Toll-like receptors (TLRs) function as the immune system's “sensory system.” These transmembrane pattern recognition receptor complexes sense and recognize pathogen-associated molecular patterns (PAMPs) or endogenous danger associated molecular patterns (DAMPs) (1, 2). Although TLRs, in particular TLR-2 and TLR-4, are involved in regulation of host defense, recent studies provide evidence that excessive TLR-mediated signaling is involved in the pathogenesis of multiple diseases including atherosclerosis, thrombosis, ischemic/reperfusion injury, inflammatory bowel disease (IBD), necrotizing enterocolitis (NEC), erythematosus, diabetes, Alzheimer disease, psoriasis, chronic obstructive pulmonary disease, asthma (1, 3, 4), and skin diseases (2). TLRs also play an integral role in autoimmune diseases, including autoimmune colitis, multiple sclerosis, and systemic lupus erythematosus (2). To date, a limited number of validated therapeutic options exist for these diseases.
More recently researchers have become interested in the therapeutic potential of blocking of excessive TLR signaling and its clinical applications, especially in inflammatory conditions (1). Significant attention has also been given to TLR blockers that can competently bind TLRs, but do not induce the structural rearrangement essential for intracellular signal transduction. These TLR antagonists inhibit activation of the TLR-mediated signaling cytokine cascade and can prevent or attenuate uncontrolled adaptive immune responses (5).
In general, TLR antagonists are natural agonists or “agonist like molecules” that function as immune system regulators. These include small molecules, aptamers, oligonucleotides, peptides, proteins, and antibodies that interact with TLRs, but do not transduce intracellular TLR signaling. Examples of small molecule inhibitors (SMIs) include (dsRNA, ssRNA, and CpG-DNA) which are synthetic or naturally derived chemical agents that inhibit TLR signal transduction. Despite good bioavailability the poor specificity and targeting capability of SMIs limit their application (6).
Eritoran and TAK-242 (Resatorvid) are examples of TLR antagonists which bind the extracellular and intracellular Toll/IL-1 receptor (TIR) domains respectively (7). Unfortunately, neither of these agents is currently being considered for therapeutic use in humans due to failed clinical trials (8). Despite the disappointing results of Eritoran and TAK-242 in human trials, the importance of TLR signaling multiple diseases continues to fuel the search for clinically effective TLR antagonists (8). Table 1 provides a brief summary of currently available TLR-2/4 inhibitors, their classification, site of action, applications, and limitations (9–16). Sparstolonin B (SsnB) was shown to inhibit TLR-2 and TLR-4 ligand-induced inflammation (6, 17–20). SsnB is structurally different from other small-molecule TLR-4 antagonists, making it an intriguing candidate for researchers to investigate regarding its role in various inflammatory conditions (6, 17).
The aim of this review is to discuss current studies addressing SsnB and its application in different diseases. Specifically, evidence indicating the potential of this inhibitor to regulate inflammation in various diseases will be discussed.
TLR STRUCTURE, SIGNALING, AND FUNCTION
TLRs are pattern recognition receptors that were first detected in Drosophila embryos (21). Subsequent to their discovery, homologous TLRs receptors have been identified in almost mammals including humans. TLRs are type 1 transmembrane proteins (TM) with a tripartite domain architecture: an extracellular recognition domain/ectodomain rich in leucine repeats, a single pass TM domain, a cytoplasmic TIR downstream signaling domain, which senses the ligands and TIR homologue domain to trigger downstream signaling by binding to adaptor protein myeloid differential factor 88 (MyD88) (2). Upon detecting a PAMP or DAMP, TLRs recruit adapter proteins and undergo either homodimerization or heterodimerization and initiate a complex process of downstream signal transduction events leading to expression of inflammatory cytokines and Interferon (IFN) (22).
TLRs have been shown to activate both nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) pathways via MyD88, a common adaptor molecule recruited toward the TIR domain of TLRs (Fig. 1) (2). The TLR signaling pathway is very similar to the interleukin (IL)-1R family which also requires a TIR domain-containing adaptor protein (TIRAP), protein kinase, and transcription factor to transfer the signal (6). The cytoplasmic domain of TLR-4 is commonly referred to as a TIR domain on the basis of its extensive homology to the corresponding domains of all members of the Toll, TLR, and IL-1R families (23). The importance of the TIR domain for TLR-4 signaling was demonstrated by the constitutive activation of endogenous cytokine genes by a protein containing the TIR and transmembrane domains of TLR-4 fused to the extracellular domain of CD4 (CD4/TLR-4) (24). TLR-2 must form heterodimers with TLR-1 or TLR-6 to transmit a signal (19). The TLR-2/6 heterodimer recognizes di-acylated lipopeptides (19), whereas TLR-1/2 can recognize tri-acylated lipopeptides (25). TLR-4 interacts with its ligands via the coreceptor soluble cluster of differentiation 14 (CD14) and MD2 (26, 27). For example, in macrophages, lipopolysaccharide (LPS) binds to CD14, which then binds the TLR-4/MD-2 complex. The interaction of interleukin-1 (IL-1) with its receptor (IL-1R) or PAMPs/DAMPs with TLRs induce the recruitment of the adaptor protein MyD88 to the receptors, which is followed by the recruitment of interleukin receptor-associated kinase 4 to MyD88 via interactions between the N-terminal death domains of these proteins (28). These activate TNF receptor-associated factor 6 (TRAF6), which activates the I-κB kinase (IKK) complex and MAPK (29, 30).
MAPK subsequently phosphorylates c-jun N-terminal kinase (JNK) and p38 MAPK, which activate transcriptional factor AP-1 (3). NF-κB essential modulator stabilizes the IKK complex. This allows the phosphorylation of IκB, which is subsequently degraded by ubiquitination. Loss of IκB allows for NF-κB activation, resulting in its nuclear translocation and transcription stimulation of pro-inflammatory cytokines. NF-κB is also triggered independently from MyD88 (4).
Upon pathogen recognition TLRs recruit the cytoplasmic protein MyD88, TLR-4 is unique since it activates both the MyD88 dependent (D) and independent (I) pathway (31). TLR-4 activation of the D pathway recruits MyD88 and Mal/TIRAP to the TLR-4-TIR domain resulting in the activation of TRAF6, early phase activation of NF-κB and mitogen-activated protein kinase and the subsequent induction of pro-inflammatory cytokines (32). TLR-4-mediated activation of the I pathway recruits Toll-IL-1R domain-containing adaptor that activate IRF3 leading to IFN-production (33). Pretreatment with D-specific agonists results in increased TNF and IFN-γ production. It also results in upregulation of distal portions of both the inflammatory cytokine and IFN-γ limbs of the I pathway. For example, pretreatment with D-specific agonists primes inflammatory cytokine and IFN-responses to subsequent treatment with LPS. However, when cells are treated first with a D-specific agonist, despite proximal inhibition of the D pathway, LPS can still access the up-regulated distal pathways via the proximal, noninhibited I pathway. Although the D and I pathways share some distal intermediaries (e.g., TNFR-associated factor 6 and NF-κB), the complexity of responses suggests multiple intermediaries may be involved. Further studies will be required to pinpoint complex interactions between the D and I pathways.
TLRs represent the host's first line of defense against invading pathogens (e.g., bacteria and viruses). At present, more than a dozen TLRs have been identified, with the first nine being well characterized (34). Differential subcellular localizations of these homologous TLRs correlate with the type of molecular patterns they recognize; TLR-1, 2, 4, 5, and 6 are mainly located on the plasma membrane where they recognize bacterial, fungal, and protozoan pathogens, whereas TLR-3, 7, 8, and 9 are located on endosomal/lysosomal membranes that bind to viral RNAs or DNAs (35, 36). TLRs are extensively expressed on the membranes of lymphocytes, macrophages, monocytes, dendritic cells, and natural killer cells (37). Furthermore, among TLRs, TLR-2, and TLR-4 exhibit the widest distribution, broadest pathogen recognition ability and greatest cytokine and chemokine release. Research has shown that the signaling initiated by TLRs is a double-edged sword. It may trigger confinement or elimination of invading organisms or in some circumstances, a prolonged, exaggerated inflammatory response resulting in tissue and organ damage (38). Generally, the primary role of TLR-4 is beneficial for induction of an inflammatory response that provides protection from invading bacteria and promotes mucosal integrity (39). However, in some experiments, TLR-4 can be maladaptive, actually causing tissue destruction and ulceration. Therefore, understanding the molecular mechanisms which regulate these responses could lead to development of new therapeutic strategies which target TLR-4 activity in inflammatory diseases. ALL abbreviations used in this section are summarized in Table 2.
HISTORY, DISCOVERY, AND FUNCTION OF THE SPARSTOLONIN B
Sparstolonin B is an oxygen-mixed anthracene compound isolated from the tubers of both Sparganium stoloniferum and Scirpus yagara. These flowering plants belong to the cyperaceous family, which contains many active compounds found in traditional Chinese medicine such as “SanLeng;” SanLeng has been used for treatment of several inflammatory diseases by alleviating stagnated blood, promoting the circulation of qi, and relieving dyspepsia and pain (40) along with treating abscesses, congestion, and amenorrhea (41). Its structure was elucidated as 8, 5′-dihydroxy-4- phenyl-5, 2′-oxidoisocoumarin by nuclear magnetic resonance spectroscopy and x-ray crystallography (22) (Fig. 2). Structural analysis revealed SsnB to be a polyphenol compound with core components of both xanthone and isocoumarin compounds. A wide range of studies have shown that polyphenols possess antioxidant and anti-inflammatory properties (8). Xanthones have anti-oxidation, immunomodulation, and cholesterol-lowering benefits (42, 43), and isocoumarins have anticoagulation, antitumor, and anti-inflammation. SsnB's intrinsic chemical properties may therefore act by several mechanisms to combat inflammatory conditions and could have a possible therapeutic application in numerous areas such as cancer, nervous diseases, cardiac diseases, and sepsis. Unlike other TLR inhibitors, SsnB is a potentially safe, nontoxic pharmaceutical agent, which has been used for the treatment of several pathological conditions. In terms of toxicity, even at concentrations as high as 100 mM, SsnB did not exhibit cytotoxic effects on various cell types, including mouse peritoneal macrophages, human umbilical vein endothelial cells (HUVECs), human aortic smooth muscle cells, and monocytic THP-1 cells (44, 45).
SsnB effectively inhibits inflammatory cytokine expression in mouse macrophages stimulated with LPS (a TLR-4 ligand), Pam3CSK4 (a TLR-1/TLR-2 ligand), and Fsl-1 (a TLR-2/TLR-6 ligand), but not by poly (I:C) (a TLR-3 ligand) or ODN1668 (a TLR-9 ligand)(46). It also suppresses LPS-induced cytokine secretion from macrophages and diminished phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (Erk1/2), p38 and JNK in these cells (46). Additionally, co-immunoprecipitation studies show SsnB reduces the association of MyD88 with TLR-4 and TLR-2, but not with TLR-9, in human embryonic kidney (HEK) 293T cells and monocytic THP-1 cells overexpressing MyD88 and TLRs (46). This confirms SsnB selectively acts at TLR-2/4 receptors. Also SsnB appears to exhibit its inhibitory actions by binding to the active site on MyD88 and there is no significant difference in NF-κB activity in mutant MyD88 of HEK-293 cells in the presence or absence of SsnB (47). In monocytic THP-1 cells expressing chimeric receptor CD4-TLR-4, which triggers constitutive activation of NF-κB, SsnB effectively blunted NF-κB activation (46). These results provide evidence that SsnB acts as a selective TLR-2 and TLR-4 antagonist by inhibiting the early intracellular events in TLR-2 and TLR-4 signaling.
SSNB'S POTENTIAL ROLE IN INFLAMMATORY DISEASES
Inflammation following intracerebral hemorrhage (ICH) has been shown to play a vital role in causing secondary brain injury. In particular, TLR-2/4 has been shown to play a critical role in amplifying proinflammatory responses in ICH-induced secondary brain injury in mice (47, 48). In this model hemoglobin (Hgb) from the intracranial hematoma was shown to cause microglial activation and induce coprecipitation of TLR-2 and 4 resulting in exacerbation of brain injury through production of inflammatory cytokines (47). SsnB was shown to inhibit TLR-2/4 heterodimer formation in cultured bone marrow-derived dendritic cells exposed to Hgb (47). Also, SsnB attenuates NF-κB activation by inhibiting TLR-2/4 heterodimer formation in HEK-293 cells. In another ICH study by Wang et al. (49), SsnB significantly improved neurological deficit scores, brain water content after the Morris water maze test, ameliorated brain edema, and attenuated TLR-2/TLR-4-induced inflammation. It is important to note that SsnB treatment decreased the MyD88 and NF-κB expression, without affecting the expression of TLR-4 and TRIF in perihematomal tissues, suggesting that Hgb released from hematoma activates TLR-2 and 4 on macrophages and lymphocytes, causing their infiltration and subsequent release of inflammatory cytokines responsible for inflammation in secondary injury as reported by Zhong et al. in the IHC model (47). However, there was no effective control group in the study by Zhong et al. to show the activation of TLR-4 in the ICH group.
In an experimentally induced model of rat spinal cord injury, SsnB reduced inflammation and apoptosis by modulating the TLR-4/MyD88/NF-κB signaling pathway (50). Meanwhile in a rat model of lumbar intervertebral disk disease (IVDD), SsnB effectively reduced the histological score of disc degeneration, inhibited the IVDD-induced inflammatory cytokines, stress factors, and increased endplate porosity, suggesting its potential efficacy in treating degenerative joint diseases (51). SsnB suppressed protein expression of TLR-4, MyD88, and NF-κB in addition to protein kinase B (PKB, also known as Akt) expression in nicotinamide adenine dinucleotide phosphate oxidase 2 and induced phosphoinositide 3 kinase (PI3K) (51). In addition, inflammation, oxidative stress, and apoptosis observed in degenerative IVDD were attenuated by SsnB (51). Treatment with SsnB has also been reported to alleviate neuropathic pain in a chronic constriction injury model in rats by increasing the mechanical withdrawal threshold through regulation of TLR-2/4 and reducing inflammation (20). These results highlight the potentially protective benefits of SsnB administration in lumbar IVDD-induced inflammation. SsnB appears to modulate oxidative stress and apoptosis by regulating TLR-4/MyD88/NF-κB, NADPH oxidase activation, and the PI3K/Akt signaling pathway (51, 52). Unlike other compounds, SsnB can easily cross the blood brain barrier due to its high lipid solubility and low molecular weight. Collectively, these findings suggest SsnB could be a potentially important therapeutic agent in treating inflammatory conditions of the brain and spinal cord.
Sepsis is characterized by systemic inflammation causing multiple organ injury mediated by TLR-2 and TLR-4. In a mouse endotoxin model, SsnB inhibited LPS-stimulated inflammation and decreased mortality (45). LPS-induced IL-1β, monocyte chemoattractant protein-1 (MCP-1), endothelial cell adhesion molecules, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, which are key components produced by endothelial cells during inflammatory responses in atherosclerosis, also regulate ERK1/2 and Akt phosphorylation that are related to TLR-4 activation (52). Similarly, co-incubation with SsnB attenuated THP-1 monocyte adhesion to LPS-activated HUVECs (52). SsnB reduced LPS-induced cytokine release from macrophages and reduced phosphorylation of IκBα, Erk1/2, p38a, and JNK (46). Upon intraperitoneal injection with 3 mg/kg to 9 mg/kg in rats with high fat diets (HFD), SsnB inhibited LPS-induced TLR-4 expression, NF-κB activation, and subsequent inflammation in vitro(18).
SsnB also appears to be effective in treating obesity-related inflammation. Treatment with SsnB was able to lower the levels of serum triglyceride, total cholesterol induced by HFD in obese rats (18). In a peroxynitrite-induced nonalcoholic steatohepatitis model, TLR-4 recruitment occurs in response to lipid rafts and subsequent NADPH oxidase activation (53). By decreasing peroxynitrite formation, SsnB prevents NADPH oxidase activation in vivo and in vitro(53). Furthermore, SsnB attenuates steatohepatitis injury (decreased necrosis, ballooning, Mallory-Denk bodies), improves hepatic function tests, and decreases TLR-4 trafficking in the plasma membrane of HFD mice in a CYP2E1-mediated oxidative stress model (18). After intraperitoneal administration in mice, SsnB mice showed significant reduction of microRNA 21, which is a marker for liver inflammation and Kupffer cell activation, along with concomitant inhibition of macrophage infiltration in the injured liver (53). Further supporting the potential usefulness of SsnB as immune regulator, human T cell lines or peripheral blood mononuclear cells challenged with SsnB significantly inhibited HIV-1 transcription via a novel mechanism that requires the transacting responsive region and synergy with azidothymidine (54). Collectively, these results provide evidence SsnB could be a promising agent to combat severe inflammation in numerous inflammatory conditions and modulating immune response.
Anticancer and antiangiogenesis
Previous studies suggest the inflammatory components surrounding cancer cells play a crucial role in tumor growth, angiogenesis, and dissemination which is linked to TLR activation in several types of cancer (55). Several studies reported the effectiveness of SsnB in inflammation-induced tumor metastasis. This has been exemplified by its ability to counter tumor growth in vitro in melanoma B16 cells by inhibiting cell migration, matrix metalloproteinase 2 (MMP2) expression, and adhesion to the extracellular matrix components collagen I, fibronectin (56). Interestingly, treatment with SsnB in vivo inhibits LPS-activated pulmonary metastasis in mice resulting in decreased metastatic nodules, weight, and lung inflammation (56). This study also showed SsnB significantly attenuates LPS-activated increases in TNF-α, IL-6, and expression of hepatic TLR-4 (56). Additionally, SsnB blocks p38 and ERK1/2 signaling pathways and significantly abates LPS-induced migration and invasion of B16 cells (56). Overexpression of N-Myc has been shown to contribute to tumorigenesis, especially in the pediatric tumor, neuroblastoma (57). SsnB treatment has been shown to reduce the ability of human neuroblastoma cells with diverse genetic background SH-SY5Y, SKNF-1, NGP, IMR-32, and SK-N-BE-2 to form tumors (57). Kumar et al. (57) demonstrated this through multiple arrests of cell cycle progression at the G2-M phase by SsnB. SsnB also induces apoptosis in these cells by generating reactive oxygen species (ROS) (57). These studies highlight the unique ability of SsnB to inhibit tumor metastasis.
Atherosclerosis is a chronic inflammatory disease characterized by endothelial injury culminating in lipid accumulation and subsequent inflammatory responses causing vascular smooth muscle cell (VSMC) migration, proliferation, and secretion of pro-inflammatory cytokines. There is evidence that activation of TLR signaling contributes to the inflammatory process in atherosclerosis and LPS-induced inflammation in VSMCs occurs via a TLR-4-dependent mechanism (58). TLR-2/4 have been detected in human atherosclerotic plaque and are involved in neo-intimal lesions under heat-shock protein 60 stimulation. In addition, endogenous TLR-4 ligands are found in lesions of ApoE-knockout mice and human coronary bypass grafts, confirming their role in atherosclerosis (59). Li et al. (60) demonstrated LPS-induced activation of the TLR-4/NF-κB pathway upregulates MMP-9 expression and migration in human aortic VSMC suggesting a role for TLR-4/NF-κB signaling in the pathogenesis of atherosclerosis. SsnB significantly attenuates the expression of MCP-1, TNF-α, and IL-6 in VSMCs stimulated with either LPS or platelet-derived growth factor (PDGF) (61). SsnB also inhibits PDGF-induced VSMC proliferation, migration, inflammatory response, and lipid accumulation (61). SsnB also inhibits the activation of Erk1/2 and Akt signaling pathways by LPS or PGDF stimulation in VSMCs (52). SsnB also suppresses the intracellular cholesterol accumulation in VSMCs loaded with acetylated low-density lipoprotein (LDL) (61). SsnB inhibits the LPS-induced up-regulation of CD36, which is responsible for lipid uptake. Similarly, SsnB dramatically reverses the LPS-induced inhibition of ATP binding cassette sub-family member 1 that promotes the efflux of intracellular free cholesterol (61). Collectively, these studies suggest a possible therapeutic application of SsnB in treating atherosclerotic disease.
Hypoxia–reoxygenation-induced cardiomyocyte inflammation
Ischemia/reperfusion (I/R) injury during myocardial infarction results in myocardial damage due to rapid increase in cytokines, chemokines, and inflammatory cell infiltration. TLRs appear to play an important role in I/R-mediated myocardial inflammation and apoptosis (62). Increased LDL levels in H9c2 cardiomyocytes and rat ventricular tissues during hypoxia were attenuated by treatment with SsnB (61). SsnB also inhibits hypoxia-induced apoptosis in rat ventricular tissue (63). Liu et al. (61) examined the role of SsnB in protecting rat cultured left ventricular tissue slices from hypoxic injury by inhibiting the myocardial inflammatory response independently of inflammatory cell infiltration. In H9c2 cardiomyocytes, SsnB downregulates TLR-2 and TLR-4-mediated inflammatory responses during hypoxia-reoxygenation injury (61). MCP-1 and IL-6 protein levels, which were increased during hypoxia, were attenuated by treatment with SsnB. SsnB also decreased the migration of mouse macrophages to injured cardiomyocytes (46). In addition, SsnB attenuated the injury-induced upregulation of MCP-1 and high mobility group box 1 protein (HMGB1) (53). SsnB treatment also reduces the hypoxia-reoxygenation activation of ERK1/2 and JNK inflammatory signaling pathways in cardiomyocytes (63). Figure 3 shows the pathophysiological protective role of SsnB in various inflammatory diseases. Collectively, these findings suggest SsnB could attenuate cardiac inflammation during hypoxic injury.
POTENTIAL APPLICATION IN OTHER INFLAMMATORY DISEASES
TLR-2 and -4 have been implicated in multiple inflammatory diseases and targeting these receptors with SsnB could reasonably have therapeutic potential. For example, TLR-4 appears to play an important role in inflammatory pulmonary conditions like asthma and chronic obstructive pulmonary disease exacerbations. There is also a strong correlation between cigarette smoke exposure and increased expression of TLR-4 and TLR-9 (64). In addition, cigarette smoke-induced neutrophil recruitment is dependent in part on TLR-4/MyD88/IL1R1 signaling (65).
TLR-4 has also been shown to play a critical role in the development of NEC. Consistent with this idea, the inhibition of TLR-4 activation enhances enterocyte proliferation and inhibits enterocyte apoptosis (66). Recent studies from our laboratory suggest activation of TLR-4 by short chain fatty acids appears to play an important role in intestinal epithelial inflammation (67). TLR-4 is also expressed on platelets and plays a crucial role in platelet number and function, specifically in the isolation of infection and release of inflammation-inducing cytokines (60). A study conducted by Ding et al. (68) demonstrated that platelet function is impaired in a mouse model of hemorrhagic shock and that TLR-4 plays a crucial role in platelet function impairment. The inhibition of TLR signaling appears to have beneficial effects on IBD progression (69). TLR-2-p, a TLR-2 TM domain-derived peptide, interacts directly with TLR-2 in cell membranes, inhibiting TLR-2-TLR-6/1-mediated activation of monocytes. This leads to significant amelioration of dextran sodium sulfate -induced colitis in mice (70). Additionally, in type 1 diabetes, TLR-4 receptor expression is key in inducing islet β cell injury with the activation of HMGB1 (71). Collectively, these results provide evidence that attenuating TLR signaling represents a potential therapeutic target in the multiple inflammatory conditions.
In summary, TLR-targeted therapies have potential to prevent and treat inflammatory diseases mediated by TLR activity and signaling. Several agents targeting TLRs activation, especially for TLR2/4, have been discovered. SsnB is a natural TLR antagonist which demonstrates multiple potentially beneficial anti-inflammatory effects on different inflammatory conditions. This review provides an overview of how SsnB's interaction with TLR 2/4 can regulate inflammation in various conditions and its potential role in developing new therapies. Although the in vivo and in vitro experimental studies described in this review greatly enhance our understanding of the molecular mechanism(s) by which SsnB attenuates TLR-4 mediated inflammation, additional studies are needed to translate these findings to the clinical arena.
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