Matrix metalloproteinases: Expression, regulation and role in the immunopathology of tuberculosis

Abstract Mycobacterium tuberculosis (Mtb) leads to approximately 1.5 million human deaths every year. In pulmonary tuberculosis (TB), Mtb must drive host tissue destruction to cause pulmonary cavitation and dissemination in the tissues. Matrix metalloproteinases (MMPs) are endopeptidases capable of degrading all components of pulmonary extracellular matrix (ECM). It is well established that Mtb infection leads to upregulation of MMPs and also causes disturbance in the balance between MMPs and tissue inhibitors of metalloproteinases (TIMPs), thus altering the extracellular matrix deposition. In TB, secretion of MMPs is mainly regulated by NF‐κB, p38 and MAPK signalling pathways. In addition, recent studies have demonstrated the immunomodulatory roles of MMPs in Mtb pathogenesis. Researchers have proposed a new regimen of improved TB treatment by inhibition of MMP activity to hinder matrix destruction and to minimize the TB‐associated morbidity and mortality. The proposed regimen involves adjunctive use of MMP inhibitors such as doxycycline, marimastat and other related drugs along with front‐line anti‐TB drugs to reduce granuloma formation and bacterial load. These findings implicate the possible addition of economical and well‐tolerated MMP inhibitors to current multidrug regimens as an attractive mean to increase the drug potency. Here, we will summarize the recent advancements regarding expression of MMPs in TB, their immunomodulatory role, as well as their potential as therapeutic targets to control the deadly disease.

Mtb and walls off the bacteria in dense cellular masses known as granulomas or tubercular lesions. 7,8 Although host innate immune response is one of the important determinants of the disease, the possible outcome of the infection varies among susceptible individuals and the factors involved therein are not well understood. 9,10 Recent studies have suggested a new concept of TB immunopathology that directly involves inhibition of matrix metalloproteinase (MMP) activity to hinder matrix destruction and reduce the morbidity and mortality associated with TB. 11,12 Matrix metalloproteinases (MMPs, also known as matrixins) are secreted or membrane-bound endopeptidases belonging to the metzincin superfamily, collectively capable of degrading all components of ECM. The prefix "metallo-" refers to the reliance of these enzymes on zinc ions to carry out the hydrolysis of protein substrates, and their structure has been reviewed in detail. 13,14 The first MMP was reported by Gross and Lapiere in 1962 as a collagenase engaged in tail resorption during the tadpole metamorphosis. 15 Currently, MMPs consist of 23 members in human and are expressed in almost all organs and tissues. 16  in catalytic domain. 23 In vitro, these proMMPs can be activated by chemical agents, such as sodium dodecyl sulphate, oxidized glutathione and thiol-modifying agents 24 ; however, in vivo activation of proMMPs is more complicated and is conducted by other MMPs or other classes of proteinases such as plasmin and neutrophil elastases. 25 In healthy tissues, MMPs are occasionally expressed and their biological activity is tightly regulated by various mechanisms.
Activity of activated MMPs is regulated by endogenous inhibitors called tissue inhibitors of metalloproteinases (TIMPs) that bind active and latent forms of MMPs. 26 Matrix metalloproteinases activity is implicated in non-infectious and chronic lung diseases such as asthma and COPD. [27][28][29] Mtb infection leads to disturbance in the balance between MMPs and TIMPs, and also alters extracellular matrix deposition as well as the cell behaviour of monocyte-microglial networks. 30,31 MMPs are secreted by Mtb-infected macrophages and monocytes, and also by uninfected stromal cells stimulated through intercellular networks. 32 Many studies have demonstrated the involvement of MMP-1, the major human collagenase, and its activator MMP-3 in driving pathology in pulmonary TB. 30,33,34 In this review, we will focus on the recent studies demonstrating the immunomodulatory roles of MMPs and their potential as therapeutic targets to hamper the pulmonary matrix destruction and reduce the morbidity and mortality associated with TB.

| E XPRE SS I ON OF MMPS IN TB
The majority of MMPs are expressed in diseased conditions wherein the tissues are inflamed and undergo repair and remodelling, while some of the members such as MMP-2, MMP-19 and MMP-28 are evident in normal tissues indicating their roles in homeostasis. 35 Many immune cells express low levels of MMPs in the resting state, and expression of MMPs is upregulated by exogenous stimuli, cytokines and cell-cell interaction. 36 This regulation is mainly carried out by TIMPs as unstimulated human peripheral blood monocytes, B cells and T cells express higher levels of TIMP-1, TIMP-2 and TIMP-4. 37 Pulmonary epithelial cells are also a significant source of MMPs as they express many MMPs including MMP-1, MMP-2, MMP-7 and MMP-9. 38 In many pathological conditions, cell migration is closely linked to degradation of the ECM and the activated MMPs are considered as a prerequisite for invasion and metastasis of cancerous cells. 39 Many studies have analysed the expression of MMPs in the pathophysiology of TB ( Table 1). Infection of THP-1 cells with Mtb leads to increased expression of MMP-9. This MMP-9 induction is regulated by receptor-mediated signalling pathways. 40 In TB patients, plasma concentrations of various MMPs may vary between the genders and this expression may not associate with the severity of the disease. Sathyamoorthy et al found significantly higher plasma concentrations of MMP-1 and MMP-8 in male TB patients as compared to females. This increased concentration of the MMPs was inversely correlated with body mass index. 41 Similarly, plasma MMP-3 was also significantly higher in men as compared to women in a number of clinical conditions including both infectious and non-infectious diseases. 42 MMPs, like MMP-1, cause lung extracellular matrix destruction, and MMP-10 is known as a key activator of MMP-1. In a recent study, MMP-10 secretion was increased in Mtb-infected macrophages while inhibition of MMP-10 activity decreased collagen breakdown. MMP-10 expression was also increased in both induced sputum and bronchoalveolar lavage fluid (BALF) as compared to control subjects and patients with other respiratory diseases. 43 This Mtb-driven MMP-10 secretion was inhibited in a dose-dependent manner by p38 and extracellular signal-related kinase mitogen-activated protein kinase blockade. In vivo and in vitro, Mtb infection leads to increased expression and activity of MMP-1, MMP-2, MMP-3 and MMP-9. 44 This study also reported the involvement of miR-223 in MMP expression through BMAL1 modulation. Azikin et al evaluated the levels of MMP-9 in children who lived in the same house with a person having active TB. 45 There were no significant differences between the expression levels of MMP-9 in the group of exposed and Mtb infected children, and the levels of MMP-9 were not influenced by sex, age, nutritional status and the status of BCG immunization. In a related study, M avium also induced the secretion of MMP-1 in duodenal biopsy tissues, as well as in blood samples as compared to negative controls. 46 48 The serum values of IL-1β, MMP-1 and MMP-13 after surgery were lower than those of before treatment.
Like other tissues of the body, inflammation of central nervous system (CNS) also results in increased MMP secretion and it can also affect the permeability of blood brain barrier (BBB). 49,50 Increased expression of MMP-1 and MMP-3 has been reported in the patients with TB of CNS. 30,51 Similarly, in a recent study, MMP-9 upregulation has been noticed in the brain biopsies of the patients having TB meningitis. 52 This enhanced activity of MMP-9 in the brain tissues may be involved in the damage of BBB, oedema and the inflammatory cell exudation. Li et al analysed the expression of MMP-9 in the pathophysiological process of TB meningitis in a mouse model. 53 The data

| REG UL ATI ON OF MMP E XPRE SS I ON AND AC TIVIT Y
Since MMPs might cause significant damage to the host tissues, therefore, their expression is strictly regulated. This regulation involves several levels including gene expression, zymogen activation, compartmentalization and inhibition of active enzyme. 35 Initially, MMPs are believed to be regulated at the transcriptional level by a variety of physiological factors including cytokines, tumour promoters, growth factors, hormones, chemokines and cell-cell or cell-ECM communications. 54 MMP promoters contain cis-acting elements that can be bound and regulated by several transcription factors such as activator protein 1 and NF-kB. 55 Expression of MMPs is further regulated at the post-transcriptional level. 56 Studies also have uncovered the contribution of epigenetic modifications in regulation of MMPs. 57 MMP activity is also controlled through their compartmentalization in a certain intracellular or extracellular location. 58 In addition, many intracellular pathways have been discovered which are actively involved in MMP regulation.

| Role of TIMPs in MMP regulation in TB
Tissue inhibitors of metalloproteinases are well known to downregulate the activity of MMPs by binding their latent and active forms.
TIMPs are constitutively expressed in many tissue fluids including cerebrospinal fluid (CSF). 59,60 There is a considerable connection in the biochemical properties of various TIMPs, although there are some MMPs having specificity to substrates. 16

| Kallikrein-kinin system in MMP regulation
Kallikrein-related peptidases (KLKs) are a subgroup of serine proteases with either trypsin-like or chymotrypsin-like activity. Tissue KLKs are expressed in various tissues and consist of 15 proteases, KLK1 to KLK15. KLKs perform many physiological and pathological functions. The kallikrein-kinin system appears to play a direct role in promoting anti-fibrotic responses and collagen degradation. 67,68 As KLKs are able to convert kininogens into bradykinin, these kinins then bind to bradykinin receptor 1 (B1R) and bradykinin receptor 2 (B2R). B1R is generally latent, but it is upregulated in inflammation or by the members of cytokine family including IL-1β and TNF-α, F I G U R E 1 Role of bradykinin signalling in expression and regulation of MMPs in non-TB conditions. Bradykinin stimulation can lead to activation of intracellular signalling pathways which in turn induces various MMP secretion. Both B1R and B2R can activate ERK1/2 kinases eventually leading to increased expression of MMP-1 and MMP-9 in breast cancer and tracheal smooth muscles, respectively. Similarly, exogenous bradykinin treatment of granulosa cells induced MMP-3 and MMP-20 explaining their role in ovulation while B2R is constitutively expressed in many tissues of the body. 69 Bradykinins play a pivotal role in the modulation of airway inflammation by stimulation of cytokine expression and recruitment of inflammatory cells. 70,71 Many showed reduced interstitial fibrosis. The increased interstitial fibrosis in B2 -/mice was associated with decreased activity of MMP-2 suggesting the protective role of bradykinin and MMP-2. 73 Similarly, involvement of B2R in the release of MMP-2 from tracheal smooth muscle cells of guinea pig has been reported. 74 In a recent study, B1R agonist, Lys-des[Arg9]-bradykinin (LDBK), increased the proliferation of oestrogen-sensitive breast cancer cells. B1R was also involved in the expression of MMP-2 and MMP-9 via ERK-dependent pathway. 75 Similarly, B2R has been reported to regulate MMP-9 secretion via MAP kinases (ERK1/2) signalling in trabecular meshwork cells. 76 On the other hand, MMPs can also regulate the function of KLKs. 77 These studies suggest a potential interaction of KLKs and MMPs in various physiological and pathological conditions. It is likely that this B1R and B2R signalling is involved in the MMP secretion in TB but no study has been reported yet. Investigations of these signalling pathways may reveal bradykinin receptors as therapeutic targets in TB treatment.

| IL-12 and IL-17 cytokines mediated induction of MMPs
Cytokines are the important regulators of immunity and inflammation. These cytokines including TNF-α and IFN-γ generally upregulate the expression and activation of MMPs in monocytes and macrophages. 78 For example, in neutrophils, MMP-9 is stored as gelatinase granules and it is released following stimulation with IL-8 or TNF. 79  Historically, Th1 cells have been believed to be essential in the

| NF-κB and MAPK regulation of MMPs
In silencing had no effect on MMP-1, which shows epigenetic modification of histone acetylation also plays a role in expression of MMP-3.

Taken together, multiple pathways are involved in MMP regulation but
it has yet to be established that which signalling pathway can be effectively targeted to minimize the TB pathology.

| Role of MMPs in pulmonary granuloma and cavitation
Tuberculosis is primarily a disease of lung, and following infection with Mtb, pulmonary granulomas are formed in humans and develop heterogeneous microenvironments, often featuring hypoxia and central necrosis. 103 The same lesions can be recapitulated in nonhuman primate and rabbit models of the disease. 104 Historically, pulmonary granuloma formation has been considered as a host protective response; however, recent studies suggest that Mtb uses secreted virulence factors to induce granuloma formation to create a favourable niche for its dissemination and replication. 105,106 Moreover, recent studies have demonstrated a wide variation in the distribution of drugs within TB granulomas, with very few agents able to penetrate the central regions of the granuloma. 107 This differential ability of drugs to penetrate TB granulomas has been incorporated into modern TB drug development programmes to select a more efficient combination. 108 However, the mechanisms contributing to this differential penetration of drugs are not fully understood yet, and novel approaches to improve TB drug delivery and efficacy are urgently needed. The standard treatment of TB has remained unchanged for many decades, and multidrug-and extensively drug-resistant strains are emerging progressively, leading to high mortality rates among patients even after commencement of TB treatment. [109][110][111][112] Therefore, it urges the development of new drugs to accomplish the sustainable development goals, aiming to reduce 90% of TB incidence rate by 2030. 113 The role of MMPs in pulmonary physiology and pathology is gaining attention, and several studies show that they are associated with lung tissue destruction and inflammatory lung disorders including chronic obstructive pulmonary disease (COPD) and emphysema.

Mtb infection induces the production of MMPs both in vitro and in
vivo. 40,114 Many MMPs, specifically MMP-1, have been shown to contribute in TB pathology in human lungs. 82 Although the primary function of MMPs is thought to be matrix cleavage and tissue remodelling, many evidences suggest these enzymes also play a major role in angiogenesis, cell motility, apoptosis, regulating immunity, inflammation and host defences. 115  hosts. 119 In contrast to MMP-9 which plays a role in ECM deposition and granuloma formation, MMP-1 is believed to degrade ECM as its primary function, thus contributing in spread of Mtb and cavitary disease development (Figure 3). 22 However, the pathogenesis and role of other MMPs are incompletely understood. 120 The understanding of MMP role is important for designing the rational therapies to minimize immune-mediated host damage and improve outcomes in TB.

| Animal models of pulmonary granuloma and cavitation
Mouse, rabbit and guinea pig are most commonly used animal models of TB. 121,122 These species of animals have contributed a lot to the understanding of TB immunopathology. In recent years, majority of in vivo Mtb investigations have been carried out by using mouse model, and different mouse strains and different infection methods have been created. 123 Mtb infection has been reported to upregulate MMP-9 expression in Mtb-infected mouse. 124 In another study, MMP-9 knockout mice exhibited reduced cellular recruitment to the lung granuloma. 125 These studies implicate that Other animal models such as guinea pig, rabbits and non-human primates (NHPs) are preferred which exhibit caseation necrosis as seen in human. [129][130][131] Guinea pigs have contributed significantly to understand the immunopathology of pulmonary TB. Although the guinea pigs are relatively economical model and produce TB lesions more similar to human in terms of lung pathology, even then they lack some characteristics features of human TB. 132 In this regard, rabbits develop cavitary TB same as seen in human and offer a good opportunity to study the factors causing this type of disease. Moreover, rabbits are also susceptible to bovine TB caused by M bovis. 133 Rabbit model is suitable for the investigation related to drug penetration and distribution in the lung tissue, and also to evaluate the response to chemotherapy. 134

| Role of MMPs in TB/HIV co-infection
It is believed that almost half of patients surviving pulmonary TB with apparent recovery suffer substantial pulmonary impairment. 143,144 Furthermore, cured pulmonary TB is a major cause of chronic lung disease globally. 145  whereby, immune response restoration impairs pulmonary function.

| INHIB ITI ON OF MMPS: UNLE A S HING THEIR THER APEUTI C ROLE S
Host-directed therapy with MMP inhibitors has been investigated in several inflammatory conditions such as multiple sclerosis. 156 Encouraging results have been obtained in experimental models of meningococcal and pneumococcal meningitis, where MMP inhibition resulted in decreased morbidity and mortality. 157