Lymphatic filariasis (LF) is characterized by dysfunction of lymphatics that can lead to severe and often irreversible lymphedema and elephantiasis [1], [2]. The critical factor in the development of host pathology appears to reflect the parasite-mediated initiation of a cascade of events that leads to tissue fibrosis and scarring [2], [3]. It is assumed that both parasite products and the host inflammatory response lead to lymphatic dysfunction and lymphangiogenesis that, in turn, predisposes infected individuals to secondary bacterial and fungal infection [2], [4], [5]. Host-parasite interactions as well as secondary infections then trigger inflammatory reactions in the skin and subcutaneous tissue with underlying fibrosis and cellular hyperplasia processes resulting in lymphedema and elephantiasis [2], [4]. Typically in Wuchereria or Brugia infections, disease manifests years after exposure, while clinically asymptomatic infection is not only more common but can also occur at a relatively young age [1].

Although lymphatic dysfunction and localized/systemic immunologic and inflammatory responses are important features of lymphatic pathology [6], perturbations in extracellular matrix (ECM) architecture and subsequent remodeling are also associated with filarial disease [7]–[9]. Chronic inflammation, as seen in LF disease, causes an excessive accumulation of ECM components (such as collagen) that can contribute to fibrotic scarring [2]. Patent filarial infections are typically associated with Type 2 and regulatory cytokine responses, but some of these are also pro-fibrotic, especially IL-5, IL-13, and TGF-β [10], which are known to influence collagen deposition and ECM remodeling [11].

The turnover of collagen and other ECM proteins is controlled by a large family of proteolytic enzymes called matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of metalloproteinases [TIMPs]), produced by a variety of cell types including macrophages, granulocytes, epidermal cells, and fibroblasts [12]–[14]. Tissue immunopathology is known to be associated with dysregulation of MMPs and TIMPs in several infections, including viral, bacterial, spirochetal, protozoan, fungal, and parasitic infections [15]. The MMP family consists of more than 26 different proteases that differ in their tissue expression/localization and target specificity, while the TIMP family consists of four ubiquitously expressed proteins (TIMP1–4) [12]–[14]. In addition, other factors such as fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) are known mediators of tissue fibrosis [11].

Although the importance of tissue fibrosis in the pathology associated with LF is well known [3], the molecular mechanisms underlying the fibrotic process in filariasis has not been well established. We therefore sought to delineate the role of the factors known to regulate fibrosis and tissue remodeling in filarial disease development. Our data suggest that while elevated circulating levels of MMPs and TIMPs are characteristic of filarial disease, it is the increased ratios of certain MMPs to TIMPs (associated with elevated pro-fibrotic cytokines) that is specifically associated with the pathogenesis of disease in LF.

The dynamics of the ECM in tissues are orchestrated by the interplay among matrix breakdown, matrix deposition, and reorganization. MMPs are a family of zinc-metalloendopeptidases responsible for the turnover of ECM [12]–[14]. MMPs are tightly regulated at multiple levels including transcriptional and translational regulation as well as by TIMPs [12]. Therefore, tissue homeostasis is achieved by a tight balance of MMP proteolysis to TIMP production. When this balance is altered by inflammation and other mechanisms, dysregulated MMP activity ensues [12]. Altered MMP/TIMP expression ratios have been associated with many diseases including: those associated with tissue destruction, such as cancer invasion and metastasis [16], rheumatoid arthritis [17], osteoarthritis [18]; those associated with fibrosis, such as liver cirrhosis [19], scleroderma [20], systemic sclerosis [21]; and those associated with weakening of the extracellular matrix, such as dilated cardiomyopathy and epidermolysis bullosa [12].

Although some parasitic infections can induce fibrosis, few studies have actually examined the role of MMPs and TIMPs in human parasitic infections. MMPs and TIMPs have been shown to be associated with disease activity in human neurocysticercosis [22], leishmaniasis [23], schistosomiasis [24], and eosinophilic meningitis [25]. In addition, the regulation of MMP activity has been demonstrated in animal models of Schistosoma mansoni, Toxoplasma gondii, Angiostrongylus cantonensis, Opistorchis viverrini, and Mesocestoides cortii [15], [26]. Interestingly, although skin and subcutaneous fibrosis and scarring is a characteristic feature associated with longstanding LF and specifically elephantiasis, no study has examined the regulation of MMPs and TIMPs in LF infection.

We utilized a cohort of clinically well defined individuals from an area endemic for LF to examine the role played by MMPs and TIMPs in filarial disease pathogenesis. We examined the expression pattern of the pro-fibrotic factors using a two-step comparison with the rationale that factors truly reflective of infection-driven pathogenesis (as opposed to general factors) would be significantly different between individuals with lymphedema/elephantiasis with active infection and clinically asymptomatic but actively infected individuals but not between those with disease (CP) without active infection and uninfected, endemic normal individuals. Our examination of the baseline levels of MMPs and TIMPs revealed that both groups of proteins were non-specifically elevated in individuals with chronic pathological consequences of LF infection irrespective of their circulating filarial antigen level. Therefore, this suggests that filarial disease per se (and not the presence of viable parasites) is associated with these elevations in ECM modulators; however, it also known that TIMPs mediate blocking of MMP-induced proteolytic activity by noncovalently binding to the MMP active site in a 1∶1 stoichiometric ratio. Therefore, we also examined the ratios of various MMPs to TIMPs in our cohort of patients [27]. Not surprisingly, we found evidence of an imbalance between MMPs and TIMPs. Our data would therefore imply that altered ratios of MMP/TIMP are an important underlying factor in the pathogenesis of tissue fibrosis in filarial lymphatic disease. This process is reflective of imbalances in the levels of MMP and TIMPs seen in other diseases with tissue fibrosis, including systemic sclerosis [21], scleroderma [20], amyotrophic lateral sclerosis [28], coronary artery disease [29], endometriosis [30], rheumatoid arthritis [17], viral hepatitis [31], and a variety of cancers [16]. Our data also suggest that currently available synthetic MMP inhibitors could potentially be of benefit in ameliorating pathology or preventing the development of pathology in active infection [32]. In the context of a role for infection per se, we observed that the presence of active infection significantly enhanced the levels of most MMPs and TIMPs, suggesting that changes in ECM remodeling are occurring prior to the onset of clinically evident lymphedema.

Interestingly, among the MMPs with enhanced baseline expression in filarial lymphedema are the two major collagenases in the MMP family, MMP-1 (or collagenase-1) and MMP-8 (or collagenase-2) [12]. While collagenases are known to degrade collagen, MMP-9 is also a gelatinase that can degrade collagen as well as gelatin [12]. As these proteolytic enzymes mainly target collagen and because collagen has been previously shown to be altered in filarial lymphatic pathology [7]–[9], it is interesting to note that these enzymes are specifically elevated in chronic pathology individuals irrespective of their infection status. In addition to their role in ECM remodeling, TIMPs are also known to have MMP-independent functions [27]. TIMP-3 is an antagonist of vascular endothelial growth factor receptor-2 (VEGFR-2) [33], while TIMP-2 has been shown to antagonize VEGF signaling [34], resulting in inhibition of angiogenesis. Because lymphangiogenesis is a well described feature of filarial lymphedema [2], it is of interest to note the significantly deceased production of TIMP-3 as well as decreased levels of MMP/TIMP-2 ratios in filarial lymphedema. The decreased levels of TIMP-3 and the decreased MMP/TIMP-2 ratios therefore, could potentially signify an important role for these enzymes in promoting VEGF-mediated angiogenesis and/or lymphangiogenesis in response to inflammation, as has been described in neuroinflammatory disorders such as multiple sclerosis or experimental autoimmune encephalomyelitis [27]. We plan to explore the functional activity of MMPs and TIMPs in future experiments.

While MMPs and TIMPs are major regulators of fibrosis, PDGF, FGF, epidermal growth factor (EGF), VEGF, and bone morphogenic proteins (BMPs) are also known to influence tissue fibrosis [11]. By examining expression of PDGF-AA and FGF-2 in filaria-infected and filaria-uninfected individuals, we were able to show that neither PDGF nor FGF was associated with filarial lymphedema. These data suggest that while filarial lymphedema is characterized by alterations in certain pro-fibrotic factors such as MMPs/TIMPs, it is not associated with a generic increase in the circulating levels of other factors known to influence tissue fibrosis.

Although persistent and progressive fibrosis is postulated to be a hallmark of LF disease, the data on the known pro-fibrotic cytokines in filarial disease are limited. By quantifying IL-5, IL-13, and TGF-β levels in the four well defined groups, our data indicate clearly that these particular cytokines are associated with development of overt pathology in actively infected individuals. Because pro-fibrotic cytokines are known to influence tissue fibrosis by regulating the levels of MMPs and TIMPs [35]–[38], we also examined the association between these factors. In agreement with studies in animal models of parasitic infection showing association between MMP/TIMP levels and Type 2 cytokines [39]–[42], our examination of filaria-infected individuals also reveals a significantly positive association between MMP-1/TIMP-4 and MMP-8/TIMP-4 ratios (both of which were specifically elevated in CP Ag+) and Type 2 (and pro-fibrotic) cytokines. Our study clearly implicates a tissue-fibrosis promoting role for IL-5 and IL-13 in filaria-induced lymphatic pathology.

Our study clearly identifies a novel role for MMPs and TIMPs as well as Type 2 cytokines in filarial infection-driven morbidity associated with a persistent and progressive tissue fibrosis. While requiring validation in future studies, these results point to potential therapeutic interventional targets in ameliorating filarial lymphedema and possibly even elephantiasis.