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

Arthritis Research & Therapy volume 26, Article number: 224 (2024) Cite this article

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.

Background

Osteoarthritis (OA) is a major contributor to adult disability and an increasing societal burden, with an estimated 595 million people living with OA worldwide [1]. Pain drives OA patients to seek clinical care; yet, current strategies for OA analgesia do not provide adequate pain relief [2].

The role of synovial inflammation in OA pathology has been recognized for some time [3], and a number of studies have shown a positive correlation between synovial inflammation and pain [4, 5]. More recently, a link between OA synovitis and sensitization of the nervous system was also identified [6]. Of the various types of immune cells that have been found in the OA synovium, macrophages represent the predominant cell type [7], and a novel imaging approach demonstrated that numbers of activated macrophages in OA knees positively correlated with pain severity [8]. In addition, elevated levels of soluble macrophage markers in both the synovial fluid and in the blood have been associated with symptom severity as well as disease progression [9, 10], which could be due to the fact that macrophages are able to produce a number of pro-algesic factors, including cytokines and chemokines, that can signal to nociceptors to promote sensitization [11].

As joint-innervating nociceptors become activated, long-term changes can be enacted at the level of the dorsal root ganglia (DRG), where the cell bodies of these neurons are located. We and others have shown that chemokines produced in the DRG can in turn promote the accumulation of macrophages at this site [12,13,14,15], in addition to the immune cell changes within the joint itself. Preclinical studies have provided evidence that targeting macrophages in the DRG may be sufficient to provide analgesia. For instance, in a model of nerve injury, depletion of macrophages at the site of nerve injury did not abate pain, while systemic depletion reduced macrophages in the DRG and pain-related behaviors in both male and female mice [16]. In addition, in the mouse or rat monoiodoacetate (MIA) model of joint pain, systemic ablation of macrophages reduced pain-related behaviors [13, 17]. However, each study only focused on characterizing the effects of systemic depletion in one anatomical location – either in the synovium [17] or the DRG [13], and the effect of depletion on joint damage was not assessed in either of those studies.

While targeting macrophages in the joint and/or in the DRG is potentially beneficial in terms of pain relief, the impact of macrophage depletion on the joint remains unclear. Intra-articular macrophage depletion approaches that began from an early stage were shown to promote synovitis in some models [18,19,20], while in one, intra-articular depletion of macrophages from an early stage reduced osteophyte formation [21]. Here, we aimed to examine the acute effects of systemic macrophage depletion on structural joint changes, pain-related behaviors, and cellular and molecular changes in the DRG in surgically induced OA in mice of both sexes (destabilization of the medial meniscus (DMM) in males, and partial medial meniscectomy (PMX) in females). We focused the intervention on late-stage disease, i.e., time points when both joint structural damage and pain-related behaviors had already developed, in order to provide information on whether targeting macrophages in established OA could be beneficial.

Methods

Animals

Macrophage Fas-Induced Apoptosis (MaFIA) mice were purchased from Jackson laboratories (strain name: C57BL/6-Tg(Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6)2Bck/J; RRID: IMSR_JAX:005070) and bred in house (homozygous x homozygous). When male mice became 12 weeks old, destabilization of the medial meniscus (DMM) surgery was performed on right knee joints, as previously described [22]. Since young female mice do not develop appreciable joint damage in the DMM model [23], partial meniscectomy surgery (PMX) was performed in the right knee joint of 12-week old female MaFIA mice, as previously described [24]. Animals were anesthetized using isoflurane for surgical procedures. Animals were housed at Rush, had unrestricted access to food and water, and were kept on a 12-hour light cycle. All animal experiments were approved by our Institutional Animal Care and Use Committee.

Macrophage depletion

MaFIA mice were weighed and given 10 mg/kg AP20187 (Tocris, Cat. #6297) or vehicle solution (4% Ethanol, 1.7% Tween-80, 10% PEG-400, water) to achieve macrophage depletion as previously described [25]. This otherwise inert pharmacologic agent, AP20187, binds to the CSF1R-eGFP transgenic receptor containing a Fas-cytoplasmic domain, and induces Fas-driven apoptosis in CSF1R + cells, resulting in systemic macrophage depletion. For male MaFIA mice at 8-weeks post DMM surgery, vehicle (n = 10) or AP20187 (n = 13) was administered intraperitoneally (i.p.) once daily for 5 consecutive days, at approximately the same time each day (Fig. 1). During the treatment period, mice were housed with softer bedding and dripping water. Vehicle and AP20187 groups were housed in separate cages. Within one to three days before and one to three days after the 5-day depletion period, mice were subjected to pain-related behavior testing. Following the last behavioral test post depletion (3–4 days after the last depletion injection), mice were sacrificed for downstream analyses. The same procedure was done in a different cohort of male MaFIA mice at 16 weeks post DMM surgery (Vehicle (n = 6), AP20187 (n = 6)) and in female MaFIA mice at 12 weeks post PMX surgery (Vehicle (n = 8), AP20187 (n = 10)).

Fig. 1
figure 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

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Behavior testing

Male and female MaFIA mice were evaluated for mechanical allodynia and knee hyperalgesia, while weight bearing was tested on female mice only. Only one behavior test was performed per day, and testers were blinded to animal groups. Mice were evaluated before and after the 5-day treatment period.

Mechanical allodynia in the hind paws was measured using von Frey fibers and the 50% withdrawal threshold was calculated via the up-down method, as described previously [12, 26].

Knee hyperalgesia was measured by pressure application measurement (PAM) testing, which applies a range of forces directly to the knee joint to determine a quantitative withdrawal threshold [27, 28]. Briefly, mice were restrained by hand and the knee was held in flexion at a similar angle for each mouse. Then, the PAM transducer was pressed against the medial side of the ipsilateral knee and an increasing amount of force was applied up to a maximum of 450 g. The force at which the mouse tried to withdraw its knee was recorded. If the mouse did not withdraw, the maximum force of 450 g was assigned.

Weight bearing asymmetry was assessed in female MaFIA mice, utilizing a custom voluntarily accessed static incapacitance (VASIC) method where mice were trained to perform a string-pulling task while freely standing on a Bioseb static incapacitance meter platform (Harvard Apparatus) in a custom-built plexiglass chamber (previously described in [29]). To accustom mice to the task, mice were trained one week prior to the first test, as described. Weight bearing was recorded once mice had one hind limb placed on each load cell and were pulling a hanging string to receive a Cheerio reward. Three readings were taken per animal and averaged.

Joint histology

Following behavior testing, mice were sacrificed and right-side (ipsilateral-affected) knees were collected for histologic analysis. Knees were formalin fixed, then decalcified in EDTA for 3 weeks, and embedded in paraffin. Six-micron thick sections from the center of the joint were stained with Toluidine Blue or Safranin-O for the evaluation of cartilage damage based on Osteoarthritis Research Society International recommendations [30, 31]. These analyses were done semi-quantitatively, and tissue sections were stained altogether when possible. For cartilage degeneration, medial femoral condyles and tibial plateau were scored for severity of cartilage degeneration. For each cartilage surface, scores were assigned individually to each of three zones (inner, middle, outer) on a scale of 0–5, with 5 representing the most damage. The maximum score for the sum of femoral and tibial cartilage degeneration is 30. We also measured osteophyte width, one section with the major osteophyte was assessed using Osteomeasure software (OsteoMetrics) as described [15, 32]. The synovial pathology was evaluated as changes in synovial hyperplasia, cellularity, and fibrosis, which were evaluated at the synovial insertion of medial femur and medial tibia separately as described [33]. Both joint spaces were visible, except for capsule insertion in some instances, in which the score was considered 0 for that quadrant. Synovitis scoring was performed by 1 independent observer blinded to the groups. Synovial hyperplasia was defined as thickness of the lining layer with a score range of 0–3. Cellularity was defined as the cell density of the synovial sublining with a score range of 0–3. Fibrosis was defined as the extracellular matrix density in the synovium with a score range of 0–1. Each synovial pathology was reported as a sum score of the medial tibial and medial femoral quadrants and reported per knee.

Joint microcomputed tomography

Micro-computed tomographic (µCT) imaging was performed on a subset of intact knee joints using a high-resolution laboratory imaging system (µCT50, Scanco Medical AG, Brüttisellen, Switzerland) in accordance with the American Society of Bone and Mineral Research (ASBMR) guidelines for the use of µCT in rodents [34]. Scans were acquired using a 10 µm3 isotropic voxel, 70 kVp and 114 µA peak x-ray tube potential and intensity, 500 ms integration time, and were subjected to Gaussian filtration. The subchondral bone analysis was performed by manually contouring the outline of the entire tibial epiphysis. A lower threshold of 375 mg HA/cm3 was used to evaluate trabecular bone volume fraction (BV/TV, mm3/mm3).

Joint immunohistochemistry

For TRAP and F4/80 staining, a subset of knee joints were evaluated.

Paraffin sections were used to evaluate osteoclasts in the subchondral bone by enzymatic TRAP staining [35]. Within the tibial epiphysis, we counted the total number of osteoclasts (# osteoclasts/mm), which was normalized by bone surface (BS). All measurements were performed using Osteomeasure software (OsteoMetrics, Decatur, GA, USA).

Immunohistochemistry was performed by deparaffinization, antigen retrieval, blocking with normal goat serum, incubating with primary anti-mouse F4/80 antibody (1:100, Abcam ab6640, RRID: AB_1140040) overnight, and developed with DAB staining the next day. Images were taken at 4x and 10x. For quantification, F4/80 images (at 10x) of the medial femoral, medial meniscus, and medial tibial synovium were input into FIJI/Image J (version 1.0). A threshold was set for positive signal detection, and number of regions of interest (ROIs) were counted. For each sample, the ROIs for all three locations in the medial synovium were summed.

DRG immunohistochemistry

DRG samples were collected from the lumbar spine of a separate cohort of WT female sham and PMX mice 12 weeks after surgery (n = 3 mice/group) and cryopreserved in O.C.T. Section (12 μm) were fixed in 4% PFA, blocked with 5% normal goat serum for 1.5 h, and incubated with primary antibody, rat-anti-mouse F4/80 (Abcam #6640), at 1:200 dilution overnight at 4 °C. Sections were washed 3 times with PBST (1% Tween-20) and incubated with fluorescent secondary antibody, AF546 anti-rat IgG (1:500), for 1 h at room temperature. Sections were washed 3x with PBS-T, counterstained with DAPI, and mounted with VectaShield Mounting medium. Immunofluorescence images were acquired with Fluoview FV10i confocal microscope at ×10 and ×30 magnification (Olympus Fluoview FV10- ASW Ver.04.02). F4/80 + signal was quantified semi-automatically by using the ‘Analyze particles’ function in FIJI (ImageJ, version 1.0). Briefly, after subtracting background and setting a positive signal threshold, F4/80 + particles were identified, and artifacts were manually eliminated, resulting in a signal count. Three 30x tissue areas per mouse were analyzed, the mean F4/80 + signal count was calculated per mouse, and the number of F4/80 + signal per mouse was normalized to tissue area (mm2). The scorer was blinded to groups.

Spinal immunohistochemistry

For spinal column evaluation the spinal column was collected in a subset of animals. This was done by hydrophobic exclusion, i.e., flushing the intact spine (vertebrae intact), with phosphate buffered saline (PBS), placing the spine in PFA overnight, then 30% sucrose overnight-3 days at 4 °C, and embedding in OCT. Upper lumber spinal column was cryosectioned at 10 μm and stained for chicken anti-mouse Green Fluorescent Protein (GFP) (Abcam) and DAPI (nuclei dye stain, Sigma). CSF1R-GFP + cells were manually counted in 5 separate areas of the spinal column from 60x images for Vehicle (n = 3) or AP20187 (n = 3) male MaFIA mice 8 weeks post DMM or from Vehicle-treated (n = 3) naïve mice as controls.

Flow cytometry

Ipsilateral L3-5 DRGs were dissected from DMM or PMX mice and pooled from two mice (3 ipsilateral affected DRGs per mouse = total 6 DRGs), i.e., when the mouse number n = 10, flow cytometry sample number n = 5. To yield at least 1 million cells, pooling of 6 DRGs was necessary [15]. Ipsilateral lumbar levels 3–5 DRG were selected because these are the knee and hind-paw innervating DRGs [36]. After dissection, DRGs were digested with collagenase type IV (1.6 mg/mL) and DNase I (200 µg/mL) shaking for 1 h at 37 °C. Following digestion, cells were counted, and 1 million cells were stained with an immune cell panel of anti-mouse antibodies: PE-CD45, AF700-CD3, BV711-CD11b, PE/Cy7-MHCII, PerCP/Cy5.5-Ly6G, APC-F4/80, BV421-CD163, BV605-CCR2 (BioLegend), endogenous signal for GFP-CSF1R, and Aqua-Live/Dead stain (ThermoFisher). After staining, sample data were acquired through an LSR Fortessa flow cytometer. For peripheral blood, additional antibodies were used for T cells, BV421-CD3, AF700-CD8, and PE-Cy7-CD4, and tissue macrophage markers F4/80, MHCII, and CD163 were excluded. Stained sample data were collected through the LSR Fortessa and analyzed by FlowJo software (version 10). See Supplementary Table 1 for detailed information on flow cytometry antibodies.

DRG bulk RNA-sequencing

A separate cohort of male MaFIA mice that underwent DMM surgery were treated 8 weeks after surgery with either vehicle (n = 5) or AP20187 (n = 5), as described above. Age-matched naïve mice (20-weeks old) were treated with vehicle (n = 5). After 5 consecutive days of treatment, mice were sacrificed and L3-5 ipsilateral DRGs were collected and lysed in Trizol. RNA was extracted using an RNeasy micro kit (Qiagen) and sent to LC Sciences for sequencing. RNA Integrity Number must have been greater than 6.5 to continue with Poly(A) RNA sequencing. Two samples had RNA integrity numbers less than 6.5 but were determined to be of sufficient quality based on their Electropherogram profile.

Poly(A) RNA sequencing library was prepared following Illumina’s TruSeq-stranded-mRNA sample preparation protocol. Paired-ended sequencing (150 bp) was performed on Illumina’s NovaSeq 6000 sequencing system (LC Sciences). HISAT2 was used to map reads to the Mus musculus reference genome (https://ftp.ensembl.org/pub/release-107/fasta/mus_musculus/dna/) [37]. The mapped reads of each sample were assembled using StringTie with default parameters, and StringTie and ballgown were used to estimate the expression levels of all transcripts and perform expression abundance for mRNAs by calculating FPKM (fragment per kilobase of transcript per million mapped reads) value. mRNA differential expression analysis was performed by R package DESeq2 [38] between two different groups (DMM AP vs. DMM vehicle; DMM vehicle vs. Naïve vehicle). The raw sequence data and counts matrices have been submitted to NCBI Gene Expression Omnibus (GEO) with accession number GSE246252.

The focus of our analysis here was to identify genes related to immune cell function that were regulated in OA (DMM-vehicle vs. naïve-vehicle) and were regulated after macrophage depletion (DMM-AP vs. DMM-vehicle). To do this, we generated a screening list of 180 genes related to immune cell function: 91 genes were selected as a result of a previous study that investigated immune changes in DRGs through qPCR [39], and to this list we added the remaining genes from the Il, Ilr, Ccl, Ccr, Cxcr, Cd, and Csf families, as well as other genes related to innate immune function in OA (S100a8, S100a9, Tlr2, Tlr4). Genes were selected for Table 1 if they were (1) expressed in the current study above 0.5 FPKM by samples in at least one treatment group, and (2) if they were regulated in the current study (DMM-vehicle vs. naïve-vehicle) or if they were regulated in at least 2 other published DRG datasets from OA models available from the literature [14, 39, 40]. The complete list of genes and the results of the differential expression analysis are provided in Supplemental Tables 2 and 3.

Table 1 Regulation of genes related to immune cell function in the dorsal root ganglia (DRG) in mouse models of OA
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Statistical analysis

Statistical calculations were performed using GraphPad Prism 9. Paired two-tailed t-tests were used for before and after macrophage depletion comparison for MaFIA mice pain-related behavior studies. For mechanical allodynia, since the von Frey fibers are on a log scale, the data was first log-transformed and then a paired student’s t test was used for pairwise comparison. Unpaired two-tailed student’s t-test was used for histology analysis, except for synovitis scores, where Mann-Whitney test was used. For flow cytometry, Mann-Whitney test was used. Unpaired two-tailed t tests were used in all other analyses. Mean ± 95% Confidence Interval (CI) is shown in all graphs unless otherwise stated, and p values are stated on each graph. P values were considered significant if less than 0.05.

Results

Systemic macrophage depletion reduces pain-related behaviors in both sexes

To determine if the acute depletion of macrophages in the DRG changes pain-related behaviors in established OA, we utilized MaFIA transgenic mice [25] to perform conditional macrophage depletion at time points after surgery when mice had developed joint damage, macrophage infiltration in the DRG [12, 14] (Suppl. Figure 1), and pain-related behaviors.

In brief, we tested mechanical allodynia and knee hyperalgesia in male MaFIA mice 7 weeks after DMM, followed by 5 consecutive days of treatment with AP20187 (10 mg/kg i.p.) or vehicle (Fig. 1A). We confirmed macrophage depletion when mice were taken down following the last behavioral test, 3 days after the last injection, by evaluating CSF1R-GFP + cells by flow cytometry of DRGs (Suppl. Figure 2) and peripheral blood (Suppl. Figure 3). We also checked if AP20187 depletion affected CSF1R + cells in the dorsal horn of the spinal cord, and found no difference in GFP + CSF1R + cells in the lumbar dorsal horn between AP20187 and vehicle-treated male DMM mice (Suppl. Figure 4). This suggests acute macrophage depletion has no effect on CSF1R + microglia in the central nervous system, as others have reported [16].

Immediately after macrophage depletion, osteoarthritic male mice, 8 weeks post DMM, had significantly improved mechanical allodynia (Fig. 2A) and knee hyperalgesia (Fig. 2B).

Fig. 2
figure 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

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We next asked if macrophage depletion would have the same effect in female mice with experimental OA. We performed PMX surgery in 12-week old female MaFIA mice, and depleted macrophages 11 weeks post PMX (Fig. 1C). This resulted in significantly attenuated mechanical allodynia (Fig. 2C), knee hyperalgesia (Fig. 2D). In female mice, we also assessed weightbearing deficits and found that macrophage depletion resulted in improved weight bearing on the ipsilateral knee (Fig. 2E) in AP20187-treated vs. vehicle-treated mice.

Acute macrophage depletion effects in the joint

We checked F4/80 + macrophage levels in the synovium in mice 8 weeks post DMM (3 days after the end of depletion) and found a significant decrease in F4/80 + synovial macrophages in knees from AP20187 vs. vehicle-treated mice (Suppl. Figure 5 A, B). Since CSF1R is reportedly expressed on osteoclasts [41], we evaluated if osteoclasts were affected by AP20187 treatment by staining for tartrate-resistant acid phosphatase (TRAP) in mice of both sexes. In addition, we evaluated whether depletion affected overall bone mass using micro-CT, because one study also reported changes in bone morphology within one week of depletion [18]. Eight weeks post DMM in male mice, acute macrophage depletion did not change the number of TRAP + osteoclasts (Suppl. Figure 5 C, D) nor the bone volume to trabecular volume (BV/TV) ratio (Suppl. Figure 5E). Likewise, we found no difference in the number of TRAP + osteoclasts (Suppl. Figure 5 F, G) or in BV/TV (Suppl. Figure 5 H) 12 weeks post PMX in female mice.

We next examined cartilage degeneration and synovitis scores. As seen in representative images of knee histopathology at 8-weeks post DMM from vehicle or AP20187 treated male mice (Fig. 3A), macrophage depletion did not affect the severity of medial cartilage degeneration (Fig. 3B), osteophyte width (Fig. 3C), or synovitis (Fig. 3D), including no difference in medial synovial hyperplasia (Fig. 3E), cellularity (Fig. 3F), or fibrosis (Fig. 3G). We found the same lack of effect in AP20187-treated female mice 12 weeks after PMX surgery compared to vehicle (Fig. 3H): there was no difference in medial cartilage degeneration (Fig. 3I), osteophyte width (Fig. 3J), or synovitis (Fig. 3K), including no difference in medial synovial hyperplasia (Fig. 3L), cellularity (Fig. 3M), or fibrosis (Fig. 3N). These data suggest acute macrophage depletion did not affect subchondral bone mass, the degree of cartilage degeneration, or synovitis in male or female mice with established OA.

Fig. 3
figure 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

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CSF1R + cells and neutrophils are reduced in the DRG in both sexes after macrophage depletion

To determine the immune cell changes in the DRG with and without macrophage depletion we used flow cytometry to examine DRG immune cell phenotypes. The representative DRG gating strategy is shown in Suppl. Figure 2 A. In DRGs from male mice, there was no change in the frequency of CD45 + leukocytes (Fig. 4A), CD11b + myeloid cells (Fig. 4B), or CD3 + T cells (Fig. 4C) between vehicle and AP20187 treated mice at 8-weeks post DMM. As expected, CSF1R+ (Fig. 4D, Suppl. Figure 2B) monocyte/macrophages were decreased. Ly6G + neutrophils (Fig. 4E) were also significantly decreased after macrophage depletion at 8-weeks post DMM surgery.

For female DRGs, there was a significant decrease in CD45 + leukocytes (Fig. 4F), and no difference in CD11b + myeloid cells (Fig. 4G) or CD3 + T cells (Fig. 4H) after macrophage depletion with AP20187 compared to vehicle, treated 12 weeks post PMX. CSF1R + monocyte/macrophages (Fig. 4I, Suppl. Figure 2 C) and Ly6G + neutrophils (Fig. 4J) were significantly decreased after macrophage depletion. Altogether, this shows that AP20187 treatment reduced CSF1R + cells (macrophages) as well as neutrophils in the DRG in both males and female with experimental OA, while overall CD45 + leukocytes were decreased in females only.

Fig. 4
figure 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

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Effect of macrophage depletion on DRG macrophage phenotypes

Finally, we aimed to characterize the functional phenotypes of DRG macrophages by evaluating the M1-like activated/pro-inflammatory MHCII + macrophages and M2-like anti-inflammatory CD163 + macrophages. There was a trending decrease in F4/80 + macrophages in AP20187-treated mice compared to vehicle in males post DMM (Fig. 5A). Under F4/80 + macrophages, we first looked at total F4/80 + MHCII + macrophages and F4/80 + CD163 + macrophages. We then made quadrants of MHCII and CD163 positive and negative populations, resulting in CD163 + MHCII-, CD163 + MHCII+, CD163-MHCII-, and CD163-MHCII + macrophage populations. In male DRGs, total MHCII + macrophages were significantly decreased at 8-weeks post DMM (Fig. 5B), while total F4/80 + CD163 + macrophages did not change (Fig. 5C). There were also no differences in CD163-MHCII- (Fig. 5D) or CD163 + MHCII- (Fig. 5G) at either timepoint. However, there was a significant decrease in the CD163-MHCII+ (Fig. 5E) and CD163 + MHCII+ (Fig. 5F) populations at 8-weeks post DMM in AP20187-treated vs. vehicle mice. This suggests a stronger decrease in MHCII + M1-like macrophages than CD163 + M2-like macrophages in DMM male mice DRGs.

In female DRGs, similar to males, there was a trending decrease in total F4/80 + macrophages in the DRG in AP20187-treated mice compared to vehicle (Fig. 5H). Total MHCII + macrophages were significantly decreased at 12-weeks post PMX (Fig. 5I) and total F4/80 + CD163 + macrophages did not change (Fig. 5J). There was no difference in CD163-MHCII- (Fig. 5K) or CD163 + MHCII- (Fig. 5N) populations, and a significant decrease in CD163-MHCII+ (Fig. 5L) and CD163 + MHCII+ (Fig. 5M) populations following depletion. This suggests, just like in males, that macrophage depletion with AP20187 compared to vehicle results in a significant decrease in MHCII + M1-like macrophages with little to no effect on CD163 + M2-like macrophages.

Fig. 5
figure 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

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Effect of macrophage depletion on pain-related behaviors and DRG cells in late-stage OA

To determine if macrophage depletion is also analgesic in late-stage OA, we used a new cohort of male mice and depleted macrophages 15 weeks post DMM (Suppl. Figure 6 A). Again, we found significant alleviation of mechanical allodynia (Suppl. Figure 6B) and knee hyperalgesia (Suppl. Figure 6 C). When we examined the DRGs by flow cytometry, we found similar patterns as before, with stronger decreases in MHCII + macrophages than in CD163 + macrophages following depletion, although we were limited in sample size in this cohort (n = 6 mice/group for behavioral tests resulted in n = 3/group for flow cytometry) (Suppl. Figure 6D-O).

Effect of macrophage depletion on DRG gene expression

To understand the molecular changes in the macrophage-depleted DRG, we conducted bulk RNA-sequencing analyses of DRGs from male mice 8-weeks after DMM surgery, with vehicle or with AP20187 treatment, and age-matched naïve controls treated with vehicle (n = 5 mice/group) (Suppl. Tables 2,3). We selected a screening list of 180 immune-related genes to focus on based on DRG molecular studies from the literature [12, 15, 39, 42,43,44,45,46]. Here, in DMM mice treated with vehicle compared to vehicle-treated naïve mice, we found Ccl2, Ccl3, Ccl7, Cd200r1, Cx3cr1, Cxcl10, Il1b, Il25, Il36g, S100a8, S100a9, and Tlr2, were all upregulated compared to naïve (Table 1, DMM-veh vs. naïve-veh). We also compared our results to other available published datasets [14, 39, 40], and looked for genes consistently regulated in at least 2 of the 5 OA datasets (4 published datasets or the newly presented DMM-veh vs. naïve-veh dataset here) (Table 1). Across the different datasets, Ccl2, Ccl3, Ccr5, Cx3cr1, Cd200r1, Csf1r, Il1b, and Il25 were consistently upregulated in the DRG with OA.

After macrophage depletion, chemoattractant genes such as Ccl2 and Ccl7 were further upregulated (Table 1, DMM-AP vs. DMM-veh), presumably as a response to return macrophages to the DRG, while the pro-inflammatory genes Il1b and Cxcl10 were downregulated (Table 1, Suppl. Figure 7 A, B, DMM-AP vs. DMM-veh), suggesting that the decrease in these genes contributed to the associated reduction in pain-related behaviors.

Finally, we also examined gene expression patterns to determine if M1-like or M2-like macrophage patterns changed with depletion. We found that signature genes expressed by CD163 + macrophages [47] were upregulated (Cd163, Fcrls, Mrc1), while genes expressed by MHCII + macrophages were downregulated (Ccr2, H2-Aa) (Table 2, Suppl. Figure 7 C-G), supporting the flow cytometry results that suggested that MHCII + macrophages were reduced with depletion and contributed to pain-related behaviors (Fig. 5).

Table 2 Macrophage marker genes regulated in the DRG after AP20187 treatment
Full size table

Acute macrophage depletion effects on body weight

As seen previously with systemic macrophage depletion [16, 25, 48], we did observe a significant drop in bodyweight in all groups of OA MaFIA mice treated with AP20187 compared to the respective Vehicle mice, while no difference in bodyweight was observed when AP20187 was administered to WT naïve mice (Suppl. Figure 8).

Discussion

In summary, targeting macrophages systemically in established experimental OA, when joint damage and pain-related behaviors have developed, resulted in reduced pain-related behaviors in both sexes. We observed no acute adverse effects on joint degeneration, synovitis, or osteophytes. Systemic depletion of CSF1R + cells reduced MHCII + M1-like pro-inflammatory macrophages in the DRG, while CD163 + MHCII- M2-like macrophage numbers were unchanged. In addition, numbers of Ly6G + neutrophils were also reduced following treatment, however, microglia in the dorsal horn of the spinal cord were not acutely affected by this depletion protocol. After examining differentially expressed genes in the DRG in various OA datasets across the literature [14, 39, 40], we confirmed the upregulated expression of several inflammatory chemokines and cytokines in the DRG, but only Il1b and Cxcl10 were reversed as a result of macrophage depletion in male DRGs at 8-weeks post DMM. Upregulation of these cytokines in the DRG has been implicated by previous work investigating other types of painful conditions [16, 46, 49,50,51,52], and together with the work here suggests these cytokines may play a role in supporting OA persistent pain via DRG macrophages.

Our observation that systemic depletion of CSF1R + cells attenuated pain-related behaviors is consistent with previous studies in other rodent models of knee OA. In the rat MIA model, an intravenous injection of Clophosome restored grip strength [17], and in the mouse MIA model, systemic ablation of macrophages through diphtheria toxin targeting of CSF1R + cells reduced hind paw mechanical allodynia and weight bearing asymmetry [13]. Repeated intra-articular injection of clodronate liposomes from week 2 after surgery also prevented the development of knee hyperalgesia and hind paw mechanical allodynia in the rat anterior cruciate ligament transection and destabilization of the medial meniscus (ACLT/DMM) surgery model [20]. This observation contrasts with a study using a mouse nerve injury model, where only systemic depletion, and not local nerve injury site depletion, of macrophages reduced hind paw mechanical allodynia [16]. Together, these results suggest that both targeting DRG and knee macrophages could be beneficial for reducing OA pain-related behaviors.

Because of previous concerns regarding the effects of macrophage depletion on joint pathology [18,19,20], we examined several different aspects of joint damage in this study. In both male and female mice, systemic depletion of macrophages did not result in worsening of cartilage degeneration, synovitis, osteophyte width, or subchondral bone mass when assessed approximately one week later. However, as others have noted [16, 25, 48], mice did lose a significant percentage of body weight as a result of the depletion. Our findings may have differed from previous work due to a difference in the location and/or timing of depletion. For instance, in a closed fracture model, intra-articular depletion of macrophages 2 days prior to injury resulted in increased synovitis and reduced bone mineral density one week later, whereas depletion at the time of injury had no effect on either measure [18]. Similarly, in the rat ACLT/DMM surgery model, repeated intra-articular depletion of synovial macrophages beginning 2 weeks post surgery resulted in increased synovial fibrosis, vascularization, and perivascular edema by 12 weeks post surgery [20]. Finally, when macrophage depletion was performed systemically at the time of surgery in a mouse model of OA induced by high fat diet and DMM surgery, this promoted increased synovitis by 9 weeks post depletion [19]. In contrast, in the collagenase-induced OA model, intra-articular depletion of macrophages prior to the injection of collagenase reduced synovial inflammation and fibrosis as well as osteophyte formation by days 7 and 14 [21]. Similarly, systemic depletion at the time of injury in an annular puncture induced disc herniation mouse model reduced ectopic bone formation in herniated discs and adjacent cortical bones 20 days later [48]. The local environment of immune cells changes rapidly following joint injury [53], and thus avoiding critical periods when macrophages are needed for resolution of joint inflammation may at least partially explain the differing effects observed. Here, systemic macrophage depletion was performed at a time point in the model when knee inflammation had already peaked [23, 24, 54, 55], which may explain why we did not observe any worsening of damage with depletion.

We have previously demonstrated that increased numbers of F4/80 + macrophages can be found in the DRG 8–16 weeks post DMM [12, 14]. Here, the reduction in pain-related behaviors observed following systemic ablation of CSF1R + cells was associated with a decrease specifically in the pro-inflammatory MHCII + macrophage population in the DRG in both sexes, as well as with downregulated expression of Il1b, Cxcl10, and M1 marker genes (Ccr2, H2-Aa) in the DRG. This is consistent with another study, where injection of M1-like macrophages adjacent to the DRG induced mechanical allodynia of the hind paw in naïve mice, while injection of M2-like macrophages reduced mechanical hypersensitivity in the MIA model [13]. However, it contrasts with the response in the DRG after systemic ablation in a model of disc herniation where increased numbers of F4/80 + macrophages and increased expression of inflammatory cytokines was observed in the DRG 6 days after injury [48]. Likewise, this previous study also found that systemic ablation promoted increased numbers of neutrophils at the site of disc herniation [48], while in the current study we found that depletion of CSF1R + cells resulted in a reduction of the Ly6G + neutrophil population within the DRG.

The homeostatic role of DRG macrophages, and the roles of macrophage infiltration and macrophage proliferation in the DRG in response to a peripheral injury have only begun to be explored [16, 47]. Recently, CD163- macrophages were shown to be replenished more rapidly by circulating monocytes than CD163 + macrophages [47]. Here, we found no differences in CD163 + MHCII- macrophages after macrophage depletion in males or females with OA, and upregulated gene expression of Cd163 in the DRG following depletion. In previous work [47], this population has been suggested to be a recently described tissue resident subset termed ‘TLF macrophages’ that are maintained primarily through self-renewal instead of through monocyte input [56].

There were several limitations in this study. While we used two different time points in the male DMM model in addition to the female PMX model, we did not collect tissues at different time points following macrophage depletion. In future work, it will be interesting to investigate the relationship between depletion of CSF1R + cells and the resulting changes in other cell populations such as neutrophils in the DRG. In addition, we assessed synovitis and F4/80 + cells in the joint, but we did not quantify changes in macrophage sub-populations in the joint. Finally, we only examined short-term effects on joint damage, but a strength of this study was that we intervened at time points when joint damage had already developed, which gave us some insight into the effects of targeting macrophages therapeutically.

Conclusions

In conclusion, acute systemic macrophage depletion in mid- to late-stages of surgical models of OA alleviated pain-related behaviors and had no acute effect on joint damage severity in mice of both sexes. We gained mechanistic insights from DRG immune phenotyping, where M1-like MHCII + macrophages were more affected by macrophage depletion than M2-like CD163 + MHCII- macrophages in both sexes. RNA sequencing analyses revealed that the pro-inflammatory gene Il1b was consistently upregulated in the DRG with OA here and in other published datasets, and this gene was downregulated in the DRG with macrophage depletion. Future studies will continue to investigate the molecular mechanisms of DRG macrophages and how they contribute to persistent pain during OA progression.

Data availability

Sequence data that support the findings of this study have been submitted to NCBI Gene Expression Omnibus (GEO) with accession number GSE246252. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

References

  1. Collaborators GBDO. Global, regional, and national burden of osteoarthritis, 1990–2020 and projections to 2050: a systematic analysis for the global burden of disease study 2021. Lancet Rheumatol. 2023;5(9):e508-22.

    Article  Google Scholar 

  2. Neogi T. The epidemiology and impact of pain in osteoarthritis. Osteoarthritis Cartilage. 2013;21(9):1145–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Altman RD, Gray R. Inflammation in osteoarthritis. Clin Rheum Dis. 1985;11(2):353–65.

    Article  CAS  PubMed  Google Scholar 

  4. Hunter DJ, Guermazi A, Roemer F, Zhang Y, Neogi T. Structural correlates of pain in joints with osteoarthritis. Osteoarthritis Cartilage. 2013;21(9):1170–8.

    Article  CAS  PubMed  Google Scholar 

  5. Dainese P, Wyngaert KV, De Mits S, Wittoek R, Van Ginckel A, Calders P. Association between knee inflammation and knee pain in patients with knee osteoarthritis: a systematic review. Osteoarthritis Cartilage. 2022;30(4):516–34.

    Article  CAS  PubMed  Google Scholar 

  6. Neogi T, Guermazi A, Roemer F, Nevitt MC, Scholz J, Arendt-Nielsen L, et al. Association of joint inflammation with pain sensitization in knee osteoarthritis: the multicenter osteoarthritis study. Arthritis Rheumatol. 2016;68(3):654–61.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Griffin TM, Scanzello CR. Innate inflammation and synovial macrophages in osteoarthritis pathophysiology. Clin Exp Rheumatol. 2019;37(Suppl 120):57–63.

    PubMed  PubMed Central  Google Scholar 

  8. Kraus VB, McDaniel G, Huebner JL, Stabler TV, Pieper CF, Shipes SW, et al. Direct in vivo evidence of activated macrophages in human osteoarthritis. Osteoarthritis Cartilage. 2016;24(9):1613–21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Daghestani HN, Pieper CF, Kraus VB. Soluble macrophage biomarkers indicate inflammatory phenotypes in patients with knee osteoarthritis. Arthritis Rheumatol. 2015;67(4):956–65.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Haraden CA, Huebner JL, Hsueh MF, Li YJ, Kraus VB. Synovial fluid biomarkers associated with osteoarthritis severity reflect macrophage and neutrophil related inflammation. Arthritis Res Ther. 2019;21(1):146.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Miller RJ, Malfait AM, Miller RE. The innate immune response as a mediator of osteoarthritis pain. Osteoarthritis Cartilage. 2020;28(5):562–71.

    Article  CAS  PubMed  Google Scholar 

  12. Miller RE, Tran PB, Das R, Ghoreishi-Haack N, Ren D, Miller RJ, et al. CCR2 chemokine receptor signaling mediates pain in experimental osteoarthritis. Proc Natl Acad Sci U S A. 2012;109(50):20602–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Raoof R, Martin Gil C, Lafeber F, de Visser H, Prado J, Versteeg S, et al. Dorsal root ganglia macrophages maintain osteoarthritis pain. J Neurosci. 2021;41(39):8249–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Miller RE, Tran PB, Ishihara S, Syx D, Ren D, Miller RJ, et al. Microarray analyses of the dorsal root ganglia support a role for innate neuro-immune pathways in persistent pain in experimental osteoarthritis. Osteoarthritis Cartilage. 2020;28(5):581–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Geraghty T, Obeidat AM, Ishihara S, Wood MJ, Li J, Lopes EBP, et al. Age-associated changes in knee osteoarthritis, pain-related behaviors, and dorsal root ganglia immunophenotyping of male and female mice. Arthritis Rheumatol. 2023;75(10):1770–80.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Yu X, Liu H, Hamel KA, Morvan MG, Yu S, Leff J, et al. Dorsal root ganglion macrophages contribute to both the initiation and persistence of neuropathic pain. Nat Commun. 2020;11(1):264.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sakurai Y, Fujita M, Kawasaki S, Sanaki T, Yoshioka T, Higashino K, et al. Contribution of synovial macrophages to rat advanced osteoarthritis pain resistant to cyclooxygenase inhibitors. Pain. 2019;160(4):895–907.

    Article  CAS  PubMed  Google Scholar 

  18. Bailey KN, Furman BD, Zeitlin J, Kimmerling KA, Wu CL, Guilak F, et al. Intra-articular depletion of macrophages increases acute synovitis and alters macrophage polarity in the injured mouse knee. Osteoarthritis Cartilage. 2020;28(5):626–38.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wu CL, McNeill J, Goon K, Little D, Kimmerling K, Huebner J, et al. Conditional macrophage depletion increases inflammation and does not inhibit the development of osteoarthritis in obese macrophage Fas-induced apoptosis-transgenic mice. Arthritis Rheumatol. 2017;69(9):1772–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Blackler G, Lai-Zhao Y, Klapak J, Philpott HT, Pitchers KK, Maher AR, et al. Targeting STAT6-mediated synovial macrophage activation improves pain in experimental knee osteoarthritis. Arthritis Res Ther. 2024;26(1):73.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Blom AB, van Lent PL, Holthuysen AE, van der Kraan PM, Roth J, van Rooijen N, et al. Synovial lining macrophages mediate osteophyte formation during experimental osteoarthritis. Osteoarthritis Cartilage. 2004;12(8):627–35.

    Article  PubMed  Google Scholar 

  22. Glasson SS, Blanchet TJ, Morris EA. The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse. Osteoarthritis Cartilage. 2007;15(9):1061–9.

    Article  CAS  PubMed  Google Scholar 

  23. von Loga IS, Batchelor V, Driscoll C, Burleigh A, Chia SL, Stott B, et al. Does pain at an earlier stage of chondropathy protect female mice against structural progression after surgically induced osteoarthritis? Arthritis Rheumatol. 2020;72(12):2083–93.

    Article  Google Scholar 

  24. Knights CB, Gentry C, Bevan S. Partial medial meniscectomy produces osteoarthritis pain-related behaviour in female C57BL/6 mice. Pain. 2012;153(2):281–92.

    Article  PubMed  Google Scholar 

  25. Burnett SH, Kershen EJ, Zhang J, Zeng L, Straley SC, Kaplan AM, et al. Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene. J Leukoc Biol. 2004;75(4):612–23.

    Article  CAS  PubMed  Google Scholar 

  26. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods. 1994;53(1):55–63.

    Article  CAS  PubMed  Google Scholar 

  27. Leuchtweis J, Imhof AK, Montechiaro F, Schaible HG, Boettger MK. Validation of the digital pressure application measurement (PAM) device for detection of primary mechanical hyperalgesia in rat and mouse antigen-induced knee joint arthritis. Methods Find Exp Clin Pharmacol. 2010;32(8):575–83.

    Article  CAS  PubMed  Google Scholar 

  28. Miller RE, Ishihara S, Bhattacharyya B, Delaney A, Menichella DM, Miller RJ, et al. Chemogenetic inhibition of pain neurons in a mouse model of osteoarthritis. Arthritis Rheumatol. 2017;69(7):1429–39.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Obeidat AM, Wood MJ, Adamczyk NS, Ishihara S, Li J, Wang L, et al. Piezo2 expressing nociceptors mediate mechanical sensitization in experimental osteoarthritis. Nat Commun. 2023;14(1):2479.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Miller RE, Tran PB, Ishihara S, Larkin J, Malfait AM. Therapeutic effects of an anti-ADAMTS-5 antibody on joint damage and mechanical allodynia in a murine model of osteoarthritis. Osteoarthritis Cartilage. 2016;24(2):299–306.

    Article  CAS  PubMed  Google Scholar 

  31. Glasson SS, Chambers MG, Van Den Berg WB, Little CB. The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the mouse. Osteoarthritis Cartilage. 2010;18(Suppl 3):S17-23.

    Article  PubMed  Google Scholar 

  32. Obeidat AM, Ishihara S, Li J, Adamczyk NS, Lammlin L, Junginger L, et al. Intra-articular sprouting of nociceptors accompanies progressive osteoarthritis: comparative evidence in four murine models. Front Neuroanat. 2024;18:1429124.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Obeidat AM, Kim SY, Burt KG, Hu B, Li J, Ishihara S, et al. A standardized approach to evaluation and reporting of synovial histopathology in two surgically induced murine models of osteoarthritis. Osteoarthritis Cartilage. 2024;32(10):1273-1282.

  34. Bouxsein ML, Boyd SK, Christiansen BA, Guldberg RE, Jepsen KJ, Muller R. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J Bone Min Res. 2010;25(7):1468–86.

    Article  Google Scholar 

  35. Ko FC, Karim L, Brooks DJ, Bouxsein ML, Demay MB. Bisphosphonate withdrawal: effects on bone formation and bone resorption in maturing male mice. J Bone Min Res. 2017;32(4):814–20.

    Article  CAS  Google Scholar 

  36. Miller RE, Kim YS, Tran PB, Ishihara S, Dong X, Miller RJ, et al. Visualization of peripheral neuron sensitization in a surgical mouse model of osteoarthritis by in vivo calcium imaging. Arthritis Rheumatol. 2018;70(1):88–97.

    Article  CAS  PubMed  Google Scholar 

  37. Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015;12(4):357–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Martin Gil C, Raoof R, Versteeg S, Willemen H, Lafeber F, Mastbergen SC, et al. Myostatin and CXCL11 promote nervous tissue macrophages to maintain osteoarthritis pain. Brain Behav Immun. 2024;116:203–15.

    Article  CAS  PubMed  Google Scholar 

  40. Bangash MA, Alles SRA, Santana-Varela S, Millet Q, Sikandar S, de Clauser L, et al. Distinct transcriptional responses of mouse sensory neurons in models of human chronic pain conditions. Wellcome Open Res. 2018;3:78.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Mun SH, Park PSU, Park-Min KH. The M-CSF receptor in osteoclasts and beyond. Exp Mol Med. 2020;52(8):1239–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Miller RE, Belmadani A, Ishihara S, Tran PB, Ren D, Miller RJ, et al. Damage-associated molecular patterns generated in osteoarthritis directly excite murine nociceptive neurons through toll-like receptor 4. Arthritis Rheumatol. 2015;67(11):2933–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Miller RE, Ishihara S, Tran PB, Golub SB, Last K, Miller RJ, et al. An aggrecan fragment drives osteoarthritis pain through toll-like receptor 2. JCI Insight. 2018;3(6):e95704.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Tran PB, Miller RE, Ishihara S, Miller RJ, Malfait AM. Spinal microglial activation in a murine surgical model of knee osteoarthritis. Osteoarthritis Cartilage. 2017;25(5):718–26.

    Article  CAS  PubMed  Google Scholar 

  45. van der Vlist M, Raoof R, Willemen H, Prado J, Versteeg S, Martin Gil C, et al. Macrophages transfer mitochondria to sensory neurons to resolve inflammatory pain. Neuron. 2022;110(4):613–26 e9.

    Article  PubMed  Google Scholar 

  46. Ebbinghaus M, Uhlig B, Richter F, von Banchet GS, Gajda M, Bräuer R, et al. The role of interleukin-1β in arthritic pain: main involvement in thermal, but not mechanical, hyperalgesia in rat antigen-induced arthritis. Arthritis Rheum. 2012;64(12):3897–907.

    Article  CAS  PubMed  Google Scholar 

  47. Lund H, Hunt MA, Kurtovic Z, Sandor K, Kagy PB, Fereydouni N, et al. CD163 + macrophages monitor enhanced permeability at the blood-dorsal root ganglion barrier. J Exp Med. 2024;221(2):e20230675.

    Article  CAS  PubMed  Google Scholar 

  48. Xiao L, Matharoo J, Chi J, Ma J, Chen M, Manley B, et al. Transient depletion of macrophages alters local inflammatory response at the site of disc herniation in a transgenic mouse model. Osteoarthritis Cartilage. 2023;31(7):894–907.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Chen Y, Yin D, Fan B, Zhu X, Chen Q, Li Y, et al. Chemokine CXCL10/CXCR3 signaling contributes to neuropathic pain in spinal cord and dorsal root ganglia after chronic constriction injury in rats. Neurosci Lett. 2019;694:20–8.

    Article  CAS  PubMed  Google Scholar 

  50. Lee HL, Lee KM, Son SJ, Hwang SH, Cho HJ. Temporal expression of cytokines and their receptors mRNAs in a neuropathic pain model. Neuroreport. 2004;15(18):2807.

    CAS  PubMed  Google Scholar 

  51. Li M, Shi J, Tang JR, Chen D, Ai B, Chen J, et al. Effects of complete Freund’s adjuvant on immunohistochemical distribution of IL-1beta and IL-1R I in neurons and glia cells of dorsal root ganglion. Acta Pharmacol Sin. 2005;26(2):192–8.

    Article  CAS  PubMed  Google Scholar 

  52. Steain M, Gowrishankar K, Rodriguez M, Slobedman B, Abendroth A. Upregulation of CXCL10 in human dorsal root ganglia during experimental and natural varicella-zoster virus infection. J Virol. 2011;85(1):626–31.

    Article  CAS  PubMed  Google Scholar 

  53. Knights AJ, Farrell EC, Ellis OM, Song MJ, Appleton CT, Maerz T. Synovial macrophage diversity and activation of M-CSF signaling in post-traumatic osteoarthritis. bioRxiv. 2023:2023.10.03.559514.

  54. Jackson MT, Moradi B, Zaki S, Smith MM, McCracken S, Smith SM, et al. Depletion of protease-activated receptor 2 but not protease-activated receptor 1 may confer protection against osteoarthritis in mice through extracartilaginous mechanisms. Arthritis Rheumatol. 2014;66(12):3337–48.

    Article  CAS  PubMed  Google Scholar 

  55. Sambamurthy N, Zhou C, Nguyen V, Smalley R, Hankenson KD, Dodge GR, et al. Deficiency of the pattern-recognition receptor CD14 protects against joint pathology and functional decline in a murine model of osteoarthritis. PLoS One. 2018;13(11):e0206217.

    Article  PubMed  PubMed Central  Google Scholar 

  56. Dick SA, Wong A, Hamidzada H, Nejat S, Nechanitzky R, Vohra S, et al. Three tissue resident macrophage subsets coexist across organs with conserved origins and life cycles. Sci Immunol. 2022;7(67):eabf7777.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We would like to thank the Rush University Flow cytometry core and the Comparative Research Center.

Funding

National Institutes of Health (NIH), National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) funding supported this work: (T32AR073157 and F32AR081129, T.G.); (F31AR083277, N.S.A.); (R01AR060364, R01AR064251, P30AR079206, A.M.M.); (R01AR077019, R01AR077019-03S1, R.E.M.).

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Authors and Affiliations

  1. Department of Internal Medicine, Division of Rheumatology, Rush University Medical Center, Chicago, IL, USA

    Terese Geraghty, Shingo Ishihara, Alia M. Obeidat, Natalie S. Adamczyk, Rahel S. Hunter, Jun Li, Lai Wang, Anne-Marie Malfait & Rachel E. Miller

  2. Chicago Center on Musculoskeletal Pain, Chicago, IL, USA

    Terese Geraghty, Shingo Ishihara, Alia M. Obeidat, Natalie S. Adamczyk, Rahel S. Hunter, Jun Li, Lai Wang, Hoomin Lee, Frank C. Ko, Anne-Marie Malfait & Rachel E. Miller

  3. Department of Anatomy & Cell Biology, Rush University Medical Center, Chicago, IL, USA

    Hoomin Lee & Frank C. Ko

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Contributions

Conceptualization: T.G., A.M.M., R.E.M. Investigation: T.G., S.I., A.M.O., J.L., L.W., N.S.A, H.L., F.K. Formal analysis: T.G., R.H., A.M.O., H.L., F.K., R.E.M. Visualization and data presentation: T.G., R.E.M. Writing: T.G. A.M.M., R.E.M. Reviewing and editing, T.G., A.M.M., R.E.M. Funding acquisition: T.G., A.M.M., R.E.M.

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Correspondence to Rachel E. Miller.

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All animal experiments were approved by the Rush University Institutional Animal Care and Use Committee.

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T.G., S.I., A.M.O., J.L., L.W., N.S.A, R.H., H.L., F.K., and R.E.M have no competing interests. A.M.M. has received consulting fees from LG, Averitas, and Orion.

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Geraghty, T., Ishihara, S., Obeidat, A.M. et al. Acute systemic macrophage depletion in osteoarthritic mice alleviates pain-related behaviors and does not affect joint damage. Arthritis Res Ther 26, 224 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13075-024-03457-9

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13075-024-03457-9

Keywords

Arthritis Research & Therapy

ISSN: 1478-6362

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