**Matrix Metalloproteinase**

Editors

**Magnus S. Agren ˚ Ulrich auf dem Keller**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editors* Magnus S. Agren ˚ University of Copenhagen Denmark

Ulrich auf dem Keller Technical University of Denmark Denmark

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *International Journal of Molecular Sciences* (ISSN 1422-0067) (available at: https://www.mdpi.com/ journal/ijms/special issues/matrix metalloproteinase).

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## **Contents**

## **About the Editors**

**Magnus S. Agren ˚** is Affiliate Professor of Surgery at the Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark, and active at the Copenhagen Wound Healing Center and Digestive Disease Center, Bispebjerg Hospital. Professor Agren received his doctoral degree (dr.med.sci.) in Experimental Pathology at the University of ˚ Linkoping, Sweden 1990. He subsequently was a postdoctoral fellow at the University of Miami ¨ School of Medicine, Miami, Florida, USA from 1991–1993 with affiliation to the Departments of Dermatology (Professor William Eaglstein), Biochemistry (Professor Fred W. Woessner, Jr.), and Molecular and Cell Biology (Professor Gary Grotendorst). Since 1993, Dr. Agren has worked as ˚ Senior Scientist and manager in both academia and industry. He was appointed Associate Professor at University of Linkoping 1994 and became Professor at the University of Copenhagen 2011. ¨ Dr. Agren' s research has focused on mechanisms and novel treatments for wound healing in ˚ the skin and the gastrointestinal tract. In these fields he has developed reliable and translatable cell and animal wound healing models, studied pathological wound healing, and worked on the identification of biomarkers of normal and aberrant wound healing. His specific scientific interests include the role of zinc, extracellular matrix metabolism and fibroblast phenotypes in wound healing. Worth mentioning among his scientific achievements are the demonstration of the physiological requirement of MMPs in epidermal repair, beneficial effect of MMP inhibitors in colonic wound repair, and the detrimental role of senescent fibroblasts in chronic cutaneous wound healing. Professor Agren has supervised 12 basic scientists and medical doctors, and has hosted ˚ several visiting researchers from continental Europe. He has published 140 wound-healing articles in internationally renowned and peer-reviewed medical journals. His current H-index is 31 (Web of Science). Finally, Professor Agren has edited a textbook series on Wound Healing Biomaterials (34 ˚ chapters), authored 4 book chapters and holds 3 patents. Dr. Agren is Scientific Advisor for several ˚ Scandinavian and international companies in the health care sector. He is Section Editor of Acta Dermato-Venereologica, and acts on the Editorial Board of Wound Repair and Regeneration and Journal of Wound Care. Dr. Agren organized the 7th Joint meeting of European Tissue Repair Society ˚ (ETRS) with the Wound Healing Society and the 25th Annual Meeting of ETRS in Copenhagen, 2015, and served as the President of ETRS from the year 2016 to 2017.

**Ulrich auf dem Keller** is Professor (with special responsibilities) and head of the Section for Protein Science and Biotherapeutics at the Department of Biotechnology and Biomedicine at Technical University of Denmark. Professor auf dem Keller received his diploma in Biochemistry in 2000 from the University of Tubingen, Germany and his PhD in 2005 from ETH Zurich, Switzerland, after completing his doctoral studies supported by a Boehringer Ingelheim Fonds PhD fellowship in the Institute of Cell Biology with Prof. S. Werner. After a short postdoctoral period in the same department he joined in 2006 Prof. C. M. Overall's laboratory at the University of British Columbia, Vancouver, Canada as a recipient of a Research Fellowship from the German Research Foundation (DFG) to work on protease proteomics. In 2009 he returned to ETH Zurich as a Senior Scientist and Junior Group Leader, where he applied newly developed advanced proteomics methods to elucidate the function of proteases in healthy and diseased skin. In 2017, Prof. auf dem Keller moved to Denmark to establish a research program on proteolytic networks in skin homeostasis, inflammation and repair with a focus on matrix metalloproteinases (MMP) and supported by a Young Investigator Award from the Novo Nordisk Foundation. Professor auf dem Keller is a pioneer in mass spectrometry-based proteomics technologies and has published several highly cited papers on MMP substrate discovery and global analysis of proteolysis in the healing skin wound. He served as president of the International Proteolysis Society from 2017 to 2019 and is the current president of the ETRS.

## *Editorial* **Matrix Metalloproteinases: How Much Can They Do?**

#### **Magnus S. Ågren 1,2,\* and Ulrich auf dem Keller 3**

Received: 16 March 2020; Accepted: 9 April 2020; Published: 12 April 2020

**Abstract:** Zinc-dependent matrix metalloproteinases (MMPs) belong to metzincins that comprise not only 23 human MMPs but also other metalloproteinases, such as 21 human ADAMs (a disintegrin and metalloproteinase domain) and 19 secreted ADAMTSs (a disintegrin and metalloproteinase thrombospondin domain). The many setbacks from the clinical trials of broad-spectrum MMP inhibitors for cancer indications in the late 1990s emphasized the extreme complexity of the participation of these proteolytic enzymes in biology. This editorial mini-review summarizes the Special Issue, which includes four review articles and 10 original articles that highlight the versatile roles of MMPs, ADAMs, and ADAMTSs, in normal physiology as well as in neoplastic and destructive processes in tissue. In addition, we briefly discuss the unambiguous involvement of MMPs in wound healing.

**Keywords:** extracellular matrix; inflammation; wound healing; cytokines; proteinases; interstitial collagens

More than half a century ago, Gross and Lapière discovered a true collagenase, which was the first vertebrate matrix metalloproteinase (MMP) responsible for the resorption of the tail in the metamorphosing tadpole [1]. We now know that vertebrate collagenase belongs to the metzincins, which is a clan of metalloendopeptidases found in all living organisms [2,3]. The metzincins, with their third ligand being histidine or aspartate in the active site, comprise not only the MMP family, which has 23 members in humans [4], but also other metalloproteinases, such as adamlysins or reprolysins, including ADAMs (a disintegrin and metalloproteinase domain; 21 members in humans) and ADAMTSs (a disintegrin and metalloproteinase thrombospondin domain), consisting of 19 secreted enzymes and at least 7 ADAMTS-like proteins that are devoid of catalytic activity [5,6], astacins (e.g., meprins, bone morphogenetic protein-1), leishmanolysins, serralysins, and snapalysins [2].

Research activities that followed the discovery by Gross and Lapière focused in the beginning on the critical role of these proteinases in extracellular matrix (ECM) remodeling in homeostatic balance and imbalance [7]. Thus, it turned out that MMPs are necessary for multiple and diverse physiological processes, such as reproduction, morphogenesis, embryonic development, bone remodeling, angiogenesis, and tissue repair, but they can also contribute to tissue destruction during cancer development and spreading and in arthritis/osteoarthritis and fibrotic diseases. The association of pathologies with MMP overexpression was also the impetus for the intense exploration of synthetic MMP inhibitors (MMPIs), especially those targeting cancer diseases, in the mid and late 1990s [8,9]. The results of randomized controlled trials were overwhelmingly disappointing for small-molecule MMPIs, due to their poor oral bioavailability, lack of efficacy, dose-limiting toxicities, and undesired musculoskeletal side effects [10]. These first-generation synthetic MMPIs targeted

broadly by mimicking MMPs' natural substrate (usually collagen) [8], but later innumerable substrates were identified [11]. Unfortunately, these early MMPIs also inactivate proteinases unrelated to the disease but necessary for one or more physiological processes [8]; the importance of ADAMs and ADAMTSs was unknown at the time. Today, there is consensus that MMPs, ADAMs, and ADAMTSs function in many cell-signaling pathways, in which they are probably even more important than in ECM remodeling [11,12]. In parallel with our increased understanding of the diverse biological roles of these proteinases, the current goal is the development of highly selective inhibitors [8,13] although there is still no approved MMPI available. The only exception is low-dose oral doxycycline (Periostat®), which was approved in 1998 as an adjunct for the treatment of adult periodontitis. The mechanism of the beneficial doxycycline at subantimicrobial doses (20 mg b.i.d.) involves inhibiting collagenase (MMP-8) activity [14].

The content of this Special Issue highlights the multiple biological functions of these proteolytic enzymes and includes four review articles [4,15–17] and 10 original articles [18–27]. The articles address the role of MMPs, ADAMs, and ADAMTSs, in normal physiology as well as in neoplastic and tissue destructive processes.

Reflecting their pleiotropic activities, MMPs are closely associated with direct and indirect cell communication by modifying cell adhesion via integrin interactions and by activating or inactivating cytokines/chemokines or other signaling biomolecules and their cognate receptors. The specific roles of MMPs in these complicated signaling networks are highlighted by Young et al. [17].

A specific example of MMP-dependent cytokine signaling was elucidated by Sammel et al. [27]. They showed that not only ADAM-10 but also ADAM-17 and several other MMPs can shed the interleukin (IL)-11 receptor to induce transsignaling, a process where soluble receptor fragments interact with the ligand to act on cells not responsive to the ligand alone. Functional redundancy ensures a robust response, even in the absence of individual members of the shedding machinery. Although the current study remains at the level of cell culture and overexpression systems, it warrants further investigation of this elaborate proteinase network in animal models and in samples from patients with disturbances in individual components.

Another example of the importance of MMPs as proteinases with essential functions that have evolved within robust networks with redundant activities is presented by Rogerson et al. in their studies on ADAMTS [26]. In their research study in genetically manipulated mice, Rogerson et al. identified ADAMTS-9 as a novel aggrecanase that, in the absence of the aggrecanolytic ADAMTS-4 and ADAMTS-5, is highly increased in its abundance and might assume the critical functions of ADAMTS-4 and ADAMTS-5 in normal skeletal development [26]. ADAMTS-4 and ADAMTS-5 are thought to contribute to osteoarthritis by degrading the proteoglycan aggrecan in articular cartilage [28]. ADAMTSs are highly conserved in mice and humans, but it remains to be explored if similar redundancies also contribute to human pathologies.

The way in which membrane-tethered MMPs can interact with soluble members of the MMP family was demonstrated by Albrechtsen et al. at the University of Copenhagen [18]. By identifying basigin, an inducer of soluble MMPs, as a novel shed substrate of ADAM-12, they revealed a new function of the proteinase within the MMP network. This mechanistic insight has the potential to help devising novel strategies for inhibiting aberrant MMP activity in carcinogenesis, although in vivo validation is required.

Yip et al. [16] thoroughly reviewed the literature on MT4-MMP (MMP-17), another membrane-anchored MMP that has only been poorly studied [4]. MT4-MMP is one of the two members (the other one is MT6-MMP) of the family that is tethered to the membrane by a glycosylphosphatidylinositol anchor and shows specific activity in processing only a few ECM components. These appear to be important in many diseases, particularly in several types of cancer, which again demonstrates the strong need to better understand every member of the MMP family as a potential target for therapy.

Several studies in this Special Issue corroborate findings that show that MMPs have to be tightly controlled to prevent detrimental activity in disease. This might also be achieved by posttranslational modification (PTM) of the proteinase or the substrates. Many PTMs in MMPs and their target proteins have been identified, but their dynamic relationships are still poorly understood. In a review article, Madzharova et al. [4] summarize the current knowledge of the PTM (glycosylation, phosphorylation, and glycosaminoglycans)-mediated control of MMP activity and review the technologies used to study this topic.

As an additional layer of the control of MMP activity, soluble MMPs can also activate each other, e.g., by propeptide removal. For instance, the stromelysin MMP-3 is incapable of cleaving triple-helical fibrillar collagens but increases collagenolysis via the activation of collagenases [29]. Mirastschijski et al. [24] has convincingly demonstrated the profound effect of the lack of MMP-3 on tumor necrosis factor-α (TNF-α)-initiated collagenolysis in the skin of MMP-3-deficient mice, a finding that supports the indirect pathological role of MMP-3 in skin collagen catabolism through the activation of the human collagenase MMP-1 [30]. The authors did not demonstrate a direct link between murine MMP-13 and collagenase activity.

Oku et al. [25] have studied the effect of inflammation on cancer metastasis and the involvement of matrix metalloproteinase-9 (MMP-9). They used exogenous TNF-α to mimic the in vivo conditions and found that TNF-α upregulated MMP-9 at the transcriptional and translational levels in gastric cancer and mesothelial cell lines. The peritoneal barrier was modeled in vitro by mesothelial cells grown on a basement membrane matrix. TNF-α increased cancer cell invasion of the mesothelium via a process that was inhibited by MMP-9 gene silencing but not by MMP-2 gene silencing and was rescued by the addition of MMP-9 (Figure 1).

**Figure 1.** Tumor necrosis factor-α (TNF-α) stimulates gastric carcinoma cells and peritoneal mesothelial cells to secrete matrix metalloproteinase-9 (MMP-9), which promotes cancer cell invasion [25].

Huber et al. [22] set out to elucidate the cellular mechanisms involved in the differential effect of high-molecular-weight heparin (HMWH) compared with that of other anticoagulants on MMP-9 blood levels. For this purpose, monocytic and T and B lymphocytic cell lines were cocultured. The researchers demonstrated that HMWH increased IL-16 and sICAM-1 secretion by T lymphocytes, which in turn increased IL-8 and MMP-9 production by monocytes (Figure 2).

**Figure 2.** Proposed model of the induction of matrix metalloproteinase-9 (MMP-9) production in monocytes by high-molecular-weight heparin (HMWH)-treated T cells. T cells secrete the mediators IL-16 and sICAM-1, which induce monocytic IL-8 production. Together, these factors induce continuous IL-8 secretion as well as enhanced MMP-9 production by monocytes [22].

To investigate the emerging functions of MMPs as immune modulators, Bates et al. [19] mimicked the influence of inflammatory processes initiated by *Porphyromonas gingivalis*in vitro, causing periodontal destruction. They developed a 3-cell coculture model that includes monocyte-derived dendritic cells, CD4+ T lymphocytes, and primary gingival keratinocytes. MMP-7 and MMP-12 production differed significantly between the single cell cultures and the cocultures.

Dreschers et al. [20] addressed the clinical problem of premature delivery due to intrauterine infection. They hypothesized that detrimental persistence of inflammation is caused by reduced apoptosis of neonatal monocytes following phagocytosis of the infectious agent, e.g., *Escherichia coli*. The reduced apoptosis of infected neonatal monocytes compared with that of infected adult monocytes was attributed to increased shedding of CD95L by MMP-9. TACE (ADAM-17) was not involved in this process. One drawback of the study was the use of the general MMP inhibitor chlorhexidine; thus, the involvement of other MMPs could not be ruled out [31].

Tuberculosis remains a serious infectious disease, causing 2 million deaths every year at a global level. In a comprehensive review of MMP involvement in tuberculosis, Rohlwink et al. [15] summarize the current knowledge of the functions of MMPs in tuberculosis infections in both the lung and the brain. They outline the critical activities of MMPs in ECM degradation and pathogen release in the lung as well as in modulating immune responses. The use of various MMPIs in preclinical models of pulmonary and central nervous system tuberculosis is also reviewed. The ambiguous roles of MMPs in disease progression, particularly in childhood tuberculous meningitis, underscore the need for an increased understanding of how to balance MMP activity in treatment strategies.

Neuropathic pain is very difficult to treat, and there is an unmet medical need for effective therapeutic approaches. Kwan et al. [23] evaluated the effect of MMP-2/MMP-9 inhibition on neuropathic pain (Figure 3). They used an orally bioavailable MMP-2/MMP-9 inhibitor (AQU-118) in the spinal nerve ligation rodent model of neuropathic pain and discovered a novel relationship between elevated MMP-2 mRNA expression levels and caspase-3-mediated cell death, once again highlighting the complex interactions of MMPs with other proteinases within the proteinase network.

**Figure 3.** MMP inhibitor (MMPI) targets (increased apoptosis, increased cytokine activation, and decreased myelin basic protein levels) in neuropathic pain [23].

Snake venom metalloproteinases belong to the adamlysins, and their antithrombotic effects are well-known [32]. Huang et al. [21] tested the therapeutic effect of the metalloproteinase SP, which was isolated and purified from the venom of a moccasin snake (*Agkistrodon acutus*), in animal models of induced thrombosis and pulmonary embolism. The protein prolonged the coagulation time and inhibited platelet aggregation and thrombosis. Mechanistically, metalloproteinase SP cleaved the α, β, and γ chains of fibrinogen.

During wound healing, MMPs are involved in multiple cellular, molecular, and biochemical processes [33]. To decipher the role of MMPs, we used synthetic nondiscriminative MMPIs in animal and human acute injury wound-healing models [34–39]. The main conclusion of these studies is that neoepithelium formation is severely impaired by blocking MMP activity, while, paradoxically, increased collagen deposition in skin or peritoneal or intestinal wounds does not result from inhibiting MMP activities [34–37]. Without the protective epithelium, there is an increased susceptibility to infection. This presents a therapeutic challenge for nonhealing chronic cutaneous wounds, which often present with excessive MMP activity [33,40,41]: therapy should block the activity of pathogenic MMPs but not affect the activity of the MMPs required for reepithelialization [39]. The MMPs responsible for reepithelialization are unknown, but mouse models indicate that MMP-9 is one candidate [42], and in vitro studies suggest MMP-1 and MMP-7 as important candidates as well [43,44]. MMP-10 is highly upregulated in keratinocytes of the migrating tip in wounds with dermal involvement [39,45] and can cleave several cell adhesion and bioactive proteins [46]. Nonetheless, MMP-10-deficient mice did not show severe deficits in epithelial healing [47], indicating that the role of redundancy in the interconnected MMP network needs to be explored. Another clinical example is the regeneration of

the mucosal epithelium of anastomotic wounds after resection of diseased colorectal tissue. While they are beneficial for normal anastomotic wound healing [9,48], broad-spectrum MMPI therapies are detrimental to anastomotic wound repair under complicated conditions [49]. Under these conditions, nonselective MMPIs severely delay epithelial coverage resulting in increased invasion by pathogenic microorganisms, abscess formation, and anastomosis insufficiencies [49]. Additionally, inhibition of the antimicrobial MMP-12 may contribute to weakening of the host defense [50]. The use of more selective MMPIs or local delivery of the MMPI to avoid systemic side effects may be a solution [37,51].

In conclusion, the present Special Issue has shed some light on the complex functions of MMPs, ADAMs, and ADAMTSs, in physiological and pathological processes. The new experimental and collected data that are provided here add to the current knowledge. The identification of novel substrates has been critical to the recent advances of the MMP research field [52], and novel high-throughput degradomics technologies have been instrumental in extending the classical view on MMPs as simple tissue degraders to precise signaling scissors modulating complex immune responses. The use of these promising technologies together with valid disease models and clinical studies has the potential to translate into effective therapeutic modulators inhibiting detrimental MMP activities and enhancing their beneficial effects as targets and antitargets in diseases [12,53].

**Funding:** Ulrich auf dem Keller acknowledges support by a Novo Nordisk Foundation Young Investigator Award (NNF16OC0020670).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Post-Translational Modification-Dependent Activity of Matrix Metalloproteinases**

#### **Elizabeta Madzharova, Philipp Kastl, Fabio Sabino and Ulrich auf dem Keller \***

Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark<EMAIL_ADDRESS>(E.M<EMAIL_ADDRESS>(P.K<EMAIL_ADDRESS>(F.S.)

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +45-45-25-2791

Received: 27 May 2019; Accepted: 18 June 2019; Published: 24 June 2019

**Abstract:** Due to their capacity to process different proteins of the extracellular matrix (ECM), matrix metalloproteinases (MMPs) were initially described as a family of secreted proteases, functioning as main ECM regulators. However, through proteolytic processing of various biomolecules, MMPs also modulate intra- and extracellular pathways and networks. Thereby, they are functionally implicated in the regulation of multiple physiological and pathological processes. Consequently, MMP activity is tightly regulated through a combination of epigenetic, transcriptional, and post-transcriptional control of gene expression, proteolytic activation, post-translational modifications (PTMs), and extracellular inhibition. In addition, MMPs, their substrates and ECM binding partners are frequently modified by PTMs, which suggests an important role of PTMs in modulating the pleiotropic activities of these proteases. This review summarizes the recent progress towards understanding the role of PTMs (glycosylation, phosphorylation, glycosaminoglycans) on the activity of several members of the MMP family.

**Keywords:** MMPs; PTMs; glycosylation; phosphorylation; glycosaminoglycans

#### **1. MMP Domain Structure and Classification**

Matrix metalloproteinases (MMPs) comprise a family of 23 distinct secreted or membrane-anchored endopeptidases in humans that belong to the metzincin superfamily of metalloproteases. MMPs were initially described as regulators of the extracellular matrix due to their capacity to degrade ECM proteins like collagen, gelatin, laminin, aggrecan, fibronectin, elastin, and proteoglycans. Based on structure and substrate specificity, the MMPs are divided into collagenases (MMP1, MMP8, MMP13), gelatinases (MMP2, MMP9), stromelysins (MMP3, MMP10, MMP11), matrilysins (MMP7, MMP26), membrane-type MMPs (MMP14, MMP15, MMP16, MMP17, MMP24, MMP25), and other non-classified family members (MMP12, MMP19, MMP20, MMP21, MMP23, MMP27, MMP28). MMPs share a multidomain structure consisting of signal peptide, zymogenic pro-peptide domain, a catalytic domain, a variable linker 'hinge' region and a hemopexin domain. Some MMPs show variation in the domain arrangements, e.g., MMP7 and MMP26 are lacking the linker and hemopexin domain, MMP23 has a unique cysteine-rich and immunoglobulin-like instead of a hemopexin domain, and the two gelatinases MMP2 and MMP9 have additional fibronectin type-II-related domains. Membrane-type MMPs are anchored to the membrane either via glycosylphosphatidylinositol (GPI) or with help of a transmembrane domain (Figure 1). Additionally, MMPs are distinguished by the highly conserved HExGHxxGxxH motif in the catalytic domain, which contains three histidines that coordinate the zinc molecule in the active site, and the PRCGxPD motif identified in the pro-domain whose cysteine residue coordinates with the active zinc molecule to inhibit proteolysis. Each of these domains is associated with a specific function and very frequently post-translationally modified. This suggests an important role of PTMs in modulating the pleiotropic activities of MMPs [1,2].

**Figure 1.** MMP multidomain organization. Based on their domain structure, MMPs can be subdivided into eight different groups. All MMPs share a common structure comprising a signal peptide (SP), a pro-domain (Pro), containing a thiol group (SH), a catalytic domain (Catalytic) with a zinc (Zn) binding site, a linker region (Hinge) and a hemopexin domain (Hemopexin), which has a disulfide bond (S-S). Exceptions to this are the two gelatinases, which contain three fibronectin repeats (Fi) within their catalytic domain and furin-activated MMPs which have a furin-recognition site (Fu) within their pro-domain and MMP21 with an additional vitronectin-like insert (Vn). Some membrane-type MMPs are anchored to the membrane via glycosylphosphatidylinositol (GPI), whereas some MT-MMPs have transmembrane (TM) and cytosolic domains (Cy). In type II MT-MMPs, an N-terminal signal anchor (SA), a cysteine array (CA) domain, and an immunoglobulin-like (Ig-like) domain are present. Adapted by permission from "Springer Nature: New functions for the matrix metalloproteinases in cancer progression. *Nat. Rev. Cancer* **2002**, *2*, 161–174. Egeblad & Werb, Copyright (2002)".

#### **2. MMP Substrates and Function**

MMPs are primarily extracellular proteases, supporting the initial concept that they are generally associated with degradation and regulation of the ECM, thereby influencing many fundamental cellular events involving ECM remodeling [2–5]. More recently, it was observed that they also can irreversibly process growth factors, cell-surface receptors, cytokines, and chemokines, as well as other MMPs, other proteases and protease inhibitors and even act inside the cell [6]. The activation or inactivation of bioactive molecules by MMP proteolytic processing unraveled unexpected roles of these proteases in the regulation of extra- and intracellular signaling pathways [4,7–9]. Hence, the field of MMP research progressed from perceiving these enzymes solely as regulators of ECM to the conception that MMPs are functionally implicated in the regulation of multiple physiological and pathological signaling processes [8,10]. Accordingly, the physiological relevance of their function is emphasized by a direct association between altered expression and/or dysregulation of MMPs and development of pathological conditions, such as chronic inflammatory diseases, vascular diseases, neurological disorders, and cancer [10,11].

#### **3. Multilayered Regulation of MMP Activity**

Since MMPs regulate major physiological processes, a strict spatiotemporal control of their activity is essential to avoid possible detrimental activities of these proteases. Primarily, expression of MMPs is regulated at the transcriptional level, keeping these enzymes at very low levels in normal tissue homeostasis. MMPs share cis-regulatory elements in their promoter sequences, which allow induction of their expression by stimuli—e.g., in the form of growth factors, cytokines, or hormones. The cooperation between these cis-regulatory elements, coupled with the integration of multiple signaling pathways, provides a wide range of potential interactions between transcriptional regulators, ensuring tissue-specific expression of diverse MMP family members and facilitating a strict control of MMP transcriptional activity. In addition, transcription of MMP encoding genes is regulated by epigenetic mechanisms, such as DNA methylation or histone acetylation [4,5,11,12]. At the post-transcriptional level, regulation of MMP expression is mediated by modulation of mRNA stability and miRNA-based mechanisms that interfere with MMP expression by either transcriptional inhibition or mRNA degradation [13–16]. An important level of MMP regulation is achieved at the post-translational level, since most MMPs are secreted as inactive pro-enzymes. The 'cysteine-switch' region in the pro-peptide domain shields the active site, thereby preventing substrate access and maintaining the enzyme in an inactive state. The activation of proMMPs occurs upon direct proteolytic cleavage of the pro-domain (within the secretory pathway or extracellularly), or induction of conformational changes that disrupt the chelating cysteine residue and enable auto-proteolysis for removal of the pro-domain region [9,12]. Moreover, allosteric activation of proMMPs can be triggered by interaction with ECM components and cell surface molecules and by low-density lipoprotein receptor-related protein (LRP1)-mediated endocytosis [12,17–20]. Upon activation, mature MMPs are exclusively controlled by endogenous inhibitors, such as tissue inhibitors of metalloproteases (TIMPs) and α2-macroglobulin [21,22].

#### **4. PTMs—An Additional Level of Protein Regulation**

The enormous functional complexity of the cellular proteome is regulated by diverse mechanisms, including transcription, alternative splicing, translation, and PTMs [23]. Among these regulatory mechanisms, PTMs provide a significant genome-independent expansion and diversification of the proteome, thereby creating a continuously fine-tuned regulatory network implicated in many cellular processes [24–26]. Based on the type of modification, PTMs can be assigned to several categories: chemical modifications, including methylation, phosphorylation, acetylation, and oxidation; polypeptide modifications, including ubiquitination, SUMOylation and other ubiquitin-like protein conjugation; modifications by complex molecules, including glycosylation, lipids (e.g., acylation, prenylation) and extended structures (e.g., glycosaminoglycans (GAGs)); and modifications of the amino acids or of the polypeptide backbone, including deamidation, eliminylation, and protein cleavage through proteolysis [27–30]. By reversible or irreversible addition of these functional groups PTMs can modify protein function by altering protein structure, subcellular localization, protein–protein interactions, and degradation, thereby influencing many cellular processes in health and disease [31–36].

Like many enzymes, MMPs as well as their substrates are modified by a variety of PTMs. Different domains of the MMP structure are associated with specific functions and undergo different, highly specific PTM modifications, which suggests an important role of PTMs in modulating the pleiotropic activities of MMPs (Figure 2) [1]. Understanding the influence of specific PTMs on the activity of MMPs is necessary to fully understand MMP regulation. In this review, we provide an overview of different PTMs with a focus on glycosylation, phosphorylation, and interaction with extracellular GAGs, and describe their effects on activity of various MMP family members.

**Figure 2.** Possibilities of PTM crosstalk with MMPs. PTMs can regulate the activity of MMPs on the protease level by (**a**) activating the protease to allow cleavage of the target protein, (**b**) inactivating the protease, or (**c**) modulating its substrate specificity. Additionally, PTMs may regulate substrate cleavage by MMPs, through (**d**) directly modifying the cleavage motif (catalytic interaction), or substrate domains outside the cleavage site (non-catalytic interaction), guiding MMPs to cleave the same (*cis*) (**e**) or another (*trans*) protein (**f**).

#### *4.1. Glycosylation of MMP*

Glycosylation describes the enzymatic reaction that links saccharides to lipids, other saccharides or proteins and represents the most abundant and diverse PTM. The most common forms of protein glycosylation are the *N*- and *O*-linked glycosylation [37,38]. *N*-linked glycosylation is a glycosidic linkage of glycans to the side chains of asparagine (Asn) residues. It is initiated by a single oligosaccharyltransferase complex with a transfer of *N*-acetylglucosamine (GlcNAc) phosphate at the consensus Asn-Xaa-Ser/Thr sequence (X, indicating any amino acid excluding proline) on the cytosolic side of the endoplasmic reticulum (ER) and extensively modified further in the lumen of the ER and Golgi prior to sorting to secretory vesicles [37,39–41]. *O*-linked glycosylation is a glycosidic linkage of glycans to the side chains of serine/threonine (Ser/Thr) or tyrosine (Tyr) residues. *O*-linked glycosylation shows a higher diversity than *N*-glycans, resulting from the complex synthesis of *N*-acetylgalactosamine (GalNAc)-type *O*-linked glycans in the Golgi, which can be initiated by up to 20 different polypeptide GalNAc-transferase isoforms that may compete for the same glycosylation site and the same glycan. Upon transfer of a GalNAc moiety from a donor substrate uridine diphosphate (UDP)-GalNAc to Ser/Thr residues, the *O*-linked glycan is branched out by different core-forming enzymes and further extended by fucosylation and sialylation to the final *O*-linked glycan structure [41–45]. Protein glycosylation regulates essential biological processes, such as protein folding, secretion, cell adhesion, and inter- and intra-cellular trafficking [46–50]. Consequently, alterations in glycosylation patterns are often associated with different pathological conditions like neurodegenerative diseases, diabetes, inflammatory conditions, and cancer [51,52]. The majority of MMPs are glycosylated, and *N*- and *O*-linked glycosylation are present across the MMP family [39]. The following section summarizes the current structural and functional information about glycosylation of different MMPs.

#### 4.1.1. MMP9

MMP9 is the most extensively glycosylated MMP. This protease contains two *N*-linked glycosylation sites, Asn38 and Asn120 (asparagine residues 38 and 120), in the pro-domain and the catalytic domain, respectively [53]. These *N*-linked glycans have been described as core-fucosylated biantennary structures, partially sialylated with variable fucosylation branches [54]. Despite their similar composition, the individual functions of these *N*-linked glycosylations are distinct, primarily due to their location within the protease. Since *N*-linked glycosylation is generally required for protein secretion, initially a potential role of these two glycans was related to the secretion of MMP9 [55]. Indeed, abrogation of MMP9 glycosylation at Asn120 reduced the efficiency of its secretion by increasing the interaction between MMP9 and calreticulin (a protein that prevents misfolded proteins from entering the secretory pathway), thereby inducing MMP9 retention in the ER. The glycosylation at Asn38 does not influence the secretion of MMP9, but Asn38-glycosylation-deficient MMP9 shows strong amino-acid dependency towards interaction with calreticulin, likely affecting MMP9 secretion in an *N*-glycosylation-independent manner [56]. Additionally, *N*-glycosylation at Asn38 was commonly associated with the activation of proMMP9, yet the process of MMP9 activation occurs independently of the glycosylation at Asn38. Recent molecular dynamics simulations suggest that the glycosylation at Asn38 is indirectly involved in the activation of proMMP9 by inducing conformational changes within the pro-domain, enabling MMP3 to access the two cleavage sites for proteolytic activation [57]. Moreover, interactions between the glycosylation at Asn38 and galectin 3 decrease the proteolytic activation of MMP9, whereas interactions with galectin 8 enhance the MMP3-mediated processing, suggesting that the presence of *N*-glycosylation is important for a fine-tuned regulation of MMP9 activity [58,59]. Furthermore, MMP9 has a proline-rich linker sequence between the active site and the hemopexin domain, which contains 14 *O*-linked glycans (denominated OG domain) [60]. Based on their glycan composition, these *O*-linked glycans comprise a heterogenic mixture, varying from core-1 (Galβ1-3GalNAc) to core-2 (Gal-(GlcNAc-)GalNAc) structures and further elongated to larger glycans [61]. The presence of these *O*-linked glycans increases the domain flexibility of MMP9, allowing the protease to adopt multiple enzyme conformations and facilitating individual movements of the catalytic and the hemopexin domain. This influences the recognition, binding, and processing of substrates, cell receptors, and endogenous inhibitors [62,63]. A study by den Steen et al. described that the OG domain is indispensable for correct orientation of the hemopexin domain for MMP9 internalization and degradation by LRP-1 and LRP-2, as well as inhibition by endogenous TIMP1 [60]. Consequently, deletion of this linker region significantly reduced the affinity towards TIMP1 and disrupted the interactions with LRP-1 and LRP-2 [60]. However, since deletion of the OG domain did not affect the activity of MMP9, the authors reported that the OG domain functions as a regulator of extracellular bioavailability of the protease, rather than as a regulator of its activity [60]. An independent analysis of MMP9 lacking the OG domain performed by Vandooren et al. showed a reduced gelatinolytic activity of the truncated protease [64]. Furthermore, a study by Dufour et al. reported reduced MMP9-induced cell migration in endothelial cells expressing MMP9 without the OG domain, which was independent of the proteolytic activity of the enzyme [26].

#### 4.1.2. MMP14

Similar to MMP9, the transmembrane collagenase MMP14 (MT1-MMP) contains *O*-linked glycosylation sites (Thr291, Thr299, Thr300, and Ser301) identified in the linker region. The presence of these *O*-linked glycans does not affect zymogen activation, collagenase activity or the autolytic processing of MMP14. However, the *O*-linked glycosylation is important for the formation of a stable complex between MMP14, TIMP2, and proMMP2, which is required for subsequent cell-surface activation of MMP2. Being unable to recruit TIMP2, which connects the catalytic domain of MMP14 and the hemopexin domain of proMMP2, the glycan-deficient MMP14 cannot present a stable trimeric complex on the cell surface and thereby activate MMP2 [65–67]. Interestingly, although it has not appeared that glycosylation could directly affect MMP14 activity, recent reports describe increase of MMP14 activity upon alterations in the glycosylation pattern. Indeed, hyper-glycosylation of MMP14 led to higher proteolytic activity and promoted tumor growth [68].

#### 4.1.3. MMP1

MMP1 has two potential glycosylation sites in the active site, but only *N*-glycosylation at Asn120 has been experimentally confirmed. Comparison between the glycosylated and non-glycosylated MMP1 showed no significant differences in activity, substrate specificity or inhibitory profiles of the two proteoforms. However, the presence of specific glycan motifs (e.g., α1,3-fucosylated LacdiNAc) can initiate MMP1 selectin-mediated binding to the surface of activated cells through a selectin/glycan interface and therefore may have a profound effect on cell migration [39,69].

#### 4.1.4. MMP2

MMP2 has two potential *N*-linked glycosylation sites, Asn573 and Asn642, in the hemopexin domain [70,71]. The function of these *N*-linked oligosaccharides remains unclear. However, there are strong implications regarding their involvement in the regulation of MMP2, since the hemopexin domain is involved in MMP2 activation/inhibition, localization of its catalytic activity, and induction of cell signaling upon interaction of MMP2 with cell-surface receptors [71,72].

#### 4.1.5. MMP3

Based on the consensus sequence for *N*-linked glycosylation, MMP3 also has two potential *N*-linked glycosylation sites, Asn120 in the catalytic domain and Asn398 in the hemopexin domain. However, only a small portion of MMP3 (~20%) is glycosylated, and no correlation has been established between these glycans and the function of the protease [39,73].

#### 4.1.6. MMP13

MMP13 was shown to be potentially glycosylated at two asparagine residues (Asn117 and Asn152) in the catalytic domain. The *N*-linked glycosylation site at Asn117 has been experimentally verified, but the function of this *N*-linked glycan has not been determined since no differences were observed between glycosylated and non-glycosylated recombinant MMP13 [74,75].

#### 4.1.7. MMP17

MMP17 (MT4-MMP), a glycosylphosphatidylinositol-anchored matrix metalloproteinase, has two *N*-linked glycans at Asn137 and Asn318 in the catalytic site and the linker region, respectively. The presence of these *N*-linked oligosaccharides stabilizes the dimeric form of MMP17 by promoting non-covalent interactions or facilitating folding and formation of disulfides [76–78].

#### *4.2. Phosphorylation of MMPs*

Protein phosphorylation is one of the most important PTMs for regulation of biological processes [79]. This dynamic PTM is characterized by the reversible enzymatic addition of a phosphate group to amino acid side chains of serine (Ser), threonine (Thr), or tyrosine (Tyr), resulting in alterations of protein structure, stability, and dynamics [79]. Hence, phosphorylation can initiate different conformational changes associated with differential activity and binding specificity, leading to an activation or deactivation of proteins [80,81]. Furthermore, the dynamic nature of phosphorylation resulting from the opposing activities of kinases (which add phosphate groups) and phosphatases (which remove phosphate groups) is functionally compatible with the modulation of intramolecular interactions, implicated in essential cellular processes, such as cell division, cellular proliferation and differentiation, apoptosis, and signal transduction [79]. Consequently, dysregulation of phosphorylation dynamics is often related with pathologies, e.g., tumor formation, chronic inflammatory diseases, autoimmune diseases, and neurodegenerative disorders [81,82].

As many secreted and extracellular proteins, the MMPs are phosphorylated. Ser-, Thr-, and Tyr phosphorylation sites are identified across the different structural domains of the MMPs. Yet, the biological significance of these phosphate groups is overall poorly characterized. Among the MMPs, MMP2 and membrane-type MMP14 metalloproteinase are the ones with most extensively studied phosphorylation-dependent effects.

#### 4.2.1. MMP2

MMP2 contains 29 potential phosphorylation sites distributed across the pro-peptide domain, collagen-binding domain, collagenase-like domain-1 and -2, and hemopexin domain. However, only five of the predicted phosphorylation sites (S32, S160, Y271, T250, and S365) have been confirmed by mass spectrometry (MS). The phosphorylation of MMP2 noticeably diminishes its proteolytic activity, while dephosphorylation increases MMP2 activity. This is possibly due to the conformational changes observed in the secondary structure of dephosphorylated MMP2, with α-helices 50% longer and β-strands 17% shorter than phosphorylated MMP2. However, it is still unclear if protease activity is directly influenced by these conformational changes [83,84].

#### 4.2.2. MMP14

MMP-14 phosphorylation is known to play an important role in fine-tuning the activity of MMP14 at the cell surface and in the activation of intracellular signaling, and it is critical for regulating the pro-metastatic function of this metalloproteinase. This membrane-type metalloprotease has nine potential phosphorylation sites in its multidomain structure (PhosphoSitePlus [85], UniProt [86]). The cytoplasmic domain of MMP14 regulates its internalization and trafficking, thereby modulating enzymatic activity at the cell surface. A study by García-Pardo et al. reported that this domain was essential for MMP14 mediated cellular invasion and migration [72]. The cytoplasmic domain of MMP14 contains two phosphorylation sites, at Thr567 and Tyr573, which have a significant influence on MMP14-induced cellular invasion and migration. A study by Williams et al. in fibrosarcoma cells showed that substitution of the residue Thr567 by alanine increased retention at the cell surface and reduced internalization of the protease significantly compromising invasion and migration. Contrarily, mimicking protease phosphorylation by substitution of Thr567 by glutamic acid reduced retention of MMP14 at the cell surface, increased efficiency of internalization, and was correlated with an increase in migration and invasion [87]. A study by Moss et al. showed that phospho-mimetic Thr567 mutants exhibit higher collagenolytic activity and three-dimensional growth within a collagen matrix, thereby promoting enhanced matrix invasion in ovarian cancer cells [88]. In addition, phosphorylation of Thr567 impacted the integrity of cell monolayer, cell motility and multicellular aggregate dynamics in ovarian cancer cells, promoting metastasis-associated behaviors [89]. Furthermore, a study by Nyalendo et al. reported that phosphorylation at Tyr573 influenced cell migration, suggested

by the ability of a phospho-defective mutant to inhibit migration of cells endogenously expressing MMP14 [90]. Additionally, epidermal growth factor (EGF)-induced phosphorylation of Tyr573 prompted internalization of MMP14 together with pericellular collagen, establishing an environment for expansive growth in three–dimensional collagen matrix, whereas lack of responding to EGFR signaling triggered invasive growth. Therefore, the phosphorylation of Tyr573 modulates cell surface dynamics of MMP14, thereby regulating the transition between invasive and expansive growth [91].

#### 4.2.3. Extracellular Phosphorylation of MMPs

A subset of MMPs (MMP1, 12, 13, 14, 16, 24, and 27) are tyrosine phosphorylated by the extracellular vertebrate lonesome kinase (VLK). These phosphotyrosines are found exclusively within the hemopexin domains of the MMPs. Interestingly, proteins distantly related to MMPs, which contain hemopexin-like domains, were found to be tyrosine phosphorylated at identical positions by VLK. The structural conservation of these phosphorylation sites suggests that they play a role in regulating MMP activity, a potential function, which still remains largely undiscovered [92,93]. Additionally, a study by Bordoli et al. showed that co-expression of VLK with MMPs supported their tyrosine phosphorylations that have been extensively observed in vivo. Bordoli also showed that by introducing a mutation in the ATP binding site of VLK or deletion of the proline glycine-rich domain (a conserved domain close to the kinase domain), the phosphorylation of the MMPs and other co-expressed substrates in the extracellular environment was eliminated. Similarly, shRNA-mediated downregulation of VLK expression and substitution of Tyr360 with phenylalanine in MMP1 reduced MMP13 and MMP1 tyrosine phosphorylation, respectively [93]. While VLK generates the majority of extracellular phosphotyrosines, additional secreted kinases have been identified, which may also be responsible for the extracellular protein phosphorylation events observed in vivo. This indicates a major impact of the secreted kinome on tissue homeostasis and disease pathogenesis. Discovery of novel extracellular kinases, identification of their substrates, and dissection of the regulatory mechanisms involved will provide us with a better understanding of their functions [94–96].

#### *4.3. Glycosaminoglycans*

An additional layer of regulation of extracellular proteolysis is mediated by interactions of MMPs with glycans in the extracellular space rather than direct glycosylation or phosphorylation of the protease or substrate. Glycosaminoglycans (GAGs) are extracellular glycans, which are composed of linear, unbranched repeats of disaccharide units. Heparan sulfate (HS) is a GAG-family member expressed by virtually every cell of a multicellular organism composed of repeats of glucoronic acid (GlcA) and *N*-acetylglucosamine (GlcNAc) dimers, which in most cases are attached to a cell-membrane associated core protein. During GAG-chain synthesis HS undergoes several sulfotransferase-mediated *N*- and *O*-sulfations. These modifications do not undergo completion, which results in strongly negatively charged sugar chains with variable length and degree of sulfation interrupted by stretches of unmodified, neutral regions. The sulfated regions bind basic peptide motifs of many signaling molecules, including morphogens (Hedgehogs, Wnts, TGFs), cytokines, and most chemokines [97–99]. This electrostatic interaction leads to partial neutralization of the protein surface and can have different effects on the biological functions of the interacting protein by facilitating, enhancing, or inhibiting the interaction with other proteins. It is important to note that these interactions, even though they are mainly based on electrostatics, are in most cases very specific and do not occur randomly. This is shown by the fact that many proteins contain specific GAG-binding motifs, with which they exclusively bind HS, even though they contain many lysine and arginine residues (for an in-depth review of the molecular basis of HS-protein interactions see Xu & Esko, 2014 [100]). Examples of HS-binding proteins are members of the fibroblast growth factor (FGF) family. Here, HS is part of the trimeric FGF/FGF-receptor/HS co-receptor complexes and activates FGF signaling in a sulfation pattern-dependent manner [101–104]. HS-induced oligomerization has been shown in many other processes including dimerization of amyloid precursor protein (APP) complexes or nearly all

chemokines [105–108]. Interestingly, HS does not only mediate protein complex formation, but many proteins binding to HS, including the aforementioned candidates, are also known to undergo proteolytic processing on or near the cell surface. This indicates a crucial role of HS in regulating extracellular cleavage processes.

#### 4.3.1. GAG-regulated Substrate Proteolysis

Signaling processes in the extracellular space usually involve at least one form of substrate proteolysis. Therefore, co-factors have to act as decision makers to ensure regulated processing of signaling proteins. Multiple cases have been reported, where this is mediated by substrate binding to GAGs. For example, similar to *N*-glycans, GAGs can modulate the stability of substrates. FGF2 binding to heparin, an extremely sulfated HS variant expressed by connective-tissue type macrophages, stabilizes FGF2 and protects it from degradation by proteolysis [109,110]. The same stabilizing effect has been shown for the degradation of stromal cell-derived factor 1 (SDF1)/CXCL12 and C-terminal processing of interferon-γ [111,112]. Despite this stabilizing effect GAGs have been demonstrated to play additional important roles in extracellular proteolysis. In the case of shedding of morphogens from the Hedgehog (Hh) family, HS acts as an assembly platform for protease-release complexes [113]. Here, HS recruits Hh co-factors that co-localize proteases and allow for cleavage of the substrate (Figure 3) [114]. Furthermore, direct HS binding of the N-terminal cleavage site of Hh also has a stabilizing effect by inhibiting N-terminal Hh processing and reduces its release in vitro and in *Drosophila* in vivo [114,115]. An example of how GAG binding can positively regulate substrate cleavage (Figure 3) was shown for the cleavage of viral capsid proteins. Binding of the human papillomavirus viral capsid protein L1 to highly sulfated HS is essential to induce a conformational change and leads to cleavage of L1 by the human trypsin-like serine proteinase kallikrein 8, a process which is required for virus internalization [116].

**Figure 3.** Heparan sulfate (HS)-protein interaction as a post translational regulator of extracellular proteolysis. Electrostatic interactions of proteins with negatively charged HS affect extracellular proteolysis at three different levels: (1) by mediating co-localization of reactants; (2) on the protease level by affecting protease activity/accessibility; or (3) on the substrate level by modulating accessibility to cleavage sites. The substrate cleavage site is indicated by a red circle, green substrate/protease color denotes activity; red substrate/protease color denotes inactivity/inaccessibility.

#### 4.3.2. GAG-regulated MMP Activity

GAGs also regulate extracellular proteolysis at the protease level by modulating activation, localization, and protease–substrate interactions (Figure 3 middle). Many secreted vertebrate MMPs associate to the cell surface via binding to HS, including MMP9, which is specific, since excess soluble heparin extracts and solubilizes MMP2, MMP7, MMP9, and MMP13 [117,118]. Moreover, heparin, a highly sulfated form of HS, affects protease activity by increasing TIMP3 affinity to MMP2, MMP7, and MMP9, and HS affects MMP9 expression and plasma levels, depending on its sulfation and expressing cell type [119–123]. Finally, in vivo studies showed that MMPs in secretory granules of mast cells interact with heparin and that loss of heparin sulfation reduces MMP stability [124,125]. Like all MMPs, the wound repair and immune reaction associated MMP7 has to be converted into its proteolytic active form by removal of its inhibitory pro-sequence. MMP7 is able to activate itself by intermolecular autolytic processing. This is amplified by binding of MMP7 to highly sulfated GAGs (e.g., heparin or chondroitin sulfate), which significantly increases MMP7 auto-processing and also increases cleavage of specific physiological substrates [19].

#### **5. Conclusions**

MMP biology has been revolutionized with the recognition of extracellular proteolysis not as a simple mechanism of ECM degradation but as a regulatory mechanism for precise cellular control of biological processes. The paradigm shift for MMP functions from ECM degradative proteases to important regulators of essential cellular processes has highlighted the physiological relevance of these proteases, directly implicated by the relationship between MMP expression and disease development [4,8]. Hence, the multilayered regulation of MMPs emphasizes the tremendous importance of the balance between synthesis of active enzymes and their inhibition, which is pivotal to avoid the destructive activity of these proteases. Characterization of these regulatory mechanisms will aid the development of new therapeutics for various pathologies. MMP activity is regulated at the transcriptional level, post-translationally by pro-enzyme activation, by PTMs, and through extracellular inhibition by TIMPs and by non-specific proteinase inhibitors. Among these regulatory mechanisms, PTMs have recently obtained wide attention in the MMP community. MMPs are modified by PTMs at multiple sites (Table 1), which affects the activity of individual MMPs to a different extent, but the biological relevance of many of them is still unknown. However, many of the studies referenced in this review have been performed prior to the rapid progression of proteomics method development within the last decade. Many of the challenges that still limit our understanding of PTM function in MMP biology can now be approached by modern technologies of state-of-the-art proteomics. Advances in MS-based methods including multiplexed chemical labeling, novel label-free quantification strategies, improvement in PTM enrichment, more robust PTM analysis workflows and streamlined bioinformatics strategies will contribute to reliable identification and quantification of high numbers of PTMs [126,127]. The remaining challenge lies in defining the functional role of the physiologically relevant PTMs to understand PTM-dependent activity of MMPs in complex biological systems [128].

**Table 1.** *Cont*.

**Funding:** Ulrich auf dem Keller acknowledges support by a Novo Nordisk Foundation Young Investigator Award (NNF16OC0020670). Philipp Kastl is supported by a research fellowship from the German Research Foundation (project no. 415888450), and Fabio Sabino is recipient of a H.C. Ørsted COFUND Postdoc Fellowship (COFUNDfellowsDTU, grant agreement no. 713683).

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **Abbreviations**

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Matrix Metalloproteinases in Pulmonary and Central Nervous System Tuberculosis—A Review**

**Ursula K. Rohlwink 1, Naomi F. Walker 2, Alvaro A. Ordonez 3,4, Yifan J. Li 5, Elizabeth W. Tucker 4,6,7, Paul T. Elkington 8, Robert J. Wilkinson 9,10,11 and Katalin A. Wilkinson 9,10,\***

Received: 30 January 2019; Accepted: 3 March 2019; Published: 18 March 2019

**Abstract:** Tuberculosis (TB) remains the single biggest infectious cause of death globally, claiming almost two million lives and causing disease in over 10 million individuals annually. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes with various physiological roles implicated as key factors contributing to the spread of TB. They are involved in the breakdown of lung extracellular matrix and the consequent release of *Mycobacterium tuberculosis* bacilli into the airways. Evidence demonstrates that MMPs also play a role in central nervous system (CNS) tuberculosis, as they contribute to the breakdown of the blood brain barrier and are associated with poor outcome in adults with tuberculous meningitis (TBM). However, in pediatric TBM, data indicate that MMPs may play a role in both pathology and recovery of the developing brain. MMPs also have a significant role in HIV-TB-associated immune reconstitution inflammatory syndrome in the lungs and the brain, and their modulation offers potential novel therapeutic avenues. This is a review of recent research on MMPs in pulmonary and CNS TB in adults and children and in the context of co-infection with HIV. We summarize different methods of MMP investigation and discuss the translational implications of MMP inhibition to reduce immunopathology.

**Keywords:** tuberculosis; matrix metalloproteinases; tuberculous meningitis; HIV-TB-associated IRIS; extracellular matrix breakdown; adult; pediatric; lung; central nervous system

#### **1. Introduction**

Tuberculosis (TB) remains one of the top 10 causes of death globally, with 1.6 million deaths attributed to TB in 2017 [1]. Although the overall incidence of the disease is decreasing, there were over 10 million new cases of TB reported in 2017 [1], suggesting that more work needs to be done on disease prevention and cure. Tuberculous meningitis (TBM) is the most severe form of this disease and leaves many surviving adults and children with severe neurological impairment [2]. The inflammatory processes occurring during TB infection in the lungs and the neuro-inflammatory response as a result of dissemination to the brain play key roles in disease outcome. In pulmonary TB (pTB), matrix metalloproteinases (MMPs) are considered important in cavitary lung disease and the subsequent spread of *Mycobacterium tuberculosis* (*Mtb*) from the lung parenchyma into the airways from whence they can be expectorated to perpetuate infection [3].  In this review, we discuss the current status of MMP research in pulmonary and central nervous system (CNS) TB, and their potential dual role in pathology as well as neurodevelopment and recovery. We also summarize potential novel treatment strategies that may target MMPs.

#### *MMPs*

MMPs are a superfamily of zinc- and calcium-dependent proteolytic enzymes that is well conserved across species with 25 vertebrate MMPs (24 of which are found in humans) characterized to date [4]. Several MMPs, including MMP-1, -3, -7, and -8, are located within a single gene cluster, highlighting their essential role in fundamental physiologic processes [4]. Their primary function in humans is degradation of the extracellular matrix (ECM) [5], which not only contributes to health by providing tissue homeostasis but also has a role in many pathologic conditions [4]. MMPs fall under the general category of metalloproteinases (MPs) of the Metzincin family, which function both on cell surfaces (as sheddases, which release growth factors, death receptors, and death-inducing ligands), and within the ECM [6]. They are divided into five sub-families, namely collagenases (MMP-1, -8, and -13), gelatinases (MMP-2 and MMP-9), stromelysins (MMP-3 and MMP-10), elastases (MMP-7 and MMP-12) and membrane-type MMPs (MT-MMP-1 to -5) [7,8].

MMPs are synthesized and released as proenzymes, known as zymogens, that are subsequently activated when the zinc-thiol interaction between the catalytic and the pro-peptide domain is disrupted by cleavage of the pro-peptide domain [8–10]. Due to the destructive nature of excessive activity, MMPs are controlled by gene expression (transcriptional and post-transcriptional regulation), proenzyme activation, and innate inhibitors. They are inhibited by α2-macroglobulin in the plasma or tissue inhibitors of metalloproteinase (TIMPs) in the tissue [4]. TIMPs are endogenous protein regulators and show tissue-specific, constitutive, or inducible expression [11]. There are four members of the TIMP family (TIMPs 1-4), which bind MMPs non-covalently to inhibit activity. TIMP-3 acts on both MMPs and tumor necrosis factor (TNF)-α converting enzyme (TACE) [9,11]. However, despite preference for specific MMPs, they are able to inhibit all MMPs, binding them in a 1:1 molar stoichiometry [11].

Physiologically, MMPs have an important role in development (blastocyst implantation, embryonic development, nerve growth), reproduction (ovulation, endometrial cycling, cervical dilatation), and maintenance of homoeostasis (wound-healing, bone remodeling, angiogenesis, and nerve regeneration) [4]. However, in various pathologies, MMPs have demonstrated a role in both immuno-protection and tissue destruction. They contribute to activation of immune mediators and leukocyte migration to sites of inflammation and infection by modulating cytokine and chemokine activity [12]. They are also implicated in numerous pathologies such as cancer invasion and metastasis, hypertension, rheumatoid and osteo-arthritis, CNS diseases, and may cause direct tissue destruction at excessive concentrations [10,12]. In the lung, a number of MMPs contribute to tissue homeostasis, such as MMP-7, -16, -19, -21, -24, -25, and -28 [4]. For example, lung epithelial cells express MMP-7 that is essential in normal wound repair and plays a part in the chemotactic recruitment of neutrophils [13]. In the CNS, MMPs are normally undetectable or present at very low levels

(except for MMP-2) [10,14]. They can play a beneficial role in the CNS through their involvement with tissue repair after injury [9,14] and are also found to be elevated postnatally in rat and mouse CNS, suggesting pivotal roles in neuroplasticity and CNS development [15,16] such as synaptogenesis, synaptic plasticity, and long-term potentiation [17].

#### **2. Methods of Investigation**

Quantification of MMPs can be an analytical challenge for several reasons. In TBM, a number of methods of MMP investigation have been used, including ELISA, zymography and reverse zymography for serum, and cerebrospinal fluid (CSF) (Table 1). ELISAs for MMPs are, however, often limited by the absence of differentiation between active and inactive or degraded forms of MMP, and multiplexed assays are often complicated by cross-reactivity between analytes due to the protein's common domains [8]. Additionally, the method of blood collection can impact the concentration of MMPs, with higher concentrations reported in serum compared to plasma (both heparin and EDTA) [8] due to release from leukocytes and platelets during clot activation. For MMP-1, -8, and -9, plasma is recommended [18]. Brain tissue or granuloma samples have been examined using immunohistochemistry [19,20] and MMP gene expression using PCR [20,21]. Newer techniques such as analysis of gene polymorphisms, near-infrared optical imaging, and HPLC (high performance liquid chromatography) together with inductively-coupled plasma mass spectrometry (ICPMS) are evolving and promising analytic techniques that could improve the ability to profile all MMPs [8]. In addition to human data, in vitro cell and animal models have also offered valuable insights.

**Table1.***Cont*.

**Table 1.***Cont*.

36

**Table1.***Cont*.

37

factor-BB (PDGF-BB). Platelet factor-4 (PF4). Pentraxin-3 (PTX3). Receiver operating

Expressed, and Secreted (RANTES). Membrane type (MT). Monocyte

Albumin quotient (Q-alb).

chemoattractant

 protein 1 (MCP-1).

characteristics

 (ROC). Tumour necrosis factor (TNF). Regulated upon Activation, Normal T cell

Haematoxylin

 and Eosin (H&E). Single nucleotide

polymorphism

 (SNP).

#### **3. MMPs in pTB**

Destructive pulmonary pathology is the hallmark of human tuberculosis, and *Mtb* is relatively unique amongst pathogens for its ability to drive progressive destructive pulmonary pathology, including cavitation, in immunocompetent adults. Cavities are critical in *Mtb* pathogenesis, as they drive transmission (the most highly infectious patients are those with cavities), are immune-privileged sites with high bacterial burden, and are poorly penetrated by anti-mycobacterial drugs, leading to higher risk of treatment failure and relapse [54]. MMPs have been implicated in the pathology of pulmonary tissue damage and cavitation, as collectively, they can degrade all fibrillary components of the ECM (Figure 1). Previous reviews of MMPs in TB and mechanisms of cavitation have presented substantial evidence of MMP activity in pTB disease [3,55], summarized below:

As MMPs have different cellular sources and substrate specificity, it is unsurprising that specific MMPs have been associated with different roles within the same disease. For example, MMP-1 has been most strongly implicated in the development of pulmonary cavities, whereas MMP-9 has been implicated in *Mtb* dissemination.

Here, we review in detail more recent (since 2013) studies of pTB that have further delineated cellular sources of MMPs in TB, mechanisms by which MMP activity is regulated in TB, and the importance of the cellular and extracellular environment. We also describe studies that have evaluated MMPs and matrix degradation products as TB biomarkers (Table 1, Section A).

**Figure 1.** MMPs contribute to extracellular matrix (ECM) destruction in pulmonary TB (pTB). Lung TB granulomas comprise mycobacterium tuberculosis (*Mtb*) infected macrophages (purple), dendritic cells (beige), T (blue) and B (green) lymphocytes, and neutrophils (orange). These cells secrete numerous MMPs including MMP-1, -3, -7, -9, and -10, which may act in a proteolytic cascade. Evidence demonstrates their association with lung ECM breakdown, necrosis, and pTB disease severity. MMP-1 drives degradation of fibrillary type I, III, and IV collagen and is the key pulmonary collagenase. Neutrophil derived MMP-8 also contributes to collagen degradation, and both MMP-1 and -8 are involved in cavity formation, which facilitates the transmission of *Mtb* by releasing the bacilli into the airways. Hypoxia augments monocyte and neutrophil MMP secretion acting through the hypoxia inducible factor (HIF)-1α transcription factor, an important regulator of the host response to oxygen deprivation. *Mtb* also promotes MMP activity via cellular networks involving immune and stromal cells. For example, lung fibroblasts have demonstrated upregulated MMP gene expression in response to *Mtb* infection or in the presence of monocytes infected with *Mtb* and MMP-1 secretion in response to *Mtb*-induced TNF-α.

#### *3.1. Collagen Degradation is an Early Pathological Event Promoted by Cell-Matrix Adhesion in TB*

Fibrillar type I, III, and IV collagen are major structural components of the human lung. In order to develop cavities, these collagen fibers must be cleaved. MMP-1 (also known as interstitial collagenase, collagenase-1) has emerged as the key MMP causing collagen degradation in pTB. In a recent study, of all the MMPs evaluated, MMP-1 was the most significantly increased in induced sputum of TB patients compared to respiratory symptomatic patients and healthy controls, along with MMP-3, which activates MMP-1 [57]. Further, MMP-1 positively correlated with chest radiograph inflammation score in HIV-uninfected patients and with sputum acid fast bacilli score and cavity frequency in HIV-infected and uninfected patients [57]. Procollagen III N-terminal propeptide (PIIINP), a matrix degradation product released during Type III collagen turnover, was increased in sputum of TB patients compared to controls and correlated with sputum MMP-1 but not with other sputum MMPs or cytokines measured, suggesting that MMP-1 is the key collagenase degrading Type III collagen in the lung [30].

Kubler et al. [32] investigated cavitary formation in a novel rabbit model of pTB, demonstrating increased MMP -1, -3, -7, -12, and -13 transcription in abnormal lung tissue. MMP-1, followed by MMP-3, was the most highly upregulated MMP with increased expression in the cavity walls compared to granulomatous tissue, and MMP-1 was found to be highly abundant in cavitary areas, supporting human data implicating MMP-1 in cavity formation. In contrast, TIMP-3 expression was lower in the cavitary compared to granulomatous areas of lung.

Al Shammari et al. [58] examined lung biopsy specimens from pTB patients and demonstrated that collagen and elastin absence mapped to areas of caseous necrosis. In transgenic mice expressing human MMP-1 on the scavenger receptor A promoter/enhancer, infection with a pathological strain of *Mtb* (recently isolated from a patient) resulted in MMP-1 expression in the lung and development of granuloma with central areas of tissue destruction containing amorphous cellular debris, consistent with caseous necrosis. Wild-type or MMP-9-expressing mice infected with the same strain of *Mtb* did not develop this pathology despite a similar extent of pulmonary inflammation, mycobacterial burden, and cytokine profiles. The primary pathological difference was loss of collagen within the MMP-1-expressing mouse granulomas. In a 3D extracellular matrix *Mtb* infection model, the addition of collagen, but not gelatin, improved monocyte survival after *Mtb* infection, suggesting that loss of collagen may promote cell death in TB. Together, this work supports the hypothesis that MMP-1 activity leads to collagen degradation as a prelude to caseous necrosis in TB rather than as a consequence, and that the integrity of the ECM may be an important determinant of the host immune response to *Mtb* [58].

Following up on this, Brilha et al. [59] found that the ECM regulated MMP activity in TB. Culture filtrate from *Mtb*-infected monocytes (CoMtb) increased monocyte adhesion to the extracellular matrix. Adhesion to Type I collagen increased MMP-1 secretion from *Mtb*-infected monocytes by 60%, MMP-7 by 57%, and MMP-10 secretion by 90%. Similarly, adhesion to fibronectin (but not Type IV collagen) increased MMP-1 secretion by 63% and MMP-10 secretion by 55%. Type I collagen adhesion increased *Mtb*-driven TIMP-1, but TIMP-2 decreased. Monocyte migration, adhesion, and type I collagen degradation were dependent on αVβ3-integrin activation, and integrin αVβ3 cell surface expression increased in *Mtb*-infected monocytes following adhesion to type I collagen and fibronectin. This suggests that *Mtb* infection promotes immune cellular interaction with the ECM propagating ECM destruction. Whilst plasma concentrations of TIMP-1 are elevated in pTB in adults and children [60,61], the concentration of antiproteases at the site of disease is evidently insufficient to prevent matrix destruction, and TIMP-1 concentrations in pulmonary secretions are reduced in TB [27]. In cell culture experiments, the increase in MMP secretion is not countered by an increase in TIMP-1 secretion [23], consistent with *Mtb* skewing the protease-antiprotease balance towards matrix breakdown.

In addition to surface-bound collagenases, MT-MMPs may drive collagen degradation in TB. MT-MMP-1 (also known as MMP-14) is expressed in TB granulomas [39]. Increased MT-MMP-1 transcript abundance was found in the sputum of pTB patients compared to controls. *Mtb* infection increased monocyte cell surface MT-MMP-1 expression and resulted in collagen degradation, which was largely MT-MMP-1-dependent. CoMtb similarly upregulated MT-MMP-1, which was p38 MAPK dependent. MT-MMP-1 expression increased cell migration in a cellular model of TB infection, therefore MT-MMP-1 may contribute to both collagenase activity and cell migration in TB [39].

#### *3.2. Neutrophil-Derived MMP-8 Drives TB Immunopathology*

Neutrophils are abundant in the lungs in pTB disease and can be both helpful and harmful in TB [62–64]. Neutrophil-derived MMP-8 (neutrophil collagenase) has now emerged as the second main collagenase contributing to TB immunopathology. A systematic investigation of the role of MMP-8 in TB [38] demonstrated that neutrophils secrete MMP-8 in response to *Mtb* infection via NF-κβ and in response to CoMtb, resulting in collagen degradation. Furthermore, elevated MMP-8 found in sputum from pTB patients correlated well with neutrophil activation markers and TB severity score and was associated with cavitation and involved with collagen breakdown compared to control sputum. Neutrophils containing MMP-8 were found at the inner wall of pulmonary cavities in TB patients and at the center of cavities in areas of caseous necrosis. Pro-catabolic AMP-activated protein kinase (AMPK) phosphorylation was identified as a key regulator of neutrophil-derived MMP-8, but there was no evidence of involvement of Akt/phosphoinositide-3-kinase (PI3K) pathway and mTOR/p70S6 kinase [38]. A further study found elevated sputum and plasma MMP-8 in patients with and without HIV infection, and two studies have implicated MMP-8 in pulmonary immunopathology associated with TB-associated immune reconstitution inflammatory syndrome (TB-IRIS—see below) [57,65]. In HIV-uninfected patients, elevated plasma MMP-8 was found in TB patients compared to respiratory symptomatic patients and healthy controls and was found to differ by gender in TB patients, with elevated MMP-8 in men compared to women [35]. Although men had a longer duration of cough than women, the association with gender was independent of cough duration. Taken with the MMP-1 data, these findings suggest that multiple collagenases contribute to lung matrix destruction

in TB, potentially with macrophage-derived MMP-1 causing initial matrix breakdown and then neutrophil-derived MMP-8 augmenting lung tissue damage once early cavitation occurs.

#### *3.3. Hypoxia Drives Collagenase Activity in pTB*

TB granulomas in several animal models have been shown to be hypoxic [66], but the interplay with matrix turnover in human TB has only recently been investigated. The MMP-1 promoter sequence has numerous putative hypoxia response elements, which are hypoxia-inducible factor (HIF)-1-binding sites as well as NF-κB and activated protein-1 (AP-1)-binding sites [67]. HIF-1α is a heterodimeric transcription factor, which is a key regulator of the host response to oxygen deprivation. Positron emission tomography–computed tomography (PET-CT) study of human pTB lesions with the hypoxia-specific tracer [18F] fluoromisonidazole ([18F]FMISO) has demonstrated severe hypoxia in radiologically abnormal areas [67]. Experimental hypoxia or HIF-1α stabilization using dimethyloxalyl glycine (DMOG) increased expression and secretion of MMP-1 in *Mtb*-infected macrophages without affecting *Mtb* growth. A similar effect of experimental hypoxia on MMP-1 secretion in response to *Mtb*-stimulation (via CoMtb) on normal human bronchial epithelial cells was observed. *Mtb* increases HIF-1α accumulation, even in normoxia, and HIF-1α is found in TB granuloma co-localizing with epithelioid macrophages and multinucleate giant cells [67], demonstrating a complex interplay between *Mtb* infection, hypoxia, and matrix breakdown, which together can be predicted to cause lung cavitation.

Ong et al. [68] recently demonstrated that hypoxia (or HIF-1α stabilization) exacerbates neutrophil-derived *Mtb*-driven MMP-8 secretion. The formation of neutrophil extracellular traps (NETs) in response to direct infection of neutrophils by *Mtb* was decreased in the presence of hypoxia, but neutrophil phagocytosis of *Mtb* was not affected. Furthermore, hypoxia increased neutrophil survival following *Mtb* infection compared to normoxia and increased neutrophil-driven matrix destruction, suggesting that hypoxia in pTB lesions may facilitate *Mtb* survival and promote tissue destruction.

#### *3.4. MMP and Cytokine Networks Enhance Tissue Damage in TB*

Cellular networks lead to a tissue degrading phenotype in pTB, upregulating MMPs by a number of pathways without a compensatory increase in TIMPs. Cell-cell networks involving respiratory epithelial cells and fibroblasts in addition to immune cells result in synergistic MMP upregulation in TB [3].

MMP-10 (stromelysin-2) was not formerly well studied in TB. However, as a key activator of MMP-1, recent work demonstrated that MMP-10 drives collagenase activity in cell culture, and its upregulation in macrophages was induced by virulent *Mtb* in an ESAT-6-dependent manner via p38 and ERK MAPK [40]. Elevated MMP-10 was found in respiratory fluids (induced sputum and BAL fluid) in TB patients compared to respiratory symptomatic controls, suggesting it may play an important role in human disease operating within a proteolytic cascade.

A recent study by Fox et al. [41] identified a novel role for platelets in amplifying *Mtb*-driven MMP-1, -3, -7, and MMP-10, and interleukin (IL)-1β secretion from monocytes without increasing TIMP-1 and -2 levels. Similar to MMP-10, MMP-3 can activate MMP-1, and thus a predicted outcome was increased collagenase activity. When *Mtb* was co-cultured with monocytes and platelets in a fluorescence model using dye-quenched (DQ) collagen, significantly increased type 1 collagen degradation was found compared to incubation with monocytes or platelets alone. Platelet-derived mediators (P-selectin, RANTES, platelet-derived growth factor (PDGF)-BB) were present in BAL fluid from TB patients at increased concentrations compared to BAL from non-TB respiratory symptomatic patients and correlated with multiple MMPs (including MMP-1, -8, -9) and IL-1β.

IL-17 is expressed in human TB granulomas [42]. Supplementation with IL-17 enhanced CoMtb-induced MMP-3 secretion from human distal small airway epithelial cells without compensatory TIMP-1/2 upregulation. MMP-9 secretion was inhibited by IL-17 while MMP-1, -7, and -8 were unchanged. IL-17 enhanced CoMtb-driven MMP-3 upregulation in normal human bronchial epithelial cells via p38 MAPK-dependent and the PI3K pathway. In fibroblasts, CoMtb-driven MMP-3 secretion was augmented by both IL-17 and IL-22 [42].

In pleural TB, MMP-1, MMP-9, and TNF-α concentrations were elevated in pleural fluid compared to pleural fluid from heart failure controls [34]. MMP-1 and -9 were secreted by pleural mesothelial cells in response to *Mtb*-stimulation. TNF-α strongly correlated with MMP-1 secretion and was found to be a potent stimulus of MMP-1 upregulation by pleural mesothelial cells in response to *Mtb*. In pericardial TB, concentrations of MMP-1, MMP-2, and TIMP-1 were significantly elevated in pericardial fluid, while MMP-7 and MMP-9 were higher in blood of patients with and without HIV-infection [69]. Differential abundance was also detected at mRNA level for MMP-2, MMP-9, TIMP-1, and TIMP-2.

In summary, *Mtb* promotes MMP activity and collagen degradation via cellular networks, likely involving monocytes, macrophages, neutrophils, platelets, and epithelial cells in the airways and mesothelial cells in the pleural space. In addition to previously described roles for monocyte-derived Oncostatin M and TNF-α enhancing MMP-1 secretion from fibroblasts [70], *Mtb*-driven (ESAT-6-dependent) MMP-10 and IL-17-augmented MMP-3 may propagate collagenase activity in cellular networks via MMP-1 activation. An outstanding question is the relative contribution of direct cellular infection versus intercellular networks in causing lung cavitation in vivo.

Furthermore, whilst macrophages and stromal cells are established sources of MMPs in TB, the role of T cells in MMP-related pathology is unclear. T cells are critical to an efficacious host immune response to *Mtb*, yet they also contribute to immune-mediated tissue damage. To exemplify, a greater purified protein derivative (PPD) response associates with subsequent development of pulmonary TB [71] and immune checkpoint inhibition for cancer associates with development of active TB [72,73]. In contrast, in advanced HIV-induced immunocompromise, pulmonary cavitation rarely occurs—despite high mycobacterial load within the lung—unless immune reconstitution takes place with therapy. The knowledge gap defining what comprises a protective T cell response versus what contributes to MMP-mediated tissue damage is a challenge to the development of rational strategies that may limit pathology after treatment initiation or alternatively prevent cavitation to break the cycle of transmission.

#### *3.5. Intracellular Regulation of MMP Activity in pTB*

Regulation of MMP-1 along with MMP-3 and MMP-9 by the PI3K pathway in TB has been investigated in normal human bronchial epithelial cells stimulated by intercellular networks [74]. Inhibition of the PI3K p110α subunit led to increased MMP-1, whereas downstream inhibition of Akt, mTOR, and p70S6 kinase led to reduced MMP-1. MMP-3 was inhibited by blockade at all three points in the pathway, whereas MMP-9 was increased by blockade at PI3K p110α subunit, decreased by blockade at Akt, and increased by blockade at mTOR or p70S6 kinase, indicating that regulation was complex and possibly involved epigenetic mechanisms [74].

Subsequent work has demonstrated epigenetic control of *Mtb*-driven MMP-1 and -3 expression by changes in histone acetylation in macrophages and normal human bronchial epithelial cells in a cell-type specific manner [75]. *Mtb* also inhibits negative regulatory pathways in directly infected human macrophages that function to limit MMP-1 secretion by inducing microRNAs that target PI3K, mTOR-containing complex 1 (mTORC1), and MAPK-interacting kinase (MNK) pathways, which together inhibit MMP-1 gene expression [76]. Therefore, *Mtb* not only increases collagenase activity by inducing macrophage MMP-1 and -10 secretion via p38 and ERK MAPK, it also effectively switches off inhibition of MMP-1 gene expression by interfering with PI3K and MNK pathway- regulation of MMP-1 inhibition [76].

Andrade et al. [37] observed elevated plasma MMP-1 and heme oxygenase (HO)-1 in TB patients compared to latently TB-infected asymptomatic controls. There was an inverse relationship between plasma MMP-1 and HO-1, and active TB patients were found to be either plasma MMP-1-high, plasma HO-1 low, or plasma MMP-1-low, HO-1-high. HO-1 is an antioxidant that catalyses the first step of the oxidative reaction that degrades the heme group contained in several proteins (including haemoglobin and myoglobin) and is immunomodulatory [77]. In vitro, both MMP-1 and HO-1 were induced in a dose-dependent fashion by *Mtb*. HO-1 was determined to be ESAT-6-dependent and required replicating bacilli, whereas MMP-1 secretion did not. In murine macrophages, HO-1-induced carbon monoxide (CO) production inhibited MMP-1 at the transcriptional level by inhibiting activation of c-JUN/AP-1, suggesting that *Mtb*-driven HO-1 may serve to limit MMP-1 [37]. Together, these findings demonstrate the complexity of MMP regulation with a balance between stimulatory and inhibitory pathways.

#### *3.6. MMPs as Biomarkers of TB Disease*

MMPs have been evaluated as diagnostic biomarkers of active TB. The previously described study by Sathyamoorthy et al. [35] reported that MMP-8 was the most discriminatory MMP for active TB in plasma, reporting an area under the curve (AUC) of 0.77 (0.80 for men and 0.72 for women) in comparison to non-TB respiratory symptomatic patients by receiver operating characteristics (ROC) curve analysis.

Following the demonstration that plasma PIIINP correlated with radiographic inflammation score and sputum MMP-1 in pTB, PIIINP was explored alongside other matrix degradation products as a peripheral biomarker of pTB disease [30]. Plasma PIIINP was combined with procollagen III C-terminal propeptide (PIIICP), MMP-2, MMP-7, MMP-8, and body mass index (BMI) in a TB prediction model in comparison to a mixed group of non-TB respiratory symptomatics and healthy controls. The final model, which included only those variables predictive after adjustment for all the others, included only MMP-8 and PIIINP, giving an AUC of 0.82 (95% confidence interval CI 0.742–0.922, *p* < 0.001) by ROC curve analysis.

In the aforementioned study by Andrade et al. [37], elevated plasma MMP-1 and HO-1 were highly discriminatory for active TB compared to latent TB. However, the comparator group of latent TB patients was asymptomatic. In an analysis including patients with other granulomatous pulmonary conditions, MMP-1 was significantly elevated in active TB (*n* = 18) compared to patients with sarcoidosis (*n* = 48) but not compared with patients with non-TB mycobacterial infection (*n* = 11). TIMP-1 has been evaluated in active TB compared to patients with pneumonia. However, the sensitivity was only 62.3% for a specificity of 45.95% [60]. It is possible that a panel of biomarkers that combine elements of tissue remodeling (MMPs, TIMPs, and matrix degradation products) may be more helpful than focus on a single component given the complexity of their interactions.

A limited number of studies to date have evaluated MMPs as biomarkers of treatment response in pTB. Ugarte-Gil et al. examined sputum MMPs longitudinally in relation to culture conversion and TBScore (which is associated with mortality risk) in a study of Peruvian patients [31]. Elevated sputum MMP-2, -8, -9, and TIMP-2 at diagnosis (but not TBScore) and elevated sputum MMP-3, MMP-8, and TIMP-1 at two weeks were associated with two-week sputum culture positivity. A study of Taiwanese patients found a modest association between elevated plasma MMP-8 and sputum culture positivity, both tested at two months [36]. An association between elevated serum MMP-1 and -8 over the first six weeks of treatment and delayed sputum culture conversion has been reported [78]. Further studies to evaluate MMPs and matrix degradation products as biomarkers of TB disease are in progress.

#### *3.7. MMPs in TB-IRIS*

TB is the leading cause of death among HIV-1-infected persons, and the World Health Organization (WHO) African region accounted for 72% of the global burden of HIV-associated TB in 2017 [1]. There were 464,633 cases of HIV-TB notified in 2017 globally, and 84% were on antiretroviral therapy (ART), which is an essential, life-saving intervention for HIV infection. However, ART-mediated immune system recovery in the setting of *Mtb* infection may lead to a pro-inflammatory state in the form of IRIS, presenting as TB-associated IRIS (TB-IRIS), which frequently complicates

the otherwise beneficial dual therapy for HIV-1 and TB [79]. Two forms of TB-IRIS are recognized: paradoxical, which occurs in patients established on antituberculosis therapy before ART, but who develop acute, recurrent, or new TB symptoms and pathology after ART initiation; and unmasking TB-IRIS in patients not receiving treatment for TB when ART is started but who present with active TB within three months of starting ART [80]. The most common and most frequently studied form of HIV-associated IRIS is the paradoxical TB-IRIS with incidence in adults ranging between (19–57%) and lower incidence in children [81]. A recent randomized controlled trial demonstrated that prednisone therapy during the first four weeks after the initiation of ART for HIV infection resulted in a lower incidence of TB-IRIS than placebo without evidence of an increased risk of severe infections or cancers [82]. The pathophysiology of paradoxical TB-IRIS remains incompletely defined. The majority of studies evaluating MMPs have been conducted in adults, and this section of the review focuses on studies related to adult TB-IRIS.

The hypothesis that dysregulated MMP activity may play a role in immunopathology and tissue destruction in TB-IRIS patients was investigated for the first time by Tadokera et al. [83] through a comprehensive analysis of MMP-1, -2, -3, -7, -8, -9, -10, -11, -12, and -13 as well as TIMP-1 and -2 gene expression in *Mtb* stimulated peripheral blood mononuclear cells (PBMC) from 22 patients who developed paradoxical TB-IRIS compared to 22 similar HIV-TB co-infected, non-IRIS control patients. Protein secretion in the form of MMP pro-enzyme content was also investigated using multiplex assays in peripheral blood mononuclear cell (PBMC) culture supernatants and serum samples using the Luminex platform and ELISA for TIMP-1 and -2. Transcript levels were increased in TB-IRIS patients for MMP-3, -7, and -10, while protein secretion was higher for MMP-1, -7, -8, and -10 in stimulated cultures compared to the control patients. Serum MMP-7 concentration was elevated in TB-IRIS, and two weeks of clinically beneficial corticosteroid therapy [84] decreased this level, although not significantly.

The association between the development of paradoxical TB-IRIS and increasing MMP-1, -2, -3, -8, and -9 concentrations during ART was investigated in plasma from a cohort of 148 HIV-infected adults with advanced HIV and TB before ART initiation and four weeks later [65]. This study found that median concentrations of MMP-1, -2, and -3 decreased, while MMP-8 and -9 increased significantly during ART, and a greater early MMP-8 increase was associated with both TB-IRIS development and decreased long term lung function following TB cure. The results were broadly consistent with the Tadokera study [83], although in that study, MMP-8 transcript abundance was not increased in IRIS in *Mtb*-stimulated PBMC. This is possibly because MMP-8 is predominantly secreted by neutrophils and present in pre-synthesized granules, thus these changes would not be identified by RNA analysis of PBMC [38].

A large systematic study of HIV-associated TB that comprised of (i) a cross-sectional study of HIV-1-infected and uninfected TB patients and controls, and (ii) a prospective cohort study of HIV-1-infected TB patients at risk of TB-IRIS evaluated plasma and sputum MMP concentrations using Luminex multiplex assays [57]. The results were analyzed in conjunction with plasma PIIINP (determined by ELISA), a degradation product of collagen type III known to be released by matrix destruction in pTB [30]. MMP activity differed between HIV-1-infected and -uninfected TB patients. In HIV-uninfected TB patients, MMP activity was prominent in the pulmonary compartment and MMP-1 was dominant, whereas HIV-1-infected TB patients had reduced pulmonary MMP-1, -2, and -9 concentrations and reduced cavitation but increased plasma PIIINP compared to HIV-1-uninfected TB patients. TB-IRIS was associated with elevated extrapulmonary extracellular MMP concentrations (measured in plasma) both before and during TB-IRIS onset. Plasma MMP-8 was the most significantly increased and correlated with plasma PIIINP concentration as well as neutrophil count. This was likely driven by *Mtb* antigen, as TB-IRIS patients with a positive urinary LAM (lipoarabinomannan) result had higher plasma MMP-3, -7, and -8 concentrations compared to urine LAM negative TB-IRIS patients. Consequently, the compartmentalization of MMP activity differs between immunocompetent adults, where it is primarily within the lung, whereas in advanced HIV infection, MMP dysregulation

is more systemic. In an in vitro culture model, *Mtb*-induced matrix degradation was suppressed by the MMP inhibitor doxycycline, suggesting MMP inhibition as a possible novel host-directed therapeutic strategy for preventing and/or treating TB-IRIS [57].

Overall, these studies indicate that TB-IRIS is associated with a distinct pattern of MMP gene and protein activation. In addition to the beneficial effect of corticosteroid therapy [82], modulation of dysregulated MMP activity may represent a novel therapeutic approach to alleviate TB-IRIS in HIV-TB patients undergoing treatment.

#### **4. MMPs in TBM**

#### *4.1. The Blood Brain Barrier (BBB)*

The BBB is a highly-selective permeability barrier comprised of specialized capillary endothelial cells with tight junctions, astrocytic foot processes, and pericytes [85]. It is integral in maintaining homeostasis in the CNS by the stringent regulation of molecular access to prevent entry of toxins and organisms into the brain. Increased permeability of the BBB is considered a hallmark of neuroinflammation and CNS infection [85], and research has implicated MMPs in BBB compromise [86,87]. Astrocytes, which form an integral component of the BBB, are known to release MMPs in response to disease and may therefore contribute to local damage [12]. Research into BBB disruption in TBM is sparse, but evidence indicates that MMP levels are increased in TBM [19,43,44,46,47], suggesting that they play a key role in BBB-breakdown, thereby contributing to the pathogenesis of TBM. The level and presence of specific MMPs and their inhibitors in the cerebrospinal fluid (CSF) and blood of patients may therefore be markers of BBB compromise. However, human studies have not shown association between MMP concentrations and the CSF-serum albumin index, an accepted index of BBB breakdown [47,52,53].

#### *4.2. MMPs in Neuro-Inflammation*

As summarized in Figure 2, MMPs contribute to ECM destruction in CNSTB. The two MMPs that have been the focus of human TBM research to date include MMP-2 and MMP-9 and their respective inhibitors, TIMP-2 and TIMP-1. MMP-2, also known as gelatinase-A, is a constitutively-expressed molecule that is present in large quantities in the normal brain and is found in astrocytes and CSF [9]. MMP-9, or gelatinase-B, is normally absent or present at low levels in the brain. It is found to be up-regulated during neuroinflammation [6], specifically in conditions such as viral and bacterial meningitis [88,89]. Together, these gelatinases digest tenascins, proteoglycans, fibronectin, type IV collagen, and laminin found in basement membranes of the BBB and ECM of the CNS, and thus are likely to contribute to the degradation of the BBB and tissue destruction [6,19,46]. This BBB degradation drives cerebral vasogenic oedema and facilitates further influx of blood-derived inflammatory cells into the CNS. The resultant chronic fibrosis and adhesions (due to exudate formation) contribute to the complications and neurological sequelae of TBM discussed below. In contrast to MMPs, TIMPs are widely expressed in many mammalian tissues. In the CNS, TIMP-1 expression is restricted to regions of persistent neuronal plasticity, such as the hippocampus, cerebellum, and olfactory bulb [11].

Cell culture models suggest that astrocyte and microglial secretion of MMPs does not occur with direct *Mtb* infection of these cells but rather in response to *Mtb*-infected monocytes as part of a leukocyte-astrocyte and leukocyte-microglia interaction, leading to the release of MMP-9, -1, and -3, respectively [12,20,21]. This network-dependent MMP expression is driven by pro-inflammatory mediators including TNF-α and IL-1β and IL-1β's synergistic interaction with IFN-γ in the case of MMP-9. The transcription of MMP-9 is regulated by MAPK and NF-κβ [12], while MMP-1 and -3 transcriptional regulation occurs through p38 MAPK pathway, NF-κβ and AP-1 [20,90]. In contrast to other MMPs, MMP-2 secretion from microglia is inhibited by cytokines, such as TNF-α, through p38, caspase 8, and NF-κβ signaling [91]. Additionally, TIMP expression is not influenced by these cellular networks, suggesting a possible shift to matrix degradation in response to TB. MMP-3 may also be

released by apoptotic neurons, resulting in the activation of microglia [12] and contributing to BBB breakdown by digesting the basal lamina and tight-junction proteins, specifically claudin-5, occludin, and laminin-α1 [92]. The breakdown of the BBB facilitates an influx of neutrophils that exacerbate BBB damage by secreting MMP-9 (also activated by MMP-3) through a monocyte-neutrophil network regulated by MAPK and Akt-PI3K [21,92]. MMP-9 is also reported to specifically target myelin basic protein in the white matter, which can negatively affect neuronal myelination [93]. The source of MMP secretion in the CNS is therefore likely a combination of infiltrating monocytes and neutrophils as well as resident astrocytes and microglia.

**Figure 2.** MMPs contribute to extracellular matrix (ECM) destruction in CNS TB. *Mtb* bacilli cross the protective blood brain barrier (BBB) through various mechanisms, including in infected monocytes. In the presence of these infected monocytes and driven by TNF-α and interleukin (IL)-1β, brain microglia and astrocytes respond as part of a leukocyte-microglia and leukocyte-astrocyte interaction by secreting MMP-1, -2, and -9, and MMP-1 and -3, respectively. These MMPs contribute to the breakdown of ECM proteins of the brain parenchyma, including fibronectin, laminin-α1, collagen type IV, and proteoglycans, and are pro-apoptotic to neurons (indicated in red text as "cell death") with the resultant tissue destruction adding to the cerebral inflammatory response. MMP-9 is also known to attack myelin basic protein (MBP), an integral component of the myelin sheath insulating neurons. Further, MMPs degrade claudin-5 and occludin, thereby compromising the tight BBB and contributing to the influx of proteins and water resulting in vasogenic edema, as well as further blood-derived inflammatory cells, including neutrophils. Neutrophils secrete MMP-9 in the parenchyma as well as in TB granulomas (tuberculomas), in which *Mtb*-infected microglia have also been found to secrete MMP-1 and -3. Contrary to the other MMPs, MMP-2 expression is suppressed through cytokines like TNF-α. Up-arrow indicates increased concentrations, down-arrow indicates decreased concentrations.

#### *4.3. MMPs in TBM*

The role of MMPs in TBM is summarized in Table 1, section B. Elevated MMP-9 concentrations have been found in the CSF of TBM patients with the degree of elevation correlated with the severity of disease [50], neurological compromise [19,44], and brain tissue injury [43,44]. Matsuura et al. demonstrated that concentrations of MMP-2 and -9 as well as TIMP-1 (but not TIMP-2) in the CSF were increased in patients with subacute (involving tubercle bacilli or mycotic organisms) meningitis compared with controls, and MMP-9 levels associated with CNS complications (such as disturbances of consciousness and psychiatric symptoms) [43]. Similarly, a study by Lee et al. demonstrated that increased MMP-2 and -9 levels in the CSF persisted during the late course of TBM and were associated

with the development of complications [46]. Price et al. demonstrated that the imbalance of MMP-9 versus TIMP-1 in the CSF of TBM patients correlates with morbidity and mortality [94]. Although Green et al. found that adjunctive dexamethasone was associated with a significant decrease in CSF MMP-9 concentrations, they did not find an association between a decline in MMP-9 concentrations and improved outcome [47].

However, the association of elevated MMP-2 and -9 concentrations with poor outcome is not consistent across TBM studies, and numerous studies have demonstrated no association between MMP concentrations and outcome [45,48,52,53]. In fact, Mailankody et al. demonstrated an association between elevated MMPs and a favorable outcome, and the authors postulated that the MMP contribution to a leaky BBB may be favorable to anti-TB drug penetration and could therefore offer an advantage to recovery [52]. In a pediatric TBM study, the beneficial effect of MMPs was corroborated by a 2.2 times greater likelihood of a better outcome associated with an increase in MMP-9 concentrations in the early weeks after hospitalization [53]. It is known that MMP-9 can mediate both protective and pathological immunity at different concentrations and in different age groups [94], and this raises questions about the role of MMP-9 in pathology—and potentially recovery—at different time points during the disease course. Similar challenges have been encountered in therapeutically targeting MMPs in oncology, reflecting the complexity of protease networks in human disease [95].

TBM is often associated with localized TB granulomas (tuberculomas) [96], which demonstrate elevated MMP-1, -3, and -9 expression [19–21]. In brain biopsies from patients with CNS TB, MMP-1 secretion was highest in the granuloma, and elevated concentrations of MMP-1 and -3 were associated with microglia in the peri-granuloma region and surrounding brain parenchyma and p38 positive microglia infiltrating necrotizing TB granulomas [20,91]. Using similar samples, Ong et al. also demonstrated MMP-9 secreting neutrophil presence in these CNS TB granulomas [21].

#### *4.4. MMPs in TBM-IRIS*

TBM-IRIS is a frequent, severe complication of ART in HIV-associated TBM patients. A prospective study of HIV-infected ART-naive patients with TBM showed that 47% (16/34) of TBM patients developed TBM-IRIS [97]. Patients who went on to develop TBM-IRIS had higher CSF neutrophil counts compared with non-TBM-IRIS patients and were more likely to have *Mtb* cultured from CSF at TBM presentation. The relative risk of developing TBM-IRIS was 9.3 (CI: 1.4–62.2) if the CSF was *Mtb* culture positive, indicating that the condition is driven by *Mtb* antigen load. This study also showed that a combination of IFN-γ and TNF-α concentrations measured in the CSF may predict TBM-IRIS, potentially paving the way for CSF immunodiagnostics. Subsequently, the concentration of 40 immune mediators in CSF (33 paired with blood) from HIV-infected patients with TBM was determined at TBM diagnosis, at initiation of antiretroviral therapy (ART, day 14), 14 days after ART initiation, at presentation of TBM-IRIS, and 14 days thereafter [49]. Among others, MMP-1, -2, -3, -7, -9, -10, -12, and -13, and TIMP-1 and -2 were analyzed using Luminex multiplex assays in CSF and plasma of patients who developed TBM-IRIS (*n* = 16) compared to those who did not (TBM-non-IRIS; *n* = 18).

Patients who developed TBM-IRIS had higher concentrations of MMP-1, -3, -7, and -10 in their blood, while MMP-9 and TIMP-1 were higher in CSF relative to blood at the time of TBM diagnosis and at the time of starting ART, as well as at the time of developing TBM-IRIS symptoms, when their concentrations increased further. However, CSF MMP-9 concentrations did not decrease following anti-TB and corticosteroid therapy and continued to rise following ART initiation, suggesting that more potent and specific therapy may be needed for the management of TBM-IRIS [98]. Overall, both at TBM diagnosis and at two weeks after ART initiation, TBM-IRIS was associated with severe compartmentalized inflammation in the CSF, elevated concentrations of cytokines, chemokines, neutrophil-associated mediators, and MMPs compared with TBM-non-IRIS, and driven by a high baseline *Mtb* antigen load [49]. A longitudinal whole-blood microarray analysis of HIV-infected patients with TBM confirmed the more abundant neutrophil-associated transcripts in patients who went on to develop TBM-IRIS from before symptom development through to TBM-IRIS symptom

onset. This study also demonstrated a significantly higher abundance of transcripts associated with canonical and noncanonical inflammasomes in patients with TBM-IRIS compared to non-IRIS controls [51]. These findings, reflected at the protein and molecular level, suggest a dominant role for the innate immune system in the pathogenesis of TBM-IRIS, offer mechanistic insights into the disease, and inform potential future directions for host-directed therapy. It is notable that when patients with HIV-associated TBM were compared to those with HIV-associated cryptococcal meningitis, MMP-1 and -3 were more elevated in the former and MMP-10 in the latter, suggesting that MMP profiles may differ across meningitides of varying etiologies [99].

#### *4.5. Adults and Children*

Before the era of HIV, the most important risk factor for TBM was age, with young children (aged 0–4) bearing the brunt of the disease [100,101]. This is possibly due to children having an immature immune system less capable of halting TB dissemination after primary infection [100,101]. However, apart from their role in neuro-inflammation, MMPs also play a unique role in normal brain development. Several animal studies have shown that MMPs, specifically MMP-2, -3, and -9, have a crucial role in the developing brain. For example, mice lacking MMP-9 have maladapted neuronal circuitry with simplified dendritic morphology, abnormal synaptic structure and function, and enhanced excitability [102]. MMP-3 and -2 are expressed in the cortical neurons of young mice and are required for appropriate axonal outgrowth and orientation [16,103]. Van Hove et al. also found that MMP-3 deficient mice developed deficits in balance and motor performance related to abnormal cerebellar development [104]. Despite the high prevalence of TBM in children, MMPs during pediatric TBM infection have not been well studied. In our recent pediatric TBM study [53], the positive association between increasing MMP-9 concentrations and good outcome may suggest a role in recovery and ongoing neurodevelopment, possibly including angio- and myelino-genesis, the growth of axons and synaptic plasticity. Similarly, the positive correlation found between MMP-2 and TIMP-2 may also suggest a physiological role in remodeling ECM in the developing CNS [53]. However, further studies with appropriate comparison with adults are required.

#### **5. Treatment**

#### *5.1. MMP Inhibition in pTB*

Multiple host-directed therapies centered on modulating protease activity have been evaluated in animal models (Table 2) as adjuncts to antibacterial treatment in TB with the prospect of beneficial immune modulation leading to enhanced bacterial clearance and the reduction of tissue damage and inflammation, thereby potentially shortening treatment duration [105]. This is challenging given the complexity of the host immune response. For example, multiple drugs that target upstream inflammatory pathways can have an effect on MMP expression by indirectly inhibiting their activity (e.g., prednisone, doxycycline, vitamin D, rapamycin) [106]. Similarly, non-specific immunomodulation of the host response with corticosteroids has been evaluated in pTB, with most studies showing no added benefit compared to standard treatment alone [107]. Conversely, in cases of extra-pulmonary TB, such as TBM and TB pericarditis, adjuvant use of corticosteroids has been shown to reduce mortality and morbidity, leading to their common use in current clinical practice [108]. Doxycycline, a tetracycline antibiotic, has been shown to non-specifically inhibit multiple MMPs independently of its antimicrobial activity [109]. However, doxycycline also has immunomodulatory effects on other host inflammatory pathways, confounding its potential MMP-specific effects [110]. Walker et al. reported that doxycycline modulated MMP expression in *Mtb*-infected cells and reduced mycobacterial growth in vivo [28]. However, additional studies are necessary to evaluate the potential of doxycycline as MMP-inhibitor therapy in TB. Indirect inhibition of MMPs has also been reported with a phosphodiesterase-4 inhibitor (CC-3052) in a rabbit model of TB [111]. CC-3052 plus isoniazid significantly reduced the extent of immune pathology and the expression of MMPs compared with antibiotics alone.

A more specific approach to evaluating MMP-inhibition in pTB has also been attempted with multiple specific MMP inhibitors (e.g., batimastat, cipemastat, marimastat) using different animal models of pTB. However, these studies have generated conflicting data, and the differences in treatment regimens, bacterial burden, host immune response, and variables measured make direct comparisons difficult (Table 2).

Initial experiments focused on monotherapy with MMP inhibitors in mouse models of pTB. Batimastat (BB-94), a potent broad-spectrum inhibitor, showed promising results in cancer models and was one of the first MMP inhibitors to enter clinical trials [112]. However, its poor solubility and side effects associated with intraperitoneal dosing led to its discontinuation. In pTB models, Hernandez-Pando et al. used *Mtb*-infected Balb/c mice to show a trend towards increased mortality if the treatment with batimastat was started immediately after infection with *Mtb* [113]. However, the increased mortality and histological differences were not evident between treated and control groups if batimastat was initiated a month after infection. Overall, there was a shift towards a type-2 cytokine profile and delayed granuloma formation in treated mice compared to controls. Subsequent studies by Izzo et al. using batimastat in C57BL/6 mice infected with *Mtb* showed reduced bacterial burden and smaller granulomas with more collagen deposition at earlier stages (day 40), but no differences were seen at day 60 post-infection [114]. However, a follow up study by the same group did not observe a reduced bacterial burden in the same mouse model when batimastat was initiated right after *Mtb* infection [115].

Recently, Urbanowski et al. used cipemastat, an orally available MMP inhibitor, in a rabbit model of cavitary pTB with repeated aerosol exposures to *Mtb* [116]. Compared to controls, monotherapy with cipemastat during week five to ten post-infection did not reduce the rates of cavitation evaluated by CT, mitigate disease severity, or visibly affect the collagen structure of the cavitary wall (examined post-mortem). Conversely, the cipemastat-treated group had a trend toward worse cavitary disease. Ordonez et al. used cipemastat monotherapy in the C3HeB/FeJ mouse model that develops cavitary lesions after aerosol infection with *Mtb* [117]. Similar to rabbits, cipemastat-treated mice had worse cavitation and more disease severity compared to controls.

While monotherapy with MMP inhibitors suggested worse outcomes, the use of multiple MMP inhibitors (marimastat, batimastat, or SB-3CT) in combination with anti-TB therapy have had synergistic effects in reducing the *Mtb* burden in a pTB mouse model [118]. While no effect on bacterial burden was observed with marimastat treatment alone, its combination with isoniazid significantly reduced lung colony forming units compared to isoniazid alone. Marimastat also reduced vascular leakage surrounding TB granulomas and increased the concentration of isoniazid in *Mtb*-infected lungs compared to controls. Similarly, targeting MMP-9 with a specific antibody in addition to anti-TB therapy (rifampin, pyrazinamide, and isoniazid) reduced pulmonary bacterial burden and significantly lowered relapse rates in a cavitary mouse model of pTB [117]. Therefore, MMP inhibition in conjunction with antibiotic therapy may be beneficial, whilst MMP inhibition monotherapy seems to be harmful, further demonstrating the complexity of events in vivo and the importance of performing translational preclinical studies that most closely reflect the intended therapeutic use in patients.

*Int. J. Mol. Sci.* **2019**, *20*, 1350

#### *5.2. MMP-Inhibition in CNS TB*

Given the important role MMPs have in the degradation of the BBB and tissue destruction in CNS TB, MMPs are a possible target for adjunctive treatment. In vitro CNS TB studies have demonstrated that dexamethasone leads to a decrease in MMP-1 and -3 mRNA expression and secretion without impacting TIMP expression. This has been suggested as a mechanism by which steroids improve short-term mortality from TBM [20]. NF-κβ inhibition and anti-TNF-α have been shown to decrease neutrophil expression of MMP-9, which is postulated as one mechanism by which to decrease CNS destruction [21]. Given the pro-inflammatory network involvement in MMP-9 expression, it has been suggested that targeting this network may offer opportunities for MMP regulation without directly interfering with the role of MMPs in the host defense against *Mtb* [12]. In TBM-IRIS, protein and transcriptome studies have highlighted the role of a neutrophil driven and inflammasome-associated inflammatory response in disease pathogenesis [49,51]. ECM destruction due to neutrophil secretion of MMP-9 is suggested to contribute to brain tissue damage, and pyroptosis driven by caspase-1 secretion may cause further injury. Agents specifically targeting the inflammasome could therefore offer another potential avenue for host-directed therapy.

Although MMP secretion may be affected by several medications, there has also been interest in developing drugs to specifically target MMPs. Paul et al. evaluated the effect of the MMP inhibitor Batimastat (BB-94) on BBB breakdown (as measured by Evans Blue leakage) and increased intracranial pressure (ICP—measured via catheter) using a rat model of meningococcal meningitis. The disruption of the BBB (and subsequent pathological changes) was ameliorated in animals treated with BB-94 in a dose-dependent manner [120]. Majeed et al. used an MMP-9 specific inhibitor SB-3CT [50,121], which appeared more effective than dexamethasone in that it suppressed MMP-9 and enhanced the effect of anti-TB drugs on clearing TB bacilli [121].

#### **6. Conclusions**

Evidence suggests that MMPs are an important component of the inflammatory response to Mtb infection and the consequent breakdown of the ECM in both the lungs and the brain. Their potential role in brain development suggests that MMPs may be relevant in both normal physiology and pathophysiology. However, they form part of an elaborate network of inflammation and injury and constitute one element of a complex inflammatory process in TB. A better understanding of what contributes to MMP-mediated tissue damage and the role of MMPs in the broader disease context is needed to develop targeted treatment strategies that may limit pathology or prevent cavitation and transmission without the risk of unexpected effects.

**Author Contributions:** Conceptualization: U.K.R., R.J.W., K.A.W.; Methodology: U.K.R.; N.F.W., Y.J.L.; Software and visualisation: A.A.O.; Resources: P.T.E., R.J.W., K.A.W.; Data curation, writing of the original draft: U.K.R., N.F.W., A.A.O., Y.J.L., E.W.T., K.A.W. All authors contributed to reviewing and editing the manuscript.

**Funding:** NFW is supported by a National Institute for Health Research Academic Clinical Lecturership, the British Infection Association, and a Starter Grant for Clinical Lecturers (The Academy of Medical Sciences UK, Wellcome, Medical Research Council UK, British Heart Foundation, Arthritis Research UK, Royal College of Physicians and Diabetes UK). AO is supported by NIH grants R01-EB020539 and R01-HL131829. EWT is supported by the Johns Hopkins All Children's Hospital Foundation Institutional Grant Program. PTE is supported by the Medical Research Council Global Challenges Research Fund: Bioengineering to combat the tuberculosis epidemic (MR/P023754/1). RJW and KAW are supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK, the UK Medical Research Council, and the Wellcome Trust (FC00110218). RJW also receives support from the Wellcome (104803 and 203135), National Research Foundation of South Africa (96841) and National Institutes of Health (U01AI115940).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

1. World Health Organisation. *Global Tuberculosis Report*; WHO: Geneva, Switzerland, 2018.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **MT4-MMP: The GPI-Anchored Membrane-Type Matrix Metalloprotease with Multiple Functions in Diseases**

#### **Cassandre Yip, Pierre Foidart, Agnès Noël and Nor Eddine Sounni \***

Laboratory of Tumor and Development Biology, Groupe Interdisciplinaire de Génoprotéomique Appliqué-Cancer (GIGA-Cancer), University of Liège, Liège 4000, Belgium<EMAIL_ADDRESS>(C.Y<EMAIL_ADDRESS>(P.F<EMAIL_ADDRESS>(A.N.) **\*** Correspondence<EMAIL_ADDRESS>Tel.: +3243662570

Received: 24 December 2018; Accepted: 12 January 2019; Published: 16 January 2019

**Abstract:** MT4-MMP (or MMP17) belongs to the Membrane-Type Matrix Metalloproteinase (MT-MMP) family. This family of proteases contributes to extracellular matrix remodeling during several physiological processes, including embryogenesis, organogenesis, tissue regeneration, angiogenesis, wound healing, and inflammation. MT4-MMP (MMP17) presents unique characteristics compared to other members of the family in terms of sequence homology, substrate specificity, and internalization mode, suggesting distinct physiological and pathological functions. While the physiological functions of MT4-MMP are poorly understood, it has been involved in different pathological processes such as arthritis, cardiovascular disease, and cancer progression. The *mt4-mmp* transcript has been detected in a large diversity of cancers.  Given its contribution to different pathologies, particularly cancers, MT4-MMP represents an interesting therapeutic target. In this review, we examine its biological and structural properties, and we propose an overview of its physiological and pathological functions.

**Keywords:** MT4-MMP; cancer; diseases

#### **1. Introduction**

The integrity of interstitial compartments is crucial for tissue homeostasis. The perturbation of the extracellular matrix and its related components destabilizes this balance, leading to pathogenesis. Matrix Metalloproteinases (MMPs) are the main remodeling enzymes of the extracellular matrix. This protease family counts more than 20 members, and most of them are secreted in the extracellular microenvironment. The membrane-type MMP (MT-MMPs) are associated to the membrane by a transmembrane domain, an amino-terminal link, or a glycosylphosphatidylinositol anchor (GPI). Together, secreted or attached to the membrane, MMPs can directly cleave almost all components of the extracellular matrix (ECM). However, the GPI anchor confers to MMPs a unique location in the lipid raft, giving access to a specific set of substrates. Only two MT-MMPs display this anchor: MT4-MMP (MMP-17) and MT6-MMP (MMP-25). In this review, we focus on MT4-MMP. Discovered more than 20 years ago, this protease aroused interest only a decade ago [1–4]. MT4-MMP exhibits unique characteristics, which distinguishes it from other MMPs. Unlike the others, it is unable to activate pro-MMP2 and cleaves only a few ECM components [5,6]. Its sensitivity to tissue inhibitors of metalloproteinases (TIMPs) is also different, with MT4-MMP being more sensitive to TIMP1 than TIMP2 [6,7]. These differences are probably due to the least degree of sequence identity [1]. MT4-MMP has been involved in inflammation and angiogenesis, contributing to associated pathologies such as osteoarthritis and atherosclerosis, as well as thoracic aortic aneurysms and dissection (TAAD) [2,8,9]. Interestingly, inflammation and angiogenesis are two pillars of tumor development. The *mt4-mmp* transcript has been detected in prostate carcinomas, oral carcinomas, osteosarcomas, gastrointestinal adenocarcinomas, embryonal carcinomas, leukemias, lung carcinomas, glioblastomas, cervical carcinomas, melanomas, adrenal adenocarcinomas, and thyroid carcinomas [3,10–12]. MT4-MMP was first described in breast cancers [1], in which it has been more widely investigated compared to the other cancers. The pro-angiogenic and pro-metastatic functions of MT4-MMP have been highlighted in breast cancer [4,13]. MT4-MMP-mediated metastatic dissemination has been also pointed out in colon cancer and head and neck cancer [3,11]. All these data propel MT4-MMP onto the stage of future potential therapeutic treatments.

#### **2. Characteristics of the MMP family**

The MMPs are endopeptidases characterized by the presence of a zinc ion in the catalytic domain. Twenty-four members have been identified and are separated into two different groups: The soluble MMPs (MMP-1, -2, -3, -7, -8, -9, -10, -11, -12, -13, -19, -20, -21, -22, -27, and -28) and the MMPs linked to the membrane by a transmembrane domain (MMP-14, -15, -16, and -24), a glycosylphosphatidylinositol (GPI) anchor (MMP-17 and -25), or an amino-terminal signal peptide (MMP-23A and -23B). The groups are shown in Figure 1.

**Figure 1.** Classification of different Matrix Metalloproteinases (MMPs) according to their structure. Matrix Metalloproteases are either soluble (MMPs) or membrane-tethered (MT-MMPs). MMP14 (MT1-MMP), MMP15 (MT2-MMP), MMP16 (MT3-MMP), and MMP24 (MT5-MMP) are attached to the cell membrane by a transmembrane domain. MMP17 (MT4-MMP) and MMP25 (MT6-MMP) are linked to the cell membrane by a glycosylphosphatidylinositol anchor (GPI).

The MMPs share common structures including: (1) The pre-domain, an N-terminal sequence driving the MMP to the endoplasmic reticulum (ER); (2) the pro-domain, keeping enzymes in an inactive form; and (3) the catalytic domain, implicated in the recognition and cleavage of substrates. These are shown in Figure 2.

**Figure 2.** Structural domains of MT4-MMP, including the pre-domain or signal peptide (amino acids 1 to 41), the pro-domain (42–128), the catalytic domain with a zinc ion (129–297), a linker (298–333) containing the furin site (R–X–K/R–R), the hemopexin domain (334–535), and the glycosylphosphatidylinositol (GPI) anchored to the membrane (572–605).

The catalytic domain is characterized by a consensus sequence "HEXXHXXGXXH", which allows the linking of a zinc ion. The presence of a zinc ion facilitates the binding of H2O molecules, thus providing the hydrolytic reactions of peptides and substrates [14]. Except for MMP-7, -26, and -23, all MMP family members display an hemopexin domain known to play a role in substrate recognition, proteolytic activity, and inhibitor binding. The GPI-anchored MT4-MMP displays unique features as compared to other MT-MMP members [15]. First, MT4-MMP is distantly related in its amino acid sequence to the other members. The catalytic domain displays less than 40% sequence identity, while the sequence identity is more than 65% among the other MMP members [1]. Second, MT4-MMP is unable to process pro-MMP2 into its active form, in contrast with MT1-, MT2-, MT3-, and MT5-MMP [5,6,16]. The pro-MMP2-activating MT-MMPs contain eight amino acids located in the catalytic domain, the so-called "MT-loop", which are lacking in MT4-MMP [17]. It has been reported that the pro-MMP2 activation is impaired when the MT-loop of MT1-MMP is deleted or inhibited by neutralizing antibodies [18]. These results are consistent with the capacity of the MT-Loop of MT1-MMP to interact with the fibronectin-like domain of pro-MMP2. Furthermore, a mutation in the MT-Loop of MT1-MMP impairs pro-MMP2 activation [19]. Thirdly, unlike other MMPs, MT4-MMP has a small repertoire of substrates among the ECM, with the exception of weak hydrolyzing capacities against fibrinogen, fibrin, and gelatin [5,6]. However, MT4-MMP is efficient in the cleavage of proTNF, ADAMTS4, α-2-macroglobulin, low density lipoprotein receptor-related protein, osteopontin, and cartilage oligomeric matrix protein, as detailed in Table 1 [2,5,6,8,20,21]. Interestingly, a soluble MT4-MMP form has been abundantly detected in the media of cells overexpressing active MT4-MMP, but not in the media of cells overexpressing its inactive form [22]. These data suggest that MT4-MMP is able to process itself by an active autocleavage. Finally, MT4-MMP is most potently inhibited by TIMP1 rather than TIMP2 or TIMP3, unlike the pro-MMP2-activating MT-MMPs that are efficiently inhibited by TIMP-2, -3, and -4, but not by TIMP-1 [6,7].

**Table 1.** Overview of identified substrates of MT4-MMP.

#### **3. Biosynthesis and Tra**ffi**cking**