Acute systemic macrophage depletion in osteoarthritic mice alleviates pain-related behaviors and does not affect joint damage

Abstract

Background: Osteoarthritis (OA) is a painful degenerative joint disease and a leading source of years lived with disability globally due to inadequate treatment options. Neuroimmune interactions reportedly contribute to OA pain pathogenesis. Notably, in rodents, macrophages in the DRG are associated with onset of persistent OA pain. Our objective was to determine the effects of acute systemic macrophage depletion on pain-related behaviors and joint damage using surgical mouse models in both sexes.

Methods: We depleted CSF1R + macrophages by treating male macrophage Fas-induced apoptosis (MaFIA) transgenic mice 8- or 16-weeks post destabilization of the medial meniscus (DMM) with AP20187 or vehicle control (10 mg/kg i.p., 1x/day for 5 days), or treating female MaFIA mice 12 weeks post partial meniscectomy (PMX) with AP20187 or vehicle control. We measured pain-related behaviors 1-3 days before and after depletion, and, 3-4 days after the last injection we examined joint histopathology and performed flow cytometry of the dorsal root ganglia (DRGs). In a separate cohort of male 8-week DMM mice or age-matched naïve vehicle controls, we conducted DRG bulk RNA-sequencing analyses after the 5-day vehicle or AP20187 treatment.

Results: Eight- and 16-weeks post DMM in male mice, AP20187-induced macrophage depletion resulted in attenuated mechanical allodynia and knee hyperalgesia. Female mice showed alleviation of mechanical allodynia, knee hyperalgesia, and weight bearing deficits after macrophage depletion at 12 weeks post PMX. Macrophage depletion did not affect the degree of cartilage degeneration, osteophyte width, or synovitis in either sex. Flow cytometry of the DRG revealed that macrophages and neutrophils were reduced after AP20187 treatment. In addition, in the DRG, only MHCII + M1-like macrophages were significantly decreased, while CD163 + MHCII- M2-like macrophages were not affected in both sexes. DRG bulk RNA-seq revealed that Cxcl10 and Il1b were upregulated with DMM surgery compared to naïve mice, and downregulated in DMM after acute macrophage depletion.

Conclusions: Acute systemic macrophage depletion reduced the levels of pro-inflammatory macrophages in the DRG and alleviated pain-related behaviors in established surgically induced OA in mice of both sexes, without affecting joint damage. Overall, these studies provide insight into immune cell regulation in the DRG during OA.

Keywords: Dorsal root ganglia; Macrophages; Neuroimmune; Osteoarthritis; Pain; Sensitization.

Fig. 1

Macrophage depletion experimental design schematic. A Male MaFIA mice had DMM surgery at 12 weeks of age. At 7 weeks after DMM, mice were tested for mechanical allodynia and knee hyperalgesia, followed by 5 once daily injections (i.p.) of vehicle solution or AP20187 (10 mg/kg). Pain-related behaviors were tested afterward, one test per day. Then mice were sacrificed for downstream analyses. B Female MaFIA mice had PMX surgery at 12 weeks of age and were tested for mechanical allodynia, knee hyperalgesia, and static weightbearing at 11 weeks after PMX. After 5 daily i.p. injections of Vehicle or AP20187, mice were tested for the same pain-related behaviors and then sacrificed for downstream analyses

Fig. 2

Macrophage depletion alleviated pain-related behaviors in mice of both sexes with OA. A Mechanical allodynia and (B) Knee hyperalgesia withdrawal thresholds in male MaFIA mice at 8 weeks after DMM surgery showing before and after treatment with Vehicle (in blue, n = 10) or AP20187 (in red, n = 13). The left graph shows individual mouse data points before and after treatment, and the right graph shows the mean +/- 95% CI of each group’s withdrawal thresholds before and after treatment. C Mechanical allodynia and (D) Knee hyperalgesia withdrawal thresholds in female MaFIA mice at 12 weeks after PMX surgery showing before and after Vehicle (in dark yellow, n = 8) or AP20187 (in purple, n = 10) treatment. E Static weight bearing in same cohort of female MaFIA mice at 12 weeks after PMX surgery showing before and after Vehicle (in dark yellow, n = 8) or AP20187 (in purple, n = 10) treatment. Statistical analysis by paired t-test. P values stated on graphs; considered significant if p < 0.05

Fig. 3

Acute systemic macrophage depletion does not affect joint damage severity. A Representative Safranin-O stained Vehicle (cartilage degeneration score = 11) or AP20187-treated (cartilage degeneration score = 11) male MaFIA mouse knee images at 8 weeks after DMM surgery; 2x magnification. B Medial cartilage degeneration, (C) Osteophyte width (micrometers), (D) Synovitis medial sum score, (E) Synovial medial hyperplasia, (F) Synovial medial cellularity, and (G) Synovial medial fibrosis scored for male MaFIA mice at 8 weeks after DMM surgery from Vehicle (in blue, n = 10) and AP20187 (in red, n = 13) groups. H Representative Safranin-O stained Vehicle (cartilage degeneration score = 21) or AP20187-treated (cartilage degeneration score = 20) female MaFIA mouse knee images at 12 weeks after PMX surgery; 2x magnification. I – N Same as in (B) – (G) but showing scores for female MaFIA mice at 12 weeks after surgery from Vehicle (in yellow, n = 8) or AP20187 (in purple, n = 10) treated mice. Scale bar is 500 μm. Statistical analysis for cartilage degeneration and osteophyte width by two-tailed unpaired t-test, and for synovitis scoring, Mann-Whitney test. Graphs show mean +/- 95% CI. P values stated on graphs; considered significant if p < 0.05

Fig. 4

Decreased Ly6G + neutrophil and CSF1R + macrophage populations in the DRG after macrophage depletion. Frequency of (A) CD45 + leukocytes, (B) CD11b + myeloid cells, (C) CD3 + T cells, (D) CSF1R + monocyte/macrophages, (E) Ly6G + neutrophils in the DRG of male MaFIA mice at 8 weeks after DMM surgery for Vehicle (blue, n = 5) or AP20187 (red, n = 7). F – J Same as in (A) – (E) but showing frequencies of each cell population for female MaFIA mice at 12 weeks after surgery for Vehicle (yellow, n = 4) or AP20187 (purple, n = 6) treated mice. Statistical analysis by Mann-Whitney test. Error bars show mean +/- 95% CI. P values stated on graphs; significant if p < 0.05

Fig. 5

Decreased MHCII + macrophages, but no change in CD163 + macrophages in the DRG after systemic macrophage depletion in both sexes. A Frequency of total F4/80 + macrophages, (B) F4/80 + MHCII+, (C) F4/80 + CD163+, (D) CD163-MHCII-, (E) CD163-MHCII+, (F) CD163 + MHCII+, (G) CD163 + MHCII- macrophage populations in the DRG in male MaFIA mice at 8 weeks after DMM surgery for Vehicle (blue, n = 5) or AP20187 (red, n = 7). H – N Same as in (A) – (G) but showing frequencies of each cell population for female MaFIA mice at 12 weeks after surgery for Vehicle (yellow, n = 4) or AP20187 (purple, n = 6) treated mice. Statistical analysis by Mann-Whitney test. Error bars show mean +/- 95% CI. P values stated on graphs; significant if p < 0.05