C/EBPβ Contributes to Cancer-Induced Bone Pain by Inhibiting CD200/CD200R1 in the Spinal Cord.

1
Introduction
Advanced cancer patients often suffer excruciating pain due to bone metastases, causing bone destruction and metastatic bone pain [1]. Non‐steroidal and opioid drugs commonly used in clinics only partially alleviate cancer‐induced bone pain (CIBP) while exhibiting unignorable side effects [2]. Therefore, it is urgent to explore the potential mechanism of CIBP and find new therapeutic targets.
Neuroinflammation refers to a pro‐inflammatory state in the central nervous system (CNS), characterized by glial cell activation and the generation of multiple pro‐inflammatory factors [3, 4]. It has been testified that spinal microglia‐mediated neuroinflammation is critical in the progression of CIBP [5]. Under peripheral nociceptive stimulation, neurons release multiple biomolecules to activate microglia surface receptors and promote microglia proliferation in the spinal cord. Activated microglia further release pro‐inflammatory cytokines to interact with neurons, increasing neuronal hyperexcitability and central sensitization [6, 7]. Thus, neuronal‐microglia interactions play an important role in regulating spinal neuroinflammation. CD200, a transmembrane glycoprotein in the CNS. Its receptor, CD200 receptor 1 (CD200R1), is mainly expressed on microglia in the CNS [8]. Normally, CD200 interacts with CD200R1 to maintain the resting state of microglia [9]. When signal transduction between CD200 and CD200R1 is disrupted, microglia are increasingly activated and release pro‐inflammatory cytokines such as IL‐1β, TNF‐α, and IL‐6, leading to neuroinflammation of the CNS [10]. It has been reported that blocking the spinal CD200R1 promoted microglia activation toward a pro‐inflammatory type after spinal cord injury [11]. Moreover, intrathecal injection of CD200 fusion protein (CD200Fc), a CD200R1 agonist, could significantly alleviate chronic constriction injury (CCI)‐induced mechanical and thermal hyperalgesia [12]. However, the role of the CD200/CD200R1 signaling pathway in the progression of CIBP remains unclear.
Transcription factor CCAAT/enhancer binding protein β (c/EBPβ) regulates the expression of pro‐inflammatory genes by binding to promoters or enhancers, which is essential for microglia activation [13]. Recent studies have reported that the upregulated c/EBPβ promoted spinal neuroinflammation in amyotrophic lateral sclerosis [14]. In the spinal cord of postsurgical pain mice, the protein level of c/EBPβ was significantly elevated, accompanied by microglia activation and pro‐inflammatory cytokines generation. Intrathecal injection of a small interfering RNA (siRNA) of c/EBPβ significantly alleviates operative stress‐induced mechanical allodynia [15]. Moreover, c/EBPβ binds to the promoter of CD200R1 to negatively regulate the protein expression. The protein level of CD200R1 in primary microglia was significantly reduced after lipopolysaccharide (LPS) intervention, which was reversed by c/EBPβ knockdown. Besides, overexpression of c/EBPβ reduced the basic level of CD200R1 in microglia [16]. However, the underlying mechanism of spinal c/EBPβ in the pathological process of CIBP is still unclear. Based on previous research, we explored whether c/EBPβ negatively regulates CD200R1 to induce neuroinflammation in the spinal cord and finally leads to CIBP.

2
Materials and Methods
2.1
Animals
6–8 weeks male C57BL/6J mice were chosen in this study. All mice were purchased from Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. The rearing environment was maintained at 23°C ± 2°C, relative humidity 50% ± 10%, and a 12 h light to dark cycle. Two to five mice were housed in each cage with free access to food and water. All experimental procedures strictly complied with the ARRIVE Guidelines for Reporting Animal Research [17] and were approved by the Experimental Animal Care and Use Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (No. TJH‐202307034).

2.2
Cell Cultures
The cell culture method was based on published articles [18]. Mouse Lewis lung cancer (LLC) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) high glucose medium (Servicebio) containing 10% fetal bovine serum (Servicebio) and 1% penicillin and streptomycin (Servicebio) and placed in the 37°C incubator with 5% CO2. Cells were lightly digested with 0.25% trypsin and diluted in PBS with a final concentration of 2 × 105 cells/μL. The cell suspension was kept on ice for subsequent injection.

2.3
CIBP Model
The specific procedure of the CIBP model construction was consistent with the published research [19]. Mice were anesthetized with 3% isoflurane and placed in the supine position. After shaving and disinfecting with 10% indophor, a superficial incision (0.5–1 cm) was performed near the left knee. The patellar ligament was exposed and a blunt separation was made to further expose the condyles of the distal femur. A 25‐gauge needle was inserted into the femoral cavity at the left femoral intercondylar notch and then replaced with a 10 μL microinjection syringe containing a 5 μL LLC cells suspension. The syringe contents were slowly injected into the medullary cavity and left for 2 min to prevent tumor cell leakage. The injection hole was filled with sterile bone wax after the syringe was removed and the skin incisions were sutured with 4–0 silk thread. Mice in the sham group were injected with 5 μL PBS solution. All mice were placed in a warm blanket until full recovery.

2.4
Behavioral Tests
The behavioral tests were evaluated following the previous reports. von Frey filaments (Stoelting, USA) were used to assess the mechanical allodynia [18] and the hot plate (Ugo Basile, Italy) was used to assess thermal hyperalgesia [20]. All the behavioral tests were started blindly at 9:00 AM.
Mice were placed into individual plastic boxes (8 cm × 8 cm × 15 cm) on a metal mesh rack for 30 min to habituate the testing environment before mechanical allodynia detection. von Frey filaments (0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1, and 1.4 g) were pressed against the middle of the hind paw and maintained for 3–5 s. The stimuli gradually increased to identify the positive response. Mice paw lifting, rapid paw retraction, or licking were considered positive reactions. Each hind paw was tested three times at intervals of 5 min. The average of the measured values was considered the paw withdrawal threshold (PWT).
Mice were also placed in the apparatus with a plastic cylindrical box (approximately 20 cm in diameter) for 30 min before thermal hyperalgesia measurement. The hot plate device consists of a plastic cylinder on a heated metal plate maintained at 55°C ± 1°C. Mice were placed individually inside the cylinder, and the thermal withdrawal latency (TWL) was recorded by a stopwatch when paw flinching, shaking, or jumping was observed. The experimental time was limited to 30 s to avoid hind paw damage.

2.5
Intrathecal Injection
The procedure of intrathecal injection was conducted as described previously [21]. Mice were placed in a prone position while conscious, and their hip bones were gently but firmly stabilized with the non‐dominant hand. A 10 μL Hamilton syringe was inserted into the intervertebral space after determining the site of the lumbar 4/5 level. The tail reflexive flick indicated the syringe had entered the subarachnoid space. The volume of each intrathecal injection was 10 μL for 30 s. After checking the locomotor activity, all mice were returned to their home cages.

2.6
Drug Administration
CD200Fc (R&D Systems, USA), a CD200R1 agonist, was dissolved in sterile saline. To investigate the role of spinal CD200R1 in the progression of CIBP, CD200Fc (1, 5, 10 μg, 10 μL) or vehicle was intrathecally injected into CIBP mice. The drug dosage was determined based on the preliminary experimental results. To explore whether a single dose of CD200Fc could attenuate mechanical allodynia of CIBP, CD200Fc (1, 5, 10 μg, i.t.) was injected on 14 days after surgery. The PWT was assessed before injection and 1, 2, 4, 6, 8, and 12 h after injection. Since frequent thermal stimulation in mice could decrease the basal thermal threshold, TWL was not performed after the single administration of CD200Fc. Then, to assess whether repeated doses of CD200Fc could reverse established CIBP, CD200Fc (1, 5, 10 μg, i.t.) was injected once a day from 14 to 18 days. Behavioral tests were performed before and 6 h after daily administration. To identify whether repeated doses of CD200Fc could prevent the development of CIBP, CD200Fc (1, 5, 10 μg, i.t.) was given once a day from 1 to 14 days. Behavioral tests were performed before and 3, 7, 14, and 21 days after surgery.

2.7
Adeno‐Associated Viruses (AAVs) Construction
The AAV2/9 serotype was selected to construct the targeted AAVs based on previous research and the requirements for high affinity to spinal cord tissue, along with long‐term expression [22]. AAVs were manufactured by Obio Technology (Shanghai, China), and the details of the AAVs and shRNA sequences were provided in Table S1. All AAVs were diluted in PBS to a final titer of 1 × 1012 v.g./mL. Since the viruses were equipped with enhanced green fluorescent protein (EGFP) elements, immunofluorescence was used to observe green fluorescence in the spinal dorsal horn to determine the successful expression of AAVs. All AAVs were intrathecally injected into the mice's subarachnoid space as described previously [23].

2.8
Western Blotting
Total protein from spinal lumbar segments was extracted using radio‐immunoprecipitation assay (RIPA) lysis buffer containing protease inhibitors and phosphatase inhibitors (Boster, China). The concentrations of protein supernatants were measured by the BCA kits (Boster, China). Equal amounts of protein (40 μg) were separated on 10% or 12.5% SDS‐PAGE wet‐electrotransferred to 0.45 μm PVDF membranes (Millipore, USA). The membranes were placed in protein‐free rapid blocking buffer to block at room temperature (RT) for 30 min and incubated overnight at 4°C with primary antibodies. After washing with Tris‐buffered saline and Tween 20 (TBST) for 30 min, samples were incubated with corresponding secondary antibodies at RT for 1 h. Protein bands were visualized by the SuperLumia ECL Plus HRP Substrate Kit (K22030, Abbkine, USA) and imaged with an image analysis system (Bio‐Rad, ChemiDoc XRS+, USA). Image Lab software (Bio‐Rad Laboratories) was used to quantify the intensity of protein blots. The ratio of each band intensity to the internal standard (β‐actin) was used for statistical analysis. The data are presented as the ratio relative to the sham group and the blot density of the sham group was set to 1.

2.9
Immunofluorescence
Mice were anesthetized with 3% isoflurane and intracardially perfused with PBS and 4% ice‐cold paraformaldehyde (PFA). The spinal lumbar segments (L4‐L6) were collected and postfixed in 4% PFA overnight at 4°C. After dehydrating in 20% and 30% sucrose solution, the tissues were embedded in OCT medium. All spinal segments were sliced 20 μm thick on a freezing microtome (CM1900, Leica, Germany). Spinal sections were infiltrated by PBS containing 0.3% Triton X‐100 for 15 min, blocked with 5% donkey serum at RT for 1 h, and incubated with primary antibodies overnight at 4°C. To investigate the activation of microglia, the tissues were incubated with rabbit anti‐Iba1 (1:100, ab178846, Abcam, UK). To explore the cellular localization of molecules, the spinal sections were incubated in a mixture of primary antibodies (Table S2). The sections were washed with PBS and then incubated with the corresponding secondary antibodies (Table S3) at RT for 2 h. The stained sections were photographed under a fluorescence microscope (DP70, Olympus, Japan) and analyzed by Image J version 1.8 software. The mean fluorescence intensity of each mouse was measured by calculating the average fluorescence intensity of three slices. Each slice was spaced 40 μm apart to prevent duplicate calculations of the same cell. The quantitative measurement of immunoreactivity was obtained by calculating the positive immunofluorescent area of the spinal dorsal horn. The percentage of overlapped area from the total marker+ area was used to represent the degree of colocalization in the spinal dorsal horn. All fluorescence quantifications were conducted by a researcher who was unknown of the experimental groups.

2.10
Hematoxylin Eosin (HE) Staining
Under deep anesthesia with 3% isoflurane, the left femur of mice was removed from the left hind limb and fixed in 4% PFA at 4°C for 48 h. The fixed femur was placed into EDTA decalcification solution until the tissue softened. After washing in running water, the femur tissue was immersed in ethanol (80%, 90%, 95%, and 100%) for dehydration. Then the tissue was embedded with wax mixture and sliced into 4 μm thick sections. The sections were baked, dewaxed, and placed into ethanol (100%, 95%, 85%, and 75%). After washing with ultrapure water, the tissues were placed in hematoxylin dye solution for 4 min and then in eosin dye solution for 30 s. Finally, the femur sections were observed and captured by a microscope (DP70, Olympus, Japan).

2.11
Statistical Analysis
All statistical data were analyzed by GraphPad Prism version 8.0 and the normality of the data distribution was assessed using the Shapiro–Wilk test. Normally distributed data (p > 0.05) are expressed as mean ± SEM and analyzed with parametric tests. Behavioral data were analyzed by two‐way analysis of variance (ANOVA) followed by a Bonferroni post hoc test. Western blotting and immunofluorescence data were evaluated by one‐way ANOVA followed by Bonferroni post hoc test (group ≥ 3) or unpaired Student's t‐test (group < 3). Non‐normally distributed data (p ≤ 0.05) are presented as median with interquartile range and analyzed using the Mann–Whitney test. p < 0.05 indicated the differences between groups were statistically significant.

3
Results
3.1
Reduced CD200/CD200R1 Signaling Transduction in the Spinal Cord of CIBP Mice
In this study, the CIBP model was constructed by transplanting LLC cells into the femoral marrow. PWT and TWL were evaluated at baseline and 3 days, 7 days, 14 days, and 21 days after surgery. As shown in Figure 1A, the baseline PWT and TWL between the sham and CIBP groups were similar. However, on the 7 days, 14 days, and 21 days after surgery, the PWT and TWL of CIBP mice were significantly decreased compared with the sham group. These findings confirmed that TCI induced significant mechanical allodynia and thermal hyperalgesia in mice. Then, to further investigate the femoral destruction caused by tumor cell implantation (TCI), femurs from sham and CIBP mice were harvested on 21 days after surgery for HE staining. In the sham mice, the microstructure showed a normal structure, with a complete trabecular structure and uniform distribution of bone marrow cells. In contrast, severe destruction was observed in CIBP femoral marrow; specifically, the bone marrow cells were replaced by tumor cells and the trabecular structure disappeared (Figure 1B). Thus, the HE staining indicated that TCI induced remarkable bone destruction. Collectively, the CIBP model was successfully established.
Then we detected the protein expression and cellular localization of CD200 and CD200R1 in the spinal cord of CIBP mice. As shown in Figure 1C–E, compared with the sham group, the protein levels of spinal CD200 and CD200R1 were markedly decreased on 14 and 21 days after TCI. Moreover, dual‐label immunofluorescence analysis at 14 days post‐operation further indicated that in the spinal dorsal horn of sham and CIBP mice, CD200 was mainly co‐labeled with NeuN (a marker of neurons) and CD200R1 was mainly co‐labeled with Iba‐1 (a marker of microglia) (Figure 1F–I). These results confirm that in the spinal cord of CIBP mice, both CD200 and CD200R1 are down‐regulated, with CD200 predominantly expressed in neurons and CD200R1 primarily localized to microglia.

3.2
CD200Fc, A CD200R1 Agonist, Attenuated the Pain Behaviors of CIBP Mice
To validate the role of spinal CD200R1 in CIBP mice, a CD200R1 agonist CD200Fc was given intrathecally (i.t.) to sham and CIBP mice. Firstly, a single dose of CD200Fc (1, 5, 10 μg, i.t.) or vehicle was injected on 14 days after TCI. PWT was tested at 1, 2, 3, 6, 8, and 12 h after CD200Fc treatment. To avoid possible thermal injury induced by frequent heat exposure, TWL assessment was not performed in this study. As shown in Figure S1A, there was no statistical difference in PWT between the sham + vehicle group and the sham + CD200Fc 10 μg group, suggesting CD200Fc did not affect the PWT of mice. Besides, compared with the CIBP + vehicle group, 10 μg CD200Fc had a significant analgesic effect at 4 h after administration, reaching a peak at 6 h and continuing until 8 h, while 5 μg only took effect at 6 h after administration. No analgesic effect was observed in 1 μg CD200Fc. Then, to explore the effect of repetitive doses of CD200Fc on CIBP mice, CD200Fc (1, 5, 10 μg, i.t.) was injected once a day from 14 to 18 days after TCI. PWT and TWL were evaluated before and 6 h after treatment. As shown in Figure S1B,C, both 5 μg and 10 μg CD200Fc remarkably elevated the PWT and TWL of CIBP mice for 5 consecutive days. However, 1 μg CD200Fc did not affect CIBP mice. These results testified that CD200Fc could significantly reverse the nociceptive behavior of established CIBP mice.
Moreover, to further examine the preventive effect of CD200Fc in the pathological process of CIBP, CD200Fc (1, 5, 10 μg) was given once daily from 1 to 14 days after TCI. PWT and TWL were assessed before and 3, 7, 14, and 21 days after TCI. As shown in Figure S1D,E, only 10 μg CD200Fc inhibited the development of mechanical allodynia in CIBP mice on 7 and 14 days, and thermal hyperalgesia on 14 days after TCI. These data indicated that CD200Fc could delay the progression of mechanical allodynia and thermal hyperalgesia in CIBP mice.

3.3
Overexpression of CD200R1 Suppressed Nociceptive Behaviors in CIBP Mice
The aforementioned results confirmed that activating CD200R1 could alleviate pain behaviors in CIBP mice. To further investigate the role of CD200R1 in the progression of CIBP, we constructed AAVs to overexpress CD200R1 (AAV‐CD200R1) in the spinal cord. The details of AAV‐CD200R1 and the control virus (AAV‐Vector) are shown in Figure 2A and Table S1. The AAVs were intrathecally injected into mice and CIBP surgery was performed 14 days after AAVs injection. Both PWT and TWL were assessed before AAV injection, before and 3, 7, 14, and 21 days after surgery. The spinal lumbar segments were collected at 21 days after surgery for western blotting and immunofluorescence analysis (Figure 2B).
The EGFP fluorescence was detected on 21 days after TCI (35 days post‐AAVs injection), corresponding to 21 days after TCI. Immunofluorescence data showed that the spinal dorsal horn had remarkable green fluorescence, confirming that the AAVs were successfully expressed in mice (Figure 2C). Behavioral results showed that compared with the CIBP + AAV‐Vector group, the PWT and TWL of the CIBP + AAV‐CD200R1 group were significantly elevated at 7, 14, and 21 days after CIBP construction (Figure 2D). Besides, no statistical difference was observed between the Sham + AAV‐Vector group and the Sham + AAV‐CD200R1 group, suggesting AAV injection did not affect the pain threshold of mice. These data verified that CD200R1 overexpression effectively suppressed the mechanical allodynia and thermal hyperalgesia in CIBP mice.

3.4
Overexpression of CD200R1 Restored Spinal Neuroinflammation in CIBP Mice
To further determine the effect of overexpressing CD200R1 on spinal neuroinflammation in CIBP mice, the protein level of microglia marker Iba‐1 and pro‐inflammatory cytokines (IL‐1β, TNF‐α, IL‐6) in the spinal cord of CIBP mice were detected by western blotting. Immunofluorescence staining was used to investigate microglia activation. As shown in Figure 2E,F, AAV‐CD200R1 notably restored the expression of CD200R1 in the spinal cord of CIBP mice. Besides, the spinal protein levels of Iba‐1, IL‐1β, TNF‐α, and IL‐6 were largely increased after CIBP, which was reduced by AAV‐CD200R1. Immunofluorescence analysis showed that compared with the Sham + AAV‐Vector group, the microglia were significantly activated in the spinal dorsal horn of the CIBP + AAV‐Vector group, while the microglia activation was inhibited by AAV‐CD200R1 (Figure 2G,H). These data suggest that the CD200R1 overexpression effectively restored spinal neuroinflammation in CIBP mice.

3.5
Knockdown of CD200R1 Induced Nociceptive Behaviors in Naïve Mice
The aforementioned results confirmed that overexpression of CD200R1 significantly alleviated pain perception and neuroinflammation in the spinal cord of CIBP mice. To further determine the role of CD200R1 in the progression of pain behaviors, we constructed AAVs to knock down CD200R1 (AAV‐sh.CD200R1) in the spinal cord. The structure of AAV‐sh.CD200R1 and the control virus (AAV‐sh.NC) are shown in Figure 3A. Behavioral tests were conducted before and 7, 14, 21, and 28 days after AAVs injection. Spinal segments were obtained at 28 days after AAVs injection for western blotting and immunofluorescence analysis (Figure 3B).
Immunofluorescence data showed that the fluorescence of EGFP was observed in the spinal dorsal horn, suggesting the AAVs were effectively expressed in the spinal cord of mice (Figure 3C). As shown in Figure 3D, compared with the control group, AAV‐sh.CD200R1 significantly reduced the PWT and TWL of naïve mice. These behavioral data suggested that knocking down CD200R1 in the spinal cord induces significant mechanical allodynia and thermal hyperalgesia in naïve mice.

3.6
Knockdown of CD200R1 Induced Spinal Neuroinflammation in Naïve Mice
Based on the results that AAV‐sh.CD200R1 induced pain behavior in naïve mice, we further investigated whether knocking down CD200R1 could induce microglia‐mediated neuroinflammation in the spinal cord. As shown in Figure 3E,F, compared with the control group, AAV‐sh.CD200R1 notably decreased the protein level of CD200R1. Moreover, the spinal expression of Iba‐1, IL‐1β, TNF‐α, and IL‐6 was significantly upregulated after AAV‐sh.CD200R1 injection. Immunofluorescence analysis indicated that AAV‐sh.CD200R1 promoted the activation of microglia in the spinal dorsal horn in naïve mice (Figure 3G,H). These results testify that knocking down CD200R1 induced remarkable spinal neuroinflammation in the naïve mice.

3.7
Expression and Cellular Localization of c/EBPβ in the Spinal Cord of CIBP Mice
Previous studies have identified that transcription factor c/EBPβ regulates the activation of microglia and various pro‐inflammatory cytokines, which is a critical mechanism of chronic pain. To investigate the effect of c/EBPβ in CIBP, we determined the protein expression and cellular localization of c/EBPβ in the spinal cord of CIBP mice. As shown in Figure 4A,B, compared with the sham group, the spinal expression of c/EBPβ was significantly elevated on 14 days and sustained on 21 days after TCI. Immunofluorescence analysis of 14 days after surgery showed that the spinal c/EBPβ of CIBP mice was mostly co‐localized with microglia, some with neurons and few with astrocytes (Figure 4C,D). The above finding indicates that the c/EBPβ is upregulated and mainly expressed on microglia in the spinal cord of CIBP mice.

3.8
Knockdown of c/EBPβ Reversed Nociceptive Behaviors in CIBP Mice
To examine the effect of spinal c/EBPβ in CIBP mice, the AAVs were constructed to knock down the expression of c/EBPβ (AAV‐sh.c/EBPβ, Figure 4E). The AAVs were intrathecally injected into mice and CIBP surgery was performed 14 days after AAVs injection. Behavioral tests were performed before AAVs injection, before and on day 3 after 3, 7, 14, and 21 days after TCI. The lumbar segments were removed at 21 days after TCI for western blotting and immunofluorescence analysis (Figure 4F).
Significant green fluorescence was observed in the spinal dorsal horn of CIBP mice at 21 days post‐operation (35 days after AAVs injection), confirming the successful expression of AAVs (Figure 4G). Behavioral results showed that compared with the CIBP + AAV‐sh.NC group, intrathecal injection of AAV‐sh.c/EBPβ effectively reversed the decreased PWT and TWL of CIBP mice (Figure 4H). Moreover, there was no statistical difference between the Sham + AAV‐sh.NC group and Sham + AAV‐sh.c/EBPβ group, which demonstrated that the injection of AAVs did not affect the pain threshold of mice. These results suggest that knocking down c/EBPβ could inhibit the development of nociceptive behaviors in CIBP mice.

3.9
Knockdown of c/EBPβ Upregulated CD200R1 and Suppressed the Neuroinflammation in the Spinal Cord of CIBP Mice
To determine the effect of knocking down c/EBPβ on the protein level of CD200R1 in the spinal cord of CIBP mice, we detected the spinal expression of c/EBPβ and CD200R1 in the AAV‐sh.c/EBPβ group and the control group. Western blotting analysis indicated that the spinal protein level of c/EBPβ was markedly reduced after AAV‐sh.c/EBPβ treatment compared with the sham + AAV‐sh.NC group (Figure 5A,B), suggesting that AAV‐sh.c/EBPβ effectively inhibited the expression of c/EBPβ in the spinal cord. Besides, AAV‐sh.c/EBPβ significantly elevated the decreased level of CD200R1 in the spinal cord of CIBP mice (Figure 5A,B). These data show that knocking down c/EBPβ promotes the spinal protein expression of CD200R1 in CIBP mice.
To further investigate whether knocking down c/EBPβ affected the spinal neuroinflammation of CIBP mice, pro‐inflammatory cytokines were evaluated by western blotting and the activation of microglia was assessed by immunofluorescence. As shown in Figure 5A,B, the expression of Iba‐1, IL‐1β, TNF‐α, and IL‐6 were significantly increased in the CIBP + AAV‐sh.NC group, which was inhibited by AAV‐sh.c/EBPβ. The immunofluorescence analysis also confirmed that AAV‐sh.c/EBPβ obviously suppressed the activation of microglia in the spinal dorsal horn of CIBP mice (Figure 5C,D). Above analysis verifies that knocking down c/EBPβ remarkably inhibited the spinal neuroinflammation in CIBP mice.

3.10
Overexpression of c/EBPβ Promotes the Development of Nociceptive Behaviors in Naïve Mice
The aforementioned results demonstrated that silencing c/EBPβ not only attenuates pain behaviors but also hinders microglia‐mediated neuroinflammation in the spinal cord of CIBP mice. To explore the contribution of proteins to pain progression, the AAVs to overexpress c/EBPβ (AAV‐c/EBPβ) were constructed and the specific structures were displayed in Figure 6A. The PWT and TWL were measured before and 7, 14, 21, and 28 days after AAVs injection. The lumbar tissues were collected at 28 days after AAVs injection (Figure 6B).
The EGFP fluorescence in the spinal dorsal horn proved effective expression of the AAVs (Figure 6C). As shown in Figure 6D, compared with the control group, intrathecal injection of AAV‐c/EBPβ statistically reduced the PWT and TWL of naïve mice. The behavioral analysis determines that overexpressing c/EBPβ induced significant mechanical allodynia and thermal hyperalgesia in naïve mice.

3.11
Overexpression of c/EBPβ Suppressed CD200R1 and Induced Neuroinflammation in the Spinal Cord of Naïve Mice
To investigate whether overexpressing c/EBPβ affects the protein level of CD200R1, western blotting was used to assess the expression of CD200R1. As shown in Figure 6E,F, AAV‐c/EBPβ significantly upregulated the expression of c/EBPβ while downregulating the expression of CD200R1 compared with the control group. Additionally, intrathecal injection of AAV‐c/EBPβ considerably elevated the protein level of Iba‐1, IL‐1β, TNF‐α, and IL‐6. Similarly, immunofluorescence staining showed that AAV‐c/EBPβ significantly activated the microglia in the spinal dorsal horn of CIBP mice (Figure 6G,H). These results indicate that overexpressing c/EBPβ suppressed the expression of CD200R1 and induced spinal neuroinflammation in naïve mice.

4
Discussion
This study indicated that (1) the deficient CD200/CD200R1 pathway in CIBP mice contributes to the progression of CIBP mice, as CD200R1 overexpression alleviates nociceptor behaviors and neuroinflammation. (2) Elevated microglial c/EBPβ in CIBP promotes pain behaviors and neuroinflammation by suppressing CD200R1, as demonstrated by the reversal of pathology following c/EBPβ knockdown and the recapitulation of CIBP‐like phenotypes upon its overexpression in naïve mice. In summary, these findings indicated that the transcription factor c/EBPβ plays a pivotal role in CIBP by downregulating CD200R1 expression in spinal microglia, thereby disrupting CD200/CD200R1 signaling transduction and promoting neuroinflammation.
CD200 is a transmembrane glycoprotein that binds to the corresponding receptor CD200R1 through the N‐terminal immunoglobulin‐like domain. Both CD200 and CD200R1 are members of the immunoglobulin superfamily. CD200 is widely distributed in various cell types throughout the body, including vascular endothelial cells, follicular dendritic cells, B cells, T cells, and neurons. CD200R1 is restrictedly expressed in myeloid cells such as macrophages, mast cells, neutrophils, and plasmacytoid dendritic cells. Notably, CD200 is predominantly expressed in neurons, while CD200R1 is mainly expressed in microglia in the central nervous system [11, 24]. Previous studies have demonstrated that CD200 binds to CD200R1 to regulate the communication between neurons and microglia. Under physiological conditions, the activated CD200/CD200R1 signaling pathways maintain the rest state of microglia. When the connection between CD200 and CD200R1 is disrupted, microglia are activated and then release various pro‐inflammatory cytokines such as IL‐1β, TNF‐α, and IL‐6 [25]. It has been confirmed that microglia‐mediated neuroinflammation is critical for the initiation and maintenance of chronic pain [6]. Hence, this experiment was designed to investigate the specific mechanism of the CD200/CD200R1 signaling pathway in the spinal cord of CIBP mice. Our experimental results show that both CD200 and CD200R1 are downregulated in the spinal cord of CIBP mice. One possible speculation about the decrease in CD200 is that neurons in the spinal cord undergo various severe injuries. Since CD200 is mainly expressed in neurons, the spinal protein level of CD200 was decreased with the aggravation of neuron damage. It has been indicated that the surrounding glia could transmit CD200 to neighboring neurons. The lack of supply from glia may be another reason for the decrease in CD200. Besides, Dentesano et al. also found that the CD200R1 was reduced after germinal matrix hemorrhage. However, few studies have seen an increase in the expression of CD200R1 after injury, which was contrary to our results [26]. Zhao et al. found the expression of CD200R1 in the brain was increased from 1 day to 7 day after middle cerebral artery occlusion [8]. Hernangómez et al. indicated that the protein level of CD200R1 was elevated after CCI [12]. The discrepancy between these results may be due to differences in the rodent model and sample sites.
To further investigate the effect of activating CD200R1, the CD200R1 agonist CD200Fc was intrathecally injected into the CIBP mice. Our data analysis showed that CD200Fc effectively reversed the mechanical allodynia and thermal hyperalgesia of CIBP mice. However, due to the activation effect of CD200Fc on all subtypes of CD200R (R1–R4), the AAV overexpressing CD200R1 was constructed to more accurately focus on the role of activating CD200R1. Similar to CD200Fc, the AAV‐CD200R1 efficiently alleviated the nociceptive behavior of CIBP mice and restored spinal neuroinflammation in CIBP mice. Meanwhile, knocking down CD200R1 in the spinal cord significantly reduced the PWT and TWL in naïve mice, increased the expression of neuroinflammatory molecules and activated microglia in the spinal cord. The currently published studies have also confirmed the protective effect of activating CD200R1. Hernangómez et al. found that intrathecal CD200Fc administration not only attenuated the mechanical and thermal hyperalgesia but also suppressed the activation of microglia and astrocytes in the spinal dorsal horn of rats with nerve injury [12]. Besides, CD200Fc decreased the mRNA levels of pro‐inflammatory factors (IL‐1β, TNF‐α, and IL‐6) and increased the mRNA levels of anti‐inflammatory factors (IL‐4 and IL‐10). Lago et al. verified that intraspinal injection of CD200 remarkably improved functional recovery, and reduced neuronal loss and microglia activation after spinal cord injury by binding with CD200R1 [11]. Additionally, the CD200‐CD200R1 signaling pathway could prevent spontaneous bacterial infections, promote the resolution of neuroinflammation, and restore neurological function after stroke (Ritzel et al. 2019). The above results and our experimental analysis demonstrate that the CD200/CD200R1 signaling pathway is protective in inhibiting CNS neuroinflammation.
It has been verified that c/EBPβ is a negative regulatory transcription factor of CD200R1 via binding to the promoter of CD200R1. The mRNA and protein levels of CD200R1 in BV2 cells co‐cultured with lipopolysaccharide (LPS) were significantly reduced in vitro research, which was not observed when knocking down c/EBPβ. Additionally, overexpression of c/EBPβ could reduce the basal expression level of CD200R1 in microglia [16]. Based on the above results, we speculate that c/EBPβ is the upstream mechanism of CD200R1 downregulation in the spinal cord of CIBP mice. c/EBPβ is a member of the transcription factor c/EBP family, which regulates the transcription of multiple pro‐inflammatory cytokines and promotes microglia activation [27]. Elevation of c/EBPβ was observed in both CCI‐induced [28] and HIV‐associated neuropathic pain [29]. Inhibiting the upregulation of c/EBPβ significantly alleviated mechanical allodynia and microglia‐related neuroinflammation in both studies. Given the obscure role of spinal c/EBPβ in CIBP, we detected the expression and cellular localization of c/EBPβ in the spinal cord of CIBP mice. Our analysis found that the spinal protein level c/EBPβ was remarkably increased and was mainly expressed in microglia in CIBP mice. Our results also confirmed that specific knockdown of c/EBPβ in the spinal cord could alleviate mechanical allodynia and thermal hyperalgesia in CIBP mice. These results are similar to the current study findings. It has been reported that the spinal c/EBPβ was elevated in neuropathic pain rats [29] and postsurgical pain rats [15]. Intrathecal injection of c/EBPβ siRNA effectively reversed the mechanical allodynia induced by nerve injury and surgical procedures. In addition, the downregulated c/EBPβ could not only restore the protein level of CD200R1 in the spinal cord of CIBP mice but also suppress the expression of pro‐inflammatory cytokines and activation of microglia. Furthermore, overexpression of c/EBPβ in the spinal cord could inhibit the spinal protein level of CD200R1 and promote the elevation of neuroinflammatory cytokines and microglia, ultimately inducing pain behavior in naïve mice. Hence, c/EBPβ regulates the protein level of CD200R1 and then affects the signaling transduction between CD200 and CD200R1, which is pivotal in inducing neuroinflammation. It has been identified that c/EBPβ binds to the consensus sequence of CD200R1 to inhibit the transcription of CD200R1 and overexpressed c/EBPβ could inhibit the basal level of CD200R1 in LPS‐treated microglia [16]. In addition, our experimental results showed an interesting temporal dissociation: while significant pain behaviors in CIBP mice emerged as early as 7 days post‐TCI, the protein levels of C/EBPβ, CD200, and CD200R1 showed marked increases only after 14 days. The observed temporal pattern suggests that c/EBPβ‐mediated regulation of CD200/CD200R1 signaling primarily contributes to the maintenance rather than initiation of chronic pain.
Our research findings prove that c/EBPβ negatively regulates CD200R1 in the process of activating microglia and inducing spinal neuroinflammation in CIBP mice. However, our study still has some limitations. Only male mice were selected based on established literature indicating potentially elevated pain sensitivity in female rodents [30, 31, 32]. The potential sexual dimorphism in the c/EBPβ‐CD200R1 regulatory relationship remains an important scientific question for future investigation. Besides, AAVs in this experiment were delivered into the spinal cord of mice through intrathecal injection, which did not target the dorsal horn and increased the infection in other parts of the spinal cord. Compared to the point‐to‐point spinal injection, the intrathecal administration is not precise enough. In addition, the AAVs constructions were equipped with general promoters to ensure stable gene expression but lacked cell‐type specificity, and we failed to further explore the co‐localization analysis between the EGFP and Iba‐1 to verify the cellular identity of transduced cells, which constitutes another constraint of our experiment. The construction and application of AAVs require further improvement in subsequent research. Of note, there are still few studies on the c/EBPβ and CD200/CD200R1 signaling pathways in chronic pain. Future investigations could focus on elucidating cell‐type‐specific mechanisms, particularly the molecular alterations on c/EBPβ‐mediated transcriptional programs in activated microglia, as well as the precise molecular mechanisms of CD200/CD200R1‐regulated neuron–microglia crosstalk. It has also been confirmed that the activated CD200/CD200R1 signaling promotes M2 polarization of microglia and reduces M1 polarization by increasing the phosphorylation of phosphatidylinositol 3‐kinase (PI3K)/protein kinase B (Akt) and inhibiting NF‐κB transcription in the hippocampus of postoperative cognitive dysfunction mice. Therefore, the downstream mechanisms linking CD200/CD200R1 signaling to microglia‐mediated neuroinflammation also need to be further investigated.
Collectively, this study provides evidence that the c/EBPβ‐mediated inhibition of the CD200/CD200R1 signaling pathway serves as a potential pathway for facilitating microglial activation and neuroinflammation in the spinal cord, which participates in the progression of CIBP. These findings identify the c/EBPβ‐CD200/CD200R1 axis as a potential therapeutic target for CIBP management.

Author Contributions

Dan‐Yang Li: methodology, investigation, writing – original draft; Lin Liu: methodology, investigation, writing – original draft; Dai‐Qiang Liu: visualization, supervision, writing – review and editing; Long‐Qing Zhang: visualization, supervision, writing – review and editing; Ya‐Qun Zhou: conceptualization, supervision, writing – review and editing; Wei Mei: conceptualization, supervision, writing – review and editing.

Funding
This work was supported by the National Natural Science Foundation of China, 82271291, 82071556, 82001198, 82101310. National Key Research and Development Program of China, National Key Research and Development Program of China, 2020YFC2005303. Wuhan Talents Excellent Youth Program, 24‐2RSC09033‐09. Key Research Project of Tongji Hospital, 2023A20, 2024A19, 2024A07.

Ethics Statement
The ethics approval statement was labeled as No. TJH‐202307034. All animal procedures were conducted in accordance with protocols approved by the Experimental Animal Care and Use Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.

Conflicts of Interest
The authors declare no conflicts of interest.

Supporting information

Figure S1: CD200Fc attenuated the pain behaviors of CIBP mice. (A) A single dose of CD200Fc (5 and 10 μg) attenuated the mechanical allodynia of CIBP mice. (B‐C) Repetitive doses of CD200Fc (5 and 10 μg) reversed the mechanical allodynia and thermal hyperalgesia of CIBP mice. (D‐E) Preventive injection of CD200Fc (10 μg) delayed the decline of PWT and TWL in CIBP mice. ***p < 0.001 vs Sham + vehicle group, #p < 0.05, ##p < 0.01, ###p < 0.001 vs CIBP + vehicle group, n = 6. The red arrow indicates the administration time.

Table S1: Construction Details of AAVs.

Table S2: Primary antibodies used for Western blotting and immunofluorescence.

Table S3: Secondary antibodies used for Western blotting and immunofluorescence.