pharmacology & therapeutics · trypanosomiasis leishmaniasis toxoplasmosis blood–brain...

Pharmacology & Therapeutics 133 (2012) 257–279

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Pharmacology & Therapeutics

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Associate Editor: Mauro Teixeira

Matrix metalloproteinases as therapeutic targets in protozoan parasitic infections

Nathalie Geurts, Ghislain Opdenakker, Philippe E. Van den Steen ⁎Laboratory of Immunobiology, Rega Institute for Medical Research, University of Leuven, Leuven, Minderbroedersstraat 10, B3000 Leuven, Belgium

Abbreviations: 15-HETE, 15(S,R)-hydroxy-eicosatetrmotif); BBB, blood–brain barrier; CL, cutaneous leishmaexperimental autoimmune encephalomyelitis; ECM, extnonenal; Hz, hemozoin; ICAM-1, intercellular cell adhessaccharide; ML, mucocutaneous leishmaniasis; MMP, maND, neurologically affected dogs; NF-κB, nuclear factor kpost-kala-azar dermal leishmaniasis; pRBC, parasitizedtomatic dogs; TGF-β, transforming growth factor-β; ThT cell; UD, uninfected dogs; UM, uncomplicated malaria⁎ Corresponding author. Tel.: +32 16 33 73 63; fax: +

E-mail address: [email protected]

0163-7258/$ – see front matter © 2011 Elsevier Inc. Alldoi:10.1016/j.pharmthera.2011.11.008

a b s t r a c t
a r t i c l e i n f o
Keywords:
Matrix metalloproteinasesInhibitorParasiteMalariaTrypanosomiasisLeishmaniasisToxoplasmosisBlood–brain barrierCerebrospinal fluid
Matrix metalloproteinases (MMPs) are associated with processes of tissue remodeling and are expressed inall infections with protozoan parasites. We here report the status of MMP research in malaria, trypanosomi-asis, leishmaniasis and toxoplasmosis. In all these infections, the balances between MMPs and endogenousMMP inhibitors are disturbed, mostly in favor of active proteolysis. When the infection is associated with leu-kocyte influx into specific organs, immunopathology and collateral tissue damage may occur. These patholo-gies include cerebral malaria, sleeping sickness (human African trypanosomiasis), Chagas disease (humanAmerican trypanosomiasis), leishmaniasis and toxoplasmic encephalitis in immunocompromised hosts. De-struction of the integrity of the blood–brain barrier (BBB) is a common denominator that may be executedby leukocytic MMPs under the control of host cytokines and chemokines as well as influenced by parasite prod-ucts. Mechanisms by which parasite-derived products alter host expression of MMP and endogenous MMP in-hibitors, have only been described for hemozoin (Hz) in malaria. Hence, understanding these interactions inother parasitic infections remains an important challenge. Furthermore, the involved parasites are also knownto produce their own metalloproteinases, and this forms an extra stimulus to investigate MMP inhibitorydrugs as therapeutics. MMP inhibitors (MMPIs) may dampen collateral tissue damage, as is anecdoticallyreported for tetracyclines as MMP regulators in parasite infections.

© 2011 Elsevier Inc. All rights reserved.

Contents

1. Matrix metalloproteinases: multidomain proteins covering multiple functions. . . . . . . . . . . . . . . . 2572. Matrix metalloproteinases as pharmacotherapeutic targets: intrinsic conformational dynamics in the focus . . 2593. The (patho)physiology of matrix metalloproteinases in infections: host defense and tissue damage . . . . . . 2604. Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260275

aenoic acid; actMMP, activated MMP; ADAM(-TS), a disintegrin and metalloprotease (-with a thrombospondin type 1-likeniasis; CM, cerebral malaria; CNS, central nervous system; CSF, cerebrospinal fluid; CVL, canine visceral leishmaniasis; EAE,racellular matrix; ELISA, enzyme-linked immunosorbent assay; HAT, human African trypanosomiasis; HNE, 4-hydroxy-2-ion molecule-1; IFN-, interferon-; IHC, immunohistochemistry; IL-, interleukin-; KA, kala-azar; KO, knockout; LPS, lipopoly-trix metalloproteinase; MMPI, MMP inhibitor; MS, multiple sclerosis; MT-MMP, membrane-type matrix metalloproteinase;appa-light-chain-enhancer of activated B cells; nHz, natural hemozoin; NO, nitric oxide; OD, oligosymptomatic dogs; PKDL,red blood cell; RBC, red blood cell; ROS, reactive oxygen species; RT-PCR, real-time polymerase chain reaction; SD, symp-, T helper; TIMP, tissue inhibitor of metalloproteinase; TJ, tight junction; TNF-α, tumor necrosis factor-α; Treg, regulatory; VL, visceral leishmaniasis; VWF, von Willebrand factor; WB, Western blot; WBC, white blood cell.32 16 33 73 40.

n.be (P.E. Van den Steen).

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http://dx.doi.org/10.1016/j.pharmthera.2011.11.008

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Fig. 1. Schematic representation of the domain organization of MMPs, ADAMs andADAM-TSs. A prototypic MMP incorporates an N-terminal signal peptide, a propep-tide to retain catalytic inactivity, a catalytic domain and the conserved Zn2+ bindingmotif and a C-terminal hemopexin domain (except MMP-7, -23 and -26). MMP-23 orCA-MMP (cysteine array-MMP) is localized in the membrane via a signal anchor andthis proteinase has a unique cysteine-rich array and an immunoglobulin (Ig)-like do-main. Gelatinases are distinguished by an additional gelatin-binding domain withthree fibronectin-like repeats. In addition, MMP-9 is the only MMP containing aserine-, threonine- and proline-rich O-glycosylated domain; in other MMPs only ashorter linker peptide is present. Membrane-type MMPs (MT-MMPs) are anchoredonto the cell surface by a C-terminal transmembrane domain (some MT-MMPs alsopossess a C-terminal cytoplasmic domain), or by a glycosylphosphatidylinositol(GPI) linker. The interaction between the conserved cysteine in the propeptide andthe catalytic Zn2+ ion retains the latency of the pro-enzyme. Activation of MMPsthrough removal of the propeptide according to the ‘cysteine-switch mechanism’ al-lows the Zn2+ ion to interact with a water molecule and with the substrate. RASI-1,rheumatoid arthritis synovial inflammation-1. Based on (Nagase et al., 2006; Ra &Parks, 2007). ADAMs are multidomain proteins composed of a propeptide, a metallo-protease, a disintegrin, a cysteine-rich and an Epidermal Growth Factor (EGF)-likedomain. Membrane-anchored ADAMs possess a transmembrane and cytoplasmic re-gion. ADAM-TSs have a variable number of thrombospondin type 1-like motifs.Based on Klein and Bischoff (2011).

258 N. Geurts et al. / Pharmacology & Therapeutics 133 (2012) 257–279

1. Matrix metalloproteinases:multidomain proteins covering multiple functions

Every mammalian host is in constant danger of infections causedby pathogens, such as viruses, bacteria, fungi or parasites. Host de-fense against these pathogens requires a well-regulated inflammato-ry response marked by leukocyte migration into the site of infection,killing of the microorganisms, resolution of inflammation and finallyhealing and repair of the tissue architecture. Host-derived MMPs arekey players in these processes (Parks et al., 2004). However, uncon-trolled activity of these enzymes contributes to chronic inflammationand enhanced tissue destruction (Hu et al., 2007), and may therebyhelp pathogens to disseminate throughout the body and invadeimmune-privileged sites.

Matrix metalloproteinases (MMPs) represent a large family ofover 20 different secreted or membrane-bound endopeptidases im-portant in many physiological as well as pathological conditions(Sternlicht & Werb, 2001; Parks et al., 2004; Nagase et al., 2006; Huet al., 2007). It has long been thought that MMPs participate inthese processes simply by degrading extracellular matrix (ECM) mole-cules (e.g. collagen, laminin, fibronectin) and by releasing cryptic epi-topes from the ECM. However, a much more extensive spectrum ofbiological functions has been discovered given that these enzymes acton various biomolecules, including cytokines, hormones and chemo-kines, thereby regulating immune responses. This is exemplified bythe positive feedback between MMP-9 and IL-8, where IL-8 triggersneutrophil chemotaxis and degranulation. The released MMP-9, on itsturn, cleaves and potentiates IL-8 (Van den Steen et al., 2000). Further-more,monocyte chemoattractant protein-3 (MCP-3)was identified as asubstrate of MMP-2 and cleaved MCP-3 acts as an antagonist thatdampens inflammation (McQuibban et al., 2000). Other examples in-clude several other chemokines, proTNF-α, IFN-β, serine protease in-hibitors (Opdenakker et al., 2001a,b; Sternlicht & Werb, 2001; Mott &Werb, 2004). In addition, MMPs were found to process membrane-bound as well as intracellular substrates (Cauwe et al., 2007; Cauwe &Opdenakker, 2010).

MMPs are structurally composed of an NH2-terminal prodomainand a catalytic domain containing a conserved Zn2+-binding motifwith the zinc ion liganded to three histidine residues in the sequenceAHEXGHXXGXXH (Nagase & Woessner, 1999) (Fig. 1). In addition,most MMPs possess several conserved protein domains, such as theC-terminal hemopexin domain (absent in MMP-7, -23 and -26) im-portant for binding of particular substrates, endogenous inhibitorsand cell surface receptors (Piccard et al., 2007). Gelatinases have anadditional fibronectin-like domain involved in the binding of dena-tured collagens or gelatin. The presence of an O-glycosylated (OG)domain is a unique feature of MMP-9 (Van den Steen et al., 2006a).Based on their primary structure and substrate specificity, MMPscan be categorized into collagenases, gelatinases, stromelysins, matri-lysins, membrane-type MMPs (MT-MMPs) and other MMPs (Fig. 1).MT-MMPs are linked to the cell surface by a transmembrane domainor a glycosylphosphatidylinositiol anchor (Visse & Nagase, 2003). Inthe related enzyme families of A Disintegrin And Metalloproteases(ADAMs) and ADAMs with a thrombospondin type 1-like motif(ADAM-TS), also other domains are present, including a disintegrindomain, a cysteine-rich domain (only in ADAMs), and an Epider-mal Growth Factor (EGF)-like domain. ADAM-TSs have a variablenumber of thrombospondin type 1-like repeats (Klein & Bischoff,2011).

A few MMPs, e.g. MMP-2, are mostly constitutively expressed atrelatively low levels in normal resting tissues, implying functionalroles in tissue homeostasis, e.g. constantly ongoing ECM remodeling.Most MMPs, however, are not only induced in inflammation, repairand remodeling processes, but also in several disease states. MMP ex-pression levels are precisely regulated in a spatial and temporal fash-ion, by genetic and epigenetic factors, as well as at the (post)

259N. Geurts et al. / Pharmacology & Therapeutics 133 (2012) 257–279

transcriptional level by hormones, growth factors, cytokines, cell-to-cell and cell-to-matrix interactions (Nagase & Woessner, 1999;Clark et al., 2008). MMPs are synthesized as pro-enzymes or zymo-gens and removal of the NH2-terminal propeptide is required togain catalytic activity. This process, described as the cysteine-switchmechanism, is accomplished by the disruption of the coordination be-tween the cysteine thiol group of the unique sequence motifPRCGXPD in the propeptide and the catalytic Zn2+. The unlocked ac-tive site can subsequently bind to substrates and the zinc ion becomesavailable for binding a water molecule that is essential for catalysis(Van Wart & Birkedal-Hansen, 1990). This activation process can becatalyzed by proteases and other MMPs, or alternatively by chemicaltreatment with organomercurials, urea, detergents and by reactiveoxygen species (ROS). In addition, some MMPs are activated intracel-lularly by furins (Ra & Parks, 2007). Furin is a type I membrane,subtilisin-like serine protease present in the trans-Golgi networkthat proteolytically activates a large number of proprotein substrates,including the MT-MMPs.

During normal physiological processes such as embryogenesis,precursor or stem cell mobilization and wound healing (Heissig etal., 2002; Page-McCaw et al., 2007), MMP activities are under tightcontrol by a combination of zymogen activation and inhibition. En-dogenous regulators of inhibition are primarily the tissue inhibitorsof metalloproteinases (TIMPs), which form complexes with bothpro- and activated MMP forms and whose expression is also regulatedby a network of signaling molecules (Brew & Nagase, 2010). Also, thegeneral protease inhibitor α2-macroglobulin is an important inhibitorof MMPs and blocks their activity in plasma and tissue fluids (Baker etal., 2002). This balance between activation and inhibition is essentialto avoid uncontrolled MMP activities which may cause a number ofpathological conditions such as tumor progression and metastasis incancer, vascular (e.g. stroke, atherosclerosis), neurodegenerative (e.g.Alzheimer disease, Parkinson disease) and autoimmune (e.g. rheuma-toid arthritis (RA), multiple sclerosis (MS)) diseases (Paemen et al.,1994; Folgueras et al., 2004; Burrage et al., 2006; Yong et al., 2007;Zhao et al., 2007; Rosenberg, 2009). Hence, therapeutic strategies tar-geting the MMP/TIMP balance are needed.

2. Matrix metalloproteinases as pharmacotherapeutictargets: intrinsic conformational dynamics in the focus

To intervene with uncontrolled disease-promotingMMP activities,several strategies were employed to generate potent synthetic MMPblockers. Most broad-spectrum small synthetic inhibitors – such ashydroxamates, carboxylate inhibitors and thiol inhibitors – containa zinc-chelating group that targets the conserved catalytic site (Huet al., 2007). Despite tremendous efforts, severe side-effects andproblems with bioavailability and specificity are the main causes ofpoor performances of these compounds in clinical trials. Indeed,treatment with broad-spectrum inhibitors can be either beneficial,or it can hinder recovery and even induce unacceptable side-effectsand disease. For example, in cancer, therapeutic treatment withMMPIs is intended to reduce metastasis, but it may cause tendinitisas side-effect (Drummond et al., 1999). Furthermore, MMPs are multi-functional enzymes as they not only cleave ECM but also act on variousbiomolecules. Hence, besides being target molecules, several MMPsmay be anti-targets or counter-targets at different stages of disease(Overall & Kleifeld, 2006). These pleiotropic actions of MMPs furthercomplicate research approaches for the development of clinically usefulMMPIs (Hu et al., 2007; Tu et al., 2008; Sela-Passwell et al., 2010). Prob-lems of genetic instability and unpredictability of cancer versus the hostgenetic stability and advantages of short-term treatment in acute in-flammation and infections also suggest thatMMPIsmay bemore usefulin acute inflammatory pathologies (Hu et al., 2007). Thesemay certainlyalso include infection-related immunopathologies.

Interestingly, antibiotics such as tetracyclines and chemicallymodified tetracyclines are able to bind metal ions such as zinc andthereby inhibit MMPs (Paemen et al., 1996). They also affect MMPgene expression and cross biological barriers such as the blood–brain barrier (BBB) and are therefore evaluated as agents to treat neu-rological disorders. For example, minocycline is a promising thera-peutic for MS (Brundula et al., 2002; Metz et al., 2004; Chen et al.,2011). Importantly, doxycycline is clinically approved for therapy ofperiodontitis, and its effectiveness appears mainly due to the inhibi-tion of collagenases (e.g. MMP-8) rather than to its antibacterial ef-fects (Sorsa et al., 2011).

One of the major bottlenecks in developing efficient drug candi-dates is the high structural homology between different MMP catalyt-ic domains in addition to a limited understanding of the dynamicnature of these enzymes (Sela-Passwell et al., 2010). These issues en-couraged researchers to design more elaborated selective MMPIs.

Novel classes of promising MMPIs are the mechanism-based in-hibitors, also known as the suicide inhibitors, e.g. SB-3CT(Whittaker et al., 1999; Brown et al., 2000a). Characteristic for thesecompounds is the thiirane group, which coordinates with the activesite zinc ion, leading to a conformational rearrangement that resultsin a covalent attachment to the active site glutamate and loss of enzy-matic activity. The group of Mobashery has developed a suicide inhibi-tor, selective for MMP-2 (Ikejiri et al., 2005).

Nowadays, major advances in the field of MMP crystallographyand nuclear magnetic resonance (NMR) spectroscopy and studies ofMMP structural dynamics and intrinsic flexibility boosted future per-spectives for selective and efficient inhibition (Bertini et al., 2009a;Sela-Passwell et al., 2010). Recent studies have identified exosites orallosteric protein regions as secondary binding sites that non-enzymatically may participate in MMP activation and substrate inter-action and hydrolysis. Studies on a prototypic collagenase, MMP-1,point to the importance of the hemopexin domain to locally unwindthe triple-helical fibrillar collagen to enable hydrolysis by the enzyme(Chung et al., 2004; Bertini et al., 2009b). For MMP-12, theinflammation-associated metalloelastase secreted by macrophages,NMR footprinting identified exosites on the hemopexin domain andon the periphery of the catalytic domain that endorse substrate inter-action and degradation (Bhaskaran et al., 2008; Palmier et al., 2010).Flexibility in the structure of the enzyme allows independent move-ments of the enzyme terminal domains and exosites, which will facili-tate interaction of the substrate with the active site. This isexemplified by the flexible nature of the OG domain in MMP-9(Rosenblum et al., 2007).

Also the natural MMP inhibitors, TIMPs, are the subject of detailedinvestigations. For instance, Lee et al. have recently shown that a tri-ple mutation in TIMP-1 affects its binding interface flexibility andthereby enhances its potential to inhibit MT1-MMP functions in acell-based environment (Lee et al., 2010). Such engineering ofTIMPs might also contribute to our further understanding of the mo-lecular interactions and conformational dynamics between MMPsand potential therapeutic inhibitors.

Function blocking antibodies are also attractive and promising al-ternative candidates and may specifically interact with multiple allo-steric regulatory MMP domains. For example, by specifically binding asection of the catalytic domain and not the zinc-binding site or the fi-bronectin domain, REGA-3G12 is the most selective inhibitor ofMMP-9 (Martens et al., 2007). Because of their potential high selec-tivity, the development of MMP blocking antibodies is highlyencouraged.

The identification of novel MMP topological allosteric sites impor-tant in MMP activities is a critical challenge to advance the (re)inno-vation of pharmacotherapeutics that selectively target multiplesubstrate recognition sites. Indeed, binding to the active site as wellas to particular exosites will improve the specificity and efficiencyof highly needed MMPIs.


3. The (patho)physiology of matrix metalloproteinases ininfections: host defense and tissue damage

Upon entry of a pathogen, the host innate immune system is im-mediately challenged with and triggered by pathogen-associated mo-lecular patterns (PAMPs), including lipopolysaccharides (LPS),glycolipids, glycoproteins and nucleic acids. These PAMPs are recog-nized by pattern recognition receptors (PRRs), such as the Toll-likereceptors (TLRs). The first-line host immune response to infection islocal inflammation, mediated by the recruitment of leukocytes tothe site of infection together with the release of a massive amountof defense molecules, e.g. ROS, defensins, cytokines and MMPs.MMPs are essential for leukocyte traffic through tissue barriers bydigesting ECM molecules. In addition, these enzymes modulate cyto-kine and chemokine activity, thereby establishing gradients that driveinflammatory cell recruitment and reciprocally providing negativefeedback loops that dampen inflammation. Finally, when the infec-tion is cleared, MMPs initiate tissue repair processes (Korpos et al.,2009). Although MMPs are crucial for a normal inflammatory re-sponse, uncontrolled activity of these proteases after infection endsup in severe tissue damage, microbial dissemination and immunopa-thology in the host that might lead to death. In addition to inducingMMP secretion by host cells, pathogens themselves also may producemetalloproteases which are required for virulence, e.g. falcilysin bymalaria parasites (Murata & Goldberg, 2003), gp63 by Leishmaniaparasites (McGwire et al., 2003) and toxolysin 4 secreted from Toxo-plasma gondii micronemes (Laliberte & Carruthers, 2011). Hence,metalloprotease inhibition may not only influence host pathwaysbut could also affect the parasites' metabolism (Elkington et al.,2005). In addition to MMP inhibition by antibiotics such as tetracy-clines, which inhibit both MMP gene expression and enzyme activity,synthetic low molecular weight MMPIs have been tested as potentialtherapeutics in infectious diseases. Still, blocking MMP activity withsynthetic inhibitors remains a major therapeutic challenge.

3.1. Protective versus detrimental matrixmetalloproteinases in bacterial and viral infections

Bacteria, e.g. belonging to the intestinal flora, contribute to normalhealthy physiology in man. During infections with pathogenic bacte-ria, several MMPs are upregulated and can be either protective or det-rimental for the host. For example, the beneficial role of MMP-7 hasbeen demonstrated in gastrointestinal infection of mice with Salmo-nella typhimurium (Wilson et al., 1999). MMP-7 is co-expressedwith α-defensins in the Paneth cells of the small intestinal cryptsand it protects the intestinal mucosa by activating these α-defensins, thereby igniting their bactericidal function. In addition,TIMP-1 knockout (KO) mice were resistant against Pseudomonas aer-uginosa and this was due to uninhibited protease activity since MMP-9, MMP-7 and MMP-3 were found to be essential for resistance (Leeet al., 2005). In contrast, in mycobacteria-induced tuberculosis in zeb-rafish, MMP-9 was found to promote granuloma formation by accel-erating macrophage migration, thereby favoring bacterial expansion(Volkman et al., 2010). In addition, BBB damage and neuronal injuryin human bacterial meningitis were associated with MMP-8 and -9,probably expressed by infiltrating granulocytes as well as by activat-ed brain parenchymal cells (Leppert et al., 2000). Hence, in the searchfor therapeutic strategies, it is important to keep in mind that MMPscan be targets as well as anti-targets, depending on the type ofinfection.

An overreaction of the immune system to a systemic bacterial in-fection, often by gram-negative bacteria, can evolve into severe sepsiswith organ dysfunction and finally septic shock. A unique outer mem-brane, enriched in LPS, surrounds gram-negative bacteria. In mice, ad-ministration of LPS and the subsequent exaggerated inflammatoryresponse causes an endotoxic shock and this is an appropriate

model to study immunopathology in sepsis. MMP-9 deficient micewere resistant against endotoxin shock (Dubois et al., 2002) and ele-vated levels of MMP-9 were found in plasma from patients with se-vere sepsis. MMPs might be relevant targets during sepsis andseveral studies have already demonstrated the positive effect of theuse of MMPIs (Hu et al., 2005; Vanlaere & Libert, 2009). BesidesMMP-9, TIMP-1 levels were increased in severe sepsis and it was sug-gested that TIMP-1 might be a useful marker to predict mortality inseptic shock (Hoffmann et al., 2006).

MMPs are also involved in the pathogenesis of viral infections.Chronic immune activation and inflammation as well as immunodefi-ciency cause a homeostasis imbalance between MMPs and TIMPs inhuman immunodeficiency virus (HIV) infections. Increased MMP-2,-7 and -9 levels in cerebrospinal fluid (CSF) are hallmarks of HIV-associated dementia. In addition, HIV infection of macrophages stimu-lated MMP-2 release which caused neuronal death by cleavage ofstromal cell derived factor-1α into a highly neurotoxic protein(Conant et al., 1999; Zhang et al., 2003). Human T-lymphotropicvirus 1 infection, another retrovirus that causes neuropathology, wasalso associated with increased levels of MMP-9 and TIMP-3 in CSF ofinfected individuals, implicating this enzyme in the breakdown ofthe BBB (Lezin et al., 2000). In addition, MMP-9 promoted the entryof West Nile Virus into the central nervous system (CNS) and resis-tance in MMP-9 KO mice correlated with an intact BBB (Wang et al.,2008). Interestingly, as reviewed by Westermann et al., MMPs werereported to have several roles in virus induced myocarditis, includingremodeling of the myocardium, ventricular dysfunction and modula-tion of inflammation (Westermann et al., 2010).

In conclusion, targeting excess activity of MMPs with MMPIs,whether or not in combination with antibiotics, antiviral or anti-inflammatory drugs, might be of therapeutic interest to reduce pa-thology and to increase survival in bacterial and viral infections(Mastroianni & Liuzzi, 2007; Vanlaere & Libert, 2009). However, asexemplified by some bacterial infection models, MMPs might alsocontribute to protection. It will therefore be of paramount importanceto 1) investigate which MMPs contribute to protection vs. pathologyand 2) to monitor and obtain sufficient control of the proliferationof the pathogen, e.g. by combining the MMPI with potent antimicro-bial agents.

3.2. Matrix metalloproteinase biology in parasitic infections

The symbiotic relationship between host and parasite determinesthe outcome of the infection. Indeed, on an evolutionary scale, mostparasites have had the time to adapt to the host immune systemand they have developed mechanisms to modify immune responses.Most parasites evade the host's immune defenses by hiding intracel-lularly. However, Trypanosoma brucei has managed to survive extra-cellularly via several sophisticated mechanisms. We will give anoverview of the current knowledge of the role of MMPs and TIMPsin parasitic infections.

3.2.1. Challenges for the understanding of matrixmetalloproteinase regulation and function in malaria

Malaria is a deadly infectious disease that affects 5–10% of theworld's population and causes between 300 and 500 million clinicalcases and around 1 million deaths each year. The high degree of mor-bidity and mortality together with the financial costs for malariatreatment result in an enormous socio-economic impact (Kappe etal., 2010; www.who.int/topics/malaria/en/, 2011). Once diagnosedwith malaria, a patient should be treated immediately with safe andeffective antimalarial drugs, e.g. artemisinin-based combination ther-apies, to render complete recovery. However, drug resistant parasites,severe side-effects from long term use and low efficiency to cure im-munopathological complications demand the search for new strate-gies (Wells et al., 2009). At the same time, the development of an


effective malaria vaccine remains a great challenge (Crompton et al.,2010). Human malaria is caused by five species of the genus Plasmo-dium, of which P. falciparum is the most virulent and abundant one insub-Saharan Africa. After the bite of an infected female Anophelesmosquito, sporozoites enter the bloodstream and migrate to theliver where they infect hepatocytes. Upon asymptomatic exoerythro-cytic schizogony, merozoites are released into the bloodstream andinvade red blood cells (RBCs). In the erythrocytes, the parasites mul-tiply and burst out to infect fresh RBCs, resulting in an exponential in-crease in parasite numbers. This erythrocytic schizogony isaccompanied with the release of parasite-derived products, e.g.hemozoin (Hz), and with the induction of fever and malaria-associated pathology (Schofield & Grau, 2005). Hz or malaria pigmentis a crystalline detoxification product of heme formed in the food vac-uole of parasites during hemoglobin digestion. Meanwhile, aminoacids are released for the parasite's own metabolism. Natural Hzhelps to catalyze the production of ROS thatmay generate lipoperoxidesand other breakdown products from polyunsaturated fatty acids(PUFAs), e.g. the highly reactive 4-hydroxy-2-nonenal (HNE) and15(S,R)-hydroxy-eicosatetraenoic acid (15-HETE). Phagocytosis and ac-cumulation of the malaria pigment into macrophages and neutrophilicgranulocytes cause a multidimensional immunomodulation (Arese &Schwarzer, 1997; Schwarzer et al., 1999; Hanscheid et al., 2007;Schwarzer et al., 2008).

An optimal immune response is able to clear a malaria infectionand is mediated by Th1-associated proinflammatory responses, char-acterized by interferon-γ (IFN-γ)-induced macrophage activationand induction of antiparasitic effects by tumor necrosis factor-α(TNF-α), interleukin (IL)-6, IL-1 and nitric oxide (NO). This proin-flammatory state, however, needs to be balanced in order to avoid de-velopment of severe pathology. Once parasite replication is undercontrol, a regulatory T (Treg) cell-tuned anti-inflammatory responsetakes over, characterized by cytokines such as IL-10, IL-4 and TGF-β.In parallel, CD4+ Th2 cells provide help for B cell maturation intoantibody-producing plasma cells to facilitate resolution of patent in-fection (Beeson et al., 2008; Taylor-Robinson, 2010).

People living in malaria-endemic areas gradually develop clinicalimmunity, often characterized by the chronic presence of low levelsof parasites without clinical symptoms. This semi-immunity, whichis short-lived and only slowly acquired through frequent reinfection,involves both cell-mediated immune effector mechanisms and anti-body responses (Riley et al., 1992; Perlmann & Troye-Blomberg,2002; Artavanis-Tsakonas et al., 2003; Beeson et al., 2008). In areasof high transmission, mainly infants and young children developlife-threatening complications such as cerebral malaria (CM), severemalaria anemia (SMA), blackwater fever and metabolic acidosis.Pregnant women, especially primigravidae, often suffer from placen-tal malaria. In low transmission areas, primary infection might occurin adulthood, resulting in similar complications and also in malaria-associated acute respiratory distress syndrome (Snow & Marsh,1998; Schofield & Grau, 2005).

Despite the onset of an array of immunological responses inducedby a malaria infection, the parasites manage to survive in the hostmainly by hiding intracellularly inside the RBC and by avoiding pas-sage through the spleen. By encoding variant cell surface proteins(e.g. P. falciparum erythrocyte membrane protein-1 (PfEMP-1)), theparasite enables the infected RBCs to engage multiple host receptorsexpressed by vascular endothelial cells, e.g. intercellular cell adhesionmolecule-1 (ICAM-1), vascular cell-adhesion molecule-1, endothelialcell-selectin and CD36. These sophisticated escape mechanisms intovarious organs cause the mechanical blockage of blood vessels andlocal inflammation, leading to organ-specific disease syndromessuch as placental malaria and CM (Schofield & Grau, 2005;Pasternak & Dzikowski, 2009).

In the brain, parasite-triggered production of proinflammatory cy-tokines causes enhanced adhesion molecule expression by activated

endothelial cells. This exacerbates the sequestration of parasitizedred blood cells (pRBC) in brain capillaries and post-capillary venules,thereby causing vascular obstruction and hypoxia. pRBC sequestra-tion, however, may not be sufficient to cause CM. Host immune re-sponses also play a major role in CM pathogenesis, by recruitmentand activation of leukocytes and platelets into brain microvesselsand by their accompanied excessive release of potent proinflamma-tory mediators, such as IFN-γ, TNF-α and NO. In human CM, the ma-jority of cells adhering to the cerebral microvascular endothelium arepRBCs, whereas in murine CM, the pattern is reversed in that cytoad-hesion of pRBCs is less prominent and mainly sequestration of leuko-cytes and platelets is observed (Hunt & Grau, 2003; Coltel et al., 2004;Schofield & Grau, 2005). This inflammatory cascade favors further cellsequestration as well as BBB dysfunction. In mice, dramatic leakage ofthe BBB is accompanied by the occurrence of cerebral edema, where-as brain edema in humans is more limited (Hunt et al., 2006; Medana& Turner, 2006; Mishra & Newton, 2009).

The BBB serves as a protective shield that separates the CNS fromthe systemic blood circulation. This physiological barrier is formed bycapillary endothelial cells stitched together by tight junctions (TJs)which mediate strict selective control of molecular transport on theroute across the BBB. Indeed, these TJs, composed of transmembraneproteins such as occludin, claudin and junctional adhesion molecules,drive most molecular traffic in a transcellular way rather than movingparacellularly through TJs. Below the endothelial cells is a small peri-vascular space bordered by the basal lamina. The latter consists of aninner endothelial basement membrane and an outer parenchymalbasement membrane, created by networks of ECM (glyco)proteins in-cluding laminins, fibronectin and type IV collagen. Only during in-flammatory conditions, such as in experimental autoimmuneencephalomyelitis (EAE), the endothelial and parenchymal mem-branes are microscopically distinguishable due to accumulation ofleukocytes between both membranes, thereby forming perivascularcuffs. The parenchymal membrane is interconnected to astrocyteendfeet by the transmembrane receptor dystroglycan, and togetherthis unit forms the glia limitans which delineates the border of thebrain parenchyma. Astrocytes and microglial cells in the brain paren-chyma provide support and protection to the neurons. The blood–CSFbarrier is established by the fenestrated endothelia of the choroidplexus. These endothelial cells not only cover a barrier function, butthey also secrete the CSF. The endothelial TJs of the choroid plexus,however, do not restrict leukocyte diapedesis. Hence, under normalphysiological conditions, a limited number of activated lymphocytesare exceptionally allowed to access the CNS to undertake immune-surveillance (Engelhardt & Sorokin, 2009; Sorokin, 2010; Wilson etal., 2010). This brain-monitoring is essential to deal with infectionsor inflammatory conditions, which require leukocyte influx into thebrain. Transmigration of leukocytes from blood into the CNS involvesmultiple steps mediated by adhesion molecules, cytokines, chemo-kines and MMPs. Leukocyte migration across the BBB, which takesplace at the level of the post-capillary venules, is likely to occur viaa transcellular pathway, leaving the TJs intact. Subsequently, theirtrafficking into the brain parenchyma is regulated by the biochemicalcomposition of both the endothelial and parenchymal basementmembranes. Disruption of these biological barriers is mediated bythe action of MMPs as described in many neurological diseases(Yong et al., 2001; Engelhardt & Wolburg, 2004; Prendergast &Anderton, 2009; Rosenberg, 2009).

Both in murine and human CM, leukocytes, platelets and pRBCsusually do not actively transmigrate to the brain parenchyma but re-main intravascularly adhering to the endothelium (Schofield & Grau,2005). This adhesion to brain endothelial cells together with an im-balance between pro- and anti-inflammatory cytokines causes signif-icant changes in the expression and function of intercellularjunctional proteins which might lead to increased permeability ofthe BBB to plasma proteins (Adams et al., 2002; Combes et al., 2006;

Table 1Expression and function of MMPs/TIMPs/ADAM(-TS)s in malaria (Plasmodium spp.).

Model Mmpa-MMPb/Timpa-TIMPb expression patternc Functional data/associations Comments References

1. IN VITROHuman monocytes+trophozoites,nHz or control meals

nHz/15-HETE→NF-κB ↑→ IL-1β ↑→TNF-α ↑→ MMP-9↑MMP-9 and TNF-α→mutually correlated (Prato et al., 2005):+ rhTNF-α: MMP-9↑+ anti-hTNF-α: MMP-9↓TNF-α/MMP-9 ↑→ IL-1β dependent (Prato et al., 2008):+ rhIL-1β: MMP-9↑+ anti-rhIL-1β: TNF-α↓, MMP-9↓IL-1β/TNF-α/MMP-9 ↑→NF-κB dependent (Prato et al., 2010):+ NF-κB inhibitors: IL-1β↓, TNF-α↓, MMP-9↓ (Prato et al., 2010)+ Pg-FET and constituents: NF-κB↓→MMP-9↓ (Dell'agli et al., 2010)(zymography, WB, RT-PCR, assay of matrix invasion, ELISA, EMSA,cDNA macroarray)

Matrix invasion ↑ by Hz-fed monocytesMMP(-9) inhibition: → soluble TNF-α ↓

Hz-enhanced MMP-9 effects, such as (IL-1β)/TNF-α shedding and disruption of basal laminacould be involved in CM pathogenesis.

Dell'agli et al.,2010; Giribaldi etal., 2010; Prato etal., 2005; Prato etal., 2008; Prato etal., 2010

Human recombinantproMMP-9+sHz

β-Ht-induced autocatalytic processing of MMP-9 prodomain→moreefficient activation of truncated MMP-9 by MMP-3 (zymography,WB, Edman degradation, activity assays)

β-Ht (sHz)-primed MMP-9 activation might berelevant in human CM, where Hz particles weredetected in brain microvasculature→activatedMMP-9 might contribute to disruption of theBBB in CM.

Geurts et al., 2008

(Murine LPS-stimulatedmacrophage-like) RAW 264.7 cellstreated with latex beads, β-Ht,HNE or 15-HETE

HNE-induced Mmp-9/MMP-9↑; Timp-1↓15-HETE-induced Mmp-9↓ (microarray, RT-PCR, ELISA)

Steady-state MMP-9/TIMP-1 imbalance maylead to increased proteolysis of the ECM; al-though 15-HETE treatment resulted in elevationof IL-1β mRNA, Mmp-9 expression wasdownregulated.

Schrimpe & Wright,2009a; Schrimpe &Wright, 2009b

HMEC-1 cells+nHZ total gelatinolytic activity ↑proMMP-9 and actMMP-9↑MMP-1, MMP-3↑; TIMP-2↑ (fluorogenic gelatin conversion assay,zymography, WB, microvessel-like structuresformation assay)

↑ Number of microvessel-like structures ProMMP-9 might be activated by MMP-3 in theendothelium and activated MMP-9 may con-tribute to BBB disruption; morphologicalchanges in nHz-treated cells are suggested to beconsistent with the upregulation of MMPs.

Prato et al., 2011

Human umbilical veinendothelial cells and platelets,P. falciparum cultures

Flow adhesion experiments By cleaving VWF multimers, addition ofADAM-TS13 cleared pRBCs that are tetheredvia platelet-decorated ULVWF strings onactivated endothelium

Endothelial cell activation and release of ULVWFmultimers enables adhesion of pRBC via CD36-dependent platelet bridging, a mechanism thatmay contribute to sequestration.

Bridges et al., 2010

2. IN VIVOPlasma samples from P. bergheiANKA-infected mice

MMP-9↑Treatment DPTA/NO donor: MMP-9↓ (ELISA usingLuminex be

Administration of NO prevents the developmentof experimental CM and reduces the induction ofMMP-9 and its BBB-damaging capacity.

Gramaglia et al.,2006

Mouse infection models:C57Bl/6+P. berghei ANKA: CM-susc.C57Bl/6+P. berghei NK65: no CMBALB/c+P. berghei ANKA: CM-resist.C57Bl/6 MMP-9KO+P. berghei ANKA:CM-susc.

Brain in CM P-9+ CD11b+ cellsBrain in CM p-8,-13,-3, Timp-1↑; Mmp-12↓, MMP-2,-9↑,actMMP-9↑ h ‘net’ gelatinolytic activity associated withblood vesseBrain in CMSpleen in CactMMP-9↓Liver in CMMt2-mmp↓Lungs in CM

MMP-9KO in C57Bl/6±ANKA:→ no effect on CM→ no effect on ‘net’ gelatinolytic activity

Limited effect of MMP-inhibition on CM patho-genesis suggest that some MMPs may bedisease-promoting (MMP-8 in brain), while

Van den Steen etal., 2006b

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withads): MM: Mm, hig
ls in brainresistant: Mmp-3, Timp-1↑, little gelatinolytic activity
M: Mmp-3,-8,-13, Timp-1,-3, Mt1-mmp↑, TACE↑; MMP-2↓,

: Mmp-7,-8,-12,-13, Mt1-mmp, Timp-1,-3↑;; TACE↑; MMP-9↑: Mmp-8,-9↑, Mmp-11↓

MMP-9 not essential in CM pathogenesisIn situ zymography±EDTA:→ Abrogation of ‘net’ gelatinolytic activityin CM-susceptible miceMMP inhibition with BB-94:→ Limited effect, delay of CM with 1 dayN.B.: single treatment on day 6

others could be protective (MMP-3 in brain).

Kidneys in CM: Mmp-11, Mt2-mmp↓; Mt1-mmp↑; MMP-2↓;MMP-9↑, (RPAs, RT-PCR, (in situ) zymography, IHC)

Brain P. berghei ANKA-infectedC57Bl/6 mice (CM susc.)

MMP-7↑ in cerebral neocortex of infected mice (IF, WB) Increased MMP-7 proximal to the neuronalsynapse may influence synaptic structure andfunction.

Szklarczyk et al.,2007

3. CLINICAL STUDIESCSF of Vietnamese adults witha variety of neurologic andinfectious diseases (malaria)

MMP-9 in CSF is similar in CM patients and in controls(radial immunodiffusion assay, ELISA)

MMP-9 is linked to a loss of BBB integrity inmeningitis, but unchanged levels of MMP-9 inCSF of CM patients means no significant BBBbreakdown in this population.

Brown et al., 2000b

Brain samples of P. falciparum-infectedpatients with CM vs. without CM

VEGF-induced MMP-1↑ in astrocytes and Dürck's granulomas in brainof CM patients (IHC, WB)

Similar VEGF, Flt-1, CTGF and MMP-1 labelingpatterns in patients who died with CM

Ongoing angiogenesis may lead to neurologicalsequelae and fatal brain edema during CM.

Deininger et al.,2003

Whole blood from convalescent(negative for parasites) vs. acutelyill Kenyan children

Mmp-9↑ in blood from acutely ill subjects (microarray, RT-PCR) Neutrophils and macrophages produce MMP-9,neutrophilia has been linked to acute malaria.

Griffiths et al., 2005

Serum samples from Gabonesechildren with SM, UM vs. HCnegative for parasites

MMP-8↑ SM and UM vs. HCTIMP-1↑ SM vs. UM vs. HCTIMP-2↓ SM and UM vs. HCMMP-9 levels are equal in SM, UM and HC (ELISA)

→ TIMP-1 levels correlated significantly withclinical parameters in SM→ TIMP-1 levels correlated significantly withMMP-8 level→ MMP-8 levels correlated significantly withneutrophil numbers

MMP-8 may mediate BBB damage, TIMP-1 mayhave a protective role in the pathogenesis of SMby compensating the activity of MMP-9 andMMP-8, thereby preventing further detrimentalMMP activities.

Dietmann et al.,2008

CSF of CM patients vs. uninfectedpatients

MMP-1,-2,-3,-7,-8,-9,-10 and TIMP-1,-2,-4 in CSF is similar in CMpatients and in controls (ELISA)

Green et al., 2011

Plasma from patients withsymptomatic P. falciparum orP. vivax infection vs. healthycontrols with negative blood slide

ADAM-TS13 antigen↓ADAM-TS13 activity↓ (ELISA, FRET assay+VWF proteolysis assayfor ADAM-TS13 activity)

Endothelial cell perturbation, ADAM-TS13 defi-ciency and increased levels of active and ULVWFmultimers may contribute to malaria-relatedthrombocytopenia.

de Mast et al., 2009

Plasma from children withsevere malaria vs. healthy children

ADAM-TS13 antigen↓ADAM-TS13 activity↓ (ELISA, FRET assay+VWF proteolysis assayfor ADAM-TS13 activity)

Time-dependent inhibition of ADAM-TS13activity:→ Mixing studies of malaria and controlplasma→ Spiking of patient plasma with rec. humanADAM-TS13

The presence of ULVWF multimers is associatedwith a significant reduction in plasma levels andactivities of the VWF-cleaving protease ADAM-TS13. This is mediated by an unidentified inhib-itor. The ULVWF multimers may be involved inmediating the pathophysiology of severe P. fal-ciparum malaria.

Larkin et al., 2009

Plasma from P. falciparum-infected patients with SMand UM vs. HC

ADAM-TS13 activity↓: SMbUM=HC (ELISA, FRET assay+VWFproteolysis assay for ADAM-TS13 activity)

ADAM-TS13 activity correlated with overalldisease severity, including coma

Presence of low ADAM-TS13 activity combinedwith high VWF concentrations can contribute tothrombocytopenia. ULVWF multimers couldcause platelet accumulation in the microvascu-lature which would lead to cytoadherence ofpRBCs.

Löwenberg et al.,2010

↑ indicates increased levels; ↓ indicates decreased levels; 15-HETE, 15(S,R)-hydroxy-eicosatetraenoic acid; ADAM(-TS): a disintegrin and metalloprotease (-with a thrombospondin type 1-like motif); actMMP, activated MMP; BB-94, broad-spectrum MMP and TACE-inhibitor from British Biotech.; BBB, blood–brain barrier; β-Ht, β-hematin; CM, cerebral malaria; CSF, cerebrospinal fluid; CTGF, connective tissue growth factor; DPTA/NO, dipropylene-triamine NONOate; ECM,extracellular matrix; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; Flt-1, Fms-related tyrosine kinase/vascular endothelial growth factor receptor-1(VEGFR-1); HC, healthy control; HMEC-1, human microvascular endothelial cell line; HNE, 4-hydroxy-2-nonenal; Hz, hemozoin; IF, immunofluorescence; IHC, immunohistochemistry; IL-1β, interleukin-1 β; KO, knockout; LPS, lipopolysac-charide; MMP, matrix metalloproteinase; MT-MMP: membrane-type matrix metalloproteinase; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; nHz, natural hemozoin; Pg-FET, Punica granatum-fraction enriched intannins; pRBCs, parasitized red blood cells; rec., recombinant; resist., resistant; RPA, ribonuclease protection assay; RT-PCR, real-time polymerase chain reaction; sHz, synthetic hemozoin; SM, severe malaria; susc., susceptible; TACE,TNF-α converting enzyme; TIMP, tissue inhibitor of metalloproteinases; TNF-α, tumor necrosis factor-α; UL, ultra-large; UM, uncomplicated malaria; VEGF, vascular endothelial growth factor; VWF, vonWillebrand factor; WB, Western blot.

a Mmp/Timp refers to mRNAs.b MMP/TIMP refers to proteins.c bold lettering indicates the specific molecules under study.

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Jambou et al., 2010). Evidence for structural changes in the BBB dur-ing CM is supported by human and mouse studies. For example,Brown et al. observed loss of endothelial cell junction proteins zonaoccludens-1 and occludin in post-mortem brain samples of adult ma-laria patients (Brown et al., 1999). Progressive deterioration of theBBB integrity was also observed in a murine CM model (Polder etal., 1992; Medana et al., 2000). In addition, CD8+ T cells are essentialin experimental CM by damaging endothelial cells via perforin- andgranzyme-dependent mechanisms (Nitcheu et al., 2003; Haque etal., 2011). As a result, increased paracellular transport is expected tocause leakage of plasma constituents and malarial antigens as wellas other harmful molecules into the brain compartment from whichthey are normally excluded. These generalized alterations in theBBB might contribute to pathological hallmarks such as perivascularedema, microhemorrhages, glial activation and redistribution and as-trocyte apoptosis. Later, lesions known as Dürck's granulomas, char-acterized by patchy gliosis around the ruptured vessel accompaniedby the presence of parasite debris that is phagocytosed by host mono-cytes and glial cells, may become apparent in the brain parenchyma(Turner, 1997). Finally, these neurological sequelae may contributeto neuronal dysfunction and cause coma and death (Patnaik et al.,1994; Deininger et al., 2002; Coltel et al., 2004; Hunt et al., 2006;Medana & Turner, 2006).

In several neurological disorders, MMPs derived from infiltratingleukocytes or from cells of the CNS cleave matrix proteins that sup-port BBB integrity and neuronal survival (Yong, 2005). For example,breakdown of the BBB and the subsequent leakage of microvesselswere observed after intraparenchymal injection of MMPs(Rosenberg et al., 1992). In MS patients, IFN-β treatment confers pro-tection from relapses of MS. From in vitro studies, it was suggestedthat beneficial effects of IFN-β may result from diminished gelatinasesecretion by human T cells, which reduces their potential to travelacross the BBB (Leppert et al., 1996). In addition, in cerebral ischemia,MMP-2, -3 and -9 damage and open the BBB by attacking proteins inendothelial TJs, e.g. occludin and claudin-5, as well as components ofthe basal lamina, e.g. laminin and collagen type IV (Yong et al., 2001;Rosenberg & Yang, 2007). Treatment with synthetic MMPIs wasreported to block the opening of the BBB (Mun-Bryce & Rosenberg,1998; Rosenberg et al., 1998). Furthermore, tetracyclines, which areantibiotics able to inhibit MMPs at the level of transcription and activity(Uitto et al., 1994; Paemen et al., 1996), were shown to possess po-tential as therapies for MS by targeting leukocyte transmigration(Brundula et al., 2002). In a mouse model of EAE, knocking outboth MMP-2 and MMP-9 genes confers protection against damageto the brain parenchyma (Agrawal et al., 2006). Hence, MMPs andTIMPs may be relevant for malaria pathogenesis either by degradingBBB substrates or as effectors and regulators of the immune re-sponse. Table 1 gives an overview on the MMP/TIMP expression pat-terns and functions in malaria infections. In addition, published dataon ADAM(-TS)s are included.

Expression analysis of blood cell samples from Kenyan childrenwith acute malaria revealed an upregulated MMP-9mRNA expressionwhich was associated with malaria-linked neutrophilia and inter-preted as a proinflammatory response (Griffiths et al., 2005). Howev-er, in Gabonese children with uncomplicated and severe malaria,MMP-9 serum levels were unaltered compared to healthy controls,and TIMP-2 in healthy controls was higher than in malaria patients.Levels of MMP-8 and TIMP-1 were significantly increased in patientswith severe and uncomplicated malaria. Interestingly, TIMP-1 levelscorrelated significantly with clinical disease severity and were higherin children classified with CM. Moreover, TIMP-1 also correlated withMMP-8, and MMP-8 was associated with neutrophil numbers(Dietmann et al., 2008). These results suggest that TIMP-1 andMMP-8 are markers of malaria severity and TIMP-1 may prevent fur-ther MMP-induced damage by counterbalancing the activity of MMP-9 and, to a lesser extent, MMP-8 activity.

During experimental CM, increased MMP-9 levels were observedin plasma samples on day 6 of infection. This induction was counter-regulated by the administration of exogenous NO. Interestingly, NOtreatment was accompanied by significantly reduced vascular leakand hemorrhage into the brain. Hence, NO protects from develop-ment of CM and inhibits MMP-9 mediated BBB damage (Gramagliaet al., 2006). An increased expression of various MMPs and TIMPswas also observed in different organs in experimental CM. However,their exact role in malaria infection is not yet clear. Interestingly,TIMP-1 mRNA levels were more increased in the brain of CM-resistant Balb/c mice compared to CM-susceptible C57Bl/6 mice.This may possibly suggest a protective function of TIMP-1 in thebrain. Taken together, these data provide evidence that MMPs andTIMPs are critically involved in malaria pathogenesis (Van denSteen et al., 2006b).

Hz and its association with malaria pathophysiology are well-documented, although controversy exists whether it acts as an activa-tor or suppressor of the immune system (Hanscheid et al., 2007;Schwarzer et al., 2008). Activation of the immune system by Hz wasevidenced by various studies. For example, β-hematin, a synthetic crys-tal chemically and structurally identical to native Hz (Egan, 2002), wasshown to induce the activation and migration of leukocytes (Huy et al.,2006). In addition, uptake of synthetic or native Hz by host cells wasfound to stimulate release of proinflammatory molecules, e.g. TNF-α,Il-1β and macrophage inflammatory protein-1α (Pichyangkul et al.,1994; Jaramillo et al., 2004; Jaramillo et al., 2009). In contrast, severalstudies reported on the impairment of activities such as oxidativeburst and repeated phagocytosis (Schwarzer et al., 1992), antigen pro-cessing (Scorza et al., 1999) and dendritic cell maturation (Skorokhodet al., 2004) by Hz. Interestingly, in human CM, Hz particles havebeen detected in the brain microvasculature (Silamut et al., 1999;Grau et al., 2003). We found that a direct interaction in vitro betweenβ-hematin and the hemopexin domain ofMMP-9 significantly facilitatedactivation of MMP-9 by MMP-3, a well-known activator of MMP-9(Geurts et al., 2008). This Hz-mediated priming of MMP-9 activitymight contribute to an accelerated BBB disruption during CM. Interest-ingly, activated MMP-9 was detected in the brain in an experimentalmodel for CM (C57Bl/6 mice infected with P. berghei ANKA). Intense‘net’ gelatinolytic activity was observed in microcapillaries and largerblood vessels in the brain of CM-susceptible mice. This activity was ab-rogated by ethylenediaminetetraacetic acid (EDTA) and therefore likelywas the result of MMP activity. Only little activity was detected in thebrains of infected CM-resistant Balb/c mice. Hence, these results indi-cate that intense MMP activity is associated with CM (Van den Steenet al., 2006b).

As also recently reviewed by Prato and Giribaldi, several in vitrostudies are in line with TNF-α-dependent induction of MMP-9mRNA and protein by monocytes fed with natural Hz (nHz) or trea-ted with 15-HETE, a lipoperoxidation derivative generated by Hz(Prato et al., 2005; Prato et al., 2008; Giribaldi et al., 2010; Pratoet al., 2010; Prato & Giribaldi, 2011). Uptake of Hz was also accompa-nied by an increased matrix invasion ability (Prato et al., 2005). Inter-estingly, besides the release of TNF-α by the converting enzyme(TACE) (the main enzyme responsible for the release of TNF-α fromcell-bound precursors) (Mohan et al., 2002), membrane-boundTNF-α can also be released in vitro by several MMPs, includingMMP-9, thereby closing a positive feedback loop. This mutual inter-dependence between TNF-α and MMP-9 has been described previ-ously (Gearing et al., 1995; Bond et al., 1998). However, the mainsheddase of membrane-bound TNF-α is TNF-α converting enzyme(TACE), also named ADAM-17 (Black et al., 1997; Moss et al., 1997;Mohan et al., 2002). Its expression was found to be increased at day6 in both liver and spleen of mice with experimental CM (Van denSteen et al., 2006b). Increased plasma levels of TNF-α is one of thehallmarks of CM, together with BBB breakdown (Medana & Turner,2006).


Hence, sinceMMP-9 production and activation increase BBB perme-ability and infiltration of leukocytes into the brain in multiple diseases,e.g. MS (Mun-Bryce & Rosenberg, 1998; Muroski et al., 2008), MMP-9might also play an important role in CM pathogenesis. Recently, Pratoet al. demonstrated the induction of activated MMP-9 and an increasein both MMP-1, MMP-3 and TIMP-2 protein expression after stimula-tion of the human microvascular endothelial cell line HMEC-1 withnHz (Prato et al., 2011). This nHz-mediated production of activatedMMP-9 by endothelial cells might reflect a mechanism that contributesto BBB breakdown during CM.

Schrimpe et al. found, however, a down-regulation in macrophageMMP-9 expression after 15-HETE treatment (Schrimpe & Wright,2009b), but they observed lower TIMP-1 levels together with an in-duced MMP-9 expression by HNE, another lipoperoxidation productof Hz (Schrimpe & Wright, 2009a). Interestingly, tannins from thefruit rind of Punica granatum inhibited the TNF-α induced expressionand secretion of MMP-9 by THP-1 cells. Suppression of excess host-inflammatory responses together with the previously describedanti-parasitic activity (Dell'agli et al., 2009) could explain the benefi-cial effect of the fruit rind of P. granatum, which is presently distribut-ed in India as a herbal formulation for the therapy and prophylaxis ofmalaria (OMARIA, Orissa Malaria Research Indigenous Attempt)(Dell'agli et al., 2010). Furthermore, the most efficient drug againstcomplicated malaria, i.e. artemisinin, is able to block the expressionof MMP-9 (Prato et al., 2010; Wang et al., 2011). Although MMP-9 ex-pression and activation were observed in the brain in a specific exper-imental model of CM, no effect on the development of CM andsurvival was found in MMP-9 KO mice, indicating no essential rolefor this single MMP in CM pathogenesis. Compared to wild typeCM-susceptible mice, the ‘net’ gelatinolytic activity was also not al-tered in the brain of MMP-9 KO mice (Van den Steen et al., 2006b).This suggests that this EDTA-inhibitable activity also originates fromother proteinases, such as MMP-2. In addition, clinical studies byBrown et al. provide evidence for a role of MMP-9 in loss of BBB integ-rity only in bacterial and tuberculous meningitis, but not in CM. In-deed, the albumin index together with the unaltered levels ofMMP-9 in CSF of infected patients even implied only a minimal de-gree of BBB breakdown in CM (Brown et al., 2000b). Recent workby Green et al. also demonstrates that MMP/TIMP expression is notaltered in CSF of CM patients compared to controls (Green et al.,2011).

Increased MMP-7 protein expression levels were observed nearthe neuronal synapse in the neocortex from P. berghei ANKA-infected mice and thus might influence synaptic structure and func-tion (Szklarczyk et al., 2007). In contrast, we detected upregulatedMMP-7 mRNA expression only in the liver from P. berghei ANKA sus-ceptible mice. Moreover, even though several MMPs, MT-MMPs andTIMPs were increased in diverse organs of experimental CM, MMP-inhibition had only a limited effect on CM pathogenesis and survival,suggesting that some MMPs might be disease-aggravating, whileothers might be protective (Van den Steen et al., 2006b).

Analysis of post-mortem brain samples from P. falciparum-infectedpatients revealed MMP-1 protein accumulation in astrocytes of theBBB and in macrophages/microglial cells of Dürck's granulomas. Inaddition, similar expression patterns of vascular endothelial growthfactor, Fms-related tyrosine kinase 1, connective tissue growth factorand MMP-1 were observed, reflecting ongoing angiogenesis in thebrains of patients who died with CM (Deininger et al., 2003).

In addition, the MMP-related enzyme ADAM-TS13, which is avon Willebrand factor (VWF)-specific cleaving protease, mighthave a protective role against severe malaria. This enzyme is de-creased in plasma from patients with severe malaria (de Mast etal., 2009; Larkin et al., 2009; Löwenberg et al., 2010), resulting inultra-large von Willebrand multimers. These multimers mediatethe bridging of platelets to activated endothelium, andP. falciparum-infected erythrocytes may sequester on these platelets

in a CD36-mediated fashion. This process is inhibited by ADAM-TS13 (Bridges et al., 2010).

An overview of the possible interactions between MMPs/TIMPs/ADAM-TS13 and the BBB during a malaria infection is given in Fig. 2.

In conclusion, these data highlight the altered (MT-)MMP/TIMP/ADAM-TS13 expression patterns accompanied with a Plasmodium in-fection, particularly in CM. Hence, MMPs and their natural inhibitorsare involved in malaria and uncontrolled MMP activities might reflectmechanisms that contribute to the onset of malaria pathology, for ex-ample by breaking down components of the BBB that is characteristicfor CM. However, direct or conclusive evidence of these enzymes forplaying an important role in disruption of the BBB during malaria in-fection is lacking. Studies using MMP KO mice or (specific) MMPIsmight yield new insights concerning any functional role of these en-zymes in malaria pathogenesis.

3.2.2. Matrix metalloproteinases as therapeutic targetsagainst BBB damage and infiltration by African trypanosomes

Trypanosomiasis includes a group of diseases caused by protozoanparasites of the genus Trypanosoma. African trypanosomes (T. brucei)are responsible for one of the most important parasitic infectionsthroughout numerous countries in sub-Saharan Africa. These flagel-lated unicellular parasites are the causative agents of human sleepingsickness (human African trypanosomiasis, HAT) in man and Naganain cattle. These diseases lead to enormous consequences for publichealth and economic development in the affected areas. In 1986, theWHO estimated that 70 million people were at risk of HAT and in1998, 40,000 cases were reported. It is believed that many cases re-main undiagnosed in Africa each year. Due to great improvementsin HAT control, a 69% reduction has been observed in reported casesbetween 1997 and 2006. The estimated number of actual cases is cur-rently 30,000 (www.who.int/mediacentre/factsheets/fs259/en/,2010). African trypanosomes are parasites dwelling extracellularlyin blood and CSF after they have been transmitted through the biteof an infected tsetse fly (Glossina species). After injection, metacyclictrypomastigotes transform into proliferating long slenders in the cir-culation and even cross the BBB and enter the CSF. These long slen-ders change into short stumpy forms which need to be ingested bya tsetse fly in order to survive (Matthews & Gull, 1994). Parasites ofthe T. b. brucei sub-species are not pathogenic for human hosts, asthey are efficiently lysed by serum factors, such as the lipoproteinapoL-1 (Pays et al., 2006). This parasite is used to study experimentaltrypanosomiasis in mice. HAT is caused by T. b. rhodesiense (acute) orT. b. gambiense (chronic) which are resistant against normal humanserum-mediated lysis (De Greef & Hamers, 1994). Trypanosomesmodulate host-immune responses in such a way that parasite aswell as host viability is benefited. Upon infection, macrophage activa-tion, antibody production and Th1 polarized cytokine responses aretriggered, leading to control of parasite growth (Vincendeau &Bouteille, 2006; Tabel et al., 2008). In trypanotolerant C57Bl/6 wildtype mice infected with T. congolense, IL-10 produced by Tregs wasfound to be a key cytokine in counterbalancing excessive IFN-γ pro-duction by activated macrophages, thereby preserving host tissuefrom damage in experimental African trypanosomiasis (Guilliams etal., 2007). African trypanosomes have developed sophisticated mech-anisms to establish a chronic infection in the mammalian host, in-cluding antigenic variation of the variant surface glycoprotein coat(Pays, 2006), polyclonal B cell activation (Sacco et al., 1994) and theinduction of a generalized state of immunosuppression (Darji et al.,1992).

The first or hemolymphatic stage, before the parasites have enteredthe CNS, is associated with symptoms like intermittent fever, malaiseand generalized lymphadenopathy. The second ormeningoencephaliticstage of HAT is manifested when trypanosomes manage to cross theBBB and invade the CNS. They first pass through the highly vascularizedepitheliumof the choroid plexus into the CSF. Later, they enter the brain

Fig. 2. BBB alterations and MMPs/TIMPs/ADAM(-TS)s in (cerebral) malaria. The BBB is composed of endothelial cells and their TJs and of the underlying basal lamina. The latterconsist of the endothelial basement membrane, underlying the endothelial cells, and the parenchymal basement membrane that borders the brain parenchyma. Astrocytes andmicroglial cells in the brain parenchyma provide support and protection for neurons. Note that endothelial cells and some astrocyte endfeet are enlarged in the figure. Adherenceof pRBC onto brain endothelial cells via EMP-1 induces inflammation marked by recruitment and activation of leukocytes and platelets. CD36 is not expressed on brain endotheliumbut its surface expression by platelets can modulate sequestration of (p)RBCs. Leukocytes adhere to endothelial cell surface receptors, e.g. ICAM-1 and P-selectin through lympho-cyte function-associated antigen 1 (LFA-1) and P-selectin glycoprotein ligand-1 (PSGL-1), respectively. The excessive release of proinflammatory cytokines will cause endothelialcell damage and BBB dysfunction and may be further exacerbated by the presence of Hz. MMPs might contribute to the enhanced BBB permeability by attacking endothelial TJs anddegrading basement membrane, thereby supporting leakage of plasma proteins and malarial antigens across the BBB into the brain compartment. However, leukocytes and pRBCswere assumed not to penetrate brain parenchyma. MMP-9 positive myeloid cells were observed in the brain from CM-susceptible mice. One of the hallmarks of CM pathogenesisare Dürck's granulomas which are usually seen in older hemorrhages. These granulomas are characterized by gliosis associated with the ruptured vessels and the presence of par-asite debris that is phagocytosed by monocytes and glial cells. In post-mortem brain samples of CM patients, increased expression of MMP-1 was found associated with Dürck's gran-ulomas. In addition, increased levels of MMP-8 and TIMP-1 were observed in serum samples of human patients with severe and uncomplicated malaria. In severe malaria, ultra-large von Willebrand factor (ULVWF) strands, secreted by the endothelium, bridge platelets to the endothelium, thereby facilitating parasite sequestration. Accumulation ofthese ULVWF multimers results from malaria-associated deficiency in ADAM-TS13, the VWF-cleaving metalloprotease.


parenchyma by traversing the BBB. This coincides with the induced ex-pression of host endothelial receptors, e.g. ICAM-1. The accumulation ofparasites in the brain, paralleled by the recruitment of leukocytes,causes chronic encephalopathy. Hence, treatment of the second stagerequires drugs that can enter the brain to reach the parasite. Patientslose weight, suffer from anemia and have a wide range of mental,motor and sensory symptoms and circadian pattern (i.e. sleep) distur-bances. In addition, infiltration of inflammatory cells and activation ofastrocytes and microglia characterize the induced neuropathology.Without treatment, the disease is invariably fatal (Enanga et al., 2002;Kristensson et al., 2010; Rodgers, 2010).

In mice and rats, trypanosomes cross the BBB and enter the brainparenchyma not only in a transcellular, but also in a paracellular man-ner (Kim, 2008). Brucipain, a cystein protease secreted from African

trypanosomes, was suggested to enable parasite traversal of the BBB(Grab & Kennedy, 2008). In addition, lymphocyte-derived IFN-γ wasfound to be a key regulator in parasite penetration into the brain pa-renchyma (Masocha et al., 2004). In contrast to the findings ofMulenga et al., who reported that trypanosomes cross the BBB with-out any loss of TJs (Mulenga et al., 2001), other groups have founddisparate results. Philip et al. observed extensive dye leakage duringchronic trypanosomiasis in a rat model, indicating progressive dam-age of the BBB as the disease advanced (Philip et al., 1994). Recently,imaging of the whole brain by applying the magnetic resonance im-aging technique indicated significant BBB dysfunction in a murinemodel of CNS stage trypanosomiasis (Rodgers et al., 2011).

Although controversy exists whether the BBB is disrupted,channeling of leukocytes and parasites through the basal lamina

image of Fig.�2


into the brain at second stages of HAT is a similar and interrelatedprocess and may involve the action of MMPs. Hence, the role ofMMPs herein was investigated by several groups. Table 2 summarizesthe current knowledge on the MMP/TIMP expression profiles andfunctions during African trypanosomiasis.

In the CSF of T. b. gambiense-infected patients with second-stageHAT, MMP-2 and MMP-9 were significantly induced and were corre-lated with the presence of parasites and leukocytes in the CSF. Thissuggested that gelatinase activity assisted in the infiltration into theCNS (Hainard et al., 2011). In addition, these authors determined 3markers, i.e. ICAM-1, MMP-9 and heart-type fatty acid binding pro-tein (H-FABP), which provided a powerful panel for staging HAT.Both MMP-2 and MMP-9 were assigned a crucial role for leukocytepenetration of the outer parenchymal basement membrane into thebrain parenchyma by the cleavage of the transmembrane anchor pro-tein β-dystroglycan (Agrawal et al., 2006). Indeed, MMP inhibitionduring EAE resulted in the perivascular arrest of lymphocyte infiltra-tion. In the brain of T. b. brucei-infected mice, a massive increase inparasite and leukocyte numbers was, among other things, accompa-nied by a significantly induced mRNA expression of MMP-3 andMMP-12 (Masocha et al., 2006). Treatment with minocycline down-regulated this increased enzyme expression and was associatedwith prolonged survival of the animals due to a considerably sup-pressed influx of parasites and leukocytes into the brain. Minocyline,however, had no effect on peripheral parasite levels. Hence, by digest-ing components of the ECM and basement membranes, both MMPsmight contribute to trypanosome and leukocyte neuroinvasion.Fig. 3 represents the possible contribution of MMPs in the develop-ment of HAT.

In summary, excessive MMP expression levels are involved inopening the BBB to enable African trypanosomes and leukocytesenter the brain and cause cerebral pathology. Indeed, treatmentwith minocycline reduced MMP expression and ameliorated survivalof T. brucei-infected mice. Therefore, MMPsmay be interesting targetsfor inhibition in HAT, whether or not supplemented with trypanoci-dal drugs. A major challenge is to improve the design of specificMMPIs that may be of therapeutic value in the fight against the break-down of the BBB.

3.2.3. Yin-yang of matrix metalloproteinases in Chagas diseaseAmerican trypanosomiasis or Chagas disease is caused by the in-

tracellular protozoan parasite T. cruzi and is transmitted by infectedfeces of a blood-sucking triatomine bug. The feces are deposited onthe skin of the host and after scratching because of the bite of thebug, the parasite can enter the body. If treatment is initiated soonafter infection, Chagas disease is curable. Today, an estimated 10 mil-lion people are infected with T. cruzi in the Americas (principally LatinAmerica), whereas more than 25 million are at risk of the disease. In2008, Chagas disease caused death of more than 10,000 people(www.who.int/mediacentre/factsheets/fs340/en/, 2010). Infectionwith this parasite is often lethal in children and infants, whereasadults develop chronic disease. Upon the infectious bite of the triato-mine bug, a local edematous swelling occurs which is called a ‘cha-goma’. By entering the blood circulation, infectious metacyclictrypomastigotes invade a variety of cells, including macrophages, fi-broblasts, skeletal and heart muscle cells, neuronal and epithelialcells. Intracellular trypomastigotes differentiate into replicative amas-tigotes which escape into the cytosol. Following repeated mitosis, theamastigotes turn back into trypomastigotes, which burst out of thecell, enter the bloodstream and invade other cells or are taken up byan insect vector. During the acute stage of infection, symptoms arelargely absent or mild and may include fever, lymphadenopathy,local swelling and diarrhea. A major characteristic mark of acute Cha-gas disease is called Romana's sign, which is manifested after trypo-mastigotes have penetrated the mucous membrane of the eye and ischaracterized by edema of one or both eyelids (Boscardin et al.,

2010; Rassi et al., 2010). To establish a chronic infection, a good bal-ance between parasite survival and host immune responses is essen-tial. Hence, infection-triggered proinflammatory cytokines, such as IL-12, IFN-γ and TNF-α, act to activate macrophages to control parasitesthrough the production of NO and ROS, which exert potent trypano-cidal actions. In addition, a CD8+ T cell response is induced andcauses tissue destruction and fibrosis. This proinflammatory responseis tempered by the Th2-related anti-inflammatory cytokines IL-10 andTGF-β (Tarleton, 2007; Boscardin et al., 2010). The importance of an-tibodies in controlling chronic infection was evidenced by transfer ofserum of T. cruzi-infected mice, showing significantly reduced parasi-temia and prolonged survival (Krettli & Brener, 1976). Furthermore,pathogen persistence is favored by the induction of polyclonal B cellactivation and Tregs (Gutierrez et al., 2009). Chronic stage disease istriggered by excessive inflammation. The most serious manifestationof chronic Chagas disease is the progressive decline of cardiac func-tion. About 30% of chronically infected patients develop cardiac dis-ease and up to 10% suffer from digestive, neurological or mixeddamage (Rassi et al., 2010). Chagasic cardiomyopathy is featured bymassive inflammatory cell infiltration into the myocardium. In addi-tion, lymphocytes from infected mice and chagasic patients recognizeself-epitopes, suggesting a possible autoimmune basis for develop-ment of Chagas disease cardiomyopathy (Cunha-Neto et al., 2006).Chronic infection and progressive destruction of the heart musclealong with intense fibrosis can cause heart failure (Rassi et al., 2009).

The myocardial ECM comprises a complex architectural networkof highly organized matrix proteins such as type I and III collagen,and it is this well-organized microenvironment that provides struc-tural integrity to adjoining myocytes and contributes to overall leftventricular function. In addition to a fibrillar collagen network, themyocardial ECM also contains a large reservoir of bioactive molecules.Following myocardial infarction, MMPs and their endogenous inhibi-tors, TIMPs, contribute to the process of remodeling of the cardiacECM, which results in repair of the necrotic area and myocardial scar-ring. However, ECM remodeling by an MMP/TIMP imbalance causesthe loss of structural integrity of the heart and is characteristic forseveral cardiomyopathies, including viral myocarditis, ischemia andreperfusion injury and heart failure. For instance, MMP-2 is implicatedin the processing of various intracellular substrates, e.g. troponin I,during myocardial oxidative stress injury (Wang et al., 2002). This pro-cess of extensive degradation and adverse remodelingmay lead to dys-function and dilatation, resulting inmyocardial hypertrophy and failure(Rutschow et al., 2006; Spinale, 2007).

T. cruzi-induced myocarditis is characterized by a strong inflam-matory reaction accompanied by a diffuse infiltration of activated Tcells and monocytes. This immune response is essential to mediateparasite killing, but will also contribute to the pathology associatedwith Chagas disease (Teixeira et al., 2002). The migration of infectedimmune cells throughout the ECM requires the help of MMPs. Indeed,besides degrading ECM, MMPs are shown to be important immuno-modulators by their action on cytokines and chemokines(Opdenakker et al., 2001b; Parks et al., 2004). In addition, MMPs arealso essential for the remodeling of the cardiac ECM. Hence, the in-volvement of these enzymes in cardiac lesions typical of chronic Cha-gas disease was studied in T. cruzi-infected cardiomyocytes. Anoverview of the MMP/TIMP expression profiles and functions inAmerican trypanosomiasis is represented in Table 2.

Although T. cruzi infection led to a decreased production of pro-and activated MMP-9 in hepatocytes in vitro (Nogueira de Melo etal., 2004), both MMP-2 and MMP-9 were induced by T. cruzi in cul-tured cardiomyocytes. The expression of both enzymes was, however,abrogated after treatment with 15-deoxy-Δ12,14 prostaglandin J2,whereas the number of intracellular parasites was increased(Hovsepian et al., 2011). 15-Deoxy-Δ12,14 prostaglandin J2, derivedfrom the dehydration of prostaglandin D2, is a negative feedback regu-lator of inflammation and may function as a natural ligand for

Table 2Expression and function of MMPs/TIMPs in trypanosomiasis (Trypanosoma spp.).


1. IN VITROT. cruzi-infected primary murine hepatocytes proMMP-9↓ in cell cytoplasm of infected cells

actMMP-9↓ in infected SN (zymography, gelatinaseactivity assays, WB, IHC)

T. cruzi infection might lead to reduction in Kupffer cell numbers orother MMP-producing cells, or inhibition of MMP-9 signalingpathways.

Nogueirade Melo etal., 2004

T. cruzi-infected primary murine cardiomyocytes Mmp-2,-9/MMP-2,-9↑ in cultured neonatalcardiomyocytes (RT-PCR, zymography)Pre-treatment with 15dPGJ2:→ MMP-2,-9↓: PPARγ-dependent(PPARγ-siRNA silencing)

MMPs are suggested to have an important role in induction ofchagasic acute myocarditis.

Hovsepianet al., 2011

2. IN VIVOBrain tissue of T. b. brucei-infected C57Bl/6 mice Mmp-3 and -12↑ in T. b. brucei-infected brain

(RT-PCR)Mice treated with MMP inhibitor minocycline:→ Mmp-3,-8,-12↓→ increased survival rate→ Impedes trypanosome invasion in brain

MMPs might degrade components of ECM and basementmembranes, leading to invasion of T. b. brucei and leukocytes intothe brain.

Masochaet al., 2006

Hearts from T. cruzi-infected C57Bl/6 mice Mmp-2,-9/MMP-2,-9↑ in T. cruzi-infected hearttissueTimp-1,-3↑ in T. cruzi-infected heart tissue(zymography,IHC, RT-PCR)

Mice treated with MMP inhibitor doxycycline:→ Mmp-2,-9↓→ Increased survival rate→ Reduced myocarditis→ No effect on parasitemiaImportant role for MMPs in induction ofT. cruzi-induced acute myocarditis

Overexpression of TIMPs may favor fibrosis. Gutierrezet al., 2008

3. CLINICAL STUDIESCSF from T.b.gambiense-infected patients inhemolymphatic stage (S1) vs. meningoenceph-alitic stage (S2) (staging based on WBC and/orparasites in CSF)

MMP-2,-9↑ in CSF from patients with S2HAT (ELISA, mBSA)

MMP-2 and MMP-9 highly correlated withpresence of WBC and parasites in CSF

Leukocyte and parasite infiltration into CNS might result from BBBdestruction induced by MMP-2 and MMP-9; combination of MMP-9/ICAM-1/H-FABP provide a panel with powerful staging markersfor HAT.

Hainard etal., 2011

↑ indicates increased levels; ↓ indicates decreased levels; 15dPGJ2, 15-deoxy-Δ12,14 Prostaglandin J2; actMMP, activated MMP; BBB, blood–brain barrier; CNS, central nervous system; CSF, cerebrospinal fluid; ECM, extracellular matrix; ELISA,enzyme-linked immunosorbent assay; HAT, human African trypanosomiasis; H-FABP, heart-type fatty acid binding protein; ICAM-1, intercellular cell adhesion molecule-1; IHC, immunohistochemistry; mBSA, multiplex bead suspensionassay; MMP, matrix metalloproteinase; PPAR-γ, peroxisome proliferator-activated receptor-γ; RT-PCR, real-time polymerase chain reaction; siRNA, small interfering RNA; SN, supernatant; TIMP, tissue inhibitor of metalloproteinases;TNF-α, tumor necrosis factor-α; WB, Western blot; WBC, white blood cell.


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Fig. 3. BBB alterations andMMPs in African trypanosomiasis. Second stage HAT is induced when Trypanosoma parasites cross the BBB, accompanied by influx of leukocytes after theyhave attached to the endothelial cell surface. Although controversy exists on whether the tight junctions are disrupted, degradation of the basal lamina is required for the parasites andleukocytes to gain entry into the brain parenchyma. It was demonstrated that several MMPs, e.g. MMP-2,-3,-9 and -12 were upregulated in the brain during HAT. This suggests that leu-kocytes may pave the way for parasites to enter the CNS by expressing MMPs that digest basal lamina proteins.


peroxisome proliferator-activated receptor-γ or through peroxisomeproliferator-activated receptor-γ-independent pathways. In line withthese results, Gutierrez et al. observed an increased expression of bothMMP-2 andMMP-9 in T. cruzi-infected heart tissue, whichwas associat-edwith leukocyte infiltration. By treating T. cruzi-infectedmicewith theMMP inhibitor doxycycline, MMP-2 andMMP-9 expression in heart tis-sue was abrogated and this was accompanied by significantly less se-vere myocarditis and an increased survival rate, without any effect onparasitemia levels (Gutierrez et al., 2008). Henceforth, both enzymesmight contribute to pathogenic chagasic myocarditis by disruptingECM architecture, favoring inflammatory cell infiltration and modulat-ing immune responses. MMPs are also associated with viral myocardi-tis, although their role is still controversial. For example, Cheung et al.recently reported an increased cardiac inflammatory response inMMP-9 KO mice infected with coxsackievirus, suggesting that MMP-9is involved in the antiviral immune response (Cheung et al., 2008).However, Heymans et al. ascertained that MMP inhibition preventedcardiac injury and dysfunction during coxsackievirus-induced myocar-ditis (Heymans et al., 2006). Hence, this inconsistency of MMPs inviral myocarditis needs further investigation and illustrates the com-plexity of research of MMPs in infectious diseases, including parasiteinfections.

TIMP-1 and TIMP-3 mRNA expression was found to be upregu-lated in T. cruzi-infected heart tissue (Gutierrez et al., 2008). Althoughthis increased TIMP expression may reflect a compensatory responseto the infection-induced inflammation, it was insufficient to counter-balance MMP activities and avoid severe myocarditis. Consequently,it was suggested that TIMP overexpression favors fibrosis, a feature

of dilated cardiomyopathy secondary to chagasic myocarditis. Fibrosisis characterized by the formation of a poorly structured myocardialmatrix due to abnormal ECM accumulation. TIMPs were reported tofavor cardiac fibrosis, both by inhibiting MMPs and by inducing fibro-blast proliferation (Vanhoutte & Heymans, 2010).

In conclusion, these data underline the essence of fine-tuningMMP as well as TIMP activity for positive cardiac remodeling. As fu-ture perspectives, it will be important to discriminate specific MMPtargets in severe myocarditis to improve the (re)search for appropri-ate inhibitors. Furthermore, it is essential to evaluate the timing of theadministration of eventual therapeutic MMPIs in order to restore ahealthy MMP/TIMP balance that resolves the excessive inflammationwhile avoiding fibrosis.

3.2.4. Imbalance of matrix metalloproteinases andtissue inhibitors of metalloproteinases in leishmanial pathology

Leishmaniasis encompasses a group of diseases caused by differ-ent species of Leishmania parasites endemic in 88 countries ofwhich 72 are developing countries. These protozoan parasites devel-op intracellularly within macrophages after they have been transmit-ted to their host by female sandflies of the genus Phlebotomus in theOld World and the genus Lutzomyia in the New World. Leishmaniasisis endemic in areas of the tropics, subtropics and southern Europe. Itis estimated that worldwide 12 million people are currently infected,about 350 million people are threatened and an incidence of 1–2 mil-lion new cases each year is observed (Desjeux, 2004; www.who.int/leishmaniasis/en/, 2011). The promastigote form is injected into thehost where it subsequently invades macrophages and loses its flagella

image of Fig.�3


to become a replicative amastigote form. These amastigotes multiplyin a parasitophorous vacuole until the cell bursts. In humans, threeclinical manifestations are evident: visceral, cutaneous and mucocu-taneous leishmaniasis. Visceral leishmaniasis (VL), also known asdumdum fever or kala-azar (KA), is caused by L. donovani and L. infan-tum in the Old World and L. chagasi in the NewWorld. VL, with an es-timated 60,000 deaths per year (Desjeux, 2004), is the most severeand often fatal form of leishmaniasis, particularly in India, Bangla-desh, Nepal, South Sudan and Brazil. Canine visceral leishmaniasis(CVL) and VL have a great impact on Brazilian public health since do-mestic dogs are the major VL reservoirs. CVL is suggested to be an ap-propriate model to study human leishmaniasis. The hallmarkhistopathologic alteration is the accumulation of infected mononucle-ar cells, mainly in lymphoid organs. Parasites are found in macro-phages throughout the viscera including spleen and liver. Symptomsare characterized by fever, splenomegaly, hepatomegaly and anemia.The fatality rate in developing countries can be 100% without medicaltreatment of the disease. Survivors acquire long-lasting immunity, al-though post-kala-azar dermal leishmaniasis (PKDL) may develop,which is a dermal manifestation of VL characterized by hypo-pigmented parasite-laden macules, papules or nodules in the skin(Stanley & Engwerda, 2007).

Cutaneous leishmaniasis (CL), caused by L. tropica in the OldWorld and L. mexicana in the New World, is the most common formof the disease. This relatively mild skin disease is characterized by in-flamed ulcers on hands, feet, legs and face, which heal spontaneouslyif not infected by bacteria. Most cases do not show systemic symp-toms, although lymphadenopathy, hepatomegaly and fever can occur.In mucocutaneous leishmaniasis (ML), caused mainly by L. braziliensis,chronic ulcers can lead to destruction of mucous membranes of thenose, mouth and throat cavities. Lymphadenopathy typically precedeslesion ulceration and may be accompanied by systemic symptoms likefever and hepatomegaly. Generally, intramuscular or intravascularinjection of pentavalent antimony compounds is used as treatmentstrategy, although this is fraught with problems of toxicity anddrug resistance (Herwaldt, 1999; David & Craft, 2009).

A protective immune response against Leishmania parasites de-pends on a Th1-tailored environment with IL-12 and IFN-γ being crit-ical cytokines. IFN-γ activates macrophages to kill parasites by theNO/ROS pathway. A defective Th1 response results in a non-healingdisease phenotype associated with upregulation of suppressive cyto-kines, e.g. IL-4 by Th2 cells and IL-10 and TGF-β by Tregs (Alexander &Bryson, 2005; Mansueto et al., 2007). However, in VL, the cytokineprofile is not characteristically polarized, since both Th1 and Th2 re-sponses are activated and IL-4 was even shown to promote resistancein experimental VL (Satoskar et al., 1995). In addition, polyclonal Bcell activation leads to disease exacerbation in VL (Deak et al.,2010). PKDL is the reoccurrence of KA that may appear on the skinafter many years and this condition is associated with high levels ofIL-10 in blood and skin (Gasim et al., 1998).

Leishmania infection activates macrophages resulting in the re-lease of several leishmanicidal agents, including peptidases, which,in excess, can cause severe tissue/organ damage. Summarized inTable 3 are the altered expression patterns of MMPs/TIMPs in differ-ent clinical manifestations of leishmaniasis. In VL, mainly the tissuearchitecture of liver and spleen is disrupted, and an increase in acti-vated MMP-9 was observed in macrophage-hepatocyte co-culture su-pernatants. Treatment with phenanthroline, a non-specificmetalloproteinase inhibitor, significantly reduced the release of he-patic damage markers, such as alanine aminotransferase and aspar-tate aminotransferase, indicating that MMPs might participate inliver damage by breaking down the architectural support for hepato-cyte growth and survival (Costa et al., 2008).

CVL manifests itself as a chronic systemic disease with three dif-ferent clinical outcomes: the asymptomatic form, the oligosympto-matic form with symptoms like fever, moderate weight loss,

cutaneous ulcerations and discrete anemia; and the classical symp-tomatic form, characterized by disseminated cutaneous ulcerations,blindness and severe weight loss, progressing towards a general mor-bidity state and death. Asymptomatic dogs have low parasitemia,whereas the cutaneous ulcerations in symptomatic dogs are differen-tiated by a diffuse inflammatory infiltrate with a high parasite burden(Reis et al., 2009). In serum samples from dogs naturally infected byL. chagasi, both proMMP-2 and proMMP-9 as well as activatedMMP-9 were upregulated, and mature and proMMP-9 were positive-ly correlated in infected dogs (Melo et al., 2011). These results indi-cate that the multisystemic inflammatory lesions in leishmaniasismay result from increased MMP expression.

In addition to the classical symptoms, other disorders may appear.Garcia-Alonso et al. reported neurological pathology in brain fromdogs with CVL. Leukocytes and deposits of Leishmania antigen and an-tibodies were detected in the CSF from Leishmania-infected dogs withneurological symptoms (Garcia-Alonso et al., 1996; Melo et al., 2009).These findings may be explained by loss of barrier integrity. Hence, itwas investigated whether MMPs were implicated in Leishmania-induced neurological complications. Elevated proMMP-2 andproMMP-9 expression levels were found in the encephalon fromdogs infected with L. chagasi. However, these levels did not correlatewith disease severity (Machado et al., 2010). Nevertheless, in a recentstudy from the same research group, both the latent and activatedforms of MMP-9 were detected in CSF from dogs with CVL(Marangoni et al., 2011). Since no correlation was found with thelevels of MMP-9 in serum, the authors suggested that MMP-9 wasgenerated in the nervous tissue and perhaps mediated injury ofbrain barriers, thereby permitting the passage of antibodies and leu-kocytes into the CNS.

Leishmania parasites are deposited in the skin and cause cutane-ous lesions, characterized by a robust inflammatory infiltrate of leu-kocytes. These lesions usually evolve slowly to healing. The healingof CL is associated with a predominant Th1 response, whereas aTh2 environment results in a chronic and destructive mucocutane-ous form of leishmaniasis (Pirmez et al., 1993). Wound healing isa complex dynamic process, involving inflammation, re-epithelialization, angiogenesis and matrix deposition. The role ofMMPs in all stages of wound healing has been well-documented(Gill & Parks, 2008).

In tissue samples from patients with ML, mucosal lesion progres-sion is related to neutrophil chemotaxis. These neutrophils exhibitedintense MMP-9 staining, and excessive MMP-9 release contributes tolesion expansion (Boaventura et al., 2010). However, in dermal tissuelesions from L. donovani-infected patients with KA and PKDL, themRNA expression of MMP-9 was comparable to that of control tissue,but TIMP-1 and TIMP-3 were significantly elevated in PKDL incomparison with KA (Ansari et al., 2008). These data suggest thatTIMP-1 and TIMP-3 may contribute to lesion pathology in PKDL. In-terestingly, it has been demonstrated that IL-10, the most prominentcytokine found in PKDL, stimulates TIMP-1 production in humanmononuclear phagocytes while decreasing metalloproteinase biosyn-thesis (Lacraz et al., 1995). In accordance with these data, overexpres-sion of TIMP-1 and TIMP-3 was also demonstrated in intestinal andcutaneous lesions of graft-versus-host disease (Salmela et al., 2003),suggesting that high TIMP levels may inhibit wound healing. In skintissue fragments from CL patients under treatment, increased MMP-2 levels together with a high MMP-2 to TIMP-2 ratio was reportedto be essential for successful cutaneous wound re-epithelialization(Maretti-Mira et al., 2011). In lesions from poor responders, however,low MMP-2 mRNA and a strong gelatinolytic activity were observedand wound healing was impaired. A role for MMP-2 in wound healingwas already described by Jackson et al., who found that the serineprotease activated protein C dampens excessive MMP activity and se-lectively induces MMP-2 activation to enhance angiogenesis and re-epithelialization (Jackson et al., 2005).

Table 3Expression and function of MMPs/TIMPs in leishmaniasis (Leishmania spp.).


1. IN VITROL. chagasi (VL)-infectedmacrophage-hepatocyte co-cultures

actMMP-9↑ in infected co-culture supernatants(zymography, WB)

Treatment of co-cultures with metallopeptidaseinhibitors (phenantroline): Most efficient decreasein AST/ALT enzyme release

Production of activated MMP-9 may participate in he-patocyte damage, since the ECM architectural support isessential for the growth and survival of hepatocytes.

Costa et al.,2008

Analysis of serum samples fromL. chagasi-infected dogs (CVL)

proMMP-2↑ in infected dogsproMMP-9 and actMMP-9↑ in infected dogs(zymography)

Strong positive correlation between pro- andactMMP-9 in infected dogs

Conversion of proMMP-9 to actMMP-9 confirms thepresence of systemic inflammation in dogs naturallyinfected with L. chagasi.

Melo et al.,2011

2. IN VIVOHemisphere of encephalon from SD,OD and ND infected with L. chagasi(CVL) vs. neuronal tissue from UD

MMP-2,-9+ cellsproMMP-2↑: OD>SD>ND=UDproMMP-9↑: OD=SD=ND>UD (IHC, zymography)

ProMMP-9 and proMMP-2→no good indicatorsfor neurological alterations

Machadoet al., 2010

Analysis of CSF from L. chagasi-infecteddogs (CVL)

pro- and actMMP-9↑ in some dogs No correlation between MMPs in serum andCSF→MMP-9 produced in brain tissue

MMP-9 is produced in the central nervous system andmight cause BBB disruption.

Marangoniet al., 2011

3. CLINICAL STUDIESDermal tissue lesions in L. donovani-infectedpatients with PKDL and KA vs. normaltissue from PKDL

Timp-1↑: PKDL>KA=controlTimp-3↑: PKDL>KA; PKDL=control; KA=controlMmp-9: PKDL=KA=control (RT-PCR)

TIMP-1 and TIMP-3 may have an importantimplication for distinct PKDL pathology.

Ansari etal., 2008

Mucosal tissue samples from L. braziliensis-infected ML patients vs. normal mucosalsamples

MMP-9+ neutrophils ↑ in ML lesions (IHC) Lesion progression is related to neutrophil recruitment,suggesting that MMP-9 is involved in lesiondevelopment.

Boaventuraet al., 2010

Skin tissue fragments from L. braziliensis-infected CL patients (good and poor treat-ment responders) vs. normal human skinsamples

Mmp-2/MMP-2↑ in CL lesions from good vs. poor respondersMmp-2:Timp-2↑ in CL lesions from good vs. poor respondersGelatinase activity↑ in CL lesions from poor vs. goodresponders (RT-PCR, in situ zymography, IHC)

High MMP-2:TIMP-2 ratio associated withsuccessful healing; strong positive correlationbetween gelatinolytic activity and 3 cytokines(IFN-γ, TGF-β, IL-10) in lesions from poorresponders

Increased MMP-2 levels were reported to berequired for cutaneous wound re-epithelialization.

Maretti-Mira et al.,2011

↑ indicates increased levels; ↓ indicates decreased levels; actMMP, activated MMP; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BBB, blood–brain barrier; CL, cutaneous leishmaniasis; CSF, cerebrospinal fluid; CVL, caninevisceral leishmaniasis; ECM, extracellular matrix; ELISA, enzyme-linked immunosorbent assay; IFN-γ, interferon-γ; IHC, immunohistochemistry; KA, kala-azar; LCL, localized cutaneous leishmaniasis; ML, mucocutaneous leishmaniasis;MMP, matrix metalloproteinase; ND, neurologically affected dogs; OD, oligosymptomatic dogs; PKDL, post-kala-azar dermal leishmaniasis; RT-PCR, real-time polymerase chain reaction; SD, symptomatic dogs; TGF-β, transforming growthfactor-β; TIMP, tissue inhibitor of metalloproteinases; UD, uninfected dogs; VL, visceral leishmaniasis; WB, Western blot.


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Altogether, MMPs and TIMPs play pleiotropic roles in leishmania-sis causing tissue destruction and lesion progression e.g. in liver, skinor brain, but also favoring wound healing. This again emphasizes theneed of a correctly-regulated balance between MMPs and TIMPs forpositive wound healing and remodeling (Xue et al., 2006). Hence,the determination of MMP and TIMP profiles may have diagnosticand prognostic value in various forms of leishmaniasis and selectivecontrol of MMP activity may be a valuable therapeutic approach topromote the healing process during leishmaniasis.

3.2.5. Matrix metalloproteinases astargets and anti-targets in toxoplasmosis

Toxoplasmosis is caused by T. gondii, an obligate intracellular pro-tozoan with a worldwide distribution and a broad host range. Toxo-plasma infects up to a third of the world's population, and thisparasite can be life-threatening during pregnancy and in immuno-compromised individuals (Munoz et al., 2011). In humans, infectionoccurs by oral ingestion of tissue cysts or oocysts through consump-tion of undercooked meat or contact with infected feces of cats. In ad-dition, toxoplasmosis can be passed on from the mother to the fetusby transplacental transmission, resulting in abortion or fetal abnor-malities. Following oral infection, viable bradyzoites or sporozoitesare released from tissue cysts or oocysts, respectively, and activelycross the intestinal epithelial barrier before they disseminate intoother tissues. Subsequently, after invading host cells, they differenti-ate into tachyzoites and develop and multiply inside a parasitophor-ous vacuole until the cell lyses (Montoya & Liesenfeld, 2004). Tissuecysts are formed when tachyzoites differentiate into latent brady-zoites which are protected by a cyst wall. Neutrophils, lymphocytesandmacrophages are permissive to parasite growth and these “Trojanhorses” contribute to parasite dispersal throughout the body intomuscles, brain, placenta or eyes. In these organs, parasite growth isunder control of a regulated inflammatory response. Indeed, one ofthe hallmarks essential to control T. gondii infection is the induction– coordinated by macrophages, NK cells, dendritic cells and epithelialcells – of a well-balanced Th1 immune response (TNF-α, IFN-γ, CD4+

and CD8+ T cells). This proinflammatory state is equilibrated byT. gondii triggered anti-inflammatory cytokines such as IL-10 andTGF-β (Munoz et al., 2011). Hence, in immunocompetent humans, in-fections are clinically asymptomatic and the parasite establishes alifelong state of latency by forming cysts containing the slow replicat-ing bradyzoites in neuronal tissues and skeletal muscles. However,when parasites invade mesenteric lymph nodes and liver parenchy-ma, individuals may develop acute influenza-like symptoms, such aslymphadenopathy, fever and muscle pain. When retina or braincells are involved, serious lesions can develop, maybe because theseanatomical sites have less immune control. Normally, the durationof the chronic stage is limited by the host's immunological system.However, in immunocompromised patients, such as those with HIV/AIDS, reactivation of the parasite ‘latent’ stage can cause severe dam-age to brain and eyes leading to toxoplasmic encephalitis or necrotiz-ing retinochorioditis, respectively. Chronic toxoplasmosis can alsocausemyocarditis, leading to permanent heart damage and pneumonia.In immunocompromised patients, the combination of pyrimethamineand sulfadiazine is the treatment of choice for toxoplasmosis (Weiss &Dubey, 2009; Munoz et al., 2011).

In order to reach immune-privileged sites, T. gondii has to dissem-inate through the ECM and cross biological barriers such as the intes-tine, the BBB and the placenta. This is the point where MMPs mightcome into play. Table 4 schematically recapitulates the current statusof MMP/TIMP/ADAM research in toxoplasmosis.

Infection of THP-1 cells with T. gondii results in a strong decreaseof proMMP-2, proMMP-9 and TIMP-2 expression and secretion, butthe expression and activation of proMT1-MMP is induced (Buacheet al., 2007). In addition, an increase in MT1-MMP expression wasalso reported in T. gondii-infected RAW cells, and secretion of

activated MMP-9 was enhanced. Upon treatment with an MMP inhibi-tor, in vitro migration of infected macrophages through matrigel wassignificantly reduced, assuming that MT1-MMP and MMP-9 mighthelp infected leukocytes to traverse biological barriers (Seipel et al.,2010). Interestingly, increasing levels of MT1-MMP correlated withthe shedding of CD44, a cell adhesion molecule involved in cell–celland cell–matrix interactions. CD44 binds ECM components such astype I collagen, fibrin and laminin and MT1-MMP is co-expressed withCD44 on migrating cells and metastatic tumor cells. Hence, processingof CD44 by MT1-MMP is critical to detach cells from the ECM and stim-ulate cell motility (Seiki, 2003). Moreover, intact CD44 serves as a dock-ing platform for anchoring proteolytically active MMP-9 and this cellsurface-localized MMP-9 is shown to activate TGF-β and thereby pro-vides a mechanism for invasion and angiogenesis (Yu & Stamenkovic,2000). In conclusion, these data suggest that T. gondii infection modu-lates cell migration throughout the body by inducing activation ofMT1-MMP and shedding of CD44 by this membrane-bound MMP.This is accompanied by an increased secretion of activated MMP-9,and this protease might facilitate access into immune-privileged sites.In addition, thework by Seipel et al. also demonstrated increasing levelsof activated ADAM-10 at the cell surface of T. gondii-infected macro-phages, and it was suggested that this enzyme, together with otherMMPs, might contribute to facilitate the access of infected leukocytesinto immune-privileged sites (Seipel et al., 2010).

Inflammation of the small intestine following oral infection withT. gondii-containing cysts has been studied in susceptible C57Bl/6wild type mice, that develop acute severe ileal necrosis and finallysuccumb to infection (Liesenfeld et al., 1996). The immunopatho-genesis is mediated by massive infiltration of leukocytes and lym-phocytes associated with an excessive release of proinflammatoryagents (so called ‘cytokine storm’), including IL-12, IFN-γ, TNF-αand NO (Liesenfeld et al., 1999). In addition, IL-23 is essential fordevelopment of ileitis and it induces the upregulation of MMP-2and MMP-9 in the small intestine after T. gondii infection. An addi-tional zymolytic band on gelatin zymography, corresponding to theactivated form of MMP-2, was also observed 5 days post infection(Munoz et al., 2009). This MMP-mediated intestinal pathology re-sembles the role of MMPs in the inflammatory and remodeling pro-cesses associated with inflammatory bowel disease (Gao et al.,2005). The induction of MMPs by IL-23 has been shown by Lan-gowski et al., who observed IL-23-induced upregulation of MMP-9during tumor development (Langowski et al., 2006). Although thenumber of parasitophorous vacuoles containing T. gondii tachyzoitesin the ileum remained unchanged, MMP-2 KO mice, unlike MMP-9KO mice, were protected against T. gondii-induced intestinal pathologyand early death. Necrosis of the small intestinewas also prevented afterprophylactic or therapeutic treatment with MMPIs, again without anyeffect on the numbers of parasites in the small intestinal lamina propria.Hence, infection-induced MMP-2, and not the number of parasites pre-sent in the small intestine, is an important factor in T. gondii-induced il-eitis andmay be a suitable target for treatment. Interestingly, cells in thelamina propria, but not granulocytes, seem to secrete MMP-2, in agree-ment with previous findings that neutrophils do not produce MMP-2(Opdenakker et al., 2001a). This inflammatory character of MMP-2 inthe intestine of T. gondii infection is, however, quite surprising. Indeed,during colitis, which is an inflammatory bowel disease, MMP-2 hasbeen shown to protect against tissue damage and tomaintain gut barrierfunction (Garg et al., 2006). In addition, MMP-2 is generally consideredan enzyme involved in dissipating proinflammation by processing sev-eral chemokines into antagonists with anti-inflammatory properties,e.g. the cleavage of MCP-3 (McQuibban et al., 2000). This is in contrastto the classical proinflammatory roles of MMP-9, e.g. the processingand potentiation of IL-8 (Van den Steen et al., 2000).

Inmurine Toxoplasma encephalitis, leukocytes, and in particular CD4+

andCD8+T cells, are recruited to the brain. These lymphocytes play a cru-cial role in host survival from chronic T. gondii infections by the

Table 4Expression and function of MMPs/TIMPs/ADAMs in toxoplasmosis (Toxoplasma gondii).


1. IN VITROT. gondii-infected THP-1 cells Mmp-2,-9, Timp-2↓, Mt1-mmp↑

proMMP-2, proMMP-9, TIMP-2↓proMT1-MMP↑, actMT1-MMP↑ ((reverse) zymography,ELISA, RT-PCR, WB)

MT1-MMP activation by T. gondii probably explains parasitedissemination and access to immune-privileged sites.

Buache etal., 2007

T. gondii-infected (macrophage-like) RAW 264.7 cells

actMMP-9↑ in infected supernatantsMT1-MMP↑ in T. gondii-infected cellsADAM-10↑ in T. gondii-infected cells(FC, migration assays, IP and zymography)

MMP inhibitor I→ Abolished invasiveness of T. gondii-infectedmacrophages over 3D ECMMMPs might facilitate the access of infectedleukocytes to immune-privileged sites

Increasing levels of MT1-MMP→shedding of CD44, a dockingmolecule for MMP-9

Seipel etal., 2010

2. IN VIVOIleum tissue from T. gondii-infected mice

Mmp-2,-9/MMP-2,-9↑ in small intestineactMMP-2↑ in small intestineIL-23p19KO: miceMmp-2,-9/MMP-2,-9↓no actMMP-2→ Significantly reduced intestinal pathology(zymography,ELISA, IHC, RT-PCR)

MMP-2KO mice (compared to MMP-9KO and WT mice)→ Protected against the development of intestinalimmunopathology and early death; MMP-2 mediatesimmunopathology in T. gondii-induced ileitisTreatment with gelatinase inhibitors (doxycycline andMMPI RO28-2653)→ Ameliorated intestinal pathologyTreatment with gelatinase inhibitors protects miceagainst T. gondii-induced immunopathology

Selective blockage of gelatinases may be a safe and effectivestrategy in prevention and treatment of intestinal inflammation.

Munoz etal., 2009

Brain tissue from T. gondii-infected mice

Mmp-8,-10 and Timp-1↑ in brainMMP-8↑ in brain infiltrating CD4+/CD8+ T cellsMMP-10↑ in brain infiltrating CD4+ T cellsTIMP-1↑ in CNS-resident astrocytes and in braininfiltrating CD4+/CD8+ T cells (RT-PCR, IHC, ELISA)

TIMP-1 deficient mice→ Little morphological changes in tissue structure→ ↑ CD4+ T cells in brain→ Reduced parasite burden in brainTIMP-1 is associated with inhibition of pathogenclearance without development of adverse pathology

MMP-8 and -10 production by brain-infiltrating T cells implies arole for MMPs in brain tissue penetration; TIMP-1 is associatedwith inhibition of pathogen clearance.

Clark etal., 2010

3. CLINICAL STUDIESSerum from pregnant womenwith or without T. gondiiinfection vs. healthy non-pregnant women

MMP-12↑ - > associated with ↑ elastin degradationproducts:Pregnant women with toxoplasmosis>healthypregnant women>healthy non-pregnant women (WB, IP)

Interaction between MMP-12 and elastin in the serum of infectedpregnant women suggests that MMP-12 mediates damage toelastin and contributes to T. gondii-associated pathology duringpregnancy.

Chou &Lai, 2011

↑ indicates increased levels; ↓ indicates decreased levels; ADAM: a disintegrin and metalloprotease; actMMP, activated MMP; CNS, central nervous system; CSF, cerebrospinal fluid; ECM, extracellular matrix; ELISA, enzyme-linked immu-nosorbent assay; FC, flow cytometry; IF, immunofluorescence; IHC, immunohistochemistry; IP, immunoprecipitation; KO, knockout; MMP, matrix metalloproteinase; MT-MMP, membrane-type matrix metalloproteinase; RT-PCR, real-timepolymerase chain reaction; TIMP, tissue inhibitor of metalloproteinases; WB, Western blot.


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prevention of reactivation of latent infections (Gazzinelli et al., 1992). Pro-tection by CD4+ and CD8+ T cells is mainly mediated by IFN-γ, whichstimulates anti-parasitic effector mechanisms and regulates chemokineexpression and leukocyte recruitment (Strack et al., 2002).

Recently, an increase in brain-infiltrating T cells producing MMP-8 and MMP-10 was reported in the brain tissue of T. gondii-infectedmice (Clark et al., 2010). In conjunction, infection-induced TIMP-1 ex-pression was observed in invading T cells and CNS-resident astro-cytes. Histological analysis showed changes in tissue morphologyand evidence for astrocyte activation was found. In contrast, littlemorphological changes and no sign of astrocyte activation were ob-served in infected TIMP-1 KO mice. Further analysis of T. gondii infec-tion in TIMP-1 KO mice revealed an increase in CD4+ T cells togetherwith a significantly reduced parasite burden in the brain of thesemice, whereas peripheral parasite burden was not altered in the ab-sence of TIMP-1. Hence, increased expression of TIMP-1 during infec-tion may inhibit pathogen clearance by limiting MMP-mediatedlymphocyte penetration into the CNS, and this may reflect a parasiteevasion strategy or a host-mediated dampening of inflammation. In-deed, brain inflammation in infected TIMP-1 KO mice occurred withreduced or absent perivascular cuffing with more cells infiltratingthe brain parenchyma, probably reflecting uninhibited MMP-mediated degradation of the basal lamina. Taken together, thesedata indicate a beneficial role of MMPs in pathogen clearance, whichmight resemble the resistance of TIMP-1 KO mice to P. aeruginosa, dueto uninhibitedMMP activity (Lee et al., 2005). Further studies are, how-ever, essential to examine the effect of TIMP antagonists or MMP in-ducers or agonists in these infection models. Fig. 4 illustrates thedescribed interactions between (MT-)MMPs, TIMPs and ADAM-10 dur-ing toxoplasmic encephalitis.

In a recent clinical study, increased concentrations of MMP-12 andelastin degradation products were detected in the serum of pregnantwomen infected with T. gondii. Co-immunoprecipitation of MMP-12with elastin suggested that MMP-12 might mediate the pathologicaldegradation of elastin in pregnant women with toxoplasmosis(Chou & Lai, 2011). Furthermore, elevated levels of MMP-12 mightcontribute to leukocyte recruitment and additional tissue damage.Hence, this enzyme might be a useful marker in the pathogenesis ofT. gondii infection during pregnancy.

As a conclusion, the data discussed in this section provide a morefunctional insight into the enhanced expression of MMPs in toxoplas-mosis. Indeed, studies on both MMP and TIMP KO mice indicated thatMMPs might be useful targets as well as anti-targets in the pathologyof a T. gondii infection, depending on the organ affected. In particular,MMP-2 was found to be a key contributor in the T. gondii-induced in-testinal pathology and it might also be interesting to study this en-zyme in other inflammatory bowel diseases. In contrast, MMPinhibition by TIMPs proved permissive for the pathogenesis of toxo-plasmic encephalitis, as MMPs are required for an efficient removalof T. gondii parasites from the brain by the immune system. Thesearch for and evaluation of chemical MMP blockers or inducersthat resolve the infection and indirectly clear the parasites would bea justified focus for future research.

4. Conclusions and future perspectives

Parasite infections with Plasmodium, Trypanosoma, Leishmania andToxoplasma species all induce increased levels of several MMPs, eitherdirectly or indirectly by influencing cytokine levels. These infectionscause various forms of (meningo)encephalitis, often including themigration of leukocytes and/or parasites across the BBB. In all theseexamples, the activity of MMPs is invoked for both the migrationand the cleavage of structural elements of the BBB, and this furthercontributes to tissue breakdown and pathology. Therefore, additionalfunctional studies of the different MMPs in these different parasite in-fections may uncover important roles for particular MMPs in the

pathogenesis of these world-level infectious diseases. Moreover, allfour parasite species possess metalloproteinase target molecules.This implies that dual-specificity MMPIs, which inhibit pathogenicMMPs and parasitic metalloproteases, might be particularly usefulfor therapy. However, the inhibition of host-protective MMPs shouldbe avoided. Furthermore, while at some point uncontrolled MMP ac-tivity is detrimental, excessive TIMP upregulation can also be harmfulby favoring fibrosis, e.g. in cases of cardiomyopathy. Another issue isthe interaction of MMPs, either direct or indirect, with various bio-molecules, including cytokines and chemokines. This is exemplifiedby the positive feedback loop between MMP-9 and TNF-α as dis-cussed by Prato et al. (2005). In general, several MMPs are able tocleave particular cytokines and chemokines, with sometimes impor-tant pathological effects, but this still remains to be investigated inprotozoan parasitic diseases. Alternatively, cytokines might also influ-ence MMP expression, as shown by the decreased expression ofMMP-2 and MMP-9 in T. gondii-infected IL-23 KO mice (Munoz etal., 2009). Since balances in pro- and anti-inflammatory cytokinesare crucial in the outcome of protozoan parasitic infections, it mightbe essential to delineate the effects of specific MMPs on the levels ofcytokines in view of pharmacological intervention.

Besides the MMPs, the related ADAM(-TS)s family of metallopro-teinases might also be an interesting class of proteins to study interms of protozoan parasitic infections. Indeed, TACE or ADAM-17,ADAM-TS13 and ADAM-10 have already been studied in malariaand toxoplasmosis, but these enzyme families contain several otherinteresting members for further studies. Hence, although data onADAM(-TS)s in the field of protozoan parasitic infections are scarce,it might be a fascinating future challenge to further unravel the func-tional roles of these transmembrane proteases in several parasiticdiseases.

Another critical aspect is the present lack of knowledge of theroles played by parasitic agonists and of the parasite molecules thatinfluence the expression of MMPs and their endogenous inhibitors.For instance, similarly to the role played by natural Hz as an MMP ag-onist in malaria, nucleic acids, metabolites or specific glycoproteins ofparasitic origin may be discovered as switches of immunopathologi-cal events in infections. Such molecular identifications may yield fu-ture drug targets and become instrumental to fight these diseases.In view of the fact that many discussed parasite infections hatch with-in the CNS, ideal MMPIs should possess pharmacological properties tocross the BBB, as is the case for tetracyclines.

In summary, although proof of concept is still in high demand, thecurrently available data suggest that MMPs might play importantroles in several protozoan diseases. It is of crucial importance to pin-point the specific roles of the individual MMPs and to evaluate thebalances and imbalances with TIMPs. This will help to discriminatebetween targets and anti-targets in each protozoan disease. Such in-formation, together with more elaborated insights into MMP struc-ture and biophysics, will pave the way towards the (re)development of useful specific and valid MMPIs as therapeutic agentsin parasitic infections (Hu et al., 2007). It is hoped that this reviewwill emphasize the need for more functional data on the role ofMMPs/TIMPs in parasitic infections. Studies with KO mice on well-defined genetic backgrounds, complemented with the use of MMPIsshould help researchers to further unravel the diverse roles of theseproteases with the aim to design effective therapeutic strategies fortreatment of parasitic infections.

Acknowledgments

The present study was supported by the Fund for ScientificResearch-Flanders (FWO-Vlaanderen), the Geconcerteerde Onder-zoeksActies (GOA 2007–2011) and a CREA-grant (CREA-09/023)from the Research Fund of the University of Leuven. NG is a ResearchAssistant of the FWO-Vlaanderen and PVDS is a Research Professor of

Fig. 4. BBB alterations and MMPs/TIMPs/ADAMs in toxoplasmic encephalitis. During T. gondii infection, increased secretion of activated MMP-9 was associated with an enhancedexpression and activation of MT1-MMP (MMP-14) and ADAM-10 in vitro. This altered expression profile may explain parasite dispersal throughout the body. Control of murineToxoplasma encephalitis is achieved by a well-orchestrated proinflammatory immune response, characterized by infiltrating leukocytes, in particular IFN-γ producing CD4+ andCD8+ T cells. Increased expression of MMP-8 and MMP-10 was observed in CD4+ and CD8+ brain infiltrating T cells. In addition, TIMP-1 expression was also induced in invading Tcells and CNS-resident astrocytes. It is hypothesized that the MMPs might facilitate lymphocyte trafficking into the CNS in order to prevent reactivation of chronic T. gondii infec-tion. Since TIMP-1 KO mice demonstrate a reduced parasite burden in the brain and less perivascular cuffing with more cells trafficking into the brain parenchyma, T cells gainaccess into the CNS in a manner that is regulated by TIMP-1. This might reflect a parasite evasion strategy or could be interpreted as a host response to minimize braininflammation.


the Research Fund of the University of Leuven. The authors have noconflicting financial interests.

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