Identification of WT161 as a Potent Agent for the Treatment of Colitis by Targeting the Nucleotide-Binding Domain-Like Receptor Family Pyrin Domain Containing 3 Inflammasome

Inflammatory bowel diseases (IBD), including Crohn’s disease and ulcerative colitis (UC), are chronic and recurrent intestinal inflammatory disorders. Numerous studies have revealed that the nucleotide-binding domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome plays a pivotal role in the pathogenesis of IBD, and inhibition of the NLRP3 inflammasome alleviates colitis in experimental animals. Our previous study showed that C646, an inhibitor of histone acetyltransferase p300, has a protective role in dextran sulfate sodium (DSS)-induced colitis by targeting the NLRP3 inflammasome, making us further study the inhibitors of histone deacetylases (HDACs) in the treatment of colitis. In this study, we have shown that WT161, an inhibitor of HDAC6, exerts a protective role in a colitis model, blocks NLRP3 inflammasome activation, disrupts ASC speck formation, and decreases the expression of NLRP3. This study uncovers a new inhibitor of the NLRP3 inflammasome and suggests its potential application in the treatment of active IBD.


INTRODUCTION
Inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis (UC), are chronic and recurrent intestinal inflammatory disorders (Hodson, 2016). Although the precise pathogenesis of IBD remains unclear, it is now widely suggested that genetic-environment-mediated dysregulation of the mucosal immune response contributes to its occurrence. The morbidity and burden of IBD are increasing worldwide (Kaplan, 2015;Kaplan and Ng, 2016). The limitations of drugs currently used for IBD treatment, such as aminosalicylic acid, corticosteroids, biologicals, and immunosuppressive agents (Lamb et al., 2019), have prompted researchers to develop new drugs that are suitable for long-term use with fewer side effects. Small-molecule inhibitors have attracted increasing interest as potential agents for the treatment of IBD.
Numerous studies have revealed that the nucleotide-binding domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome plays a pivotal role in the pathogenesis of IBD (Kanneganti, 2017;Mao et al., 2018). The NLRP3 inflammasome is a cytosolic protein complex that consists of NLRP3, apoptosisassociated speck-like protein containing a CARD (ASC) and caspase-1 (Agostini et al., 2004). Once activated, NLRP3 recruits ASC and caspase-1 to form a large complex, leading to the conversion of precursors prointerleukin-1β and prointerleukin-18 into active interleukin (IL)-1β and IL-18, respectively, as well as inducing cell death. Researchers have indicated that single-nucleotide polymorphisms in the NLRP3 gene are associated with susceptibility to UC (Hanaei et al., 2018;Zhou et al., 2021), and NLRP3 inflammasome activity is increased in some patients with UC and Crohn's disease (McAlindon et al., 1998;Coccia et al., 2012). Moreover, selective blockade of the NLRP3 inflammasome could ameliorate colonic inflammation in a colitis model Song et al., 2021). Thus, the NLRP3 inflammasome may be a promising target for future treatment of IBD.
Pharmacological inhibition of the NLRP3 inflammasome plays a protective role in colitis (Bauer et al., 2010). In our previous study, we found that the histone acetyltransferase inhibitor C646 inhibits NLRP3 inflammasome activity and exerts anti-inflammatory effects in dextran sulfate sodium (DSS)-induced colitis mice by targeting the NLRP3 inflammasome (Xu et al., 2021). Furthermore, Magupalli et al. (2020) found that histone deacetylase 6 (HDAC6) promotes the activation of the NLRP3 inflammasome (Magupalli et al., 2020), which draws our focus to HDAC6 inhibitors. Based on these studies, we suspect that the HDAC6 inhibitor WT161 can inhibit the activation of NLRP3 inflammasome and alleviate colitis in DSS-treated mice.
In this study, we found that WT161 exerts a protective role in a colitis model, blocks NLRP3 inflammasome activation, disrupts ASC speck formation, and decreases NLRP3 expression. This study uncovers a new inhibitor of the NLRP3 inflammasome and suggests its potential application in the treatment of active IBD. WT161 (cat. no. 1206731-57-8)   ,  ELISA kits (Thermo Fisher).

Mice
All procedures were performed in accordance with the Institutional Animal Care and Use Committee of the Central South University. Male C57BL/6 mice (6-8 weeks old) were purchased from Hunan SJA Laboratory 100 Animal Co. Ltd (Changsha, China), and all animal experiments were carried out in the Department of Laboratory Animals of Central South University under SPF conditions.

Histological Analysis
The 0.3-0.5 cm distal colonic segment close to the rectum was fixed in 4% paraformaldehyde for 24 h for histological analysis. The slides were prepared and stained with H&E and examined under a Nikon ECL IPSE Ci biological microscope. The images were collected using a Nikon DS-U3 digital camera. Histological scores were evaluated according to a previously published study (Obermeier et al., 1999). Epithelium (E): 0 (normal), 1 (loss of goblet cells), 2 (loss of goblet cells in large areas), 3 (loss of crypts), 4 (loss of crypts in large areas); infiltration (I): 0 (no infiltrate), 1 (infiltrate around crypt basis), 2 (infiltrate reaching to L. muscularis mucosae), 3 (extensive infiltration reaching the L. muscularis mucosae, and thickening of the mucosa with abundant edema), and 4 (infiltration of the L. submucosa). The histological scores were the sum of the epithelium and infiltration scores.

Colon Explant Culture
The next 0.3-0.5 cm distal colonic segment (with the same weight) close to the rectum was washed with saline briefly and then washed three times with cold RPMI-1640 (Gibco) containing penicillin G (200 μg/ml) and streptomycin (200 μg/ ml) to remove the residual bacteria. They were then cultured in RPMI-1640 supplemented with 10% FBS, penicillin G (200 μg/ ml), and streptomycin (200 μg/ml) at 37°C with 5% CO 2 for 24 h. Pro-inflammatory cytokines in the supernatant were detected by ELISA.

Cell Culture
Peritoneal macrophages from healthy SPF C57BL/6J mice (6-8 weeks old) were elicited by i.p. injection of 3 ml 3% thioglycolate broth. Macrophages were harvested 3 days later by i.p. instillation and retraction of peritoneal lavage with 10 ml RPMI-1640 medium. Cells were purified by adherence and cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified incubator with 5% CO 2 .

Western Blot
For cell proteins, cells were lysed with CLB supplemented with a protease inhibitor cocktail and phenylmethylsulfonylfluoride (PMSF), and then the cell lysates were centrifuged at 12,000 g for 5 min at 4°C. For tissue protein analysis, the mouse colon and sodium dodecyl sulphate (SDS) lysate were lysed at a ratio of 10 mg:200 μl followed by ultrasound and grinding, centrifuged at 12,000 g for 10 min, and the supernatant was aspirated. After protein quantitative analysis, the extracts were boiled and dissolved in SDS loading buffer for western blot. Equal amounts of extracts (all proteins in the supernatant of each well, 10 μg protein extracted from cell lysate or colonic tissue) were separated by 10% SDS-PAGE, and then transferred onto 22mm PVDF membranes (Merck Millipore, ISEQ00010) for immunoblot analysis. The signal intensity was detected using the ImageJ software. The data were standardized to β-actin expression levels.

ASC Oligomerization
Peritoneal macrophages were seeded overnight in six-well plates (2 × 10^6/well). The next day, the cells were primed with LPS (100 ng/ml) for 3 h, followed by DMSO or WT161 (5 μM, dissolved in DMSO) for 30 min, and then stimulated with 10 μM nigericin for 1 h. After stimulation, cells were rinsed in PBS and 100 μl ice-cold Triton buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100) with 0.1 mM phenylmethylsulfonylfluoride (PMSF). The cells were centrifuged at 6,000 g for 15 min at 4°C. The supernatant was resuspended in SDS loading buffer as a soluble sample. Insoluble samples (pellets) were washed twice with Triton buffer and resuspended in 200 μl Triton buffer containing 0.1 mM PMSF. Disuccinimidyl suberate (2 mM) was added to the resuspended pellets and incubated at 37°C for 30 min. The samples were then re-centrifuged at 6,000 g for 15 min at 4°C and suspended in SDS loading buffer. Both samples were then collected for western blot. The extracts (10 μg) from each sample were separated by 10% SDS-PAGE.

ASC Speck Formation
The peritoneal macrophages were cultured overnight on chamber slides. The following day, the cells were primed with LPS (100 ng/ ml) for 3 h, followed by treatment with DMSO or WT161 (5 μM, dissolved in DMSO) for 3 h, then stimulated with 10 μM nigericin or 5 mM ATP for 1 h. Cells were then fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.1% Triton X-100. After blocking with 5% BSA, cells were incubated with anti-ASC antibody (1:200) overnight at 4°C in PBS containing 5% BSA and DyLight 488-labeled secondary antibody (1:50) for 1 h in PBS containing 5% BSA. After thorough washes, the slides were mounted with mounting medium containing DAPI. Analyses were carried out using a fluorescence microscope (Nikon Ti2-U).

Statistical Analysis
All results are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA with Bonferroni's test or two- way ANOVA with Bonferroni's multiple comparisons test for the groups test in GraphPad Prism software 8.0. Statistical significance was defined as p < 0.05, with increasing levels of confidence displayed as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

WT161 Alleviated Intestinal Inflammation in a DSS-Induced Colitis Model
To investigate the function of WT161 in the treatment of NLRP3related diseases, DSS-induced colitis was used. Based on previously reported pharmacokinetic properties, WT161 was formulated for i.p. administration once a day at a dose of 10 or 20 mg/kg in 3% DSStreated mice from day 1 to day 10. Body weight, grossly bloody stools, and stool consistency were recorded daily. Compared to that of the control group, the WT161 treatment group showed improved weight loss ( Figure 1A). Accordingly, WT161 reduced the disease activity index (DAI) score, which included weight loss, diarrhea, and gross bleeding in a dose-dependent manner ( Figure 1B). Colon length, which reflects the severity of colitis, was longer after WT161 treatment ( Figures 1C,D). Consistently, histopathological analysis (hematoxylin and eosin [H&E] staining) showed that WT161 administration decreased the loss of goblet cells and alleviated disruption of crypt structure and mucosal epithelium ( Figure 1E), which was also confirmed by the lower histological injury score ( Figure 1F). Taken together, these data indicate that WT161 exerts a protective role in a DSS-induced colitis animal model.

WT161 Decreased the Levels of Pro-Inflammatory Factors in the Colonic Tissue of DSS-Induced Colitis Mice and Activated Peritoneal Macrophages Induced by LPS + DSS
Aberrant and excessive activation of some cytokines is involved in the pathogenesis of IBD (Neurath, 2014), among which IL-1β and IL-6 have been regarded to play a prominent role in the initiation and maintenance of colonic inflammation, respectively (Neurath, 2014). We then evaluated the effect of WT161 on IL-1β and IL-6 in the supernatant culture of colonic explants isolated from DSS-induced colitis mice and activated peritoneal macrophages induced by LPS + DSS. The levels of IL-1β and IL-6 in the supernatant were detected by ELISA after 24 h of colonic tissue culture. WT161 decreased the release of IL-1β ( Figure 2A) and IL-6 ( Figure 2B) in the colonic explants, and similar results were found in LPS + DSS-activated peritoneal macrophages ( Figures 2C,D). Taken together, WT161 disturbed the release of IL-1β and IL-6, thereby inhibiting inflammation.

WT161 Decreased NLRP3 Inflammasome Activation in Peritoneal Macrophages
Considering that IL-1β mainly depends on the activation of the NLRP3 inflammasome (Mao et al., 2018) and the fact that HDAC6, the target of WT161, is indispensable for the activation of inflammasomes (Magupalli et al., 2020), we investigated the effect of WT161 on NLRP3 inflammasome. The NLRP3 inflammasome can be activated by a wide variety of unrelated stimuli such as nigericin. ATP and MSU are other commonly used activators of the NLRP3 inflammasome by acting on the plasma membrane P2X7 purinergic receptor to promote Ca 2+ and Na + influx or by causing lysosomal rupture, respectively. WT161 reduced the release of IL-1β upon nigericin, ATP, and MSU stimulation ( Figure 3A). We observed a reduction in cleaved caspase-1 (p10) and mature IL-1β (p17) ( Figures  3B,C) upon nigericin stimulation in the supernatant by WT161 treatment in a dose-dependent manner. Upon treatment with NLRP3 activators, NLRP3 undergoes initial self-oligomerization, after which NLRP3 recruits ASC, leading to ASC oligomerization, which is critical for subsequent caspase-1 activation. Thus, we tested ASC oligomerization to further evaluate the effect of WT161 on the activation of the NLRP3 inflammasome. Using disuccinimidyl suberate crosslinking, we found that WT161 effectively inhibited the formation of ASC polymers (Figures 3D,E). This result was confirmed by immunofluorescence ( Figures 3F,G).

WT161 Inhibited the Expression of NLRP3 and Negatively Modulated NF-κB Signaling
Finally, we aimed to explore the effect of WT161 on the expression of NLRP3, ASC, and caspase-1. The priming process of NLRP3 activation is induced by various pathogenassociated molecular patterns or cytokines, leading to activation of NF-κB signaling, which induces the expression of IL-1β and NLRP3 (Bauernfeind et al., 2009). We treated macrophages with WT161 before LPS stimulation and analyzed the mRNA expression of NLRP3, ASC, and caspase-1. Intriguingly, the qPCR results showed that WT161 significantly reduced the mRNA level of NLRP3, with no effects on ASC and caspase-1 ( Figures 4A-C). Consistently, western blot analysis showed that WT161 only decreased the NLRP3 protein expression, without affecting ASC and caspase-1 expression in the LPS-treated peritoneal macrophages (Figures 4D,E). Moreover, WT161 reduced the LPS-induced increase in p-p65 and p-p65/p65 ( Figures 4D,F), a direct indicator of activated NF-κB signaling. To further demonstrate this effect, we collected the protein extracted from colonic tissue isolated from the DSStreated mice and detected the expression of these proteins. We observed that DSS induced the expression of NLRP3 in the colon tissues, whereas the WT161-treated group had a lower expression of NLRP3 ( Figures 4G,H). However, ASC and caspase-1 were not affected by WT161 ( Figures 4G,H). Taken together, these results imply that WT161 inhibits the expression of NLRP3 and negatively modulates NF-κB signaling during the priming process.

DISCUSSION
In this study, we found that the HDAC6 inhibitor WT161 exerts a protective effect in a DSS-induced colitis murine model and blocks NLRP3 inflammasome activation, as IL-1β release and ASC oligomerization were inhibited in the WT161-treated group. WT161 decreased the NLRP3 expression by inhibiting the NF-κB signaling. Thus, our study suggests that WT161 could be a promising agent for the treatment of active IBD. WT161 has been regarded as a remarkable anti-tumor agent for several types of cancers, including multiple myeloma (Hideshima et al., 2016;McClure et al., 2018;García-Guerrero et al., 2021), breast cancer (Hideshima et al., 2017), osteosarcoma (Sun et al., 2021), and retinoblastoma (Sun et al., 2019) by affecting the regulation of CD38, growth factor receptors, PTEN, or X-linked inhibitor of apoptosis protein (XIAP) in tumor cells. However, the role of WT161 in the pathology of IBD or inflammatory diseases has not yet been explored. In this study, we found that WT161 ameliorated DSS-induced colitis by targeting the NLRP3 inflammasome, expanding its potential application in inflammatory diseases.
Network of cytokines including IL-1β, TNF-α, IL-6, IL-10, IFN-γ, and IL-18, have been reported to regulate mucosal inflammation in colitis, and IL-6, TNF-α are viewed as therapeutic targets in IBD (Neurath, 2014(Neurath, , 2017Giraldez et al., 2021). In this study, we focused on the role of WT161 on the NLRP3 inflammasome, so we detected the downstream effector of NLRP3 inflammasome, IL-1β, and the nonspecific effector, IL-6, in vitro and in vivo. As TNF-α is the most frequent studied cytokine in mucosal inflammation, we did not further detect it. We supposed that WT161 could down-regulate TNF-α expression since it could suppress NF-κB signaling.
Although the role of the NLRP3 inflammasome in murine IBD models with NLRP3, ASC, or caspase-1 deficiency remains controversial (Siegmund et al., 2001;Allen et al., 2010;Bauer et al., 2010;Dupaul-Chicoine et al., 2010;Zaki et al., 2010;Bauer et al., 2012;Mao et al., 2018), inhibitors targeting the NLRP3 inflammasome, such as MCC950, oridonin, and INF39, have anti-inflammatory effects in colitis models . Consistent with previous studies, in an animal colitis model, WT161 improved weight loss and DAI score induced by DSS in this study. Moreover, the WT161-treated group showed longer colon length and mitigated intestinal inflammatory injury. The improvement in efficiency was positively related to the dose of WT161. Thus, WT161 exerted protective effects in a DSSinduced murine model. The role of the NLRP3 inflammasome in murine IBD models remains controversial and has been extensively discussed (Mao et al., 2018;Chen et al., 2021). Researchers have attempted to analyze and explain this contradiction. The NLRP3 inflammasome is an important part of innate immunity that resists the invasion of pathogenic microorganisms, and defect or excessive inhibition of inflammasomes leads to more severe colitis. In addition, IL-18 promotes epithelial repair, and the defect or excessive inhibition of inflammasomes leads to lower IL-18 levels. In addition, researchers have found that the intestinal flora plays a role in the controversial data from studies of NLRP3 inflammasome and IBD. In short, the reasons for this are complex, and further studies are needed. Targeting the NLRP3 inflammasome might be an effective way to treat IBD despite contradictions (Song et al., 2021).
Although we speculate that WT161 modulates NF-κB signaling and inhibits the expression of NLRP3, resulting in the inhibition of NLRP3 inflammasome activation based on the results of this study, the function of WT161 as an inhibitor of HDAC6 should not be neglected. Acetylation participating in NLRP3 inflammasome activation (Zhao et al., 2019;Liu et al., 2020) regulates NLRP3 acetylation a good orientation to identify NLRP3 inflammasome inhibitors. HDAC regulate the protein acetylation process, together with histone acetyltransferase. Thus, these corresponding inhibitors could be effective agents for regulating NLRP3 inflammasome activation. These effects have been reported in the recent studies.
The study of Junya Kaneko showed that Ky-2, a hybridcompound HDAC inhibitor, regulates M1 macrophage polarization by downregulating the expression of NLRP3 and the IL-1β-encoding gene in THP-1 cells (Kaneko et al., 2018). IL-6 is an important pro-inflammatory cytokine secreted by M1 macrophages (Atri et al., 2018). The release of IL-6 was inhibited after WT161 treatment ( Figures 2B,D). Whether WT161 influences macrophage polarization in vivo and in vitro needs to be further explored. In addition, Maartje et al. reported that butyrate, an inhibitor of class I HDAC, decreases the production of IL-1β in MSU-stimulated peritoneal macrophages by inhibiting NLRP3 activation (Cleophas et al., 2016). In this study, we found that treatment with the selective HDAC6 inhibitor WT161 effectively decreased the release of IL-1β and ASC oligomerization upon NLRP3 activation. Mechanistically, WT161 decreased NLRP3 expression and negatively modulated NF-κB signaling. Our data are consistent with a previous report that HADC6, the target HADC of WT161, promotes NLRP3 activation by mediating an aggresome-like mechanism and is indispensable for the assembly of inflammasomes (Magupalli et al., 2020). Given that HDAC6 exerts its promotive effects by mediating the microtubule transport of trans-Golgi networklocalized NLRP3 (Magupalli et al., 2020), whether WT161 affects NLRP3 assembly through the same mechanism remains to be investigated. We speculate that WT161 might affect the ubiquitin-binding of HDAC6, the critical activity of HDAC6 required for NLRP3 inflammasome activation, but this hypothesis requires more data for testing.
Overall, our data suggest that WT161 alleviated colonic inflammation in DSS-induced colitis mice as an effective inhibitor of the NLRP3 inflammasome. WT161 not only decreased NLRP3 expression by suppressing NF-κB signaling but also inhibited ASC formation. In a colitis model, WT161 ameliorated intestinal injury, suggesting its potential use in the treatment of IBD. Although further mechanisms remain to be elucidated, our studies have identified WT161 as a therapeutic agent for the treatment of NLRP3-related inflammatory diseases such as IBD.

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 author.

ETHICS STATEMENT
The animal study was reviewed and approved by Institutional Animal Care and Use Committee of the Central South University.