3. Natural products that target innate immune system

Chinese herbal formulas with anti-diabetic effects are well developed such that a number of these formulas have commonly been used in diabetic patients since ancient times. The most frequently used 10 Chinese herbs in the period from 2004 to 2009, for the treatment of T2DM, include Radix Astragali, Rhizome Dioscoreae, Radix Rehnabbiae, Radix Salviae Miltiorrhizae, Radix Puerariae, Rhizoma Coptidis, Fructys Lycii, Poria, Rhizoma Alismatis, and Fructus Corni [43]. However, it is unclear whether these Chinese herbs have as potent anti-inflammatory effects as those of Western anti-diabetic drugs.

Glycyrrhiza plants (licorice) have been used as herbal medicine worldwide for over 4000 years [44]. Several studies have reported that Chinese and Japanese herbal medicines or their components regulate innate immunity. Among Glycyrrhiza plants, G. uralensis is one of the most used herbal medicines in Asian countries. Various components have been isolated from licorice, for example triterpene saponins, flavonoids, isoflavonoids, and chalcones. Therefore, we have focused on the anti-inflammatory effects of G. uralensis and its components on innate immune responses.

#### 3.1. Glycyrrhizin

mice [34, 35]. Among B cell subsets, marginal zone (MZ) B cells express high density of RP105. We have showed that RP105 is indispensable for TLR4-dependent plasma cell differentiation and IgM production in MZ B cells [36]. Additionally, M1 macrophages of murine epididymal white adipose tissue (eWAT) highly express RP105 [32]. This expression is markedly increased by HFD supplementation [32]. Furthermore, HFD-induced obesity, adipose tissue inflammation, and insulin resistance are severely attenuated in RP105 KO mice compared with wildtype (WT) and TLR4 KO mice. In contrast to TLR4, RP105 is not activated by palmitic acid [32]. Our results suggest that ligands and signaling pathways involved in RP105-mediated adipose tissue inflammation do not completely overlap with those utilized by TLR4. Future investigations will determine an endogenous ligand and a signaling pathway of RP105 in adipose

Inflammasomes are cytoplasmic receptors and play an important role in the host defense against microbial infection. Activation of NLRP3 inflammasome is regulated by various sterile stimuli, including cholesterol crystals, β-amyloid, palmitic acid, and ceramides. It is generally accepted that two signals are required for NLRP3 inflammasome activation. One is an NF-κBdependent priming step that induces the transcription of pro-IL-1β and NLRP3. Another is an activation step that induces the activation of caspase-1. Normal activation of NLRP3 inflammasome contributes to host defense, but several studies suggest that excessive activa-

Islet amyloid polypeptide (IAPP) is deposited in the pancreas and associated with the loss of β cell function in T2DM. The observation of NLRP3-dependent IL-1β production by macrophages in response to IAPP implied a potential role for NLRP3 in promoting IL-1β secretion in T2DM [37]. Interestingly, an anti-diabetic drug glyburide inhibits NLRP3 activation by macrophages in response to IAPP. Direct involvement of NLRP3 in obesity has been confirmed in studies that NLRP3 KO mice fed HFD display reduced caspase-1 activation and pro-IL-1β

C-type lectin receptors elicit inflammation and innate immune responses through activation of multiple signaling cascades. Mincle recognizes cord factor, a mycobacterial glycolipid, and transduces activation signals by associating with the Fc receptor common γ-chain, which contains immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domain. The phosphorylated ITAM recruits Syk (spleen tyrosine kinase), leading to activation

The expression level of Mincle is increased by various cellular stresses and stimuli. Mincle expression is upregulated in patients with rheumatoid arthritis and is increased in microglia, neuron, and endothelial cells in the brain after ischemic stroke [41, 42]. Furthermore, Mincle is highly expressed in inflammatory M1 macrophages in adipose tissue and involved in the induction of adipose tissue fibrosis and insulin resistance [22, 23]. These results suggest that

tion leads to the development of obesity-associated inflammation.

expression in adipose tissue compared with WT mice [38].

tissue.

2.2. NLRP3 inflammasome

124 Biological Activities and Action Mechanisms of Licorice Ingredients

2.3. C-type lectin Mincle

of NF-κB and MAP kinases [39, 40].

Glycyrrhizin (GL), a triterpene saponin, is considered to be the major biological active ingredient of G. uralensis. It has been reported that GL inhibits LPS-induced TLR4 internalization [45]. Our experiments confirmed that GL suppressed lipid A moiety of LPS-induced IL-6 production in mouse macrophages [24]. Furthermore, GL treatment significantly suppressed the production of inflammatory cytokines, including TNF-α and IL-6, in LPS-injected mice [24]. We further demonstrated that GL attenuated lipid A-mediated activation of NF-κB and MAPKs, including JNK, p38, and ERK. It was suggested that GL might be incorporated into lipid bilayers and suppress the plasma membrane integrity [45]. LPS binds to MD-2 and this triggers homodimerization of the TLR4/MD-2 complex, resulting in the induction of signal transduction [46]. Our data demonstrated that GL inhibited LPS binding to the complex in a dose-dependent manner. Accordingly, LPS-induced TLR4 homodimerization was suppressed by GL stimulation. Moreover, GL inhibited not only LPS- but also TLR9 ligand CpG-DNAinduced inflammatory responses (our unpublished data), as suggested previously [45]. Thus, GL inhibits the activation of multiple TLRs at the plasma membrane by altering membrane integrity.

GL also inhibits the activation of another PRR, NLRP3 inflammasome, induced by various stimuli, including adenosine triphosphate (ATP), monosodium urate (MSU) and nigericin [25]. LPS is a potent inducer of the priming of the NLRP3 inflammasome. However, the inhibitory activity of GL on NLRP3 inflammasome is independent of its effect on TLR4 activation [25]. Intriguingly, activation of another inflammasome, absent in melanoma 2 (AIM2) inflammasome, was suppressed by GL stimulation [25], suggesting that GL inhibits multiple inflammasomes activation. However, a high concentration of GL is required for these inhibitory effects on inflammasomes. Therefore, GL may not be suggested to be a potential therapeutic agent for the treatment of obesity-associated inflammation.

### 3.2. Isoliquiritigenin

ILG, another component of G. uralensis, is a flavonoid with a chalcone structure. It has a wide variety of biological activities, including anti-allergic, anti-tumor growth, and anti-platelet aggregation activity. We recently demonstrated that ILG potently inhibited activation of both TLR4/MD-2 and NLRP3 inflammasome [24, 25]. ILG significantly suppressed LPS-induced production of inflammatory cytokines not only in vitro but also in vivo [24]. Whereas GL stimulation affects the binding of LPS to TLR4/MD-2, the amount of bound LPS was not decreased by ILG stimulation. However, LPS-induced homotypic interaction of TLR4 was potently inhibited by ILG stimulation in a dose-dependent manner. Additionally, ILG blocked lipid A-induced NF-κB and MAPKs activation [24]. This may be due to the inhibition of TLR4 homodimerization. Beside this, it has been reported that ILG interacts with IKK directly and inhibits its kinase activity [47]. Thus, ILG suppresses TLR4/MD-2-mediated immune responses in multiple steps, at the receptor level and the downstream signaling level (Figure 1).

#### 3.2.1. Inhibitory effects of isoliquiritigenin on PRR-mediated adipose tissue inflammation

In addition to the inhibitory effects of ILG on the TLR4/MD-2 complex, we demonstrated that it was highly effective in inhibiting NLRP3 inflammasome activation by various stimuli including ATP, MSU, and nigericin [25]. These responses were suppressed by a low concentration of ILG (10∼30 μM). The IC50 for the inhibitory effect of ILG is 0.4936 μM. Furthermore, ILG was more effective in inhibiting NLRP3 inflammasome activation than GL (IC50, 358.9 μM) and parthenolide, a known inhibitor of NLRP3 inflammasome. Contrary to GL, ILG did not inhibit poly(dA:dT)-induced activation of the AIM2 inflammasome. As ILG suppresses NLRP3- but not AIM2-induced formation of ASC pyroptosome, it is unlikely that ASC is a molecular target of ILG. IAPP is a unique polypeptide constituent of amyloid deposited in pancreatic islets [48, 49]. This deposition is associated with disease progression of T2DM and triggers NLRP3 inflammasome activation in islet macrophages [37]. The sulfonylurea drug glyburide inhibits IAPP-induced activation of NLRP3 inflammasome [37]. It is noteworthy that a low concentration of ILG is more effective in inhibiting IAPP-induced activation of NLRP3 inflammasome than that of glyburide [25] (Figure 1).

The above observations led us to investigate the inhibitory effects of ILG on TLR4- and NLRP3 associated inflammation in obesity. ILG supplementation remarkably improved obesity, hyperlipidemia, hepatic steatosis, and insulin resistance in HFD-fed wild-type mice [25].

Isoliquiritigenin: A Unique Component That Attenuates Adipose Tissue Inflammation and Fibrosis... http://dx.doi.org/10.5772/66727 127

activity of GL on NLRP3 inflammasome is independent of its effect on TLR4 activation [25]. Intriguingly, activation of another inflammasome, absent in melanoma 2 (AIM2) inflammasome, was suppressed by GL stimulation [25], suggesting that GL inhibits multiple inflammasomes activation. However, a high concentration of GL is required for these inhibitory effects on inflammasomes. Therefore, GL may not be suggested to be a potential thera-

ILG, another component of G. uralensis, is a flavonoid with a chalcone structure. It has a wide variety of biological activities, including anti-allergic, anti-tumor growth, and anti-platelet aggregation activity. We recently demonstrated that ILG potently inhibited activation of both TLR4/MD-2 and NLRP3 inflammasome [24, 25]. ILG significantly suppressed LPS-induced production of inflammatory cytokines not only in vitro but also in vivo [24]. Whereas GL stimulation affects the binding of LPS to TLR4/MD-2, the amount of bound LPS was not decreased by ILG stimulation. However, LPS-induced homotypic interaction of TLR4 was potently inhibited by ILG stimulation in a dose-dependent manner. Additionally, ILG blocked lipid A-induced NF-κB and MAPKs activation [24]. This may be due to the inhibition of TLR4 homodimerization. Beside this, it has been reported that ILG interacts with IKK directly and inhibits its kinase activity [47]. Thus, ILG suppresses TLR4/MD-2-mediated immune responses in multiple steps, at the receptor level and the downstream signaling

3.2.1. Inhibitory effects of isoliquiritigenin on PRR-mediated adipose tissue inflammation

In addition to the inhibitory effects of ILG on the TLR4/MD-2 complex, we demonstrated that it was highly effective in inhibiting NLRP3 inflammasome activation by various stimuli including ATP, MSU, and nigericin [25]. These responses were suppressed by a low concentration of ILG (10∼30 μM). The IC50 for the inhibitory effect of ILG is 0.4936 μM. Furthermore, ILG was more effective in inhibiting NLRP3 inflammasome activation than GL (IC50, 358.9 μM) and parthenolide, a known inhibitor of NLRP3 inflammasome. Contrary to GL, ILG did not inhibit poly(dA:dT)-induced activation of the AIM2 inflammasome. As ILG suppresses NLRP3- but not AIM2-induced formation of ASC pyroptosome, it is unlikely that ASC is a molecular target of ILG. IAPP is a unique polypeptide constituent of amyloid deposited in pancreatic islets [48, 49]. This deposition is associated with disease progression of T2DM and triggers NLRP3 inflammasome activation in islet macrophages [37]. The sulfonylurea drug glyburide inhibits IAPP-induced activation of NLRP3 inflammasome [37]. It is noteworthy that a low concentration of ILG is more effective in inhibiting IAPP-induced activation of NLRP3 inflammasome

The above observations led us to investigate the inhibitory effects of ILG on TLR4- and NLRP3 associated inflammation in obesity. ILG supplementation remarkably improved obesity, hyperlipidemia, hepatic steatosis, and insulin resistance in HFD-fed wild-type mice [25].

peutic agent for the treatment of obesity-associated inflammation.

126 Biological Activities and Action Mechanisms of Licorice Ingredients

3.2. Isoliquiritigenin

level (Figure 1).

than that of glyburide [25] (Figure 1).

Figure 1. Schematic diagram of ILG-mediated suppression of PRR activation in macrophage. NLRP3 inflammasome is activated by various DAMPs, including IAPP and free FAs. The inflammasomes assemble into oligomeric complex with ASC and activate caspase-1. Activated caspase-1 processes pro-IL-1β into mature IL-1β. ILG potently inhibits IAPP-induced NLRP3 inflammasome activation. The LPS sensor TLR4/MD-2 also recognizes free FAs such as palmitic acid. ILG inhibits TLR4/MD-2-induced inflammation and fibrogenic responses in multiple steps, at the receptor level and the downstream signaling level. Mincle recognizes a mycobacterial glycolipid TDM, also named cord factor. TDM-stimulated inflammatory and fibrogenic responses are attenuated by ILG treatment. Because HFD-induced adipose tissue inflammation and fibrosis are greatly improved in Mincle-deficient mice compared with WT mice, it is suggested that Mincle may recognize an endogenous ligand derived from inflamed tissues, resulting in the induction of inflammation and fibrosis.

Furthermore, ILG supplementation inhibited IL-1β and caspase-1 production in eWAT from wild-type mice fed with HFD for 4 weeks. At this time point, TNF-α production was not increased in eWAT from HFD-fed mice compared with that from normal diet-fed mice. Thus, inflammasome activation occurs in eWAT at an early time point during obesity before TNF-αassociated inflammation.

Because ILG inhibits the priming and activation steps of the inflammasome via TLR4 and NLRP3, respectively, the therapeutic effects of ILG on HFD-induced adipose tissue inflammation and insulin resistance may be attributed to the inhibition of not only NLRP3 but also TLR4 pathways. Indeed, TLR4 is shown to have a critical role in the pathogenesis of obesity-induced inflammation using TLR4-deficient mice [13, 29]. Therefore, ILG is a potential therapeutic agent that targets NLRP3- and TLR4-associated adipose tissue inflammation in obesity.

#### 3.2.2. Inhibitory effects of isoliquiritigenin on PRR-mediated adipose tissue fibrosis

In addition to inflammation, fibrosis may have an important role in adipose tissue dysfunction [9]. Because TLR4 signaling in immune cells has a key role in the development of obesity- and endotoxin-mediated adipose tissue fibrosis [50], we examined whether ILG attenuated TLR4 stimulated expression of fibrosis-related genes in peritoneal macrophages and stromal vascular fraction (SVF) of obese eWAT. Lipid A stimulation increased the expression of fibrosisrelated genes, such as TGF-β and TIMP-1 (tissue inhibitor of metalloproteinase-1) in these cells. These increases were significantly attenuated by ILG stimulation (Figure 1).

Mincle stimulation is also crucial for fibrogenesis in SVF of obese adipose tissue [22]. The SVF from HFD-fed mice was stimulated with a Mincle ligand trehalose-6,6′-dimycolate (TDM), a mycobacterial cell wall glycolipid [22, 51]. TDM stimulation significantly increased TIMP-1 and PDGF-B mRNA expression in the SVF and these increases were significantly attenuated by ILG stimulation [51] (Figure 1).

Finally, we examined whether ILG improved HFD-induced adipose tissue fibrosis. Histological analysis revealed that HFD treatment induced extensive interstitial fibrosis in eWAT, which was markedly suppressed by ILG supplementation (0.5% w/w in HFD) [51]. HFD treatment increased collagen 1, TGF-β, TIMP-1, and PDGF-B mRNA expression in eWAT. These expressions were markedly decreased by ILG supplementation. These results highlight that ILG is a promising therapeutic candidate for HFD-induced adipose tissue fibrosis by suppressing the activation of innate immune sensors.
