The provisional matrix is a transitory ECM found during the early stages of development, tissue repair, and disease which is later replaced by a tissue-specific ECM. It is formed by fibrin, fibrinogen, fibronectin, HA, and versican, a large chondroitin sulfate proteoglycan. HA provides a "glue" core to bind all other components and place it in the pericellular space, due to its interaction with a specific membrane receptor, CD44. Several growth factors can stimulate the expression of these macromolecules. The provisional matrix has a viscoelastic property that allows it to create spaces within the ECM, providing means for cell migration. For example, the migratory routes of NCCs during early embryonic development highly express high levels of versican (Barker and Engler, 2017; Chester and Brown, 2017; Wight, 2017) and tenascin (Riou et al., 1992). It is well known that collagens type I, III, IV, V, VI, fibronectin, HSPG, laminin, and tenascin are expressed during palatogenesis in vivo (Ferguson, 1988; Dixon et al., 1993a) and EGF or TGF-α can stimulate their expression on mouse embryonic palatal mesenchymal cells in vitro (Dixon et al., 1993b). The intrinsic "internal shelf force" for palatal elevation has been attributed to HA since it is the most abundant GAG in palatal ECM before shelf elevation (Ferguson, 1988). It is produced on the cell membrane surface by specific enzymes (HA synthases—Has 1-3) and these are differentially expressed in palatal mesenchyme and epithelium during palatogenesis (Galloway et al., 2013). In TGFβ-3 null mice, expression of all Has forms is decreased, leading to reduced amounts of HA and impaired shelf elevation (Galloway et al., 2013). Recently, Has2 has been described to be a crucial HA synthase in NCC-derived mesenchyme during craniofacial development and palatogenesis (Lan et al., 2019). Also, FGFs induce HA synthesis by mouse embryonic palatal mesenchymal cells in vitro (Sharpe et al., 1993). Fibronectin is found during embryonic development in areas characterized by cell migration (Schwarzbauer and DeSimone, 2011). It appears that fibronectin arrangement is vital for cell migration and palatal shelf elevation. In this case, Rac1 and cell density modulates fibronectin deposition in mid-palate (Tang et al., 2015). Moreover, Rac1 is downregulated by retinoic acid, leading to the cleft palate as a consequence of the disarrangement of fibronectin and cell migration as well (Tang et al., 2016).

Cellular communication is a well-known mechanism in which cells can communicate with each other and modify cell behavior

through soluble factors. Intercellular communications occur via direct cellular interactions in which cell surface proteins act as mediators able, or not, to bind to the ECM (juxtacrine signaling). Alternatively, cells release local mediators into the ECM to create self-control signals (autocrine signaling) and send information to neighboring cells (paracrine signaling) or reach target cells in long distances via hormones (endocrine signaling) (Ansorge and Pompe, 2018). The local mediators are peptides or growth factors which control many cellular activities. During development, a combination of cell–cell interactions occurs, as well as the secretion of mediators named morphogens, which induce specific cell differentiation in a distinct spatial order and morphogen gradient-dependent manner (Inomata, 2017). The main morphogens are retinoic acid, HH, TGF-β, BMPs, and Wnt/β-catenin.

 Also, other morphogens and growth factors have emerged in palatogenesis, such as connective tissue growth factor (Tarr et al., 2018) and retinoic acid (Okano et al., 2014; Mammadova et al., 2016). Dysregulation of these pathways through genetic variations in individual genes has been suggested as strongly associated with CL/P (Pauws and Stanier, 2007; Krejci et al., 2009; Tang et al., 2013; Okano et al., 2014; Reynolds et al., 2019).

During the last decade, knowledge of new types of RNA with regulatory functions, located in non-coding regions of DNA, has improved our understanding of gene expression regulation (Scherrer, 2018). Many different microRNAs have been identified to temporally and spatially regulate morphogens and transcription factors during palatogenesis (Eberhart et al., 2008; Seelan et al., 2014; Ding et al., 2016; Reiss and Bhakdi, 2017; Schoen et al., 2017).

# ECM REMODELING

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The extracellular microenvironment is dynamically modeled and remodeled by soluble or EV-associated proteases secreted into the ECM or membrane-anchored proteases, which are classified as cross-linkers and remodeling proteases (Sanderson et al., 2019). Of the ECM cross-linkers, lysyl oxidases (LOX) and transglutaminases (TGs) are the major families responsible for establishing cross-links between the central core matrisome proteins. Moreover, an intricate balance between proteases and their inhibitors maintains the ECM homeostasis; abnormal ECM remodeling (excessive or inefficient) is involved in the development and/or progression of several pathologies due to modifications in macromolecule composition (posttranscriptional control and posttranslational modifications), biophysical (architecture), and biomechanical properties (stiffness).

#### ECM Cross-Linkers

Post-translational modifications (cross-links) in collagen– collagen, collagen–ECM, and ECM–ECM interactions are relevant for the integrity, stiffness, and rigidity of the microenvironment. Once formed, cross-links formed are immature but become more stable due to multivalent cross-links that generate insoluble protein polymers that are resistant to proteolytic degradation, improving the biomechanical properties of the collagen network. Procollagen maturation takes place when both N- and C-termini are classically removed by ADAMTS and BMP-1/TLD (BTPs), respectively, but this cleavage may also be mediated by meprins, forming tropocollagen (**Figure 3A**). Subsequently, three main pathways promote the final fibrillogenesis: the LOX-mediated, TG-mediated, and sugar-mediated cross-linking pathways (**Figure 3B**). The latter pathway constitutes a non-enzymatic glycosylation reaction that occurs as the consequence of prolonged exposure to reducing sugars (e.g., ribose and glucose), producing advanced glycation end products (AGEs), which are associated with aging and diabetic complications (Benkovics et al., 2017; Cruz et al., 2018).

The LOX and LOX-like (1–4) proteins are a family of copper-dependent amine oxidase enzymes that catalyze the formation of unstable aldehydes by the oxidation of the ε-amino groups of lysine/hydroxylysine in collagens and lysine in elastin, forming covalent cross-linkages in collagen–collagen and collagen–elastin complexes, respectively. LOX is secreted as a proenzyme into the ECM, but is also found intracellularly, and is then processed by BMP-1, the same enzyme that cleaves fibrillar procollagens (**Figure 3**). As such, there is a direct relationship between the collagen process and its crosslinking, suggesting a major role for LOX in ECM orientation (Rodriguez-Pascual and Rosell-Garcia, 2018).

The TGs [formed of nine members, including tissue TG (tTG) or TG2, which is the most abundant in tissues] belong to a multi-functional family of calcium-dependent acyl-transferase enzymes that catalyze transamidation of glutamine and lysine to form covalent bonds both inside and outside of the cell. These cross-links form between collagen–collagen, collagen– ECM, or ECM–ECM and can involve fibronectin, mainly, and also nidogen/entactin, osteonectin, osteopontin, laminin, vitronectin, fibrinogen, and heparan sulfate proteoglycan. Initially, the TG catalyzes the formation of an isopeptide bond between y-carboxamide groups of glutamine residue side chains and the ε-amino groups of lysine residue side chains with subsequent release of ammonia. Subsequently, glutamine and lysine residues are available to bind with peptides or proteins, and the intra- or inter-molecular ε-(γ-glutamyl)lysine cross-links take place. In the presence of water, TGs are also able to hydrolyze glutamine residues to glutamic acid by deamidation (**Figure 3B**). These cross-links exhibit high resistance to physical, chemical, and proteolytic degradation, mainly by matrix metalloproteinases (MMPs). Physiologically, TGs generate biological polymers that are indispensable for creating barriers and stable structures in several systems, while pathologically, they contribute to generating fibrotic matrices (Eckert et al., 2014; Savoca et al., 2018).

### ECM Remodeling Enzymes: Metalloproteinases

Most of the ECM remodeling enzymes belong to the metzincin family (Stöcker et al., 1995), which share numerous similarities, including multiple domains, zinc-dependent endopeptidases, a typical structural profile and tertiary configurations of the catalytic domain (the secondary structure contains three histidines bound to zinc, at the catalytic site, and a methionine, or "Met-turn"). This family comprehends vertebrate matrixins (MMPs), adamalysins (ADAMs—a family of disintegrin and metalloproteinase, mainly sheddases; and ADAMTSs—a family of disintegrins and metalloproteinases with thrombospondinlike motifs, mainly formed of proteoglycanases and procollagen N-propeptidases), astacins (BMP-1/Tolloid-like protease 1 and Meprins, mainly formed of procollagen N- and C-propeptidases), and pappalysins (main bioavailability of IGFs) (Cerdà-Costa and Gomis-Rüth, 2014) and encodes around 200 genes, identified in humans and mice, comprising around 1/3 of proteases, the largest proteolytic enzyme group existent (López-Otín and Bond, 2008). Among them, MMPs are classically recognized to degrade all ECM components, but other metalloproteinases have been recognized to play essential roles in ECM maturation and to generate bioactive molecules. As a result of the extensive study of ECM remodeling enzymes over the last six decades, other biological functions have also been attributed to them, due to their broad spectrum of substrates, identified in both subcellular and extracellular compartments (Bond, 2019). Other enzymes, such as urokinasetype plasminogen activator, cathepsins, and heparanases, are also indispensable in these processes, and growth factor bioavailability within the ECM is protease-dependent. As a result of the extensive study of ECM remodeling enzymes over the last six decades, other biological functions have also

FIGURE 3 | Collagen type I assembly and degradation by metalloproteinases. (A) The early fibrillogenesis process on fibrillar procollagens starts by the excision of both the N- and C- termini by classical Procollagen N-peptidases (sub-group of the ADAMTS family) and Procollagen C-peptidases (BMP-1). They cleave procollagens I, II, III, and V (ADAMTS2/14 and BMP-1), where ADAMTS3 can cleave only procollagen II. Recently, both meprins have been reported to be able to remove both N- and C- terminus as well. (B) After N- and C- termini removal from procollagen, mature collagen is the target for ECM cross-linker enzymes (LOX and TG), which will form collagen fibrils. Under high glucose conditions, a non-enzymatic cross-link takes place, named AGE. (C) The level of collagen fibrillogenesisis controlled by proteoglycans (SRLPs), which can interact with collagen fibrils, resulting in the modification of the diameter of collagen fibers. Fibromodulin, decorin, and lumican prevent or delay the cleavage of collagen by MMP-1 and MMP-13. Lumican acts on MMP-14 enzymatic inhibition (Pietraszek et al., 2014) and gene expression (Niewiarowska et al., 2011; Malinowski et al., 2012). Decorin inhibits MMP-2 and MMP-9 gene expression and activity (Neill et al., 2012). MMP-14 can cleave human decorin (Mimura et al., 2009) and thelumican core (Li et al., 2004), MMP-13 and ADAMTS-4 also cleave decorin (Shu et al., 2019). (D) Among the metalloproteinases, only MMPs can cleave fibrillar collagens. The collagenases generate 1/4 and 3 ⁄4 fragments (gelatin) that are further cleaved by gelatinases (MMPs-2 and -9) and MMP-13. MMP-13 also removes the N-terminal telopeptide from collagen. Indirectly, metalloproteinases may modulate collagen fibrillogenesis by processing LOX and SRLPs. BMP-1 and ADAMTS2/14 activate proLOX in distinct sites and seem to be essential for LOX-collagen binding (Rosell-García et al., 2019). Periostin increases the proteolytic action of BMP-1 on proLOX (Maruhashi et al., 2010). BMP-1 promotes the maturation of SLRPs (von Marschall and Fisher, 2010), as well as the MMPs, after C-terminal excision. Fibronectin increases BMP-1 activity against biglycan and procollagen I (Huang et al., 2009). AGE: advanced glycation end products; ADAM: adisintegrin and metalloproteinase; ADAMTS: adisintegrin and metalloproteinase with thrombospondin motifs; LOX: lysyl oxidase; MMP: matrix metalloproteinase; SLRP: small leucine-rich proteoglycans; TG: transglutaminase.

been attributed to metalloproteinases, due to their broad spectrum of substrates, identified in both subcellular and extracellular compartments (Bond, 2019). We will focus only on metalloproteinases currently known to have a role in palatogenesis and CL/P.

Among metzincins, ADAMs, ADAMTSs, and MMPs are closely related in structure, regulation, and activation. However, they have different substrates and, therefore, distinct functions under physiological and pathological conditions. Structurally, the N-terminal ectodomain of most secreted MMPs is composed of pre-, pro-, and catalytic domains (metalloproteinase domain) and contains a furin region in all ADAM, ADAMTS, and membrane-anchorage MMPs. Complementary domains confer proteolytic specificity and localization. As such, MMPs are the most studied metalloproteinases and act in many cellular functions (e.g., proliferation, migration, differentiation, among others) due to their cleavage of substrates in the ECM, on the cell surface and intracellularly (cytoplasm and nucleus) to produce bioactive molecules. However, few studies have been conducted to understand the interrelationship between these metalloproteinases and how they work together to control cell behavior.

#### MMP Family

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Over the years, the "degradative" activity of MMPs during physiological and pathological processes has led to their association with tissue destruction, due to their unique ability to cleave fibrillar collagens (Sprangers and Everts, 2019). However, "omic" studies and a better understanding of ECM dynamics support a broader role for MMPs in pathological and physiological events (Rodríguez et al., 2010). Several other core matrisome, ECM-associated proteins, and cell surface proteins, cleaved by MMPs, reveal hidden sequences and unblock the diverse cell functions (Butler and Overall, 2009; Deryugina and Quigley, 2010; Bauvois, 2012; Mannello and Medda, 2012).

Extensive reviews focus on MMPs regulation at both intra and extracellular levels and have been extensively reviewed. At the gene transcription level, signals from the ECM (cytokines, growth factors, EMMPRIN/ECM metalloproteinase inducer/CD147, integrins, ECM proteins, cellular stress, morphological changes, among others) significantly impact MMP expression. Mutations and polymorphisms in MMP genes (particularly in promoter regions), together with epigenetic modifications, have been shown to modulate MMP expression. Post-transcriptional regulation includes changes in mRNA stability through microRNAs, decoy RNAs, and degradation pathways. MMPs are targets of several PTMs that are crucial for correct cellular localization (via insertion of the GPI-anchor), intracellular activation of membrane-anchoring MMPs by furins, and insertion of carbohydrates (N and O-glycosylation) (Boyd, 2006; Reuben and Cheung, 2006; Vincenti, 2007; Clark et al., 2008).

In the ECM, soluble proenzymes are activated by other proteinases, mainly active MMPs, via the "cysteine switch mechanism." The catalytic activity may be inhibited by their endogenous inhibitors in the ECM (TIMPs) or on the cell membrane (RECK). Additionally, their proteolytic activity may be modulated by allosteric control in exosites outside of the catalytic site and also by interactions with proteoglycans and GAGs (Pietraszek-Gremplewicz et al., 2019). For example, MMP-2 interacts with syndecan-2 on the cell surface, which blocks the activation of proMMP-2. In some cases, proenzymes are associated with TIMP (low concentration) for correct activation; for example, the MMP-14/TIMP-2/proMMP-2 ternary complex. MMPs may be associated with other ECM components, such as proteoglycans and GAGs, leading to their specific localization in the cellular perimeter or at ECM sites distant from the cell secretion point (Van den Steen et al., 2001; Hernandez-Barrantes et al., 2002; Ra and Parks, 2007; Gabison et al., 2009; Hadler-Olsen et al., 2011; Piperi and Papavassiliou, 2012; Rietz and Spiers, 2012; Li et al., 2014; Arpino et al., 2015; Karamanos et al., 2019).

Due to their unique ability to cleave fibrillar collagens, collagenases MMP-1, -8, and -13 are the main enzymes for collagen turnover and generate gelatin (classical 3/4 and 1/4 fragments) (**Figure 3D**). MMP-2 and -9, along with membraneanchored MMPs (MMP-14 and MMP-16), may also cleave fibrillar collagen with different affinities, as is the case of collagen I and III, which are preferentially cleaved by MMP-1, whereas collagen II is the preferred substrate for MMP-13. Additionally, stromelysins (MMP-3 and -10) can degrade just collagen III, but not collagen I or II (Sprangers and Everts, 2019). The interaction of the N-terminal site of specific SRLPs (decorin, fibromodulin, and lumican) with collagen fibrils forms a layer that can prevent or delay the collagen cleavage by MMPs (Geng et al., 2006). At the same time, different SRLPs are substrates or inhibitors of MMPs and ADAMTS (Shu et al., 2019; **Figure 3C**).

#### ADAMTS Family

N-glycosylation, O-fucosylation, and C-mannosylation are the most frequent post-translational modifications (located at the ancillary domains) to control ADAMTS activity. These modifications control secretion, localization, activation, and catalytic functions. Cleavage of the central thrombospondin type 1 sequence repeat (TSR—probably attached to the cell membrane) regulates both the proteinase activity and the localization of these enzymes. The proenzymes can be activated intracellularly, on the cell surface or within ECM by furinlike pro-protein convertases and then autocatalytic activation. Similarly to ADAMs, ADAMTSs are selectively inhibited by TIMPs, where TIMP-3 is the principal inhibitor (Bekhouche and Colige, 2015; Kelwick et al., 2015; Dancevic et al., 2016).

Although ADAMTS-like homologues lack protease domains, ancillary domains are present and may be involved in the regulation of ADAMTS due to competitive binding to substrates. Furthermore, they may have ADAMTS-independent functions in ECM, cell–EMC, or cell–cell interactions. Recently, an unexpected interaction between LOX-ADAMTSLs was reported, suggesting a role in microfibril assembly (Aviram et al., 2019). Mutations or deletions in ADAMTS have been implicated in many pathologies and syndromes, where they modulate tissue morphogenesis and remodeling, in cancer, inflammation, in the central nervous system, and in cartilage and vascular biology (Kintakas and McCulloch, 2011; Stanton et al., 2011; Lisi et al., 2014; Dubail and Apte, 2015; Hubmacher and Apte, 2015; Kelwick et al., 2015; Rodríguez-Manzaneque et al., 2015; Dancevic et al., 2016; Fu and Kong, 2017; Itoh, 2017; Lemarchant et al., 2017; Yang et al., 2017; Mead and Apte, 2018).

#### THE ROLES OF MMPS AND TIMPS IN PALATOGENESIS

The development of the facial primordia requires remodeling of the ECM, which is mediated in part by MMPs. During embryonic development, the process of morphogenesis involves MMPmediated changes in the composition of the ECM that further allow for cell migration and differentiation, cell–cell interactions, and tissue resorption. MMPs act on ECM remodeling during palatal shelves orientation and during EMT events for palatal fusion (Brown et al., 2002).

Early studies in mice have provided biological evidence for the roles of MMPs and TIMPs in embryonic development (Gack et al., 1995; Iamaroon et al., 1996; Blavier and DeClerck, 1997). Several MMPs, TIMPs, and RECK mRNA and proteins are expressed, in association with enzymatic activity, throughout the stages of murine palate development. These expressions share the same specific spatial and temporal distribution patterns in areas in which their preferred substrates are also present

(Mansell et al., 2000; Morris-Wiman et al., 2000a,b; Blavier et al., 2001; Brown and Nazarali, 2010; de Oliveira Demarchi et al., 2010; Gkantidis et al., 2012).

The secretion of GAGs in the palatal mesenchyme is related to an increase in water content, and the specific accumulation of collagen I in the nasal side of the palatal mesenchyme may be necessary to generate internal forces required for shelf elevation. Furthermore, MMP expression increases in both the medial and oral epithelium before shelf elevation (Morris-Wiman et al., 2000a; de Oliveira Demarchi et al., 2010), as does MMP gelatinolytic activity in the basement membrane and beneath the mesenchyme of the nasopharyngeal epithelial folds that form during palatal shelf reorientation from vertical to horizontal position (Gkantidis et al., 2012; **Figure 4A**).

At ED13 and ED14, the secreted inhibitors, TIMP-1 and TIMP-2, display a similar spatial distribution to the MMPs and are widely expressed in the epithelial basement membrane. TIMP-3 is strongly expressed in the palatal epithelium although weakly expressed in the medial mesenchyme (Morris-Wiman et al., 2000a). Of the four TIMPs, TIMP-2 is the most abundant (Mansell et al., 2000), whereas TIMP-3 has different roles than the other TIMPs. Animals lacking the Timp3 gene develop several pathologies associated with increased ECM degradation and loss of tissue integrity due to unregulated MMP, ADAM, and ADAMTS activity (Sahebjam et al., 2007). The anchored-membrane MMP inhibitor, RECK, is also expressed in the mesenchyme (de Oliveira Demarchi et al., 2010). RECK expression has been implicated in tissue integrity since its absence leads to extensive disarrangement of the connective tissue and embryos die in utero before craniofacial development (Oh et al., 2001). Expression of TIMP-3 and RECK in the different sites of the developing palatal epithelium suggests that they function in the maintenance of palatal tissue integrity by regulating epithelial–mesenchymal interactions (**Figure 4B**).

Matrix metalloproteinases are fundamental for the removal of the basement membrane and are expressed by epithelial cells in the EMT program to detach from MES and migrate to the adjacent mesenchyme to allow palatal fusion (Horejs, 2016). The participation of the membrane-anchored MMPs (MMPs -14, -16, and -25) appear to be crucial in this process (Shi et al., 2008; Brown and Nazarali, 2010). These observations are reinforced by the results of in vitro studies showing that addition of synthetic MMP inhibitors (Blavier et al., 2001), or the silencing of MMP genes (e.g., MMP-25) (Brown and Nazarali, 2010) in palatal cultures can prevent palatal fusion. Furthermore, Tgfβ-3- and Egfr-knockout mice, which display a cleft palate phenotype, have decreased or absent MMP expression in the MEE or MES (Miettinen et al., 1999; Blavier et al., 2001). Heparanase has also been detected in the MEE and MES and co-localized with MMPs -2, -3, and -9 (Hirata et al., 2013). MMP-25 can cleave only collagen IV and, in terms of substrates, displays more similarities to MMP-3 than other MT-MMPs (English et al., 2001). High gelatinolytic activity and laminin expression have also been found in the MEE and MES (Gkantidis et al., 2012). Furthermore, MMP-3 cleaves E-cadherin (Lochter et al., 1997) and MMP-25 is co-localized with E-cadherin in cell–cell junctions (Radichev et al., 2010), a crucial step for epithelial EMT cells to acquire a mesenchymal phenotype. Taken together, the basement membrane and epithelial cell–cell junction degradation require the cooperative proteolytic actions of MMPs and other ECM degrading enzymes in the MEE and MES cells (**Figures 4C,D**).

TGFβ-3 is expressed explicity in the palatal epithelium, and is co-expressed with MMPs (Blavier et al., 2001; Brown and Nazarali, 2010). In Tgfβ-3 null mice, TIMP-2 and MMP-13 expression in the palatal epithelia were significantly decreased, whereas no changes in expression were noted for MMP-14 (Blavier et al., 2001). Similarly, the incubation of palatal culture with a TGFβ-3-neutralizing antibody decreases MMP-25 expression in the palatal epithelia (Brown and Nazarali, 2010). Collectively, these observations suggest that both MMP-13 and MMP-25 are downstream targets of TGFβ-3 (**Figure 4C**). MMP-13 is specifically expressed in sites of bone formation in vivo (Fuller and Chambers, 1995; Gack et al., 1995; Mattot et al., 1995; Stahle-Backdahl et al., 1997), and in physiological situations that require rapid and effective remodeling of collagenous ECM.

While MMP and TIMP expressions appear to be critical throughout all stages of palatal development, knockout mice for Timp1, Timp2, Timp3, Timp4, Mmp2, Mmp9, Mmp13, Mmp14, Mmp16, and Mmp25 do not develop cleft palate (Paiva and Granjeiro, 2014; Soria-Valles et al., 2016). Interestingly, combined double knockout of Mmp14 and Mmp16 in mice leads to severe structural and craniofacial defects, including severe dysfunction in palatal shelf formation and cleft palate in 80% of the embryos (Shi et al., 2008). Among the membrane-anchored MMPs, MMP-16 is closely related to MMP-14 in terms of molecular structure and expression patterns in remodeling tissues (Szabova et al., 2005; Shi et al., 2008). While these findings suggest that potential compensatory mechanisms exist to overcome the loss of function of individual MMP genes, they also suggest that the loss of specific MMPs, in combination, may impair embryonic and palate development. Moreover, while individual MMP genes may not contribute a major gene effect to non-syndromic CL/P (NSCLP) susceptibility, they may act as modifiers on the background of other genes.

# CLEFT LIP AND/OR PALATE

Orofacial clefts result from the failure of developing embryonic facial and palatal processes to ultimately merge or fuse. A multidisciplinary team including surgical, dental, speech, genetic, and nutrition experts are typically involved in patient care to mitigate the feeding, swallowing, breathing, speech, and hearing complications inherent to the condition. Orofacial clefts can be categorized as syndromic or non-syndromic (also termed isolated), based on the presence of additional structural abnormalities. Over 500 syndromes, including chromosomal abnormalities, have been reported in association with orofacial clefts, comprising 30% of all cleft cases. The remaining 70% of cases are all non-syndromic (i.e., isolated), and may segregate in families or appear in sporadic cases (Mossey et al., 2017).

Non-syndromic orofacial clefts are the most common of congenital craniofacial disabilities, affecting approximately 1 in

FIGURE 4 | Continued

FIGURE 4 | Schematic representation of consecutive steps during secondary palatogenesis and the molecules expressed in palatal mesenchyme ECM and palatal epithelium. Collagens type I and III, fibronectin, GAGs (hyaluronic acid, heparan sulfate, and chondroitin-4-sulfate), proteoglycans (biglycan, decorin), and TGF-β are the most abundant ECM molecules found in provisional ECM during palatogenesis. MMPs are the major protein responsible for ECM remodeling in both the ECM mesenchyme and basement membrane, but also expressing heparanases as well. Expression of TIMPs and RECK occurred, and they are crucial for ECM remodeling balance. (A) Most ECM components are widely present in palatal mesenchyme. MMP expression is high in the oral and medial epithelia before shelve elevation. (B) The increased expression of collagen I and decorin in the nasal region of the palatal mesenchyme may help with palatal elevation. Decorin can bind to collagen I and cause contraction of collagen in vitro. In the medial epithelium, the specific expression of collagen IX and CSPG is vital for adequate palatal shelve adhesion. (C) TGF-β3 modulates the expressions of TIMP-2, MMP-13, MMP-25, and CSPG in the MEE. TGF-β3 knockout mice downregulated these genes. mir-127 is upregulated, leading to repression of MMP-13 expression (Warner et al., 2015). (D) Other MMPs are required for MES disruption and are involved in basement membrane degradation by EMT cells. CSPG: chondroitin sulfate proteoglycans; EMT: epithelial-to-mesenchymal transition; HA: hyaluronic acid; Has: hyaluronan synthases; M: medial site; m: medial region; MEE: medial edge epithelia; MES: medial epithelial seam;mx: maxillary region; n: nasal region; N: nasal site; o: oral region; O: oral site; TGF-β: transforming growth factor beta.

700 live births worldwide each year, and have been divided historically into cleft lip, with or without cleft palate (CL/P), and cleft palate only (CP), due to the distinct developmental origins of the lip and the palate (**Figure 5**). While their reported prevalences vary considerably according to ancestral origin, Asians are the most frequently affected population, with birth prevalence rates as high as 1 in 500 live births, followed by Caucasians with a prevalence rate of about 1 in 1000 live births, and African populations showing the lowest prevalence rates at approximately 1 in 2500 live births (Kadir et al., 2017; Mossey et al., 2017; Ishorst et al., 2018). The presence of CL/P also differs by sex and laterality, with CL/P being more common in males than females at a 2:1 ratio, and CP being more common in females, meanwhile unilateral left CL/P is more common than unilateral right CL/P at a 2:1 ratio.

The etiology of non-syndromic orofacial clefts is complex, with both genetic and environmental factors contributing to the condition. While the identification of the genes involved in the syndromic forms of clefting has been mostly successful, much remains to be learned about the factors involved in nonsyndromic cases. Genetic studies to date, using both familybased and case-control research approaches, have identified several genes and loci that may play a role in susceptibility to oral clefts (Vieira, 2008). Of these, the evidence is particularly strong for MSX1, IRF6, FOXE1, MAFB, WNT3, WNT9B, CRISPLD2, FGFR1, FGF8, BMP4, and the 8q24 chromosome region. Additional variants in numerous other genes (e.g., TGF-β3, TGF-α, MMP3, VAX1, ABCA4, AXIN2) have also been suggested as candidates for oral clefts with population-specific effects (Dixon et al., 2011; Beaty et al., 2016).

### ECM REMODELING ENZYMES AS CANDIDATE GENES FOR OROFACIAL CLEFTS

Matrix metalloproteinases and TIMPs have been considered plausible candidate genes for CLP, based on their expression

patterns in craniofacial tissues and their roles in tissue remodeling and morphogenesis during early embryogenesis; however, a functional role for any individual MMP or TIMP in palate development remains unknown (Brown et al., 2002; Verstappen and Von den Hoff, 2006).

In humans, significant associations between polymorphisms in MMP and TIMP genes with NSCLP have been reported (Blanton et al., 2004; Nikopensius et al., 2011; Letra et al., 2012; **Figure 6**). A large genome scan of multiplex NSCLP families first suggested evidence for linkage within a region on chromosome 16p13.3, in the same location for MMP25 (Blanton et al., 2004). A later large and comprehensive study evaluated 45 polymorphisms spanning all biologically relevant MMP and TIMP genes for their association with NSCLP; significant associations (P < 0.0004) were noted for variants in MMP3 (rs3025058, rs522616) and TIMP2 (rs8179096), when considering CL/P, CP, and all cleft cases combined. Additional nominal associations were also found for variants in MMP16 (rs7828497, P = 0.01) and MMP10 (rs17293607, P = 0.06) in CL/P and all cleft cases. For CP, variants in MMP3, MMP14, and TIMP1 also showed nominal associations (P < 0.05). Of note, certain allele combinations (i.e., haplotypes) involving these MMP and TIMP gene variants were also significantly associated with NSCLP (Letra et al., 2012).

Two polymorphisms, -1171 5A/6A (rs3025058) and -709 A/G (rs522616), located in the MMP3 gene promoter were significantly associated with NSCLP and shown to have functional effects on gene transcription and protein function. The 5A/6A polymorphism consists of a common adenine insertion/deletion polymorphism (5A/6A) at position -1171 of the gene promoter and modulates transcription and local expression of the MMP-3 protein. The 6A allele showed an approximately twofold lower amount of gene product, compared with the 5A allele (Ye et al., 1996), and this difference in promoter activity was attributed to a likely differential binding of the transcriptional repressor to the 6A allele (Mercapide et al., 2003).

A positive regulatory element has also been described for MMP3 -709 A/G, for which significantly higher (∼3.4-fold) promoter activity was found in the presence of allele A. This suggested that allele A can enhance promoter activity, possibly by augmenting transcription factor binding. Furthermore, this variant also appears to be modulated by the concomitant occurrence of the -1171 5A/6A variant. When analyzing the transcriptional effects of haplotypes containing both the -1171 5A/6A and -709 A/G variants, a 1.5-fold decrease in activity was observed for the combination of 5A\_A alleles, in comparison with the 5A\_G haplotype. Although speculative, this finding may represent a negative feedback loop effect, in an attempt to limit

transcription in the presence of the two "high transcription" alleles, 5A and A. In contrast, a fourfold increased activity was found with the 6A\_G promoter. The 6A\_A haplotype was the least active promoter, suggesting potential gene downregulation with this allelic combination (Letra et al., 2014). Taken together, these observations indicate that the -709 A/G variant may directly regulate MMP3 promoter activity, although its function was shown to the driven by the 1171 5A/6A alleles in the background.

Polymorphisms in the TIMP2 gene have also been associated with NSCLP in different populations. A promoter variant in TIMP2 (-180C/T; rs8179096) was strongly associated with oral clefts in a Brazilian population, whereas additional variants of unknown significance (intronic) were associated with NSCLP in US and Northeastern European populations (Nikopensius et al., 2011; Letra et al., 2012). Furthermore, functional analysis suggests that this variant has distinct allele-dependent effects, with the T allele presenting a 2.5-fold increased promoter activity. Furthermore, both C and T alleles were found to be putative binding sites for NFκB, a key transcription factor involved in the innate immune system. While C and T alleles reduced binding capability when NFκB consensus binding oligo diverges from protein in the same reaction, introduction of a mutant NFκB immunized C and T alleles from binding abolition (Letra et al., 2014). Additional studies are still necessary to unveil the exact mechanisms by which MMPs and TIMPs might contribute to NSCLP; nonetheless, allelic polymorphisms in these genes and their interactions may partly explain the variance in individual susceptibility to oral clefts.

Few studies have described roles for other ECM remodeling or cross-link enzymes during palatogenesis and their potential association with CL/P. ADAMTS-9 and -20 have been shown to participate in versican remodeling during palatogenesis (Enomoto et al., 2010); furthermore, ADAMTS-9 null mice die in utero (Dubail and Apte, 2015). During physiological palate formation, ADAMTS9 expression in the palatal shelves was restricted to microvascular endothelial cells, derived from the mesoderm, whereas CNC-derived mesenchymal cells express ADAMTS20; in contrast, the expressions of ADAMTSs 4 and 5 were not detected during palatogenesis (Enomoto et al., 2010). More recently, a genome-wide association study in both dogs and humans independently identified ADAMTS20 as a potential candidate gene for clefting. A region on chromosome 27 in dogs was found to segregate with a complex phenotype of cleft palate and syndactyly; follow-up whole genome sequencing studies then identified a frameshift mutation in the ADAMTS20 gene as potentially etiologic. Furthermore, four novel risk variants in ADAMTS20 were identified in Guatemalan cases with NSCLP (Wolf et al., 2015).

Additional genes with roles in collagen maturation and other ECM molecules have also been suggested to play a role in NSCLP. The LOXL3 gene located on chromosome 2p13.1 and mutations in this gene have been reported in association with Stickler syndrome (MIM #108300), an autosomal recessive disorder characterized by ocular, auditory, skeletal, and orofacial abnormalities (Alzahrani et al., 2015). In mice, the deletion of Loxl3 causes neonatal mortality due to impaired collagen assembly and cross-linking, as well as spine deformity and cleft palate (Zhang et al., 2015). Furthermore, its ortholog in zebrafish is required for adequate craniofacial morphogenesis, as its loss of function results in abnormal chondrogenesis with micrognathia or agnathia phenotype (van Boxtel et al., 2011). Recently, a missense mutation in LOXL3 (rs17010021; p.Ile615Phe) was reported to contribute to an increased risk of NSCLP (10-fold) in a European population (Khan et al., 2018).

#### NEW APPROACHES TO STUDYING PALATOGENESIS AND CL/P IN VITRO

Studies of both palatogenesis and CL/P are usually carried out in animal models (mouse, chicken, zebrafish, and rat, respectively), especially mice, given the similarity of biological events in these animals to humans (Van Otterloo et al., 2016; Lough et al., 2017). In vitro, the most common method of study employs murine embryonic palatal cell culture and ex vivo palate organ culture (**Figure 7A**; Abbott, 2019).

Three-dimensional (3D) cell culture represents a promising approach to better elucidate cell behavior, ECM remodeling, tissue remodeling, and tissue fusion, and ultimately to investigate clinical applications for tissue engineering (Ong et al., 2017). 3D constructs are more similar to the tissue microenvironment than classical 2D cell cultures (plastic-based) as they demonstrate more realistic cell morphology and physiology; furthermore, observation of 3D cultures over time is considered to represent 4D systems (Bissell, 2017). Thus, over the last decade, numerous reviews have illustrated the switch from 2D to 3D cell cultures. Several methods to create 3D structures have been described, such as cell aggregates, spheroids or organoids (Alhaque et al., 2018), seeded onto decellularized matrices (Taylor et al., 2018), and cell sheets (Kim et al., 2019). Among these, cell spheroid is a technology in which cells are cultured in suspension to create a 3D structure using cell–cell interactions and "scaffold-free" strategy (**Figure 8B**).  Understanding the ECM and its soluble factors are essential to comprehend embryonic development and tissue repair and will contribute to the discovery of new therapeutic tools. Proteomics is an appropriate strategy to characterize ECM components under physiological and pathological conditions. A significant challenge for studying the ECM constitutes its solubilization and protein recovery. An optimized protocol developed by Naba et al. (2017) permitted the digestion of proteins into peptides that could be analyzed by mass spectroscopy; web tools were allowed the annotation and relative quantitation of the ECM proteins. These protocols allow a faster analysis of differentially expressed proteins in the ECM, eliciting the identification of biomarkers and therapeutic targets.

Stem cells from many tissue origins, especially MSCs, have been widely employed in 3D constructions due to their effortless isolation from various tissues of the body, potential for differentiation into mesenchymal and nonmesenchymal lineages, and therapeutic use (Han et al., 2019).

Cell spheroids to study epithelial palate fusion are available, and strategies employed consist of creating mesenchymal spheroids from human umbilical-derived MSCs undergoing

MSC: mesenchymal stem cell; N: nasal site; o: oral region; O: oral site.

osteogenic differentiation, covered with ECM to mimic basement membrane, which are then seeded with human progenitor epithelial keratinocytes. This 3D organotypic model of human

forming the functional "building blocks"; spheroids can be embedded into scaffolds/biomaterials. (C) Both sing cells and spheroids and/or signalling molecules are the components of bioink in association with a hydrogel, and then it is bioprinted in a specific 3D engineered bone graft.

palatal shelves can maintain cell viability for around 4 weeks, expresses alkaline phosphatase, and is responsive to EGF, leading to increased epithelial proliferation and the prevention of epithelial fusion between spheroids (**Figure 7B**; Wolf et al., 2018). A potential bias relies on the fact that the MSCs used in the spheroid generation do not have the same embryonic origin as the palatal mesenchyme or the epithelial cells.

# STATE-OF-THE-ART OF CLEFT PALATE REPAIR AND PALATAL RECONSTRUCTION

# Bone Regeneration

Bone repair is a mechanism in which bone development program is recapitulated to form new intramembranous or endochondral bone at injured sites. Intense ECM bone remodeling by MMPs is required (Paiva and Granjeiro, 2017) as well as the action of several growth factors (TGF-β, BMPs, FGFs, IGFs, PDGFs, and VEGFs), which are implicated in driving osteochondral differentiation, regulation of bone formation, and are responsible for triggering intracellular pathways, such as Wnt (Houschyar et al., 2019). Additionally, other metalloproteinases are important for collagen maturation and mineralization. PPAP-A increases osteoblast proliferation in vitro and bone formation in vivo by augmenting IGF bioavailability upon cleavage of IGFBP-4 (Qin et al., 2006). PPAP-B expression was associated with postnatal skeletal growth, bone mass, and structure due to cleavage of IGFBP-5, the most abundant IGF in bone (**Figure 9A**; Amiri and Christians, 2015; Christians et al., 2019).

In bone and dentin matrices, BTPs can cleave the acidic domain of SIBLINGs (small integrin-binding ligand, N-linked glycoproteins), specifically DMP-1 and DSPP (dentin sialophosphoprotein—generating DPP/dentin phosphoprotein), leading to an increase in the binding affinity of these proteins for calcium, which is necessary for mineralization and improving ECM stiffness (Steiglitz et al., 2004; von Marschall and Fisher, 2010; Tsuchiya et al., 2011). BMP-1 is highly expressed in both endochondral and intramembranous ossification sites during development and contributes to an increase in bone repair (Grgurevic et al., 2011). In addition, BMP-1 expression is modulated by mutations in both alpha procollagen chains (Lindahl et al., 2011) and BMP-1-knockout mice develop an osteogenesis imperfecta-like phenotype due to inefficient procollagen processing and bone mineralization (**Figure 9B**; Muir et al., 2014). Although meprins also act on procollagen maturation and DSPP cleavage, meprin-β is inhibited in tissues with high concentrations of calcium and is, probably, not crucial in hard tissues since mice knocked out for meprins do not demonstrate alterations in bone and tooth development (Arnold et al., 2017).

Several ADAMs (8, 9, 10, 12, 15, 17, and 19) and ADAMTS (1, 4, and 5) are secreted by osteoblasts and bone tissue. Recently, ADAM17 has been reported to be a target of RUNX2 during osteoblastic differentiation, where ADAM17 is suppressed by RUNX2 (Araya et al., 2018). Human mutations in ADAMTS10 and 17 are associated with related syndromes involved in alterations in skeletal development (Dagoneau et al., 2004; Morales et al., 2009). Recently, knockout mice for both Adamts10 and 17 were shown to develop the recapitulate syndromic phenotype in human (**Figure 9C**; Mularczyk et al., 2018; Oichi et al., 2019). Reduced hypertrophic zone and increased deposition of fibrillin-2 alter the growth plates during endochondral ossification resulting in adults with short statures. Fibrillins

motifs; DMP-1: dentin matrix acidic phosphoprotein; DSPP: dentin sialophosphoprotein; IGFBP: insulin-like growth factor binding protein; IGF: insulin-like growth

are glycoproteins involved in microfibril formation and elastin deposition. Also, treatment with BMP-2 can rescue terminal chondrocyte differentiation, suggesting that ADAMTS17 is important for ossification through the modulation of BMP signaling (Oichi et al., 2019).

factor; KO: knockout mice; PAPP: pregnancy-associated plasma protein.

Extracellular matrix cross-linkers also contribute to bone formation and regeneration. In LOX knockout mice, general ECM architecture is profoundly affected in several tissues, and littermates die soon after birth. In bone, early and late osteoblastic differentiation and onset of mineralization are decreased in these mice as well as the gene expression of LOX isoforms (LOXL1-4) and osteoblastic markers (collagen type I, bone sialoprotein, and RUNX2) (Pischon et al., 2004).

The crosstalk between inflammatory cells, MSCs, chondrocytes, osteoblasts, osteoclasts, and osteocytes is necessary (Liu et al., 2018), and the success of bone regeneration results from the balance of the osteoprotegerin/RANK/RANKL axis. Most bone defects regenerate spontaneously; however, extensive bone loss due to trauma or aging-related fractures, metabolic bone disease, and congenital malformations do not regenerate per se, underscoring the search for better drug candidates. Biomaterials, biomolecules, and stem cells have been investigated to support bone repair, overcoming the limitations of autologous and allogeneic grafts (Collignon et al., 2017).

# First Generation Palatal Reconstruction: Standard Surgical CL/P Management and Cleft Palate Repair

The treatment of CL/P patients is primarily surgical (plastic surgery for the lip and a bone graft for the palate) followed

by orthodontic treatment in multiple stages over the years. The management is dependent on the cleft site, extension, and affected tissues (Farronato et al., 2014). The term "palatal reconstruction" is used to define any intervention able to restore the barrier between the oral and nasal cavities, and physiological functions. As such, several approaches, such as the lip and/or palate closure through plastic surgery, several types of bone graft, or a combination of these have been employed. More recently, the use of bioengineered bone has allowed palatal reconstruction (**Figure 10**). The decision with regard to the best treatment of choice depends on the type of CL/P and the extension of the tissue loss (Gupta et al., 2012).

Autogenous bone grafts are the gold standard for alveolar bone and cleft palate repairs. Throughout treatment stages, surgical procedures may require several grafts (e.g., primary, secondary, and tertiary graft). The most common bone donor sites are the anterior iliac crest, proximal tibia, mandibular symphysis, calvaria, and ribs. However, the bone amount and volume available from these sites are restricted, and a most advantageous site is still under debate. The use of autogenous bone graft has many drawbacks related to morbidity at the donor site, and patients may present chronic pain at the site, paresthesia of the thigh, and hypertrophic scar. The loss of the graft due to local inflammation, bone resorption, and the development of oronasal fistulas is also frequently associated with unsuccessful repair (Borba et al., 2014; Tavakolinejad et al., 2014; Shafi et al., 2015).

#### Second Generation of Palatal Reconstruction: Biomaterials and Growth Factors

Bone graft options available, such as allogeneic bone (Shirani et al., 2017), xenogeneic bone (Thuaksuban et al., 2010), and alloplastic graft (Kumar et al., 2013; Seifeldin, 2016; Sharif et al., 2016), still do not promote effective palatal reconstruction, stimulating the search for better alternatives. To overcome the limitations of autologous and allogeneic bone grafts, studies have investigated the use of the association, or not, of osteoconductive biomaterials, such as hydroxyapatite and tricalcium phosphate, with autologous bone graft or growth factors, such as BMP, which is known to stimulate bone formation and repair (Horch et al., 2006; Weijs et al., 2010; Lazarou et al., 2011; Benlidayi et al., 2012; De Ruiter et al., 2015; Takemaru et al., 2016;

Martín-Del-Campo et al., 2019). After the systematic review of 29 studies, just two were used for qualitative and quantitative analysis (Segura-Castillo et al., 2005; Dickinson et al., 2008). However, considering some aspects of randomization, allocation concealment, blinding, outcome data, and reporting, the authors concluded that the two selected articles presented a high risk of bias and that conclusions concerning the benefits of the interventions, compared to traditional iliac grafting, could not be made (Guo et al., 2011).

Growth factors such as BMPs (2, 4, and 7) and "natural adjuvant" platelet concentrates, denominated platelet-rich plasma (PRP) and platelet-rich fibrin (PRF), have been employed in palate reconstruction (Behnia et al., 2012; Seifeldin, 2016). Interestingly, PRF does not affect the expression of the RUNX2, BMP-2, or RANKL, but induces the expression of OPG, leading to the increase of OPG/RANKL, suggesting that PRF could boost osteoblastic differentiation (Sumida et al., 2019).

Clinical studies of alveolar bone repair in CL/P patients tested the association of BMPs or platelet concentrates with the gold standard of autologous bone graft. BMPs make relevant contributions to bone embryogenesis and repair (Salazar et al., 2016) and the inhibition of BMP receptors (Lai et al., 2016), mutations (Sahoo et al., 2011), and gene polymorphism (Savitha et al., 2015) are involved in cleft development. The association of bone graft with human recombinant BMP-2 (hrBMP-2) in vitro and in vivo demonstrated the production of mature bone (Shimakura et al., 2003). Furthermore, clinical studies corroborate that BMPs are, at least, just as efficient as autologous bone graft for the repair of alveolar/palate cleft (Chin et al., 2005; Canan et al., 2012; Ayoub et al., 2016; Hammoudeh et al., 2017). Very recently, 10 years of follow-up evidenced the safe use of BMP-7 for the reconstruction of the alveolar cleft (Ayoub and Gillgrass, 2019).

Evaluating the use of growth factors for the reconstruction of the alveolar cleft, Van Hout group accessed 291 studies using BMP-2, BMP-7, TGF-β, PDGF, IGF, FGF, VEGF, and PRP (van Hout et al., 2011). Only six articles met the authors' criteria for full analysis, who concluded that BMP-2 improved the quantity of bone formation during the reconstruction of the alveolar cleft, in association with reducing pain at the donor site, a reduction in surgery time, hospital stay, and overall cost. A recent study reported a similar effectiveness of autologous bone and rh-BMP2 during the maxillary reconstruction of cleft lip and palate patients (Scalzone et al., 2019).

A growing field of investigation aims to evaluate the use of cell-based therapies to treat alveolar and palate cleft (Gładysz and Hozyasz, 2015; Brozek et al., 2018) due to the ability of stem cells to differentiate into active osteoblasts that drive bone regeneration. Trabecular bone was enhanced after the treatment of 16 out of 17 alveolar clefts with MSCs together with hrBMP-2, but long-term follow-up studies are still needed (Fallucco and Carstens, 2009).

Two recent systematic reviews addressed the role of BMP2 (Scalzone et al., 2019) and tissue-engineered bone replacement materials (Kamal et al., 2018) in the efficient treatment of palate and alveolar cleft. Both studies concluded that there was no statistical difference between the autologous bone graft and the alternative materials. The meta-analysis comparing the bone repair with iliac crest bone graft (ICBG) versus BMP-2, acellular dermis matrix membrane, and cranium or rib grafts, indicated that ICBG is still the best choice for treatment (Wu C. et al., 2018). The association of BMP-2 with a collagen sponge provides similar results to those of ICBG, and PRP associated with ICBG increased bone retention for skeletally mature patients. However, better-designed controlled studies are required for long-term analysis of alveolar cleft reconstruction, with follow-ups of greater than 12 months (Seifeldin, 2016; Liang et al., 2018; Wu C. et al., 2018). Furthermore, the development of a consensus for standardized protocols, using multicenter studies, is still needed (de Ladeira and Alonso, 2012).

#### Third Generation of Palatal Reconstruction: Bone Bioengineering

Tissue bioengineering represents a new therapeutic alternative for palatal reconstruction, associating the use of stem cells, biomaterials/scaffolds, and signaling molecules. Two main approaches are dominant: the "top-down" approach, in which classically cells are seeded on or into biomaterials for recreating a new 3D tissue in vitro; and the "bottom-up" approach, which uses 3D construction techniques (described in the last section) to improve cell secretion of growth factors for tissue regeneration in vivo (**Figure 8**; Baptista et al., 2018). Furthermore, Good Manufacturing Practice (GMP) and animal-free supplements are crucial for clinical applications. The success of the bioengineered bone graft is directly related to the osteogenic potential of stem cells, biomaterial/scaffold specific properties, and adequate external signaling from growth factors.

Stem cells from different origins and potentialities represent the cutting-edge technology for palatal reconstruction (Zuk, 2008; Gładysz and Hozyasz, 2015). However, few studies of bone bioengineering approaches in animal models of palatal defects have been carried out. A large number of research articles have addressed bone bioengineering approaches for alveolar bone defects created in animal models to reproduce alveolar cleft or mid-palate cleft. However, here we consider only animal models of the mid-palate cleft and the use of human cells. Conejero et al. (2006) demonstrated that rat palatal criticalsize defect filled with autogenous engineered graft (fat-derived stem cells previously differentiated into osteoblasts/osteocytes and seeded onto poly-L-lactic acid absorbable scaffolds) could regenerate bone at 6 or 12 weeks after surgery; bone defects filled only with the scaffold or scaffold plus undifferentiated cells had a fibrotic tissue with no bone. Another study used an autogenous multi-layered graft, which was simultaneously bioengineered for palate bone and oral mucosa in a rabbit palatal critical-size defect (Martín-Piedra et al., 2016). Initially, individual cell layers of adipose tissue-derived MSCs were seeded onto fibrin-agarose hydrogels and induced toward osteogenic differentiation; and with fibroblasts and keratinocytes seeded onto fibrin-agarose hydrogels, maintained in epithelial culture medium and under air–liquid conditions. Subsequently, the oral mucosa layer was placed on the top of the osteogenic layer and compressed to induce nanostructured fusion of the mucosal

stroma (fibroblasts) and the osteogenic layer. The 3D multilayered graft was able to integrate with host tissue, and achieved partial bone differentiation; the authors suggested that complex multi-layer constructions could increase the maturation times compared to monolayers in vivo.

Bone-marrow stem cells (BMSCs) are the "gold standard" for several cell- and tissue-based clinical applications. These are, to date, the most studied stem cells and their properties are well known, but it is not clear whether the application of BMSCs or other MSCs in craniofacial bone regeneration requires handling ex vivo and/or pre-differentiation before clinical application (Shanbhag et al., 2019). For alveolar cleft repair in CL/P patients, scaffold-free BMSCs are safe, but this material is not suitable for extensive bone defect repair (Bajestan et al., 2017). The associations of BMSCs with commercial demineralized bone matrix (Osteoset DBM) (Behnia et al., 2009), tricalcium phosphate (Du et al., 2017), platelet-derived growth factor on biphasic hydroxyapatite/tricalcium phosphate (Behnia et al., 2012), or PRF membrane (Mossaad et al., 2019), have been shown to contribute to bone repair. However, the use of BMSC does not reduce the morbidity caused by iliac crest donor site handling even when using minimally invasive techniques. Thus, other sources of MSCs are necessary to eliminate this side effect.

The MSCs found in adult dental tissues display cranial NCC properties (Liu and Cheung, 2016; Niibe et al., 2017; Cui et al., 2018), as these embryonic cells are more similar to palate forming cells than BMSCs. In the oral cavity, human MSCs have been isolated and characterized from odontogenic and non-odontogenic origins, permitting the harvesting of healthy tissues during dental surgical procedures.  Recently, palatal periosteum-derived MSCs cells, cultivated under serum- and xeno-free conditions, and cells were able to retain stem cell properties (Naung et al., 2019).  However, these reports presented a limited source of tissue, and were assessed in specific situations, and therefore more effective cell sources are still required.

Five MSC populations are found in dental tissues: (I) in the dental follicle [dental follicle progenitor stem cells (DFPSCs)]; (II) in the apical papilla [stem cells from apical papilla (SCAPs)]; (III) in the ligament [periodontal ligament stem cells (PDLSCs)]; (IV) in the adult dental pulp [dental pulp stem cells (DPSCs)]; and (V) in the dental pulp of deciduous teeth [stem cells from exfoliated deciduous teeth (SHEDs)] (Baniebrahimi et al., 2018). Of these, SHEDs, which have exfoliative characteristics, are the most easily obtained odontogenic tissue, via a little or noninvasive procedure. The pulp tissue can be obtained during the period of the changing of the child's teeth, between 5 and 12 years of age, with insignificant ethical implications and provides a suitable alternative for pediatric regenerative medicine (Taguchi et al., 2019). SHEDs display high proliferative capacity, multi-lineage differentiation, secretion of immunomodulatory molecules. DPSCs, similarly to SHEDs, could be an alternative source of cells from teenagers or during adulthood during dental procedures being and are easily harvested from third molars routinely indicated to exodontia (Yamada et al., 2019). Both DPSCs and SHEDs allow cell sheets (Pedroni et al., 2018; Lee et al., 2019) and 3D spheroids cultures (Wang et al., 2010; Xiao et al., 2014). The high regenerative potential of SHEDs and DPSCs could be explained by its particular secretome content, including many types of paracrine soluble molecules and EVs, identified as immunogenic, pro-neurogenic, and proangiogenic (Kichenbrand et al., 2018; Marei and El Backly, 2018; Yusof et al., 2018). SHED secretome profile is also modulated during osteogenic differentiation leading to increase angiogenic potential (Mussano et al., 2018).

Concerning bone repair, SHEDs are better for forming new bone in a calvaria critical-size defect model, when compared to other dental MSCs and BMSCs. Recently, human SHEDs seeded onto dense collagen hydrogels, which were primed with FGF-2 and submitted to hypoxia conditions before implantation, improved intramembranous bone formation in an immunodeficient calvaria critical-size bone defect mouse model (Novais et al., 2019). Most of the 56 articles thoroughly analyzed in a systematic review reported good results and the relevance of human DPSCs for bone engineering in animal models or human clinical treatments (Leyendecker Junior et al., 2018). Similarly, a narrative review of 39 studies also concluded that DPSCs and SHEDs were of value for bone tissue regeneration (Cristaldi et al., 2018). Since 2005, many countries have started to collect and store healthy exfoliated teeth, creating biobanks, which are of low cost in comparison to umbilical cord banks (Campanella, 2018). Unfortunately, ex vivo manipulation of stem cells is still required, representing a challenge since this is a high cost and time-consuming procedure.

Little information is available regarding the use of MSCs harvested from CL/P patients. Bueno et al. (2011) isolated and characterized MSCs from orbicularis oris muscle (denominated by the authors as "cleft lip and palate muscle-derived stem cells"), usually discarded during the initial surgery repair (cheiloplasty) of CLP patients. These cells are able to express classical MSC cell surface proteins and differentiate into osteogenic, adipogenic, chondrogenic, and skeletal muscle cell lineages in vitro. The cells, when seeded onto collagen membranes, display the ability to repair bone in an immunocompromised rat critical-size cranial defect model. The analysis of DNA variants affecting the gene expression (cis-eQTLs) of cleft lip and palate muscle-derived stem cells allowed the discovery of a new susceptibility locus for NSCLP was discovered (rs1063588), coincident with the MRPL53 gene (Masotti et al., 2017). Bueno et al. (2011) compared the gene expression profile of SHEDs from healthy donors and CL/P patients and verified that 87 genes presented differential expressions, with more than a half being glycoproteins related to the ECM (collagens, MMPs, integrins, and adhesion proteins).

This study showed that MSCs might be a powerful tool for genetic and "omic" studies of CL/P. Later, the same group showed that low power laser therapy could enhance the osteogenic potential of DPSCs from CL/P patients (Pinheiro et al., 2017). These results support the need for the study of MSCs from CL/P patients to better understand cell behavior, ECM secretion, and remodeling, and employ this knowledge to drive new strategies based on bone bioengineering for palatal reconstruction.

Another growing field related to regenerative medicine that could be applied to palate reconstruction is bioprinting. Spheroids are potential building blocks in 3D bioprinting, in a large-scale process for bone and cartilage tissue production. Growing evidence shows that 3D spheroids formed from MSC present increased angiogenic and chemotactic signaling (Costa et al., 2017). 3D bioprinted cryogels, formed from chitosan (CS)/gelatin-based scaffolds for personalized palate reconstruction, have been designed by CL/P computed tomography data (Hixon et al., 2017). A 3D bioprinted bioresorbable scaffold (polycaprolactone—approved by the Korean Ministry of Food and Drug Safety for clinical use) seeded with autologous BMSCs from the iliac crest in the operation room, was implanted in a 10-year-old Korean boy with a history of previously repaired unilateral CL/P presenting a cleft alveolus and an oronasal fistula. At 6 months after transplantation, the new bone formed reached around 45% of the total defect volume, suggesting that this new technology could be a promising alternative (Ahn et al., 2018).

Three-dimensional additive manufacture allows the production of biomaterials/scaffolds used successfully for bone bioengineering have to be biocompatible, specific porosity, chemical and topographical characteristics and surface properties for osteoconduction, biomechanical properties, biodegradability, and radiolucency, to induce osteogenesis (osteoinductivity) and vascular ingrowth. In a new vision, biomaterials may trigger immunological host responses to stimulate tissue regeneration (Franz et al., 2011). As we have previously seen, the provisional matrix is secreted by embryonic palatal cells to drive palatogenesis. Among biopolymers used as biomaterials/scaffolds for bone bioengineering, collagen type I, CS, and HA are the most commonly employed. CS is a polysaccharide chitin-derived present in invertebrate exoskeletons which displays many promising characteristics such as biocompatibility, antibacterial activity, biodegradability, porosity, immunomodulatory properties, promotes cell adhesion, proliferation, migration, and ultimately enhances bone regeneration due to stimulation of osteoblast differentiation and mineralization (Farhadihosseinabadi et al., 2019).

As we have seen previously, LOX and TGs are crucial enzymes for collagen–collagen cross-links. These enzymes have been employed to create cross-links in collagen-based biomaterials (Fortunati et al., 2014; Cai et al., 2017). This modern approach can replace the traditional chemical method employing aldehydes, isocyanates, and carbodiimides. Although this method creates strong stable cross-links, undesirable consequences such as cytotoxicity, calcification, and foreign body response are well known for these chemicals (Sorushanova et al., 2019). Specifically in bone tissue, TG induces the oligomerization of SIBLINSs, and calcium-binding proteins (osteopontin and bone sialoprotein), which may drive mineral nucleation or calcium crystal growth (Forsprecher et al., 2009).

# CONCLUDING REMARKS

Although several MMPs and their inhibitors TIMPs are expressed during palatogenesis, the cell membrane-anchored MMPs appear to represent the principal pericellular collagenases (MMP-14 and MMP-16) and may be crucial for the development of CL/P. However, recent studies have demonstrated roles for other metalloproteinases, such as ADAMTS in this pathology. There seems to be a crucial compensatory effect between MMPs to ensure that the various stages of palate formation can occur. However, more studies should be carried out on other metalloproteinases to better understand the complexity of ECM remodeling, the generation of bioactive molecules, and the relationship between them.

Cleft therapy still is dependent on bone grafts, mainly of the ICBG, but new approaches have been under evaluation. Administration of BMP-2 and PDGF combined with bone graft is promising, but recent systematic reviews support the need for better-designed randomized controlled clinical trials with long-term follow-up (>12 months). The development of in vitro models using stem cells from CL/P patients may be an exciting approach for further studies of the biology of these cells as well as their potential use in new individualized therapeutic approaches for palatal reconstruction. 3D culture has been used to recapitulate the critical events of development, such as cell–cell interaction, differentiation, growth and cell fusion, which, coupled with "omics" analysis and computational biology, promote considerable advances in tissue remodeling and repair. Additionally, the emerging and exciting field of EVs may provide critical information via "omics" analyses to understand how this component of ECM contributes to the palatogenesis of CL/P.

The development of new biomaterials that simulate a provisional matrix during palatogenesis and provide the controlled release of growth factors may aid to improve new therapeutic approaches based on bone bioengineering. Emerging cell reprogramming or trans-differentiation technologies could provide other unusual sources of therapeutic cells. The former consists of differentiated adult cells, such as fibroblasts, that are forced to overexpress transcription factors that regulate pluripotency generating inducible pluripotent stem cells (iPSCs) displaying the same characteristics as embryonic stem cells (Spyrou et al., 2019). The second approach consists of the direct conversion of a fully differentiated cell type into another one; for example, the conversion of fibroblasts into osteoblasts (Cho and Ryoo, 2018). Furthermore, another possibility is to use EVs released by MSCs as potential cell-free tools for bone regeneration (Praveen Kumar et al., 2019). Finally, we now have a vast knowledge of craniofacial development, CL/P and CL/P treatment, associated with many advances in cell and material engineering technologies. So, multidisciplinary efforts must be made to achieve advances in the quality of life of CL/P patients.

# AUTHOR CONTRIBUTIONS

fcell-07-00340 December 12, 2019 Time: 15:1 # 21

KP contributed to the design, writing, and financial support of the manuscript. CM, PS, AL, and JG contributed to the design and writing of the manuscript.

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We are grateful for financial support from the São Paulo Research Foundation (FAPESP KBSP fellowship 2017/26813-9).

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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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# Unraveling the ECM-Immune Cell Crosstalk in Skin Diseases

Oindrila Bhattacharjee1,2†, Uttkarsh Ayyangar 1,2†, Ambika S. Kurbet 1,2†, Driti Ashok 2 and Srikala Raghavan2 \*

*1 School of Chemical and Biotechnology, Sastra University, Thanjavur, India, 2 Institute for Stem Cell Biology and Regenerative Medicine, GKVK Campus, Bangalore, India*

The extracellular matrix (ECM) is a complex network of proteins and proteoglycans secreted by keratinocytes, fibroblasts and immune cells. The function of the skin ECM has expanded from being a scaffold that provides structural integrity, to a more dynamic entity that is constantly remodeled to maintain tissue homeostasis. The ECM functions as ligands for cell surface receptors such as integrins, dystroglycans, and toll-like receptors (TLRs) and regulate cellular signaling and immune cell dynamics. The ECM also acts as a sink for growth factors and cytokines, providing critical cues during epithelial morphogenesis. Dysregulation in the organization and deposition of ECMs lead to a plethora of pathophysiological conditions that are exacerbated by aberrant ECM-immune cell interactions. In this review, we focus on the interplay between ECM and immune cells in the context of skin diseases and also discuss state of the art therapies that target the key molecular players involved.

#### Edited by:

*Charles D. Little, University of Kansas Medical Center, United States*

#### Reviewed by:

*Lidija Radenovic, University of Belgrade, Serbia Nagaraj Balasubramanian, Indian Institute of Science Education and Research, India*

#### \*Correspondence:

*Srikala Raghavan<EMAIL_ADDRESS>
*†These authors have contributed equally to this work and are co-first authors*

#### Specialty section:

*This article was submitted to Cell Adhesion and Migration, a section of the journal Frontiers in Cell and Developmental Biology*

> Received: *12 January 2019* Accepted: *09 April 2019* Published: *07 May 2019*

#### Citation:

*Bhattacharjee O, Ayyangar U, Kurbet AS, Ashok D and Raghavan S (2019) Unraveling the ECM-Immune Cell Crosstalk in Skin Diseases. Front. Cell Dev. Biol. 7:68. doi: 10.3389/fcell.2019.00068* Keywords: extracellular matrix, skin diseases, immune cells, crosstalk, therapeutics

# INTRODUCTION

Homeostasis in the skin, the largest organ of the body, is critical for maintaining its structural, protective and regulatory functions. Alterations in this homeostasis, as seen in major skin disorders such as Atopic Dermatitis (AD), Psoriasis, Epidermolysis Bullosa (EB), and skin cancer, lead to impaired skin hydration, thermoregulation, and increased susceptibility to infections (Mellerio, 2010; Romanovsky, 2014; Nyström and Bruckner-Tuderman, 2018). An important role in exacerbating the disease condition is played by dysregulated interactions between two important goal keepers of skin function, the immune cells and the extracellular matrix (ECM). Immune cells play a critical role in the breakdown, synthesis, and remodeling of ECM components that are important for physiological processes such as mammary gland remodeling, lung branching morphogenesis and wound healing (Allori et al., 2008; Rozario and DeSimone, 2010). ECM components such as laminin and collagens, on the other hand, act as ligands for immune cells and facilitate their adhesion and trafficking (Simon and Bromberg, 2017). Proteolytic enzymes secreted by the immune cells generate fragmented laminin and collagen peptides that can serve as chemotactic signals for the recruitment of immune cells (Bonnans et al., 2014). Moreover, ECM components and their physical properties can drive specific activation states of immune cells such as macrophages (McWhorter et al., 2015). In certain chronic disease conditions, excess ECM deposition results in fibrosis that can later develop into cancer (Bonnans et al., 2014). Therefore, maintaining a balance between remodeling by the immune system and ECM dynamics becomes critical to maintain homeostasis.

Skin diseases are largely incurable and therefore remain a major burden; globally posing an adverse socio-economic and psychological impact on the affected populations. The prevalence estimates of psoriasis can go as high as 1% in the U.S. to 8.5% in Norway (Parisi et al., 2013). AD affects up to one-fifth of the population in developed countries (Weidinger and Novak, 2016).  The prevalence of EB has risen to 11.07 per million live births globally and the numbers continue to rise (Fine, 2016). Commonly used treatment regimes for skin diseases include, the use of topical steroids and anti-inflammatory medications to control disease progression (Thomas et al., 2018; Yu et al., 2018). However, there is a paucity of curative therapies, which necessitates further understanding of the mechanisms that exacerbate the disease condition. Thus, focusing on the crosstalk between ECM and immune cells in skin diseases may help identify better targets for therapeutic interventions. In this review, we highlight the bidirectional interactions between ECM and immune cells in the major skin diseases and discuss the therapeutic strategies for dampening this "War of the Titans."

# THE SKIN AND ITS ECM

Mammalian skin comprises of a stratified epithelium known as the epidermis and a connective tissue layer termed the dermis. The epidermis is made up of specialized cells containing keratin intermediate filaments known as keratinocytes and is stratified into different layers, namely, basal (stratum basale), suprabasal (stratum spinosum), granular (stratum granulosum), and cornified (stratum corneum) layers (**Figure 1**) (Fuchs and Raghavan, 2002; Breitkreutz et al., 2013). The basal keratinocytes comprise a pool of proliferating stem cells, which undergo terminal differentiation and eventually lose their nuclei to form the outermost cornified layer (Blanpain and Fuchs, 2006). Each of these epidermal layers is characterized by the expression of different structural proteins. The basal keratinocytes express keratin 5 and 14, which are replaced by keratin 1 and 10 as they differentiate into the spinous layer. The granular layer, as the name suggests, contains keratohyalin granules made up of lipids and precursors of proteins such as loricrin, filaggrin, and involucrin. These proteins and lipids are covalently crosslinked by transglutaminase to form the outermost layer, the stratum corneum that is 10–30 cell layers thick. The stratum corneum is composed of flattened, and enucleated cells embedded in matrix that is rich in proteins and insoluble lipids (Candi et al., 2005). Mutations in any of the above mentioned genes can lead to skin diseases, some of which are discussed later in the review (Chandran, 2013). The dermis is a highly vascularized tissue comprising of fibroblasts, adipocytes and various pools of innate and adaptive immune cells (Mueller et al., 2014). It is divided into a thin upper papillary dermis with loosely arranged collagen fibrils and a thick lower reticular dermis with dense collagen packing. Furthermore, the dermal and hypodermal compartments of the skin also comprise of triglyceride rich adipocyte depot called the dermal white adipose tissue (DWAT) and subcutaneous white adipose tissue (SWAT), respectively (Driskell et al., 2014).

ECM components in the skin are present in the basement membrane and interstitial matrix (Frantz et al., 2010). The BM is a non-cellular array of structural ECM components that separates epidermis from dermis. It is composed of different subtypes of laminins and collagens, primarily laminin 5 (laminin-332) and collagen IV, which are non-covalently connected through perlecan and nidogen (Breitkreutz et al., 2013). This forms a highly dynamic fibrous structure that also functions as a diffusion barrier and serves as a repository of growth factors (Jayadev and Sherwood, 2017). The interstitial matrix is localized to the interstitial spaces in the dermal compartments. These primarily comprise of other collagen subtypes that also impart structural stability to the tissue (Bonnans et al., 2014). Additionally, there are non-structural ECM components called matricellular proteins that are synthesized by the epidermal and dermal cells during development and under non-homeostatic conditions. Matricellular proteins are involved in modulating cell function by interacting with structural ECM proteins as well as with various receptors, hormones and proteases. These interactions are a function of local availability of growth factors and overall composition of the ECM. Tenascin-C (TNC), thrombospondin (TSP), a secreted protein, acidic and rich in cysteine (SPARC), osteopontin (OPN), decorin, periostin (POSTN), and cysteine-rich protein 61 (CCN-1) are some of the major matricellular proteins (Bornstein, 2009). While basement membrane components are relatively stable, matricellular proteins undergo rapid turnover (Bonnans et al., 2014).

Organization of the BM components is mediated through transmembrane heterodimeric cell surface receptors called integrins that are expressed by the basal keratinocytes (Hynes, 2002; Andriani et al., 2003). Integrins are composed of α and β subunits. There are 18 α and 8 β subunits that form 24 different combinations. In the skin epidermis, α2β1, α3β1, and α6β4 are the main integrin pairs that are expressed (Watt and Fujiwara, 2011). Integrin β4 and β1 are components of multi-protein cellsubstratum junctional complexes known as hemidesmosomes and focal adhesions, respectively. These structures facilitate the adherence of basal keratinocytes to its underlying BM either via intermediate filaments (in hemidesmosomes) or via actin cytoskeleton network (in focal adhesions) (Tsuruta et al., 2011). ECM-integrin signaling at the BM is indispensable for several cellular processes such as cell proliferation, differentiation, adhesion, migration, and apoptosis (Hegde and Raghavan, 2013). Any disruptions in the ECM-integrin interactions lead to adhesion defects (Nelson and Larsen, 2015). Recent work from our laboratory uncovered the importance of ECM-integrin interaction in embryonic skin homeostasis. We showed that loss of integrin β1 in embryonic skin results in ECM degradation that is exacerbated through a sterile inflammatory response (Kurbet et al., 2016).

#### IMMUNOMODULATORY FUNCTIONS OF ECM

ECM molecules are potent regulators of immune cell function (**Table 1**). Alterations in the structure and composition of ECM in disease conditions provide immune cells with physical and biochemical cues that, in turn, affect their activation states. Several studies have shown that both enzymatically

digested ECM components as well as intact ECM proteins can modulate activation, fate determination, and chemotaxis of immune cells (Lu et al., 2011; Hallmann et al., 2015; Simon and Bromberg, 2017).

#### SKIN IMMUNE CELL REPERTOIRE AND ITS ROLE IN MAINTAINING ECM DYNAMICS

The skin contains a diverse population of immune cells that continuously surveil the organ to protect from assaults and maintain homeostasis. The epidermal and dermal compartments of the skin have distinct populations of immune cells that perform multiple functions including antigen cross-presentation to effector cells, tolerance induction, mediation of inflammation, promotion of growth and repair, regulation of stem cell quiescence, and maintenance of epidermal differentiation (Di Meglio et al., 2011). Langerhan cells and dendritic epidermal T cells are primarily located in the epidermis while, distinct populations of other dendritic cells and macrophages are present in the dermis (Heath and Carbone, 2013). Mast cells and T cell populations including αβ, γδT, Tregs, Th17, and memory T cells get recruited to both the epidermis and dermis depending on the homeostatic status of the skin (Matejuk, 2018). Details of the expression markers, location, and regulatory roles of immune cells in maintaining skin homeostasis are listed in **Table 2**.

#### TABLE 1 | Immune functions of ECM.

Immune cells are one of the most important regulators of ECM degradation, synthesis, assembly and remodeling. Mechanisms by which immune cells accomplish this include–(i) synthesis of enzymes that remodel ECM components, (ii) synthesis of cytokines and growth factors that induce ECM synthesis or degradation, and (iii) synthesis of ECM components.

(i) Synthesis of enzymes that remodel ECM components: Metalloproteinases are enzymes secreted by the immune cells that cleave ECM proteins leading to alterations in the physical and biochemical properties of the tissue. Degradation of ECM by metalloproteinases aids in the trafficking of immune cells, generates bioactive peptides, and releases the sequestered growth factors (Mott and Werb, 2004). Metalloproteinases include MMPs, (matrix metalloproteinases) adamlysins including ADAMS (a disintegrin and metalloproteinases), ADAMTs (ADAMs containing a thrombospondin motif), Meprins as well as serine proteases such as plasmins, granzymes, and cathepsins. These enzymes cleave collagens (MMP-1, -2, -3, -7, -8, -9, -10, -11, -12, -13, -14, -16, -19, -25, -26; ADAM-10, -12 and -15; Meprin a and b), elastins (MMP-2, -3, -7, -9, -10, -11, - 12), gelatins (MMP-2, -3, -7, -9, -12, -14, -15, -16, -17, -23A, - 23B, -24, -25; ADAM-9, -10, -12, -15), aggrecans (MMP-3, -13; ADAMTs), and laminins (MMP-3, -7, -11, -12, -14, -15, -19, Meprin α and β) (Bonnans et al., 2014). Several studies have highlighted the

#### TABLE 2 | Immune cell repertoire in skin.

*(Continued)*

TABLE 2 | Continued

important roles played by metalloproteinases, derived from immune cells, in remodeling the ECM thereby driving the pathophysiology and severity of various diseases. The T cell repertoire, particularly Th1 cells secrete gelatinases, MMP-2 and -9 that further stimulate the release of gelatinases from macrophages (Oviedo-Orta et al., 2008). In squamous cell carcinoma, mast cells, macrophages and neutrophils secrete MMP-9 that exacerbates the disease condition by increasing keratinocyte hyperproliferation and metastasis (Coussens et al., 2000). Dendritic and Langerhans cells in skin secrete MMP-2 and -9 to facilitate their trans-epithelial migration for antigen presentation (Ratzinger et al., 2002). Monocytes and lung resident macrophages are the primary source of MMP-8 that eventually contributes to fibrosis in idiopathic pulmonary fibrosis (Craig et al., 2014). Likewise, MMP-8 and -12 is synthesized by neutrophils and macrophages in cystic fibrosis accelerating disease progression (Wagner et al., 2016). Increased expression of MMP-9 by splenic and infiltrating T cells correlates with the growth in mammary tumors (Owen et al., 2003). Furthermore, in UVB irradiated skin, neutrophil elastase causes the degradation of ECM components by activating MMP-1, and MMP-2 (Takeuchi et al., 2010).

(ii) Synthesis of cytokines and growth factors that induce ECM synthesis or degradation: Cytokines (IL-4, IL-13, and IL-33) and growth factors (TGF-β) secreted by immune cells stimulate the synthesis of ECM components (Verrecchia et al., 2001; Fujitsu et al., 2003; Rankin et al., 2010). In mouse dermal fibroblasts, TGF-β signaling results in the activation of SMAD2/3, which, in turn, increases the expression of ECM, related genes such as Col1a1 and Col3a1 (Verrecchia et al., 2001; Li et al., 2003). IL-13 produced by innate lymphoid cells stimulates the differentiation of fibroblasts to myofibroblasts increasing collagen synthesis and its accumulation in fibrosis (Fichtner-Feigl et al., 2006). In bronchial asthma, Th2 cytokines IL-4 and IL-13 stimulate the synthesis of periostin from bronchial fibrocytes, leading to sub-epithelial fibrosis (Takayama et al., 2006; Aoudjehane et al., 2008). On the other hand, IL-6 and TNF-α produced by inflammatory immune cells can reduce the synthesis of MMP-2, which, in turn, protects liver hepatocytes from fibrosis (Bansal et al., 2005).

(iii) Synthesis of ECM components: Immune cells can themselves be a source for various ECM proteins. Macrophages and T cells present in tuberculosis associated granulomas produce OPN that aid in their chemotaxis, adhesion, and proliferation (O'regan et al., 1999). In a subset of patients with systemic stenosis, circulating CD14+ monocytes overexpress versican, which is associated with the aggressive fibrosis (Masuda et al., 2013). TAMs (tumor associated åmacrophages) modify the architecture of the tumor matrix by synthesizing proteoglycans, fibronectin, OPN, SPARC and various collagen subtypes (Liguori et al., 2011).

Taken together, these studies point to a critical role played by immune cells in modulating ECM dynamics during normal development and in disease states.

# SKIN DISEASES

#### Atopic Dermatitis

Atopic dermatitis (AD) is a chronic, relapsing and TH2 cell/IgE driven inflammatory skin disorder characterized by intense pruritus and eczematous lesions (Bieber, 2008). The onset of AD is caused by barrier dysfunction, due to heritable mutations in the filaggrin (FLG) gene, and environmental triggers culminating in loss of skin hydration, enhanced sensitization to allergens and susceptibility to pathogenic agents (Weidinger and Novak, 2016). Clinical manifestations of AD occur both in children (Childhood AD) and adults (Adult AD) with similar clinical symptoms but with discrete pathophysiologies. While childhood AD is characterized by additional Th17/Th22 driven cascades along with tight junction and lipid metabolism disorders, AD in adults is associated with a Th1 specific response with aberrations in the epidermal differentiation complex (Brunner et al., 2018). AD usually precedes other allergic disorders like food allergies, allergic rhinitis, and asthma, commonly referred to as the "Atopic March," the mechanism for which remains undefined (Dharmage et al., 2014).

Abnormalities in BM composition, suggesting ECM dysfunction, have been reported in AD. Affected skin in AD patients has decreased BM thickness, along with reduced expression of collagen-IV and integrin α6 in the BM zone (Kim et al., 2018). A report by Shin et al. (2015) also suggested that the loss of the regenerative capability of the skin correlated with lower expression of p63 protein, a putative stem cell marker, in AD affected skin (Shin et al., 2015). Several SNPs (single nucleotide polymorphisms) in LAMA3 gene correlated with increased susceptibility to AD (Stemmler et al., 2014). In acute AD, an increase in the expression of hyaluronan (HA), an extracellular polysaccharide, and hyaluronan synthase 3 (HAS3), an epidermal specific enzyme responsible for the synthesis and extracellular transport of hyaluronan, is observed (Ohtani et al., 2009). Increased expression of HA has been associated with abnormal keratinocyte differentiation, a hallmark of AD (Malaisse et al., 2014).

IL-4 and IL-13 are important cytokines known to play a critical role in AD pathogenesis. In situ hybridization studies on skin biopsy samples show a greater number of IL-13 positive cells in asymptomatic, acute and chronically affected AD patients compared to unaffected individuals (Hamid et al., 1996). Optimum expression of IL-13 is critical to maintain epidermal barrier homeostasis since both excess and insufficient levels of IL-13 provoke epidermal barrier dysfunction (Strid et al., 2016). IL-4 and IL-13 downregulate the expression of filaggrin, involucrin, loricrin, and the production of antimicrobial peptides. This exacerbates the skin barrier dysfunction and predisposes ADaffected skin to infection by microbes (Howell et al., 2007; Kim et al., 2008; Kisich et al., 2008). IL-4 is also shown to repress the expression of fibronectin in immortalized human keratinocytes (Serezani et al., 2017). In a human skin equivalent model system, IL-13 expression leads to the loss of BM structure and regenerative capability of the skin, recapitulating the AD phenotype (Shin et al., 2015). Furthermore, IL-13 treated human keratinocytes attract CD4+ CCR4+ T cells in vitro, providing a mechanism for a feed-forward loop that recruits more Th2 cells to acute AD lesions (Purwar et al., 2006). A critical correlation exists between IL-13 and MMP-9 mediated BM protein remodeling in AD. IL-13 is co-expressed with MMP-9 in skin samples from atopic eczema patients and it also increases MMP-9 synthesis in cultured keratinocytes (Purwar et al., 2008). IL-4 and IL-13 treated keratinocytes show an overexpression of TNC aggravating the inflammatory response (Ogawa et al., 2005). While mast cells and CD4+/CD8+ T cells are well known sources of IL-13 in AD affected skin, other immune cells including macrophages and dendritic cells can also be potential sources, which warrants further investigation (Obara et al., 2002). Taken together, these studies highlight the critical role played by IL-4 and IL-13 in driving AD pathogenesis.

Immune cell mediated remodeling of ECM in AD has been widely reported. BM remodeling matrix metalloproteinases (MMPs) including MMP-1, -3, -7, -8, and -9 is involved in AD pathophysiology (Harper et al., 2010). AD patients have enhanced serum levels of MMP-8 and -9 (Devillers et al., 2007). An immediate early effect in increase of MMP-9 by langerhans cells is observed upon treatment of skin with hapten, a well-known model for contact hypersensitivity (Kobayashi, 1997). The hyperplasic epidermis in AD secretes thymic stromal lymphopoeitin (TSLP), an IL-2 family cytokine, that activate DCs. DCs, in turn, produce MMP-3 and -9 that aid in their migration to lymph nodes where they activate naïve T cells (Ratzinger et al., 2002). Additionally, degranulation product of epidermal mast cells in AD-affected skin is associated with increased expression of MMP-7 (Landriscina et al., 2015). These reports indicate an indirect immune cell mediated BM remodeling in the context of AD. However, focused studies addressing this crosstalk are limited.

Monocytes and macrophages also play an important role in AD pathogenesis. An insightful review on the role of macrophages in AD has been previously published (Kasraie and Werfel, 2013). In AD skin, the epidermis overexpresses monocyte chemoattractant protein 1 (MCP-1) and, this results in the recruitment of monocytes in a CCR-2 dependent manner (Vestergaard et al., 2004). Acute and chronically inflamed AD skin show enhanced infiltration of heterogeneous pools of macrophages (Kiekens et al., 2001). Immunohistochemistry and ELISA analysis of AD skin and peripheral blood show increased CD163 expressing macrophages. Their role in aggravating disease condition is suggested by in vitro studies where the macrophages show an impaired TLR-2 signaling and increased secretion of pro-inflammatory cytokines such as IL-1β, IL-8, and IL-6 (Niebuhr et al., 2009). Given the preponderance of evidence on ECM-macrophage crosstalk in diseases, it is tempting to speculate that these heterogeneous pools of macrophages may play an important role in ECM remodeling.

Interestingly, recent studies point to the importance of ECM driven cascades in AD pathogenesis. Mitamura et al., showed that IL-13 upregulates the synthesis of POSTN which, in turn, increases IL-24 expression in the epidermal keratinocytes. IL-24, in turn, downregulates the expression of filaggrin, that contributes to the barrier dysfunction seen in atopic dermatitis (Mitamura et al., 2018). Another study showed that, the binding of TNF superfamily member 14 (TNFSF-14) to its receptor HVEM (herpes virus entry mediator) on keratinocytes resulted in the synthesis of POSTN and development of AD. Blocking this pathway by HVEM deletion and HVEM specific antibody resulted in suppression in AD symptoms (Herro et al., 2018). The importance of OPN in AD is highlighted in several studies. Its expression is elevated in the serum of patients with allergic contact dermatitis (Reduta et al., 2015). In disease conditions, immune cells such as T cells, NKT cells, and plasmacytoid dendritic cells synthesize OPN that acts as a bridging molecule between the innate and adaptive immune system (Clemente et al., 2016). OPN promotes a pro-inflammatory environment by enhancing production of IL-12 by macrophages and suppressing IL-27 production from conventional dendritic cells, thereby activating Th1 and Th17 cells (Clemente et al., 2016). OPN binds to CD44 receptors on mast cells leading to its recruitment and degranulation (Bulfone-Paus and Paus, 2008). Thus, studies on matricellular proteins POSTN and OPN have highlighted therapeutically targetable pathways that are the key drivers of AD symptoms.

#### Psoriasis

Psoriasis is a skin disorder associated with pathological changes such as appearance of erythematous lesions, scaling of the epidermis, inflammation, and vascular abnormalities (Nestle et al., 2009). Lesions appear as red and scaly plaques in areas like the elbow, joints, lower back, and the scalp (Boehncke and Schön, 2015). It is classified into plaque, guttate, erythrodermic, and pustular types with plaque psoriasis (or psoriasis vulgaris) being the most common type associated with about 90% of the cases (Dubois Declercq and Pouliot, 2013). Based on the age of onset of the disease, psoriasis is classified as Type I (occurs in individuals below 40 years of age) and Type II (sets in after 40 years of age) (Langley et al., 2005). Type I is more common, runs in families and is associated with more severe phenotypes compared to Type II (Schmitt-Egenolf et al., 1991). The exact cause of this disease is still unknown. However, a combination of genetic and environmental factors such as infection, injury, and various administered drugs may be responsible for disease onset (Langley et al., 2005).

Cell death due to injury or trauma is thought to trigger the keratinocytes to produce antimicrobial peptides like LL-37 that activate plasmacytoid dendritic cells (pDCs) (Morizane and Gallo, 2012). The pDCs secrete IL-12 and IL-23 and mount a T cell mediated inflammatory response. The T cells, primarily Th1, Th17, and Th22, secrete the cytokines IFNγ and TNF that promote inflammation in psoriatic skin. An increase in the infiltration of neutrophils, dendritic cells and T cells in the lesional skin of psoriatic patients evokes a crosstalk between the activated keratinocytes and immune cells (Baliwag et al., 2015; Albanesi et al., 2018). Although, T cells and dendritic cells primarily drive psoriasis, interesting roles for macrophages and neutrophils have recently emerged. Depletion of macrophages by clodronate treatment leads to alleviation of the psoriatic phenotypes in mice (Stratis et al., 2006). Neutrophil derived cytokines such as IL-1β, IL-6, IL-17, and IL-23 are associated with specific gene expression changes in keratinocytes that result in epidermal hyperproliferation and abnormal differentiation (Terui et al., 2000; Tecchio et al., 2014). In contrast, antibody mediated neutralization of neutrophils does not reduce inflammation. Interestingly, neutrophils in psoriatic skin form web-like extracellular traps, referred to as NET (Neutrophil extracellular trap) that is composed of chromatin and antimicrobial components such as LL-37, neutrophil elastase, and myeloperoxidase (Fuchs, 2007). The formation of NET is associated with NETosis, which involves killing of pathogens through the release of IL-17 and antimicrobial peptides (Hu et al., 2016).

Several studies have underscored the contribution of ECM proteins in the pathogenesis of psoriasis. A disorganized BM (laminin) in the psoriatic skin is associated with keratinocytederived plasminogen activating factor (McFadden et al., 2012). Laminin-332 and -511 are overexpressed in the BM of psoriatic skin lesions. Furthermore increased expression of laminin-511 in HaCat cells stimulated proliferation and inhibited apoptosis providing a mechanism for the observed epidermal hyperplasia (Natsumi et al., 2018). Additionally, alteration in laminin α1 chain along with overexpression of fibronectin in the papillary dermis and T cell lymphokines are correlated with abnormal cellular morphology (Vaccaro et al., 2002). While laminins are shown to facilitate neutrophil recruitment and enhance the phagocytic ability of dendritic cells, its role in immunomodulation in the context of psoriasis remains elusive (Wondimu et al., 2004; García-Nieto et al., 2010; Simon and Bromberg, 2017).

CCN-1, a matricellular protein is overexpressed in the lesional and non-lesional psoriatic epidermis in response to proinflammatory cytokines IL-17 and TNF-α. It binds to integrin α6β1 on keratinocytes and activates PI3K/Akt/NF-κB pathway and result in the upregulation of ICAM, HLA-DR, and HLA-ABC genes. These genes, in turn, increase the immune cellkeratinocyte interaction and mount an inflammatory response. Its role in promoting epidermal hyperplasia and inflammation was further demonstrated in IL-23 induced psoriatic skin lesions, where the knockdown of CCN-1 ameliorated the phenotypes (Sun et al., 2015). Other studies have shown that CCN-1 stimulates secretion of the cytokines IL-1β and IL-8 by psoriatic keratinocytes via the p38 MAPK and JNK/NF-κB signaling pathways, respectively underscoring its role in regulation of inflammation (Sun et al., 2017; Wu et al., 2017). Furthermore, CCN-1 promotes CCL-20 chemokine synthesis from hyperplasic epidermis of psoriatic skin that causes increased recruitment of immune cells (Li H. et al., 2017). Taken together, these studies highlight an important role played by CCN-1 in regulation inflammation in psoriatic skin.

OPN, expressed by epithelial cells, is shown to act as a potent pro-inflammatory stimulant and activates immune cells such as T-cells, dendritic cells, and macrophages/monocytes (Clemente et al., 2016). Interaction of OPN with integrin and CD44 leads to the promotion of Th1 response and suppression of Th2 response. In psoriasis, OPN is highly expressed in the PBMCs (peripheral blood mononuclear cell), skin and plasma. Buommino et al showed that OPN secreted by PBMCs enhances the expression pro-inflammatory cytokines such as IL-1β, TNF-α, and IFN-γ. TNF-α upregulates OPN expression while anti-TNF-α antibody treatment abrogates its synthesis (Buommino et al., 2012).

Overall, these studies suggest that ECM components play a critical role in psoriatic skin by acting as co-stimulatory factors for keratinocytes and immune cell activation leading to chronic progression of the disease.

#### Epidermolysis Bullosa

Epidermolysis bullosa (EB) is a group of rare heritable diseases characterized by blistering and fragile skin. It is associated with anomalies such as palmar-plantar thickening, syndactyly, dysphagia, gingival hyperkeratosis, cardiomyopathy, and osteoporosis (Maldonado-Colin et al., 2017). The blistering results from mechanical stress that eventually gives rise to chronic wound conditions (Medeiros and Riet-Correa, 2015). EB is caused by mutations in hemidesmosomal and extracellular matrix genes and the mode of inheritance can be either dominant or recessive. EB has been broadly categorized into simplex (EBS), junctional (JEB), dystrophic (DEB) types, and kindler syndrome (Shinkuma, 2015; Barna et al., 2017). The severity of disease is a function of the location of the split and the genes affected. So far mutations in at least 19 genes are reported to cause different types of EBs (Vahidnezhad et al., 2018). The site of splitting associated with various mutations is illustrated in **Figure 2**.

**EBS** is caused by mutations in the genes coding for either keratin 5 (KRT5) or keratin 14 (KRT14) accounting for the four major subtypes. KRT5/14 form a stable cytoskeletal network and are involved in maintaining tissue integrity (Coulombe and Lee, 2012). Mutations in KRT5/14 result in rupture of basal keratinocytes leading to increased susceptibility to infection and dehydration. The least severe form of EBS is Weber-Cockayne type characterized by mild blistering restricted to feet and hands whereas the most severe form known as Dowling-Maera type is marked by extensive blistering, which however seems to get better with age. The other two subtypes are Koebner type and EBS with mottled pigmentation (Chung and Uitto, 2010a). Additionally, mutation in the plectin (PLEC) gene, a hemidesmosomal protein, is associated with a rare type of EBS known as Ogna type (Pfendner et al., 2005). The features common to all the subtypes are thickened palm and soles (palmoplantar keratoderma), nonscarring blisters, alopecia and dystrophic nails (Chung and Uitto, 2010a). EBS is the most prevalent and mildest type of EB, and exhibits an autosomal dominant inheritance pattern (Coulombe et al., 2009). Mouse knockouts for Krt5 and Krt14 serve as good models for studying EBS disease pathogenesis (Coulombe et al., 1991; Peters et al., 2001).

**JEB** is an autosomal recessive form of EB caused by mutations in the laminin-332 chains encoded by LAMA3, LAMB3, LAMC2 genes, and COL17A1 genes that code for collagen XVII/BP180 protein (Sawamura et al., 2010). Collagen-XVII is an important structural element of the hemidesmosomal complex that together with laminins in the BM, maintain the dermal-epidermal adhesion. JEB can be further classified into two subtypes; Herlitz and non-Herlitz based on its severity. Patients with the Non-Herlitz subtype display blisters primarily in the feet, elbows and knees, which are non-life threatening (Yancey and Hintner, 2010). The Herlitz subtype, on the other hand, causes severe disease, and is associated with widespread blistering of the lining of oral and digestive tracts, syndactyly and alopecia, and restricts life expectancy to 1 year after birth (Yuen et al., 2011).

Col17a1 knockout mice display the blistering pattern associated with the non-Herlitz subtype and show prolonged survival; whereas Lama3 and Lamc2 knockouts recapitulate the Herlitz type of EB, exhibiting severe skin blistering resulting in perinatal lethality (Bubier et al., 2010; Natsuga et al., 2010). Two other rare autosomal recessive forms of EB include EB with pyloric atresia (EB-PA) and EB with muscular dystrophy (EB-MD). EB-PA is caused by mutations in ITGA6 and ITGB4 genes whereas EB-MD is associated with mutations in the PLEC gene (Shimizu et al., 1999; Yancey and Hintner, 2010). Itga6 and Itgb4 knockout mouse models recapitulate the dermal-epidermal detachment leading to development of EB-PA (Georges-Labouesse et al., 1996; Mencía et al., 2016).

**DEB** is associated with mutations in Col7a1 gene resulting in the loss of collagen-VII protein. Collagen-VII is an anchoring fibril protein crucial for dermal-epidermal integrity and is present in the BM on the side of papillary dermis (Chung and Uitto, 2010b). The two major subtypes of DEB based on mode of inheritance and severity include autosomal dominant (DDEB) and recessive (RDEB) types. Unlike EBS and JEB, DEB is associated with extensive scarring (Shinkuma, 2015). RDEB, the more severe subtype is characterized by pseudosyndactyly, alopecia with scarring, and esophageal stenosis that leads to dysphagia. This disease is further complicated by the development of aggressive squamous cell carcinoma in juvenile patients (Shinkuma, 2015). The conditional Col7a1 KO mice recapitulate all of the phenotypes of RDEB and has proven to be an invaluable tool to understand disease progression as well as to test therapeutic interventions (Natsuga et al., 2010).

**Kindler syndrome** is the only skin disease known to be associated with actin cytoskeleton related abnormalities and is characterized by variable planes of cleavage in the skin (Medeiros and Riet-Correa, 2015). Keratinocytes express kindlin-1, a protein that regulates cellular processes such as proliferation, migration and cell-substratum adhesion (Rognoni et al., 2016). Mutations in the FERMT1 gene, coding for the kindlin-1 protein, lead to this rare fragile skin blistering disorder with symptoms similar to other EB subtypes (Lai-Cheong et al., 2009). Kindlin-1 deficient mice display skin atrophy without blister formation (Ussar et al., 2008). The loss of kindlin-1 from keratinocytes results in dysregulated integrin signaling and focal adhesion turnover (Margadant et al., 2013).

Another form of EB called aquisita **(EBA)** is caused by the production of autoantibodies against collagen-VII protein. The two subtypes of EBA are classical and the inflammatory types. The classical form is characterized by trauma induced blistering in adults whereas the inflammatory form starts in childhood and show more widespread blistering that is not restricted to the injured sites (Kasperkiewicz et al., 2016).

The null and conditional KO mouse models that have been discussed above recapitulate various aspects of disease progression of the EB class of skin disorders and are being used as platforms to develop therapeutic interventions for these devastating diseases (Georges-Labouesse et al., 1996; Cao et al., 2001; Peters et al., 2001; Bruckner-Tuderman et al., 2010).

Insights into the role of inflammation and immune cells in EB pathophysiology have come from the studies on Krt5, Itgb4, and Col7a1 knockouts that cause EBS, EB-PA, and DEB, respectively. Increased expression of chemokines and cytokines such as CCL-2, CCL-19, CCL-20, IL-6, and IL-1β is reported in Krt5 knockout skin. The cytokine expression is associated with a 2-fold increase in the recruitment of Langerhans cells to the EB affected skin (Lu et al., 2007; Roth et al., 2009). Loss of Itgb4 in mice leads to severe blistering associated with Th2 inflammatory response, which suggests involvement of immune cells in EB pathophysiology (Han et al., 2018). Knockout of Col7a1 recapitulates RDEB and creates a chronic wound condition underscoring its role in maintenance of epidermal-dermal integrity. Collagen-VII deficient wounds display suprabasal expression of α6β4 that lead to a dysregulated α6β4-laminin-332-JNK-STAT3. This, in turn, delays wound re-epithelialization and prolongs the presence of inflammatory CD11b cells in skin (Nyström et al., 2013). Interestingly, depletion of collagen-VII from immunodeficient mice (NSG mice) results in a less severe disease phenotype. Taken together, these studies emphasize the critical role played by the immune cells in exacerbating the disease condition (Esposito et al., 2016).

Blisters in the bullous diseases are usually associated with increased infiltration of cytotoxic T cells, B cells, neutrophils and other immune cells as well as their secreted cytokines (Hussein et al., 2007). Previous work has suggested that there is a substantial increase in the levels of cytokines such as IL-1, IL-6, IL-15, and TNF-α, and chemokines such as CXCL-1, CXCL-2, CXCL-3, CCL-5, CCL-27 in the blister fluids of EB patients compared to healthy controls. Moreover, the cytokine and chemokine expression profile varies between EB subtypes such that early DEB and JEB blisters expressed lower levels of cytokines and chemokines compared to that of early EBS (Alexeev et al., 2017). In EBA, several groups have shown that autoantibody production against COL7A1 is mediated through a milieu of neutrophils, macrophages, B cells and T cells and these phenotypes are ameliorated by the depletion of neutrophils and T cells (Sitaru et al., 2010; Iwata et al., 2013; Bieber et al., 2016).

MMPs secreted by immune cells can also influence the recruitment pattern of other immune cells to the injured tissue. Studies have shown that the severity of RDEB is a function of the type of MMP synthesized, its expression level and the balance between MMPs and TIMPs (Bodemer et al., 2003). Interestingly, a SNP (single nucleotide polymorphism) in the MMP1 gene has also been implicated in increasing disease severity by influencing the expression of collagen-VII (Titeux et al., 2008). Likewise the correlation between MMP-9 and blister formation was shown in a study where treatment with doxycycline derivative, an inhibitor of MMP-9, alleviated the phenotype (Lettner et al., 2013). Collectively MMPs can serve as effective therapeutic targets.

Since DEB is a disorder of the basement membrane zone (BMZ), it is not surprising that, in RDEB patients that lack collagen-VII, two major BM components collage-IV and laminin-332 display a disrupted distribution pattern and do not co-localize at the BMZ (Onetti Muda et al., 1996). The disruption precedes blister formation suggesting that it could be a direct consequence of the lack of collagen-VII. Dermalepidermal splitting also leads to an increase in the expression of TNC at the site of the split, which could be in response to the cytokines secreted either by epidermis or the recruited immune cells (Schenk et al., 1995). These studies suggest that TNC can serve as a potential link between the immune and ECM components in DEB.

Interaction between collagen-VII and cochlin is required to mount an immune cell mediated antibacterial response. In RDEB patients, absence of this interaction leads to increased bacterial colonization that further exacerbates the disease condition (Nyström et al., 2018). Lack of collagen-VII in RDEB destabilize the BMZ and cause the release of TGF-β associated with ECM increasing its bioavailability. TGF-β mediated activation of dermal fibroblasts, in turn, cause excessive deposition of ECM components (Nyström and Bruckner-Tuderman, 2018). In a recent case study, monozygotic twins suffering from RDEB exhibited distinct phenotypes due to changes in the expression of TGF-β target genes, despite having similar loss of collagen-VII. The more affected individual displayed increased fibrosis, which was ameliorated by treatment with the anti-fibrotic the matricellular protein decorin (Odorisio et al., 2014). These data point to the complexity of managing and treating RDEB, a genetic disease that is associated with loss in ECM organization which is further exacerbated by TGF-β mediated alteration in ECM dynamics. The involvement of matricellular proteins in mounting an immune response in EB provides an additional avenue for therapeutic intervention. Taken together, while EBs remain one of the most challenging diseases to manage and treat, and future treatment paradigms will need to take into account the immune-ECM interactions.

#### Skin Cancers

Skin cancers are broadly classified as melanoma and nonmelanoma types. Melanoma is an aggressive skin cancer that originates from melanocytes; the pigment producing cells in the skin (Villanueva and Herlyn, 2008). Non-melanoma skin cancers include Squamous cell carcinoma (SCC), Basal cell carcinoma (BCC), and Merkel cell carcinoma (MCC). SCCs originate from interfollicular stem or progenitor cell population in the epidermis and BCCs from the progenitor cells present in interfollicular epidermis and upper infundibulum (Donovan, 2009; Wang et al., 2011). MCCs, on the other hand, are the cancer of neuroendocrine (merkel) cells present in the basal layer of the epidermis (Munde et al., 2013). Other aggressive forms of skin cancer include cutaneous T and B lymphomas, a varied group of extranodal lymphoproliferative disorders associated with abnormal accumulation and proliferation of T and B lymphocytes in skin (Pandolfino et al., 2000; Siegel et al., 2012). Cutaneous T cell lymphomas are the most common with 65% incidence rate as compared to B cell lymphomas with a 25% incidence rate (Sokołowska-Wojdyło et al., 2015). All skin cancer types show a positive correlation with exposure to UV radiation as well as mutations in genes associated with tumor suppression, DNA repair, apoptosis and cell cycle regulation (D'Orazio et al., 2013; Kandoth et al., 2013).

Tumor resident and recruited immune cells play a dynamic role in regulating cancer progression. The tumor and its stroma recruit different populations of immune cells by releasing chemotactic factors and cytokines resulting in a chronic inflammatory condition (Binnewies et al., 2018). The immune cell repertoire, associated with the tumor microenvironment includes monocytes, macrophages, neutrophils, dendritic cells, natural killer cells, T and B cells (Cavallo et al., 2011). Factors such as CCL-2, CCL-5, CCL-17, CCL-22, CXCL-12, M-CSF, VEGF, and TGF-β synthesized by the tumor stroma aid in the recruitment of monocytes and subsets of T cells (Owen et al., 2003; Hiratsuka et al., 2006; Yang et al., 2008). In melanomas, the chemokines CCL-2, CCL-3, CCL-5, CXCL-9, and CXCL-10 recruit CD8+ T cells (Harlin et al., 2009). In a recent study it was shown that, tumor derived factors such as colony stimulating factor-1 (CSF-1), CCL-2, IL-34, and VEGF recruit circulating macrophages to the melanomas (Pieniazek et al., 2018). In human melanomas, CCL-2, also known as MCP-1, is associated with an increased inflammatory response and recruitment of macrophages and T cells (Ilkovitch and Lopez, 2008). Similarly in BCCs, CXCL-9, CXCL-10, and CXCL-11 recruit CXCR-3 expressing effector and memory T cells (Lo et al., 2011). In BCCs, the chemokines, CCL-17, CCL-18, and CCL-22 that recruit Tregs are overexpressed in the tumor islands and peritumoral skin. The stromal cells in turn synthesize CCL-17, which suggests a mechanism for the Treg infiltration seen in the stroma surrounding the BCC nests (Omland et al., 2016). In MCC, a recent study has shown that over expression of CCL-17 and its receptor CCR-4, aid in recruitment of CD4+ Tregs, Th2, and Th17 cells (Rasheed et al., 2018). Macrophage derived CCL-18 in cutaneous T cell lymphomas (CTCL) recruit T lymphocytes to the skin and is associated with enhanced invasion (Wu et al., 2014). Overall, the tumor and its stroma recruit a plethora of immune cells that contributes to tumor progression.

Dynamic crosstalk between the stroma and immune cells, change the tumor milieu from a tumor suppressive (or immunogenic) to tumorigenic (or immunosuppressive) state. Specifically, tumor associated macrophages (TAMs) switch from pro-inflammatory M1 macrophages, that are antigen presenting cells, to anti-inflammatory M2 macrophages, that are tissue remodeling and tumor growth promoting cells (Tariq et al., 2017). M1 macrophages phagocytose tumor cells, produce reactive oxygen (ROS) and nitrogen (RNS) species and secrete pro-inflammatory cytokines such as IL-6, IL-23, IL-12, and TNF-α that activate Th1 response (Komohara et al., 2014; Noy and Pollard, 2014). In contrast, M2 macrophages express anti-inflammatory cytokines like IL-10 and TGF-β that play a role in promoting tumor growth (Aras and Raza Zaidi, 2017). Depletion of M2 macrophages by treating with clodronate in CTCL xenograft mouse models, reduces angiogenesis and tumor growth (Wu et al., 2014). Human keratinocytes can inactivate T cell response by downregulating signaling pathways critical

for T cell recruitment and cytotoxicity (Bronte and Zanovello, 2005; Bluth et al., 2009). In SCCs and BCCs, immunosuppression is mediated by preventing the recruitment of T cells to the cancer site through the activation of EGFR-Ras-MAPK signaling pathway (Pivarcsi et al., 2007). Interestingly, Tregs are known to suppress immune response by producing IL-10 and TGF-β and compete for T-cells-activating pro-inflammatory cytokines (Jarnicki et al., 2006). They are critical for inducing tolerance to self-antigens and also inhibit the antigen presenting ability of DCs (Sakaguchi et al., 2009). The transcription factor FOXP3 is important in regulating Treg function. Increased FOXP3 expressing Tregs correlates with poor disease outcome in SCCs, BCCs and melanomas (Moreira et al., 2010). In BCCs, recruited Tregs build an immunosuppressive niche by locally synthesizing TGF-β (Omland et al., 2016). In MCC, M2 macrophages and Tregs present in the tumor maintain an immunosuppressive state contributing to a pro-tumorigenic microenvironment (Gaiser et al., 2018). Interestingly, increased recruitment of CD8+ T cells to the skin is associated with better prognosis in MCCs (Touzé et al., 2011).

Taken together, there is growing evidence that tumor microenvironment in cancers develop immune suppressive mechanisms to evade the T cell mediated cytotoxicity that can promote metastasis.

The ECM in cancer is highly remodeled and disorganized due to aberrant deposition by activated fibroblasts and enzymatic remodeling by the tumor and immune cells (Karagiannis et al., 2012). Changes in ECM dynamics promote tumor growth by regulating proliferation, survival, adhesion and migration (Pickup et al., 2014). The BM in melanomas has reduced thickness compared to healthy controls due to the absence of collagen-VII (Schmoeckel et al., 1989). A study of the BM in BCC affected skin showed aberrations in bullous pemphigoid antigens possibly due to its abnormal synthesis by the tumor associated cells (Stanley et al., 1982). Similarly, the BM structure in aggressive SCCs is irregular and discontinuous (Sakr et al., 1987). Matrix metalloproteinases such as MMP-1, -2, -3, -8, -9, -10, -13, -15, -16, and -26 can facilitate tumor growth, invasion, and angiogenesis in BCC, SCC and melanomas (Pittayapruek et al., 2016). In a recent study, expression profiling of BCCs showed an increase in ECM remodeling proteins including MMP-1, -3, -8, -9, and Cathepsin-K indicating the important role played by these enzymes in cancer progression (Ciazynska et al., 2018). In organ culture models of BCC the increased expression of MMP-2 and MMP-9 correlates with degradation of collagen-I and -IV at the tumor front (Gozdzialska et al., ´ 2016). Likewise, the expression of ADAM-10, -12, and -17 was specifically observed in deep invasion area of BCC biopsies and correlated with the loss of collagen-XVII (Oh et al., 2009). The TAMs in BCCs induce COX-2 expression, which, in turn, results in a COX-2 dependent release of MMP-9, VEGF, and FGF that promotes tumor invasion (Tjiu et al., 2009). Interestingly, MMP-9 producing inflammatory macrophages and neutrophils are associated with BM degradation in BCC patients (Boyd et al., 2008).

Similarly, in SCCs the γ2 chain of laminin-332 is overexpressed in the invasive front (Hamasaki et al., 2011). In a DMBA (7,12-dimethylbenz-anthracene)-TPA (12-Otetradecanoyl-phorbol-13-acetate) induced mouse model of SCCs, overexpression of collagen-XVII, integrin α6β4 and the γ2 chain of laminin, promoted tumorigenesis (Moilanen et al., 2017). Increased laminin-332 expression with high MMP-2 and MMP-14 activity is seen in invasive SCCs with poor prognosis (Marinkovich, 2007). Reduced expression of collagen-VII in SCCs enhances invasion, dysregulates epithelial differentiation and drives epithelial to mesenchymal transition (Martins et al., 2009). Loss of collagen-VII expression (in RDEB) results in an upregulation of TGF-β signaling and increased angiogenesis in SCCs underscoring the critical role played by collagen-VII in suppressing TGF-β signaling (Martins et al., 2016). Transgenic mice lacking MMP-9 reduced the tumor invasiveness in a mouse model of SCC whereas, transplanting MMP-9 producing bone marrow cells increased tumor invasiveness (Coussens et al., 2000). The cleaved oligosaccharides generated by heparinize digestion of heparin sulfate chains is shown to play an important role in tumor progression in BCCs and SCCs (Pinhal et al., 2016). The abundance and stiffness of collagen also play an important role in melanoma progression where it stimulates proliferation and differentiation in a YAP/PAX3/MITF dependent manner (Miskolczi et al., 2018). In melanoma and BCCs, cathepsin K expression is associated with a fibromucinous stroma around the tumor nests, indicative of ECM degradation (Ishida et al., 2013). Interestingly, the matricellular protein POSTN is associated with metastatic progression in melanoma by attenuating adhesion and promoting migration (Fukuda et al., 2015). Tumor fronts of aggressive BCCs and SCCs overexpress TNC (Schalkwijk et al., 1991; Dang et al., 2006). Likewise, CCN-1 and CCN-2 are overexpressed in keratinocytes of BCCs in a YAP dependent manner promoting cell proliferation and survival (Quan et al., 2014).

The crosstalk between immune cells and ECM is thought to be critical in driving skin cancer pathogenesis. In patient samples of SSCs, an increase in CD163+ TAMs is associated with enhanced expression of MMP-9 and MMP-11, which promotes angiogenesis and tumor growth. The same study also reports the activation of IL-4 dependent STAT-6 signaling in TAMs that induces differentiation of Th2 cells, further maintaining an immune-suppressive environment (Pettersen et al., 2011). In a DMBA-TPA induced mouse model of SCC, Gr1+ neutrophils produce remodeling enzymes including myeloperoxidase (MPO) and elastase, which are associated with poor disease outcome (Gasparoto et al., 2012). TNF-α and TGF-β secreted by macrophages synergistically increase melanoma migration in a NF-κB dependent manner through the release of MMP-1 and MT1-MMPs (Li R. et al., 2017). Hyaluronic and proteoglycan link protein (HAPLN-1) is an ECM protein highly expressed in young fibroblasts compared to aged fibroblasts and is important for maintaining stability of hyaluronan and collagen (Ecker et al., 2019). Since T cells use collagen fibrils to infiltrate tumors, downregulating HAPLN-1 expression in melanoma models results in decreased CD3+ T cell infiltration associated with increased tumor cell extravasation. On the other hand treatment with recombinant HAPLN-1 increases the T cell recruitment and reduces the

tumor growth. The association between collagen fiber stability and T cell migration has been suggested as one of the reasons for aggressive melanomas in aged individuals (Kaur et al., 2018). In CTCL, T lymphocytes localize toward the epidermis by expressing integrin α3β1 allowing them to bind with laminin-332 and migrate through the BM, which interestingly does not result in ECM disorganization (Wayner et al., 1993). Tumor derived OPN binds to macrophages through integrin α9β1 resulting in the overexpression of COX-2 and MMP-9 and promotes angiogenesis in melanomas (Kale et al., 2014). In melanoma models, IFN-γ induced by TSP-1 overexpression is critical for macrophage recruitment and polarization to the M1 state (Martin-Manso et al., 2008). This points to an important role played by TSP-1 in suppressing tumor growth.

The ECM-immune cell interactions have been well studied in the context of cancer since the ECM is highly remodeled by both the tumor and immune cells to facilitate the tumor growth. The tumor microenvironment can switch from being anti-tumorigenic to pro tumorigenic depending on dynamic interactions between the tumors; ECM and immune compartments and can result in aggressive cancer with poor prognosis. Understanding the signaling events regulating this process has led to the development of therapies targeting the immune system as evidenced by advent of immunotherapy in treating cancer. **Figure 3** summarizes the ECM-immune interactions in all of the skin diseases discussed in this review.

#### CURRENT STATE OF THE ART THERAPEUTIC INTERVENTIONS

Currently available treatment approaches for skin diseases involve treatment with anti-inflammatory drugs, analgesics, topical steroids, and anti-histamines (Grassberger et al., 2004; Abraham and Roga, 2014). Conventional therapies for the treatment of the diseases discussed in this review are listed in the **Table 3**. With the advent of techniques such as nanomedicine, gene therapy (genetic engineering), stem cell transplantation, and cell and protein replacement therapy, curative treatment of skin diseases seem achievable and have been discussed below.

#### Nanomedicine

Nanomedicine involves the use of particles within the nanometer range, for the detection and treatment of diseases. They are effective drug delivery agents for targeted therapy that enhance drug bioavailability and facilitate controlled drug release for long-term effect. Various nanoparticles as delivery agents for treatment of skin diseases include liposomes, solid lipid nanoparticles, polymeric micelles and nanospheres,

#### TABLE 3 | Conventional treatment strategies.

#### TABLE 3 | Continued

*(Continued)*

TABLE 3 | Continued