Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Mar;36(3):864-875.
doi: 10.1002/jor.23671. Epub 2017 Aug 29.

Chemokine receptor-7 (CCR7) Deficiency Leads to Delayed Development of Joint Damage and Functional Deficits in a Murine Model of Osteoarthritis

Affiliations
Free PMC article

Chemokine receptor-7 (CCR7) Deficiency Leads to Delayed Development of Joint Damage and Functional Deficits in a Murine Model of Osteoarthritis

Nisha Sambamurthy et al. J Orthop Res. .
Free PMC article

Abstract

Elevated chemokine receptor Ccr7 is observed in knee osteoarthritis (OA) and associated with severity of symptoms. In this study, we confirmed that CCR7 protein expression is elevated in synovial tissue from OA patients by immunohistochemical staining. We then investigated whether Ccr7 deficiency impacted structural and functional joint degeneration utilizing a murine model of OA. OA-like disease was induced in male C57BL/6 and Ccr7-deficient (Ccr7-/- ) mice by destabilization of the medial meniscus (DMM). Functional deficits were measured by computer integrated monitoring of spontaneous activity every 4 weeks after DMM surgery up 16 weeks. Joint degeneration was evaluated at 6 and 19 weeks post-surgery by histopathology, and subchondral bone changes analyzed by microCT. Results showed reduction in locomotor activities in DMM-operated C57BL/6 mice by 8 weeks, while activity decreases in Ccr7-/- mice were delayed until 16 weeks. Histopathologic evaluation showed minimal protection from early cartilage degeneration (p = 0.06) and osteophytosis (p = 0.04) in Ccr7-/- mice 6 weeks post-DMM compared to C57BL/6 controls, but not at 19 weeks. However, subchondral bone mineral density (p = 0.03) and histologic sclerosis (p = 0.02) increased in response to surgery in C57BL/6 mice at 6 weeks, while Ccr7-/- mice were protected from these changes. Our results are the first to demonstrate a role for Ccr7 in early development of functional deficits and subchondral bone changes in the DMM model. Understanding the mechanism of Ccr7 receptor signaling in the initiation of joint pathology and disability will inform the development of innovative therapies to slow symptomatic OA development after injury. Published 2017. This article is a U.S. Government work and is in the public domain in the USA. J Orthop Res 36:864-875, 2018.

Keywords: animal model; bone remodeling; chemokines; murine behavioral analysis; osteoarthritis.

Conflict of interest statement

Conflicts of interest: All other authors report no conflict of interest related to this manuscript.

Figures

Figure 1
Figure 1
Schematic for the experiments included in this study. Function: Mice underwent activity monitoring on the LABORAS™ platforms pre-operatively, and then underwent DMM or sham surgery, or were left unoperated/näve (n = 7–10 mice per treatment group). Activity was measured every 4 weeks up to 16 weeks post-operatively. Structure: Mice were then sacrificed at 19 weeks post-operatively for micro CT (n = 5) and histology (n = 7–10) of cartilage and bone structure. Additional mice (n = 5 per group) were subjected to DMM or sham (or näve), and then sacrificed 6 weeks post-surgery for structural analysis at an earlier stage of disease. Inflammation: Another set of mice underwent DMM surgery, and then were sacrificed in groups of 20–30 at 1, 2, and 4 weeks post-DMM. The anterior synovium and intra-articular fat pad were dissected together, and then tissues from 4 to 5 mice pooled per sample resulting in 5–6 samples per time point. mRNA was extracted and analyzed as described.
Figure 2
Figure 2
Immunostaining for CCR7 in human synovial tissues. Synovial biopsies obtained from donors and patients were stained using an anti-human CCR7 antibody (Materials and Methods). Representative sections from an asymptomatic donor, early OA patient undergoing meniscal arthroscopy, and an advanced OA patient are shown in A. Isotype control is shown in inset. Staining was quantified using automated image analysis and percent fractional area stained presented in B. Percent area stained was significantly greater in the meniscal arthroscopy patients than in asymptomatic donors.
Figure 3
Figure 3
Comparison of spontaneous behaviors after DMM surgery compared to control groups in the two strains. Spontaneous activity was measured as described every 4 weeks up to 16 weeks post-DMM in the two strains of mice. Time spent climbing (A–C), distance traveled (D–F), and locomotion (G–I), are depicted compared with sham and näve controls at the 4, 8, and 16 week time points. In wild-type mice, time spent climbing post-DMM was significantly decreased by 8 weeks (B) compared to controls. A decreasing trend was also seen at 8 weeks in distance traveled (E). Reduction in activity post-DMM compared to control groups was not observed in CCR7−/− mice until 12 weeks (decrease in locomotion, Supplemental Table S1), and was statistically significant by 16 weeks for distance traveled (F) and locomotion (I). Medians, and IQRs are displayed. p values from Mann–Whitney U test compared to controls. n = 7–10 mice per group.
Figure 4
Figure 4
Longitudinal activity patterns in wild-type and Ccr7−/− mice subjected to DMM surgery. Spontaneous activity was measured pre-operatively and every 4 weeks up to 16 weeks post-operatively using LABORAS platforms (details in materials and methods). Median (IQR) time spent climbing (A), in locomotion (B), and distance traveled (C) in 14 h is presented. In wild-type mice, time spent climbing (A) decreased slightly although non-significantly after DMM surgery compared to baseline up to 8 weeks post-operatively. CCR7−/− mice increased climbing activity compared to baseline up to 8 weeks with decreases thereafter. Similar longitudinal patterns were seen with locomotion (B) and distance travelled (C). (*p < 0.01 compared to pre-op levels in same strain, #p <0.05 for trend difference between strains using linear mixed effects models).
Figure 5
Figure 5
Ccr7 deficient mice show reduced cartilage degeneration at 6 weeks, but not 19 weeks after DMM surgery. Groups of 5–10 mice were subjected to DMM or sham surgery at 10–12 weeks of age, or left unoperated (näve). At 6 weeks and 19 weeks post-surgery, knee joints were dissected and prepared for histologic analysis. Total cartilage degeneration scores in WT (black squares) and Ccr7 knockout mice (red circles) at (A) 6 weeks and (B) 19 weeks post-surgery are shown. **p < 0.025 compared to DMM operated mice of the same strain (Kruskal–Wallis followed by Mann–Whitney U-test). Representative photomicrographs of knee histopathology are depicted in panels C–E. (C) Wild-type at 6 weeks post-DMM, (D) wild-type at 19 weeks post-DMM, (E) Ccr7−/− at 6 weeks post-DMM, (F) Ccr7−/− at 19 weeks post-DMM.
Figure 6
Figure 6
Histologic bone changes post-DMM in wild-type and Ccr7−/− mice. Osteophyte size (panels A and B) and subchondral bone sclerosis (panels C and D) were scored as described. Osteophyte size and bone scores in WT and Ccr7−/− mice (A and C) at 6 weeks post-surgery and (B and D) 19 weeks post-surgery are shown. p-values from Kruskal–Wallis followed by Mann–Whitney U-test. Representative histopathology is shown of the medial tibial plateau from (E) an unoperated WT knee at the 6 week time point (no osteophyte, subchondral bone score of 0) and (F) a DMM-operated WT knee at 6 weeks (osteophyte = 200 micron, subchondral bone score = 5). White arrow = subchondral bony sclerosis, red arrow = marginal osteophyte.
Figure 7
Figure 7
Subchondral bone volume and mineral density measured by microCT in wild-type and Ccr7 deficient mice. MicroCT scans of näve joints at 10–12 weeks of age (time 0), and näve and operated (sham or DMM) joints at 6 and 19 weeks after surgery were performed. n = 5 mice per group (final numbers analyzed are 3–5 per group, as some scans contained artifacts that precluded analysis). Panels A and C describe trends in bone mineral density (BMD) and bone volume fraction (%BV/TV) in wild-type mice. Panels B and D indicate trends in BMD and % BV/TV in Ccr7−/− mice. Representative 2D microCT slices were chosen from 10 to 12-week-old (time 0) un-operated wild-type (E) and Ccr7−/− (F) mice.
Figure 8
Figure 8
Synovial response to DMM injury in both strains of mice. Synovial hyperplasia in wild-type (WT) and Ccr7−/− mice at 6 weeks (A) and 19 weeks (B) post-surgery. Hyperplasia was scored as described just superior to the medial meniscus. No significant differences were observed between groups. Gene expression analysis of CD68 transcript levels in knee synovial capsular tissue up to 4 weeks post-DMM in WT (C) and Ccr7 deficient mice (D). mRNA copy number measured by droplet digital PCR and normalized to number of TBP transcripts. *p < 0.05 compared to left un-operated side, Wilcoxon matched-pairs signed-rank test. #p < 0.017 compared to baseline levels, Mann–Whitney U-test.

Similar articles

See all similar articles

Cited by 5 articles

Publication types

Feedback