TNFAIP3 ameliorates the degeneration of inflammatory human nucleus pulposus cells by inhibiting mTOR signaling and promoting autophagy

Upregulation of TNFAIP3 expression in human nucleus pulposus tissues

To detect whether TNFAIP3 is expressed in IVDD, we tested the TNFAIP3 expression level in human NPCs with different grades of degeneration in IVDD patients. The control group was obtained from patients of lumbar vertebral fracture (LVF) without IVDD. The results of immunohistochemistry revealed that the expression of TNFAIP3 protein in human NPCs of patients with IVDD was significantly increased compared with the control counterparts (Figure 1A, 1B). LPS treatment mimicked the introduction of IVDD as in our previously established LPS-induced IVDD cell model (Aging 2019, 11:7294), which also increased the expression of TNFAIP3 with IVDD introduction (Figure 1C). The results demonstrated that TNFAIP3 expression is upregulated in IVDD patients and LPS-stimulated NP cells, suggesting that TNFAIP3 may be involved in the process of IVDD.

Figure 1. The expression of TNFAIP3 in human nucleus pulposus tissues. (A, B) TNFAIP3 expression was measured by immunohistochemistry from the IVD section from LVF and IVDD patients (magnification × 100, n>5). (B) Human NPCs expressing TNFAIP3 were enumerated from the above sections, results are expressed as a percent of the total number of NPCs. (C) TNFAIP3 expression was measured at the mRNA level by qPCR from the primary cell culture of the NPCs from LVF patients. Con and LPS, cell culture representatively treated with no and with LPS, data are shown by the mean ± standard deviation from 3 independent replicates. *** P<0.01, ** P<0.05, * P>0.05 by t-test. TNFAIP3, tumor necrosis factor α-induction protein 3; IVD, intervertebral disc; LVF, patients with lumbar vertebral fracture as non-IVDD controls; IVDD, patients with intervertebral disc degeneration; LPS, lipopolysaccharide; Con, normal human NPCs culture from LVF patients with no LPS treatment; LPS, the normal human NP cell culture treated with LPS at 200 ng/mL for 24 h.

TNFAIP3 reduces pro-inflammatory cytokines expression in LPS-stimulated human NPCs

As a key molecule in the negative regulation of inflammatory response, TNFAIP3 limits the expression of inflammatory cytokines such as TNF-α, IL-1β [15]. As shown in (Figure 2A), primary human NPCs isolated from patients of lumbar vertebral fracture (LVF) were mainly polygonal, irregular fusiform, and the 2^nd passaged cells were used for the experiment. The results of fluorescence microscopy showed that the transfection efficiency reached 80% after 48 hours of transfection of NPCs with Ad-TNFAIP3 (Figure 2B). To further confirm the effect of TNFAIP3 on human NPCs, the mRNA and protein levels of TNFAIP3 in LPS-stimulated human NPCs were detected. The expression of TNFAIP3 was higher in LPS-stimulated NPCs compared with non-treated cells. TNFAIP3 siRNA decreased TNFAIP3 levels compared with LPS-stimulated NPCs (Figure 2C–2E). However, there was no difference in the expression of TNFAIP3 between the LPS group and the LPS transfected with the adenovirus vector without TNFAIP3 group, indicating that the adenovirus vector does not affect cell function. We then detected whether the knocking down of TNFAIP3 expression affects the expression of pro-inflammatory cytokines TNF-α and IL-1β. ELISA assay of the supernatants of these cell cultures displayed that LPS increased the expression of TNF-α and IL-1β, but was reduced back to a normal level with overexpressing TNFAIP3 in the human NPCs. In contrast, both TNF-α and IL-1β were significantly up-regulated in TNFAIP3 knockdown NPCs (Figure 2F, 2G). Thus, these results indicated that TNFAIP3 decreased the expression of pro-inflammatory cytokines (TNF-α, IL-1β) in LPS-stimulated human NPCs.

Figure 2. TNFAIP3 reduces pro-inflammatory cytokines expression in LPS-stimulated human NPCs. (A) Human primary nucleus pulposus cells isolated from patients with lumbar fractures (LVF), amplification × 100. (B) Human nucleus pulposus cells were transfected with Ad-TNFAIP3 for 48 h. (C–E) TNFAIP3 responses to inflammatory stimulation. After treatment with LPS for 24 h, the mRNA and protein expression levels of TNFAIP3 were measured by RT-qPCR and Western blot, respectively. (F, G) After transfected with TNFAIP3-His and TNFAIP3-siRNA for 24 h, then stimulated with LPS for 24 h, the expression levels of TNF-α, IL-1β were tested by ELISA. Data are represented by the mean ± standard deviation of 3 independent experiments. *** P<0.01, ** P<0.05, * P>0.05 by the Student t-test. Error bars indicate standard deviations. NC, negative control (adenovirus vector without TNFAIP3); TNFAIP3-His, Adenovirus with TNFAIP3 overexpression; TNFAIP3-siRNA, Adenovirus with TNFAIP3 knockdown. TNFAIP3-His +LPS, human NPCs transfected with TNFAIP3-His and then treated with LPS. TNFAIP3-siRNA +LPS, human NPCs transfected with TNFAIP3-siRNA and then treated with LPS.

TNFAIP3 promotes autophagy in LPS-stimulated human NPCs

Cell autophagy is involved in the pathogenesis of degenerative diseases, including osteoarthritis and IVDD [13]. To determine whether TNFAIP3 regulates autophagy in inflammatory human NPCs, the autophagosomes was observed by the transmission electron microscopy (TEM). The number of autophagosomes in TNFAIP3-His+ LPS treatment group were more than that in TNFAIP3-siRNA + LPS or LPS treatment group (As shown in red arrow in Figure 3A). Western blot results showed that the protein expression of LC3 II was significantly increased and P62 expression was decreased in TNFAIP3-His + LPS compared with TNFAIP3-siRNA + LPS or LPS treatment group (Figure 3C–3E), indicating the activation of autophagy in human NPCs. Furthermore, the LC3 puncta calculation experiments were performed by Fluorescence-based detection of LC3 isoforms in human NPCs. The results showed that the LC3 puncta were significantly increased in the TNFAIP3-His and TNFAIP3-His + Torin1 treatment group (Figures 3F, 5G). These results demonstrated that TNFAIP3 may induce autophagy in the LPS-stimulated human NPCs.

Figure 3. TNFAIP3 promotes autophagy in LPS-stimulated human NPCs. (A) The effect of TNFAIP3 on autophagy in LPS-stimulated human NPCs. Morphological observation of autophagosome under transmission electron microscope, amplification × 15,000, n>3. (B) The number of autophagosomes. (C–E) The NPCs were the primary cell cultures from LVF patients without IVDD. Con, no LPS treatment; LPS+TNFAIP3-His and LPS+ TNFAIP3-siRNA, TNFAIP3 and its siRNA were delivered into the NPCs by adenovirus expressing TNFAIP3-His and TNFAIP3-siRNA, respectively. Western blot analysis of LC3II and P62 expression in human NPCs (F) Human NPCs were transfected with adenovirus containing GFP-LC3 and the formation and distribution of GFP-LC3 punctate were observed under confocal microscopy, amplification × 800. Data are represented by the mean ± standard deviation of 3 independent experiments. *** P<0.01, ** P<0.05, * P>0.05 by the Student’s t-test.

TNFAIP3 inhibits mTOR signaling in the inflammatory human NPCs

As mTOR is a major negative regulator of autophagy, we hypothesized that TNFAIP3 could inhibit mTOR signaling for the introduction of autophagy. The mTOR phosphorylation at S2448 in the inflammatory NPCs was detected by Western blot. As expected, LPS increased the phosphorylated mTOR level. In contrast, phosphorylated mTOR at S2448 significantly increased in TNFAIP3 knockdown cells and decreased in TNFAIP3 overexpression cells, suggesting TNFAIP3 specifically inhibits mTOR signaling (Figure 4A, 4B). Thus, we infer that TNFAIP3 may decrease phosphorylation level of mTOR after LPS stimulation in human NPCs.

Figure 4. Knockdown of TNFAIP3 increases mTOR signaling in LPS-treated human NPCs. (A) The mTOR signaling was measured by Western blot in LPS-stimulated human NPCs at the phosphorylation level of mTOR at S2448. (B) Relative phosphorylation level of the mTOR. Data are represented by the mean ± standard deviation of 3 independent replicates. *** P<0.01, ** P<0.05, * P>0.05 by the Student’s t-test.

TNFAIP3 promotes autophagy by inhibiting mTOR signaling in inflammatory human NPCs

To understand the molecular mechanism of TNFAIP3 on autophagy induction in inflammatory human NPCs. Torin1, a selective ATP-competitive mTOR inhibitor that can strongly induce autophagy (J Biol Chem 2009; 284:8023-32) was used to evaluate the effect of TNFAIP3 on both mTOR signaling and autophagy in IVDD. Again, LPS induced inflammatory response with higher expression of pS2448-mTOR, which was further enhanced by TNFAIP3 knockdown. However, Torin1 treatment at 20 nM for 1 day shutdown both the increase to non-inflammatory levels (Figure 5A, 5B). Thus, TNFAIP3 inhibits mTOR signaling in this cell model. After Torin1 treatment, western blot results showed that Torin1 reversed the level of LC3 protein in TNFAIP3-siRNA cells + LPS (Figure 5C, 5E), but the protein expression of P62 was not significant in Torin1 treatment (Figure 5C, 5F), indicating the activation of autophagy in human NPCs. In addition, the LC3 puncta were increased in the Torin1 treatment group compared with TNFAIP3-siRNA + LPS group (Figure 5G). The above results suggested that TNFAIP3 induces autophagy through inhibiting mTOR signaling.

Figure 5. Inhibition of mTOR signaling ameliorates autophagy in the inflammatory human NPCs. (A, B) Torin1 decreases mTOR signaling in inflammatory human NPCs. The phosphorylation level of mTOR at S2448 was measured by Western blot (A). Torin1 treated cells at 20 nM for 24 h. (B) With its quantification normalized to mTOR level. (C, D) Inhibition of mTOR signaling increased autophagy by Western blot for LC3II protein level. (D) With its quantification normalized to GAPDH for TNFAIP3. (E, F) The relative quantification of LC3II and P62 protein. (G) Human NPCs were transfected with adenovirus containing GFP-LC3 and the formation and distribution of GFP-LC3 punctate were observed under confocal microscopy, amplification × 800. Data are represented by the mean ± standard deviation of 3 independent replicates. *** P<0.01, ** P<0.05, * P>0.05 by the Student t-test.

TNFAIP3 inhibits inflammatory response and promotes extracellular matrix expression in human NPCs

To further evaluate the function of TNFAIP3 on the inflammatory response and extracellular matrix production in human NPCs and its underpinning mechanism. We continued to test inflammatory cytokines (IL-1β and TNF-α) production and extracellular matrix of aggrecan and type II collagen under the condition of mimicking IVDD. As we previously established the LPS-stimulated IVDD model in human primary NPCs, LPS reduced matrix protein production of aggrecan and type II collagen as previously reported (Aging 2019, 11:7294). We found that over-expression and knockdown of TNFAIP3 respectively increased and decreased the expression of both aggrecan and type II collagen (Figure 6A–6C), indicating TNFAIP3 maintains the NPCs function. Furthermore, inhibition of mTOR signaling further increased the production of both aggrecan and type II collagen. In addition, the ELISA assay was used to test the pro-inflammatory cytokines such as IL-1β and TNFα. The results showed that LPS treatment induced the expression of both factors. However, TNFAIP3 reduced the expression of both factors under inflammatory conditions by LPS induction. Moreover, inhibition of mTOR signaling reversed TNFAIP3 inhibition on both IL-1β and TNF-α production (Figure 6D, 6E). These data indicated that TNFAIP3 diminished the levels of inflammatory cytokines and enhanced the synthesis of extracellular matrix in inflammatory human NPCs.

Figure 6. TNFAIP3 inhibits the inflammatory response and promotes the synthesis of the extracellular matrix. (A, C) TNFAIP3 enhanced matrix production by the inhibition of mTOR signaling. Matrix production was measured by Western blot for the levels of COL2A1 and Aggrecan (A) With their quantification normalized to GAPDH (B, C). (D, E) TNFAIP3 decreased the protein level of inflammatory cytokines IL-1β (D) and TNF-α (E) in the supernatants of the NPCs culture as quantified by ELISA. Data are represented by the mean ± standard deviation of 3 independent replicates. *** P<0.01, ** P<0.05, * P>0.05 by the Student t-test. Torin1 treated cells at 20 nM for 24 h.

Cell collection and culture

The collection and use of human NP specimens have been approved by the Ethics Committee of Chongqing Medical University before surgery. All patients signed written informed consent. All samples were obtained after surgery at the First Affiliated Hospital of Chongqing Medical University. The control group was obtained from patients with lumbar vertebral fracture (LVF) (n>5), and the degenerative group was obtained from patients with intervertebral disc degeneration (IVDD) (n>15). Pfirrmann classification was used to define degeneration grades of IVDD by MRI scans before surgery [36]. Pfirrmann grade I or II was defined as a relatively normal discs in patients with LVF, and Grades III-IV was defined as degenerative disc cells in patients with IVDD.

The NPCs isolated from patients' inter-vertebral disc tissue after surgery was quickly digested with 0.25% trypsin solution at 37° C for 30 mins, then digested with 0.2% type II collagenase for 4 hours. Tissue debris was removed with a 200 μm filter after digested. The mixture was centrifuged at 1,000 × g for 5 min, and then the cells were cultured with DMEM/F 12 medium (Gibco, USA) containing 10% FBS (Corning, USA) and 1% penicillin-streptomycin at 37° C, 5% CO₂. The 2^nd passaged NPCs were used for following experiments.

Cell treatment and transfection

The human NPCs were cultured with DMEM/F-12 medium with 10% FBS and transfected Ad-TNFAIP3 which was constructed with pHB Ad-EF1-MCS-GFP system for 24 h, and then were treated with 200 ng/ml LPS (Sigma, USA), simultaneously stimulated with Torin1 (20 nM) for 24 h. Over-expression TNFAIP3 (TNFAIP3-His), knockdown TNFAIP3 (TNFAIP3-siRNA) and Adenovirus vector without TNFAIP3, (Negative control, NC) were designed and purchased from HANBIO (Shanghai). Cells were transfected according to the manufacturer's instructions.

Immunohistochemistry (IHC)

The human NP tissues collected from patients with lumbar vertebral fracture (LVF) and IVDD were fixed in 4% paraformaldehyde at 4° C for 48 h, and then were embedded in paraffin. According to the manufacturer's protocol, the IHC was performed by immunohistochemical kit (Wuhan Boster Biological Technology, Ltd., Wuhan, China), Briefly, the tissues samples were baked and dewaxed. Then antigen was retrieved. Next, the tissues were incubated with rabbit primary anti-TNFAIP3 (EPR2663, Abcam) at 4° C overnight. Then, the tissues were incubated with the corresponding secondary antibody IgG-HRP (155000) for 60 mins. DAB staining was added under the microscope and counterstained with hematoxylin for 2 mins. The sample was sealed with a neutral resin for 2 d. The images were collected with a microscope (Leica DM4 M, GERMARY).

The observation of autophagosomes by TEM in NPCs

The transmission electron microscopy (TEM) was used to observe the autophagosomes in NPCs. The samples were fixed in 4% glutaraldehyde after centrifugation twice, then cut into thin sections (60-80 nm), mounted on a copper grid, and post-stained with uranyl acetate and lead citrate. Images were captured using the JEOL JEM-1400PLUS.

The detection of autophagy activity by GFP-LC3 assay

The GFP-LC3-adenovirus was used to detect the autophagy activity in NPCs. After transfected for 24 h, the levels of autophagy were evaluated with green fluorescent puncta of autophagosomes through laser confocal microscopy.

The detection of inflammatory cytokines by ELISA

The ELISA assay was used to detect inflammatory cytokine in NPCs. Samples supernatants were collected from each group of cells after treated for 48 h. The samples supernatants were centrifuged at 1,200 × g for 5 min to remove cell debris and stored at -80° C until use. The levels of TNF-α and IL-1β in cultured supernatants of human NPCs was precisely measured with human ELISA kits according to the manufacturer's instructions, respectively. The optical density was determined with a BIO-RAD Microplate reader (CA, USA), and the absorption was measured at 450 nm. A standard curve for each measurement was established using a cytokine standard provided by the ELISA Kit. Each experiment was performed three times independently.

The detection of protein level by Western blot

Cell protein was dissociated using RIPA lysis buffer, and then cell protein concentration was measured by BCA Protein Assay Kit (Solarbio, PC0020). The proteins (35-40 μg) were separated using Nu PAGE 6-12% Bis-Tris-SDS gel (Invitrogen) and transferred to PVDF and NC membrane (0.45, 0.22 mm). Membranes were blocked with 4% Nonfat-Dried Milk for 2 h at room temperature and incubated with indicated primary antibodies anti-TNFAIP3 (EPR2663, Abcam), anti-COL2A1 (ab 34712, Abcam), anti-Aggrecan (ab 3778, Abcam), anti-LC3A/B (CST, D3U4C), anti-TNFAIP3 (ab 92324, Abcam), anti-phospho-mTOR (S 2448) (AP0094, Abclonal), anti-mTOR (A 2445, Abclonal), at 4° C overnight. All antibodies were used according to the manufacturer's recommendations. After washing the membranes three times with Tris-buffered saline containing 0.1% Tween 20 (TBST), the membranes were incubated in the appropriate secondary antibody (1:5,000 dilution) (Jackson Research, PA) for 2 h at room temperature, and then washed 3 times with PBS for 5 minutes each time. Finally, the membranes were measured by an ECL plus reagent (Solarbio, PE0010) and the results were quantified using ImageJ software (National Institutes of Health).

The detection of mRNA level by RT-qPCR

Collected the cells from Con group, LPS group, LPS+NC group, LPS+TNFAIP3-His group, LPS+TNFAIP3-siRNA group, the mRNAs of human NPCs were isolated using TRlzol Reagent (Invitrogen, USA) according to the manufacturer's instructions. Genes expression was determined by real-time PCR using Prime-Script® RT reagent kit (Takara, DRR037A) and SYBR® Green Real-Time PCR Master Mix (TOYOBO, QPK-201). The relative expression of the gene from the control was evaluated by the 2-ΔΔCT method. The Primers for target gene were as follow: (Table 1).

Statistical analysis

All experiments were performed in three independent replicates, and the results were shown by the mean ±SD. Statistical analysis was performed by t-test using software SPSS 19.0. P-value was defined as significant, *** indicates p < 0.01, ** indicates p < 0.05, * indicates p > 0.05. Error bars indicate ±SD.