TRPV1 alleviates osteoarthritis by inhibiting M1 macrophage polarization via Ca2+/CaMKII/Nrf2 signaling pathway

Osteoarthritis (OA) is the major course of joint deterioration, in which M1 macrophage-driven synovitis exacerbates the pathological process. However, precise therapies for M1 macrophage to decrease synovitis and attenuate OA progression have been scarcely proposed. Transient receptor potential vanilloid 1 (TRPV1) is a cation channel that has been implicated in pain perception and inflammation. In this study, we investigated the role of TRPV1 in the M1 macrophage polarization and pathogenesis of OA. We demonstrated that TRPV1 expression and M1 macrophage infiltration were simultaneously increased in both human and rat OA synovium. More than 90% of the infiltrated M1 macrophages expressed TRPV1. In the rat OA model, intra-articular injection of capsaicin (CPS), a specific TRPV1 agonist, significantly attenuated OA phenotypes, including joint swelling, synovitis, cartilage damage, and osteophyte formation. CPS treatment markedly reduced M1 macrophage infiltration in the synovium. Further mechanistic analyses showed that TRPV1-evoked Ca2+ influx promoted the phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) and facilitated the nuclear localization of nuclear factor-erythroid 2-related factor 2 (Nrf2), which ultimately resulted in the inhibition of M1 macrophage polarization. Taken together, our findings establish that TRPV1 attenuates the progression of OA by inhibiting M1 macrophage polarization in synovium via the Ca2+/CaMKII/Nrf2 signaling pathway. These results highlight the effect of targeting TRPV1 for the development of a promising therapeutic strategy for OA.


Introduction
Osteoarthritis (OA) is the most common form of joint deterioration, manifesting several symptoms that weaken the quality of life, such as pain, joint stiffness, and dysfunction 1 . OA increases socioeconomic burden due to growing prevalence and incidence 2 . However, there is no fundamental treatment of OA as its pathogenesis is not sufficiently clarified.
Synovial inflammation (synovitis) is highly correlated with the progression and severity of OA 3,4 . Recently, OA is well established as a low-grade inflammatory disease primarily mediated by the activity of innate immune cells, especially macrophages 5 . Macrophages can be generally classified as inflammatory M1 macrophages and antiinflammatory M2 macrophages 6 . Emerging evidence reveals that the M1 macrophage polarization in the synovium plays a critical role in the development of OA 7,8 . However, conditional depletion of macrophages failed to mitigate OA and instead resulted in increased inflammatory cytokines in joint synovial fluid 9 , highlighting the difficulty of targeting the highly complex immune microenvironment in OA. Therefore, a targeted approach to inhibit M1 macrophage polarization and alleviate OA is awaiting to be developed.
Transient receptor potential vanilloid 1 (TRPV1) is a nonspecific cation channel that can be activated by various thermal, mechanical, and chemical stimuli 10 . The role of TRPV1 in pain perception has been investigated in a variety of diseases [11][12][13] . Intra-articular injection of a TRPV1 agonist significantly suppresses pain in an OA model and OA patients 14,15 . Furthermore, TRPV1 activation is also involved in regulating immune cell activity, including the activation of CD4-positive T cells and the proinflammatory phenotype polarization of macrophages (M1) 16,17 . In contrast, a recent study has shown that TRPV1 modulates M1/M2 macrophage polarization and decreases the levels of M1 macrophage markers, inducible nitric oxide synthase (iNOS) and interleukin-6 (IL-6), to promote dopaminergic neurons survival in a Parkinson's disease model 18 . This apparent divergent role of TRPV1 on M1 macrophage polarization remains to be explored. Interestingly, the immunoreactivity of TRPV1 is increased in the OA synovium, with its majority localized to CD68positive macrophages 19 . However, whether TRPV1 can regulate M1 macrophage polarization and the role of TRPV1 in OA synovitis remain unknown.
In this study, we examined the human and rat OA synovium and observed a significant increase of TRPV1 expression and M1 macrophage infiltration. In a rat OA model of the radial transection of the medial meniscus, intra-articular injection of capsaicin (CPS), a specific TRPV1 agonist, significantly decreased M1 macrophage polarization in synovium and reduced synovitis, and sequentially attenuated cartilage deterioration and osteophyte formation. Further, TRPV1 inhibited M1 macrophage polarization via the Ca 2+ /calcium/calmodulindependent protein kinase II/nuclear factor-erythroid 2related factor 2 (Ca 2+ /CaMKII/Nrf2) signaling pathway. These findings suggest that TRPV1 is a novel therapeutic target reducing synovitis and alleviating OA.

Clinical specimen
The study protocol was approved by the Ethical Committee of the Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School (2009022). Normal synovial tissues were obtained from three patients (56-71 years old) without OA history who underwent lower limb amputation due to a traffic accident. OA synovial tissues were obtained from agematched six patients (59-77 years old, Kellgren-Lawrence grade 4) who suffered from OA and underwent total knee arthroplasty. All of the patients accepted informed consent before their enrollment.

Animals
All animal experiments were authorized and performed in accordance with the Animal Care and Use Committee of Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University (2019AE02010). We have complied with all relevant codes of ethics.
Adult male Sprague-Dawley (SD) rats (2 months, n = 36) were obtained from the Animal Center of the Nanjing Medical University (Jiangsu, China). The number of rats in each group was chosen based on the calculation formula reported by Charan and Kantharia 20 . After a 1-week acclimation period, 24 rats underwent surgery for the radial transection of the medial meniscus to generate an OA model 21 , while the remaining 12 rats received a sham operation. Then, the rats were randomly grouped into either the 4 or 8 weeks of the sham, OA, or OA with CPS treatment (OA + CPS) groups (n = 6, each group). Intraarticular injection of 50 μL of 50 μM CPS (#ab141000, Abcam, Cambridge, USA) was administered twice a week for 4 or 8 weeks in the OA + CPS group, while rats in the OA group were injected with 50 μL phosphate-buffered saline (PBS). During this procedure, the knee diameter of the rats in each group was monitored by an electronic vernier caliper.
Cell culture RAW264.7 cells were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Science (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin and streptomycin (Gibco) at 37°C and 5% CO 2 condition. The cells were induced for the M1 macrophage polarization with 50 ng/mL lipopolysaccharide (LPS) (#L2630, Sigma-Aldrich, St. Louis, MO, USA). To assess the role of TRPV1 in M1 macrophage polarization, 50 μM CPS was used to activate TRPV1. To study the regulation of the CaMKII phosphorylation, cells were induced by LPS with or without CPS for 5, 15, and 30 min. To evaluate the Nrf2 nuclear translocation, the cells were pretreated with 500 μM ethylenediaminetetraacetic acid (EDTA) (#1340, Biofroxx, Germany), 20 μM KN-93 (#HY-15465, MCE-MedChemExpress, New Jersey, USA), or 5 μM ML385 (#HY-100523, MCE) for 1 h, followed by the treatment of LPS and CPS for 3 h. To evaluate the expression of M1 macrophage markers, the cells were treated with LPS with or without CPS for 24 and 48 h and the messenger RNA (mRNA), and protein expression levels of M1 macrophage markers were analyzed.

Histological analysis
After rats were sacrificed, their affected knee joints were fixed in 4% paraformaldehyde (PFA) for 3 days, and the samples then underwent decalcification in 10% EDTA (#1340, Biofroxx) solution for 2 months. The decalcified tissues were then embedded in paraffin blocks and cut into 5-μm coronal slides by a microtome (Thermo, Germany). Tissue sections from each experimental group were stained by hematoxylin and eosin (H&E) (#C0105S Beyotime, Shanghai, China) and safranin O/fast green (#G1371, Solarbio, Beijing, China). The Osteoarthritis Research Society International (OARSI) scoring and synovitis scoring 22 were assessed by two individuals who were ignorant of the animals' treatment. The M1 macrophage polarization was assessed by immunofluorescence staining of the synovium.

Micro-computed tomography (micro-CT)
After fixed in 4% PFA, the microstructure of rat knee joints was analyzed using a micro-CT scanner (mCT80; Scanco Medical AG) with 80-μm voxel resolution. The reconstruction images were acquired by Scanco Medical software.

Enzyme-linked immunosorbent assay (ELISA)
The IL-6 levels in the supernatant of cultured RAW264.7 cells were detected using an IL-6 ELISA Kit according to the manufacturer's protocol (#PI326, Beyotime Biotechnology, China). The absorbance at 450 nm was measured by a microplate reader. The concentrations of IL-6 in the supernatant were normalized to the cell count.

Quantitative real-time polymerase chain reaction (qPCR)
Total RNAs were isolated using the RNA-quick Purification Kit (#RN001, ES Science, Shanghai, China) according to the manufacturer's instructions. qPCR was conducted by magnifying 20 μL of diluted complementary DNA with the SYBR Green Q-PCR Kit (#Q411-02, Vazyme, Nanjing, China) on a LightCycler 480 PCR System (Roche, Switzerland). The primer sequences of mRNAs used are shown in Supplementary Table 1.

Flow cytometry
After LPS treatment with or without CPS for 48 h, RAW264.7 cells were suspended in PBS and then stained with a mixture of fluorochrome-conjugated anti-mouse antibodies: F4/80-FITC (#123107, BioLegend, USA) and CD80-APC (#104713, BioLegend). After the cells were washed three times, they were diluted in flow buffer and run through a BD AccuriC6 Plus Flow CytoMeter (BD Biosciences, USA). Data were analyzed with the FlowJo software (version 10).

Alamar Blue assay and Cell Counting Kit-8 (CCK8) assay
The proliferation capacity of RAW264.7 cells was assessed by Alamar Blue (#A7631, Solarbio) and CCK8 (#CK04, Dojindo, Shanghai, China) assay according to the manufacturer's instructions after LPS treatment with or without CPS for 24 h. For Alamar Blue assay, the fluorescence at 545 nm upon excitation with radiation at 590 nm was measured. For CCK8 assay, the absorbance at 450 nm wavelength was detected.

5-Ethynyl-2′-deoxyuridine (EdU) assay
The EdU Reagent Kit (#C10310-1, Ribobio, Guangzhou, China) was used to evaluate cell proliferation. Briefly, RAW264.7 cells were treated with LPS with or without CPS for 24 h, followed by 10 μM EdU incubation for 2 h. The following steps were carried out according to the manufacturer's protocol, and images were obtained with a fluorescence microscope (Zeiss).

Scratch assay and Transwell migration assay
The effect of CPS on macrophage migration was assessed by in vitro Scratch assay and Transwell migration assay as previously reported 25 . Briefly, scratched wound lines were created by a 200 μL micropipette tip when the confluence of RAW264.7 cells reached 80%. Then, cells were treated by LPS with or without CPS for 48 h. For Transwell migration assay, RAW264.7 cells were seeded in the Transwell cell culture chambers with 8 μm pores, in the presence or absence of LPS or CPS. After incubation for 24 h, migrated cells were stained with crystal violet for the obtain of images.

Statistical analyses
The SPSS software (version 25.0) and GraphPad Prism software (version 8.0) were used for the statistical analysis. Quantitative data are representative of at least three independent experiments. No samples or animals were excluded from the analysis. Shapiro-Wilk method was used to estimate the normal distribution of data and Levene method was used to test the homogeneity of variance. Unpaired two-tailed Student's t test was used to compare mean values between two groups. For comparisons more than two groups, one-way analysis of variance, followed by Tukey's multiple comparison tests were used. The data were presented as mean values ± SD. P < 0.05 was considered statistically significant.

TRPV1 is highly expressed in M1 macrophage in the OA synovium
To explore whether TRPV1 can be a target to regulate macrophage polarization in OA, we first examined the expression of TRPV1 and M1 macrophage markers in the synovium of OA patients and rat OA model. Consistent with a previous study 7 , the number of CD14, CD80, and iNOS (M1 macrophage markers)-positive cells were significantly increased, and the proportion of TRPV1positive cells was significantly higher in the human OA synovium than the human normal synovium (Fig. 1A, B). Macrophage accumulation in the 4-and 8-week-old rat OA model was then assessed. Compared to the sham group, macrophage infiltration in the synovium was increased in the OA groups, with a significantly higher percentage of CD14-positive cells (Fig. 1C, D). The proportion of M1 macrophage and TRPV1-positive cells were also significantly increased in the synovium of both 4-and 8-week-old OA models (Fig. 1C, D). In addition, the increased F4/80 (macrophage marker)-positive cells were observed in OA synovium ( Supplementary Fig. 1A, C). However, the proportion of cells positive for CD163 and CD206 (M2 macrophage markers) showed no significant difference among each group ( Supplementary Fig. 1A-D). These results suggest that M1 but not M2 macrophages accumulate in OA synovium, accompanied by significantly increased TRPV1 expression.
We next examined whether TRPV1 was expressed in macrophages, particularly M1 macrophages. The colocalization of CD80 and TRPV1 in rat OA synovium was identified, indicating that TRPV1 was expressed by the highly accumulated M1 macrophages (Fig. 1E). Besides, iNOS and TRPV1 were also highly co-localized. Of note, the percentage of TRPV1 localized to CD80-and iNOS-positive cells was >90% in the synovium of both sham and OA groups, but with no statistical difference (Fig. 1E), indicating that TRPV1 may be consistently expressed by M1 macrophages. Taken together, these findings suggest that TRPV1 is expressed by M1 macrophages and its expression is increased in the OA synovium, suggesting that TRPV1 could be a potential therapeutic target to regulate M1 macrophage polarization.

TRPV1 alleviates OA by inhibiting M1 macrophage polarization
To gain insight into whether the TRPV1 activation can inhibit M1 macrophage polarization and alleviate OA in vivo, we performed intra-articular injection of a specific TRPV1 agonist, CPS, in the rat OA model. After radial transection of the medial meniscus, the knee joint diameter of the OA groups increased faster than the sham group ( Fig. 2A). However, after intra-articular injection of CPS, the diameter of rat knee joint increased much slower than the OA group, resulting in a significantly smaller knee diameter ultimately ( Fig. 2A). Besides, there was no significant difference in the rat weight among every group during the entire experiment ( Supplementary Fig. 2A).
The histological examination showed abundant cell infiltration and high levels of hyperplasia in the synovium of the 4-and 8-week-old OA groups, accompanied by significantly severe synovitis than the sham group ( Fig. 2B and Supplementary Fig. 2B). After the CPS injection, the number of infiltrated cells and synovitis scores decreased significantly, although the synovium was still hyperplastic and hypertrophic ( Fig. 2B and Supplementary Fig. 2B). Thus, intra-articular injection of a TRPV1 agonist decreased synovitis in OA rats. The percentage of macrophage in the rat synovium, particularly M1 macrophage, was significantly higher in the OA group compared to the sham group (Fig. 2C). Along with decreased synovitis scores, there was a substantial reduction in the number of CD14-, CD80-, iNOS-and TRPV1-positive cells in synovium following the CPS injection (Fig. 2C, D), indicating that TRPV1 attenuates M1 macrophage polarization.
We then investigated the role of inhibiting M1 macrophage polarization by TRPV1 activation in the development of OA. In the OA group, 4 weeks after surgery, larger area of the surface fibrillation, increased loss of cartilage extracellular matrix, and abnormal distribution of chondrocytes were observed compared to the sham group, although there was no discernible difference in the thickness of the articular cartilage ( Fig. 2E and Supplementary Fig. 2C). Eight weeks after surgery, the articular cartilage was significantly thinner compared to the sham group (Fig. 2E). However, following intra-articular injection of CPS, improved score based on the OARSI scale (Fig. 2F), increased abundance of extracellular matrix, improved chondrocyte organization, a smoother cartilage surface, and less surface fibrillation were observed when compared to the OA group (Fig. 2E). We then evaluated the osteophyte formation by micro-CT analysis followed by three-dimensional (3D) modeling. Compared to the sham group, 4-and 8-week-old OA rats developed larger periarticular osteophytes (Supplementary Fig. 2D). However, the osteophytes were smaller in the CPS injection group than in the OA group. These findings suggest that TRPV1 activation induced by the intra-articular injection of a TRPV1 agonist can inhibit M1 macrophage polarization and thus protect from the development of OA.

TRPV1 inhibits M1 macrophage polarization, proliferation, and migration in vitro
To explore the mechanism through which TRPV1 inhibits M1 macrophage polarization, we performed the subsequent experiments using LPS-treated RAW264.7 cells, a well-established M1 macrophage cell line 26,27 . First, we confirmed that the inflammatory factors, Il-1β, Il-6, Tnf-α, iNos, Il-8, and Il-18, were significantly increased in response to LPS stimulation at the mRNA level (Fig. 3A). iNOS and COX2 protein levels were also increased in LPS-treated cells compared to the vehicle control (Fig. 3B). Although activation of TRPV1 by CPS in LPS-treated RAW264.7 cells resulted in a significant reduction of inflammatory factors at mRNA and protein levels (Fig. 3A, B), it did not affect the expression of M2 macrophage markers (Supplementary Fig. 3A). Thus, we focused on the role of TRPV1 in M1 macrophage polarization. Diminished expression of iNOS in the cytoplasm of CPS-treated M1 macrophages was observed by immunofluorescence staining (Fig. 3C). IL-6, a soluble cytokine with a pleiotropic effect on inflammation and immune response, induces chondrocyte catabolism mainly via Stat3 signaling pathway 28,29 . Thus, we investigated whether TRPV1 activation can prevent the secretion of IL-6 from M1 macrophages. Using ELISA, we observed a massive secretion of IL-6 in the supernatant of M1 macrophage, which was significantly decreased by TRPV1 activation (Fig. 3D). Furthermore, we assessed the expression levels of M1 macrophage membrane markers by flow cytometry and demonstrated that the proportion of F4/80 + CD80 + cells decreased remarkably after TRPV1 activation (Fig. 3E). These data indicate that TRPV1 can prevent the polarization of RAW264.7 cells towards the M1 macrophage phenotype promoted by LPS in vitro.
We further examined the role of TRPV1 in the proliferation and migration of M1 macrophage. After the treatment of CPS, the proliferation of LPS-induced M1 macrophage was significantly impeded ( Supplementary  Fig. 3B). EdU staining also demonstrated that the proliferation ability of M1 macrophage was significantly inhibited by TRPV1 activation (Supplementary Fig. 3C). Then, we investigated the efficiency of TRPV1 activation on the migration of macrophage. Under the treatment of LPS, the migration capacity of RAW264.7 cells was significantly enhanced, whereas the migration ability was remarkably suppressed by CPS treatment ( Supplementary  Fig. 3D-F). Besides, CPS inhibited the mRNA expression of (C-X-C motif) ligand 10 (Cxcl10) and monocyte chemoattractant protein-1 (Mcp1) (Supplementary Fig.  3G), two well-established chemokines of macrophage   30 . Together, our data indicate that TRPV1 activation by CPS plays an inhibitory role in the polarization, proliferation, and migration of LPS-induced M1 macrophage.

TRPV1 promotes the nuclear translocation of Nrf2 and inhibits M1 macrophage polarization
The above results drove us to explore how TRPV1 inhibits M1 macrophage polarization. We examined whether TRPV1 could activate Nrf2, the master regulator of multiple cytoprotective responses that plays an anti-inflammatory role by repressing the transcription of proinflammatory genes and inhibiting the infiltration of immune cells 31 . Under normal conditions, Nrf2 is kept in the cytoplasm by Kelch-like ECH-associated protein-1 (Keap1), which facilitates its degradation; however, once activated, Nrf2 dissociates from Nrf2/Keap1 complex and translocates into the nucleus 32 . Therefore, we examined the nuclear level of Nrf2 after TRPV1 activation. Western blot analyses showed that the TRPV1 activation by CPS treatment significantly increased nuclear Nrf2, but not cytosolic Nrf2 (Fig. 4A, B). The rate of the nuclear localization of Nrf2 was significantly increased by TRPV1 activation (Fig. 4C). Over 80% of the cells analyzed displayed Nrf2 nuclear localization in the CPS treatment group, while the percentage in the control and LPStreated groups was <20% (Fig. 4D), substantiating our conclusion regarding the effect of TRPV1 on Nrf2 activation. However, blocking the function of Nrf2 with its specific inhibitor, ML385, resulted in a significant decrease of density and positive rate of Nrf2 in the nucleus under the TRPV1 activation (Fig. 4A-D). These results indicate that Nrf2 mediates the downstream of the TRPV1 signal.
Next, we assessed whether Nrf2 is necessary for TRPV1mediated inhibition of M1 macrophage polarization. mRNA expression of the M1 macrophage markers upregulated by LPS stimulation was significantly reduced by TRPV1 activation due to the CPS treatment, and ML385 partially blocked the effect of the CPS (Fig. 4E). Similar results were obtained in protein levels (Fig. 4F). Taken together, these results suggest that TRPV1 activation inhibits M1 macrophage polarization by activating the Nrf2 signaling pathway.

TRPV1-mediated Ca 2+ influx facilitates Nrf2 nuclear translocation
As a calcium-permeable channel, TRPV1 provides a path for the entry of calcium ions (Ca 2+ ), an important second messenger in numerous cellular signaling pathways 10,33 . We reasoned that the TRPV1-mediated Ca 2+ influx may regulate the Nrf2 nuclear translocation. Using a cell-permeable Ca 2+ probe, Fluo-4 AM 34 , we examined the intracellular Ca 2+ concentration with or without CPS and CaCl 2 under the Ca 2+ -free condition. A significantly rapid increase in fluorescence intensity was observed after 10 min in the CPS + CaCl 2 group, but not in the control, CaCl 2 -only, or CPS-only group, indicating that TRPV1evoked Ca 2+ influx (Fig. 5A, B). Of note, there was no statistically significant difference of fluorescence intensity among the control, CaCl 2 -only, and CPS-only groups (Fig.  5B), suggesting that other forms of Ca 2+ influx that could interfere with the experimental results were absent.
We then investigated whether Ca 2+ played a role in Nrf2 nuclear translocation and the M1 macrophage polarization. In the condition of TRPV1 activation, we used EDTA to chelate Ca 2+ and observed a significant decrease in Nrf2 nuclear localization ( Fig. 5C and Supplementary Fig. 4A). This result was also achieved by using Ca 2+ -free medium to fully remove Ca 2+ (Fig. 5C and Supplementary Fig. 4B). Using immunofluorescence, we confirmed that both EDTA and Ca 2+ -free medium inhibited Nrf2 nuclear translocation (Fig. 5E). It is worth noting that the ratio of Nrf2-positive cells in the nucleus in the EDTA-treated and Ca 2+ -free group were similar to the control group, suggesting that TRPV1-mediated Nrf2 nuclear translocation may occur mainly through the Ca 2+ influx via TRPV1 (Fig. 5D). Despite the TRPV1 activation, the expression of M1 macrophage markers at both the mRNA and protein levels was significantly increased after EDTA treatment ( Supplementary Fig. 5A, B). These findings suggest that Ca 2+ influx resulting from TRPV1 activation plays an indispensable role in the activation of Nrf2, which promotes the inhibition of M1 macrophage polarization.
CaMKII mediates the Nrf2 nuclear translocation by TRPV1 Given the above results, we then examined a mediator that connects TRPV1-evoked Ca 2+ influx and the Nrf2 nuclear localization. Given that CaMKII is a major Ca 2+ signal transducer involved in a variety of signaling pathways 35,36 , we reasoned that CaMKII might be the mediator between Ca 2+ signaling and Nrf2. We harvested proteins of RAW264.7 cells after 5-, 15-, and 30-min LPS induction with or without CPS treatment. Increased phosphorylation of CaMKII was observed after 15 and 30 min of the CPS treatment, along with a significant increase of p-CaMKII/ CaMKII ratio, but not in the vehicle control-and LPStreated groups (Fig. 6A), suggesting that CaMKII was activated following CPS treatment. KN-93 37 , a competitive inhibitor of p-CaMKII, significantly reduced the nuclear density of Nrf2 under the TRPV1 activation ( Fig. 6B and Supplementary Fig. 6). Besides, the Nrf2 nuclear translocation was markedly decreased after the KN-93 treatment (Fig.  6C, D). Taken together, these findings indicate that CaMKII mediates the effect of TRPV1 activation on Nrf2 nuclear translocation.

Discussion
In this study, we explored TRPV1 as a potential therapeutic target for inhibiting M1 macrophage polarization, proliferation, and migration in the pathogenesis of OA. We found that TRPV1 expression was upregulated in both human and rat OA synovium and was highly expressed by M1 macrophage. TRPV1 activation significantly decreased M1 macrophage polarization, reduced synovitis, and attenuated cartilage deterioration and osteophyte formation in the rat OA model. Furthermore, we unveiled that TRPV1 inhibited M1 macrophage polarization by Ca 2+ /CaMKII/Nrf2 signaling pathway.
Recent studies have shown that TRPV1 plays a key role in the management of pain due to its long-term desensitization effect on nociceptive nerve terminals 12,13 . A recent phase IIb clinical trial demonstrated that a single intra-articular injection of a TRPV1 agonist, CNTX-4975 (a type of CPS), provided a significant and clinically meaningful reduction of pain scores in patients with chronic OA-associated knee pain 15 . Based on this, topical application of CPS was conditionally recommended for OA in the 2019 American College of Rheumatology/ Arthritis Foundation Guideline 38 . Beyond regulating pain responses, we showed that TRPV1 was involved in the regulation of macrophage polarization as it was highly localized to M1 macrophage, and the increase of the infiltrated M1 macrophage was highly correlated with synovitis and subsequent severity of OA 19 . However, the regulatory role of TRPV1, particularly in the context of M1 macrophage polarization, in OA remains unknown. Consistent with the previous studies 7, 19 , we found markedly increased infiltration of M1 macrophages in human and rat OA synovium, accompanied by simultaneously upregulated TRPV1 expression. Approximately 90% of M1 macrophages expressed TRPV1 in both normal and OA synovium, providing a potential therapeutic target regulating M1 macrophage polarization precisely. Further, the rat OA model showed that TRPV1 activation attenuated knee joint swelling, improved synovitis scores, and reduced M1 macrophages levels, resulting in decreased cartilage degeneration and osteophyte formation. The results demonstrated that targeting TRPV1 is a potential therapeutic approach to prevent M1 macrophage polarization to reduce synovitis and slow OA progression. Further evaluation of the effect of TRPV1 on M1 macrophage polarization in the synovium of OA patients enrolled in clinical trials will contribute to a more comprehensive understanding of the therapeutic effect of targeting TRPV1 on synovitis.
Of note, TRPV1 is also found expressed in chondrocytes, and mediates the attenuation of cytokineinduced ADAMTS9 expression by mechanical strain, suggesting the decreased cartilage catabolism by TRPV1 39 . Thus, multiple pathological joint compartments and diverse processes involved in OA pathogenesis may respond to a TRPV1-targeting therapy.
Nrf2 is a redox-regulated transcription factor essential for the counteraction of oxidative stress and chronic inflammation 40,41 . Increased expression and enhanced activity of Nrf2 have been observed in the OA synovium and the damaged area of the OA cartilage, suggesting that there is a self-adaptive mechanism to promote cell survival in the OA microenvironment with increased inflammation and oxidative stress 42,43 . The current study identifies a protective role for Nrf2 in OA synovitis mediated via the inhibition of M1 macrophage polarization. TRPV1-evoked Ca 2+ influx enhanced the phosphorylation of CaMKII and then activated Nrf2, leading to the downregulation of M1 macrophage markers. However, several recent studies have shown that ultraviolet irradiation induced the TRPV1-evoked Ca 2+ influx and promoted Nrf2 degradation and cell apoptosis in human dermal fibroblasts 23,44 . The divergent effects could be explained by the dual role of Ca 2+ in living organisms. Ca 2+ signals are necessary for cell survival due to their management of a host of vital cell functions; however, Ca 2+ influx caused by cytotoxic agents or receptor overstimulation can result in cytotoxicity and trigger either necrotic or apoptotic cell death 45 . Thus, we reason that the divergence in these studies following TRPV1 activation could be attributed to the dissimilar efficacy of CPS and ultraviolet irradiation on TRPV1 activity.
In conclusion, we found that TRPV1 is a novel therapeutic target for inhibiting M1 macrophage polarization and decreasing synovitis to attenuate OA progression, unveiling a new mechanism for the topical administration of CPS for the treatment of OA. Mechanically, CPSmediated TRPV1 activation reduced M1 macrophage marker expression via the Ca 2+ /CaMKII/Nrf2 signaling pathway. Future studies are warranted to investigate the role of TRPV1 activation in chondrocytes, synovial fibroblasts, and other cells in the joint in order to generate a more comprehensive understanding of the pharmacological effects of intra-articular injection of CPS.