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. 2023 Jun 7;26(1):359.
doi: 10.3892/etm.2023.12058. eCollection 2023 Jul.

CX3CL1/CX3CR1 axis alleviates inflammation and apoptosis in human nucleus pulpous cells via M2 macrophage polarization

Xiao-Wen Gao  1 , Han-Lin Hu  1 , Ming-Hua Xie  1 , Cai-Xia Tang  2 , Jun Ou  1 , Zheng-Hao Lu  1
Affiliations

CX3CL1/CX3CR1 axis alleviates inflammation and apoptosis in human nucleus pulpous cells via M2 macrophage polarization

Xiao-Wen Gao et al. Exp Ther Med. .

Abstract

CX3C chemokine ligand 1 (CX3CL1) belongs to the CX3C chemokine family and is involved in various disease processes. However, its role in intervertebral disc degeneration (IDD) remains to be elucidated. In the present study, western blotting, reverse transcription-quantitative PCR and ELISA assays were used to assess target gene expression. In addition, immunofluorescence and TUNEL staining were used to assess macrophage infiltration, monocyte migration and apoptosis. The present study aimed to reveal if and how CX3CL1 regulates IDD progression by exploring its effect on macrophage polarization and apoptosis of human nucleus pulposus cells (HNPCs). The data showed that CX3CL1 bound to CX3C motif chemokine receptor 1 (CX3CR1) promoted the M2 phenotype polarization via JAK2/STAT3 signaling, followed by increasing the secretion of anti-inflammatory cytokines from HNPCs. In addition, HNPC-derived CX3CL1 promoted M2 macrophage-derived C-C motif chemokine ligand 17 release thereby reducing the apoptosis of HNPCs. In clinic, the reduction of mRNA and protein levels CX3CL1 in degenerative nucleus pulposus tissues (NPs) was measured. Increased M1 macrophages and pro-inflammatory cytokines were found in NPs of IDD patients with low CX3CL1 expression. Collectively, these findings suggested that the CX3CL1/CX3CR1 axis alleviates IDD by reducing inflammation and apoptosis of HNPCs via macrophages. Therefore, targeting CX3CL1/CX3CR1 axis is expected to produce a new therapeutic approach for IDD.

Keywords: CX3C chemokine ligand 1; CX3C motif chemokine receptor 1; apoptosis; inflammation; intervertebral disc degeneration; macrophage.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
CX3CL1 expression is reduced in degenerative NP tissues. Analysis of (A) mRNA and (B) protein levels of CX3CL1 in NPs via RT-qPCR and western blotting. Analysis of (C) mRNA and (D) protein levels of CX3CR1 in NPs via RT-qPCR and western blotting. All data were shown as mean ± SD from five independent experiment with each performed in triplicate. *P<0.05 vs. Control group. CX3CL1, CX3C chemokine ligand 1; NPs, nucleus pulposus tissues; RT-qPCR, reverse transcription-quantitative PCR.
Figure 2
Figure 2
The binding of CX3CL1 to CX3CR1 induces monocyte migration. (A) Detection of the intensity of macrophage marker CD68 in degenerative NPs via immunofluorescence staining. Scale bar, 50 µm. (B) Measurement of medium CX3CL1 level of HNPCs using ELISA. Analysis of (C) mRNA and (D) protein levels of CX3CL1 in HNPCs via reverse transcription-quantitative PCR and western blotting. (E-H) Analysis of immunofluorescence signal of calcein-labelled THP-1-derived monocyte to assess monocyte migration. Scale bar, 50 µm. (I) Analysis of the protein levels of total RhoA and activated RhoA (RhoA-GTP) using western blotting. All data were shown as mean ± SD from three independent experiment with each performed in triplicate. *P<0.05 vs. Control LV or Control LV medium group; **P<0.05 vs. LV-CX3CL1 medium group. CX3CL1, CX3C chemokine ligand 1; CX3CR1, CX3C motif chemokine receptor 1; CD, cluster of differentiation; NPs, nucleus pulposus tissues; LV, lentivirus.
Figure 3
Figure 3
CX3CL1/CX3CR1 axis accelerates M2 macrophage polarization via JAK2/STAT3 signaling and inhibited the inflammatory response in HNPCs. Analysis of (A) M1 (CD86) and (B) M2 (CD206) markers in NPs via RT-qPCR. (C-D) Analysis of M1 (CD86) and M2 (CD206) markers of THP-1-derived macrophages via RT-qPCR. (E) Measurement of media IL-4 and IL-10 level of HNPCs using ELISA. (F and G) Analysis the protein levels of t-JAK2, p-JAK2, t-STAT3 and p-STAT3 via western blotting. Analysis of (H) M1 (CD86) and (I) M2 (CD206) markers via RT-qPCR. (J) Measurement of media IL-4 and IL-10 level of HNPCs using ELISA. (K) Analysis of pro-inflammatory cytokines (IL-1β and IL-6) and anti-inflammatory cytokines (IL-4 and IL-10) markers in NPs via RT-qPCR. Analysis of (L) pro-inflammatory cytokines (IL-1β and IL-6), anti-inflammatory cytokines (IL-4 and IL-10) and (M) M1 (CD86) and M2 (CD206) markers in NPs of low or high CX3CL1 expression via RT-qPCR. All data are mean ± SD from three independent experiment with each performed in triplicate. *P<0.05 vs. Control or Control LV medium or Low CX3L1 groups; **P<0.05 vs. LV-CX3CL1 medium group. CX3CL1, CX3C chemokine ligand 1; CX3CR1, CX3C motif chemokine receptor 1; HNPCs, human nucleus pulposus cells; CD, cluster of differentiation; NPs, nucleus pulposus tissues; RT-qPCR, reverse transcription-quantitative PCR; t-, total; p-, phosphorylated; LV, lentivirus.
Figure 4
Figure 4
M2 macrophage-derived CCL17 reduced apoptosis of HNPCs. (A and B) Analysis of apoptosis of HNPCs using TUNEL staining, Scale bar, 50 µm. (C) Measurement of CCL17 secretion of THP-1-derived macrophage using ELISA. (D) Measurement of CCL17 overexpression efficiency in THP-1-derived macrophage via ELISA. (E) Analysis of the expression of Bcl-2 and Bax in HNPCs via western blotting. (F and G) Analysis of apoptosis of HNPCs via TUNEL staining. All data are mean ± SD from three independent experiment with each performed in triplicate. *P<0.05 vs. Control LV medium group; **P<0.05 vs. LV-CX3CL1 medium group. HNPCs, human nucleus pulposus cells; CCL17, C-C motif chemokine ligand 17; CX3CL1, CX3C chemokine ligand 1; LV, lentivirus.
Figure 5
Figure 5
Model for CX3CL1/CX3CR1 axis alleviating IDD progression. HNPC-derived CX3CL1 binds to monocyte CX3CR1 that leads to monocyte migration and M2 macrophage polarization via RhoA and JAK2/STAT3 signaling, respectively. M2-like macrophage promotes the production of anti-inflammatory cytokines from HNPCs and M2 macrophage-secreted CCL17 to inhibit apoptosis of HNPCs. CX3CL1, CX3C chemokine ligand 1; CX3CR1, CX3C motif chemokine receptor 1; IDD, intervertebral disc degeneration; HNPCs, human nucleus pulposus cells; RhoA, Ras homolog family member A; CCL17, C-C motif chemokine ligand 17.
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References

    1. Peng B, Hao J, Hou S, Wu W, Jiang D, Fu X, Yang Y. Possible pathogenesis of painful intervertebral disc degeneration. Spine. 2006;31:560–566. doi: 10.1097/01.brs.0000201324.45537.46. - DOI - PubMed
    1. Adams MA, Freeman BJ, Morrison HP, Nelson IW, Dolan P. Mechanical initiation of intervertebral disc degeneration. Spine (Phila Pa 1976) 2000;25:1625–1636. doi: 10.1097/00007632-200007010-00005. - DOI - PubMed
    1. Freemont AJ, Watkins A, Le Maitre C, Jeziorska M, Hoyland JA. Current understanding of cellular and molecular events in intervertebral disc degeneration: Implications for therapy. J Pathol. 2002;196:374–379. doi: 10.1002/path.1050. - DOI - PubMed
    1. Jia J, Nie L, Liu Y. Butyrate alleviates inflammatory response and NF-κB activation in human degenerated intervertebral disc tissues. Int Immunopharmacol. 2020;78(106004) doi: 10.1016/j.intimp.2019.106004. - DOI - PubMed
    1. Yu H, Liu Y, Xie W, Xie Q, Liu Q, Cheng L. IL-38 alleviates the inflammatory response and the degeneration of nucleus pulposus cells via inhibition of the NF-κB signaling pathway in vitro. Int Immunopharmacol. 2020;85(106592) doi: 10.1016/j.intimp.2020.106592. - DOI - PubMed