P22077 inhibits LPS-induced inflammatory response by promoting K48-linked ubiquitination and degradation of TRAF6

Results

P22077 does not affect cell viability of Raw264.7 cells and mouse peritoneal macrophages

The chemical structure of P22077 was shown in Figure 1A. To determine the cytotoxicity of P22077, MTT assay was used to evaluate the cell viability in macrophages. As shown in Figure 1B, 1C, P22077 (2.5, 5.0 and 7.5 μM) had no significant cytotoxicity both in Raw264.7 cells and mouse peritoneal macrophages. Similarly, no obvious changes were observed in cell density of P22077-treated Raw264.7 cells and mouse peritoneal macrophages (Figure 1D). Therefore, P22077 at these concentrations were selected for the subsequent cellular experiments.

Figure 1. P22077 does not affect cell viability. (A) The chemical structure of P22077. (B, C) The cytotoxicity of P22077 in Raw264.7 cells (B) and mouse peritoneal macrophages (PM) (C). (D) Raw264.7 cells and peritoneal macrophages plated treated by different concentrations of P22077 for 12h, the morphology of the cells observed under the microscope. Scale bars, 100μm. Similar results were obtained from three independent experiments and data were presented as mean ± SD of one representative experiment.

P22077 inhibits LPS-induced inflammatory response in Raw264.7 cells

The expression of inflammatory mediators was a crucial response of macrophages with LPS stimulation. To investigate the anti-inflammatory effect of P22077 in macrophages, Raw264.7 cells were exposed to different concentrations of P22077 (2.5, 5.0 and 7.5 μM) and followed by LPS stimulation. The results showed that mRNA levels of TNF-α, IL-1β, IL-6, COX2 and iNOS were highly induced by LPS (100 ng/mL), and the expression of these pro-inflammatory cytokines were significantly decreased (up to 80%) in a dose-dependent manner with pretreated with P22077 (Figure 2A, 2B). Nitric oxide (NO) is a free radical, which is an important inflammatory signaling molecule. To examine the NO production, we use the Griess reagent to investigate the concentration of nitrite which is regard as biomarker of NO in supernatant. As expected, LPS obviously increased the release of NO and this effect could be inhibited by P22077 in a dose-dependent manner with maximum effects of about 50% NO reduction (7.5 μM P22077 compared with LPS alone group) (Figure 2C). Furthermore, we measured pro-inflammatory cytokines TNF-α and IL-6 in the cell culture supernatant by ELISA, and detected pro-IL-1β by immunoblot. The results showed that pretreated with P22077 suppressed the production of LPS-induced TNF-α, pro-IL-1β and IL-6 in Raw264.7 cells (Figure 2D). Taken together, these results indicate that P22077 exhibits anti-inflammatory properties in Raw264.7 cells.

Figure 2. P22077 suppresses LPS-induced inflammatory response in Raw264.7 cells. (A, B) Raw264.7 cells were pretreated with DMSO or P22077 (2.5 μM, 5.0 μM and 7.5 μM) for 2 h, then stimulated with LPS (100 ng/ml) for another 4 h. The mRNA expressions of TNF-α, IL-1β, IL-6 (A), COX2 and iNOS (B) were analyzed by Q-PCR. (C, D) Raw264.7 cells were pretreated with DMSO or P22077 (2.5 μM, 5.0 μM and 7.5 μM) for 2 h, and then stimulated with LPS (100 ng/ml) for 10 h. Nitric oxide (NO) concentration in culture supernatant was measured by Griess assay (C). TNF-α and IL-6 in the supernatants were measured by ELISA, pro-IL-1β was detected by immunoblot (D). Similar results were obtained from three independent experiments and data were presented as mean ± SD of one representative experiment. *P<0.05, **P<0.01, ***P<0.001 vs LPS-stimulated DMSO group.

P22077 inhibits LPS-induced inflammatory response in mouse peritoneal macrophages

To further confirm the anti-inflammatory effect of P22077 in a primary macrophages, we next studied the inflammatory response of P22077-treated mouse peritoneal macrophages. Q-PCR results showed that P22077 could significantly inhibit LPS-induced TNF-α, IL-1β, IL-6, COX2 and iNOS mRNA levels compared to control group, which is similar with that in Raw264.7 cells (Figure 3A, 3B). Besides, the production of NO, TNF-α, IL-1β and IL-6 in the cell culture supernatant was also significant inhibition by P22077 (Figure 3C, 3D). These results further confirm that P22077 has significant anti-inflammatory potential by suppressing LPS-induced inflammatory mediators production in mouse peritoneal macrophages.

Figure 3. P22077 suppresses LPS-induced inflammatory response in mouse peritoneal macrophages. (A, B) Mouse peritoneal macrophages were pretreated with DMSO or P22077 (2.5 μM, 5.0 μM and 7.5 μM) for 2 h, then stimulated with LPS (100 ng/ml) for another 4 h. The mRNA expressions of TNF-α, IL-1β, IL-6 (A), COX2 and iNOS (B) were analyzed by Q-PCR. (C, D) Mouse peritoneal macrophages were pretreated with DMSO or P22077 (2.5 μM, 5.0 μM and 7.5 μM) for 2 h, and then stimulated with LPS (100 ng/ml) for 10 h. Nitric oxide (NO) concentration in culture supernatant was measured by Griess assay (C). TNF-α, IL-1β and IL-6 in the supernatants were measured by ELISA (D). Similar results were obtained from three independent experiments and data were presented as mean ± SD of one representative experiment. *P<0.05, **P<0.01, ***P<0.001 vs LPS-stimulated DMSO group.

P22077 suppresses LPS-induced NF-κB and MAPKs signaling pathways activation

NF-κB and/or MAPKs signaling pathways play an important role in the regulation of innate immunity and inflammation. To determine whether NF-κB and/or MAPKs pathways were involved into the anti-inflammatory response of P22077, we examined the key phosphorylation proteins in NF-κB and MAPKs signaling pathways by immunoblot. The results showed that P22077 treatment inhibited LPS-induced p65 and IKKα/β phosphorylation in Raw264.7 cells (Figure 4A). Besides, P22077 also significantly inhibited LPS-induced phosphorylation of ERK and p38 in Raw264.7 cells (Figure 4A). Similarly, in mouse peritoneal macrophages, we also found that P22077 inhibited LPS-induced p65, IKKα/β phosphorylation in NF-κB signaling pathway and ERK, p38 phosphorylation in MAPKs signaling pathway (Figure 4B). Furthermore, the NF-κB luciferase reporter experiment also indicated that P22077 attenuated the activity of NF-κB signaling pathway (Figure 4C). Taken together, these findings reveal that P22077 suppresses LPS-induced inflammatory response by inhibiting the activation of NF-κB and MAPKs signaling pathways.

Figure 4. P22077 inhibits the activation of NF-κB and MAPKs signaling pathways in LPS-stimulated Raw264.7 cells and mouse peritoneal macrophages. (A) Raw264.7 cells were pretreated with DMSO and P22077 (7.5 μM) for 2 h, and then stimulated with LPS (100 ng/ml) for the indicated times and analyzed the indicated proteins by immunoblot. (B) Mouse peritoneal macrophages were pretreated with DMSO and P22077 (7.5 μM) for 2 h, and then stimulated with LPS (100 ng/ml) for the indicated times and analyzed the indicated proteins by immunoblot. (C) RAW 264.7 cells were transfected with NF-κB luciferase reporter plasmid and Renilla luciferase construct phRL-TK plasmid for 24 h and pretreated with the P22077 (7.5 μM) for 2 h before stimulation with 100ng/ml LPS for 4 h and luciferase activity was measured. Similar results were obtained from three independent experiments and data were presented as mean ± SD of one representative experiment. **P<0.01 vs LPS-stimulated group.

P22077 promotes the degradation of TRAF6 in a proteasome-dependent manner

To fully understand the function of P22077 in the NF-κB signaling pathway, further study was performed to identify the molecular target of P22077. NF-κB signaling pathway was complicated, its upstream protein include TRAF6, TAK1 and other signaling proteins. As previously reported, TRAF6 is a key adaptor molecular for the activation of the NF-κB signaling pathway and USP7 is an important deubiquitinase which stabilizes TRAF6 protein. Therefore, we speculated that P22077 inhibited USP7 protein activity, and thus promoted TRAF6 degradation on upstream of NF-κB signaling pathway. To verify this hypothesis, we over-expressed Flag-TRAF6 in HEK293T cells and then treated with or without P22077, we found that P22077 obviously reduced the protein level of TRAF6 (Figure 5A). Furthermore, Western blot analysis showed that P22077 treatment decreased the levels of TRAF6 in LPS-stimulated mouse peritoneal macrophages (Figure 5B). In addition, the quantity of TRAF6 was reduced by treated P22077 in a dose-dependent manner (Figure 5C). We treated mouse peritoneal macrophages with CHX (an inhibitor of protein synthesis) in order to explore whether P22077 affecting TRAF6 synthesis or degradation, and we found P22077 could increase TRAF6 degradation (Figure 5D). Using different protein degradation inhibitors such as MG132, CQ and 3-MA, we confirmed that P22077 promoted TRAF6 degradation in a proteasome-dependent manner as only MG132 rescuing the degradation of TRAF6 (Figure 5E). These results indicate that P22077 promotes the degradation of TRAF6 in a proteasome-dependent manner.

Figure 5. P22077 promotes the degradation of TRAF6. (A) HEK293T cells were transfected with Flag-TRAF6 and HA-Ub plasmid for 24h, and then treated with or without P22077 (7.5 μM) for 6 h, the Flag-TRAF6 protein was detected by immunoblot. (B) Mouse peritoneal macrophages were pretreated with or without P22077 (7.5 μM) for 2 h, and then stimulated with LPS (100 ng/mL) for 1 and 2 h. Immunoblot assay was used to analyze TRAF6 protein. (C) Mouse peritoneal macrophages were pretreated with various dose of P22077 (0, 2.5, 5 and 7.5 μM) for 2 h, and then stimulated with LPS (100 ng/mL) for 1 h. Immunoblot assay was used to analyze TRAF6 protein. (D) Mouse peritoneal macrophages were treated with cycloheximide (100 μg/ml) in the absence or presence of P22077 (7.5 μM) for 0, 6 and 12 h. Immunoblot assay was used to analyze TRAF6 protein. (E) Mouse peritoneal macrophages were pretreated with P22077 (7.5 μM), MG132 (20 μM), Chloroquine (50μM) or 3-MA (1 mM) for 2 h, and then stimulated with LPS (100 ng/mL) for 1 h. Immunoblot assay was used to analyze TRAF6 protein. Numbers below lanes indicated densitometry of the protein presented relative to β-actin. Similar results were obtained from three independent experiments and data were presented of one representative experiment.

P22077 promotes K48-linked ubiquitination of TRAF6

Having shown that P22077 promotes TRAF6 degradation in a proteasome- dependent manner, we further studied the cellular function of P22077 toward TRAF6. Flag-TRAF6 plasmid and HA-Ub plasmid were co-transfected into HEK293T cells with following MG132 and P22077 treatment. We found that P22077 significantly increased the ubiquitination level of TRAF6 (Figure 6A). In mouse peritoneal macrophages, we also confirmed these results. Ubiquitination assay showed that TRAF6 ubiquitination was significantly increased in mouse peritoneal macrophages pretreated with P22077 (Figure 6B). In addition, the ubiquitination level of TRAF6 was elevated both in P22077 treated and USP7 knockdown cells (Figure 6C). Subsequently, to study the form of ubiquitination chains linking to TRAF6, HEK293T cells were transfected with Lys-mutated ubiquitin plasmids (K48 or K63). The mutated K48 or K63 sites ubiquitin has only one lysine in ubiquitin at position 48 or 63, respectively. Ubiquitination assay demonstrated that P22077 significantly increased K48-linked but not K63-linked ubiquitination of TRAF6 (Figure 6D). Taken together, these data indicate that P22077 promotes K48-linked polyubiquitination-dependent degradation of TRAF6.

Figure 6. P22077 promotes the K48-linked ubiquitination of TRAF6. (A) HEK293T cells were transfected with Flag-TRAF6 and HA-Ub plasmid for 24h and then treated with P22077 (7.5 μM) and MG132 (20 μM) for 6 h, immunoprecipitation assay was used to detect ubiquitination level of TRAF6. (B) Mouse peritoneal macrophages were pretreated with P22077 (7.5 μM) and MG132 (20 μM) for 2 h, and then stimulated with LPS (100 ng/mL) for 1 h. Immunoprecipitation assay was used to analysis of TRAF6 protein ubiquitination level. (C) HEK293T cells were knockdown USP7, and transfected with Flag-TRAF6, HA-Ub, and then treated with P22077 (7.5 μM) and MG132 (20 μM) for 6 h, immunoprecipitation assay was used to detect the ubiquitination level of TRAF6. (D) HEK293T cells were transfected with Flag-TRAF6, HA-K48-Ub and HA-K63-Ub plasmid for 24h and then treated with P22077 (7.5 μM) and MG132 (20 μM) for 6 h, immunoprecipitation assay was used to detect the K48 and K63 sites of TRAF6 ubiquitination. Similar results were obtained from three independent experiments and data were presented of one representative experiment.

P22077 relieves LPS-induced inflammatory response in vivo

To investigate the anti-inflammatory properties of P22077 in vivo, we induced a systemic acute inflammation mice model by LPS intraperitoneal injection [30–32]. It is characterized by overproduction of inflammatory cytokines and leads to the lethal multiple organ damage after LPS stimulation. Before LPS challenge, C57BL/6 mice were intraperitoneally injected with P22077 (15 mg/kg) or vehicle DMSO. Two hours after LPS challenge, mice were sacrificed to obtain serum and lung. As shown in Figure 7A, P22077 treatment suppressed mRNA expression of pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6 in lung tissue. In addition, compared to control group, the serum levels of NO, TNF-α, IL-1β and IL-6 were decreased in the mice treated with P22077 (Figure 7B–7C). H&E staining of lung tissue showed that P22077 treatment reduced LPS-induced pulmonary interstitial edema and inflammatory infiltrates (Figure 7D). Immunohistochemistry staining of TRAF6 in lung tissue suggested that LPS induced the expression of TRAF6, but when treated with P22077, the protein of TRAF6 was significantly reduced (Figure 7E). We also examined the key phosphorylation proteins in NF-κB and MAPKs signaling pathways in lung tissue, the results suggested that the phosphorylation of IKKα/β, p65 in NF-κB signaling pathway, and p38, ERK in MAPKs signaling pathway were significantly decreased in P22077 treated mice, as observed in Raw264.7 cells and mouse peritoneal macrophages (Figure 7F). These data indicate that P22077-treated mice were more resistant to LPS-induced inflammation and lung injury.

Figure 7. P22077 relieves LPS-induced inflammatory response in mice. C57BL/6 mice (10 mice/group) were intraperitoneally injected with DMSO or P22077 (15 mg/kg), following challenged with PBS or LPS (10 mg/kg). Mice were sacrificed after 2 h. (A) TNF-α, IL-1β and IL-6 mRNA level in lung tissue were analyzed by Q-PCR. (B) Nitric oxide (NO) concentrations in the serum were examined by Griess assay. (C) TNF-α, IL-1β and IL-6 concentrations in the serum were examined by ELISA. (D) Left lung was collected for HE staining, and representative images were displayed. Scale bars, 100 μm. (E) Left lung was collected for immunohistochemistry staining of TRAF6. Scale bars, 100 μm. (F) Lung tissue was grinded and centrifuged, and the supernatant was used to analyzed the indicated proteins by immunoblot. Similar results were obtained from three independent experiments and data were presented as mean ± SD of one representative experiment. *P<0.05, **P<0.01, ***P<0.001 vs LPS-stimulated DMSO group (A–C).

Discussion

It is still a challenge for human to overcome inflammatory disease which due to invading of exogenous pathogens, trauma and some unknown reason, particularly some widespread chronic diseases such as Alzheimer [33, 34]. Developing more effective and less toxic drugs to treat inflammatory diseases is extremely urgent. Up to now, larger number of strategies have been adopted, including reduce the activity of specific cytokines or their receptors, block cell adhesion and migration, deplete B or T lymphocytes, etc. Among different strategies, identifying small-molecule inhibitors targeting downstream of cytokine receptors to reduce production or activities of pro-inflammatory cytokines was regard as a promising method [35–37]. Post-translational modifications, such as ubiquitination and deubiquitination played a crucial role in signaling cascades. Furthermore, deubiquitination effect of some DUBs, such as A20 [38], CYLD [39, 40] and OTULIN (also known as FAM105B) [41], were found playing a critical role in inflammatory response induced by TNF-α, lipopolysaccharide (LPS) and some other stimulus. All of these imply ubiquitin proteasome system (UPS) was a potential drug targets in anti-inflammatory process. In this study, we demonstrated P22077, as an inhibitor of USP7, was a potential anti-inflammatory drug that inhibited secretion of pro-inflammatory cytokines by promoting K48-linked ubiquitination and degradation of TRAF6 (Figure 8). In our study, P22077 did not appear to impact the K63-linked ubiquitination of TRAF6. But Xiang et al. reported that USP7 regulated the K63-linked polyubiquitination of TRAF3 and TRAF6 [42], indicated that USP7 regulating polyubiquitination of TRAF6 maybe difference in different stimulus. Furthermore, our data suggest that small molecule inhibitor of DUBs maybe an excellent anti-inflammatory drug with potential clinical application.

Figure 8. Schematic of the anti-inflammatory mechanism of P22077. P22077 promotes K48-linked ubiquitination and degradation of TRAF6, and subsequently inhibits the LPS-induced NF-κB and MAPKs pathway to alleviate inflammation.

Bacterial lipopolysaccharides (LPS) consist of lipid A was a typical ligand of plasma membrane protein TLR4, which can induce host innate immune response [43]. Macrophages are key cells for inflammatory processes which involved in all phases of inflammation, regulating acute inflammatory response and chronic pathological changes [44]. In this study, we utilized two different kinds of macrophages stimulated by LPS to investigate the roles of P22077 in inflammation process. Pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), Cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) increase dramatically over the following several hours stimulated with LPS that are regard as biomarkers of inflammatory processes [45]. In addition, nitric oxide (NO) is characteristic of acute inflammation for inhibiting leukocyte adhesion to vascular endothelium [46]. Here we provided sufficient and systemic evidence on mRNA and protein level exhibiting that P22077 inhibits LPS-induced production of pro-inflammatory cytokines or mediators (TNF-α, IL-1β, IL-6, COX-2, iNOS and NO) not only in Raw264.7 cells and mouse peritoneal macrophages, but also in C57BL/6 mice. The results of lung HE staining from mice potently supported our finding. Our data confirmed that P22077 could reduce the secretion of inflammatory mediators and relieved inflammatory response in vitro and in vivo.

LPS recognized by TLR4, and then a lot of molecules participate in signaling cascade, including MyD88, TRAF, TAK1 and several downstream kinases. Among them, NF-κB and MAPKs signaling pathways were two critical pathways. Here, we detected some key signaling proteins in these two signaling pathways, and phosphorylation of NF-κB was significantly down-regulated both in Raw264.7 cells and mouse peritoneal macrophages after treatment with P22077. Moreover, NF-κB luciferase reporter gene assay also confirmed P22077 certainly inhibited NF-κB signaling pathway activation. Li et al. found that USP7 downregulated NF-κB signaling by interacting with HSCARG [23], but Colleran et al. found USP7 promoted inflammation by deubiquitination of NF-κB [22]. Based on these findings of two groups above, we have reasons to believe that USP7 affects inflammatory response with a variety of mechanisms. Furthermore, it has been reported that decreasing of USP7 expression coincided with reducing cellular TRAF6 protein [24]. Additionally, A20 also been reported participating in suppressing Toll-like receptor–induced activity of NF-κB signaling and pro-inflammatory gene expression in macrophages by removing ubiquitin chains from TRAF6 [47]. Recently, study showed that resveratrol could suppress NF-κB activation by attenuating TRAF6 expression and ubiquitination [48, 49]. So, we assumed that P22077 negatively regulated inflammation by targeting of TRAF6. Next, we verified that P22077 promoted the degradation of TRAF6 in a proteasome-dependent manner. Our study basically confirmed that P22077 could inhibit activation of NF-κB and MAPKs signaling pathways by promoting K48-linked ubiquitination and degradation of TRAF6.

In conclusion, we demonstrate that P22077 can promote K48-linked ubiquitination and degradation of TRAF6 and then inhibit the activation of NF-κB and MAPKs signaling pathways, and then negatively regulates inflammatory response. These findings provide a new insight into the small-molecule compounds for inhibition of NF-κB and MAPKs-mediated inflammation and provides a new potential anti-inflammatory drug to cure infection-related inflammatory and autoimmune diseases.

Abbreviations

LPS: lipopolysaccharide; TRAF6: tumor necrosis factor receptor-associated factor 6; USP7: ubiquitin specific protease-7; iNOS: inducible nitric oxide synthase; NO: nitrous oxide; TNF-α: tumor necrosis factor-α; IL-1β: interleukin-1β; IL-6: interleukin-6; NF-κB: Nuclear factor κB; DUBs: deubiquitinating enzymes.

Author Contributions

Xibao Zhao, Feiyang Ji and Weilin Chen participated in the research design. Xibao Zhao, Feiyang Ji, Hongrui Li, Qianqian Di, Huihui Zhu, Xingyu Ma conducted the experiments. Zizhao Zhao and Jing Ling contributed new reagents or analytic tools. Xibao Zhao, Feiyang Ji, Qianqian Di and Weilin Chen performed data analysis. Xibao Zhao, Feiyang Ji and Weilin Chen contributed to the writing of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding

This work was supported by grant from the National Natural Science Foundation of China (No. U1801283, 31870908, 31670914), grant from Zhejiang Provincial Natural Science Foundation of China (No. LZ17H100001), grant from the Shenzhen Science and Technology Innovation Commission grant JCYJ20180507182253653, Guangdong Provincial Science and Technology Program (No. 2019B030301009), Guangdong Province Basic and applied basic research fund (No. 2019A1515110086) and SZU medical young scientists program (No. 71201-000001) to Xibao Zhao.

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