C646 Protects Against DSS-Induced Colitis Model by Targeting NLRP3 Inflammasome

Numerous pieces of evidence have identified that the NLRP3 inflammasome plays a pivotal role in the development and pathogenesis of colitis. Targeting the NLRP3 inflammasome represents a potential therapeutic treatment. Our previous studies have suggested that acetylation of NLRP3 is indispensable to NLRP3 inflammasome activation, and some acetyltransferase inhibitors could suppress the NLRP3 inflammasome activation. Here, we identified that C646, an inhibitor of histone acetyltransferase p300, exerts anti-inflammatory effects in DSS-induced colitis mice by targeting the NLRP3 inflammasome. Mechanistically, C646 not only inhibits NF-κB activation, leading to the decreased expression of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and NLRP3, but also suppresses the NLRP3 inflammasome assembly by disrupting the interaction between NLRP3 and ASC. In addition, C646 attenuated the LPS-induced acute systemic inflammation model. Thus, our results demonstrate the ability of C646 to suppress the NLRP3 inflammasome activity and its potential application in the treatment of inflammatory bowel disease.


INTRODUCTION
Inflammatory bowel diseases (IBDs), comprising ulcerative colitis (UC) and Crohn's disease (CD), are chronic and complex disorders of the gastrointestinal tract that are characterized by uncontrolled intestinal inflammation and epithelial injury. However, a comprehensive understanding of the pathophysiological mechanisms of IBD is far less clear (Kaser et al., 2010). Recent studies have suggested that biological therapies inhibition cytokines promote inflammation (e.g., anti-tumor necrosis factor TNF antibodies) or modulation of lymphocyte trafficking are effective in many IBD patients, implying that targeting dysregulated immune responses may be a potential therapeutic treatment in the future (Kaser et al., 2010;Kobayashi et al., 2020;Roda et al., 2020).
The NLRP3 inflammasome, a multiple protein complex, has been observed to play a critical role in the development and pathogenesis of IBD (Tourkochristou et al., 2019;Zhen and Zhang, 2019). It consists of the nucleotide-binding domain, leucine-rich repeat, pyrin domain-containing protein 3 (NLRP3), the apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1 (Martinon et al., 2009;Swanson et al., 2019). NLRP3 can be activated by a broad range of microbial motifs, endogenous danger signals, and environmental irritants. Upon activation, NLRP3 recruits ASC and caspase-1, leading to the maturation and secretion of IL-1β and IL-18, as well as gasdermin D-mediated pyroptotic cell death. It is well known that NLRP3 inflammasome activation is a two-step process, namely, priming and activation. Pathogen-associated molecule patterns (PAMPs), such as LPS, lead to the nuclear factor-κB (NF-κB) activation and increase the expression of NLRP3 and IL-1β, which is called the priming step. Following the priming step, diverse stimuli, such as ATP or nigericin, induce the assembly of the NLRP3 inflammasome and full activation. Recent studies have illustrated that excessive NLRP3 inflammasome-induced IL-1β aggravates colitis, and the absence of NLRP3 inflammasome components has a protective role in the dextran sodium sulfate (DSS)-induced colitis model (Tourkochristou et al., 2019;Zhen and Zhang, 2019). In addition, pharmacological inhibition of NLRP3 inflammasome activation ameliorates intestinal inflammation in animal models, suggesting that the NLRP3 inflammasome is a candidate target for treatment of IBD (Bauer et al., 2010;Du et al., 2017;Zhao Y. et al., 2019;Mei et al., 2019).
Our previous study (Zhao et al., 2019a;Liu et al., 2020) and the Danica Chen group's research study  have both uncovered that acetylation of NLRP3 is required for the full activation of the NLRP3 inflammasome and have further suggested that some acetyltransferase inhibitors may suppress the NLRP3 inflammasome activation. By searching for kinds of lysine acetyltransferase inhibitors, we recently showed that SI-2 hydrochloride (SI-2) could block NLRP3 inflammasome activation . During the process, we noticed that one compound named C646, an inhibitor of histone acetyltransferase p300, not only decreased IL-1β but also TNFα secretion. Whether C646 exerts a protective function in the colitis model is an interesting question to further study. In this work, we will evaluate the effect of C646 on colonic inflammation and explore the underlying mechanisms.

Mice
Wild-type (WT) C57BL/6 male mice (6-10 weeks old, 20-24 g) were obtained from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China), and they were bred under SPF conditions. Studies were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Central South University.

Cell Culture
Primary peritoneal macrophages from C57BL/6 mice were harvested using the following method. Mice that were 6-8 weeks old were injected intraperitoneally with 3 ml of sterile 3% thioglycolate broth to elicit peritoneal macrophages. After 72 h, cells were collected via peritoneal lavage with 10-15 ml of RPMI medium 1,640 (Gibco). After being resuspended, the cells were cultured in RPMI-1640 containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37oC in a humidified incubator with 5% CO2.

ASC Oligomerization
Primary peritoneal macrophage cells were treated with indicated stimuli. Next, the cells were lysed with Triton Buffer 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, and 0.1 mM phenylmethylsulfonylfluoride (PMSF) for 10 min at 4°C. The cell lysates were centrifuged at 6000 g for 15 min at 4°C. After that, the supernatant was collected and resuspended in 200 μL Triton Buffer and 2 mM disuccinimidyl suberate (DSS). The complex was cross-linked for 30 min at 37°C. At last, cell pellets were washed twice. Samples were centrifuged, and the pellets were dissolved in sodium dodecyl sulphate (SDS) loading buffer for Western blotting.

ASC Speck Formation
Primary peritoneal macrophage cells were seeded on chamber slides overnight. On the next day, the cells were treated with indicated stimuli. Next, the cells were washed three times with PBS buffer, fixed in 4% paraformaldehyde (PFA) for 10 min, permeabilized with 0.1% Triton X-100 for 15 min, and blocked with PBS buffer containing 3% BSA. Cells were then stained with anti-ASC (1:200) at 4°C for 12 h and with DyLight 488-labeled secondary antibody (1:50) at room temperature for 60 min. Last, DAPI was used to stain nuclei. Cells and tissues were visualized using a fluorescence microscope (Nikon Ti2-U).

Immunoprecipitation and Western Blot
After stimulation, peritoneal macrophages were lysed in immunoprecipitation (IP) buffer containing 50 mM Tris HCl (pH 7.4), 50 mM EDTA, 200 mM NaCl, and 1% NP-40. Supplemented with a protease inhibitor cocktail, precleared cell lysates were then subjected to specific antibodies overnight and to protein G plus-agarose for 2 h and then washed four times with IP buffer. The immunoprecipitation complex was dissolved in sodium dodecyl sulphate (SDS) loading buffer for Western blotting.
For immunoblot analysis, cells were lysed with CLB supplemented with a protease inhibitor cocktail and PMSF, and then the cell lysates were centrifugated at 12,000 g for 5 min at 4°C. Equal amounts of extracts were separated by SDS-PAGE, and then they were transferred onto 0.22-mm PVDF membranes (Merck Millipore, ISEQ00010) for immunoblot analysis.

SDD-AGE
The oligomerization of NLRP3 Western blot was analyzed following published protocols (Hou et al., 2011;Jiang et al., 2017). Cells were lysed with Triton X-100 lysis buffer and then centrifuged at 12,000 g for 5 min at 4°C. Next, the lysis was resuspended in 5 × sample buffer (2.5 × TBE, 50% glycerol, 10% SDS, and 0.0025% bromophenol blue) and run onto vertical 1.5% agarose gel. After electrophoresis for 1 h at a constant voltage of 80 V at 4°C in the running buffer (1 × TBE and 0.1% SDS), the proteins were transferred onto 0.22-mm PVDF membranes for immunoblotting.

LPS-Induced Acute Systemic Inflammation
C57BL/6 mice were pretreated with or without C646 half an hour earlier. Next, they were injected intraperitoneally with LPS (20 mg/kg). After 8 h, the mice were terminated to collect the serum (Huang et al., 2020;Liu et al., 2020). The serum concentrations of IL-1β, TNF-α, and IL-6 were measured by ELISA, and the lungs were harvested.

DSS-Induced Colitis
For acute experimental colitis induction, C57BL/6 mice were treated with 3% DSS or saline in their drinking water for 10 days. During the experiment, body weight, stool, and body posture were monitored daily to assess the DAI (Cooper et al., 1993;Alex et al., 2009). The DAI is the combined score of weight loss compared with initial weight, stool consistency, and bleeding. The details are as follows: (a) weight loss (0 point none, 1 point 1-5% weight loss, 2 points 5-10% weight loss, 3 points 10-15% weight loss, and 4 points more than 15% weight loss), (b) stool consistency or diarrhea (0 point normal, 2 points loose stools, and 4 points watery diarrhea), and (c) bleeding (0 point no bleeding, 2 points slight bleeding, and 4 points gross bleeding). Mice were euthanized at the indicated time points, and the colon was immediately collected for colon length measurement, colon explant culture, colon Western blot analysis, and histological analysis.

Colon Explant Culture
After removing the intestines from the mice, they were briefly washed with saline and then washed three times with cold RPMI-1,640 containing penicillin G (200 μg/ml) and streptomycin (200 μg/ml). The purpose is to wash away residual intestinal bacteria. Then they were incubated in supplemented culture medium containing penicillin G (200 μg/ml) and streptomycin (200 μg/ml). The medium was collected after incubation for 24 h at 37°C with 5% CO2, and the production of pro-inflammatory cytokines was determined by ELISA.

Colonic Immunoblot Analysis
The mouse intestines and SDS lysate were lysed according to the ratio of 10 mg:200 ul, followed by ultrasound and grinding, after which they were centrifuged at 12,000 g for 10 min, and the supernatant was aspirated. After protein quantitative analysis, extracts were boiled and dissolved in sodium dodecyl sulphate (SDS) loading buffer for Western blotting.

Histological Analysis
The mouse tissue was fixed in 4% PFA at 4°C for 24 h and sectioned after being embedded in paraffin. The sections were prepared and stained with H&E using standard procedures. The slides were inspected under a Nikon ECL IPSE Ci biological microscope, and images were captured using a Nikon DS-U3 color digital camera.

Statistical Analysis
All the results are expressed as the mean ± SD, and statistical analyses were performed using GraphPad Prism software 8.0. Statistical analysis was performed using the ANOVA with the Bonferroni test for the two groups test. p > 0.05 was not a significant statistical difference; p < 0.05 was considered statistically significant, with increasing levels of confidence displayed as **p < 0.05; ***p < 0.01; ****p < 0.0001.

C646 Protects Against DSS-Induced Colitis Murine Model
We previously  showed that C646 may suppress inflammation in vitro. To further study the effect of C646 on the treatment of colonic inflammation, the DSS-induced acute colitis murine model was adopted, which could destruct epithelial cells, increase the permeability of the colon, and trigger inflammation in the gut (Alex et al., 2009).
Mice received 3% DSS in their drinking water for 10 days, and C646 was given once a day by intraperitoneal injection from day 0 to day 9 ( Figure 1A). The clinical assessments, including body weight, grossly bloody stools, and stool consistency, were recorded daily. As shown in Figure 1B, mice only administrated with DSS suffered a 20% weight loss, while for the group treated with 5 mg/kg or 10 mg/kg of C646, it improved the weight loss caused by DSS significantly. The disease activity index (DAI) is the summary of scores including weight loss, diarrhea, and gross bleeding, used to assess the severity of colitis. Similarly, C646 reduced the DAI in a dose-dependent manner ( Figure 1C). Colon length is another indicator for monitoring the severity of colitis, and we found that C646treated mice showed longer colon length than the control group ( Figures 1D,E). To further evaluate the effect of C646 in colitis, histological examination of hematoxylin and eosin (H&E) staining was carried out. We observed severe mucosal damage, gland destruction, and infiltration of mononuclear cells in the colon specimens of DSS-fed mice. In contrast, C646-treated mice exhibited intact colonic architecture without mucosal damage and with less mononuclear cell infiltration ( Figure 1F). Thus, our results demonstrated that C646 protects against DSS-induced colitis.

C646 Suppresses NLRP3 Inflammasome Activity in DSS-Induced Colitis Model
Numerous studies (Bauer et al., 2010;Tourkochristou et al., 2019;Zhen and Zhang, 2019) have suggested that the NLRP3 inflammasome is a potential target for the treatment of colitis. Then we examined whether C646 could inhibit NLRP3 inflammasome activity in the DSS-induced model. We detected the expression of NLRP3 inflammasome components and the secretion of IL-1β in colon tissue. We observed that C646 markedly reduced the expression of NLRP3 and the cleavage of caspase-1, the downstream event of inflammasome activation ( Figure 2A). Consistently, IL-1β secretion from the C646-treated group was decreased in a dose-dependent manner ( Figure 2B), Immunoblot analysis of ASC oligomerization in cross-linked cytosolic pellets of DSS-induced colitis treated with C646 or DMSO. The results of (B) and (C) were expressed as mean ± SD. Statistics were analyzed using one-way ANOVA and the Bonferroni test. **p < 0.05; ***p < 0.01; ****p < 0.0001.
FIGURE 3 | C646 suppresses the NLRP3 inflammasome at the priming and assembly steps.(A-D) Mouse peritoneal macrophages were treated with C646 and then primed with LPS, followed by stimulation with nigericin. The supernatant (SN) was subjected to ELISA assay for IL-1β, IL-6, TNF-α, and LDH. (E-H) Mouse peritoneal macrophages were primed with LPS and then treated with C646, followed by stimulation with nigericin. The supernatant (SN) was subjected to ELISA assay for IL-1β, IL-6, TNF-α, and LDH. For (A) to (H), values are mean ± SD. Statistics were analyzed using one-way ANOVA and the Bonferroni test. **p < 0.05; ***p < 0.01; ****p < 0.0001.
Frontiers in Pharmacology | www.frontiersin.org July 2021 | Volume 12 | Article 707610 and IL-6 also showed a similar pattern ( Figure 2C). ASC speck formation was another hallmark of NLRP3 inflammasome activation, and we further detected the ASC speck by using disuccinimidyl suberate cross-linking (Fernandes-Alnemri et al., 2007;Davis et al., 2011). As anticipated, C646 reduced ASC speck formation in colon tissues ( Figure 2D). Taken together, these data suggested that C646 inhibits NLRP3 inflammasome activation in the DSS-induced colitis model.

C646 Inhibits NLRP3 Inflammasome at Both Priming and Assembly Steps
To further study the inhibiting effect of C646 on the NLRP3 inflammasome, we treated primary macrophages with C646 before or after LPS priming and then stimulated them with nigericin, an NLRP3 inflammasome agonist. We observed that the secretion of IL-1β, TNF-α, and IL-6 was decreased whenever treatment with C646 was carried out and so was cell death, evaluated by LDH release (Figures 3A-H), suggesting that C646 suppresses NLRP3 inflammasome activation at both priming and activation steps. We also noticed that C646 inhibited nigericininduced NLRP3 activation in a dose-dependent manner and achieved the maximum suppressing effect at 8 μM.
To further confirm the results, ATP and MSU, another two kinds of NLRP3 inflammasome agonists, were used. C646 had a similar suppressing effect on ATP-or MSU-induced NLRP3 inflammasome activation, reflected by cytokine production and cell death (Figures 4A-H). In addition, the cleavage of caspase-1 and pro-IL-1β detected by the Western blot was shown to be decreased upon C646 treatment (Figures 4I,J). Thus, these data indicated that C646 inhibits NLRP3 inflammasome activation at both priming and activation steps.

C646 Inhibits the Activation of the NF-κB Pathway
Next, we decided to explore whether the mRNA expressions of pro-inflammatory cytokines were impaired by C646 treatment. As expected, C646 significantly inhibited the mRNA levels of IL-1β, TNF-α, and IL-6, as well as NLRP3 (Figures 5A-D). Since NF-κB signaling dominantly contributed to the production of pro-inflammatory cytokines and NLRP3 (Bauernfeind et al., 2009), we detected this pathway and observed that C646 sharply decreased the phosphorylation of p65, a key event for NF-κB activation ( Figure 5E). Consistently, the protein expression of NLRP3 and pro-IL-1β was also decreased upon C646 treatment ( Figure 5E). Thus, these data suggested that C646 inhibits the NF-κB pathway activation, which is consistent with a previous study .

C646 Inhibits NLRP3 Inflammasome Activation by Disrupting the Association Between NLRP3 and ASC
To explore the underlying mechanism of C646 inhibiting the NLRP3 inflammasome at the activation step, we first checked ASC speck formation. We found that C646 treatment significantly attenuated the percentage of cells containing ASC specks in the ATP or nigericin stimulation groups via immunofluorescence ( Figures 6A,B), and this was further demonstrated by the detection of ASC oligomerization using disuccinimidyl suberate cross-linking ( Figure 6C). Thus, C646 inhibits the formation of ASC specks.
Previous studies have suggested that interaction between NLRP3 and ASC is an upstream step for ASC speck formation. By using immunoprecipitation and immunoblotting assays, we found that C646 significantly inhibits the interaction between NLRP3 and ASC but not ASC and caspase-1 upon LPS + nigericin treatment ( Figure 6D). Recent work has suggested that NEK7, a member of the mammalian NIMA-related kinases, is essential for the activation of the NLRP3 inflammasome by interacting with NLRP3 and promoting ASC oligomerization (Franklin et al., 2014;He et al., 2016;Schmid-Burgk et al., 2016). Therefore, we investigated the NLRP3-NEK7 interaction and observed that C646 barely affected the association between them ( Figure 6E). Since oligomerized NLRP3 can recruit ASC (Swanson et al., 2019), we further explored whether C646 affects NLRP3 oligomerization, as the results showed that C646 had no effect on the oligomerization of NLRP3 ( Figure 6F). Overall, our results indicated that C646 disrupts the interaction between NLRP3 and ASC and then leads to the impairment of NLRP3 inflammasome assembly.

C646 Ameliorates LPS-Induced Acute Systemic Inflammation in vivo
We further verified the function of C646 in another inflammation model by adopting LPS-induced acute systemic inflammation (Mariathasan et al., 2006). Mice were pretreated with C646 (10 or 20 mg/kg i. p.) or saline and then challenged with LPS (20 mg/kg). As shown, the C646-treatment group decreased IL-1β, IL-6, and TNF-α levels in serum in a dose-dependent manner ( Figures  7A-C). Furthermore, C646 alleviated the endotoxemia-induced lung injury, as evaluated by histopathology ( Figure 7D). Thus, C646 shows therapeutic effects on LPS-induced acute systemic inflammation.

DISCUSSION
In the present study, we have found that C646 plays an antiinflammatory role in the DSS-induced colitis model by targeting the NLRP3 inflammasome. Mechanistically, C646 not only impairs NLRP3 and pro-inflammatory cytokine expression by affecting NF-κB activation at the priming step but also decreases NLRP3-ASC association, and then it inhibits the NLRP3 inflammasome assembly at the activation step. In addition, C646 ameliorates LPS-induced acute systemic inflammation. Thus, our study demonstrated that C646 could be a potential agent for the treatment of colitis. C646, an inhibitor of histone acetyltransferase p300, was initially discovered by virtual ligand screening (Bowers et al., Statistics of (B) were analyzed using two-way ANOVA and the Bonferroni test. **p < 0.05; ***p < 0.01; ****p < 0.0001.
Frontiers in Pharmacology | www.frontiersin.org July 2021 | Volume 12 | Article 707610 8 2010). After that, the potential application of C646 was mostly evaluated in cancer biology (Oike et al., 2014;Wu et al., 2015;Ono et al., 2016;Liang et al., 2020). Numerous studies have found that C646 could induce apoptosis, arrest the cell cycle, and block the proliferation in cancer cells (Oike et al., 2014;Wu et al., 2015). Recent works have suggested an immunomodulatory role of C646 in antibacterial  and antiviral (Zhao et al., 2015) responses. Moreover, the application of C646 has been enhanced in inflammatory lung diseases. Based on our previous study (Zhao K. et al., 2019;Liu et al., 2020), we investigated the anti-inflammatory role of C646 in the DSS-induced colitis model. We found that C646 improved the weight loss, diarrhea, and gross bleeding induced by DSS. Moreover, the C646-treated group shows longer colon length and exhibits intact colonic architecture without mucosal damage and with less mononuclear cell infiltration in comparison to the control group, suggesting a protective role of C646 in the DSS-induced murine model. Then we explored whether C646 affects the NLRP3 inflammasome activation in colon tissues. We observed that DSS induced elevated expression of NLRP3 and cleavage of caspase-1, which were attenuated by C646 treatment in colon tissues. The following secretion of IL-1β and IL-6 was also decreased. Consistently, ASC speck formation, an NLRP3 inflammasome activation marker, was impaired upon C646 treatment, indicating that C646 inhibits NLRP3 inflammasome activation in the DSS-induced murine model. We further investigated the underlying mechanism of C646 on the NLRP3 inflammasome in primary macrophages. We found that C646 inhibits NLRP3 inflammasome activity in a dose-dependent manner, and this suppressing effect was achieved both at the priming step and the activation step when given C646 before or after LPS priming. Consistent with early studies (Atreya et al., 2008), our study also observed that C646 inhibits NF-κB-induced gene expression in primary macrophages, which could be explained by the role of p300 in the regulation of the NF-κB pathway. Moreover, we found that C646 inhibits NLRP3 inflammasome assembly by disrupting the association between NLRP3 and ASC but not NLRP3 and NEK7. Since oligomerized NLRP3 is required for recruiting ASC, we further investigated this process and observed that C646 has no effect on the oligomerization of NLRP3. We speculated that C646 may interact with NLRP3 or ASC or both of them and then block the association between them. This hypothesis may need the help of structural biology to be demonstrated, which still needs further investigation. In addition, we confirmed the anti-inflammatory function of C646 in another LPS-induced systemic inflammation model in vivo. Thus, C646 shows a robust suppressing effect on the NLRP3 inflammasome.
Frontiers in Pharmacology | www.frontiersin.org July 2021 | Volume 12 | Article 707610 9 decreased intestinal epithelial integrity, suggesting a protective role of the NLRP3 inflammasome. Although the contradictory results in mice models exist, human data have shown that NLRP3 inflammasome activity is increased in UC and CD patients, and some studies even demonstrated that targeting NLRP3 activity, such as MCC950 (Bauer et al., 2010), flavonoid VI-16 (Zhao et al., 2019), carboxyamidotriazole (CAI) (Du et al., 2017), and PAP-1 (Mei et al., 2019), in IBD murine models displayed therapeutic effects. In this study, we have found that C646 suppresses NLRP3 inflammasome activity through impairing the NF-κB pathway at the priming step and inhibiting the assembly at the activation step. It is well known that the NF-κB pathway is crucial for the production of inflammatory cytokines, including IL-6 and TNF-α, both of which are important targets for the treatment of intestinal inflammation (Mao et al., 2018;Waljee et al., 2018). Considering that the use of curcumin (Gong et al., 2015;Iqbal et al., 2018;Wang et al., 2018), an agent inhibiting NLRP3 inflammasome and NF-κB activation, in UC patients was linked to clinical improvement and endoscopic remission, C646 may have potential in clinical studies for IBD treatment, but we still need more clinical data to test.
Overall, our study showed that C646 could ameliorate DSSinduced colitis, expanding the anti-inflammatory potential of this agent. Moreover, the suppressing effect on the NLRP3 inflammasome delineates a new insight into the immunomodulatory function of C646.

CONCLUSION
In summary, our study indicates the protective effect of C646 in DSS-induced colitis. This effect could be mediated by suppressing the activation of the NLRP3 inflammasome and the NF-κB signaling pathways. Our results demonstrated that C646 could be a candidate agent for IBD treatment.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Central South University. Written informed consent was obtained from the owners for the participation of their animals in this study.

AUTHOR CONTRIBUTIONS
XX, JL, XL, YZ, XY, and XR performed the experiments; XX and JL analyzed the results; KZ and LT designed the research study; XX, KZ, and LT wrote the manuscript. KZ and LT supervised the whole project.