Small leucine-rich proteoglycans orchestrate receptor crosstalk during inflammation

Abstract

Inflammation is not only a defensive mechanism against microbial invasion, but also frequently represents a critical response to tissue injury under sterile conditions. It is now well established that tissue injury leads to the release of endogenous molecules of intra- and extracellular origin acting as damage-associated molecular patterns (DAMPs). The small leucine-rich proteoglycans (SLRPs) can act as powerful DAMPs following their proteolytical release from the extracellular matrix. Recent investigations of SLRP signaling networks revealed new levels of complexity, showing that SLRPs can cluster different types of receptors and orchestrate a host of downstream signaling events. This review will summarize the evidence for the multifunctional proinflammatory signaling properties of the two archetypal SLRPs, biglycan and decorin. These secreted proteoglycans link the innate to the adaptive immune response and operate in a broad biological context, encompassing microbial defense, tumor growth and autoimmunity.

Introduction

Inflammation is a crucial process in the defense against invading pathogens. Neutrophils, macrophages and dendritic cells are capable of recognizing highly conserved microbial structures, referred to as pathogen-associated molecular patterns (PAMPs), via specific pathogen recognition receptors (PRRs).^1-3 Five classes of PRRs have been identified to date. These receptors are characterized by distinct ligand recognition and cellular location: cell surface or endosomal Toll-like receptors (TLRs), cytoplasmatic NOD-like receptors (NLRs), intracellular RIG-I (retinoic acid-inducible gene-I)-like receptors, transmembrane C-type lectin receptors (e.g., dectin-1) and AIM2 (absence in melanoma 2)-like receptors^1,4 (Fig. 1). PAMP recognition by PRRs elicits innate immunity responses by triggering the nuclear factor κB (NFκB), mitogen-activated protein kinase (MAPK), p38 or type I interferon signaling pathways.^2,4,5 These signaling events result in secretion of cytokines, e.g., tumor necrosis factor α (TNFα) or interleukin-1β (IL1β), and chemokins in order to recruit additional inflammatory cells to the site of infection.⁶ After the initial activation of an innate immunity response, the adaptive immune system provides a more specific response to control and eliminate the cause of infection. Several reports have addressed the role of receptor crosstalk linking innate and adaptive immunity.^7-9 In addition, microRNAs (miRs) have been shown to act as fine-tuners of the inflammatory response reaction.¹⁰

Figure 1. Selected innate immunity receptors sensing various pathogens and endogenous danger signals. Schematic drawing depicting distinct ligand recognition and cellular location of selected pathogen recognition receptors sensing various pathogen-associated (extracellular, brown) and damage-associated (extracellular, green) molecular patterns. Abbreviations: AIM2, absence in melanoma 2; ds, double-stranded; HMGB1, high-mobility group box-1; LPS, lipopolysaccharide; NLRP3, NLR family, pyrin domain containing 3; NOD, nucleotide-binding oligomerization domain-containing protein; TLR, toll-like receptor; RIG-I, retinoic acid-inducible gene-I; ss, single-stranded.

Over the last few decades, much has been revealed about the mechanisms of pathogen-mediated inflammation. However, inflammation may also occur during tissue injury in the absence of microbial pathogens, e.g., as a consequence of trauma, chemical damage or in ischemia-reperfusion injury.^2,6 Moreover, sterile inflammation is a common feature in many pulmonary, renal and liver diseases, resulting in organ fibrosis.² Non-pathogen-mediated inflammation is an important characteristic of atherosclerosis, tumorigenesis, gout, myocardial infarction, stroke and several autoimmune diseases (e.g., systemic lupus erythematosus).^2,11,12 With the high incidence of sterile inflammatory diseases, there is a considerable unmet medical need to resolve the mechanisms of innate immune activation in the absence of microbial pathogens.

It has become increasingly clear that tissue stress or injury leads to the release of endogenous molecules of intra- or extracellular origin acting as damage-associated molecular patterns (DAMPs).^2,13 In this review, the complexity of proinflammatory signaling of the two matrix proteoglycans decorin and biglycan, both acting as DAMPs,^14-16 will be addressed. Particular focus will be put on newly described mechanisms of how these molecules orchestrate the interaction of various receptors, which are involved in triggering sterile inflammation.^15,16 Decorin- and biglycan-mediated receptor crosstalk involves TLRs, NLRs and transforming growth factor β (TGFβ) receptors and is further fine-tuned by miR-21.^15,16 Thus, we will focus on the multifunctional proinflammatory signaling properties of decorin and biglycan. These two archetypal SLRPs, when released in a soluble form, can link the innate to the adaptive immune response and can operate in a broad biological context, such as microbial defense, tumor growth and 
autoimmunity.

Intracellular and Extracellular DAMPs

DAMPs of intra- or extracellular origin, similar to PAMPs, are recognized by ligand-specific PRR and elicit innate immunity responses^2,13,17 (Fig. 1). During cell necrosis, some exclusively intracellular components, such as heat shock proteins, purine metabolites (ATP or uric acid) and high-mobility group box-1 (HMGB1), are released into the extracellular space and thus turn into DAMPs.²

The extracellular matrix, present in almost every tissue and organ, serves as a vast reservoir of sequestered components. Upon tissue stress or injury, matrix components will be released and turn into signaling molecules.^12,18,19 Among this large group of ECM components, only a few, such as biglycan,¹⁴ decorin,¹⁶ hyaluronan,²⁰ versican,²¹ tenascin-C,²² fibrinogen and heparan sulfate fragments,²³ are capable of interacting with PRRs, thereby alerting the innate immune system to impending tissue damage. These mechanisms give rise to a rapid response reaction without the need for de novo synthesis of danger molecules. It took several years to overcome the initial doubts concerning ECM components acting as endogenous ligands of PRRs. Recent data from in vivo studies, where contaminations are less likely, together with the neutralizing effects of antibodies directed against ECM-derived ligands of TLRs and NLRs, provided a reliable basis to support their role as endogenous danger signals. For further details, please refer to more specific reviews in references 12, 18, 19 and 24.

The Family of Small Leucine-rich Proteoglycans

It is remarkable that both TLRs and NLRs contain leucine-rich repeat (LRR)-motifs, which enable them to interact with other LRR-proteins.^25,26 Several matrix proteoglycans contain LRR motifs in their protein core.²⁷ The relatively small size of the protein core (up to 42 kDa) is another hallmark of this class of proteoglycans, explaining why these compounds are referred to as small leucine-rich proteoglycans.^18,28-30 SLRPs are covalently substituted with glycosaminoglycan (GAG) side chains: chondroitin sulfate, dermatan sulfate or keratan sulfate. The various lengths and states of sulfation contribute to the structural complexity of these molecules.^28,31 According to their cysteine clusters, ear repeats, intron/exon organization and homologies at the protein and genomic levels, SLRPs are divided in five distinct classes.²⁸ Biglycan and decorin, the two best-studied SLRPs, belong to the canonical class I and are substituted with one or two chondroitin/dermatan sulfate chains. Details regarding their classification have been reviewed recently in references 28 and 31. It is well established that SLRPs play a role in the stabilization and assembly of collagens and elastic fibers.^32,33 Thus, for many years, SLRPs were considered to be merely static props of the ECM.

The finding that decorin, besides being a structural matrix component, acts as a signaling molecule gave rise to a new paradigm of how SLRPs regulate a host of biologic processes, such as differentiation, proliferation, migration and apoptosis. Decorin is capable of binding to four different receptor tyrosine kinases (RTKs), including the epidermal growth factor receptor (EGFR),³⁴ insulin-like growth factor-1 receptor (IGF-IR),^35-38 Met³⁹ and vascular endothelial growth factor receptor 2 (VEGFR2),⁴⁰ thereby impacting on fibrogenesis,¹⁹ tumor growth and metastatic spreading.^12,41,42 A summary of recent data demonstrating the intricacy of decorin signaling, which links innate immunity, inflammation and tumor growth¹⁶ is provided below.

Biglycan: A Signaling Molecule and Endogenous Ligand of TLR2 and TLR4

Despite its structural homology with decorin, biglycan is not sharing the ability of decorin to bind to EGFR.⁴³ Interaction of biglycan with other RTKs involved in decorin signaling has not been investigated up to now. Even though there is some evidence for the involvement of biglycan in inflammation,^44-46 only recently biglycan-mediated signal transduction has been described in macrophages.¹⁴

Biglycan is abundant in the ECM of various organs.⁴⁷ However, in its sequestered form, it cannot act as a signaling molecule.¹⁴ Upon tissue stress or injury, biglycan becomes proteolytically released from the matrix to signal impending danger to the immune system.¹⁹ Several proteolytic enzymes, such as bone morphogenic protein (BMP)-1, matrix metalloproteinase (MMP)-2, MMP-3 and MMP-13 have been described to cleave the biglycan protein core.^48-51 In its soluble form, intact biglycan acts as an endogenous ligand of TLR2 and TLR4 in macrophages and dendritic cells, resulting in rapid activation of p38, Erk and NFκB pathways and enhanced synthesis of proinflammatory TNFα^8,14,15 (Fig. 2). Furthermore, biglycan-mediated signaling via TLR2/4 leads to the synthesis of various chemoattractants for neutrophils and macrophages, such as macrophage inflammatory protein-1α (MIP-1α), monocyte chemoattractant protein-1 (MCP-1) and regulated upon activation, normal T-cell expressed and secreted (RANTES) ^8,14 (Fig. 2). Infiltrating macrophages stimulated by proinflammatory cytokines (e.g., IL1β) are capable of synthesizing biglycan themselves.¹⁴ Probably not all biglycan fragments proteolytically cleaved from the ECM bind to and signal through TLR2/4. Therefore, it is conceivable that de novo synthesis of biglycan by macrophages serves as an amplifying mechanism to provide the full-length “signaling” form of biglycan at sites of injury. Based on this mechanism, it appears that biglycan initiates a feedforward cycle, which is able to trigger and boost the inflammatory response reaction both in an autocrine and paracrine manner.

Figure 2. Decorin- and biglycan-driven proinflammatory signaling involves multi-receptor crosstalk. Decorin and biglycan interact with TLR2 and TLR4 and stimulate the expression of various chemo- and cytokines, mainly proinflammatory TNFα and pro-IL1β but also antiinflammatory IL-10. By clustering TLR2/4- with purinergic P2X₇/P2X₄ receptors biglycan activates the NLRP3-inflammasome and caspase-1 and induces the processing and secretion of mature IL1β. Decorin, by withdrawing active TGFβ1 from binding to its receptors, inhibits maturation of miR-21, a posttranscriptional inhibitor of PDCD4. Enhanced protein abundance of PDCD4, a translational inhibitor of IL-10, causes reduction of the IL-10 protein. Abbreviations: ASC, apoptosis associated speck-like protein containing a CARD; CXCL13, chemokine (C-X-C) ligand 13; IL, interleukin; MCP-1, monocyte chemoattractant protein-1; MIP, macrophage inflammatory protein; miR, micro-RNA; NLRP3, NLR family, pyrin domain containing 3; PDCD4, programmed cell death protein 4; RANTES, regulated upon activation, normal T cell expressed and secreted; TGF, transforming growth factor; TLR, Toll-like receptor; TNFα, tumor necrosis factor α.

Biglycan Orchestrates the Interaction of TLR2/4 with Purinergic P2X₇/P2X₄ Receptors

After some initial skepticism, the concept of soluble forms of matrix components triggering inflammation has finally caught on. Further investigations of the biglycan signaling networks provided a new surprise, showing that biglycan is capable of clustering several types of receptors and orchestrating their signaling.¹⁵ Beside triggering the synthesis of TNFα, biglycan promotes secretion of mature IL1β, a proinflammatory cytokine important both in acute and chronic inflammation.⁵² Usually the synthesis, processing and secretion of mature IL1β requires at least two steps, namely, the synthesis of pro-IL1β via TLR-dependent NFκB activation and stimulation of the NLR family, pyrin domain-containing 3 (NLRP3) inflammasome and caspase-1, which leads to cleavage pro-IL1β. Surprisingly, soluble biglycan alone is capable of stimulating the synthesis and secretion of mature IL1β. This is brought about by organizing a multi-receptor complex consisting of TLR2 and TLR4 and purinergic P2X₇ and P2X₄ receptors on the cell surface of macrophages (Fig. 2). By clustering these receptors, biglycan induces cooperativity of this newly formed receptor complex. By binding to TLR2/4, biglycan initiates the synthesis of pro-IL1β and NLRP3 inflammasome. By simultaneously signaling through the P2X₇ and P2X₄ receptor, it induces the formation and activation of the NLRP3/apoptosis associated speck-like protein containing a CARD (ASC) inflammasome and of caspase-1, which drives the maturation and secretion of IL1β (Fig. 2). Biglycan-mediated activation of the NLRP3 inflammasome involves reactive oxygen species (ROS) and heat shock protein 90 (HSP90). The clustering of TLR2/4 with P2X₇/P2X₄ receptors is presumably facilitated by the complex structure of the biglycan molecule involving both the protein core and the GAG side chains. So far, our data showed that only intact biglycan is capable of triggering proinflammatory signaling in macrophages and dendritic cells.

Interaction of Biglycan with TLR2/4 in Pathogen-mediated and Sterile Inflammation

The demonstration that biglycan-evoked modulation of TLR2/4 activity is operational in vivo, provides the most robust proof that proinflammatory effects of biglycan are not due to contamination with lipopolysaccharide (LPS) and other TLR4- and TLR2-ligands. Overexpression of soluble biglycan in healthy wild-type TLR4-, TLR2- and TLR2/TLR4-deficient mice shows that biglycan exerts proinflammatory effects via both TLRs.⁸ Furthermore, biglycan deficiency⁵³ reduces the inflammatory response both under pathogen-mediated¹⁴ and sterile conditions.^8,15 Both in TLR4- and TLR2-dependent, LPS- or zymosan-induced sepsis, lack of the biglycan gene markedly enhances survival and is associated with lower levels of circulating TNFα and IL1β as well as reduced numbers of infiltrating mononuclear cells in septic lungs.^14,15 What is more, in biglycan-deficient mice, lower levels of IL1β in plasma and the lung, the major target organ of sepsis in mice, are associated with a reduction of NLRP3-inflammasome expression and active caspase-1.¹⁵ This observation clearly indicates involvement of the inflammasome in the downstream signaling of biglycan in vivo. Thus, the role of biglycan in microbial inflammation is probably to trigger inflammation via a second TLR, which is not involved in pathogen sensing, thereby potentiating the pathogen-induced inflammatory response.

Beside its impact on the pathogen-induced immune response, protective effects of biglycan deficiency were found in experimental models of noninfectious inflammatory renal diseases, e.g., unilateral ureteral obstruction and lupus nephritis.^8,15 Lack of biglycan results in a marked reduction of TNFα and IL1β and of infiltrating mononuclear cells in these models.^8,15 These effects are NLRP3- and caspase-1-dependent.^8,15 On the other hand, overexpression of soluble biglycan caused stimulation of various chemoattractants, promotes infiltration of mononuclear cells and results in more pronounced organ damage.⁸ Thus, it appears that in sterile inflammation, soluble biglycan amplifies the inflammatory response reaction by involving TLR2/4 and the NLRP3 inflammasome.

Several authors have described the coincidence of biglycan overexpression with enhanced inflammation and severe tissue injury.^54-56 The correlation between enhanced abundance of biglycan and reduction of organ function in various sterile inflammatory renal diseases has been particularly well documented.^8,19,55-58 Upregulation of biglycan in aortic stenosis, characterized by lipid accumulation and inflammation,⁵⁹ results in enhanced synthesis of phospholipid transfer protein via stimulation of TLR2.⁶⁰ Furthermore, higher levels of biglycan in cardiac transplant coronary arteriopathy,⁵⁸ severe ocular surface disease⁶¹ and in adipose tissue⁶² suggest a role for biglycan in the perpetuation of inflammation. Further studies concerning the mechanisms of biglycan-driven inflammation in those pathophysiological processes would improve our understanding of how sterile inflammation is triggered.

Biglycan Signaling Bridges Innate and Adaptive Immunity

The importance of biglycan signaling in the immune response is further underlined by recent findings indicating that biglycan is also a component of the adaptive immune system.^8,63 In lupus nephritis (LN), a renal manifestation of systemic lupus erythematosus, biglycan, induces macrophages and dendritic cells to express chemokine (C-X-C) ligand 13 (CXCL13), the major chemoattractant for B and B1 cells. This is brought about by biglycan signaling through TLR2/4 and initiating the production of ROS.⁸ B1 cells, particularly important for autoantibody production, are involved in an early, T cell-independent immune response. By attracting B1 cells to the kidney, biglycan triggers autoantibody production without T-cell involvement.

In patients with lupus nephritis and in lupus-prone mice, the presence of circulating biglycan has been described.⁸ This is of particular relevance, because only soluble biglycan is capable of acting as a proinflammatory signaling molecule.¹⁴ Indeed, renal and plasma levels of biglycan correlate with the initiation and progression of LN. Furthermore, expression of CXCL13, recruitment of chemokine (C-X-C) motif receptor 5 (CXCR5)-expressing B/B1 cells into the kidney as well as organ damage and albuminuria strongly depend on biglycan expression.⁸

Beside chemoattracting B cells, biglycan signaling via TLR2/4 also induces the expression of RANTES, MCP-1 and MIP-1α, thereby recruiting macrophages and T cells into the kidney.⁸ In addition, biglycan plays a crucial role in MHC I- and MHC II-restricted T‑cell cross-priming by acting through TLR2/4 receptors and their adaptor proteins, myeloid differentiation factor 88 (MyD88) and TIR-domain-containing adaptor-inducing interferon β (TRIF). MHC II-dependent, antigen-specific T-cell activation is mainly mediated by TLR4.⁶³ These results were further confirmed in experimental autoimmune perimyocarditis (EAP). In this model, biglycan signals via TLR4 as a potent amplifier of specific cardiomyocyte antigen presentation to prime T cells. Biglycan initiates an autoreactive immune response without influencing postinfarction fibrosis or infiltration of antigen presenting cells.⁶³

Several other findings implicate biglycan to impact on both the innate and adaptive immune system. Biglycan stimulates the development, growth and differentiation in cells of the monocytic lineage.⁶⁴ Furthermore, biglycan strongly binds to the complement factor C1q⁶⁵ with yet-unknown biological consequences. It acts as a ligand of CD44 and thereby appears to be involved in the recruitment of CD16-negative natural killer cells.⁶⁶

Thus, biglycan signaling provides an important link between innate and adaptive immunity. This concept advances our understanding of the etiology of B- and T cell-mediated inflammatory disease entities driven by soluble components of the extracellular matrix.

Decorin as Endogenous Ligand of TLR2/TLR4 and TGFβ1 Inhibitor Triggers Dual Proinflammatory Signaling

Recently it was shown that soluble decorin induces proinflammatory signaling, thereby linking innate immunity, inflammation and tumorigenesis.¹⁶ Soluble decorin has been identified as a novel endogenous ligand of TLR2 and TLR4 in macrophages. Decorin autonomously activates Erk and p38 MAPKs and typically for a TLR2/4 ligand initiates mRNA expression and protein synthesis of pro- (Tnf and Il12b) and anti-inflammatory cytokines (Il10) (Fig. 2). In the presence of LPS, decorin enhanced the effects of LPS on the synthesis of proinflammatory TNFα and IL-12 by signaling additionally via TLR2. Surprisingly, different effects of decorin on the translational regulation of IL-10 were described, depending on whether decorin was acting on macrophages alone or together with LPS. Decorin alone increased Il10 mRNA and protein expression, whereas in combination with LPS, decorin further amplified LPS-mediated upregulation of Il10 mRNA but suppressed LPS-triggered IL-10 protein synthesis.¹⁶ The underlying mechanisms are more complex than those expected by TLR2/4 signaling alone.

As ligands of TLRs, both LPS and decorin trigger the synthesis of programmed cell death protein 4 (PDCD4),¹⁶ a translational inhibitor of IL-10.^67,68 However, LPS-mediated synthesis of PDCD4 is followed by a rapid decline of PDCD4 abundance.⁶⁸ In contrast to decorin, LPS induces active TGFβ1, which processes primary (pri-miR-21) to precursor (pre-miR-21) miR-21 and enhances mature miR-21,¹⁶ a post-transcriptional inhibitor of PDCD4.⁶⁸ Reduced PDCD4 protein causes enhancement of IL-10, thereby promoting the resolution of inflammation. Addition of decorin to LPS inhibits active TGFβ1 with a subsequent decline in miR-21, leading to a reduction of IL-10 and further switch of cytokine profile toward a proinflammatory phenotype¹⁶ (Fig. 2).

Importantly, these mechanisms appear to operate in a broad biological context, linking pathogen-mediated with sterile inflammation, as shown for sepsis and growth retardation of established tumor xenografts. In sepsis, decorin is an early response gene evoked by inflammation. Decorin is markedly elevated in septic patient plasma, as well as in the plasma and tissues of septic mice. In sepsis, decorin alone mimicks the effects of LPS by enhancing plasma and tissue levels of proinflammatory TNFα, IL-12 and PDCD4. However, when administered together with LPS, it potentiates the proinflammatory response of LPS by inhibiting active TGFβ1, miR-21 and, hence, LPS-mediated IL-10 production. In tumor xenografts, overexpression of decorin results in TLR2/4-driven synthesis of PDCD4, TNFα, IL-12 and TGFβ1/miR-21-mediated inhibition of PDCD4 suppression. This decorin-evoked signaling can shift the immune reaction to a more apoptotic and inflammatory response with strong antitumorigenic effects, resulting in a marked retardation of tumor growth.¹⁶

Thus, decorin signaling boosts the inflammatory response in sepsis and tumor development. During sepsis, these mechanisms tend to inhibit the resolution of inflammation and may cause a state of hyperinflammation. On the other hand, decorin-mediated inhibition of miR-21, immunosuppressive TGFβ1 and anti-inflammatory IL-10 along with the stimulation of proinflammatory PDCD4, TNFα and IL-12 might represent an attractive approach for cancer therapy.¹⁶ Taken together, these findings show that decorin similar to biglycan is capable of orchestrating multiple signaling pathways acting as a crucial regulator of the inflammatory response reaction.

Future Directions

Taking into account the high prevalence of sterile inflammation in disorders, such as atherosclerosis, gout, myocardial infarction, stroke, fibrotic processes of various organs, autoimmune diseases and tumorigenesis, there is a large unmet medical need to develop more specific antiinflammatory therapies. The available evidence demonstrating a direct role of ECM components as signals of tissue stress or damage markedly improved our understanding of the etiology of sterile inflammation. Among various ECM components, SLRPs are particularly suited to interact with TLR and NOD-like receptors, as both ligands and receptors contain LRRs in their protein structure. In this context, the scarcity of data addressing the role of SLRPs in the regulation of inflammation is surprising. Beside the aforementioned studies, there is also evidence for the influence of lumican on inflammatory processes.^69-73 Data regarding the role of other 14 members of SLRP family are sparse. Future studies are needed to explore the molecule-specific affinities to different receptors and orchestration of their downstream signaling events.

In spite of an increasing body of evidence regarding biglycan- and decorin-mediated regulation of inflammatory processes, several questions need to be addressed before therapeutic strategies can be developed. Only soluble and intact biglycan or decorin are capable of triggering proinflammatory signaling. Therefore, the question arises why, when and for what reason SLRPs become present in the circulation. Could their presence in circulation become a future biomarker for inflammatory diseases? Furthermore, the importance of the protein core and GAG side chains for binding and initiating crosstalk among various receptors has to be explored to a greater detail. Of particular importance for the development of therapeutic approaches appears to be the identification of LRRs as well as the adaptor molecules involved in SLRP/receptor interaction.

Glossary

Abbreviations:

CXCL13

Chemokine (C-X-C) ligand 13

DAMP

damage-associated molecular pattern

ECM

extracellular matrix

GAG

glycosaminoglycan

IL

interleukin

LN

lupus nephritis

LPS

lipopolysaccharide

LRR

leucine-rich repeat

MAPK

mitogen-activated protein kinase

MCP-1

monocyte chemoattractant protein-1

MIP

macrophage inflammatory protein

miR

microRNA

NFκB

nuclear factor κB

NLR

NOD-like receptor

NLRP3

NLR family, pyrin domain containing 3

PAMPs

pathogen-associated molecular patterns

PDCD4

programmed cell death protein 4

PRR

pathogen recognition receptor

ROS

reactive oxygen species

SLRP

small leucine-rich proteoglycan

TGF

transforming growth factor

TLR

Toll-like receptor

TNFα

Tumor necrosis factor α