Yoshikatsu Hirai

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‐1β 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‐1β 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.

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.

Serum soluble MD-1 levels increase with disease progression in autoimmune prone MRL(lpr/lpr) mice.

MD-1 is a secreted protein that forms a complex with radioprotective 105 (RP105) and this complex plays a crucial role in lipopolysaccharide (LPS) recognition by B cells. Disease progression is known to improve in RP105-deficient lupus-prone MRL(lpr/lpr) mice. Furthermore, a soluble form of the homologous MD-2 protein is present in the plasma of septic patients and can opsonize gram-negative bacteria in cooperation with Toll-like receptor (TLR) 4. We have now established a flow cytometry-based assay to detect the soluble form of murine MD-1 (sMD-1) and explored potential roles in autoimmunity. The assay was quantitative and validated with sera from MD-1-deficient mice. Interestingly, heat-inactivated murine serum diminished the ability of sMD-1 to bind RP105. The sMD-1 was secreted by bone marrow-derived macrophages from C57BL/6 mice. Autoimmune prone MRL(lpr/lpr) mice had higher levels of sMD-1 than control MRL(+/+) mice, and levels markedly increased with disease progression. Expression of MD-1 but not MD-2 mRNA increased with age in the liver and kidney of MRL(lpr/lpr) mice. Finally, immunohistochemical analyses revealed that MD-1 was present in infiltrated macrophages within perivascular lesions of the MRL(lpr/lpr) kidney. This correlation suggests that sMD-1 may contribute to pathogenesis in this autoimmune disease model.

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-β (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.

Induction and properties of DNP-specific T cells in mice sensitized to DNP-coupled mycobacterium.

Splenic T lymphocytes from mice sensitized to 100 μg of DNP‐coupled mycobacterium (DNP‐Tbc) showed in vitro proliferative response against DNP‐or TNP‐conjugated protein antigens. The increased uptake of 3H‐thymidine induced by DNP‐HSA was partially inhibited by the addition of 10–4M DNP‐EACA. DNP‐AECM‐Ficoll did not induce any significant proliferative responses in DNP‐Tbc‐primed T cell population. However, priming with DNP‐Tbc augmented anti‐DNP IgG antibody response induced with DNP‐Ficoll. The augmentation of IgG response was not due to the presence of DNP‐primed B cells or anti‐DNP antibody. The results showed that the priming with DNP‐Tbc induced DNP‐reactive T helper cells which could be triggered with DNP‐Ficoll. The possible role of mycobacterium in the induction of hapten‐specific T helper cells is discussed.