Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Dec;29(12):1720-1731.
doi: 10.1016/j.joca.2021.09.003. Epub 2021 Sep 17.

Cannabinoid receptor type 2 is upregulated in synovium following joint injury and mediates anti-inflammatory effects in synovial fibroblasts and macrophages

P Rzeczycki  1 , C Rasner  1 , L Lammlin  1 , L Junginger  1 , S Goldman  1 , R Bergman  1 , S Redding  1 , A J Knights  1 , M Elliott  1 , T Maerz  2
Affiliations

Cannabinoid receptor type 2 is upregulated in synovium following joint injury and mediates anti-inflammatory effects in synovial fibroblasts and macrophages

P Rzeczycki et al. Osteoarthritis Cartilage. 2021 Dec.

Abstract

Objective: Joint injury-induced perturbations to the endocannabinoid system (ECS), a regulator of both inflammation and nociception, remain largely uncharacterized. We employed a mouse model of ACL rupture to assess alterations to nociception, inflammation, and the ECS while using in vitro models to determine whether CB2 agonism can mitigate inflammatory signaling in macrophages and fibroblast-like synoviocytes (FLS).

Design: Mice underwent noninvasive ACL rupture (ACLR) via tibial compression-based loading. Nociception was measured longitudinally using mechanical allodynia and knee hyperalgesia testing. Synovitis was assessed using histological scoring and histomorphometry. Gene and protein markers of inflammation were characterized in whole joints and synovium. Immunohistochemistry assessed injury-induced alterations to CB1+, CB2+, and F4/80+ cells in synovium. To assess whether CB2 agonism can inhibit pro-inflammatory macrophage polarization, murine bone marrow-derived macrophages (mBMDM) were stimulated with IL-1β or conditioned medium from IL-1β-treated FLS and treated with vehicle (DMSO), the CB2 agonist HU308, or cannabidiol (CBD). Macrophage polarization was assessed as the ratio of M1-associated (IL1b, MMP1b, and IL6) to M2-associated (IL10, IL4, and CD206) gene expression. Human FLS (hFLS) isolated from synovial tissue of OA patients were treated with vehicle (DMSO) or HU308 following TNF-α or IL-1β stimulation to assess inhibition of catabolic/inflammatory gene expression.

Results: ACLR induces synovitis, progressively-worsening PTOA severity, and an immediate and sustained increase in both mechanical allodynia and knee hyperalgesia, which persist beyond the resolution of molecular inflammation. Enrichment of CB2, but not CB1, was observed in ACLR synovium at 3d, 14d, and 28d, and CB2 was found to be associated with F4/80 (+) cells, which are increased in number in ACLR synovium at all time points. The CB2 agonist HU308 strongly inhibited mBMDM M1-type polarization following stimulation with either IL-1β or conditioned medium from IL-1β-treated mFLS, which was characterized by reductions in Il1b, Mmp1b, and Il6 and increases in Cd206 gene expression. Cannabidiol similarly inhibited IL-1β-induced mBMDM M1 polarization via a reduction in Il1b and an increase in Cd206 and Il4 gene expression. Lastly, in OA hFLS, HU308 treatment inhibited IL-1β-induced CCL2, MMP1, MMP3, and IL6 expression and further inhibited TNF-α-induced CCL2, MMP1, and GMCSF expression, demonstrating human OA-relevant anti-inflammatory effects by _targeting CB2.

Conclusions: Joint injury perturbs the intra-articular ECS, characterized by an increase in synovial F4/80(+) cells, which express CB2, but not CB1. _targeting CB2 in murine macrophages and human FLS induced potent anti-inflammatory and anti-catabolic effects, which indicates that the CB2 receptor plays a key role in regulating inflammatory signaling in the two primary effector cells in the synovium. The intraarticular ECS is therefore a potential therapeutic _target for blocking pathological inflammation in future disease-modifying PTOA treatments.

Keywords: Endocannabinoid system; Macrophage; Nociception; Post-traumatic osteoarthritis; Synovitis.

PubMed Disclaimer

Figures

Figure 1:
Figure 1:
ACLR induces lingering synovitis concurrent with progressively-worsening PTOA A) Representative H&E-stained sections of Sham, CL, and ACLR limbs, with emphasis on anterior synovium. Location of high-magnification views are denoted by dashed boxes. Green arrow heads denote synovial lining thickness, which increases in ACLR limbs, while yellow arrowheads denote inflammatory infiltrate, which was not observed in CL or Sham synovia. B) Representative SafO/Fast-Green stained sections of Sham, CL, and ACLR limbs. High-magnification views are denoted by dashed boxes and represent regions of maximal articular cartilage damage. Arrowheads denote cartilage erosion and damage, which progressively worsens and becomes more widespread following injury. A-B): Scale bar is 1000 μm on whole-joint images and 100 μm for high-magnification views. C) Synovitis and PTOA severity scoring, . n=4–6 mice per group/time point. * = p<0.05 compared to CL within each time point; Wilcoxon Signed Rank Test. # = p<0.05 compared to Sham; Kruskal-Wallis test with Dunn’s post-hoc test (Sham as the control group). ‡ = p<0.05 between time points within ACLR; Kruskal-Wallis test with Dunn’s post-hoc test. D-E) Cellular density (D) and Total cellularity (E) determined using quantitative histomorphometry in anterior and posterior synovium. n=4–6 mice per group/time point. *=p<0.05 compared to CL within each time point, ‡ = p<0.05 between time points within ACLR; Two-way repeated measures ANOVA with Sidak post-hoc test (limb as within-subject factor and time point as between-subject factor). #=p<0.05 compared to Sham; one-way ANOVA with Dunnett’s post-hoc test (Sham as the control group).
Figure 2:
Figure 2:
ACLR exacerbates mechanical allodynia and knee hyperalgesia. A) Mechanical allodynia measured via von Frey testing. B) Knee hyperalgesia measured via pressure application-based Randall-Selitto testing. A, B). n=6 mice per group measured longitudinally. * = p<0.05 compared to CL within each group; # p<0.05 compared to Sham; Three-way repeated measures ANOVA (limb and time point as within-subject factors and group as between-subject factor) with Sidak post-hoc test. C) Cytokine and chemokine protein concentrations in whole knees following ACLR. n=5–8 mice per group/time point. *=p<0.05 compared to CL within each time point, ‡ = p<0.05 between time points within ACLR; Two-way repeated measures ANOVA with Sidak post-hoc test (limb as within-subject factor and time point as between-subject factor). # = p<0.05 compared to Sham; one-way ANOVA with Dunnett’s post-hoc test (Sham as the control group). D) Expression of inflammatory genes by qPCR in whole synovial tissue from ACLR and contralateral joints. Relative expression was normalized to Atpb5b mRNA levels and the mean Contralateral value for each gene was set to 1. n=6–8 mice per time point. * = p<0.05 compared to CL within each time point; Wilcoxon Signed Rank Test.
Figure 3:
Figure 3:
The CB2 receptor is upregulated by F4/80(+) synovial cells following ACL rupture. A) Immunofluorescent staining of cannabinoid receptor 2 and the monocyte/macrophage marker F4/80 in synovium. S=Synovium, F=Femur, T=Tibia, O=Osteophyte. Green: CB2, Red: F4/80, Blue: DAPI. Scale bar is 50 μm. Arrows denote regions of double positive cells, indicative of F4/80(+) cells expressing CB2 within injured synovium. B-D). Quantification of the proportion of CB2-positive cells (B), F4/80-positive cells (C), and CB2-positive cells which are also F4/80-positive (D) in synovium. n=4–6 mice per group/time point. *=p<0.05 compared to CL within each time point, ‡ = p<0.05 between time points within ACLR; Two-way repeated measures ANOVA with Sidak post-hoc test (limb as within-subject factor and time point as between-subject factor). #=p<0.05 compared to Sham; one-way ANOVA with Dunnett’s post-hoc test (Sham as the control group). E) Gene expression of CNR1 and CNR2 by qPCR in whole synovial tissue from ACLR and contralateral joints. Relative expression was normalized to Atpb5b mRNA levels and the mean Contralateral value for each gene was set to 1. n=3–8 mice per time point. * = p<0.05 compared to CL within each time point. Wilcoxon Signed Rank Test.
Figure 4:
Figure 4:
mBMDM express CB2 and exhibit anti-inflammatory responses to CB2 agonist and Cannabidiol treatment. A) Macrophage expression of Cnr1 and Cnr2 is not altered by IL-1β or cannabinoid treatment. n=5 unique isolations/cultures. B) Immunocytochemistry of cultured mBMDM demonstrates widespread CB2 protein expression, with little to no CB1. Scale bar is 50 um. C) M1- and M2-associated gene expression and composite M1/M2 polarization score of mBMDM stimulated with PBS or IL-1β and treated with DMSO, the CB2 agonist HU308, or CBD. Relative expression was normalized to Gapdh mRNA levels and the mean DMSO-treated PBS value for each gene was set to 1. n=5 unique isolations/cultures. *=p<0.05 compared to respective DMSO within PBS or within IL1β group; # = p<0.05 compared to respective PBS group within DMSO, HU308, or CBD treatment. Two-way repeated measures ANOVA with Sidak post-hoc test. D) M1- and M2-associated gene expression and composite M1/M2 polarization score of mBMDM stimulated with conditioned media (CM) from PBS-treated or IL-1β-treated FLS and further treated with DMSO or HU308. Relative expression was normalized to Gapdh mRNA levels and the mean DMSO-treated PBS-CM value for each gene was set to 1. n=3 unique isolations/cultures. *=p<0.05 compared to respective DMSO within PBS-CM or within IL1β-CM group; # = p<0.05 compared to respective PBS-CM group within DMSO or HU308 treatment. Two-way repeated measures ANOVA with Sidak post-hoc test.
Figure 5:
Figure 5:
CB2 agonist treatment induces anti-inflammatory effects in cytokine-treated human OA FLS. Inflammatory and catabolic gene expression following stimulation with 10 ng/ml IL-1β (A) or 10 ng/ml TNF-α (B) and treatment with either DMSO or the CB2 agonist HU308. n=7 unique isolations/cultures from human synovial biopsies. (*=p<0.05, Wilcoxon Signed Rank Test.)
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Scanzello CR, Role of low-grade inflammation in osteoarthritis. Current opinion in rheumatology 29 (1), 2017, 79–85. - PMC - PubMed
    1. Greene MA, and Loeser RF, Aging-related inflammation in osteoarthritis. Osteoarthritis and Cartilage 23 (11), 2015, 1966–1971. - PMC - PubMed
    1. Lieberthal J, Sambamurthy N, and Scanzello CR, Inflammation in joint injury and post-traumatic osteoarthritis. Osteoarthritis Cartilage 23 (11), 2015, 1825–1834. - PMC - PubMed
    1. Thomas NP, Wu WJ, Fleming BC, Wei F, Chen Q, and Wei L, Synovial inflammation plays a greater role in post-traumatic osteoarthritis compared to idiopathic osteoarthritis in the Hartley guinea pig knee. BMC Musculoskeletal Disorders 18 (1), 2017, 556. - PMC - PubMed
    1. Buckwalter JA, Articular cartilage: injuries and potential for healing. J Orthop Sports Phys Ther 28 (4), 1998, 192–202. - PubMed

Publication types

  NODES
INTERN 1
Note 6
twitter 2