Polyenylpyrrole Derivatives Inhibit NLRP3 Inflammasome Activation and Inflammatory Mediator Expression by Reducing Reactive Oxygen Species Production and Mitogen-Activated Protein Kinase Activation

Two polyenylpyrroles from a soil ascomycete Gymnoascus reessii were previously identified as hit compounds in screening for cytotoxicity against lung cancer cells. These compounds and various analogs, which have been previously synthesized and tested for anti-lung cancer cell activity, were tested for anti-inflammatory activity. After preliminary screening for cytotoxicity for RAW 264.7 murine macrophage cells, the non-toxic compounds were tested for anti-inflammatory activity using lipopolysaccharide (LPS)-activated RAW 264.7 cells. Compounds 1h, 1i, and 1n reduced LPS-induced nitric oxide (NO) production, with respective ED50 values of 15 ± 2, 16 ± 2, and 17 ± 2 µM. They also reduced expression of inducible NO synthase and interleukin-6 (IL-6) without affecting cyclooxygenase-2 expression. Compound 1h also reduced secretion of IL-6 and tumor necrosis factor-α by LPS-activated J774A.1 murine macrophage cells, primary mice peritoneal macrophages, and JAWSII murine bone marrow-derived dendritic cells and reduced NLRP3 inflammasome-mediated interleukin-1β (IL-1β) secretion by LPS + adenosine triphosphate-activated J774A.1 and JAWSII cells. The underlying mechanisms for the anti-inflammatory activity of compound 1h were found to be a decrease in LPS-induced reactive oxygen species (ROS) production, mitogen-activated protein kinase phosphorylation, and NF-κB activation and a decrease in ATP-induced ROS production and PKC-α phosphorylation. These results provide promising insights into the anti-inflammatory activity of these conjugated polyenes and a molecular rationale for future therapeutic intervention in inflammation-related diseases. They also show how compound 1h regulates inflammation and suggest it may be a new source for the development of anti-inflammatory agents to ameliorate inflammation- and NLRP3 inflammasome-related diseases.


Introduction
One of the geological characteristics of Taiwan is its abundant geothermal resources, which provide a special environment for the growth of certain microorganisms that have been utilized in various scientific and industrial areas [1]. Under normal growth conditions, fungi produce useful metabolites, such as penicillin and immunosuppressive compounds that have improved the quality of human life, and these organisms are still a rich source of bioactive compounds [2][3][4]. In addition, the extreme growth conditions of thermophilic fungi also allow them to synthesize novel metabolites.
Conjugated polyenes are an interesting class of widely occurring natural polyketides with useful biological properties, such as antibacterial [5], antifungal [5,6], and antitumor [7] activities. Previously, auxarconjugatin A and 12E-isorumbrin isolated from the soil ascomycete Gymnoascus reessii and demonstrated that 12E-isorumbrin had antitumor activity [8]. We then synthesized auxarconjugatin A and 12E-isorumbrin and various analogs and evaluated them for anti-tumor activity, and identified two compounds with high cytotoxicity (active at nM levels) for the human non-small cell lung carcinoma cell line A549 [9]. Since polyketides also exhibit potent immune modulating activities [10,11], making them potential resources for the discovery of new immune modulating drugs, in the present study, we evaluated these compounds for antiinflammatory activity.
In this study, we evaluated the anti-inflammatory activity of the synthesized polyenylpyrroles and analogs using LPSactivated macrophages and identified compound 1h as a nontoxic compound that can reduce inflammatory mediator expression and NLRP3 inflammasome activation. The intracellular signaling pathways affected by compound 1h in activated macrophages were also investigated.

Cell culture
The murine macrophage cell lines RAW 264.7 and J774A.1 and the C57BL/6 murine bone marrow-derived dendritic cell line JAWSII were purchased from the American Type Culture Collection. RAW 264.7 macrophages stably transfected with the NF-κB reporter gene (RAW-Blue™ cells) were purchased from InvivoGen (San Diego, CA). RAW 264.7, J774A.1, and RAW-Blue™ cells were grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (both from Life Technologies, Carlsbad, CA), while JAWSII cells were grown in RPMI-1640 medium supplemented with 20% non-inactivated FBS and 5 ng/ml of murine GM-CSF (R&D Systems). All cells were cultured at 37 °C in a 5% CO 2 incubator.
AlamarBlue® assay for cell viability RAW 264.7 cells were seeded at a density of 5000 cells in 100 µl of RPMI 1640 medium containing 10% heat-inactivated FBS in each well of 96-well flat-bottom plates and incubated for 24 h at 37 °C in a 5% CO 2 incubator, then for 24 h with the test samples, then AlamarBlue® assay kits (AbD Serotec Ltd.) were used to measure cytotoxicity using the protocol described by the manufacturer.

Enzyme-linked immunosorbent assay (ELISA)
The effects of the test samples on IL-1β, TNF-α, and IL-6 production were measured by ELISA according to the manufacturer's instructions. In brief, 50 µl of biotinylated antibody reagent and 50 µl of supernatant were added to a stripwell plate precoated with anti-mouse IL-1β, TNF-α, or IL-6 antibodies, which was then incubated at room temperature for 2 h. After washing the plate three times with washing buffer, 100 µl of diluted streptavidin-HRP concentrate was added to each well and the plate incubated at room temperature for 30 min. The washing process was repeated, then 100 µl of a premixed tetramethylbenzidine substrate solution was added to each well and the plate incubated at room temperature in the

NO production inhibitory assay
RAW 264.7 cells seeded in 24-well plates at a density of 5 × 10 5 cells/ml (1 ml) were incubated for 24 h with or without LPS (1 µg/ml) in the absence or presence of the test samples, then NO production was measured indirectly by analysis of nitrite levels using the Griess reaction.

NF-κB reporter assay
RAW-Blue™ cells, RAW 264.7 macrophages stably expressing the gene for secreted embryonic alkaline phosphatase (SEAP) inducible by NF-κB, were seeded in 60 mm dishes at a density of 5 × 10 5 cells/ml (1 ml) and grown overnight in a 5% CO 2 incubator at 37 °C. They were then pretreated with vehicle or compound 1h for 30 min, then LPS (1 µg/ml) was added and incubation continued for 24 h. The medium was then harvested and 20 µl aliquots mixed with 200 µl of QUANTI-Blue™ medium (InvivoGen) in 96-well plates and incubated at 37 °C for 15 min, then SEAP activity was assessed by measuring the optical density at 655 nm using an ELISA reader.

Western blots
Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed on ice for 5 min with lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na 2 EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM betaglycerophosphate, 1 mM Na 3 VO 4 , 1 µg/ml of leupeptin, 1 mM PMSF, protease inhibitor cocktail). The lysate was then centrifuged at 12,000 x g at 4 °C for 10 min and the pellet discarded. Proteins in the supernatant were separated by SDS-PAGE and electrotransferred to a PVDF membrane (EMD Millipore Corporation), which was then blocked by incubation for 1 h at room temperature in blocking buffer [(5% nonfat milk in PBS containing 0.1% Tween 20 (PBST)], then incubated for 2 h at room temperature with the primary antibody diluted in blocking buffer. After three washes in PBST, the membrane was incubated for 1 h at room temperature with an appropriate HRP-conjugated secondary antibody diluted in blocking buffer and developed using an enhanced chemiluminescence Western blot detection system (EMD Millipore Corporation).

Measurement of NF-κB p65 nuclear translocation
Nuclear proteins were extracted from RAW 264.7 and J774A.1 cells using a Nuclear Extract Kit (Active Motif) according to the manufacturer's instructions and nuclear NF-κB p65 activation quantified using an ELISA-based TransAM NF-κB kit (Active Motif, Tokyo, Japan) according to the manufacturer's protocol by reading the absorbance at 450 nm with a microplate absorbance reader (Bio-Rad Laboratories, Inc) and a reference wavelength of 655 nm.

Statistical analysis
All values are given as the mean ± SD. Data were analyzed by one-way ANOVA with a subsequent Scheffé test.

Synthesis of the test compounds
The polyenylpyrroles, auxarconjugatin A and 12E-isorumbrin, were previously isolated from the soil ascomycete Gymnoascus reessii and 12E-isorumbrin shown to be cytotoxic for various cancer cells [8]. We have synthesized these compounds and a range of analogs (Table 1 and Figure 1) and investigated their cytotoxicity for the human lung cancer sell line A549 [9]. The backbone of the synthesized polyenylpyrroles is shown in Figure 1. Since the 3-chloropyrrole group has been shown to play an important role in the cytotoxicity of auxarconjugatin A (compound 1b) and 12Eisorumbrin (compound 1e) [8], in some analogs, it was replaced by other 2-or 3-chlorosubstituted aromatic rings. In addition, H, Me, Et, or n-Bu was added at the different R positions ( Table 1).

Effect of polyenylpyrrole derivatives on macrophage viability
The aim of the present study was to identify non-toxic polyenylpyrrole derivatives that could be used as antiinflammatory agents. Compounds 1a-n (Table 1) at concentrations from 6.25 µM to 100 µM were evaluated for cytotoxicity against the murine macrophage cell line RAW 264.7 after 24 h treatment. As shown in Table 1, compounds 1a-g exhibited high cytotoxicity, with IC 50 values below 10 µM, indicating that they were not suitable for evaluation of their antiinflammatory activities. In contrast, compounds 1h-n were not cytotoxic at any of the concentrations tested and their antiinflammatory activity was therefore examined by measuring their ability to reducing LPS-induced NO production by RAW 264.7 macrophages. As shown in Table 1, the three most potent compounds were 1h, 1i, and 1n, with respective ED 50 values of 15 ± 2, 16 ± 2, and 17 ± 2 µM, and these were used in subsequent studies.

Compounds 1h, 1i, and 1n decrease production of NO, iNOS, and IL-6 by LPS-activated RAW 264.7 macrophages
To investigate the inhibitory effect of compounds 1h, 1i, and 1n on the LPS-induced inflammatory response, NO levels in the supernatants of RAW 264.7 macrophages incubated with DMSO (vehicle) or compound 1h, 1i, or 1n for 30 min before, and during, incubation for 24 h with LPS (1 µg/ml) were measured by the Griess reaction. The results showed that treatment with compound 1h, 1i, or 1n alone did not alter NO levels produced by non-activated cells (data not shown), but decreased NO production by LPS-activated cells in a dosedependent manner (Figure 2A). We next investigated their effect on the expression of iNOS, the NO producing enzyme, using Western blots. As shown in Figure 2B, pretreatment of RAW 264.7 macrophages for 30 min with 0-40 µM compound 1h (top panel), 1i (center panel), or 1n (bottom panel) before addition of LPS for 24 h resulted in reduced LPS-induced iNOS expression, the effect being significant at 5-40 µM compound 1h and 40 µM compound 1i or 1n, but did not affect expression of COX-2, an enzyme producing prostaglandin E2. In addition, we tested the effect of pretreatment on cytokine secretion (IL-6 and TNF-α) by LPS-activated RAW 264.7 macrophages and found that none of the three compounds altered background levels of IL-6 and TNF-α in non-stimulated macrophages (data not shown), but all three significantly decreased IL-6 production by LPS-activated cells in a dose-dependent manner, with compound 1h being more potent than 1i and 1n ( Figure 2C, upper panel), while TNF-α secretion was only slightly and nonsignificantly reduced ( Figure 2C, lower panel).

Compound 1h decreases IL-6 and TNF-α secretion by LPS-activated J774A.1 macrophages, peritoneal macrophages, and JAWSII dendritic cells
To confirm the anti-inflammatory activity of compound 1h seen with RAW 264.7 cells, its effect on LPS-induced cytokine secretion was investigated using another murine macrophage cell line J774A.1 and primary peritoneal macrophages from C57BL/6 mice and the results showed that it reduced secretion of IL-6 and TNF-α in both J774A.1 cells (Figure 3A) and peritoneal macrophages ( Figure 3B) in a dose-dependent manner. It also reduced LPS-induced IL-6 and TNF-α secretion by the murine dendritic cell line JAWSII (Figure 3C).

Compound 1h reduces IL-1β secretion by inhibiting the NLRP3 inflammasome
ATP is known to activate the NLRP3 inflammasome in LPSprimed macrophages, leading to caspase-1 activation and IL-1β secretion [28]. To examine whether compound 1h could affect NLRP3 inflammasome activation, the mouse macrophage cell line J774A.1 was used (RAW 264.7 macrophages are not suitable for studying the NLRP3 inflammasome). The full activation of the NLRP3 inflammasome requires both a priming signal (LPS) and an  activation signal (ATP), and we therefore examined the effect of compound 1h on both signaling events. As shown in Figure  4A, incubation of J774A.1 macrophages with compound 1h for 30 min before treatment for 5.5 h with LPS, followed by treatment with ATP for 30 min significantly inhibited IL-1β secretion (upper panel) and the generation of active caspase-1 (p10; lower panel) in a dose-dependent manner.
Using the same conditions, compound 1h also inhibited IL-1β secretion by primary peritoneal macrophages ( Figure 4B). In addition, to examine whether compound 1h was able to affect the ATP-mediated activation signal, we incubated J774A.1 cells with LPS for 5.5 h, then with compound 1h for 30 min before ATP stimulation and, as shown in Figure 4C, found that compound 1h inhibited the LPS-induced increase in IL-1β secretion (upper panel), but not caspase-1 activation (lower panel), but, as shown in Figure 4D, had no significant effect on IL-6 secretion. These results demonstrate that compound 1h inhibited NLRP3 inflammasome-mediated IL-1β secretion, but not IL-6 secretion, which is independent of the NLRP3 inflammasome. We also tested the ability of compound 1h to inhibit expression of NLRP3 protein (an essential component of the NLRP3 inflammasome) and of proIL-1β (IL-1β precursor) in LPS-activated J774A.1 cells by incubating the cells with different concentrations of compound 1h for 30 min before addition of LPS for another 6 h and, as shown in Figure 4E, found that it inhibited LPS-induced proIL-1β expression in a dose-dependent fashion, but increased NLRP3 expression.

Compound 1h inhibits ROS production by, and MAPK activation in, LPS-activated macrophages
ROS have been demonstrated to play important roles in LPS-mediated cytokine expression [14,26]. To test whether compound 1h exerted its anti-inflammatory effect on LPSactivated cells by downregulation of ROS production, intracellular ROS production in LPS-activated RAW 264.7 macrophages was measured. As shown in the time-course study in Figure 5A LPS stimulation of cells rapidly induced ROS production and pretreatment for 30 min with NAC (10 mM), a potent antioxidant, reduced ROS production. Pretreatment for 30 min with compound 1h (20 µM) also reduced LPS-stimulated ROS production, suggesting that its anti-inflammatory effect might be mediated partially through its antioxidative activity.
LPS is a potent inducer of macrophage activation and proinflammatory cytokine production, as it activated TLR4, which, in turn, activates many signaling pathways, including the mitogen-activated protein kinase (MAPK) signaling pathways [29]. To examine whether the effects of compound 1h on LPSinduced macrophages were associated with activation of MAPK signaling cascades, RAW 264.7 macrophages were incubated with DMSO or compound 1h (20 µM) for 30 min, then with LPS (1 µg/ml) for 0-60 min, and phosphorylation of the MAPKs, ERK1/2, JNK1/2, and p38 determined by Western blot analysis. As shown in Figure 5B, compound 1h inhibited phosphorylation of all 3 MAPKs in LPS-activated RAW 264.7 macrophages, these effects being maximal with 20-30 min of LPS stimulation. Using LPS stimulation for 20 min, these results were confirmed in J774A.1 macrophages (Figure 5C).
These results show that compound 1h inhibits activation of the MAPK signaling cascades in LPS-activated macrophages.

Compound 1h inhibits NF-κB activation in LPSactivated macrophages
In resting macrophages, NF-κB is sequestered in the cytoplasm as an inactive precursor complex by its inhibitory protein, IκB. Following LPS stimulation, IκB in the complex is phosphorylated by IκB kinase (IKK), ubiquitinated, and rapidly degraded in proteasomes, thus releasing NF-κB [30]. In determining whether compound 1h could inhibit LPS-stimulated NF-κB signaling in macrophages, we found that it inhibited phosphorylation of IKK-α and IκB-α in a dose-dependent manner and also had a inhibitory effect on IκB-α degradation in LPS-activated RAW 264.7 macrophages (Figure 6A) and J774A.1 macrophages (Figure 6B). In addition, it inhibited NF-κB nuclear translocation in LPS-activated RAW 264.7 macrophages ( Figure 6C) and J774A.1 macrophages ( Figure  6D).
Furthermore, using NF-κB-dependent alkaline phosphatase reporter cells, we demonstrated that NF-κB transcriptional activity in LPS-stimulated macrophages was also reduced by compound 1h (Figure 6E). These results show that compound 1h inhibits the activation of the NF-κB signaling cascades in LPS-activated macrophages.

Compound 1h inhibits ROS production by, and PKC-α phosphorylation in, ATP-activated macrophages
ATP-induced ROS production by NADPH oxidase is required for caspase-1 activation in, and IL-1β secretion by, macrophages [31,32]. To determine whether the inhibition of LPS-induced IL-1β secretion by compound 1h occurred via inhibition of ATP-induced ROS production, LPS-primed J774A. 1 macrophages were incubated with vehicle or compound 1h (20 µM) for 30 min before addition of ATP or PBS for 0-40 min and the results showed that compound 1h slightly reduced ATP-induced ROS production at 40 min (Figure 7A), whereas addition of compound 1h 30 min before LPS priming significantly inhibited ATP-induced ROS production ( Figure  7B), while the NADPH oxidase inhibitor, diphenylene iodonium (DPI) inhibited both processes. In addition, when LPS-primed J774A.1 macrophages were incubated with vehicle or compound 1h (20 µM) for 30 min before ATP stimulation for 0-60 min, compound 1h caused significant inhibition of ATPinduced PKC-α phosphorylation at 20-60 min (Figure 7C).

Discussion
Fungi are a valuable source of novel natural products with many biological activities [8,33]. Polyketides isolated from fungi exhibit various biological properties, such as antibacterial [5], antifungal [5,6], and antitumor [7,8] activities, but their effect on immune responses are not fully understood. It has been demonstrated that mycolactone, a natural polyketide produced by Mycobacterium ulcerans which causes the skin disease Buruli ulcer, reduces the immune response and the infiltration of inflammatory cells into the infection site [10]. In addition, a polyketide synthase-produced phenolic glycolipid isolated from Mycobacterium tuberculosis has been found to inhibit the  , upper panel; B) and levels of active caspase-1 (p10) (A, lower panel) measured by Western blotting. In (C) and (D), J774A.1 macrophages (1 × 10 6 /ml; 1ml) were incubated with LPS (1 µg/ml) for 5.5 h, then with 10-40 µM compound 1h or DMSO for 30 min in the continued presence of LPS, followed by stimulation with ATP (5 mM) for an additional 30 min, then IL-1β levels (C, upper panel) and IL-6 levels (D) in the culture medium were measured by ELISA and levels of active caspase-1 (p10) were measured by Western blotting (C, lower panel). In A and C, the fold increase is the intensity of the p10 band divided by that of the p45 band normalized to the corresponding value for the 0 LPS/0 inhibitor control. In (E), J774A.1 macrophages (1 × 10 6 /ml; 1 ml) were incubated for 30 min with DMSO or 1-40 µM compound 1h, then LPS (1 µg/ml) was added and incubation continued for 6 h, then expression of NLRP3 and proIL-1β was measured by Western blotting. The fold increase is the intensity of the band of interest divided by that of the actin band normalized to the corresponding value for the 0 LPS/0 inhibitor control. In the ELISA studies, the data are expressed as the mean ± SD for three separate experiments, while, in the Western blot studies, the results shown are representative of those obtained in three different experiments and the histogram shows the quantification expressed as the mean ± SD. *, **, and *** indicate a significant difference at the level of p < 0.05, p < 0.01, and p < 0.001, respectively, compared to the DMSO/LPS/ATP group (A, B), LPS/DMSO/ATP group (C, D), or the DMSO/LPS group (E).

Figure 5. Effect of compound 1h on ROS production and MAPK phosphorylation in LPS-activated macrophages.
In (A), RAW 264.7 macrophages (5 × 10 5 /ml; 1 ml) were incubated for 30 min with compound 1h (20 µM), N-acetyl cysteine (NAC; 10 mM), or DMSO (vehicle), then 2', 7'-dichlorofluorescein diacetate (2 µM) was added for 30 min, followed by LPS (1 µg/ml) stimulation for the indicated time, then ROS levels were measured by detection of the mean fluorescence intensity (MFI) of the fluorophore carboxyl-DCF and expressing this value relative to that at time zero. In (B), RAW 264.7 macrophages (5 × 10 5 /ml; 1 ml) were incubated for 30 min with compound 1h (20 µM) or DMSO, then LPS (1 µg/ml) was added and incubation continued for 0-60 min, then phosphorylation of ERK1/2, JNK1/2, and p38 was analyzed by Western blotting and expressed relative to actin expression and as a fold increase compared to the control group at 0 time. In (C), J774A.1 macrophages (5 × 10 5 /ml; 1 ml) were incubated for 30 min with 10-40 µM compound 1h or DMSO, then LPS (1 µg/ml) was added and incubation continued for 20 min, then phosphorylation of ERK1/2, JNK1/2, and p38 was analyzed as in B. In (A), the data are expressed as the mean ± SD for three separate experiments, while, in (B) and (C), the results are representative of those obtained in three different experiments. * indicates a significant difference at the level of p < 0.05 compared to the DMSO/LPS group.   (20 µM), the NADPH oxidase inhibitor DPI (25 µM), or DMSO (vehicle) for 30 min in the continued presence of LPS, then 2',7'-dichlorofluorescein diacetate (2 µM) was added for 30 min, followed by ATP (5 mM) for the indicated time, then ROS levels were determined by measuring the mean fluorescence intensity (MFI) of the fluorophore carboxyl-DCF and expressing this value relative to that at time zero. In (B), J774A.1 macrophages (1 × 10 6 /ml; 1 ml) were incubated with compound 1h (20 µM), DPI (25 µM), or DMSO (vehicle) for 30 min, then LPS (1 µg/ml) was added for 6 h; the cells were then incubated with 2',7'-dichlorofluorescein diacetate (2 µM) for 30 min, then with ATP (5 mM) for the indicated time and ROS levels were measured by detection of the fluorescence intensity of the fluorophore carboxyl-DCF and expressed relative to that at time zero. In (C), LPS-primed J774A.1 macrophages (1 × 10 6 /ml; 1 ml) were incubated for 30 min with 20 µM compound 1h or DMSO (vehicle) followed by ATP (5 mM) stimulation for 0-60 min, then phosphorylation of PKC-α was analyzed by Western blotting and expressed as the fold increase measured as the intensity of the PKC-α band divided by that of the actin band normalized to the corresponding value for DMSO at 0 minutes. In (A) and (B), the data are expressed as the mean ± SD for three separate experiments, while, in (C), the results are representative of those obtained in three different experiments and the histogram shows the quantification expressed as the mean ± SD. * indicates a significant difference at the level of p < 0.05 compared to the DMSO/ATP group. release of pro-inflammatory mediators by activated macrophages [11]. Commercially available polyketides include rapamycin, a potent immunosuppressant [34], and fumagillin, which has been used in the treatment of microsporidiosis [35]. These results indicate that polyketides could be a valuable source of anti-inflammatory agents.
Thus far, polyketides have been mainly isolated from fungi or bacteria, but the typically small quantities that can be obtained in this way often limit biological studies. To address this limitation, we previously synthesized a group of polyketides (Figure 1 and Table 1) consisting of two hit compounds isolated from the soil ascomycete Gymnoascus reessii, auxarconjugatin A (compound 1b) and 12E-isorumbrin (compound 1e), and several analogs and evaluated their anti-lung cancer activity [9]. In the present study, we evaluated the anti-inflammatory activities of these compounds. Although compounds 1a-g were cytotoxic, compounds 1h-n were able to inhibit LPS-induced NO production without reducing macrophage viability.
ROS have an established role in inflammatory cytokine production in response to LPS [14,36]. They have also been implicated as playing an important role in NLRP3 inflammasome activation [25,[37][38][39]. Compound 1h exhibited antioxidative activity by reducing LPS-induced ROS production. However, from the data obtained, we were unable to conclude whether it inhibits the enzymes involved in ROS production or scavenges the ROS produced. However, further studies indicated that its effect of decreasing LPS-induced production of iNOS, NO, and IL-6 may be, at least in part, due to its antioxidative activity. The effect of compound 1h on LPSinduced TNF-α secretion might be cell type-dependent, as it reduced TNF-α secretion by LPS-activated murine J774A.1 macrophages, primary mice peritoneal macrophages, and JAWSII murine dendritic cells, but not LPS-activated murine RAW 264.7 macrophages. TNF-α secretion is controlled at the transcriptional and post-transcriptional levels by NF-κB and TNF-α converting enzyme, respectively (29,30). Since compound 1h reduced LPS-induced NF-κB activation in both J774A.1 and RAW 264.7 macrophages, this suggests that NF-κB plays less of a role in TNF-α secretion by RAW 264.7 macrophages. An earlier study reported that NLRP3 mRNA expression in LPS-activated macrophages was inhibited by ROS inhibitors [40], supporting an important role of ROS in NLRP3 expression. However, our results seem to contradict this finding, as, although compound 1h reduced ROS production in LPS-activated cells, it did not reduce NLRP3 protein expression. We therefore speculate that LPS activates signaling pathways other than the ROS pathway for the regulation of NLRP3 protein expression. We also found that compound 1h was able to reduce not only conventional inflammatory responses, such as NO and IL-6 production, but also NLRP3 inflammasome-mediated IL-1β expression in LPSactivated macrophages. NLRP3 inflammasome activation required both a priming signal (e.g., from TLR4) and an activation signal (e.g., from ATP) for caspase-1 activation and IL-1β secretion [40,41]. In the LPS-mediated priming stage, although compound 1h was not able to inhibit NLRP3 expression in LPS-activated macrophages, it significantly inhibited both proIL-1β expression and ROS production. These results show that it inhibits NLRP3 inflammasome activation by reducing ROS production, but not by reducing NLRP3 protein expression. Addition of compound 1h after LPS priming only slightly reduced ATP-induced ROS production ( Figure 7A), but addition before LPS priming significantly reduced LPS+ATPinduced ROS productionOK ( Figure 7B). These results suggest that it blocks an as yet unknown signal induced by LPS that contributes to ATP-mediated ROS production. They also explain why compound 1h significantly inhibited caspase-1 activation and IL-1β secretion when added before LPS priming, but only slightly reduced IL-1β secretion when added after LPS priming. ATP-induced ROS production has been shown to activate caspase-1 through the PI3-kinase/AKT pathway [32]. However in our study, we found that compound 1h did not reduce ATP-induced AKT phosphorylation in LPS-primed macrophages (data not shown), but, instead resulted, in a reduction in ATP-induced PKC-α phosphorylation.
In summary, we have shown that compound 1h, a non-toxic polyenylpyrrole, is able to inhibit NLRP3 inflammasome activation and NO and IL-6 expression by inhibiting LPS-and ATP-induced ROS production and LPS-induced activation of MAPK and NF-κB. The proposed anti-inflammatory mechanism of compound 1h is shown in Figure 8. These results suggest that compound 1h could be a lead compound for the development of anti-inflammatory therapeutics.