Abstract
-
Objective
Intervertebral disc degeneration (IVDD) is a complex pathological process involving inflammation, oxidative stress, and immune dysregulation. Emerging evidence suggests that neuroimmune interactions contribute to IVDD progression, but the role of neuropeptide-like factors remains poorly understood.
-
Methods
We investigated whether Gαi-interacting protein (GINIP+) sensory neurons infiltrate degenerative discs and secrete TAFA chemokine like family member 4 (TAFA4), a neuron-derived cytokine known to influence macrophage activity. In vivo and in vitro models were used to assess TAFA4 expression, its regulatory effects on macrophage polarization, reactive oxygen species (ROS) production, inflammasome activation, and disc cell phenotype. Knockdown of TAFA4 was achieved via lentiviral transduction in rabbit discs and cell coculture models.
-
Results
TAFA4 was upregulated in IVDD tissues and colocalized with GINIP+ neurons. Knockdown of TAFA4 in vivo exacerbated disc degeneration, increased M1 macrophage presence, elevated ROS levels, and activated the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. In vitro, GINIP+ neurons promoted macrophage M2 polarization and interleukin (IL)-10 production while suppressing tumor necrosis factor-α and IL-1β. These effects were reversed by TAFA4 knockdown. Moreover, TAFA4 attenuated ROS-dependent NLRP3 activation and preserved anabolic marker expression (ACAN [aggrecan], COL II [type II collagen], SOX9 [SRY-box transcription factor 9]), while reducing catabolic and hypertrophic-related markers (MMP13 [matrix metalloproteinase 13], ADAMTS5 [a disintegrin and metalloproteinase with thrombospondin motifs 5], COL X [collagen type X alpha 1 chain], RUNX2 [Runt-related transcription factor 2]) in nucleus pulposus cells.
-
Conclusion
TAFA4 acts as a neuron-derived mediator of neuroimmune crosstalk in IVDD that modulates macrophage polarization and oxidative stress, thereby delaying disc degeneration. This neuron–immune axis represents a potential therapeutic target.
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Keywords: Intervertebral disc degeneration, TAFA4, GINIP+ neurons, Macrophage polarization, NLRP3 inflammasome, Reactive oxygen species
INTRODUCTION
Intervertebral disc degeneration (IVDD) is a leading pathological contributor to low back pain (LBP), which is one of the foremost causes of global disability [
1,
2]. Despite clinical significance, the pathophysiological mechanisms of IVDD remain unclear. Recent studies highlight the active involvement of immune and neural elements in IVDD pathology [
3,
4]. Degenerative discs often exhibit aberrant innervation and infiltration by immune cells, particularly macrophages, suggesting the presence of a neuroimmune crosstalk within the disc microenvironment [
5-
7]. This neuroimmune crosstalk is increasingly recognized for its regulatory influence on inflammation, cell survival, and tissue remodeling during disc degeneration [
6-
8].
Macrophages are key immune cells recruited to the degenerative intervertebral disc (IVD), with their functional phenotype—proinflammatory (M1) or anti-inflammatory (M2)—playing a crucial role in disease progression [
3,
7,
9]. An imbalance toward M1 polarization is frequently observed in IVDD and contributes to exacerbated inflammatory responses and tissue degradation [
9,
10]. Alongside immune dysregulation, oxidative stress has emerged as a significant factor in IVDD [
11,
12]. The accumulation of ROS not only induces cytotoxicity in nucleus pulposus (NP) cells but also activates the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, thereby aggravating inflammation [
13]. The ROS-NLRP3 axis has been implicated in disc degeneration by promoting matrix degradation and sustaining chronic inflammation [
13].
In addition to immune and oxidative mechanisms, disturbances in extracellular matrix (ECM) homeostasis and chondrocytelike differentiation also play pivotal roles in IVDD. Key anabolic markers such as SRY-box transcription factor 9 (SOX9), aggrecan (ACAN), and type II collagen (COL II) are progressively downregulated in degenerative discs, while catabolic enzymes such as matrix metallopeptidase 13 (MMP-13) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) are elevated, driving proteoglycan and collagen degradation. At the same time, hypertrophic markers including collagen type X alpha 1 chain (COL X) and Runt-related transcription factor 2 (RUNX2) are upregulated [
14], reflecting abnormal chondrocyte differentiation and terminal maturation. Together, these alterations contribute to progressive matrix breakdown and structural failure of the IVD.
TAFA chemokine like family member 4 (TAFA4), as known as FAM19A4, is a small, secreted protein specifically produced by nonpeptidergic C-fiber sensory neurons expressing the Gαi-interacting protein (GINIP) [
15,
16]. TAFA4 has recently emerged as a neuroimmune modulator to promote tissue repair under pathological conditions [
15-
17]. However, its role within the degenerative IVD remains unknown. In particular, whether TAFA4 participates in regulating macrophage polarization, immune balance, and ECM remodeling in IVDD has not been addressed. In this study, we investigated whether GINIP
+ sensory neurons infiltrate degenerative IVD and secrete TAFA4 and further explored the impact of TAFA4 on the advancement of IVDD.
MATERIALS AND METHODS
1. Establishment of the IVDD Animal Model
The research was authorized by the Institutional Animal Care and Use Committee of Kangtai Medical Laboratory Services Hebei Co. (approval number: MDL2024-09-05-01). Briefly, 18 male New Zealand white rabbits (6 months old) provided by Beijing Baiaosi Biotechnology Co., Ltd., (license number: SYXK2021-006) were randomly divided into 3 groups: control, IVDD and IVDD+short hairpin TAFA4 (shTAFA4). Each group included 6 rabbits. All rabbits were acclimated for one week before surgery. Rabbits in the IVDD and IVDD+shTAFA4 groups were anesthetized with an intramuscular injection of ketamine (35 mg/kg, approximately 0.8 mL for a 2.3 kg rabbit, 100-mg/mL solution) combined with xylazine (5 mg/kg, approximately 0.6 mL for a 2.3-kg rabbit, 20-mg/mL solution), with a total injection volume of approximately 1.4 mL per rabbit. Under general anesthesia, percutaneous puncture of the L4–5 IVD was performed using an 18G needle under x-ray guidance. The L4–5 IVD was targeted, and needle insertion was confirmed to penetrate the annulus fibrosus. The needle tips were inserted approximately 5 mm into the IVD, rotated 180°, and held in position for 30 seconds. In the IVDD+shTAFA4 group, lentivirus carrying shTAFA4 (1×10
7 TU per disc) was administered via peridiscal injection once per week for 4 consecutive weeks, starting one week after modeling [
18-
20]. Injections were performed under fluoroscopic guidance using a 30G needle at a volume of 10 μL per disc, consistent with previously published rabbit intradiscal injection protocols [
21,
22].
Macrophages, GINIP+ neurons, and degenerative nucleus pulposus cells (dNPCs) were isolated from New Zealand white rabbits. dNPCs were seeded onto poly-L-lysine-coated 6-well plates (2×105 cells/well) in 2.5 mL of high-glucose Dulbecco’s Modified Eagle Medium (DMEM). Cells were cultured overnight in a humidified incubator at 37°C with 5% CO2. The next day, Transwell inserts (3450, Corning, USA) were placed in each well. Macrophages (2×105 cells/well) and GINIP+ neurons (1×105 cells/well) were added to the upper chamber in 0.2 mL of medium. In the dNPCs/Mφ/GINIP++shTAFA4 group, TAFA4 expression in neurons was knocked down via lentiviral transduction before coculture. Cells were cocultured for 24 hours under standard conditions.
3. Lentiviral Vector Construction and TAFA4 Knockdown
To achieve TAFA4 knockdown in GINIP+ sensory neurons, a recombinant lentivirus carrying shRNA targeting the rabbit TAFA4 gene was purchased from Hysigen Bioscience (Suzhou, China). The validated shRNA sequence was 5´-GAGAGGTTGTTGACAGTGGA-3´. The lentiviral vector (pLV[shRNA]-U6> raTAFA4[shRNA#2]-CMV>EGFP-PGK>Puro) expresses both enhanced green fluorescent protein and puromycin resistance for downstream validation and selection. The functional viral titer was 1.10×108 TU/mL, as determined by quantitative polymerase chain reaction (qPCR). For in vivo knockdown, lentivirus was administered via peridiscal injection immediately after IVDD modeling. Injections were performed once weekly for 4 consecutive weeks under fluoroscopic guidance. For in vitro experiments, GINIP+ neurons were seeded into 24-well plates at a density of 1×105 cells per well and cultured for 24 hours. The culture medium was replaced with 2-mL fresh neuronal medium supplemented with 6-μg/mL polybrene (C0351, Beyotime, China), and lentivirus was added at a multiplicity of infection of approximately 20, corresponding to approximately 2×105 TU per well. Neurons were incubated at 37°C for 24 hours, after which the medium was replaced with fresh virus-free medium.
4. Real-Time qPCR
After total RNA isolation from NPCs using TRIzol Reagent, reverse transcription was used to generate cDNA. In the RT-qPCR method, SYBR Green was used to analyze mRNA. The relative expression of each target mRNA was estimated using the 2
−ΔΔCT method. The gene sequences primarily focused on in this study are shown in
Supplementary Table 1. β-actin expression was employed as a reference point for a quantitative study.
Fluorescence In Situ hybridization (FISH) was performed on paraffin-embedded IVD sections using a commercial FISH kit (C007, Gefan Bio, China). After deparaffinization, rehydration, and antigen retrieval, sections were treated with 200-μg/mL proteinase K in 2×SSC for 5 minutes. Hybridization was conducted by applying 10-μL hybridization mix containing 2 μL of probe (TAFA4: Cy5-labeled, sequence: 5´-TTCTCTTTGCTACCGTGTGACCTG-3´; GINIP: FAM-labeled, sequence: 5´-CTCTATGTGGCCTCGATGCTCCGG-3´) and 8-μL hybridization buffer to the target area. Slides were denatured at 85°C for 5 minutes and hybridized overnight at 37°C in a humid chamber. Posthybridization washes were performed sequentially with Wash Buffer I, Wash Buffer II, and 70% ethanol. The DAPI (4´,6-diamidino-2-phenylindole) was added and allowed to come to room temperature for 5 minutes. Fluorescent signals were visualized using a fluorescence microscope (ECLIPSE Ci, Nikon, Japan).
6. Quantification of Intracellular ROS Levels
The intracellular ROS levels were evaluated via DCFH-DA probe staining (Beyotime, China). ROS can oxidize the 2´,7´-dichlorodihydrofluorescein (DCFH) probe, forming fluorescent green DCF. Briefly, nucleus pulposus cells (NPCs) were arranged in 6-well dishes and subjected to the designated treatment. The cells were subsequently grown in 1 mL of a DMEM/F12 mixture with 10 μM DCFH-DA (2´,7´-dichlorodihydrofluorescein diacetate) and incubated in a dim setting at 37°C for 30 minutes. The cells were visualized under a fluorescence microscope, and the intensity of green fluorescence was quantified.
7. Statistical Analysis
Statistical data were analyzed using IBM SPSS Statistics ver. 22.0 (IBM Co., USA), and graphs were generated using Graph-Pad Prism ver. 9.0 (GraphPad Software Inc., USA). All results were regarded as mean±standard deviation with at least 3 independent experiments. Student t-test was used to compare 2 groups, and 1-way analysis of variance was applied for multiple comparisons. The p-value of <0.05 was considered statistically significant.
RESULTS
1. TAFA4 Is Upregulated in Degenerative IVD and Colocalizes With GINIP+ Sensory Neurons
To investigate the potential involvement of GINIP
+ sensory neurons in TAFA4 secretion during disc degeneration, a rabbit model of IVDD was established. The rabbits were divided into control and IVDD groups. Immunofluorescence staining revealed the presence of GINIP-positive neurons and TAFA4 expression within degenerative IVD tissues (
Fig. 1D–
G). Compared with Control group, both GINIP and TAFA4 were markedly increased in the IVDD group. Consistently, Western blot analysis showed higher TAFA4 protein levels in the IVDD group than in the control group (
Fig. 1A and
B). Moreover, RT-qPCR analysis demonstrated a marked upregulation of TAFA4 mRNA expression in the IVDD group, further confirming the transcriptional elevation of TAFA4 under IVDD conditions (
Fig. 1C). In addition, FISH analysis was performed to examine the expression patterns of GINIP and TAFA4 in IVD tissues. As shown in
Fig. 1H, both GINIP and TAFA4 were barely detectable in the control group, whereas the IVDD group exhibited a marked increase in the expression of both markers. Notably, merged images revealed increased colocalization of GINIP and TAFA4 mRNA in the IVDD group, indicating that GINIP
+ neurons infiltrating the degenerative IVD are a key source of TAFA4. These findings demonstrate that TAFA4 is significantly upregulated in degenerative IVD tissues and is closely associated with GINIP
+ neurons.
Imbalance between M1 and M2 macrophage polarization, characterized by favoring M1, has been shown to accelerate the progression of IVDD. To explore the potential immunomodulatory role of TAFA4 in IVDD, we established an
in vivo model comprising 3 groups: control, IVDD, and IVDD+shTAFA4 (local lentiviral knockdown of TAFA4) groups (
Fig. 2A). Immunofluorescence staining revealed that both C-C chemokine receptor type 7 (CCR7)+ (M1), and CD206
+ (M2) macrophages were increased in the IVDD group compared to control (
Fig. 2D–
G). Specifically, the proportion of CCR7
+ macrophages rose from ~9% in Control to ~23% in IVDD, while CD206
+ macrophages increased from ~4% to ~26%. In the IVDD+shTAFA4 group, CCR7
+ macrophages were further elevated to ~30%, whereas CD206+ macrophages dropped to ~8%. Moreover, we explored the role of ROS, as accumulating evidence suggests their critical involvement in oxidative stress, and disc degeneration [
11,
23,
24]. Immunofluorescence staining for ROS showed a marked increase in IVD tissues, which was further exacerbated in the IVDD+ shTAFA4 group (
Fig. 2B and
C). These findings suggest that TAFA4 may facilitate an anti-inflammatory microenvironment by promoting M2 polarization and limiting oxidative stress, whereas TAFA4 knockdown disrupts this balance, leading to M1 polarization and aggravated oxidative stress.
Next, we established a Transwell coculture system to assess how TAFA4 influences inflammatory cytokine production and macrophage phenotype under degenerative conditions (
Fig. 3A). Enzyme-linked immunosorbent assay analysis revealed that TAFA4 was minimally detectable in dNPCs, whereas its concentration increased markedly following coculture with GINIP
+ neurons and TAFA4 secretion was markedly reduced after TAFA4 knockdown (
Fig. 3B). This confirms that GINIP
+ neurons are the primary source of TAFA4. Moreover, IL-10 levels were significantly increased in the dNPCs/Mφ/GINIP
+ group compared with dNPCs/Mφ alone, but this increase was abrogated after TAFA4 knockdown (
Fig. 3C), suggesting a TAFA4-dependent upregulation of anti-inflammatory cytokine expression. In contrast, TNF-α and IL-1β levels were both reduced in the coculture relative to dNPCs/Mφ group, indicating that GINIP
+ neuronderived signals suppress proinflammatory cytokine release from macrophages (
Fig. 3D and
E). However, knockdown of TAFA4 led to a partial restoration of TNF-α and IL-1β secretion. Flow cytometry analysis further revealed that the M1/M2 ratio was reduced by approximately 10% in the presence of GINIP
+ neurons compared with dNPCs/Mφ alone, while TAFA4 knockdown restored the ratio to near baseline levels (
Fig. 3F and
G). In summary, TAFA4 secreted by GINIP
+ neurons promoted macrophage M2 polarization and suppressed inflammatory cytokine production.
Given the critical role of ROS-dependent NLRP3 activation in promoting inflammation and disc cell damage during IVDD [
13,
25] and based on our previous finding that TAFA4 reduces ROS levels
in vivo, we next investigated whether TAFA4 also modulates NLRP3 inflammasome activity under degenerative conditions. Intracellular ROS levels in dNPCs were assessed using a fluorescent ROS probe. Coculture with GINIP
+ neurons significantly reduced ROS levels compared to dNPCs/Mφ group, whereas TAFA4 knockdown partially reversed this effect (
Fig. 4A). In addition to NLRP3, the mRNA expression levels of apoptosis-associated speck-like protein (ASC), Caspase-1, and Gasdermin D (GSDMD) were measured, as these molecules are key components of the NLRP3 inflammasome signaling cascade [
26]. ASC acts as an adaptor that bridges NLRP3 and Caspase-1, which in turn cleaves and activates GSDMD to mediate pyroptosis [
26]. RT-qPCR analysis revealed that the elevated expression of NLRP3 in dNPCs was markedly suppressed in dNPCs/Mφ/GINIP
+ group, with similar reductions observed in ASC, Caspase-1, and GSDMD transcripts (
Fig. 4D). This inhibitory effect was attenuated following TAFA4 knockdown. Western blot analysis confirmed these changes at the protein level, notably showing reduced levels of cleaved GSDMD (GSDMD-N) in dNPCs/Mφ/GINIP
+ group, which were restored upon TAFA4 knockdown (
Fig. 4B and
C). Together, these results indicate that TAFA4 limits oxidative stress and suppresses ROS-NLRP3 inflammasome activation in dNPCs.
To further explore the functional impact of TAFA4 on chondrocytic differentiation and ECM homeostasis in degenerative NPCs, we next examined the expression of additional anabolic, hypertrophic, and catabolic markers. Anabolic markers COL II and ACAN, together with chondrogenic transcription factor SOX9, are typically downregulated in dNPCs [
27]. Coculture with GINIP
+ neurons significantly restored COL II, ACAN and SOX9 expression in dNPCs (
Fig. 5A,
E, and
F). However, TAFA4 knockdown impaired this restorative effect. In contrast, the catabolic enzymes MMP13 and ADAMTS5 were elevated in dNPCs, suppressed by coculture with GINIP
+ neurons, and re-elevated upon TAFA4 knockdown (
Fig. 5A,
E, and
F). In addition, hypertrophic markers COL X and RUNX2 were highly expressed in dNPCs. Their expression was markedly reduced in the presence of GINIP
+ neurons (
Fig. 5B–
D). Collectively, these findings indicate that TAFA4 sustains anabolic activity while simultaneously suppressing catabolic degradation and hypertrophy, thereby maintaining ECM homeostasis in degenerative IVD.
Encouraged by these
in vitro results, we proceeded to assess the impact of TAFA4 knockdown in an animal model of IVDD. Magnetic resonance imaging (MRI) examinations were performed at both 2 and 8 weeks. At 2 weeks, the IVDD and IVDD+ shTAFA4 groups exhibited comparable disc degeneration. However, by 8 weeks, the IVDD+shTAFA4 group exhibited marked degeneration, as evidenced by a further decrease in T2-weighted MRI signal intensity compared to the IVDD group (
Fig. 6A). Histological analyses with hematoxylin and eosin (H&E) and safranin O staining at 8 weeks showed greater structural disruption in the IVDD+shTAFA4 group compared to the IVDD group, particularly with a more disorganized NP and annulus fibrosus interface (
Fig. 6B). Quantitative histological scoring confirmed a significant increase in degeneration severity following TAFA4 knockdown (
Fig. 6C). Immunohistochemical analysis revealed elevated TAFA4 expression in the IVDD group compared to control, while COL II and ACAN were markedly reduced following TAFA4 knockdown, accompanied by a similar reduction in SOX9 expression (
Fig. 6D), which helps maintaining the function of NPCs [
28]. Mean optical density analysis supported these findings (
Fig. 6E–
H). These results highlight the role of TAFA4 in maintaining disc integrity and matrix composition during IVDD.
DISCUSSION
This study identified 2 key findings: (1) GINIP+ neurons were found to infiltrate degenerative IVDs and secrete TAFA4, providing the evidence of a neuroimmune signaling axis during degeneration; and (2) TAFA4 reduces intracellular ROS levels and suppresses the NLRP3 inflammasome activation. These results highlight TAFA4 as a novel neurogenic regulator with therapeutic potential in IVDD.
IVDD is a complex pathological process involving ECM degradation, inflammation, neurovascular ingrowth, and oxidative stress [
1,
7,
29]. Extensive work has clarified mechanical and inflammatory factors. Recently, increasing attention has turned to the significance of neuroimmune interactions in the advancement of IVDD. Previous studies have recognized neuropeptides, including substance P, calcitonin gene-related peptide, vasoactive intestinal peptide, and neuropeptide Y, as important mediators of IVDD and discogenic LBP by influencing inflammatory signaling and matrix catabolism [
30,
31]. These neurogenic factors have predominantly been associated with exacerbating degeneration [
6,
30], while evidence supporting their potential protective roles remains limited. Recent studies have suggested that a subset of nerves innervating the IVD may delay degeneration [
32,
33], but the underlying mechanisms and mediators remain largely undefined. Notably, TAFA4, a chemokine like protein secreted by GINIP
+ neurons, has recently emerged as a novel neuroimmune modulator in peripheral tissues [
15,
17], yet its role within the degenerative IVD had not previously been addressed.
Our data provide the first evidence connecting TAFA4 to IVD. Immunofluorescence and FISH analyses revealed that TAFA4 expression was elevated in degenerative discs and spatially correlated with GINIP
+ neurons. Western blot and RT-qPCR further confirmed increased TAFA4 protein and mRNA expression. Macrophages have been shown to exist as immune cells in IVD and play a central role in the progression of IVDD [
9,
10,
34]. Subsequently, we assessed the modulatory role of TAFA4 in macrophage activation and polarization.
In vivo knockdown of TAFA4 via intradiscal lentiviral injection resulted in an increased proportion of M1 macrophages and a reduction in M2 macrophages within degenerative discs. In addition, elevated ROS levels observed following TAFA4 knockdown further imply that TAFA4 may act to restrain oxidative stress, which is a known activator of NLRP3 inflammasome signaling and contributes to disc degeneration.
To explore these mechanisms, we constructed a Transwell coculture system. GINIP
+ neurons stimulated IL-10 production and suppressed TNF-α and IL-1β expression in macrophages by the secretion of TAFA4. These inflammatory factors perform essential functions during the degeneration process; IL-10 exerts anti-inflammatory effect [
35], whereas TNF-α and IL-1β contribute to catabolism [
36]. Flow cytometry further confirmed that TAFA4 promoted macrophage M2 polarization highlighting its function as a crucial neuroimmune modulator within the disc.
Furthermore, coculture with GINIP
+ neurons led to a reduction of intracellular ROS levels in dNPCs, an effect that was diminished when TAFA4 was knocked down. Since oxidative stress is known to activate the NLRP3 inflammasome pathway [
37,
38], and this combined involvement is associated with the advancement of IVDD [
13,
23,
26]. We further examined related components and found that NLRP3, ASC, Caspase-1, and GSDMD expression were markedly downregulated in dNPCs cocultured with GINIP
+ neurons, while TAFA4 knockdown in GINIP
+ neurons reduced this effect. These findings indicate that TAFA4 exerts anti-inflammatory effects by modulating macrophage polarization and inhibiting inflammasome activation induced by oxidative stress.
Although our study reveals the immunomodulatory and antioxidative roles of TAFA4 in IVDD, the downstream receptor pathways remain incompletely defined. Previous work in peripheral tissues has implicated formyl peptide receptor 1 (FPR1) and atypical chemokine receptors as potential candidates mediating TAFA4’s effects on macrophages and other immune cells [
17]. In addition, TAFA4 has been suggested to engage Gi-protein–coupled signaling [
39]. While such mechanism has not yet been directly validated in the IVD, incorporating these insights provides a plausible framework to explain how TAFA4 might orchestrate neuroimmune modulation. Future investigations should focus on identifying the specific receptors and signaling cascades through which TAFA4 acts in disc cells and macrophages, which will be essential for the rational development of TAFA4-based therapeutic interventions.
Beyond immunomodulation, TAFA4 also influenced the phenotype of NPCs. GINIP
+ neuron-derived TAFA4 restored the expression of matrix-associated markers COL II, ACAN and SOX9 in dNPCs [
27], while simultaneously suppressing the expression of catabolic enzymes MMP13 and ADAMTS5 and hypertrophic markers COL X and RUNX2. TAFA4 knockdown impaired these protective effects, highlighting its potential to restore matrix homeostasis and prevent aberrant differentiation under degenerative stress.
Finally,
in vivo validation using a rabbit IVDD model further confirmed the protective role of TAFA4. At early-stage degeneration (2 weeks), MRI evaluation revealed no significant difference in disc degeneration between the IVDD and IVDD+ shTAFA4 groups. By 8 weeks, however, TAFA4 knockdown resulted in more severe disc degeneration, as demonstrated by reduced T2 signal intensity on MRI and greater structural disruption in H&E and safranin O staining. Immunohistochemistry revealed further reductions in COL II, ACAN, and SOX9 in the IVDD+shTAFA4 group, supporting the conclusion that TAFA4 contributes to matrix integrity and the maintenance of cellular phenotype [
27,
28].
Collectively, our results suggest that TAFA4 acts as a key neuroimmune mediator in IVDD. By facilitating M2 polarization, inhibiting ROS-NLRP3 inflammasome signaling, and restoring ECM of IVD, TAFA4 mitigates the inflammatory and degenerative cascade within the disc. These findings support a novel paradigm in which nociceptive neurons contribute to tissue protection through targeted immune modulation, adding complexity to the traditional view of nerve ingrowth as mainly pathogenic.
This study has some limitations. First, although the rabbit IVDD model offers valuable mechanistic insights, anatomical and immunological differences from humans—including potential variations in TAFA4 expression across different cell types, differences in receptor distribution such as FPR1 or other candidate binding partners, and distinct macrophage polarization profiles between species—could alter ligand-receptor interactions and downstream signaling, thereby limiting direct translational applicability. Second, the molecular targets downstream of TAFA4 signaling remain incompletely characterized. Third, behavioral assessments such as pain- or gait-related analyses were not performed; although MRI, histology, and molecular assays confirmed disc degeneration, functional evaluation would have provided additional validation of the model.
Moreover, the present study focused primarily on one midterm time point; the temporal expression pattern of TAFA4 and its dynamic regulatory changes during IVDD progression were not fully explored. Future studies will perform detailed time-course analyses to elucidate how TAFA4 expression and function evolve across early, active, and late stages of degeneration. Finally, future research should delineate TAFA4 receptors and downstream effectors and evaluate whether these mechanisms can be reproduced in human-derived disc cell cultures or ex vivo patient tissues, to bridge preclinical and translational gaps.
CONCLUSION
This study identifies TAFA4 as a sensory neuron-derived factor that mediates neuroimmune communication during the IVDD process. By modulating macrophage polarization, suppressing oxidative stress and inflammasome signaling, and preserving the anabolic phenotype of NPCs while restraining aberrant differentiation and catabolic activity, TAFA4 delays IVDD. These findings not only deepen our understanding of neurogenic regulation in disc pathology but also suggest TAFA4 as a potential therapeutic target for IVDD intervention.
NOTES
-
Conflict of Interest
The authors have nothing to disclose.
-
Funding/Support
This is due to the financial support of the National Natural Science Foundation of China (Grant no. 82374566) and the National Natural Science Foundation of China (Grant no. 82274637). The authors express their gratitude for this financial support.
-
Author Contribution
Conceptualization: JHan, JHuang, ZD, YZ, YD; Data curation: JHan, ZD, ZL; Formal analysis: ZD, GY, JM; Funding acquisition: YZ, YD; Methodology: JHan, JHuang, YZ; Project administration: QJ; Visualization: JHan, JHuang; Writing – original draft: JHan, JHuang, ZD, QJ, YZ, YD; Writing – review & editing: JHan, YZ, YD.
Supplementary materials
Fig. 1.GINIP+ sensory neurons infiltrate the degenerative IVD and secrete TAFA4. (A and B) Western blot analysis of TAFA4 protein levels. (C) Relative mRNA expression of TAFA4. (D-G) Immunofluorescence staining of GINIP and TAFA4 and corresponding quantification of fluorescence intensity. Scale bar 100 μm. (H) FISH images showing colocalization of GINIP+ neurons and TAFA4 mRNA. Data are shown as mean±standard deviation *p<0.05. GINIP, Gαi-interacting protein; IVDD, intervertebral disc degeneration; TAFA4, TAFA chemokine like family member 4; FISH, fluorescence in situ hybridization; DAPI, 4´,6-diamidino- 2-phenylindole.
Fig. 2.TAFA4 knockdown promotes M1 macrophage polarization and enhances ROS accumulation within the degenerative intervertebral disc. (A) Schematic of in vivo experimental design. (B–G) Immunofluorescence staining of ROS, CCR7, and CD206 and quantification of fluorescence intensity. Scale bar 100 μm. Data are shown as mean±standard deviation. *p<0.05. **p<0.01. ***p<0.001. ****p<0.0001. ns, no significance; TAFA4, TAFA chemokine like family member 4; shTAFA4, short hairpin TAFA4; ROS, reactive oxygen species; CCR7, C-C chemokine receptor type 7; IVDD, intervertebral disc degeneration; DAPI, 4´,6-diamidino- 2-phenylindole.
Fig. 3.TAFA4 regulates macrophage polarization and suppresses proinflammatory cytokine secretion in vitro. (A) Schematic of the coculture system of dNPCs, macrophages, and GINIP+ sensory neurons. (B–E) Enzyme-linked immunosorbent assay results for TAFA4, IL-10, TNF-α, and IL-1β secretion under different coculture conditions. (F) Ratio of M1 (CCR7+CD206–) to M2 (CCR7–CD206+) macrophages. (G) Flow cytometric analysis of the CCR7 and CD206 positive macrophages. Data are shown as mean±standard deviation. *p<0.05. **p<0.01. ***p<0.001. ****p<0.0001. ns, no significance; TAFA4, TAFA chemokine like family member 4; dNPC, degenerative nucleus pulposus cell; GINIP, Gαi-interacting protein; IL, interleukin; TNF, tumor necrosis factor; CCR7, C-C chemokine receptor type 7. Fig. 3A created with BioRender.com.
Fig. 4.TAFA4 inhibits ROS-dependent NLRP3 inflammasome signaling in vitro. (A) Fluorescent probe staining results show ROS levels in dNPCs. (B and C) Western blot analysis of NLRP3, ASC, Caspase-1, and GSDMD-N. (D) Relative mRNA expression of NLRP3, ASC, Caspase-1, and GSDMD. Data are shown as mean±standard deviation. *p<0.05. **p<0.01. ***p<0.001. ****p<0.0001. TAFA4, TAFA chemokine like family member 4; ROS, reactive oxygen species; dNPC, degenerative nucleus pulposus cell; NLRP3, NOD-like receptor family pyrin domain-containing 3; ASC, apoptosis-associated speck-like protein; GSDMDN, Gasdermin D N-terminal fragment.
Fig. 5.TAFA4 restores dNPC phenotype in vitro. (A and B) Representative Western blot images of COL II, ACAN, SOX9, MMP13, ADAMTS5, COL X, and RUNX2. (C) Quantification of COL X and RUNX2 protein expression. (D) Relative mRNA expression of COL X and RUNX2. (E) Quantification of COL II, ACAN, SOX9, MMP13, and ADAMTS5 protein expression. (F) Relative mRNA expression of COL II, ACAN, SOX9, MMP13, and ADAMTS5. TAFA4, TAFA chemokine like family member 4; COL II, type II collagen; ACAN, aggrecan; SOX9, SRY-box transcription factor 9; MMP13, matrix metalloproteinase 13; ADAMTS5, a disintegrin and metalloproteinase with thrombospondin motifs 5; COL X, collagen type X alpha 1 chain; RUNX2, Runt-related transcription factor 2.
Fig. 6.TAFA4 knockdown accelerates disc degeneration and reduces matrix protein expression in vivo. (A) MRI images of rabbit intervertebral discs (IVDs). Red arrows: puncture sites. (B) Safranin O (S.O.)/fast green and hematoxylin and eosin (H&E) staining of IVD sections. Scale bar: 3 mm. (C) Histological scores. (D–H) Immunohistochemical staining and quantification of TAFA4, COL II, ACAN, and SOX9. Scale bar: 400 μm. Data are shown as mean±standard deviation. *p<0.05. **p<0.01. ***p<0.001. ****p<0.0001. ns, no significance; TAFA4, TAFA chemokine like family member 4; shTAFA4, short hairpin TAFA4; COL II, type II collagen; ACAN, aggrecan; SOX9, SRY-box transcription factor 9.
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, Jie Huang3
