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

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

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 OAStatistical 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.