Induction of multiple subroutines of regulated necrosis in murine macrophages by natural BH3-mimetic gossypol.

Macrophages are critical sentinel cells armed with multiple regulated necrosis pathways, including pyroptosis, apoptosis followed by secondary necrosis, and necroptosis, and are poised to undergo distinct form(s) of necrosis for tackling dangers of pathogenic infection or toxic exposure. The natural BH3-mimetic gossypol is a toxic phytochemical that can induce apoptosis and/or pyroptotic-like cell death, but what exact forms of regulated necrosis are induced remains largely unknown. Here we demonstrated that gossypol induces pyroptotic-like cell death in both unprimed and lipopolysaccharide-primed mouse bone marrow-derived macrophages (BMDMs), as evidenced by membrane swelling and ballooning accompanied by propidium iodide incorporation and lactic acid dehydrogenase release. Notably, gossypol simultaneously induces the activation of both pyroptotic and apoptotic (followed by secondary necrosis) pathways but only weakly activates the necroptosis pathway. Unexpectedly, gossypol-induced necrosis is independent of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, as neither inhibitor for the NLRP3 pathway nor NLRP3 deficiency protects the macrophages from the necrosis. Furthermore, necrotic inhibitors or even pan-caspase inhibitor alone does not or only partly inhibit such necrosis. Instead, a combination of inhibitors composed of pan-caspase inhibitor IDN-6556, RIPK3 inhibitor GSK'872 and NADPH oxidase inhibitor GKT137831 not only markedly inhibits the necrosis, with all apoptotic and pyroptotic pathways being blocked, but also attenuates gossypol-induced peritonitis in mice. Lastly, the activation of the NLRP3 pathway and apoptotic caspase-3 appears to be independent of each other. Collectively, gossypol simultaneously induces the activation of multiple subroutines of regulated necrosis in macrophages depending on both apoptotic and inflammatory caspases.

Taraxasterol mitigates Con A-induced hepatitis in mice by suppressing interleukin-2 expression and its signaling in T lymphocytes.

Discovery of anti-inflammatory drugs that can suppress T lymphocyte activation and proliferation by inhibiting TCR/CD3 and IL-2/IL-2R signaling is still needed in clinic, though rapamycin and other related reagents have made great success. Taraxasterol (TAS) is an active ingredient of dandelion, an anti-inflammatory medicinal herb with low in vivo toxicity that has long been used in China. Yet the action mechanism of TAS on lymphocytes remains elusive. The anti-inflammatory effects of TAS were evaluated in C57BL/6 mouse primary lymphocytes stimulated with concanavalin A (Con A) in vitro and in mouse model of Con A-induced acute hepatitis in vivo. Our results showed that TAS significantly suppressed Con A-induced acute hepatitis in a mouse model, reducing the hepatic necrosis areas, the release of aminotransferases, and the production of IL-2 and other inflammatory cytokines. Supporting this, in vitro study also showed that TAS reduced the production of IL-2 and the expression of IL-2 receptor subunit α (CD25) upon the stimulation of Con A, which was likely mediated by suppressing NF-κB activation. The downstream pathways of IL-2/IL-2R signaling, including the activation of PI3K/PDK1/mTOR, STAT3 and STAT5, were also suppressed by TAS. Consistently, Con A-induced T cell proliferation was also inhibited by TAS in vitro. Our data indicate that TAS can suppress both T lymphocyte activation and cell proliferation by down-regulating IL-2 expression and its signaling pathway thereby ameliorating Con A-induced acute hepatitis, highlighting TAS as a potential drug candidate for treating inflammatory diseases including autoimmune hepatitis.

The Signaling Pathways Regulating NLRP3 Inflammasome Activation.

The NLRP3 inflammasome is a multi-molecular complex that acts as a molecular platform to mediate caspase-1 activation, leading to IL-1ß/IL-18 maturation and release in cells stimulated by various pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). This inflammasome plays an important role in the innate immunity as its activation can further promote the occurrence of inflammation, enhance the ability of host to remove pathogens, and thus facilitate the repair of injured tissues. But if the inflammasome activation is dysregulated, it will cause the development of various inflammatory diseases and metabolic disorders. Therefore, under normal conditions, the activation of inflammasome is tightly regulated by various positive and negative signaling pathways to respond to the stimuli without damaging the host itself while maintaining homeostasis. In this review, we summarize recent advances in the major signaling pathways (including TLRs, MAPK, mTOR, autophagy, PKA, AMPK, and IFNR) that regulate NLRP3 inflammasome activation, providing a brief view of the molecular network that regulates this inflammasome as a theoretical basis for therapeutic intervention of NLRP3 dysregulation-related diseases.

Inhibition of NLRP3 Inflammasome Activation and Pyroptosis in Macrophages by Taraxasterol Is Associated With Its Regulation on mTOR Signaling.

Taraxasterol (TAS) is an active ingredient of Dandelion (Taraxacum mongolicum Hand. -Mazz.), a medicinal plant that has long been used in China for treatment of inflammatory disorders. But the underlying mechanism for its therapeutic effects on inflammatory disorders is not completely clear. Inflammasome activation is a critical step of innate immune response to infection and aseptic inflammation. Among the various types of inflammasome sensors that has been reported, NLR family pyrin domain containing 3 (NLRP3) is implicated in various inflammatory diseases and therefore has been most extensively studied. In this study, we aimed to explore whether TAS could influence NLPR3 inflammasome activation in macrophages. The results showed that TAS dose-dependently suppressed the activation of caspase-1 in lipopolysaccharide (LPS)-primed murine primary macrophages upon nigericin treatment, resulting in reduced mature interleukin-1ß (IL-1ß) release and gasdermin D (GSDMD) cleavage. TAS greatly reduced ASC speck formation upon the stimulation of nigericin or extracellular ATP. Consistent with reduced cleavage of GSDMD, nigericin-induced pyroptosis was alleviated by TAS. Interestingly, TAS time-dependently suppressed the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) and mTORC2 signaling induced by LPS priming. Like TAS, both INK-128 (inhibiting both mTORC1 and mTORC2) and rapamycin (inhibiting mTORC1 only) also inhibited NLRP3 inflammasome activation, though their effects on mTOR signaling were different. Moreover, TAS treatment alleviated mitochondrial damage by nigericin and improved mouse survival from bacterial infection, accompanied by reduced IL-1ß levels in vivo. Collectively, by inhibiting the NLRP3 inflammasome activation, TAS displayed anti-inflammatory effects likely through regulation of the mTOR signaling in macrophages, highlighting a potential action mechanism for the anti-inflammatory activity of Dandelion in treating inflammation-related disorders, which warrants further clinical investigation.

Scutellarin inhibits caspase-11 activation and pyroptosis in macrophages via regulating PKA signaling.

Inflammatory caspase-11 senses and is activated by intracellular lipopolysaccharide (LPS) leading to pyroptosis that has critical role in defensing against bacterial infection, whereas its excess activation under pathogenic circumstances may cause various inflammatory diseases. However, there are few known drugs that can control caspase-11 activation. We report here that scutellarin, a flavonoid from Erigeron breviscapus, acted as an inhibitor for caspase-11 activation in macrophages. Scutellarin dose-dependently inhibited intracellular LPS-induced release of caspase-11p26 (indicative of caspase-11 activation) and generation of N-terminal fragment of gasdermin D (GSDMD-NT), leading to reduced pyroptosis. It also suppressed the activation of non-canonical nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome as evidenced by reduced apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and decreased interleukin-1 beta (IL-1ß) and caspase-1p10 secretion, whereas the NLRP3-specific inhibitor MCC950 only inhibited IL-1ß and caspase-1p10 release and ASC speck formation but not pyroptosis. Scutellarin also suppressed LPS-induced caspase-11 activation and pyroptosis in RAW 264.7 cells lacking ASC expression. Moreover, scutellarin treatment increased Ser/Thr phosphorylation of caspase-11 at protein kinase A (PKA)-specific sites, and its inhibitory action on caspase-11 activation was largely abrogated by PKA inhibitor H89 or by adenylyl cyclase inhibitor MDL12330A. Collectively, our data indicate that scutellarin inhibited caspase-11 activation and pyroptosis in macrophages at least partly via regulating the PKA signaling pathway.