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. Author manuscript; available in PMC: 2024 Mar 5.
Published in final edited form as: Biochem Biophys Res Commun. 2022 Aug 3;624:157–163. doi: 10.1016/j.bbrc.2022.07.120

Inhibition of immunoproteasome attenuates NLRP3 inflammasome formation in tumor necrosis factor α-stimulated intestinal epithelial cell

Boran Yoon a, Yewon Yun a, Kyung Bo Kim b, Dong-Eun Kim a,*
PMCID: PMC10913474  NIHMSID: NIHMS1968700  PMID: 35944388

Abstract

Excessive release of inflammatory cytokines has been considered as a major cause of chronic inflammation, resulting in intestinal barrier disruption that leads to inflammatory bowel disease (IBD). Tumor necrosis factor α (TNFα) is one of the well-known inflammatory cytokines that activates formation of NLRP3 inflammasome, thus resulting in excessive secretion of inflammatory cytokines causing IBD. Although immunoproteasome inhibitors have been reported to inhibit inflammatory cytokine release, immunoproteasome inhibition has not yet been addressed for attenuation of NLRP3 inflammasome activity in intestinal epithelial cell. Here, we observed that NLRP3 inflammasome assembly was attenuated by peptide epoxyketone YU102, a LMP2 subunit immunoproteasome inhibitor, in intestinal epithelial cell. YU102 also inhibited maturation of active caspase-1 and secretion of IL-1β, which are subsequent inflammatory cascade after the formation of NLRP3 inflammasome. Progression of epithelial-mesenchymal transition and increase of cellular permeability, which were induced by TNFα, were also suppressed through inhibition of immunoproteasome. Furthermore, we found that YU102 does not inhibit degradation of IκBα and its following NF-κB activation that leads to transcription of NLRP3. These findings suggest that inhibition of immunoproteasome with YU102 offers a potential therapeutic premise for prevention of TNFα-induced chronic inflammation through attenuation of NLRP3 inflammasome assembly.

Keywords: Tumor necrosis factor α, NLRP3 inflammasome, Immunoproteasome inhibitor, Inflammatory bowel disease, Epithelial-mesenchymal transition

1. Introduction

Inflammatory bowel disease (IBD) is comprised of ulcerative colitis (UC) and Crohn's Disease (CD), which are immune-mediated disorders characterized by chronic inflammation in the gastrointestinal tract [1]. Although etiology of both UC and CD are still unknown, possible causes of IBD are disruption in intestinal homeostasis due to several factors, such as genetic susceptibility and inflammatory disorders, that could either initiate or relapse the diseases [2,3]. Many studies showed that intestinal barrier function was disrupted in IBD patients whether their disease state was active or quiescent [4]. Such disruption can occur due to an excessive inflammatory and immune responses against pathogen-mediated infection or various stress conditions [5]. Tumor necrosis factor α (TNFα) is a well-known major pro-inflammatory cytokine that drives downstream immune responses resulting in IBD [6].

TNFα activates transcription factor Nuclear Factor-kappa B (NF-κB) through proteasomal degradation of phosphorylated IκBα [7]. Activated NF-κB has been known to induce transcriptional expression of NF-κB-mediated NLRP3 (nod-like receptor family pyrin domain containing 3), which leads to activation of NLRP3 inflammasome formation [8]. NLRP3 associates with ASC (the adaptor molecule apoptosis associated speck-like protein containing a CARD) and pro-caspase-1 to form the NLRP3 inflammasome. Once the NLRP3 inflammasome components are assembled, pro-caspase-1 is then self-cleaved to caspase-1, which subsequently proceeds to cleave the precursors of the proinflammatory cytokines IL-1β and IL-18 to their active forms [9,10]. As NLRP3 is significantly expressed in inflammatory diseases, its excessive expression and aberrant activation has been regarded as contributor of IBD pathogenesis [11]. In addition, NLRP3-dependent excessive release of IL-1β promotes inflammation in the intestine and subsequently leads to increase in intestinal permeability, which was evidenced by elevated levels of IL-1β in IBD patients [12,13].

In addition to maturation of NLRP3 inflammasome, TNFα has been reported to induce immunoproteasomes (IP) [14,15], which is a proteasome variant that contains three immunosubunits, such as low molecular mass polypeptide-2 (LMP2), multicatalytic endo-peptidase complex subunit-1 (MECL-1), and low molecular mass polypeptide-7 (LMP7) [16]. Since the IP has been shown to mediate inflammatory responses, IP-targeting inhibitors have been applied as potential anti-inflammatory agents in preclinical and clinical trials [17-19]. Recently, KZR-616, an IP-selective linear peptide epoxyketone that targets both LMP7 and LMP2 subunits, has been under evaluation in clinical trials against systemic lupus erythematosus [20]. Another epoxyketone peptide-based chemical, YU102, an LMP2 subunit inhibitor has been previously reported to improve cognitive behaviors in mouse models of AD and suppress secretion of inflammatory cytokines from microglial cells [21]. To date, however, there is no study on the effect of LMP2 inhibition in intestinal epithelial cells under inflammatory condition inflicted by inflammatory cytokine such as TNFα.

In this study, we observed that LMP2 subunit inhibitor, YU102 is effective to maintain integrity of intestinal epithelial cells under inflammatory condition through attenuation of NLRP3 inflammasome assembly that subsequently causes secretion of inflammatory cytokine, IL-1β. Our findings suggest that YU102-targeting IP subunit may be a potential therapeutic target for ameliorating TNFα-induced NLRP3 inflammasome assembly in intestinal epithelial cells.

2. Materials and methods

2.1. Reagents and antibodies

The following antibodies were used: anti-GAPDH (ab181602), anti-E-cadherin (ab1416), anti-Vimentin (ab92547) and anti-LMP2 (ab3328) were purchased from Abcam (Cambridge, MA, USA); anti-NLRP3 (15101S) was purchased from Cell Signaling technology (Danvers, MA, USA); anti-ASC (sc-514414), horseradish peroxidase-conjugated anti-mouse immunoglobulin (sc-2031), horseradish peroxidase-conjugated anti-rabbit immunoglobulin (sc-2004) and anti-IκBα (sc-1643) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); anti-LMP7 (PA1-972) obtained from Thermo Fisher Scientific (Waltham, MA, USA); anti-NLRP3 (NBP2-12446) obtained from Novus (Cambridge, UK); DAPI and Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin (A11001), Alexa Fluor 555-conjugated goat anti-rabbit immunoglobulin (A21422) and Lipofectamine 3000 (L3000015) were supplied from Invitrogen Life Technologies (Waltham, MA, USA). Tumor necrosis factor alpha (TNFα) was purchased from R&D Systems (210-TA, Minneapolis, NM, USA). Opti-MEM (31,985,070) were supplied by GIBCO (Waltham, MA, USA).

2.2. Cell culture

Human epithelial colorectal adenocarcinoma cells (Caco-2, ATCC HTB-37) were obtained from American Type Culture Collection, ATCC. Minimum Essential Medium (MEM) with Earle's Balanced Salts was obtained from Biowest (L0415-500, Seoul, Korea). Caco-2 cells were cultured in MEM with 20% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (Welgene, Daegu, Korea) at 37 °C in 5% CO2.

2.3. Western blot analysis

Caco-2 cells were lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1.0% Tergitol®, 1 mM phenylmethanesulfonylfluoride, and 10 mM 2-mercaptoethanol). The protein concentration was determined using the Bradford assay (BioRad cat# 5000006; CA, USA). Approximately 25–40 μg of proteins were separated using gradient SDS-PAGE and later transferred to an Immobilon-P PVDF membrane (Millipore, IPVH00010; MA, USA). Membranes were blocked in TBST buffer (25 mM Tris, 150 mM NaCl, 0.1% Tween 20, and 2 mM KCl, pH 7.4) with 3% BSA at room temperature for 1 h, followed by incubation at 4 °C overnight with primary antibodies (1:500 to 1:10,000 dilutions). Followed by incubation with secondary antibodies for 2 h at room temperature, membranes were washed in TBST buffer then reacted with ECL solutions from ECL Western Blot analysis kit (Millipore, WBKLS0500) and the signals were detected using G:BOX Chemi XL and GeneSnap image program Syngene (Cambridge, UK). The protein band intensities were normalized to corresponding GAPDH, which was used as a loading control.

2.4. Immunocytochemistry

Caco-2 cells were cultured in a 24-well plate with an auto-cover glass (12 mm of diameter). Cells were fixed with 4% paraformaldehyde (158127; Sigma-Aldrich, Korea) at 4 °C, followed by permeabilization with 0.2% Triton X-100 for 20 min. After washing with PBS, cells were blocked with 3% BSA for 50 min and incubated with primary antibodies (1:200 for E-cadherin; 1:400 for Vimentin; 1:100 for NLRP3, 1:100 for ASC, and 1:100 for IκBα) at 4 °C overnight. The cells were washed with PBS and incubated with fluorescence-conjugated secondary antibodies (1:1000, Alexa 488 and Alexa 555) and DAPI (1:1000) in the dark at room temperature for 2 h. Then, the cells were mounted on a cover slip together with ProLong Gold antifade reagent (P35934; Invitrogen LIFE Technologies) and were observed using a confocal microscope (Carl Zeiss LSM 800).

2.5. Transwell permeability assay

Caco-2 cells (1 × 105) were grown on a transwell insert (0.4 μm; 3401; Corning B.V. Life Sciences; NY, USA) until a monolayer was formed. The medium in both upper and below the chambers were reconstituted with medium containing 20% FBS and 1% penicillin/streptomycin. Once the monolayer is formed, the media in the upper chambers were replaced with OPTI-MEM (31,985,070; GIBCO) and 500 ml of serum-free media containing FITC-dextran (FDA-100MG; Sigma-Aldrich Korea). Fluorescence of released FITC was measured at excitation and emission wavelength of 485 and 535 nm, respectively using a VICTOR X3 multilable plate reader (PerkinElmer; Waltham, MA, USA).

2.6. Caspase-1 activity assay

Caspase-1 activity was measured using Caspase-Glo® 1 Inflammasome Assay kit (Promega, Madison, WI, USA). Caco-2 cells were seeded in a 24-well plate at a density of 1 × 105 cells per well and incubated under various conditions. The supernatants were collected and treated with Z-WEHD-aminoluciferin, a substrate that caspase-1 exerts its enzymatic activity. The plate was incubated for 1 h at room temperature and the luminescence was recorded using a VICTOR X3 multilable plate reader (PerkinElmer; Waltham, MA, USA).

2.7. Dual luciferase assay for NF-κB

Caco-2 cells were seeded in a 24-well plate at a density of 8 × 104 cells per well. After overnight incubation, the cells were co-transfected with plasmids (from Dual-Luciferase Assay Kit, Promega) encoding NF-κB luciferase reporter (1 μg per well) and the internal control Renilla luciferase (1 μg per well) using Lipofectamine 3000. After 24 h of transfection, the cells were incubated under various conditions for 24 h, and these cells were lysed with passive lysis buffer (Promega). The cell lysates were measured using the Dual-Luciferase Assay Kit according to the manufacturer's protocol (Promega).

2.8. Real-time quantitative PCR

Total RNA was extracted using TransZol Up reagent (ET111-01, Transgene, Beijing, China) according to the manufacturer's instructions. cDNAs were synthesized from 1 μg of total RNA using ReverTra Ace™ qPCR RT Master Mix (FSQ-201, TOYOBO, Osaka, Japan) according to the provided protocol. The cDNAs were amplified by real-time PCR using QuantiNova SYBR Green PCR Kit (500) (208054, QIAGEN, Hilden, Germany). Sequences of used primers were as follows: NLRP3 forward and reverse primers, 5′- CTTCTCTGATGAGGCCCAAG-3′; 5′-GCAGCAAACTGGAAAGGAAG-3′, respectively; GAPDH forward and reverse primers, 5′-ACCACAGTCCATGCCATCAC-3′; 5′-TCCACCACCCTGTTGCTGTA-3, respectively.

2.9. Enzyme-Linked Immunosorbent Assay (ELISA)

The supernatant collected from cultured cells under various conditions were assayed for IL-1β (CSB-E08053h; CUSABIO; Huston, USA) using available ELISA kits according to the manufacturer's instruction.

2.10. Proteasome activity assay

To measure proteasome activity of Caco-2, cells were lysed in Passive Lysis Buffer (Promega) and sonicated. Samples were then centrifuged for 20 min at 13,000 g (4 °C). Aliquots of supernatant containing an equivalent amount of total protein (by the Bradford protein assay) were mixed with proteasome inhibitor assay buffer (20 mM Tris-HCl, 0.5 mM EDTA, pH 8.0) at room temperature, prior to the addition of fluorogenic substrates to a final assay volume of 100 μL. Fluorogenic substrate Ac-PAL-AMC (S-310, BostonBiochem, Cambridge, MA, US) as LMP2-specific substrate was added to the assay solution (100 μM). The fluorescence of liberated AMC was measured over a period of 60 min at excitation and emission wavelength of 360 and 460 nm, respectively, via a SpectraMax M5 microplate reader (Molecular Devices, San Jose, CA, US).

3. Results and discussion

3.1. TNFα disrupts intestinal barrier function via inducement of NLRP3 inflammasome and immunoproteasome in Caco-2 cells

To illustrate disruption in intestinal barrier function, we first examined progression of epithelial-mesenchymal transition (EMT) induced by TNFα in Caco-2 cells by monitoring fluorescence of EMT marker proteins. As shown in Fig. 1A, TNFα dramatically down-regulated expression of E-cadherin, a protein marker for epithelial cells, and upregulated expression of vimentin, a protein marker for mesenchymal cells. In addition, we monitored the leakage of the intestinal epithelial layer by detecting released FITC-dextran moving across the intestinal barrier (Fig. 1B). The cells treated with TNFα and LPS, which was used as a positive control for TNFα, for 48 h showed increased level of FITC-dextran compared to the untreated cells (i.e., control cells).

Fig. 1.

Fig. 1.

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TNFα disrupts intestinal barrier function via inducement of NLRP3 inflammasome and immunoproteasome in Caco-2 cell. (A) Immunofluorescence staining of E-cadherin (green fluorescence) and Vimentin (red fluorescence) in Caco-2 cells treated with TNFα (50 ng/ml) and LPS (10 μg/ml). The nuclei were visualized with DAPI (blue fluorescence). Scale bar: 20 μm. Fluorescence of Vimentin and E-cadherin was quantified and represented as a relative ratio (Vimentin:E-cadherin); counts are the mean ± SD of 3 independent experiments. ***p < 0.005, **p < 0.01. (B) Cell permeability assay of Caco-2 monolayers incubated in TNFα or LPS for 48 h using a transwell. After incubation, cells were treated with FITC-Dextran (100 μg/ml), and it was measured every hour. (C) Fluorescence microscopy images of NLRP3 (green fluorescence) and ASC (red fluorescence) in Caco-2 cells under treatment of TNFα (50 ng/ml) for 24 h and LPS (10 μg/ml) as a positive control to induce NLRP3 inflammasome. Scale bar: 10 μm. The fluorescent puncta of NLRP3 and ASC in the merged images were quantified and presented as a graph (red; puncta of NLRP3, green; puncta of ASC, yellow; co-localized puncta of NLRP3 and ASC); counts are the mean ± SD of 3 independent experiments. (D) Immunoblots of LMP2 and LMP7 under treatment of TNFα (50 ng/ml) for 0, 12 and 24 h in Caco-2 cells. Bar graph indicates protein levels of LMP2 and LMP7 in the Western blot analysis images, which were normalized by GAPDH, relative to control.

As TNFα has been known to induce the formation of NLRP3 inflammasome, we next tested whether TNFα indeed primes assembly of NLRP3 inflammasome in Caco-2 cells (Fig. 1C). The presence of NLRP3 and ASC were traced using red and green fluorescence, respectively. The fluorescence of NLRP3 and ASC were colocalized in cells treated with TNFα and LPS, showing yellow fluorescence, representing NLRP3 inflammasomes. In addition, we next investigated whether TNFα induces immunoproteasome in Caco-2 cells. As shown in Fig. 1D, levels of both LMP2 and LMP7 gradually increased as the TNFα exposure time increased. Thus, inflammatory cytokine TNFα induces NLRP3 inflammasome formation as well as immunoproteasome, leading to enhanced permeability through EMT in Caco-2 cells.

3.2. Immunoproteasome inhibitor attenuates formation of TNFα-induced NLRP3 inflammasome

Immunoproteasome inhibitor (YU102, shown in Fig. 2A) was first tested for the selectivity against LMP2 by proteasome activity assay in Caco-2 cell lysates. As shown in Fig. 2B, YU102 showed effective inhibition of LMP2 as the concentration increased. Next, we investigated whether YU102 affects the formation of NLRP3 inflammasome in Caco-2 cells by tracing presence of fluorescently labeled NLRP3 (red) and ASC (green) (Fig. 2C). The levels of NLRP3 and ASC were significantly increased when the cells were treated with TNFα by showing colocalized yellow puncta, which represents NLRP3 inflammasomes. However, colocalized puncta were decreased when YU102 was co-treated with TNFα. Unlike YU102, co-treatment of TNFα with proteasome inhibitor, carfilzomib (CFZ) did not decrease colocalization of NLRP3 and ASC (yellow bar graph in Fig. 2C) when compared to the cells treated with TNFα and YU102.

Fig. 2.

Fig. 2.

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Immunoproteasome inhibitor attenuates formation of TNFα-induced NLRP3 inflammasome. (A) Immunoproteasome inhibitor, peptide epoxyketone YU102. (B) Systemic and selective inhibition of LMP2 in Caco-2 cells by YU102. Cell lysates were incubated with YU102 at different concentrations and were measured using fluorogenic substrates. *p < 0.05. (C) Fluorescence microscopy images of NLRP3 (green fluorescence) and ASC (red fluorescence) in Caco-2 cells under treatment of TNFα (50 ng/ml), or TNFα (50 ng/ml) in combination with YU102 (1 μM) and Carfilzomib (1 nM) for 24 h. Scale bar: 10 μm. The fluorescent puncta of NLRP3 and ASC in the merged images were quantified and presented as a graph (red; puncta of NLRP3, green; puncta of ASC, yellow; co-localized puncta of NLRP3 and ASC); counts are the mean ± SD of 3 independent experiments. (D) Caspase-1 activity was assessed using the Caspase-Glo® 1 Inflammasome Assay kit. Cells were treated with TNFα (50 ng/ml), or TNFα (50 ng/ml) in combination with YU102 (1 μM) and Carfilzomib (1 nM) for 24 h. Caspase-1 activity was normalized to cell viability. Data presented are mean ± SD of 3 independent experiments. ***p < 0.005. (E) Levels of IL-1β in the supernatant of Caco-2 cells treated with TNFα (50 ng/ml), or TNFα (50 ng/ml) in combination with YU102 (1 μM) and Carfilzomib (1 nM) for 24 h were detected by ELISA. Data presented are the mean ± SD of 3 independent experiments. *p < 0.05.

As we observed that IP inhibitor decreases formation of NLRP3 inflammasome, we next investigated whether YU102 treatment attenuates catalytic activity of NLRP3 inflammasome as well. As shown in Fig. 2D, caspase-1 activity was elevated when the cells were treated with TNFα, reflecting the formation of NLRP3 inflammasome. On the contrary, co-treatment of TNFα with YU102 significantly inhibited caspase-1 activity. In addition, activated caspase-1 cleaves pro-IL-1β to its mature and secretory form, IL-1β. Thus, we next monitored secretion of IL-1β under same conditions. As shown in Fig. 2E, amount of secreted IL-1β was elevated when the cells were treated with TNFα but reduced when co-treated with YU102. These results indicate that YU102 is capable of attenuating TNFα-induced formation of NLRP3 inflammasome and its subsequent generation of inflammatory cytokine in Caco-2 cells.

3.3. Immunoproteasome inhibitor suppresses TNFα-induced disruption of cellular junctions

Epithelial-to-mesenchymal transition (EMT) has been attributed to the pathogenesis of CD, in which cellular junctions has been lost along with enhanced permeability in intestinal epithelial cells [22]. It has been reported that EMT progression can be activated in response to inflammatory cytokines such as IL-1β [23]. In view of the notion that YU102 attenuates formation of NLRP3 inflammasome and its following inflammatory cytokine release, we further examined if YU102 suppresses progression of EMT caused by those secreted cytokines. We observed that TNFα stimulates progression of EMT, showing decreased levels of E-cadherin and increased levels of vimentin (Fig. 3A). However, co-treatment of TNFα with YU102 dramatically retained expression of E-cadherin and low levels of vimentin. Unlike YU102, co-treatment of TNFα with CFZ failed to maintain low levels of vimentin, leading to onset of EMT process. Thus, IP inhibitor YU102 effectively prevents EMT progression, which was most likely caused by TNFα-induced IL-1β release.

Fig. 3.

Fig. 3.

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YU102 attenuates TNFα-induced intestinal barrier dysfunction in Caco-2 cells. (A) Immunofluorescence staining of E-cadherin (green fluorescence) and Vimentin (red fluorescence) in Caco-2 cells treated with TNFα (50 ng/ml), or TNFα (50 ng/ml) in combination with YU102 (1 μM) and Carfilzomib (1 nM) for 24 h. The nuclei were visualized with DAPI (blue fluorescence). Scale bar: 20 μm. Fluorescence of Vimentin and E-cadherin was quantified and represented as a relative ratio (Vimentin:E-cadherin); counts are the mean ± SD of 3 independent experiments. ***p < 0.005. (B) Cell permeability assay of Caco-2 monolayers incubated in TNFα (50 ng/ml), or TNFα (50 ng/ml) in combination with YU102 (1 μM) and Carfilzomib (1 nM) for 48 h using a transwell. After incubation, cells were treated with FITC-Dextran (100 μg/ml) and fluorescence of released FITC from the chamber was measured every hour.

To further corroborate the effect of YU102 on intestinal permeability in the cells under TNFα treated condition, we monitored the leakage of the intestinal epithelial barrier (Fig. 3B). The cells exposed to TNFα for 48 h showed increased levels of FITC-dextran compared to the control cells (untreated cells). On the contrary, the levels of FITC-dextran significantly decreased when the cells were co-treated with TNFα and YU102. Consistent with the EMT results with co-treatment of TNFα and CFZ (Fig. 3A), proteasome inhibitor failed to prevent disruption of cellular junctions by showing significant leakage of FITC-dextran. Together, these results suggest that immunoproteasome specific inhibitor is more effective to maintain cellular junctions through attenuation of NLRP3 inflammasome assembly and subsequent secretion of inflammatory cytokines.

3.4. Immunoproteasome does not entail activation of NF-κB for NLRP3 inflammasome assembly

Treatment of TNFα is a well-validated mechanism for NF-κB activation, in which TNFα triggers phosphorylation of IκBα, which leads to its proteasome-dependent degradation and translocation of NF-κB to the nucleus [24,25]. We examined whether IP inhibition may prevent degradation of IκBα thus leading to inhibition of NF-κB translocation, resulting in attenuation of NLRP3 inflammasome assembly. As shown in Fig. 4A, treatment of TNFα led to decrease in IκBα fluorescence, which was similar to the cells co-treated with TNFα and YU102. Unlike these two conditions, IκBα fluorescence was increased when the cells were co-treated with TNFα and CFZ. On the contrary to our projection, IP inhibition did not prevent degradation of IκBα, which was induced by TNFα. To further validate that IP does not entail NF-κB activation through degradation of IκBα, we monitored NF-κB activity in the cells by dual luciferase assay (Fig. 4B). TNFα treatment increased the NF-κB activity but co-treatment of TNFα with YU102 increased its activity to a greater extent. In the same context, consistent results were also obtained when mRNA levels of NLRP3 were observed (Fig. 4C). Likewise, treatment of TNFα increased mRNA level of NLRP3 and co-treatment of TNFα with YU102 led to further increase in NLRP3 mRNA level. Combined with the preceding results, we suggest that immunoproteasome does not entail activation of NF-κB for NLRP3 inflammasome assembly.

Fig. 4.

Fig. 4.

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Immunoproteasome inhibition does not revoke activation of NF-κB pathway. (A) Immunofluorescence staining of IκBα (green fluorescence) in Caco-2 cells treated with TNFα (50 ng/ml), or TNFα (50 ng/ml) in combination with YU102 (1 μM) and Carfilzomib (1 nM) for 24 h. The fluorescence of IκBα was quantified and presented as a bar graph; counts are the mean ± SD of 3 independent experiments. ***p < 0.005, **p < 0.01. (B) NF-κB activity was assessed using dual-luciferase reporter assay after 24 h treatment with TNFα (50 ng/ml) or TNFα (50 ng/ml) in combination with YU102 (1 μM) and Carfilzomib (1 nM). Counts are the mean ± SD of 3 independent experiments. *p < 0.05. (C) The mRNA expression of NLRP3 in Caco-2 cells treated with TNFα (50 ng/ml), or TNFα (50 ng/ml) in combination with YU102 (1 μM) and Carfilzomib (1 nM) for 24 h. Data are presented as the mean ± SD of 3 independent experiments. **p < 0.01, *p < 0.05.

In conclusion, our present study demonstrated that inhibition of immunoproteasome with peptide epoxyketone YU102 attenuates TNFα-induced inflammatory cascade of NLRP3 inflammasome through down-regulation of NLRP3 inflammasome assembly in Caco-2 cells. As YU102 inhibited formation of NLRP3 inflammasome, it also attenuated maturation of active caspase-1 and secretion of IL-1β. Progression of epithelial-mesenchymal transition with enhanced cellular permeability in Caco-2 cells treated with TNFα was also suppressed through inhibition of immunoproteasome. In addition, we found that attenuation of NLRP3 inflammatory cascade and its subsequent cellular EMT was not caused by degradation of IκBα and its following NF-κB activation but by inhibition of NLRP3 inflammasome assembly. Taken together, we suggest that inhibition of immunoproteasome with YU102 is applicable to modulate TNFα-induced chronic inflammation in intestinal epithelial cells.

Acknowledgements

This research was supported by grants from the National Research Foundation of Korea (NRF) funded by the Korean government (2017R1E1A1A01074656) to D.-E. K. and from the National Institutes of Health funded by the United States government (R01 AG073122) to K.B.K.

Footnotes

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Dong-Eun Kim reports financial support was provided by National Research Foundation of Korea. Kyung Bo Kim reports financial support was provided by National Institutes of Health.

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