The degenerative nucleus pulposus (NP) tissues were obtained from IDD patients [n=5 (female, 2; and male, 3); age range 29-74 years, median age 58 years] undergoing posterior lumbar interbody fusion (PLIF) due to degenerative lumbar disc disease in The Affiliated Nanhua Hospital of University of South China. The control NP tissues were obtained from lumbar vertebrae fracture patients without IDD [n=5 (female, 2; and male, 3); age range 14-65 years; median age 54 years] in The Affiliated Nanhua Hospital of University of South China. Voluteers were recruited between February and December 2021. The present study protocol conformed to the globally accepted regulations on clinical studies involving human data, and approval was conferred by the ethics committee of University of South China (approval no. 2021-ky-41). Written informed consent was obtained from all of the participants or donors' families before using the samples.

Human NP tissue sections were incubated with UV block (Pierce; Thermo Fisher Scientific, Inc.) containing 10% goat serum (Abcam) for 30 min at room temperature and then with mouse cluster of differentiation (CD)68 (1:200, cat. no. ab283654, Abcam) overnight in a humid chamber at 4˚C. After, sections were incubated with a secondary antibody (1:500, cat. no. ab150077, Abcam) for 2 h at room temperature. Nuclei were counterstained with DAPI. After washing with PBS for 15 min, images were captured at x200 magnification on an Olympus FV1000 (Olympus Corporation).

HNPCs and THP-1-derived monocytes were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM or 1640 supplemented with 0.1% nonessential amino acids, penicillin (100 U/ml), streptomycin (100 mg/ml) and 10% FBS at 37˚C for 24 h. 100 ng/ml TNF-α (MedChemExpress) or 200 nmol/l PMA (MilliporeSigma) were used for inducing apoptosis of HNPCs and the differentiation of THP-1 derived monocytes into macrophages, respectively. Control vector and CX3CL1 and CCL17 overexpression vectors were packaged and purchased from Hanbio Biotechnology Co., Ltd. The 293T cell line from ATCC was used for generating viral particles. The mass of used vectors included 10 µg pHBLV, 10 µg psPAX2 and 5 µg pMD2G. HNPCs and THP-1-derived macrophages were cultured in 6-well plate at 37˚C with 5% CO_2. When cells reached 40-50% confluence, 1 ml of serum-free medium was replaced with serum-containing medium and 2x10⁸ TU/ml viral particles [HNPC (MOI: 30) and THP-1-derived macrophage (MOI: 10)]. After 12 h, 1 ml of serum-containing medium was added to wells and the medium changed to fresh serum-containing medium at 24 h. After 48 h, puromycin was used for screening of stably transfected cells. The stably expressed cells were screened using 4 µg/ml puromycin for 7 days followed by use for different experiments. The generation used of transfected cells were controlled at third generation. Cells were incubated with CX3CR1 inhibitor JMS-17-2 (MedChemExpress) or JAK2/STAT3 inhibitor SD1029 (MedChemExpress).

Human nucleus pulpous cells and THP-1-derived macrophages were cultured in 6-well plates at 37˚C and were used for RNA extraction when the confluent reached ~95%. The RNA extraction, complementary DNA (cDNA) synthesis, and qPCR were performed according to the manufacturer's protocols. Human nucleus pulpous cells, THP-1-derived macrophages and NPs were treated and lysed for RNA extraction using the TRIzol^® (Thermo Fisher Scientific, Inc.) extraction method. Total RNA was reversed transcribed into cDNA with HiScript III RT SuperMix (R323-01, Vazyme). qPCR amplifications were performed according to manufacture manual from ChamQ SYBR qPCR Master Mix (cat. no. RQ311-02; Vazyme Biotech Co., Ltd.), which was 95˚C for 30 sec, 95˚C 5 sec, 60˚C 20 sec for additional 40 cycles. Using 18s as a control, the 2^-ΔΔCq method was applied to calculate the relative mRNA expression of target genes. The sequences of the primers were: Human CX3CL1, 5'-GCCACAGGCGAAAGCAGTA-3' and 5'-GGAGGCACTCGGAAAAGCTC-3'; Human CX3CR1, 5'-AGTGTCACCGACATTTACCTCC-3' and 5'-AAGGCGGTAGTGAATTTGCAC-3'; Human CD86: 5'-AGTGGAATAGCCTCCCTGTAACTCC-3' and 5'-CCCATAAGTGTGCTCTGAAGTGAAA-3', Human CD206: 5'-TCCGGGTGCTGTTCTCCTA-3', 5'-TCCGGGTGCTGTTCTCCTA-3'; Human IL-1β: 5'-ATGATGGCTTATTACAGTGGCAA-3' and 5'-GTCGGAGATTCGTAGCTGGA-3', Human IL-6: 5'-ACTCACCTCTTCAGAACGAATTG-3' and 5'-CCATCTTTGGAAGGTTCAGGTTG-3', Human IL-4: 5'-CGGCAACTTTGTCCACGGA-3' and 5'-TCTGTTACGGTCAACTCGGTG-3', Human IL-10: 5'-GACTTTAAGGGTTACCTGGGTTG-3' and 5'-TCACATGCGCCTTGATGTCTG-3' and 18s: 5'-TTGACGGAAGGGCACCACCAG-3' and 5'-GCACCACCACCCACGGAATCG-3'. Analysis of mRNA levels was performed using ABI PRISM7900 sequence detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.).

THP-1-derived monocytes were added to the upper chamber and culture supernatant to the lower chamber of 5.0 µm Transwell inserts (Corning, Inc.). The number of migrated cells was assessed after 24 h by incubating with calcein at 37˚C and positive cells in low chambers counted. Fluorescence microscopy images were captured at x200 magnification on an Olympus BX-53-LV2000 (Olympus Corporation).

TUNEL staining was used to evaluate cell apoptosis. The cells were fixed in 4% paraformaldehyde for 1 h at room temperature and then treated with 0.5% TritonX-100 for 10 min. After washing with PBS, the cells were incubated with the cell death detection kit (Elabscience Biotechnology, Inc.) according to the manufacturer's instructions. Nuclei were counterstained with DAPI (Beyotime Institute of Biotechnology). Fluorescence microscopy images were captured at x200 magnification on an Olympus BX-53-LV2000 (Olympus Corporation).

Total protein were extracted with RIPA cell lysis buffer (Beyotime Institute of Biotechnology) and Protease and Phosphatase Inhibitor Cocktail (MedChemExpress) on ice for 30 min and centrifuged at 13,400 x g for 10 min at 4˚C. Total protein were quantified by a BCA kit (Beyotime Institute of Biotechnology). Primary antibodies included anti-CX3CL1 (Proteintech Group, Inc.; cat. no. 10108-2-AP; 1:1,000), anti-CX3CR1 (Proteintech Group, Inc.; cat. no. 13885-1-AP; 1:1,000), BCL-2 (Proteintech Group, Inc.; cat. no. 68103-1-Ig; 1:1,000), Bax (Proteintech Group, Inc.; cat. no. 50599-2-Ig; 1:1,000), anti-STAT3 (Abcam; cat. no. ab68153; 1:200), anti-phosphorylated (p-)STAT3 (Abcam; cat. no. ab76315; 1:1,000), anti-JAK2 (Abcam; cat. no. ab108596; 1:1,000), anti-p-JAK2 (Abcam; cat. no. ab32101; 1:1,000), anti-GAPDH (Abcam; cat. no. ab8245; 1:2,000) and anti-β-actin (Abcam; cat. no. ab8226; 1:2,000). Secondary antibodies were HRP-labelled Goat anti-Mouse or anti-Rabbit IgG (H + L) (Abcam; cat. no. ab6789 or ab6721; 1:5,000). The amount of GTP bound RHOA in cell-free lysates was determined using the RHO Activation Assay kit (MilliporeSigma). Protein (30 µg) of each sample were electrophoresed via 12% SDS gel and transferred to polyvinylidene fluoride membrane (MilliporeSigma). The membranes were blocked with 5% fat-free milk for 1 h at room temperature, incubated with indicated primary antibodies overnight at 4˚C and incubated with HRP-conjugated secondary antibodies at room temperature for 2 h. Antibody binding was visualized with Tanon 5500 (Tanon Science and Technology Co., Ltd.) and BeyoECL Plus (Beyotime Institute of Biotechnology), and ImageJ software (version 1.8.0.112; National Institutes of Health) was used for analyzing the blots.

Supernatant from treated HNPCs and THP-1-derived macrophages were collected and centrifuged at 2,000 x g for 10 min. The supernatants then were used to ELISA according to the instruction of kits (cat. nos. CX3CL1, ab192145; IL-4, ab215089; IL-10, ab185986; CCL17, ab183366; Abcam).

All data were presented as means ± SD. Statistical significance was evaluated by either one-way ANOVA or Student's t-test. Student's t-test was used for assessing the differences between two groups, while one-way ANOVA with Scheffe's test was used for analyzing the difference among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

To identify whether CX3CL1 contributed to IDD, the levels of CX3CL1 were detected in NPs of IDD patients. As shown in Fig. 1A and B, the mRNA and protein levels of CX3CL1 were significantly decreased in degenerative NP tissues. However, CX3CR1 expression in degenerative NPs were unchanged (Fig. 1C and D). This indicated that CX3CL1 might possess a potential regulatory effect on IDD progression.

Macrophages have been reported to be involved in inflammatory response during IDD progression (30,31). As shown in Fig. 2A, abundant CD68 positive fluorescence staining was detected in NPs of IDD patients. Given that the tissue macrophages are mainly derived from circulating monocytes, the present study aimed to identify whether CX3CL1 was involved the regulation of monocyte migration. As shown in Fig. 2B-E, HNPC supernatant significantly promoted THP-1-derived monocytes migration and overexpression of CX3CL1 markedly promoted this effect. Furthermore, CX3CR1 antagonist JMS-17-2 significantly blocked CX3CL1 overexpression and supernatant-induced THP-1-derived monocytes migration (Fig. 2D-E). Inhibition of CX3CR1 also impaired RhoA signaling in THP-1-derived monocytes (Fig. 2F and G). These data suggested that HNPC-derived CX3CL1 bound to monocyte CX3CR1 followed by the promotion of monocyte migration via RhoA signaling.

Macrophages act as an important regulator during tissue inflammation via the conversion between pro- and anti-inflammatory phenotypes. The mRNA expression level of M2 marker CD206 significantly decreased (Fig. 3B) whereas M1 marker significantly increased in degenerative NPs (Fig. 3A). The present study next aimed to investigate the role of HNPC-derived CX3CL1 in macrophage polarization and reveal its molecular detail. As shown in Fig. 3C and D, the supernatant from CX3CL1 overexpression HNPCs significantly promoted macrophage polarization into the M2 phenotype while reducing M1-like macrophages, and JMS-17-2 incubation almost eliminated these effects. Moreover, the conditional medium from CX3CL1/CX3CR1 axis-induced M2-like macrophages significantly increased anti-inflammatory cytokines release from HNPCs (Fig. 3E). The activation of classical JAK2/STAT3 signaling is known to occur during M2 polarization (32). The present study suggested that CX3CL1 elevated the phosphorylated levels of JAK2 and STAT3 in THP-1-derived macrophage, while incubation with JMS-17-2 almost reversed these (Fig. 3F-G). Furthermore, JAK2/STAT3 signaling inhibitor SD1029 effectively prevented CX3CL1 overexpression supernatant-induced M2-like macrophages and inhibited M2-like macrophage-promotion of anti-inflammatory cytokines secretion from HNPCs (Fig. 3H-J). In addition, the present study detected increased pro-inflammatory cytokines IL-6 levels and decreased anti-inflammatory cytokines IL-4 and IL-10 levels in NPs of IDD patients (Fig. 3K). Meanwhile, increased pro-inflammatory factors and M1 marker CD86 levels was found in NPs of IDD patients with low CX3CL1 expression group (Fig. 3L and M). These data revealed that the polarization of macrophage in NPs may be dependent on CX3CL1/CX3CR1 axis.

As macrophages have been reported to be involve in the regulation of cell death (33), the present study aimed to reveal whether an anti-inflammatory type M2-like macrophage serves a role in HNPC apoptosis. As shown in Fig. 4A and B, the data from TUNEL assays indicated that the medium from CX3CL1 overexpression supernatant-incubated macrophages evidently inhibited apoptosis of HNPCs. To further identify the trigger during this process, it was found that JMS-17-2 clearly blocked the increase of CCL17 level in the medium of CX3CL1-overexpressed supernatant-cultured macrophages (Fig. 4C). In addition, the supernatant from CCL17 overexpressed macrophages significantly increased the ratio of Bcl-2 to Bax and reduced apoptosis in HNPCs (Fig. 4D-G). These results indicated that increased CCL17 secretion from M2 macrophages exerted an anti-apoptotic effect on HNPCs.