Methylene blue inhibits NLRP3, NLRC4, AIM2, and non-canonical inflammasome activation

www.nature.com/scientificreports OPEN Received: 15 May 2017 Accepted: 13 September 2017 Published: xx xx xxxx Methylene blue inhibits NLRP3, NLRC4, AIM2, and non-canonical inflammasome activation Huijeong Ahn1, Seung Goo Kang3, Sung-il Yoon3, Hyun-Jeong Ko4, Pyeung-Hyeun Kim2, Eui-Ju Hong5, Beum-Soo An6, Eunsong Lee1 & Geun-Shik Lee 1 Methylene blue (MB), which has antioxidant, anti-inflammatory, neuroprotective, and mitochondria protective effects, has been widely used as a dye and medication. However, the effect of MB on inflammasome activation has not yet been studied. Inflammasomes are multi-protein complexes that induce maturation of interleukins (ILs)-1β and -18 as well as caspase-1-mediated cell death, known as pyroptosis. Dysregulation of inflammasomes causes several diseases such as type 2 diabetes, Alzheimer’s disease, and gout. In this study, we assess the effect of MB on inflammasome activation in macrophages. As the result, MB attenuated activation of canonical inflammasomes such as NLRP3, NLRC4, and AIM2 as well as non-canonical inflammasome activation. In addition, MB inhibited upstream signals such as inflammasome assembly, phagocytosis, and gene expression of inflammasome components via inhibition of NF-κB signaling. Furthermore, MB reduced the activity of caspase-1. The anti-inflammasome properties of MB were further confirmed in mice models. Thus, we suggest that MB is a broad-spectrum anti-inflammasome candidate molecule. Methylene blue (MB, 3,7-bis(dimethylamino)-phenothiazin-5-ium chloride) is a heterocyclic aromatic chemical compound with the chemical formula C16H18N3SCl1. It has many uses in biology and chemistry, such as a dye for the textile industry, and has potent antibiotic and antioxidant properties. Since its discovery as the first synthetic anti-malarial agent by Ehrlich in 1891, MB has been used in several clinical fields for the treatment of acute and chronic methemoglobinemia, carbon monoxide poisoning, urinary tract infection, septic shock, and cardiopulmonary bypass1. MB suppresses production of superoxide radicals by acting as an alternative receptor of xanthine oxide electrons. Recently, MB has received increased attention in view of studies suggesting its usefulness in treating mitochondrial dysfunction1. It has also been studied as an agent for the treatment of Alzheimer’s disease2. MB may also provide neuroprotective functions based on its anti-inflammatory properties3. Further, expression of inflammatory genes was reduced in microglia treated with lipopolysaccharide (LPS) in the presence of MB in the culture media3. Inflammation is a protective immune response mediated by the innate immune system in response to harmful stimuli such as pathogens, damaged cells, and irritants and is tightly controlled by the host4. Insufficient inflammation can cause continuous infection of pathogens, whereas excessive inflammation can lead to chronic or systemic inflammatory diseases. Innate immune function depends on germline-encoded pattern-recognition receptors (PRRs) recognizing pathogen-associated molecular patterns (PAMPs) derived from infectious pathogens as well as danger-associated molecular patterns (DAMPs) induced from endogenous stress. Inflammasomes, which are multi-protein complexes, consist of cytosolic PRRs and sensing cytosolic PAMPs or DAMPs in myeloid cells as well as non-myeloid cells such as keratinocytes, hepatocytes, and cardiomyocytes 5–10. To assemble the inflammasome complex, sensing proteins and caspase-1 are linked by an adaptor protein known as 1 College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon, Gangwon, 24341, Republic of Korea. 2Department of Molecular Bioscience, School of Biomedical Science, Kangwon National University, Chuncheon, Gangwon, 24341, Republic of Korea. 3Division of Biomedical Convergence, College of Biomedical Science, Kangwon National University, Chuncheon, Gangwon, 24341, Republic of Korea. 4Laboratory of Microbiology and Immunology, College of Pharmacy, Kangwon National University, Chuncheon, Gangwon, 24341, Republic of Korea. 5College of Veterinary Medicine and Institute of Veterinary Science, Chungnam National University, Daejeon, 34134, Republic of Korea. 6Department of Biomaterial Science, College of Natural Resources and Life Science, Pusan National University, Gyeongsangnam-do, 50612, Republic of Korea. Correspondence and requests for materials should be addressed to G.-S.L. (email: ) Scientific Reports | 7: 12409 | DOI:10.1038/s41598-017-12635-6 1 www.nature.com/scientificreports/ apoptosis-associated speck-like protein containing a carboxy-terminal caspase recruitment domain (Asc or pycard). The sensing proteins are nucleotide-binding oligomerization domain (NOD), leucine-rich repeat (LRR)-containing protein (NLR) family members such as NLRP1, NLRP3, and NLRC4, or absent in melanoma 2 (AIM2)5,6. Upon detecting certain stimuli, NLR or AIM2 can oligomerize into a caspase-1-activating scaffold. Active caspase-1 subsequently functions to cleave the proinflammatory IL-1 family of cytokines into their bioactive forms, IL-1β and IL-18, as well as induce pyroptosis, a type of inflammatory cell death5. In addition, it has been suggested that the non-canonical inflammasome activates caspases-4, -5, and/or -11 in response to intracellular LPS, resulting in IL-1β/-18 secretion, pyroptosis, and endotoxemic death11. Although MB has been used in human and veterinary medicine for over a century, there has been no study on the role of MB on inflammasome activation. In this study, we assessed the effect of MB on several well-characterized inflammasomes such as NLRP3, NLRC4, and AIM2 and non-canonical inflammasomes in murine macrophages. In addition, we demonstrated the upstream and molecular mechanisms of MB in the context of inflammasome activation. The regulatory effect of MB were further confirmed with animal models. Thus, we conclude that MB regulates inflammasome activation and inflammatory responses. Results Methylene blue inhibits NLRP3 inflammasome activation. To assess the effect of methylene blue (MB, Fig. 1A) on IL-1β maturation resulting from inflammasome activation, LPS-primed bone marrow-derived macrophages (BMDMs) were treated with MB or ATP, a NLRP3 inflammasome trigger, as a positive control. Although ATP treatment induced IL-1β secretion resulting from NLRP3 inflammasome activation, MB alone did not (Fig. 1B). This result implies that MB alone did not activate inflammasomes. Next, we tested whether or not MB inhibits inflammasome activation. LPS-primed BMDMs were subjected to NLRP3 inflammasome activation by nigericin (NG) or ATP in the presence of increasing dosages of MB, and several readouts for inflammasome activation were observed such as secretion of maturated IL-1β and caspase-1, as well as formation of Asc pyroptosome (Fig. 1C and Supplementary Fig. 1). As the result, MB dose-dependently attenuated secretion of I (...truncated)