Gabsik Yang

Oxidized phosphatidylcholine induces the activation of NLRP3 inflammasome in macrophages

The NOD‐like receptor family, pyrin domain containing 3 (NLRP3) inflammasome is a multiprotein complex consisting of a receptor, an adaptor protein, and procaspase‐1 that induces the secretion of the mature form of IL‐1β in response to microbial infection and danger signals. Activation of the NLRP3 inflammasome induced by endogenous danger signal molecules is closely linked to the development and progress of chronic inflammatory diseases. The oxidation of phospholipids occurs upon cellular stress and damage, resulting in the accumulation of oxidized phosphatidylcholines (oxPAPC) such as 1‐palmitoyl‐2‐(5‐oxovaleroyl)‐sn‐glycero‐phosphocholine (POVPC) at inflammatory sites. In this study, we investigated whether oxidized phosphatidylcholine induces the activation of NLRP3 inflammasome in macrophages, leading to the secretion of IL‐1β. POVPC induced the degradation of procaspase‐1 to caspase‐1(p10), the cleavage of pro‐IL‐1β to IL‐1β, and oligomerization of ASC in primary mouse bone marrow‐derived macrophages. POVPC‐induced production of caspase‐1, and IL‐1β was abolished in macrophages derived from NLRP3‐ or caspase‐1‐deficient mice. In an air pouch model and a peritonitis model in mice, POVPC injection resulted in the production of caspase‐1(p10), IL‐1β, and IL‐18 in wild‐type, but not in NLRP3‐deficient, mice. POVPC‐induced inflammasome activation was mediated by mitochondrial reactive oxygen species resulting from intracellular Ca2+ signaling and mitochondrial destabilization. Our results demonstrate that endogenously produced oxidized phosphatidylcholines such as POVPC induce the activation of NLRP3 inflammasome, leading to the production of IL‐1β in macrophages. The results provide an insight to understand how the oxidized lipids endogenously produced upon cellular stress and tissue damage contribute to the inflammatory reaction at pathologic sites.

Targeting ASC in NLRP3 inflammasome by caffeic acid phenethyl ester: a novel strategy to treat acute gout.

Gouty arthritis is caused by the deposition of uric acid crystals, which induce the activation of NOD-like receptor family, pyrin domain containing 3(NLRP3) inflammasome. The NLRP3 inflammasome, composed of NLRP3, the adaptor protein ASC, and caspase-1, is closely linked to the pathogenesis of various metabolic diseases including gouty arthritis. We investigated whether an orally administrable inhibitor of NLRP3 inflammasome was effective for alleviating the pathological symptoms of gouty arthritis and what was the underlying mechanism. In primary mouse macrophages, caffeic acid phenethyl ester(CAPE) blocked caspase-1 activation and IL-1β production induced by MSU crystals, showing that CAPE suppresses NLRP3 inflammasome activation. In mouse gouty arthritis models, oral administration of CAPE suppressed MSU crystals-induced caspase-1 activation and IL-1β production in the air pouch exudates and the foot tissues, correlating with attenuation of inflammatory symptoms. CAPE directly associated with ASC as shown by SPR analysis and co-precipitation, resulting in blockade of NLRP3-ASC interaction induced by MSU crystals. Our findings provide a novel regulatory mechanism by which small molecules harness the activation of NLRP3 inflammasome by presenting ASC as a new target. Furthermore, the results suggest the preventive or therapeutic strategy for NLRP3-related inflammatory diseases such as gouty arthritis using orally available small molecules.

Suppression of NLRP3 inflammasome by oral treatment with sulforaphane alleviates acute gouty inflammation

Objective The aetiology of gout is closely linked to the deposition of monosodium uric acid (MSU) crystals and the consequent activation of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. In this study, we investigated whether oral administration of an NLRP3 inhibitor would be effective to attenuate the symptoms of gout. Methods The effects of oral administration with sulforaphane (SFN) were examined in two mouse models of acute gout induced by injection of MSU crystals into footpads or air pouch. The production of caspase-1 (p10) and IL-1β was examined by immunoblotting and ELISA as hallmarks of NLRP3 inflammasome activation. Results Oral administration of SFN attenuated MSU crystal-induced swelling and neutrophil recruitment in a mouse foot acute gout model, correlating with the suppression of the NLRP3 inflammasome activation in foot tissues. Consistently, oral administration of SFN blocked MSU-crystal-induced activation of the NLRP3 inflammasome in a mouse air pouch gout model. SFN suppressed NLRP3 inflammasome activation induced by MSU crystals, adenosine triphosphate and nigericin but not by poly(dA:dT) in primary mouse macrophages, independent of the reactive oxygen species pathway. SFN inhibited ligand-independent activation of the NLRP3 inflammasome, suggesting that SFN may act directly on the NLRP3 inflammasome complex. Conclusion Oral administration of SFN effectively alleviated acute gouty inflammation by suppression of the NLRP3 inflammasome. Our results provide a novel strategy in which oral treatment with SFN may be beneficial in preventing acute attacks of gout.

Topical Application of Dieckol Ameliorates Atopic Dermatitis in NC/Nga Mice by Suppressing Thymic Stromal Lymphopoietin Production

TO THE EDITOR Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease that is characterized by extreme pruritus. AD is considered the first stage of atopic march (Leung et al., 2004). From an immunological aspect, AD is closely linked to the disruption of Th1/Th2 cytokine homeostasis that skews to Th2 immunity (Bieber, 2008). Immunological approaches to the treatment of AD have mainly focused on stimulating Th1 immunity to recover the Th1/Th2 balance (Leung et al., 2004). Thymic stromal lymphopoietin (TSLP) was recently shown to play a critical role in the progress of AD by inducing Th2 immune responses (Soumelis et al., 2002; Zhang et al., 2009). Increased TSLP production in keratinocytes induces the expression of the OX40 ligand (OX40L) in dendritic cells (DCs), which in turn stimulates the differentiation of naı̈ve CD4 T cells into Th2 cells to produce Th2 cytokines such as IL-4, IL-5, and IL-13 (Leyva-Castillo, Hener, Michea, et al., 2013). Thus, the TSLP-OX40L axis is considered integral to the induction of a Th2 cell-mediated allergic cascade in AD (MurakamiSatsutani et al., 2014). Indeed, high levels of TSLP expression have been observed in epidermal keratinocytes of AD skin lesions (Leyva-Castillo, Hener, Jiang, et al. 2013a). Therefore, suppressing TSLP may represent a novel therapeutic approach for treating AD by restoring Th1/Th2 balance. Here, we investigated whether dieckol, a phlorotannin from Ecklonia cava (Figure 1a), can suppress TSLP production to reduce Th2 immunity and effectively

Suppression of splenic lymphocyte proliferation by Eucommia ulmoides and genipin.

We investigated the modulation of innate and adaptive immune cell activation by Eucommia ulmoides Oliver extract (EUE) and its ingredient genipin. As an innate immunity indicator, the phagocytic activity of macrophages was determined by measuring engulfed, fluorescently labeled Escherichia coli. As a surrogate marker for the respective activation of cellular and humoral adaptive immunity, concanavalin A (Con A) and lipopolysaccharide (LPS) induction of primary splenocyte proliferation was assayed in in vitro and ex vivo systems. EUE and genipin suppressed the proliferation of primary splenic lymphocytes induced by Con A or LPS, but not macrophage phagocytosis. Oral administration of EUE and genipin to mice decreased splenic lymphocyte proliferation induced by Con A or LPS. These results revealed that E. ulmoides and genipin suppressed cellular and humoral adaptive immunity, and they suggest that E. ulmoides and genipin are promising candidates for immunosuppressive drugs that target diseases that involve excessive activation of adaptive immunity.

Potentiation of skin TSLP production by a cosmetic colorant leads to aggravation of dermatitis symptoms

Certain cosmetic colorants are irritant to skin or aggravate dermatitis. Thymic stromal lymphopoietin (TSLP) plays an important role in the initiation and progress of skin inflammation and atopic dermatitis by triggering Th2 immune responses. However, the effects of cosmetic colorants on TSLP production are unknown yet. Therefore, we investigated whether cosmetic colorants regulated TSLP production and dermatitis. Lithol Rubine B (LR-B, Pigment Red 57) and its calcium salt (LR-BCA), commonly used cosmetic colorants, potentiated phorbol-12-myristate-13-acetate-induced TSLP production in keratinocytes. In addition, the topical exposure to LR-B or LR-BCA on mouse ear upregulated a TSLP inducer (MC903)-induced TSLP production and Th2 cytokine expression. Dermatitis symptoms and serum IgE and histamine levels were also aggravated by LR-B or LR-BCA, implicating the role of increased TSLP expression in acute dermatitis. LR-B or LR-BCA induced IκBα degradation and NF-κB activation in keratinocytes, leading to TSLP expression. Collectively, our results demonstrate that LR-B and LR-BCA increase TSLP expression and Th2 immune responses, thereby aggravating acute dermatitis in the compromised skin. The results further suggest that certain cosmetic colorants such as LR-B may aggravate dermatitis under pro-inflammatory conditions by upregulating TSLP production.

Licochalcone A attenuates acne symptoms mediated by suppression of NLRP3 inflammasome: Licochalcone A suppresses NLRP3 inflammasome and acne

Activation of the NACHT, LRR and PYD domains‐containing protein 3 (NLRP3) inflammasome by Propionibacterium acnes (P. acnes) is critical for inducing inflammation and aggravating the development of acne lesions. We searched for available small‐molecule inhibitors of the NLRP3 inflammasome that could be topically administered for the treatment of acne. We found that licochalcone A, a chalconoid isolated from the root of Glycyrrhiza inflate, was an effective inhibitor for P. acnes‐induced NLRP3 inflammasome activation. Licochalcone A blocked P. acnes‐induced production of caspase‐1(p10) and IL‐1β in primary mouse macrophages and human SZ95 sebocytes, indicating the suppression of NLRP3 inflammasome. Licochalcone A suppressed P. acnes‐induced ASC speck formation and mitochondrial reactive oxygen species. Topical application of licochalcone A to mouse ear skin attenuated P. acnes‐induced skin inflammation as shown by histological assessment, ear thickness measurement, and inflammatory gene expression. Licochalcone A reduced caspase‐1 activity and IL‐1β production in mouse ear injected with P. acnes. This study demonstrated that licochalcone A is effective in the control of P. acnes‐induced skin inflammation as an efficient inhibitor for NLRP3 inflammasome. Our study provides a new paradigm for the development of anti‐acne therapy via targeting NLRP3 inflammasome.

Phloxine O, a Cosmetic Colorant, Suppresses the Expression of Thymic Stromal Lymphopoietin and Acute Dermatitis Symptoms in Mice

Cosmetics are primarily applied to the skin; therefore, the association of cosmetic dyes with skin diseases or inflammation is a topic of great interest. Thymic stromal lymphopoietin (TSLP) is an interleukin 7-like cytokine that activates dendritic cells to promote Th2 inflammatory immune responses. TSLP is highly expressed in keratinocytes under inflammatory conditions, which suggests that it may play a critical role in the development of skin diseases, such as atopic dermatitis. Therefore, we investigated whether cosmetic dyes influenced the production of TSLP by keratinocytes. Phloxine O, also known as D&C Red No.27, is one of the most common red synthetic pigments and is widely used in colored cosmetics. Our results showed that Phloxine O downregulated phorbol 12-myristate 13-acetate-induced production of TSLP in a murine keratinocyte cell line (PAM212). Phloxine O also suppressed TSLP expression in KCMH-1 cells, which are mouse keratinocytes that constitutively produce high levels of TSLP. To investigate the in vivo effects of Phloxine O, we induced TSLP expression in mouse ear skin by topically applying MC903, a vitamin D3 analogue that is a well-known inducer of atopic dermatitis-like symptoms. Topical application of Phloxine O prevented MC903-induced TSLP production in mouse ear skin, attenuated the acute dermatitis-like symptoms and decreased serum IgE and histamine levels in mice. Suppression of TSLP expression by Phloxine O correlated with reduced expression of OX40 ligand and Th2 cytokines in mouse ear skin. Our results showed that Phloxine O may be beneficial to prevent dermatitis by suppressing the expression of TSLP and Th2 cytokines in skin.

Suppression of Primary Splenocyte Proliferation by Artemisia capillaris and Its Components

The host immune system is the first line of host defense, consisting mainly of innate and adaptive immunity. Immunity must be maintained, orchestrated, and harmonized, since overactivation of immune responses can lead to inflammation and autoimmune diseases, while immune deficiency can lead to infectious diseases. We investigated the regulation of innate and adaptive immune cell activation by Artemisia capillaris and its components (ursolic acid, hyperoside, scopoletin, and scopolin). Macrophage phagocytic activity was determined using fluorescently labeled Escherichia coli, as an indicator of innate immune activation. Concanavalin A (ConA)- and lipopolysaccharide (LPS)-induced splenocyte proliferation was analyzed as surrogate markers for cellular and humoral adaptive immunity, respectively. Neither A. capillaris water extract (WAC) nor ethanol extract (EAC) greatly inhibited macrophage phagocytic activity. In contrast, WAC suppressed ConA- and LPS-induced proliferation of primary mouse splenocytes in a dose-dependent manner. Similarly, EAC inhibited ConA- and LPS-induced splenocyte proliferation. Oral administration of WAC in mice decreased ConA- and LPS-induced splenocyte proliferation, while that of EAC suppressed LPS-induced splenocyte proliferation. Repeated administration of WAC in mice inhibited ConA- and LPS-induced splenocyte proliferation. Ursolic acid, scopoletin, and scopolin reduced ConA- and LPS-induced primary mouse splenocyte proliferation, while hyperoside did not show such activity. These results indicate that A. capillaris and its components, ursolic acid, scopoletin, and scopolin, suppress ConA- and LPS-induced adaptive immune cell activation. The results suggest that A. capillaris is useful as a regulator of adaptive immunity for diseases involving excessive immune response activation.