Lentinan from shiitake selectively attenuates AIM2 and non-canonical inflammasome activation while inducing pro-inflammatory cytokine production

Lentinan extracted from shiitake (Lentinula edodes) is a β-glucan that has been reported as an intravenous anti-tumor polysaccharide via enhancement of the host immune system. In this study, we determined the effect of lentinan on inflammasome activation, a multi-protein platform, in myeloid cells. Mouse bone marrow-derived macrophages were treated with lentinan with/without inflammasome triggers, and maturation of interleukin (IL)-1β, IL-18, or caspase-1 was measured as a readout of inflammasome activation. As a result, lentinan selectively inhibited absent in melanoma 2 (AIM2) inflammasome activation. In addition, lentinan up-regulated pro-inflammatory cytokines and induced expression of inflammasome-related genes through toll-like receptor 4 signaling. Furthermore, we assessed the effect of lentinan on mice treated with Listeria monocytogenes or lipopolysaccharide as an AIM2 or non-canonical inflammasome-mediated model. Lentinan attenuated IL-1β secretion resulting from Listeria-mediated AIM2 inflammasome activation and reduced endotoxin lethality via inhibition of non-canonical inflammasome activation. Thus, lentinan is suggested as an anti-AIM2 and anti-non-canonical inflammasome candidate despite its up-regulation of cytokine expression.

Lentinan up-regulates expression of pro-inflammatory cytokines and inflammasome components via toll-like receptor 4 signaling. Previously, lentinan has been demonstrated as an inducer of pro-inflammatory cytokines 17 . To determine the effect of lentinan on pro-inflammatory cytokine expression, murine macrophages were treated with increasing dosages of lentinan or LPS, a toll-like receptor 4 (TLR4) ligand, as a positive control. As a result, lentinan treatment alone induced mRNA transcription of inflammatory cytokines such as IL-1α, TNFα, IL-6, IL-1rn, IL-10, and Pro-IL-1β (Fig. 3A). Moreover, inflammasome-related genes were quantitated in response to lentinan treatment. Lentinan increased transcription of Pro-IL-1β, NLRP3, and Pro-Casp1 but had no effect on NLRC4 and AIM2 (Fig. 3A). Next, we determined which pathway mediates lentinan-induced gene expression. Based on a previous report 18 that lentinan up-regulates TLR2 and 4 expression, treatment with TAK-242, a TLR4 signaling inhibitor, resulted in reduction of cytokine production and NLRP3 mRNA expression in response to lentinan (Fig. 3B). In addition, we purified LNT with endotoxin removal resin and measured endotoxin contamination by LAL assay. Endotoxin contamination levels were not significantly different between purified LNT (0.4077 ± 0.0006 EU/mg) and intact LNT (0.4345 ± 0.0008 EU/mg). We further compared expression levels of NLRP3 and Pro-IL-1β proteins between purified and intact LNT (Fig. 3C). As a result, purified LNT induced NLRP3 and Pro-IL-1β expression similar to intact LNT. Moreover, purified LNT presented similar anti-AIM2 inflammasome properties (Fig. 3D). Taken together, lentinan stimulates production of pro-IL-1β and NLRP3 through TLR4 signaling but inhibits maturation of IL-1β induced by AIM2 inflammasome activation.
Lentinan induces priming step of NLRP3 inflammasome activation and blocks formation of Asc pyroptosome. Activation of NLRP3 inflammasome requires two steps, priming and activation. The priming step is commonly mediated by toll-like receptor (TLR) ligands such as LPS, which triggers NF-κB signaling to induce transcription of pro-IL-1β and NLRP3 19 . Based on Fig. 3, LNT could act as a priming agent for NLRP3 inflammasome due to LNT-mediated transcription and translation of pro-IL-1β and NLRP3 via TLR4 signaling. Thus, we tested the effect of LNT on priming of inflammasome activation. As shown in Fig. 4, LNT-primed BMDMs showed IL-1β and Casp1 secretion in response to NG, a NLRP3 inflammasome trigger, similar to LPS-primed BMDMs. Taken together, LNT had an adequate effect on the priming step of NLRP3 inflammasome activation.
Next, we assessed the effect of LNT on formation of Asc pyroptosome and in vitro activation of caspase-1 to determine the putative mechanism for impairment of AIM2 inflammasome by LNT. Based on the literature 20 , AIM2 recognizes intracellular dsDNA and then interacts with ASC, which leads to the formation of Asc pyroptosome. The pyroptosome then activates caspase-1 and induces IL-1β secretion 20 . As shown in Fig. 4B, LNT dose-dependently attenuated formation of Asc pyroptosome, similar to secretion of Casp-1. In addition, we (B) Lipopolysaccharide-primed bone marrow-derived macrophages (LPS-primed BMDMs) were treated with the indicated concentration of LNT or ATP (2 mM) as a positive control. Secretion of active form of IL-1β was analyzed by immunoblotting. (C) LPS-primed BMDMs were treated with the indicated dosage of LNT with/without nigericin (NG, 40 μM) or monosodium urate crystals (MSU, 800 μg/mL). Secretion of caspase-1 (Casp1) was analyzed by immunoblotting, and IL-1β or IL-18 secretion was measured by ELISA. All immunoblot data shown are representative of at least three independent experiments. Bar graph presents the mean ± SD. observed the effect of LNT on recombinant human caspase-1 (rhCasp1) activities (Fig. 4C). Activity of rhCasp-1 was blocked by the pan-capase-1 inhibitor Z-VAD-FMK but not by LNT. Thus, these data indicate that LNT disrupts pyroptosome formation upstream. β-D-Glucan extracted from barley does not regulate cytokine production and maturation. We next investigated whether or not β-glucan originating from different sources can regulate inflammasome activation and gene expression. β-D-Glucan extracted from barley (GB) has a structure consisting of β-(1,3)-glucose units ( Supplementary Fig. 1A). Similar to lentinan, GB alone did not induce IL-1β secretion in LPS-primed BMDMs ( Supplementary Fig. 1B). Next, we treated LPS-primed macrophages with GB in the presence of NG for activation of NLRP3 inflammasome, flagellin for activation of NLRC4 inflammasome, or dsDNA for activation of AIM2 inflammasome. As a result, inflammasome activations were not inhibited by GB co-treatment ( Supplementary Fig. 1C). In addition, murine macrophages did not induce pro-inflammatory cytokine production in response to GB treatment ( Supplementary Fig. 1D). Thus, the inflammasome-regulatory properties of β-glucans vary depending on the source.

Lentinan reduces Listeria-mediated IL-1β secretion in mice.
In the current study, lentinan exhibited conflicting effects on inflammatory responses. Lentinan up-regulated pro-inflammatory cytokines but blocked IL-1β secretion mediated by AIM2 inflammasome activation. To assess the role of lentinan on AIM2 inflammasome activation, we adopted Listeria monocytogenes (LM)-induced peritonitis. LM has been revealed as an AIM2 inflammasome trigger, although it also activates NLRP3 and NLRC4 inflammasomes [21][22][23] . Mice injected with LM presented increased numbers of peritoneal exudate cells (PECs) as well as elevated peritoneal IL-1β, IL-6, and IL-18 secretion (Fig. 5A). Lentinan treatment significantly attenuated LM-induced IL-1β secretion but not PECs, IL-6, or IL-18 secretion. These data suggest that lentinan has anti-AIM2 inflammasome properties. However, we further investigated the priming effect of lentinan in vivo. For this, we isolated peritoneal exudate cells (PECs) from LM-and/or LNT-injected mice and analyzed the transcription levels of pro-inflammatory cytokines and inflammasome components. As shown in Fig. 5B, LM and/or LNT treatments up-regulated expression of TNFα, IL-6, IL-1α, pro-IL-1β, and NLRP3 mRNAs in PECs, although the expression levels were lower in LNT alone treatment. This result implies that LNT selectively blocked IL-1β maturation resulting from inflammasome activation, although LNT up-regulated transcription of pro-IL-1β mRNA in PCEs. The increased PECs and IL-6 secretion (Fig. 5A) might have been mediated by LNT-mediated cytokine up-regulation. However, we cannot verify the different regulatory pathways of IL-18 secretion upon LNT treatment in vivo and in vitro at this moment. In addition, we tested the effect of LNT on peritoneal LM burden. As shown in Fig. 5C, LNT treatment did not Lentinan alleviates lethality of endotoxemia in mice. Next, we focused on LPS-induced septic shock, also called endotoxemia. The LPS-induced septic shock model is a well characterized model of NLRP3 inflammasome-mediated disease, and intracellular LPS is known to trigger non-canonical inflammasome activation 24,25 . As seen in Fig. 6A, mice injected with LPS alone presented 50% lethality while mice administrated lentinan alone did not show any mortality. Notably, lentinan injection into LPS-treated mice significantly reduced lethality in a dose-dependent manner. We further tested the effect of LNT on non-canonical inflammasome activation since LPS lethality is tightly involved in the non-canonical inflammasome 14 . We performed LPS transfection and E. coli infection into BMDMs to trigger non-canonical inflammasome activation, which activates caspase-11 and then triggers NLRP3 inflammasome activation for Casp1 and IL-1β secretion 13,14 . As shown in Fig. 6B, LPS transfection induced IL-1β secretion which was blocked by LNT treatment. In addition, LNT attenuated Casp1 secretion resulting from E. coli-mediating non-canonical inflammasome activation (Fig. 6C). This suggests that LNT ameliorated endotoxemic lethality due to LNT-mediated inhibition of non-canonical inflammasome activation.

Discussion
In this study, we confirmed the effect of lentinan, β-glucan from shiitake, on inflammasome activation and cytokine maturation. As a result, lentinan selectively inhibited IL-1β maturation in response to AIM2 inflammasome activation in murine macrophages, whereas NLRP3 and NLRC4 inflammasomes were not activated by lentinan treatment. As expected, lentinan up-regulated pro-inflammatory cytokines and stimulated TLR4 signaling, resulting in expression of the inflammasome components NLRP3 and pro-IL-1β. Unlike lentinan, β-D-glucan extracted from barley (GB) did not alter cytokine maturation nor expression in macrophages. Furthermore, lentinan ameliorated LPS-induced lethality in a dose-dependent manner and selectively attenuated IL-1β secretion in response to Listeria monocytogenes-mediated AIM2 inflammasome activation. Taken together, lentinan stimulates expression of pro-inflammatory cytokines, including NLRP3, whereas it selectively attenuates cytokine maturation in response to AIM2 inflammasome activation.
The effects of β-glucans on inflammation activation have been previously studied 26,27 . Curdlan, a β-glucan from Alcaligenes faecalis, was shown to up-regulate Pro-IL-1β mRNA expression via interaction with dectin-1 as well as induce NLRP3 inflammasome-mediated IL-1β secretion resulting from NLRP3 inflammasome activation through dectin-1/Syk signaling 26 . However, other β-glucans such as GB, paramylon, and zymosan did not modulate IL-1β secretion 26 . In the current study, lentinan regulated cytokine production and maturation while GB did not, implying β-glucans had various effects on inflammasome activation depending on source or chemical structure. The effect of lentinan on NLRP3 inflammasome activation has been previously studied, whereas the previous study used human lung cancer cell lines, A549, instead of macrophages or dendritic cells 27 . Co-treatment of lentinan with paclitaxel, an anti-cancer drug, to A549 cells induces reactive oxygen species (ROS) production and thioredoxin-interacting protein (TXNIP) expression, which are tightly associated with NLRP3 inflammasome activation 28 . Further, co-treatment of lentinan with paclitaxel has been shown to stimulate the TXNIP-NLRP3 interaction, leading to IL-1β and Casp1 maturation 28 . However, we did not observe IL-1β secretion from LPS-primed BMDMs in response to lentinan alone (Fig. 1B). Thus, the inflammasome-modulating properties of lentinan may vary depending on cell type or chemical combination.
Lentinan as a strong active macromolecule is known to enhance host-mediated anti-cancer activities in the human immune system 29 . For example, cancer patients administered lentinan present a higher survival rate due to enhanced antibody-and complement-dependent cytotoxicity against tumor cells 16 . In addition, lentinan administration has been shown to induce generation of cytotoxic T cells and NK cells, stimulating their anti-cancer activities 16 . Furthermore, lentinan was shown to stimulate cytotoxicity of macrophages against metastatic tumors in mouse studies 30 . Thus, lentinan-stimulated immunity is a key mechanism responsible for its anti-cancer properties. In this study, we observed that lentinan induced cytokine and NLRP3 expression but inhibited AIM2 inflammasome activation in macrophages. Although NLRP3 and NLRC4 inflammasomes are involved in carcinogenesis and anticancer immune responses 11 , AIM2 inflammasome has not been reported to induce cancer development. Instead of inflammasome activation, loss of AIM22 gene has been shown to result in cancer growth while mutation of AIM2 is associated with development of various cancers in humans 31,32 . Moreover, Aim2 −/− mice showed increased size and number of colon tumors in a colitis-associated cancer (CAC) model, implying that the AIM2 gene itself regulates tumor progress and prevents colorectal cancer 33 . Based on the literature, we conclude that the anti-cancer properties of lentinan are not mediated by inflammasomes since lentinan did not alter AIM2 mRNA expression nor activation of NLRP3 and NLRC4 inflammasomes.
AIM2 contains a pyrin domain, which interacts with ASC, as well as hematopoietic interferon-inducible nuclear protein (HIN)-200 domains, which sense cytosolic dsDNA 15 . These domains form an auto-inhibitory conformation before detection of the trigger while auto-inhibition is relieved by cytosolic dsDNA, resulting in caspase-1-dependent pyroptosis and release of IL-1β and -18. AIM2 inflammasome activation is critical for host defense against DNA viruses and bacteria that replicate in the cytosol 34 . For example, Listeria monocytogenes predominantly activates AIM2 inflammasome among several inflammasomes 22,35 . In addition, Listeria monocytogenes-triggered pyroptosis and cytokine secretion were shown to be diminished in Aim2 −/− macrophages 22 . However, Listeria monocytogenes trigger AIM2 inflammasome activation as well as activation of NLRP3 and NLCR4 inflammasomes. That is, NLRP3 inflammasome is activated by lysosomal disruption when Listeria monocytogenes escape to the cytoplasm of phagocytes 22 . In addition, Listeria are critical for NLRC4 inflammasome activation in the absence of LPS priming 23 , whereas LPS-primed macrophages derived from NLRC4 −/− mice do not respond to Listeria infection for inflammasome activation due to LPS priming bypassing the requirement for NLRC4 22 . Although multi-inflammasomes are involved in Listeria infection, AIM2 is the most accepted inflammasome against Listeria monocytogenes 22,23,35 . Francisella tularensis, which causes tularemia, triggers activation of AIM2 inflammasome, leading to IL-1β and 18 production in infected macrophages 36 . AIM2 inflammasome is also crucial for intestinal homeostasis 37 . In Aim2 −/− mice, intestinal epithelial cells (IEC) show diminished IL-18 secretion, which is required for IEC proliferation and tissue regeneration, during steady state 37,38 . In contrast, over-production of cytokines induced by chronic inflammasome activation leads to tissue damage and colitis-associated colorectal cancer 39 . Previously, oral administration of lentinan was shown to ameliorate susceptibility to dextran sulfate sodium (DSS)-induced colitis and attenuate intestinal inflammation 3 . Although we did not confirm the effect of lentinan in DSS-induced colitis, a well characterized inflammasome-mediated disease, lentinan might block IL-1β and -18 secretion in a colitis model 38 . Thus, lentinan is suggested as an anti-inflammasome agent, especially for AIM2 inflammasome.

Materials and Methods
Preparation of bone marrow-derived macrophages (BMDMs). BMDMs were obtained by differen-