonly used as MMP inhibitors because they haveseveral advantages over conventional tetracyclines: they induceno gastrointestinal side effects or toxicities, they attain higherconcentrations in plasma, and they cross the blood-brain bar-rier and blood-retina barrier (1). Polyphenols and catechinsderived from green tea are well known examples of naturalMMP inhibitors (28, 106).

Different broad-spectrum inhibitors have been tested in dif-ferent sepsis models, and all of them demonstrate that inhibi-tion of MMPs confers protection against septic shock (Table2). This is evidence that MMP inhibition might be of thera-peutic interest. However, broad-spectrum inhibitors are notselective, and so the protection can also be attributed to inhi-bition of other metalloproteinases, such as the ADAMs (adisintegrin and metalloproteinase), because this family is alsoinhibited. Indeed, many reports attribute the attenuated TNFresponse after treatment with broad-spectrum inhibitors to theinhibition of ADAM-17, the major TNF-converting enzyme(TACE). However, several MMPs can also shed the mem-brane-anchored TNF (41, 90). Investigation of more selectiveinhibitors is needed, not only because of medical interest (amore selective inhibitor will normally reduce the side effects),but also for elucidating the specific roles of each MMP inseptic shock.

In many studies, the inhibitor was administered for prophy-laxis. A study by Milano et al. indicated that this prophylactictreatment is essential for protection, because no protectiveeffect was seen if the inhibitor was injected 1 hour after LPStreatment (89). They concluded that inhibitors act on the earlyresponse to LPS. On the other hand, CMT-3, given 12 h afterinduction of CLP, could still prevent the sequelae of sepsis(39). Despite this discrepancy (which probably can be ex-plained by the differences between the models), these studies



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combined prove that at least some MMPs and/or ADAMsmight be involved in sepsis models. To identify the MMPs thatmediate the morbidity and mortality associated with LPS chal-lenge, more specific inhibitors or MMP mutant mouse strainsare needed. Regasepin-1 and a metalloproteinase inhibitorthat contains L-pyridylalanine inhibit MMP-8, MMP-9, andTACE, and these inhibitors could prevent the mortality asso-ciated with endotoxic shock (46, 47), indicating that at leastone of these proteases might play a role.

Interestingly, Nenan’s group provided evidence for the in-volvement of MMPs in the inflammatory response by using thereverse of inhibition. Instead of inhibiting MMPs, they instilleda peptide corresponding to the catalytic domain of recombi-nant human MMP-12 in the mouse airways and showed thatMMP-12 itself can induce an early inflammatory responsecharacterized by neutrophil infiltration, cytokine release, andgelatinase activation, followed by a delayed response consistingmainly of macrophage recruitment (98). Marimastat reducedboth early and late responses (99).

The use of MMP inhibitors during infection also seems to beprotective. CMT impeded ascension of Chlamydia muridaruminto the upper genital tract, blunted acute inflammatory re-sponses, and reduced the rate of chronic disease development(50).

STUDIES USING MMP-DEFICIENT MICE

Genetic knockouts of MMPs are very effective tools foridentifying essential functions of MMPs in different conditions.Analysis of MMP knockouts revealed surprisingly subtle phe-notypes, and only Mmp14-null mice show a severe develop-mental abnormality. Rare examples of Mmp14-null mice thatsurvived until 10 weeks old showed severe dwarfism andcraniofacial anomalies (45). However, after challenge, for ex-ample with LPS, remarkable phenotypes develop in manyMMP knockout mice.

Phenotypes of MMP-Deficient Mice in Infection Models

MMPs might indirectly contribute to the eradication of bac-teria by activating antimicrobial proteins. The prototypical ex-ample is the activation of �-defensins by MMP-7 in Panethcells of the small intestine. As a consequence, MMP-7-defi-cient mice were very sensitive to orally administered E. coli andSalmonella (157). MMPs could also be involved in the directkilling of bacteria. For instance, the C-terminal cathelicidin-like domain of MMP-12 is involved in the intracellular killingof bacteria by macrophages (42) (Fig. 3). For therapeutic pur-poses, these MMPs with antibacterial capacities should be con-sidered for use as antitargets. Many other reports, however,clearly demonstrate that MMPs are interesting targets duringinfection.

The excessive MMP activity that is observed following infec-tion is expected to cause tissue damage. Uncontrolled ECMcleavage by MMPs (for instance, of the endothelial cells or theblood-brain barrier) directly contributes to tissue damage (Fig.3). A large neutrophil influx, which is also orchestrated byMMPs (see MMPs Modulate LPS-Induced Inflammatory Me-diators below), also causes damage. Tissue damage not onlyharms the host directly, it also helps to disseminate the bacte-ria. Induction of apoptosis during infection is another mecha-nism by which the pathogen might harm the host. TNF andFasL are two important proapoptotic molecules that can bereleased from the cell membrane by MMPs (34). It is thusplausible that MMPs are involved in the apoptotic processduring infection. However, apoptosis as a response to intracel-lular bacteria is a useful way for the host to eliminate infectedcells, decreasing the spread of infection and preventing persis-tence of the pathogen (92). These examples demonstrate thatit will be very difficult to predict whether or not an MMPshould be inhibited during infections. Moreover, the outcomeof an infection depends on many parameters, such as the bac-terial species and its virulence (flagellated versus nonmotile,

TABLE 2. Effect of broad-spectrum MMP inhibitors on the outcome in models of sepsis and septic shock and after LPS injection in rodents

Model Inhibitor(s) Outcome(s)a Reference

Bolus LPS i.v. Tetracycline Protection against LPS-induced mortality and inflammatory lesions 134

Bolus LPS i.p. Tetracycline/doxycycline Improved survival; no secretion of TNF, IL-1�, or NO in the blood 89Marimastat Inhibition of TNF production but only slight delay in mortality 148GM-6001 Attenuated TNF response 139Ro 31-9790/doxycycline Improved cardiac function and less-elevated MMP-9 activity 67

CLP CMT-3/hydroxamate Less mortality; less-elevated GOT, GPT, NO, and MMP-9 levels; andreduced gelatinase activity

79

CMT-3 Reduced mortality and lung pathology, less-elevated MMP-2 andMMP-9 levels in lung

142

CMT-3 Improved survival was associated with a significant improvement inlung pathology

39

LPS intratracheally CMT-3 Less neutrophil recruitment and goblet cell metaplasia, lowerexpression levels of epithelial growth factor receptor and MMP-9

59

Corticosteroids Reduced infiltration of neutrophils and reduced activity of MMP-2and MMP-9

22

LPS intracerebrally Minocycline, BB-1101, BB-94 Reduced blood-brain barrier injury by blocking production of MMP-9 126BB-1101 Reduced blood-brain barrier opening, associated with a significant

drop in MMP-2 and MMP-994

a GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminase.



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intracellular versus extracellular life cycle), the inoculum dose,the route of infection, and the immune status and age of thehost. It is quite certain that the levels and activities of MMPsvary substantially in these different conditions, which mightexplain the conflicting results obtained with MMP mutant micein infection studies. Mice challenged with different kinds ofbacteria, different infection doses, or different infection routescan display different phenotypes. For instance, Lee et al.showed that MMP-9 plays a protective role in infection withPseudomonas (70), whereas McClellan et al. concluded theopposite (87). Other reports of studies using gram-negativebacteria in MMP mutant mice are summarized in Table 3. Thequestion of whether MMPs are involved in infections has beenanswered. The challenge researchers now face is to elucidatewhen an MMP is beneficial or detrimental during a particularinfection.

Phenotypes of MMP-Deficient Mice in Septic Shock Models

Many mutant mouse strains, including some MMP-deficientmice, show either a decreased or an increased resistance toLPS challenge (132, 155). MMP-9 deficient mice are resistantto endotoxic shock, especially when they are young (29). Somegroups may not have observed this resistance because theyused adult mice (124). TIMP-3, on the other hand, plays aprotective role, as TIMP-3-null mice were more susceptible toLPS (138). Treatment with a metalloproteinase inhibitor res-cues the TIMP-3 knockout mice. This beneficial role forTIMP-3 was also reported in the CLP model by Martin et al. (84,85). LPS instilled in the trachea led to greater accumulation ofneutrophils in the alveolar space of MMP-8 knockouts (110),pointing to an anti-inflammatory role for MMP-8 in this setting.However, by using an air pouch model, Tester et al. demonstrateda proinflammatory role for MMP-8 (146). We also describedearlier a proinflammatory role for MMP-8 in a TNF-inducedhepatitis model (152). These results demonstrate that an MMPcan have both pro- and anti-inflammatory functions.

By using MMP-3 knockout mice, Gurney et al. providedevidence that the LPS-induced opening of the blood-brainbarrier is mediated by MMP-3, which degrades tight junctionand basal lamina proteins and thereby facilitates neutrophil

influx through this barrier (38). Table 4 provides an overviewof the individual MMPs that have been investigated in modelsof infection, endotoxemia, and sepsis.

MMPs ARE INVOLVED IN TLR4 TRIGGERING

LPS, together with LPS-binding protein, CD14, and myeloiddifferentiation 2, activate TLR4. CD14 can be proteolyticallycleaved from the cell surface to form a soluble peptide. Senftet al. showed that MMP-9 and MMP-12 contribute to theshedding of CD14, at least in the lung (133). Soluble CD14may substitute for mCD14 in the activation of cells lacking thisaccessory protein (33) (Fig. 3). TLR4 is critical for the re-sponses to LPS, but mounting evidence indicates that it canalso detect endogenous ligands. This might explain why SIRScan develop in the absence of an infection and why anti-LPStherapies failed (107). Many ECM fragments can act as en-dogenous TLR4 ligands, such as fibronectin (105), heparansulfate (53), and biglycan, as well as heat shock proteins (149),hyaluronic acid (145), and fibrinogen (137). Interestingly, big-lycan-null mice have a survival advantage in LPS-inducedshock, supporting the notion that the release of ECM frag-ments is important in endotoxic shock (128). Furthermore, aSIRS response can also be induced by administration of elas-tase, the enzyme that cleaves and releases heparan sulfateproteoglycans (54). These examples highlight the importanceof the ECM in the regulation of TLR4. It is possible that theECM constrains the TLR4 function by holding TLR4 in anonsignaling conformation (12). Degradation of the ECM byproteases produced during infection or tissue injury might re-lieve this constraint on TLR4 function and at the same timecreate agonists to trigger the receptor. Of course, largeamounts of LPS might overcome this suppressive mechanism(Fig. 3). The crucial step in induction of innate immunity thusis not necessarily the stimulation of TLR4 but might be therelease of TLR4 from constitutive inhibition by ECM (12).Since MMPs cleave almost all ECM components, they mightplay an important role in the regulation of TLR4. For thera-peutic purposes, blocking MMPs might prevent septic shock bykeeping the TLR4 receptor in a quiescent state.

FIG. 3. MMPs influence the response to LPS or gram-negative bacteria via different mechanisms. (Top left) TLR4 regulation. TLR4 triggeringby bacterial products leads to the activation of macrophages, which respond to the LPS by producing many proinflammatory mediators, such ascytokines, chemokines, and MMPs. MMPs can cleave membrane-bound CD14 into its soluble form, which can be used by cells lacking thiscoreceptor (A). Cleavage of the ECM generates alternative TLR4 triggers (B) and possibly relieves the receptor from the suppressive mechanismthat is exerted by the ECM. (Top right) Tissue damage. Macrophages and neutrophils are attracted to the site of infection and eliminate thepathogen by releasing a massive amount of toxic products, such as reactive oxygen species, cytokines, and MMPs. Finally, pathogens arephagocytosed and tissue repair is initialized. The toxic products efficiently kill bacteria, but they can also be very harmful to the host if they arereleased in large amounts, as is the case in sepsis. Consequently, aberrant cleavage of the ECM by the overzealous production of MMPs leads toepithelial (A) and endothelial (B) damage. (Middle left) Cytokine activity. FasL is expressed at the cell membrane of epithelial cells in responseto LPS. By shedding, MMP-7 releases the soluble form of FasL (A), which induces apoptosis after binding its receptor Fas. Activated macrophagesproduce many cytokines. The mature IL-1�, as well as its pro form, is secreted. MMPs can activate the pro form of this cytokine (B) but can alsodegrade the mature form (C). The major TNF sheddase is TACE, but MMPs also can solubilize this cytokine (D). (Middle right) Chemotaxis.Activated epithelial cells secrete MMPs and chemokines. Chemokines can be sequestered by proteoglycans of the ECM; by cleaving theseproteoglycans, MMPs generate a chemokine gradient (A). Chemokines too can be directly activated (B) or inactivated (C) by MMPs. (Bottom left)Bactericidal activities. Paneth cells, which lie at the base of the crypts between the villi of the small intestine, produce antibacterial products, suchas lysozyme and defensins. In the mouse, MMP-7 activates these defensins and thus plays an anti-inflammatory role (A). MMP-12, on the otherhand, contains a cathelicidin-like domain with direct bactericidal activities (B).



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MMPs MODULATE LPS-INDUCEDINFLAMMATORY MEDIATORS

MMPs are involved in all phases of inflammation. They areclearly implicated in the recruitment of inflammatory cells,starting from extravasation from the capillaries to migrationthrough the ECM. The activity of cytokines, such as IL-1� andTNF, as well as the chemotactic potential of chemokines, suchas LPS-induced CXC chemokine (LIX), can be altered byMMP processing (41, 129, 150, 151) (Fig. 3). MMPs can alsoactivate or inactivate other proteinases, such as serine protein-ases, which also play a role in these inflammatory processes(110). Moreover, MMPs are also implicated in the resolutionof infection and in tissue repair. Thus, MMPs are crucial for anormal immune response, but excessive release of these pro-teinases leads to severe tissue damage. A more extensive over-view of all functions performed by MMPs during inflammationcan be found in recently published reviews (10, 17, 31, 69, 80,82, 115, 151).

Whether MMPs perform all these functions in response to

LPS has to be investigated, because the action of a certainMMP depends on the nature of the stimulus and is highly celland tissue type specific. For example, MMP-9 is important inrespiratory infection with Francisella tularensis (81) but has noobvious role in LPS-induced lung inflammation (8). Tester’sgroup showed, by using an air pouch model, that MMP-8 wasa critical mediator of neutrophil chemotaxis because it cleavesthe LPS-induced chemokine LIX. MMP-8 is not the sole LIXactivator. MMP-12 also can cleave LIX, and as with MMP-8-deficient mice, fewer neutrophils were recruited to the airpouches of MMP-12-deficient mice (24). Impaired neutrophilmigration was also observed in the corneal stroma of MMP-8deficient mice. However, in this LPS-induced model, the che-motactic molecule produced by MMP-8 seemed to be not LIXbut the tripeptide Pro-Gly-Pro (72). In this model, MMP-9 didnot play a role in neutrophil migration (72). Addition ofTIMP-2 together with LPS also reduces the neutrophil influx(35). Therefore, MMPs are involved in neutrophil influx fol-lowing LPS exposure, which forms part of the early phase of

TABLE 3. Infections with gram-negative bacteria and Chlamydia in MMP-null and TIMP-null mutants

Pathogen Mutant Route of infection Outcome(s) Reference(s)

Pseudomonas aeruginosa TIMP-1�/� Pulmonary/corneal Increased resistance 70, 108MMP-3/TIMP-1�/� Corneal MMP-3 involved in resistance in TIMP-1�/� mice 70MMP-9/TIMP-1�/� Corneal MMP-9 involved in resistance in TIMP-1�/� mice 70MMP-7/TIMP-1�/� Corneal MMP-7 involved in resistance in TIMP-1�/� mice 70MMP-2/TIMP-1�/� Corneal Not involved in TIMP-1 resistance 70MMP-12/TIMP-1�/� Corneal Not involved in TIMP-1 resistance 70MMP-7�/� Pulmonary Delays host response 57MMP-10�/� Pulmonary More severe pneumonia 57MMP-9�/� Ocular Reduced disease symptoms, fewer Langerhans

cells and neutrophils, better integrity ofbasement membrane

87

Escherichia coli MMP-9�/� i.p. More bacterial outgrowth in peritoneal cavity,increased dissemination of infection, higherperitoneal chemokine and cytokine levels

124

MMP-7�/� Oral Diminished clearance of Escherichia coli in smallbowel (�-defensins)

157

Salmonella serovarTyphimurium

MMP-3�/� i.p. Lower levels of inflammatory cytokines detectedin tissues and serum

40

MMP-9�/� i.p. Reduced severity of Salmonella-induced colitis,immune response not affected

16

MMP-7�/� Oral Higher mortality (�-defensins) 157

Citrobacter MMP-3�/� Oral Delayed clearance of bacteria and delayedappearance of CD4� T lymphocytes

71

Yersinia MMP-3�/� i.p. Slightly less susceptible 40

Francisella tularensis MMP-9�/� Pulmonary Reduced neutrophil influx, mortality, andbacterial burden

81

Helicobacter felis MMP-9�/� Oral No difference in development of gastritis 7

Chlamydia trachomatis MMP-9�/� Intravaginal Blunted acute inflammatory response 49MMP-7�/� Intravaginal Higher no. of C. trachomatis inclusion-forming

units recovered from the small intestines ofMMP7�/� mice 2 wk postinfection(�-defensins)

114



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the host’s response to LPS, by interfering with the chemokineactivity.

In a later phase, anti-inflammatory mechanisms terminatethe production of proinflammatory mediators and chemokines,and thus the inflammatory cell influx, and promote repair oftissue damage. By cleavage and inactivation of large amountsof CXC and CC chemokines, MMP-12 can destroy the recruit-ment signals produced by these chemokines. This hypothesiswas based on the observations that the numbers of polymor-phonuclear neutrophils and macrophages were not decreasedin MMP-12-deficient mice compared to wild-type mice 72 hafter LPS instillation in the lung (24). This report by Dean etal. is an elegant example of how a protease can have multipleactivities (pro- and anti-inflammatory) during different phasesof the host response.

MMP-7 might modulate IL-1� release indirectly, via thematuration of defensins, as suggested by Shi et al., who foundthat IL-1� release from LPS-activated macrophages is com-pletely blocked by mature defensins (136).

CONCLUSIONS

Animal models of septic shock have delivered proof-of-con-cept that MMPs might be of therapeutic interest. However, thedisappointing results obtained with MMP inhibitors in the can-cer field raised serious questions about the clinical applicabilityof these inhibitors. Major concerns are the lack of selectivityand the severe side effects, such as musculoskeletal pain, thatwere observed after long-term use of MMP inhibitors. How-ever, acute diseases, such as sepsis and septic shock, usuallyrequire treatment only briefly; homeostatic, beneficial MMPswill be inhibited only for a short time, and the side effects areprobably milder. Nevertheless, the search for new, more selec-tive inhibitors is crucial, because higher selectivity not only willfurther decrease the side effects but also will be useful infundamental research. Selective inhibitors will help to unravelthe important roles that individual MMPs play in inflamma-tion, infection, and septic shock. Development of an effectivetreatment requires specific targeting of MMPs at the right

time. For this purpose, we should clearly define the differentphases of inflammation, sepsis, and septic shock and identifythe specific MMP expression and activity profile of each phase.We should try to understand both the temporal control and thetissue specificity of the expression of MMPs as well as theimportance of that expression to the outcome. When we knowwhere and when a certain MMP plays a detrimental or bene-ficial role, we can try to dampen or augment it in that organ ata particular time. Of course, answering these research ques-tions will demand a heavy investment, and translation of theresults to the clinic is not guaranteed. After all, whether atreatment is effective can be answered only empirically in clin-ical trials.

ACKNOWLEDGMENTS

This study was supported by FWO Vlaanderen, Belgium, and by theIAP-6/18 initiative of Belgian Science Policy. I.V. is a research assistantof the FWO Vlaanderen.

We thank Amin Bredan for editing the manuscript and Johan De-cruyenaere for his help with the definitions of sepsis.

We report no conflicts of interest with regard to this paper.

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TABLE 4. Overview of MMP and TIMP expression, inhibition, and knockout studies of infection, endotoxemia, and sepsis

MMP orTIMP

Study typea

Infection Endotoxemia Sepsis

Expression “Specific”inhibition Knockout Expression “Specific”

inhibition Knockout Expression “Specific”inhibition Knockout

MMP-2 � (62) � (70) � (66)MMP-3 � (40, 70, 71) � (113)MMP-7 � (57, 75, 76, 86,

102, 157, 158)� (57, 70, 114, 157)

MMP-8 � (47) � (43)MMP-9 � (7, 62, 87) � (7, 16, 49, 70, 81,

87, 124)� (66, 113) � (47) � (29) � (2, 96, 118,

161)� (124)

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Ineke Vanlaere (born 1981) graduated as aMaster in Biotechnology in 2003 with thehighest possible degrees. She was awarded aspecial prize by the examination jury andreceived a grant by the major Flemish fund-ing agency (FWO) to start a Ph.D. in Pro-fessor Libert’s laboratory. She is interestedin acute inflammation, bacterial infections,and sepsis, especially the role of matrix met-alloproteinases therein.

Claude Libert (born 1964) graduated as aMaster of Sciences in 1987. He was trainedby his mentor, Professor Fiers, in the fieldsof molecular biology and genetics. He ob-tained his Ph.D. in 1993. During 1994 to1995, he was a guest at the molecular biol-ogy laboratory Istituto di Ricerca di Biolo-gia Molecolare near Rome, Italy. In 1997,he became a group leader at Flanders Insti-tute for Biotechnology (VIB) and in 2003 aprofessor at the University of Ghent, Bel-gium. He has received several awards and published over 80 peer-reviewed papers. His major interest lies in the study of the regulationof inflammation and infection using a mouse molecular geneticapproach.



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Matrix Metalloproteinases as Drug Targets in Infections ...generally referred to as systemic...

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