2. PRRs that link with adipose tissue inflammation and fibrosis

#### 2.1. Toll-like receptors

crucial role in the pathogenesis of obesity-associated metabolic dysfunction [3, 4]. The chronic inflammatory alternations are associated with dynamic changes in the composition and function of immune cells in various tissues such as adipose tissue, pancreatic islet, liver, muscle, and hypothalamus [5–7]. A large number of inflammatory immune cells infiltrate into adipose tissue during the course of obesity. M1-like macrophages, an inflammatory type of macrophage, accumulate in adipose tissue and are major sources of inflammatory mediators such as

The connective fiber content of adipose tissue dramatically increases by the upregulation of collagen expression, which in turn elevates the overall rigidity of adipose tissue and finally leads to fibrosis. The deficiency of collagen 6, a key component of the extracellular matrix in adipose tissue, significantly improves the phenotypes of obese mice, including adipocyte death and adipose tissue inflammation [9]. This implies that alterations in the extracellular matrix in adipose tissue are linked to the development of inflammation. Thus, inflammation

An obese state results in an elevation of circulating levels of fatty acids (FAs) and, subsequently, an increase in inflammation of adipose tissue [10]. Adipose macrophages play critical roles in the immune responses through several FA-sensing mechanisms, such as pattern recognition receptors (PRRs). PRRs such as toll-like receptors (TLRs) and nucleotide oligomerization domain (NOD)-like receptors (NLRs) quickly recognize pathogenic agents [11]. It is now becoming even more apparent that these PRRs are not only able to recognize microbial components but also mediate immune responses to endogenous molecules, including those arising in metabolic disorders, such as FAs. These endogenous molecules have been termed danger-associated molecular patterns (DAMPs) and have similar functions as microbial components to activate immune responses [12]. In the obese state, TLR4 may be activated by saturated free FAs, such as palmitic acid, derived from hypertrophied adipocytes as a DAMP and promote adipose tissue inflammation and insulin resistance [13–15]. Inflammasomes are multimeric protein complexes that are crucial for caspase-1, IL-1β, and IL-18 production [16]. The nucleotide-binding domain, leucine-rich repeats containing family, pyrin domaincontaining-3 (NLRP3) inflammasome also senses obesity-associated FAs and contributes to obesity-induced inflammation and insulin resistance [17, 18]. Moreover, IL-1β inhibits insulin signaling in the insulin-target organs, including adipose tissue, liver, and skeletal muscle, and also induces dysfunction and cell death of insulin-producing pancreatic β cells [19]. Macrophage-inducible C-type lectin (Mincle) recognizes not only cord factor, a mycobacterial glycolipid, but also SAP130 released from dead cells [20, 21]. Furthermore, Mincle is highly expressed in M1 macrophages in adipose tissue and involved in the induction of adipose tissue

Plant-derived natural products and their derivatives or synthetic mimics make up a considerable portion of current drugs. These products have played an important role in treating T2DM, especially in Asian countries. We previously reported that glycyrrhizin (GL) and isoliquiritigenin (ILG), components of Glycyrrhiza uralensis (G. uralensis), inhibit TLR4 signaling at the receptor level on the cell surface, resulting in inhibition of NF-κB and mitogen-activated protein kinases (MAPKs) activation [24]. Furthermore, ILG potently inhibits NLRP3

tumor necrosis factor (TNF)-α and IL-6 [8].

122 Biological Activities and Action Mechanisms of Licorice Ingredients

and fibrosis are important targets for the treatment of obesity.

fibrosis and insulin resistance during obesity [22, 23].

TLRs are transmembrane proteins that recognize conserved structural moieties of microorganisms and for the subsequent induction of pro-inflammatory responses [26]. They directly bind to their ligands and activate the NF-κB and MAPK pathways to induce the production of proinflammatory cytokines that are important for evading pathogens. It is widely suggested that TLRs also sense non-microbial endogenous ligands, such as dietary FAs [12]. Activation of TLRs by the endogenous ligands similarly induces pro-inflammatory pathways as microbial ligands in various organs, such as adipose tissue and the liver.

TLR4 is the most important TLR for LPS-mediated inflammatory responses [27]. There is a body of evidence suggesting that TLR4 is an attractive candidate for linking innate immune responses to obesity-associated dysfunction. For example, TLR4 expression is increased in inflammatory macrophages derived from obese adipose tissue [13, 28]. TLR4 KO mice or mice with a loss-of-function mutation in the TLR4 gene are protected from obesity-associated insulin resistance [13, 29]. Furthermore, hematopoietic cell-specific deletion of TLR4 ameliorates HFD-induced hepatic insulin resistance [30]. Intriguingly, saturated FAs released by adipocyte lipolysis can be endogenous TLR4 ligands and activate the NF-κB pathway on macrophages [15]. Another paper reported that resistin derived from adipose tissue directly bound to TLR4 in the hypothalamus and leads to the activation of MAPKs signaling and promoting insulin resistance through MyD88 [31], suggesting that resistin is an endogenous TLR4 ligand, which links hypothalamic inflammation with insulin resistance.

We previously demonstrated that the development of obesity-related inflammation required radioprotective 105 (RP105) rather than TLR4 [32]. RP105 was identified as a first mammalian homologue of Drosophila toll that expressed on B cells [33] and suggested to be involved in LPS-induced B-cell responses. In fact, RP105-deficient mice show reduced LPS-dependent proliferation and CD86 upregulation in B cells, albeit to a lesser extent than TLR4-deficient 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 tissue.

#### 2.2. NLRP3 inflammasome

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 activation leads to the development of obesity-associated inflammation.

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

#### 2.3. C-type lectin Mincle

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 of NF-κB and MAP kinases [39, 40].

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 Mincle is involved in the pathogenesis of various inflammation and represents a potential target molecule for the treatment of inflammatory diseases, including rheumatoid arthritis, brain infarction, and T2DM.
