Betulin Attenuates TGF-ß1- and PGE2-Mediated Inhibition of NK Cell Activity to Suppress Tumor Progression and Metastasis in Mice.

Transforming growth factor (TGF)-ß1 and prostaglandin E2 (PGE2) are humoral factors critically involved in the induction of immunosuppression in the microenvironment of various types of tumors, including melanoma. In this study, we identified a natural compound that attenuated TGF-ß1- and PGE2-induced immunosuppression and examined its effect on B16 melanoma growth in mice. By screening 502 natural compounds for attenuating activity against TGF-ß1- or PGE2-induced suppression of cytolysis in poly(I:C)-stimulated murine splenocytes, we found that betulin was the most potent compound. Betulin also reduced TGF-ß1- and PGE2-induced downregulation of perforin and granzyme B mRNA expression and cell surface expression of NKG2D and CD69 in natural killer (NK) cells. Cell depletion and coculture experiments showed that NK cells, dendritic cells, B cells, and T cells were necessary for the attenuating effects of betulin. Structure-activity relationship analysis revealed that two hydroxyl groups at positions C3 and C28 of betulin, their cis-configuration, and methyl group at C30 played crucial roles in its attenuating activity. In a subcutaneous implantation model of B16 melanoma in mice, intratumor administration of betulin and LY2157299, a TGF-ß1 type I receptor kinase inhibitor, significantly retarded the growth of B16 melanoma. Notably, betulin increased significantly the number of CD69 positive NK cells in tumor sites at early stages of post-tumor cell injection. Our data suggest that betulin inhibits the growth of B16 melanoma by enhancing NK cell activity through attenuating the immunosuppressive tumor microenvironment.

Roles of Macrophages in Advanced Liver Fibrosis, Identified Using a Newly Established Mouse Model of Diet-Induced Non-Alcoholic Steatohepatitis.

Macrophages play critical roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). However, it is unclear which macrophage subsets are critically involved in the development of inflammation and fibrosis in NASH. In TSNO mice fed a high-fat/cholesterol/cholate-based diet, which exhibit advanced liver fibrosis that mimics human NASH, we found that Kupffer cells (KCs) were less abundant and recruited macrophages were more abundant, forming hepatic crown-like structures (hCLS) in the liver. The recruited macrophages comprised two subsets: CD11c+/Ly6C- and CD11c-/Ly6C+ cells. CD11c+ cells were present in a mesh-like pattern around the lipid droplets, constituting the hCLS. In addition, CD11c+ cells colocalized with collagen fibers, suggesting that this subset of recruited macrophages might promote advanced liver fibrosis. In contrast, Ly6C+ cells were present in doughnut-like inflammatory lesions, with a lipid droplet in the center. Finally, RNA sequence analysis indicates that CD11c+/Ly6C- cells promote liver fibrosis and hepatic stellate cell (HSC) activation, whereas CD11c-/Ly6C+ cells are a macrophage subset that play an anti-inflammatory role and promote tissue repair in NASH. Taken together, our data revealed changes in liver macrophage subsets during the development of NASH and shed light on the roles of the recruited macrophages in the pathogenesis of advanced fibrosis in NASH.

Bone marrow transplantation into Abcd1-deficient mice: Distribution of donor derived-cells and biological characterization of the brain of the recipient mice.

X-linked adrenoleukodystrophy (X-ALD) is a severe inherited metabolic disease with cerebral inflammatory demyelination and abnormal accumulation of very long chain fatty acid (VLCFA) in tissues, especially the brain. At present, bone marrow transplantation (BMT) at an early stage of the disease is the only effective treatment for halting disease progression, but the underlying mechanism of the treatment has remained unclear. Here, we transplanted GFP-expressing wild-type (WT) or Abcd1-deficient (KO) bone marrow cells into recipient KO mice, which enabled tracking of the donor GFP+ cells in the recipient mice. Both the WT and KO donor cells were equally distributed throughout the brain parenchyma, and displayed an Iba1-positive, GFAP- and Olig2-negative phenotype, indicating that most of the donor cells were engrafted as microglia-like cells. They constituted approximately 40% of the Iba1-positive cells. Unexpectedly, no decrease of VLCFA in the cerebrum was observed when WT bone marrow cells were transplanted into KO mice. Taken together, murine study suggests that bone marrow-derived microglia-like cells engrafted in the cerebrum of X-ALD patients suppress disease progression without evidently reducing the amount of VLCFA in the cerebrum.

Bidirectional crosstalk between neutrophils and adipocytes promotes adipose tissue inflammation.

Chronic activation of the IL-1ß system in adipose tissue on metabolic disorders is well demonstrated. However, a mechanism for its expression and activation in the tissue has remained unexplored. Here, we demonstrate that IL-1ß transcript was enriched in neutrophils of white adipose tissue (WAT) from lean mice. Mechanistically, the interaction of neutrophils with adipocytes induced IL-1ß expression via NF-κB pathway. Lipolysis of adipocytes accumulated neutrophils prior to macrophages in WAT and produced high levels of IL-1ß via an inflammasome pathway. Leukotriene B4 (LTB4) production in WAT also contributed to neutrophil accumulation. Furthermore, an LTB4-inflammasome axis contributed to the expression of chemotactic molecules involved in high-fat diet-induced macrophage infiltration into WAT. We have identified previously unappreciated roles for neutrophils in the development of adipose tissue inflammation: robust IL-1ß production and infiltration of macrophages to initiate chronic inflammation.-Watanabe, Y., Nagai, Y., Honda, H., Okamoto, N., Yanagibashi, T., Ogasawara, M., Yamamoto, S., Imamura, R., Takasaki, I., Hara, H., Sasahara, M., Arita, M., Hida, S., Taniguchi, S., Suda, T., Takatsu, K. Bidirectional crosstalk between neutrophils and adipocytes promotes adipose tissue inflammation.

Funiculosin variants and phosphorylated derivatives promote innate immune responses via the Toll-like receptor 4/myeloid differentiation factor-2 complex.

The Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) complex is essential for LPS recognition and induces innate immune responses against Gram-negative bacteria. As activation of TLR4/MD-2 is also critical for the induction of adaptive immune responses, TLR4/MD-2 agonists have been developed as vaccine adjuvants, but their efficacy has not yet been ascertained. Here, we demonstrate that a funiculosin (FNC) variant, FNC-RED, and FNC-RED and FNC derivatives are agonists for both murine and human TLR4/MD-2. FNC-RED induced nuclear factor-κB (NF-κB) activation via murine TLR4/MD-2, whereas FNC had no TLR4/MD-2 stimulatory activity. Biacore analysis revealed that FNC-RED binds to murine TLR4/MD-2 but not murine radioprotective 105 (RP105)/myeloid differentiation factor-1 (MD-1), another LPS sensor. FNC-RED induced CD14-independent expressions of pro-inflammatory cytokines and co-stimulatory molecules in murine macrophages and dendritic cells. In contrast, FNC-RED stimulation was reduced in CD14-dependent LPS responses, including dimerization and internalization of TLR4/MD-2 and IFN-ß expression. FNC-RED-induced IL-12p40 production from murine dendritic cells was dependent on NF-κB but not MAPK pathway. In addition, fetal bovine serum augmented lipid A-induced NF-κB activation but blocked FNC-RED-mediated responses. Two synthetic phosphate group-containing FNC-RED and FNC derivatives, FNC-RED-P01 and FNC-P01, respectively, activated human TLR4/MD-2, unlike FNC-RED. Finally, computational analysis revealed that this species-specific activation by FNC-RED and FNC-RED-P01 resulted from differences in electrostatic surface potentials between murine and human TLR4/MD-2. We conclude that FNC-RED and its synthetic derivative represent a novel category of murine and human TLR4/MD-2 agonist.

Inflammatory responses increase secretion of MD-1 protein.

Radioprotective 105 (RP105) is a type I transmembrane protein, which associates with a glycoprotein, MD-1. Monoclonal antibody (mAb)-mediated ligation of RP105/MD-1 robustly activates B cells. RP105/MD-1 is structurally similar to Toll-like receptor 4 (TLR4)/MD-2. B-cell responses to TLR2 and TLR4/MD-2 ligands are impaired in the absence of RP105 or MD-1. In addition to RP105/MD-1, MD-1 alone is secreted. The structure of MD-1 shows that MD-1 has a hydrophobic cavity that directly binds to phospholipids. Little is known, however, about a ligand for MD-1 and the role of MD-1 in vivo To study the role of RP105/MD-1 and MD-1 alone, specific mAbs against MD-1 are needed. Here, we report the establishment and characterization of two anti-MD-1 mAbs (JR2G9, JR7G1). JR2G9 detects soluble MD-1, whereas JR7G1 binds both soluble MD-1 and the cell surface RP105/MD-1 complex. With these mAbs, soluble MD-1 was detected in the serum and urine. The MD-1 concentration was altered by infection, diet and reperfusion injury. Serum MD-1 was rapidly elevated by TLR ligand injection in mice. The quantitative PCR and supernatant-precipitated data indicate that macrophages are one of the sources of serum soluble MD-1. These results suggest that soluble MD-1 is a valuable biomarker for inflammatory diseases.

Glycyrrhetinic acid inhibits contact hypersensitivity induced by trichophytin via dectin-1.

Trichophyton infection is highly prevalent and tends to be recurrent. Therefore, it is important to develop new therapeutic agents. Previously, we established a mouse model of Trichophyton-induced contact hypersensitivity (CHS) and demonstrated that dectin-1 was involved in inflammation induced by trichophytin, the Trichophyton antigen. Here, we used that model to investigate glycyrrhetinic acid (GA) from plants of the genus Glycyrrhiza as a potential anti-inflammatory agent against superficial mycoses. GA suppressed swelling and the expression of inflammatory cytokines, including macrophage inflammatory protein (MIP)-2, interleukin (IL)-6, tumor necrosis factor (TNF)-α and interferon (IFN)-γ mRNA. Anti-MIP-2 antibody suppressed trichophytin-induced inflammation, and antidectin-1 antibody suppressed zymosan-induced MIP-2 production in keratinocyte cells. These results suggest that MIP-2 is produced by dectin-1 activation and is involved in inflammation associated with CHS to trichophytin. GA also suppressed zymosan-induced MIP-2 and interleukin (IL)-8, production in mouse and human macrophages and keratinocytes. Furthermore, GA suppressed the phosphorylation of spleen tyrosine kinase (Syk) and inhibitor of nuclear factor-kappa B (IκBα) and the degradation of IκBα in zymosan-simulated RAW264.7 cells. The results of this study suggest that GA suppresses inflammation induced by trichophytin, partly by the downregulation of Syk phosphorylation.

Evaluating the role of conserved amino acids in bacterial O-oligosaccharyltransferases by in vivo, in vitro and limited proteolysis assays.

Bacterial O-Oligosaccharyltransferases (O-OTases) constitute a growing family of enzymes that catalyze the transfer of a glycan from a lipid carrier to protein acceptors. O-OTases are inner membrane proteins that display limited sequence similarity, except for the Wzy_C signature domain also present in a predicted periplasmic loop of the WaaL ligase, the enzyme responsible for transferring the O antigen to the lipid A core. The mechanism of O-OTase-dependent glycosylation is poorly understood. In this work, conserved amino acid residues in the O-OTases were replaced with alanine in PglL, the O-OTase of Neisseria meningitidis. The activity of wild-type PglL and its mutant derivatives were analyzed in vivo in engineered Escherichia coli cells, and in in vitro assays. We identified two additional sites of pilin glycosylated exclusively by PglL in E. coli. Both sites are modified with phosphoglycerol (PG) by different enzymes in Neisseria gonorrhoeae and Neisseria meningitidis. Limited proteolysis experiments revealed a conformational change that is triggered upon interaction of the C-terminal region of PglL with the lipid-linked oligosaccharide (LLO) substrate. These experiments showed that Q178 and Y405 are required for optimal function, whereas H349 is essential for activity and plays a critical role in the interaction with LLO. The equivalent His residue is also essential for WaaL activity, which suggests a common mechanism for both enzymes, and supports the hypothesis that O-glycosylation and lipopolysaccharide (LPS) synthesis are evolutionarily related. These results contribute to the elucidation of the mechanism of O-OTases, which are promising targets for novel antibiotics and present an enormous potential for glycoengineering novel vaccines and therapeutics.

The RP105/MD-1 complex is indispensable for TLR4/MD-2-dependent proliferation and IgM-secreting plasma cell differentiation of marginal zone B cells.

Marginal zone (MZ) B cells mount rapid T-cell-independent (T-I) immune responses against microbial components such as LPS. While Toll-like receptor 4 (TLR4) is essential for LPS responses, MZ B cells uniquely express high levels of another LPS sensor Radioprotective 105 (RP105). However, little is known about how RP105 is used by MZ B cells. In this study, we investigated TLR4- or RP105-dependent MZ B cell responses by utilizing agonistic monoclonal antibodies (mAbs) to each receptor. Cross-linking TLR4 and RP105 at the same time with the mAbs induced robust IgM-secreting plasma cell generation as lipid A moiety of LPS. In contrast, stimulation with either mAb alone did not elicit such responses. RP105-deficient MZ B cells failed to produce IgM-secreting plasma cells in response to lipid A. TLR4 or lipid A stimulation of MZ B cells up-regulated their B lymphocyte-induced maturation protein 1 (Blimp-1) and X-box-binding protein 1 (Xbp-1) mRNA expression. RP105 stimulation alone did not give these responses and in fact decreased TLR4-mediated their expression. Compared with wild-type (WT) MZ B cells, RP105-deficient MZ B cells exhibited increased levels of Blimp-1 and Xbp-1 mRNA expression in response to lipid A. Lipid A or TLR4 plus RP105 stimulation induced massive proliferation and expression of Bcl-xL and c-Myc in WT but not RP105-deficient MZ B cells. These responses contributed to TLR4-mediated anti-apoptotic responses in MZ B cells. Thus, RP105 contributes in a unique way to the TLR4-dependent survival, proliferation and plasma cell generation of MZ B cells.

Isoliquiritigenin Attenuates Adipose Tissue Inflammation and Metabolic Syndrome by Modifying Gut Bacteria Composition in Mice.

SCOPE: Isoliquiritigenin (ILG) has been reported to attenuate adipose tissue inflammation and metabolic disorder; however, the underlying mechanisms remain to be elucidated. The aim of this study is to elucidate whether ILG shows the anti-inflammatory and antimetabolic syndrome effects through gut microbiota modification. METHODS AND RESULTS: Mice are fed a high-fat diet (HFD) with or without ILG for up to 12 weeks. The effect of ILG on body weight, blood glucose level, adipose tissue inflammation, gut barrier function, and gut microbiota composition are investigated. ILG supplementation alleviates HFD-induced obesity, glucose tolerance, and insulin resistance and suppresses inflammatory gene expression in epididymal white adipose tissue (eWAT). Moreover, ILG supplementation modifies gut bacterial composition by increasing the abundance of antimetabolic disease-associated species (e.g., Parabacteroides goldsteinii and Akkemansia muciniphila) and up-regulated genes associated with gut barrier function. Fecal microbiome transplantation (FMT) from ILG-fed donors counteract HFD-induced body and eWAT weight changes, inflammation-related gene expression, glucose tolerance, and insulin resistance, thereby suggesting that ILG-responsive gut bacteria exerts anti-inflammatory and antimetabolic syndrome effects. CONCLUSION: Alterations in gut bacteria underly the beneficial effects of ILG against adipose tissue inflammation and metabolic disorders. ILG may be a promising prebiotic for the prevention and treatment of metabolic syndrome.

The function of rhamnose-binding lectin in innate immunity by restricted binding to Gb3.

L-rhamnose-binding lectins (RBLs) have been isolated from various kinds of fish and invertebrates and interact with various kinds of bacteria, suggesting RBLs are involved in various inflammatory reactions. We investigated the effect of RBLs from chum salmon (Oncorhynchus keta), named CSL1, 2 and 3, on the peritoneal macrophage cell line from rainbow trout (Oncorhynchus mykiss) (RTM5) and an established fibroblastic-like cell line derived from gonadal tissue of rainbow trout (RTG-2). CSLs were bound to the surface of RTM5 and RTG-2 cells and induced proinflammatory cytokines, including IL-1beta1, IL-1beta2, TNF-alpha1, TNF-alpha2 and IL-8 in both cells by recognizing globotriaosylceramide (Gb3). In addition, CSLs had an opsonic effect on RTM5 cells and this effect was significantly inhibited by L-rhamnose, indicating that CSLs enhanced their phagocytosis by binding to Gb3 on cell surfaces. This is the first finding that Gb3 plays a role in innate immunity by cooperating with natural ligands, RBLs.

Structural characterization of a rhamnose-binding glycoprotein (lectin) from Spanish mackerel (Scomberomorous niphonius) eggs.

A rhamnose-binding glycoprotein (lectin), named SML, was isolated from the eggs of Spanish mackerel (Scomberomorous niphonius) by affinity and ion-exchange chromatographies. SML was composed of a non-covalently linked homodimer. The SML subunit was composed of 201 amino acid residues with two tandemly repeated domains, and contained 8 half-Cys residues in each domain, which is highly homologous to the N-terminal lectin domain of calcium-independent alpha-latrotoxin receptor in mammalian brains. Each domain has the same disulfide bonding pattern; Cys10-Cys40, Cys20-Cys99, Cys54-Cys86 and Cys67-Cys73 were located in the N-terminal domain, and Cys108-Cys138, Cys117-Cys195, Cys152-Cys182 and Cys163-Cys169 were in the C-terminal domain. SML was N-glycosylated at Asn168 in the C-terminal domain. The structure of the sugar chain was determined to be NeuAc-Galbeta1-4GlcNAcbeta1-2Manalpha1-6-(NeuAc-Galbeta1-4GlcNAcbeta1-2Manalpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAc-Asn.

Isolation and characterization of l-rhamnose-binding lectin, which binds to microsporidian Glugea plecoglossi, from ayu (Plecoglossus altivelis) eggs.

A rhamnose-binding lectin, named SFL, was isolated from the eggs of ayu (sweet fish, Plecoglossus altivelis) by affinity and ion-exchange chromatographies. SFL revealed 287 amino acid residues with 3 tandemly repeated domains, and contained 8 half-Cys residues in each domain. The lectin was shown to have a highly specific binding affinity to globotriaosylceramide (Gb3) by frontal affinity chromatography using 100 oligosaccharides. SFL was localized in several tissues and serum of both male and female ayu, such as gill, liver, ovary, testis, intestine, stomach, brain, kidney and serum. The lectin agglutinated the spores of the microsporidian Glugea plecoglossi, which is a pathogen of ayu. Although SFL bound to glycoproteins and glycolipids of G. plecoglossi spores, Gb3 could not be detected in either of them. The results suggest that SFL could interact with various glycoconjugates of pathogens to play a role in the adhesion of microorganisms invading in the body.

Quantitative analysis of the relation between soft tissue stiffness palpated from the body surface and tissue hemodynamics in the human forearm.

We investigated the quantitative relation between soft tissue stiffness palpated from the body surface and hemodynamics in the human forearm. We examined the relation between pressures and blood flow in both the main artery and vein measured by magnetic resonance imaging (MRI), the cross-sectional area of forearm measured by MRI and soft tissue stiffness. Six male volunteers participated. Two tourniquet pressures, 120 mmHg and 230 mmHg, were used to induce an occlusion of the proximal portion of the upper arm. Measurements were made at the mid-belly of the brachioradial muscle. The venous outflow ceased at tourniquet pressures of 120 and 230 mmHg. The arterial flow was interrupted at 230 mmHg. Larger increases of the cross-sectional area and soft tissue stiffness were found at 120 mmHg than at 230 mmHg. The increase of the cross-sectional area of muscle was larger than that of the surrounding connective tissue during occlusion. We propose that low-pressure compression occludes venous outflow without restricting arterial inflow and induces an increase of the cross-sectional area that reflects the intramuscular pressure; and changes in this pressure caused by fluid accumulation should be the major factor for change in stiffness.

Isoliquiritigenin Attenuates Adipose Tissue Inflammation in vitro and Adipose Tissue Fibrosis through Inhibition of Innate Immune Responses in Mice.

Isoliquiritigenin (ILG) is a flavonoid derived from Glycyrrhiza uralensis and potently suppresses NLRP3 inflammasome activation resulting in the improvement of diet-induced adipose tissue inflammation. However, whether ILG affects other pathways besides the inflammasome in adipose tissue inflammation is unknown. We here show that ILG suppresses adipose tissue inflammation by affecting the paracrine loop containing saturated fatty acids and TNF-α by using a co-culture composed of adipocytes and macrophages. ILG suppressed inflammatory changes induced by the co-culture through inhibition of NF-κB activation. This effect was independent of either inhibition of inflammasome activation or activation of peroxisome proliferator-activated receptor-γ. Moreover, ILG suppressed TNF-α-induced activation of adipocytes, coincident with inhibition of IκBα phosphorylation. Additionally, TNF-α-mediated inhibition of Akt phosphorylation under insulin signaling was alleviated by ILG in adipocytes. ILG suppressed palmitic acid-induced activation of macrophages, with decreasing the level of phosphorylated Jnk expression. Intriguingly, ILG improved high fat diet-induced fibrosis in adipose tissue in vivo. Finally, ILG inhibited TLR4- or Mincle-stimulated expression of fibrosis-related genes in stromal vascular fraction from obese adipose tissue and macrophages in vitro. Thus, ILG can suppress adipose tissue inflammation by both inflammasome-dependent and -independent manners and attenuate adipose tissue fibrosis by targeting innate immune sensors.

HIF-1α in Myeloid Cells Promotes Adipose Tissue Remodeling Toward Insulin Resistance.

Adipose tissue hypoxia is an important feature of pathological adipose tissue expansion. Hypoxia-inducible factor-1α (HIF-1α) in adipocytes reportedly induces oxidative stress and fibrosis, rather than neoangiogenesis via vascular endothelial growth factor (VEGF)-A. We previously reported that macrophages in crown-like structures (CLSs) are both hypoxic and inflammatory. In the current study, we examined how macrophage HIF-1α is involved in high-fat diet (HFD)-induced inflammation, neovascularization, hypoxia, and insulin resistance using mice with myeloid cell-specific HIF-1α deletion that were fed an HFD. Myeloid cell-specific HIF-1α gene deletion protected against HFD-induced inflammation, CLS formation, poor vasculature development in the adipose tissue, and systemic insulin resistance. Despite a reduced expression of Vegfa in epididymal white adipose tissue (eWAT), the preadipocytes and endothelial cells of HIF-1α-deficient mice expressed higher levels of angiogenic factors, including Vegfa, Angpt1, Fgf1, and Fgf10 in accordance with preferable eWAT remodeling. Our in vitro study revealed that lipopolysaccharide-treated bone marrow-derived macrophages directly inhibited the expression of angiogenic factors in 3T3-L1 preadipocytes. Thus, macrophage HIF-1α is involved not only in the formation of CLSs, further enhancing the inflammatory responses, but also in the inhibition of neoangiogenesis in preadipocytes. We concluded that these two pathways contribute to the obesity-related physiology of pathological adipose tissue expansion, thus causing systemic insulin resistance.

Differential requirements of MyD88 and TRIF pathways in TLR4-mediated immune responses in murine B cells.

LPS stimulates the TLR4/Myeloid differentiation protein-2 (MD-2) complex and promotes a variety of immune responses in B cells. TLR4 has two main signaling pathways, MyD88 and Toll/IL-1R (TIR)-domain-containing adaptor-inducing interferon-ß (TRIF) pathways, but relatively few studies have examined these pathways in B cells. In this study, we investigated MyD88- or TRIF-dependent LPS responses in B cells by utilizing their knockout mice. Compared with wild-type (WT) B cells, MyD88(-/-) B cells were markedly impaired in up-regulation of CD86 and proliferation induced by lipid A moiety of LPS. TRIF(-/-) B cells were also impaired in these responses compared with WT B cells, but showed better responses than MyD88(-/-) B cells. Regarding class switch recombination (CSR) elicited by lipid A plus IL-4, MyD88(-/-) B cells showed similar patterns of CSR to WT B cells. However, TRIF(-/-) B cells showed the impaired in the CSR. Compared with WT and MyD88(-/-) B cells, TRIF(-/-) B cells exhibited reduced cell division, fewer IgG1(+) cells per division, and decreased activation-induced cytidine deaminase (Aicda) mRNA expression in response to lipid A plus IL-4. Finally, IgG1 production to trinitrophenyl (TNP)-LPS immunization was impaired in TRIF(-/-) mice, while MyD88(-/-) mice exhibited increased IgG1 production. Thus, MyD88 and TRIF pathways differently regulate TLR4-induced immune responses in B cells.

Isoliquiritigenin is a potent inhibitor of NLRP3 inflammasome activation and diet-induced adipose tissue inflammation.

Inflammasome activation initiates the development of many inflammatory diseases, including obesity and type 2 diabetes. Therefore, agents that target discrete activation steps could represent very important drugs. We reported previously that ILG, a chalcone from Glycyrrhiza uralensis, inhibits LPS-induced NF-κB activation. Here, we show that ILG potently inhibits the activation of NLRP3 inflammasome, and the effect is independent of its inhibitory potency on TLR4. The inhibitory effect of ILG was stronger than that of parthenolide, a known inhibitor of the NLRP3 inflammasome. GL, a triterpenoid from G. uralensis, had similar inhibitory effects on NLRP3 activity, but high concentrations of GL were required. In contrast, activation of the AIM2 inflammasome was inhibited by GL but not by ILG. Moreover, GL inhibited NLRP3- and AIM2-activated ASC oligomerization, whereas ILG inhibited NLRP3-activated ASC oligomerization. Low concentrations of ILG were highly effective in IAPP-induced IL-1ß production compared with the sulfonylurea drug glyburide. In vivo analyses revealed that ILG potently attenuated HFD-induced obesity, hypercholesterolemia, and insulin resistance. Furthermore, ILG treatment improved HFD-induced macrovesicular steatosis in the liver. Finally, ILG markedly inhibited diet-induced adipose tissue inflammation and IL-1ß and caspase-1 production in white adipose tissue in ex vivo culture. These results suggest that ILG is a potential drug target for treatment of NLRP3 inflammasome-associated inflammatory diseases.

Activation and regulation of the pattern recognition receptors in obesity-induced adipose tissue inflammation and insulin resistance.

Obesity-associated chronic tissue inflammation is a key contributing factor to type 2 diabetes mellitus, and a number of studies have clearly demonstrated that the immune system and metabolism are highly integrated. Recent advances in deciphering the various immune cells and signaling networks that link the immune and metabolic systems have contributed to our understanding of the pathogenesis of obesity-associated inflammation. Other recent studies have suggested that pattern recognition receptors in the innate immune system recognize various kinds of endogenous and exogenous ligands, and have a crucial role in initiating or promoting obesity-associated chronic inflammation. Importantly, these mediators act on insulin target cells or on insulin-producing cells impairing insulin sensitivity and its secretion. Here, we discuss how various pattern recognition receptors in the immune system underlie the etiology of obesity-associated inflammation and insulin resistance, with a particular focus on the TLR (Toll-like receptor) family protein Radioprotective 105 (RP105)/myeloid differentiation protein-1 (MD-1).

The TLR family protein RP105/MD-1 complex: A new player in obesity and adipose tissue inflammation.

The radioprotective 105 (RP105)/MD-1 complex is a member of the Toll-like receptor (TLR) family of proteins. We have previously reported that this complex cooperates with the essential lipopolysaccharide (LPS) receptor TLR4/MD-2 complex and plays a crucial role in LPS responses by B cells. Recent evidences suggest that TLRs can also recognize endogenous ligands and promote non-infectious chronic inflammation. For instance, TLR4/MD-2 can be ligated by adipose tissue-derived saturated free fatty acids (FAs) and induce adipose tissue inflammation and insulin resistance. Recently, we reported that RP105 knockout (KO) or MD-1 KO mice have less high-fat diet (HFD)-induced obesity, adipose tissue inflammation and insulin resistance than wild-type (WT) or TLR4 KO mice. As RP105/MD-1 is not involved in recognition of palmitic and stearic acids, which are endogenous ligands for TLR4/MD-2, we conclude that RP105/MD-1 is itself a key regulator of diet-induced chronic inflammation in adipose tissue, obesity and insulin resistance that appears to be independent of the TLR4-dependent pathway. In this mini-review, we will highlight the significance of the RP105/MD-1 complex in adipose tissue inflammation and discuss implications for human diseases.