Intracellular NAD+ Depletion Confers a Priming Signal for NLRP3 Inflammasome Activation.

Nicotinamide adenine dinucleotide (NAD+) is an important cofactor in many redox and non-redox NAD+-consuming enzyme reactions. Intracellular NAD+ level steadily declines with age, but its role in the innate immune potential of myeloid cells remains elusive. In this study, we explored whether NAD+ depletion by FK866, a highly specific inhibitor of the NAD salvage pathway, can affect pattern recognition receptor-mediated responses in macrophages. NAD+-depleted mouse bone marrow-derived macrophages (BMDMs) exhibited similar levels of proinflammatory cytokine production in response to LPS or poly (I:C) stimulation compared with untreated cells. Instead, FK866 facilitated robust caspase-1 activation in BMDMs in the presence of NLRP3-activating signals such as ATP and nigericin, a potassium ionophore. However, this FK866-mediated caspase-1 activation was completely abolished in Nlrp3-deficient macrophages. FK866 plus nigericin stimulation caused an NLRP3-dependent assembly of inflammasome complex. In contrast, restoration of NAD+ level by supplementation with nicotinamide mononucleotide abrogated the FK866-mediated caspase-1 cleavage. FK866 did not induce or increase the expression levels of NLRP3 and interleukin (IL)-1ß but drove mitochondrial retrograde transport into the perinuclear region. FK866-nigericin-induced mitochondrial transport is critical for caspase-1 cleavage in macrophages. Consistent with the in vitro experiments, intradermal coinjection of FK866 and ATP resulted in robust IL-1ß expression and caspase-1 activation in the skin of wild-type, but not Nlrp3-deficient mice. Collectively, our data suggest that NAD+ depletion provides a non-transcriptional priming signal for NLRP3 activation via mitochondrial perinuclear clustering, and aging-associated NAD+ decline can trigger NLRP3 inflammasome activation in ATP-rich environments.

Inhibitory Effect and Mechanism of Arctium lappa Extract on NLRP3 Inflammasome Activation.

Arctium lappa (A. lappa), Compositae, is considered a potential source of nutrition and is used as a traditional medicine in East Asian countries for centuries. Although several studies have shown its biological activities as an anti-inflammatory agent, there have been no reports on A. lappa with regard to regulatory role in inflammasome activation. The purpose of this study was to investigate the inhibitory effects of A. lappa extract (ALE) on NLRP3 inflammasome activation and explore the underlying mechanisms. We found that ALE inhibited IL-1ß secretion from NLRP3 inflammasome activated bone marrow derived macrophages but not that secreted by NLRC4 and AIM2 inflammasomes activation. Mechanistic studies revealed that ALE suppressed the ATPase activity of purified NLRP3 and reduced mitochondrial reactive oxygen species (mROS) generated during NLRP3 activation. Therefore, the inhibitory effect of ALE on NLRP3 inflammasome might be attributed to its ability to inhibit the NLRP3 ATPase function and attenuated the mROS during inflammasome activation. In addition, ALE significantly reduced the LPS-induced increase of plasma IL-1ß in mouse peritonitis model. These results provide evidence of novel anti-inflammatory mechanisms of A. lappa, which might be used for therapeutic applications in the treatment of NLRP3 inflammasome-associated inflammatory disorders.

Actinidia arguta extract attenuates inflammasome activation: Potential involvement in NLRP3 ubiquitination.

ETHNOPHARMACOLOGICAL RELEVANCE: Actinidia arguta (A. arguta) has been widely used in Asian countries as a traditional medicinal herb to treat inflammation-related diseases, such as gastritis, bronchitis, and arthritis. AIM OF THE STUDY: The inhibitory effect of A. arguta leaves' extract (AA) on inflammasome activation was investigated to verify its traditional use in treating inflammation-related diseases. MATERIALS AND METHODS: Bone marrow-derived macrophages (BMDMs) primed by lipopolysaccharide (LPS) were activated by selective inflammasome stimulators, and the effect of AA on inflammasome activation was investigated. A monosodium urate crystal (MSU)-induced peritonitis mouse model was used to study the in vivo efficacy of AA on inflammasome activation. RESULTS: In the in vitro study, AA regulated NLRP3 ubiquitination and apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization, leading to the inhibition of NLRP3 inflammasome-mediated interleukin (IL)-1ß secretion. The inhibitory effect of AA on inflammasome activation in vitro was further confirmed in vivo using an MSU-induced peritonitis mouse model. CONCLUSION: AA provided scientific evidence, substantiating the traditional claims for its use in the treatment of inflammation and inflammation-mediated metabolic disorders, including gout.

Protective effects of Cinnamomum cassia (Lamaceae) against gout and septic responses via attenuation of inflammasome activation in experimental models.

ETHNOPHARMACOLOGICAL RELEVANCE: Cinnamomum cassia (C. cassia, Lauraceae family), commonly used for treating dyspepsia, gastritis, blood circulation, and inflammatory diseases is considered as one of the 50 fundamental herbs in traditional Chinese medicine. AIM OF THE STUDY: The anti-inflammatory action of an ethanol extract of C. cassia (CA), and its underlying mechanisms were explored in both in vitro cellular and in vivo murine models. MATERIALS AND METHODS: Bone marrow-derived macrophages (BMDMs) were used to study the regulatory effect of CA on inflammasome activation. A lipopolysaccharide (LPS)-induced sepsis mouse model and a monosodium urate (MSU)-induced gout model were employed to study the effect of CA on in vivo efficacy. RESULTS: CA improved the survival rate in the LPS-induced septic shock mouse model and inhibited inflammasome activation including NLRP3, NLRC4, and AIM2, leading to suppression of interleukin-1ß secretion. Further, ASC oligomerization and its speck formation in cytosol were attenuated by CA treatment. Furthermore, CA improved both survival rate of LPS-induced septic shock and gout murine model. CONCLUSIONS: CA treatment significantly attenuated danger signals-induced inflammatory responses via regulation of inflammasome activation, substantiating the traditional claims of its use in the treatment of inflammation-related disorders.

Juniperus rigida Sieb. extract inhibits inflammatory responses via attenuation of TRIF-dependent signaling and inflammasome activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Juniperus rigida Sieb. (J. rigida) is used for medicinal purposes in Asian countries to treat inflammation-related disorders, such as neuralgia, dropsy, and gout. AIM OF THE STUDY: The anti-inflammatory effects of J. rigida extract (JR) and its underlying mechanisms were explored both in in vitro cell lines and in vivo metabolic disease models. MATERIAL AND METHODS: Lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages were used to study the changes in inflammatory responses in vitro. Bone marrow-derived macrophages (BMDMs) were used to study the regulatory effect of JR on inflammasome activation. The murine model for monosodium urate (MSU)-induced peritonitis and high-fat diet (HFD)-induced type 2 diabetes were employed to study the effect of JR on in vivo efficacy. RESULTS: JR suppressed the MSU-induced in vivo inflammatory response by attenuation of proinflammatory cytokines, including interleukin (IL)-1ß, IL-6, and tumor necrosis factor-alpha (TNF-α). In the in vitro study, JR suppressed IL-1ß secretion via regulation of apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization, leading to the inhibition of inflammasome activation. JR also inhibited the LPS-stimulated release of proinflammatory mediators, such as nitric oxide (NO), TNF-α, and IL-6 in RAW264.7 cells. The inhibitory effects of JR were mediated through the regulation of the TRIF-dependent signaling pathway from JAK1/STAT1 phosphorylation. Furthermore, JR showed inhibitory effects on HFD-induced type 2 diabetes in a mouse model through the regulation of blood glucose and serum IL-1ß. CONCLUSIONS: Our results indicate that JR attenuates both LPS-stimulated and danger-signal-induced inflammatory responses in macrophages via regulation of the key inflammatory mechanisms, providing scientific support for its traditional use in the treatment of various inflammation-related metabolic disorders.

Cichorium intybus Linn. Extract Prevents Type 2 Diabetes Through Inhibition of NLRP3 Inflammasome Activation.

This study provides the scientific basis for the inhibitory effect of the aerial parts of Cichorium intybus Linn. (C. intybus) on the activation of the NLRP3 inflammasome in vitro and on high-fat diet (HFD)-induced type-2 diabetes (T2D). Lipopolysaccharide (LPS)-primed bone marrow-derived macrophages were used to study the effects methanolic extract of C. intybus leaf (CI) on inflammasome activation. An insulin resistance model (mice fed a HFD) was used to study the in vivo effect of CI on T2D. CI attenuated interleukin-1ß (IL-1ß) secretion by inhibiting the activation of the NLRP3 inflammasome in mouse bone marrow macrophages. The CI treatment attenuated the intracellular movement of NLRP3 in Triton X-100 insoluble fraction, without affecting the expression of other NLRP3 inflammasome-related proteins. Attenuated IL-1ß secretion may improve glucose metabolism in the HFD-fed insulin resistance mouse model. CI also attenuated the infiltration of M1 macrophages and increased the M2 macrophage population in white adipose tissue. Collectively, our data showed that CI inhibits IL-1ß secretion through attenuation of NLRP3 inflammasome activation, leading to an antidiabetic effect by improving glucose metabolism and inhibiting metainflammation.

Inhibition of glutamine utilization sensitizes lung cancer cells to apigenin-induced apoptosis resulting from metabolic and oxidative stress.

Recent studies have shown anticancer activity of apigenin by suppressing glucose transporter 1 (GLUT1) expression in cultured cancer cells; however, it is not clear whether apigenin can suppress glucose metabolism in lung cancer cells or sensitize them to inhibition of glutamine utilization-mediated apoptosis through metabolic and oxidative stress. We show that apigenin significantly decreases GLUT1 expression in mice. Furthermore, we demonstrate that apigenin induces growth retardation and apoptosis through metabolic and oxidative stress caused by suppression of glucose utilization in lung cancer cells. The underlying mechanisms were defined that the anticancer effects of apigenin were reversed by ectopic GLUT1 overexpression and galactose supplementation, through activation of pentose phosphate pathway-mediated NADPH generation. Importantly, we showed that severe metabolic stress using a glutaminase inhibitor, compound 968, was involved in the mechanism of sensitization by apigenin. Taken together, the combination of apigenin with inhibitors of glutamine metabolism may provide a promising therapeutic strategy for cancer treatment.

Anti-inflammatory properties of Morus bombycis Koidzumi via inhibiting IFN-ß signaling and NLRP3 inflammasome activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Morus bombycis Koidzumi (M. bombycis, Moraceae) has been used in Asian countries as a traditional medicine for the treatment of hypertension, diabetes, and inflammation-related disorders. AIM OF STUDY: Although its anti-inflammatory actions have been partly documented, scientific evidence involving its molecular mechanisms related to inflammasome activation signaling pathways remains unknown. MATERIALS AND METHODS: Lipopolysaccharide-stimulated RAW 264.7 cells and bone marrow-derived murine macrophages were used to study the in vitro effect of methanolic extract of M. bombycis (MB) on inflammatory responses. A monosodium urate crystal (MSU)-induced peritonitis murine model was used to study the in vivo effects. RESULTS: MB attenuated the production of nitric oxide and interleukin-6, through the regulation of the interferon-ß receptor signaling pathway. MB also inhibited IL-1ß secretion via attenuation of NLRP3 inflammasome activation. Furthermore, MB inhibited MSU-induced peritonitis in the in vivo murine model. CONCLUSIONS: This study provides the key molecular mechanisms involved in the anti-inflammatory effects of M. bombycis, substantiating the traditional claims of its use in the treatment of inflammation-related disorders.

Anti-Inflammatory Action of an Antimicrobial Model Peptide That Suppresses the TRIF-Dependent Signaling Pathway via Inhibition of Toll-Like Receptor 4 Endocytosis in Lipopolysaccharide-Stimulated Macrophages.

Antimicrobial peptides (AMPs), also called host defense peptides, particularly those with amphipathic helical structures, are emerging as target molecules for therapeutic development due to their immunomodulatory properties. Although the antimicrobial activity of AMPs is known to be exerted primarily by permeation of the bacterial membrane, the mechanism underlying its anti-inflammatory activity remains to be elucidated. We report potent anti-inflammatory activity of WALK11.3, an antimicrobial model peptide with an amphipathic helical conformation, in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. This peptide inhibited the expression of inflammatory mediators, including nitric oxide, COX-2, IL-1ß, IL-6, INF-ß, and TNF-α. Although WALK11.3 did not exert a major effect on all downstream signaling in the MyD88-dependent pathway, toll-like receptor 4 (TLR4)- mediated pro-inflammatory signals were markedly attenuated in the TRIF-dependent pathway due to inhibition of the phosphorylation of STAT1 by attenuation of IRF3 phosphorylation. WALK11.3 specifically inhibited the endocytosis of TLR4, which is essential for triggering TRIF-mediated signaling in macrophage cells. Hence, we suggest that specific interference with TLR4 endocytosis could be one of the major modes of the anti-inflammatory action of AMPs. Our designed WALK11 peptides, which possess both antimicrobial and anti-inflammatory activities, may be promising molecules for the development of therapies for infectious inflammation.

Anti-inflammatory effect of Impatiens textori Miq. extract via inhibition of NLRP3 inflammasome activation in in vitro and in vivo experimental models.

ETHNOPHARMACOLOGICAL RELEVANCE: Impatiens textori Miq. (I. textori, Balsaminaceae) is a traditional medicinal herb used for centuries to treat several inflammatory related skin infections and allergic disorders in Asian countries. AIM OF THE STUDY: In this study, we elucidated the effects of whole plant extracts of I. textori on inflammasome activation using in vitro and in vivo models. MATERIALS AND METHODS: LPS-stimulated murine bone marrow macrophages were used to study the regulatory effect of I. textori extract (IT) on inflammasome activation. ATP, nigericin and MSU were used as danger-associated molecules to activate the NLRP3 inflammasome. An LPS-induced acute lung injury (ALI) mouse model was used to study the in vivo effect of IT on inflammasome activation. RESULTS: IT treated at 25, 50, and 100µg/mL concentrations suppressed interleukin-1ß secretion through the attenuation of NLRP3 inflammasome activation (p<0.001 at 100µg/mL) leading to the decreased amount of ASC oligomerization and caspase-1 maturation. For the in vivo model, IT inhibited the NLRP3 expression and cell recruitment at the lung tissue in the ALI mouse model. CONCLUSION: IT exhibited potent anti-inflammatory effects via the attenuation of NLRP3 inflammasome activation supporting the traditional claims and may provide a valuable therapeutic strategy in treating various inflammation-related disorders.

Syneilesis palmata (Thunb.) Maxim. extract attenuates inflammatory responses via the regulation of TRIF-dependent signaling and inflammasome activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Syneilesis palmata (Thunb.) Maxim. (S. palmata, Asteraceae) is a traditional Korean therapeutic herb widely used to treat pain, arthritis, and other symptoms. This study provides the scientific basis for the anti-inflammatory effects of S. palmata extract (SP) in both in vitro and in vivo experimental models. MATERIALS AND METHODS: Lipopolysaccharide (LPS)-stimulated murine macrophages were used to study the regulatory effect of SP on the inflammatory mediators in vitro. Bone marrow-derived macrophages were used to study the effects of SP on inflammasome activation. Escherichia coli-induced sepsis mouse model and LPS-induced endotoxin shock model were employed to study the effect of SP on in vivo efficacy. RESULTS: SP inhibited the LPS-stimulated release of proinflammatory mediators, such as nitric oxide and interleukin (IL)-6 in RAW 264.7 cells. SP treatment also attenuated IL-1ß secretion via the inhibition of NLRP3 inflammasome activation induced by monosodium urate, ATP, and nigericin. Further, SP ameliorated the severity of NLRP3 inflammasome-mediated symptoms in LPS-induced endotoxin and E. coli-induced sepsis mouse models. Mechanistic studies revealed that inhibitory effects of SP were mediated through the regulation of TRIF-dependent signaling and inflammasome activation. CONCLUSION: This study was the first to reveal mechanistic-based evidence substantiating the traditional claims of SP in the treatment of inflammation-related disorders, such as pain and arthritis.

Lysimachia clethroides Duby extract attenuates inflammatory response in Raw 264.7 macrophages stimulated with lipopolysaccharide and in acute lung injury mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: Lysimachia clethroides Duby (LC) is a traditional medicinal herb used to treat edema, hepatitis and inflammatory diseases in China and other Asian countries. In this study, the anti-inflammatory effects of LC extract and the mechanisms underlying were explored in both in vitro cell lines and acute lung injury (ALI) animal model of inflammation in vivo. MATERIALS AND METHODS: Lipopolysaccharide (LPS)-stimulated Raw 264.7 murine macrophages were used to study the regulatory effects of LC extract on inflammatory mediators such as nitric oxide (NO) and proinflammatory cytokine expression. Western blotting or ELISA techniques were employed to estimate protein levels. RT-PCR was used for analyzing the interferon (IFN)-ß production. LPS-induced ALI mouse model in vivo was employed to study the effect of LC extract. Further high-performance liquid chromatography (HPLC) fingerprinting technique was used to evaluate the active constituents present in LC extract, compared with reference standards. RESULTS: Pre-treatment with LC extract inhibited the LPS-stimulated NO release, interleukin (IL)-1ß and IL-6 production in Raw 264.7 cells dose dependently. LC extract inhibited the LPS-stimulated IRF3 and STAT1 phosphorylation. Further, in vivo experiments revealed that LC extract suppressed the infiltration of immune cells into the lung and proinflammatory cytokine production in broncho-alveolar lavage fluid (BALF) in the LPS-induced ALI mouse model. CONCLUSIONS: Our results indicate that LC extract attenuates LPS-stimulated inflammatory responses in macrophages via regulating the key inflammatory mechanisms, providing a scientific support for its traditional use in treating various inflammatory diseases.

Carpesium macrocephalum attenuates lipopolysaccharide-induced inflammation in macrophages by regulating the NF-κB/IκB-α, Akt, and STAT signaling pathways.

Carpesium macrocephalum (CM) Fr. et Sav. (Compositae) has been used in Chinese folk medicine as an analgesic, hemostatic, antipyretic, and to suppress inflammatory conditions. In the present study we aimed to provide scientific evidence for the anti-inflammatory properties of CM extract and evaluate the intrinsic mechanisms involved in both in vitro and in vivo experimental models. In in vitro findings, CM significantly inhibited the LPS-stimulated release of proinflammatory mediators such as nitric oxide, tumor necrosis factor-alpha, prostaglandin E2, and interleukin-6 in RAW264.7 macrophages in a concentration-dependent fashion. The attenuation of inflammatory responses in LPS-activated RAW264.7 cells by CM was closely associated with the suppression of nuclear factor-kappa B (NF-κB) phosphorylation, IκB-α degradation, and phosphorylation of Akt. CM treatment also attenuated the phosphorylation of STAT through TRIF dependent pathways in LPS-activated RAW264.7 cells. In vivo studies revealed that CM extract concentration dependently suppressed the acetic acid-induced vascular permeability in mice. Considering the data obtained regulation of multiple signaling mechanisms involving TRIF and Akt/NF-κB pathways might be responsible for the potent anti-inflammatory action of CM, substantiating its traditional use in inflammatory diseases.

Anti-inflammatory and anti-allergic effects of Agrimonia pilosa Ledeb extract on murine cell lines and OVA-induced airway inflammation.

ETHNOPHARMACOLOGICAL EVIDENCE: Agrimonia pilosa Ledeb (Rosaceae, AP) has long been used as a traditional medicine in Korea and other Asian countries to treat various diseases. AIM OF THE STUDY: In the present study, the anti-inflammatory and anti-allergic effects of AP extract in in vitro cell lines and in vivo mouse model of inflammation and the molecular mechanisms involved were reported. MATERIALS AND METHODS: Using Raw 264.7 murine macrophages the effects of methanol extract of AP in lipopolysaccharide (LPS)-induced production of inflammatory mediators were measured. Further IgE-DNP-induced interleukin (IL)-4 production and degranulation in RBL-2H3 rat basophilic cell lines was also estimated. To investigate the anti-asthmatic effect of AP in vivo, airway inflammation in ovalbumin (OVA)-induced mouse model was used. RESULTS: AP attenuated the production of inflammatory mediators such as NO, PGE(2) and pro-inflammatory cytokines in LPS-induced Raw 264.7 cells. Further, AP inhibited IL-4 production and degranulation in IgE-DNP-induced RBL-2H3 cells. Furthermore, AP attenuated the infiltration of immune cells into lung, cytokines production in broncho-alveolar lavage fluid (BALF) and airway-hyperresponsiveness (AHR) on OVA-induced mouse model of inflammation. CONCLUSION: Our results showed that AP attenuated the activation of macrophages, basophils, and inhibited the OVA-induced airway inflammation. The molecular mechanisms leading to AP's potent anti-inflammatory and anti-allergic effects might be through regulation of TRIF-dependent and Syk-PLCγ/AKT signaling pathways, suggesting that AP may provide a valuable therapeutic strategy in treating various inflammatory diseases including asthma.