Complement factor D (adipsin) mediates pressure-pain hypersensitivity post destabilization of medial meniscus injury

Complement factor D (adipsin) mediates pressure-pain hypersensitivity post destabilization of medial meniscus injury | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Complement factor D (adipsin) mediates pressure-pain hypersensitivity post destabilization of medial meniscus injury Priscilla Tjandra, Bethany Andoko, Jooyoung Kim, Andreana Gomez, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6702118/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Nov, 2025 Read the published version in Arthritis Research & Therapy → Version 1 posted 11 You are reading this latest preprint version Abstract BACKGROUND: Although osteoarthritis (OA) is the leading cause of pain and disability worldwide, there is a lack of models to probe the separable mechanism of OA structural damage and knee pain. We previously identified that deletion of complement factor D (FD) results in increased pressure-pain hyperalgesia despite cartilage protection after destabilization of the medial meniscus (DMM) surgery. However, how these discordant OA phenotypes manifest is not understood. We employed a novel targeted lipidomics approach to elucidate the role of eicosanoids in FD-mediated pain. We hypothesize that the absence of Cfd (FD -/- ) will protect cartilage but cause increased pressure-pain hyperalgesia and eicosanoid dysregulation that persists throughout OA development. METHODS: Male and female FD -/- and wild-type (WT) mice were challenged with DMM or remained naïve (n=5-11/group) at 16 weeks old. Pressure-pain hyperalgesia was measured bi-weekly for 8 weeks post-DMM. A second cohort was evaluated at 2 weeks post-DMM (n=6-10/group) to investigate DMM injury response. Structural damage was scored using the Modified Mankin system. To determine changes in eicosanoid profiles, serum and synovial fluid samples were analyzed via liquid chromatography-mass spectrometry (LC-MS). Statistical analysis was performed with unpaired t-test, two-way, or three-way ANOVA with Sidak’s posthoc test. Statistical significance is defined as p<0.05. RESULTS: In contrast to WT mice, FD -/- showed no significant differences in Modified Mankin scores 8 weeks post-DMM. As expected, FD -/- hyperalgesia levels persisted until 8 weeks post DMM, similar to WT. Changes in eicosanoid profiles of pain-associated factors in FD -/- when compared to WT were found in the synovial fluid at 2 weeks and the serum at 8 weeks post-DMM. CONCLUSION: The absence of Cfd drives knee hyperalgesia in male and female mice at 2 weeks-post DMM and persists through an 8-week observation period despite observing cartilage protection. Changes of eicosanoid profiles at both time points suggest that FD drives pain acutely, and the hyperalgesia phenotype emerges early in response to DMM injury, elucidating the role of the alternative complement in mediating OA pain and structural damage. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Osteoarthritis (OA), a disease characterized by the loss of cartilage lining the joint, is the leading cause of pain and disability worldwide 1 , 2 . Although pain is the primary driver for patients to seek care, current pain management strategies are inadequate, highlighting the need for new drug targets. However, disentangling the mechanism of OA pain from injury and disease entrenchment is challenging. For example, pain severity is not always a result of structural damage to the joint 3 . Until recently, most studies that employ preclinical models to study OA pathogenesis rely on structural characterization of the joint and often omit pain and behavioral changes. To address this gap in knowledge, we and others 4 routinely perform pain phenotyping in all OA preclinical models to understand the concordant or divergent pain and structure phenotypes that manifest with OA in mice. Complement factor D (FD), also known as adipsin, is a serine protease that cleaves complement factor B to activate alternative complement signaling, a key pathway in the innate immune response 5 , 6 . FD is primarily secreted from adipose tissue, which our lab has demonstrated is a key driver of OA pathogenesis and pain 7 . Although prior studies have determined that the loss of several complement signaling factors was protective for cartilage 8 – 10 , we have recently demonstrated that FD −/− mice displayed pronounced pressure-pain hyperalgesia, despite cartilage protection with DMM 11 . This model provides the opportunity to decode the discordance between structure and pain reported clinically and establishes FD as a key factor regulating fat-cartilage crosstalk in OA pain. In the present study, we leveraged this model of discordant pain and structural damage phenotype to investigate and better understand the time course changes and mechanism of pressure-pain hyperalgesia post-DMM. Clinical data indicate that there is a dimorphism in OA, such that female patients can report more severe pain for a given amount of structural damage 12 . However, many in the field of OA believe female mice are a poor model to study OA, due to more modest structural changes in cartilage damage requiring a higher sample size to observe significant differences in histological OA scoring. Of note, several studies demonstrate that indeed female mice do develop hallmarks of OA in response to DMM 13 , 14 . Previously, increased synovial fluid C5 levels were associated with increased complement activation in male patients in late-stage knee OA compared to female patients 15 . Moreover, early complement activation has been reported to be higher in male humans and macaques 16 . These findings are also corroborated in mice and are thought to be due to restricted pathway components to promote inflammation through complement C5b-9 complex in female mice 17 . Therefore, in this study, we leverage the FD −/− model to gain insight into dimorphisms in pain regulation due to DMM, as it is known that innate differences in the modulation of pain – male mice may not adequately represent pain in female mice 4 , 18 , 19 . Therefore, a secondary aim of this study is to leverage the discordance in structural damage and pain in FD −/− mice to better understand potential sexually dimorphic pain responses due to DMM. Current treatments for pain in early OA have been limited to non-steroidal anti-inflammatory drugs (NSAID) 20 – 22 . However, the efficacy of these drugs can be ineffective in OA patients for long-term use 23 . NSAIDs have been established as modulators of the complement system 24 . This same targeted eicosanoid panel 25 demonstrated that alterations in the levels of several eicosanoid species played a key role in the transition from acute to chronic hypersensitivity in the K/BxN serum transfer model of rheumatoid arthritis through toll-like receptor (TLR) 4, another key regulator of innate immunity 26 . Previously, crosstalk between TLRs and complement signaling has been demonstrated to coordinate synergistic or exaggerated immune responses 27 . Studies have also shown that eicosanoids can be deployed downstream of complement signaling to assist with clearance and the inflammatory response to pathogens 28 . To begin to elucidate the complement-mediated mechanisms that regulate pain in DMM-induced OA immediately after injury, we used a novel targeted approach in assessing eicosanoids, lipid-derived metabolites associated with pain, that are modulated by NSAIDs 25 , 29 – 31 and can be downstream of complement signaling. As systemic soluble mediators appear to drive fat-cartilage crosstalk and OA after DMM 7 , we posit that FD −/− mice may demonstrate alterations in pain-driving and pain-relieving through eicosanoid mediators that act through these signaling molecules to modulate pain. Leveraging this novel eicosanoid profiling approach will give mechanistic insights to systemic and local cellular mechanisms related to pain that could lead to the development of novel therapeutic targets. The purpose of this study was to determine the role of FD in the onset and persistence of pain in male and female mice acutely 2 weeks after DMM, and chronically after 8 weeks of DMM. We hypothesize that the absence of FD −/− will protect cartilage but result in pressure-pain hyperalgesia, which will be detectable 2 weeks post-DMM. We also hypothesize that hyperalgesia due to loss of FD would be more pronounced in male mice compared to female mice. As an exploratory hypothesis, we sought to test if eicosanoid dysregulation due to loss of FD is related to the pressure-pain hyperalgesia phenotype, potentially indicating novel fat- and lipid-derived targets that can be probed for the development of novel therapeutic strategies. METHODS Animal Studies FD − /− mice 6 (provided kindly by J. Atkinson and X. Wu; Washington University in St. Louis) were bred and maintained at the animal facility at the University of California, San Francisco. All experimental procedures were approved by the University of California, San Francisco Institutional Animal Care and Use Committee. Male and female FD −/− and wild-type (WT) (Jackson Labs #005304) control mice were challenged with destabilization of the medial meniscus (DMM) on the left knee joints or remained naïve at 16 weeks old (n = 5–11/group) as a control. Mice were sacrificed at either 2 weeks or 8 weeks post-DMM, 18 weeks or 24 weeks old in total age, at which time serum, synovial fluid, knee joints, and DRGs were collected. A timeline of these studies is presented in Fig. 1 a. Knee Joint Assessment Knee joints were prepared according to previous methods 7 , 11 . In brief, joints were fixed in 4% paraformaldehyde for 24 hours and stored in 70% ethanol at 4C°. After analyzing bone microstructure, joints were decalcified in 10% formalin solution (Cal-Ex II) for 10 hours before being processed and embedded in paraffin wax. Knee joint sections were cut at 5µm thickness in the frontal plane and stained with Safranin-O/Fast Green or Hematoxylin and Eosin (H&E). Histological assessment was performed using Modified Mankin score, Osteophyte score, and Synovitis score as previously described 7 , 11 . Bone Microstructure Analysis Whole knee joints were scanned by micro-computed tomography (SCANCO µCT50) and imaged according to the guidelines for µCT of rodent bone (energy = 55 kVP, intensity = 114 mA, 6 µm nominal voxel size, integration time = 900ms) 32 at the Skeletal Biology and Biomechanics Core at University of California, San Francisco. Analysis of the trabecular bone in the medial and lateral tibial plateau was performed by manually contouring 2D transverse slices in the region between the growth plate and the subchondral bone. Trabecular bone volume fraction (BV/TV) and bone mineral density (BMD) were determined using the manufacturer’s analysis software. Subchondral bone was assessed similarly to exclude trabecular bone in the region between the distal surface of the femoral condyles and the growth plate. Subchondral bone thickness was determined using Fiji software as previously described 33 – 35 . Immunohistochemistry to Quantify Sensory and Sympathetic Neurites To determine the presence of and changes in neurites in the knee joint, immunohistochemistry of calcitonin gene-related peptide (CGRP) positive and tyrosine hydroxylase (TH) positive neurons was performed as described 36 . Briefly, knee joints were embedded in paraffin wax and cut in 20µm sections at the frontal plane. Sections were blocked in 10% donkey serum and Triton-X buffer before incubation with anti-CGRP and TH primary antibodies (Bio-Rad, Millipore Sigma) overnight at 4°C. Following 3 washes of TNT buffer, sections were incubated in Alexa Fluor 647® and Alexa Fluor® 594 secondary antibodies (Jackson ImmunoResearch) for three hours at room temperature. The sections were then washed three times before incubation in DAPI (Sigma Aldrich) for 5 minutes. Three final washes were done before mounting with Invitrogen Fluoromount-G Mounting Medium (Fisher Scientific). Serial tiled images were taken on 10x objective images using confocal microscopy (Leica DMi8 Inverted Microscope). The region of interest was defined as the area between the synovial lining at the medial femoral quadrant, medial tibial quadrant, lateral femoral quadrant, and lateral tibial quadrant. Images were processed in Fiji to exclude bone marrow. SNT for neuroanatomy plug-in was used to quantify positive signals by manual tracing 33 , 37 . Total pixels of positive CGRP and TH signal were reported. Pain Assessments and Behavioral Testing Pain assessments were conducted one week before DMM and 2-, 4-, 6-, and 8-weeks post-surgery in the order from least to most invasive at the same time of day. All mice were acclimatized to the behavioral suite and equipment prior to testing. Pressure-pain hyperalgesia was measured using a Small Animal Algometer 7 , 11 (SMALGO, Bioseb). Three to five trials of the surgical limb and nonsurgical limb was collected by applying a steady force to the lateral aspect of each limb until the mouse showed signs of discomfort such as squeaking, paw withdrawal, or grimacing. The average of these trials was reported and a maximum value of 450g was employed to avoid tissue damage to the joint 7 . Side-to-side limb loading was measured via static incapacitance (Bioseb). Mice were placed in the restrainer to acclimate for ~ 2 minutes. Load bearing measurements for each limb were taken once the mouse was calm, had both feet on each of the force sensors, and had its paws placed on the ramp at the front. Incapacitance is measured as the difference between the right and left limbs. Three to five trials were measured, and the average was reported. To assess mechanical allodynia, a Von Frey assay was used as described 38 , 39 . Mice were placed in a box with a wire mesh bottom and left to acclimate for 20 minutes. Once acclimated, force was applied to the mid-plantar of the surgical limb paw three times using one in a series of five Von Frey filaments ranging from 0.16g to 1.4g. Paw withdrawal was noted as either “positive” or “negative response”. This was then repeated with each of the five Von Frey filaments, with one repeating filament for a total of 6. The order of filaments was random between observers. Paw withdrawal patterns were assessed as previously described to determine the average 50% paw withdrawal threshold from the three repetitions 38 . To assess motor coordination, mice were placed on a rotarod wheel with an initial speed of 4 rpm. Using the ramp function, the speed increased to a maximum of 40 rpm in 120 seconds. The time and the maximum speed at which the mouse fell were noted. The average of three to five trials was reported 40 . Eicosanoid Profiling by Targeted Lipidomics Serum and synovial fluid 41 samples were collected and extracted from male DMM mice to determine systemic and local changes in eicosanoid profiles using a novel dual extraction method that separates proteins from metabolites from a single sample 11 . In brief, all samples were extracted with methanol, vortexed, and placed at -20°C for 30 minutes to promote protein precipitation. Next, the supernatant containing metabolites was collected and dried via vacuum concentration. A 5 µL aliquot of each sample was subjected to targeted metabolomic panel of 40 known eicosanoids, analyzed at the Quantitative Metabolite Analysis Center at the University of California, San Francisco 25 on a Shimadzu 30-AD UPLC in series with a SCIEX 7500 Triple Quadrupole Mass Spectrometer. Analytes were chromatographically separated using a Kinetex 2.6 µm Polar C18 100Å, liquid chromatography (LC) column 100 x 3.0 mm (Phenomenex, cat #00D-4759-Y0) with a mobile phase scheme of [A] water + 0.1% formic acid and [B] methanol + 0.1% formic acid. The LC method was set to a constant flow rate of 500 µL/min, and the timed linear gradient program consisted of: time = 0 min, 0.10% B, time = 0.1 min, 45% B, time = 2 min, 45% B, time = 16.5 min, 80% B, time = 16.6 min, 98% B, time = 18.5 min, 98% B, time = 18.6 min, 10% B, and time = 20.5, 10% B. Data was collected using polarity switching with the following source parameters: CUR = 40, GS1 = 60, GS2 = 70, Temp = 350 o C, ISVF = 4500V (negative mode) and 5500V (positive mode). Optimized multiple-reaction monitoring (MRM) pairs detailed in (Supplementary Tables 1 and 2) were used with a total cycle time of 0.937 seconds, dwell time of 1 ms, settling time = 15 ms, and pause time of 5.007ms. This method was based off of SCIEX’s comprehensive targeted method for lipid mediator analysis application note. Raw data was processed using a built-in SCIEX OS software package (version 2.1.6.59781) for peak picking, alignment, and quantitation. Statistical Analysis All results are reported as mean ± standard error of the mean. Results from pain and behavior assays were analyzed with three-way analysis of variance (ANOVA) stratified by time point, surgery, and strain. A separate two-way ANOVA with Tukey’s post-hoc test was run by genotype and limb to determine differences within each time point. Nonparametric Spearman’s correlations were calculated to determine correlative relationships between pressure-pain threshold and osteophyte score, synovitis score, or significant eicosanoids as identified above. Lipidomic data was analyzed using an unpaired t-test on normalized abundances between WT and FD −/− mice. All other outcomes are evaluated using two-way ANOVA with Sidak’s post hoc test with strain and surgery as main effects. Heat maps and PLS-DA plots of lipidomic data were created using MetaboAnalyst. Statistical significance is defined as p < 0.05. Statistical analyses were performed using GraphPad Prism version 10.0.0 for Mac (GraphPad Software, Boston, Massachusetts USA, www.graphpad.com ). RESULTS Male and female FD −/− mice displayed cartilage protection after DMM surgery As expected, FD −/− male and female mice were protected from DMM-induced structural damage as shown in images of the highest Mankin Scores in each group (Fig. 1 b). Concordant with previous analysis at 12-weeks post-DMM 11 , male WT mice demonstrated significantly greater Modified Mankin scores between DMM and naïve groups at 8 weeks post-DMM (p < 0.001), FD −/− mice displayed no significant differences between DMM and naïve groups (Fig. 1 c). Similarly, female WT mice exhibited greater Modified Mankin scores at both 2 and 8 weeks (p = 0.01, p = 0.001), although there were no cartilage changes in FD −/− mice. Despite cartilage protection in FD −/− mice, significant osteophyte formation was present in both strains. WT and FD −/− DMM groups of male and female mice had greater osteophyte scores compared to the naïve controls at 2 weeks (male: p < 0.001, p < 0.01; female: p < 0.001, p < 0.0001) and 8 weeks (male: p < 0.01, p = 0.02; female: p = 0.01, p < 0.01) post-DMM (Fig. 1 c). Similarly, synovitis scores were greater in all DMM groups compared to the naïve control at both 2 weeks (male: WT p < 0.01, FD −/− p < 0.001; female: WT p < 0.001, FD −/− p < 0.001) and 8 weeks (male: WT p < 0.01, FD −/− <0.001; female: WT p < 0.001, FD −/− p < 0.001) post-DMM (Fig. 2 ). Interestingly, FD −/− presented worsening synovitis scores compared to WT at 2 weeks post-DMM (p = 0.02) (Fig. 2 ). Analysis of bone changes revealed subchondral bone thickening in the lateral posterior compartment in WT (p = 0.01), but not FD −/− male at 8 weeks post-DMM (Fig. 3 ). There were no significant differences in subchondral thickness at 2 weeks post-DMM, or in female mice. There were no differences in BV/TV of the proximal tibial epiphysis due to surgery, strain, or sex (Supplementary Fig. 1). Preliminary analysis of a subset of joints to quantify CGRP + and TH + neurites in the joint did not demonstrate any clear changes between surgical groups, strain, or sex in a subset of animals (Supplementary Fig. 2). Pressure-pain Hyperalgesia was increased in FD −/− mice at 2-weeks, and persisted to 8 weeks Pain and behavior assays were conducted one week before DMM at baseline, and 2, 4, 6, and 8 weeks after surgery. The surgical limb of WT and FD −/− DMM male mice displayed decreases in pressure-pain threshold as early as 2 weeks and through 8 weeks post-DMM when compared to their contralateral limb (WT: p = < 0.001, p = 0.03, p = 0.02, p < 0.001; FD −/− : p < 0.002, p = 0.01, p < 0.01). Surprisingly, female FD −/− mice showed similar pressure-pain hyperalgesia to male FD −/− and WT mice starting from 2 weeks throughout 8 weeks post-DMM (p < 0.001 p < 0.01, p < 0.01, p < 0.01) but WT female mice did not display significantly decreased thresholds compared to the contralateral limb until 6 weeks post-DMM, which persisted to 8-weeks (p = 0.03, p < 0.01). There were no differences in pressure-pain thresholds between FD −/− and WT DMM groups when evaluated within time points (Fig. 4 a) in male mice. Male mice exhibited decreased loading of the DMM limb following injury in both FD −/− and WT mice. However, while WT maintained offloading consistently through 6 weeks (WT: p < 0.001, p < 0.05, p < 0.01), FD −/− only demonstrated significantly increased DMM limb offloading at 2 weeks post-DMM compared to naïve mice (p < 0.001). In contrast, female mice displayed more modest DMM limb offloading behavior. Only female FD −/− mice exhibited significantly different side-to-side limb loading acutely 2 weeks after DMM, and again chronically at 6 and 8 weeks post-DMM compared to naïve (p = 0.03, p < 0.01) (Fig. 4 b). There were no differences in mechanical allodynia (Fig. 4 c) or motor coordination (Fig. 4 d) between DMM and naïve groups, although male FD −/− DMM mice demonstrated significantly longer time on the rotarod than the WT group at later timepoints (p = 0.02; p = 0.02). To understand if the increased pain phenotypes in FD −/− mice were explained by changes in histology, correlation analysis was performed with pressure-pain hyperalgesia and static incapacitance with osteophyte and synovitis scores. Pressure-pain threshold was significantly correlated with osteophyte scores in WT male mice at 8 weeks post-DMM (r = -0.72; p = 0.03) (Supplementary Fig. 3a), although there were no differences detected in sensory or sympathetic neurites in the osteophyte region in the preliminary set of images evaluated (Supplementary Fig. 2). Additionally, offloading of the surgical limb was significantly correlated with osteophyte scores in WT female mice at 2 weeks post-DMM (r = -0.66; p = 0.04). However, no other significant correlations between pressure-pain threshold or static incapacitance and osteophytes were observed in male or female FD −/− mice (Supplementary Fig. 3b). In comparison, synovitis scores were significantly correlated with pressure-pain threshold in WT females at 8 weeks post-DMM (r = 0.775; p = 0.05) (Supplementary Fig. 4a). However, there were no significant correlations in any other WT or FD −/− mice between pressure-pain threshold or static incapacitance with synovitis of both sexes. (Supplementary Fig. 4). Early eicosanoid changes in FD −/− mice were detected in synovial fluid and serum To determine the role of known eicosanoids – fat-secreted bioactive lipid mediators – in the FD −/− pain phenotype, lipidomic profiles were assessed from serum and synovial fluid of the surgical limb in DMM male mice at 2 and 8 weeks post-DMM using LC-MS. PLS-DA plots demonstrate a clear separation of lipidomic profiles at 2 weeks post-DMM in the serum and synovial fluid. FD −/− mice 2 weeks post-DMM displayed significant decreases of wound healing factor 12-HHT(12-hydroxyheptadecatrienoic acid) 42 (p < 0.01) in the serum, and pain suppression factors 14,15-EET (14,15-epoxyeicosatrienoic acid) and 10,11-EpDPA (10, 11-docosahexaenoic acid) 43 – 46 (p = 0.02, p = 0.03) in the synovial fluid when compared to WT (Fig. 5 a). Systemic eicosanoid changes in FD −/− mice were distinct at 8-weeks post DMM At 8 weeks, serum and synovial fluid eicosanoid profiles showed moderate overlap as shown in the PLS-DA plots (Fig. 5 b). FD −/− mice displayed decreases in pain suppression factor (prostaglandin J2, or PGJ2) 47 (p = 0.03) and increases in pain driving factors 12-HETE (12-hydroxyeicosatetraenoic acid) and 13-HODE (13-hydroxyoctadecadienoic acid) 48 , 49 (p = 0.02, p = 0.04) in the serum compared to WT. However, there were no differences in the eicosanoid profiles in the synovial fluid at 8 weeks post-DMM (Fig. 5 b). To demonstrate the differential eicosanoid profiles in each group and over time, heat maps and PLS-DA plots of four-group comparisons between strain and time point are presented in Supplementary Figs. 5 and 6. Of note, there were no significant correlations between normalized eicosanoid abundance and pressure-pain threshold or static incapacitance in combined populations of FD −/− and WT in the serum or synovial fluid at 2 or 8 weeks post-DMM (Supplementary Fig. 7). DISCUSSION The assumption that all pain in OA is derived from peripheral structural insults or changes is a major barrier to understanding the mechanisms of OA pain. This study leverages a novel preclinical mouse model that we have recently demonstrated recapitulates the clinical reports of discordance between pain and structural damage in knee OA 11 . Through constitutive knockout of Cfd , we establish that FD is a key mediator in driving cartilage damage in the onset of knee OA as early as 2 weeks post-DMM in male and female mice. Despite cartilage protection, FD −/− animals challenged with DMM exhibited pain phenotypes that were not simply explained by histological assessments of osteophytes or synovitis, or neurite growth in the joint. These data indicate that structural damage in the joint is not the primary driver for pain in acute and early OA in the FD −/− model. Analysis of pain outcomes 2 weeks post-DMM demonstrated heightened pain sensitivity due to DMM that remained consistent throughout early onset of OA in both groups of male mice. Surprisingly, we observed that with loss of FD, male and female mice demonstrated similar pressure-pain hyperalgesia and static incapacitance, which differed dimorphically in male and female WT mice. This suggests that FD may contribute to sexual dimorphisms in the development of pain with DMM and as OA becomes entrenched. Lastly, we used an innovative targeted approach to map the role of eicosanoids in synovial fluid and serum over time, which corroborates the notion that eicosanoids may be downstream mediators of FD-driven pain in OA in mice. We discovered that loss of FD in male mice resulted in increases in pain-driving (12-HETE, 13-HODE) 48 , 49 and decreases in pain-suppressive associated (PGJ2, 14,15-EET, and 10,11-EpDPA) 43 – 47 eicosanoids after DMM when compared to WT, thus these eicosanoids may be novel targets for the development of drugs to target OA pain. Here, we demonstrate that FD drives cartilage changes in OA in male and female mice. Congruent with existing literature, FD −/− mice were protected from cartilage damage compared to WT after DMM. Recent studies, including our own, indicate that male FD −/− mice showed significantly less cartilage damage in spontaneous and DMM models of established OA with similar levels of pressure-pain hyperalgesia 12 weeks post-DMM 9 – 11 . However, previous studies focused on male mice, so little is known about the FD and alternative complement signaling in female mice. Because female mice have been reported to display more modest cartilage damage after DMM 50 , they are often excluded from OA preclinical studies. Interestingly, the present data provide evidence to the contrary. With sufficient sample sizes, WT female mice displayed statistically significant cartilage damage that was not present in the FD −/− group, indicating that the chondroprotective effect of FD is not sex-dependent. These data are important for two key reasons. First, this supports the notion that female mice challenged with DMM is an intriguing model of OA pain. Second, our findings indicate that there may be sexual dimorphisms in the mechanism of FD-mediated pain-structure discordance that should be considered. WT female mice demonstrated more pain for a given amount of cartilage damage, which is concordant with reports of increased pain severity in female patients 12 , and supports the notion that holistically phenotyping preclinical mouse studies with pain and behavior may provide more translationally relevant targets and mechanistic understanding. Our findings illustrate that early pressure-pain hyperalgesia and static incapacitance are the result of DMM injury. Consistent with previous studies 51 , pressure-pain hyperalgesia thresholds for both WT and FD −/− mice significantly decrease at two weeks in male mice and remained consistent through the 8-week observation period, congruent with our previous work 11 . In females, to our surprise, FD −/− mice demonstrated a similar decrease as males in pressure-pain threshold at 2 weeks that was maintained through 8 weeks post-DMM when compared to the contralateral limb. While we did not observe changes in mechanical allodynia in this study due to DMM or loss of FD, other studies have reported that allodynia thresholds were lowest at 2–8 weeks post-DMM before a reversal of sensitivity after 8 weeks 14 , 52 . Taken together, these findings indicate comparable pain response between FD −/− male and female mice despite female mice exhibiting less severe cartilage damage, and as such, we reject the initial hypothesis that FD −/− male mice would have a more profound pain phenotype compared to female mice. While several studies demonstrate a role for FD and alternative complement signaling in driving cartilage damage, FD’s influence on pathological changes in other joint changes after DMM is unclear. This is particularly important because it is known that complement factors are produced and activated in almost all joint tissues 53 , including the infrapatellar fat pad, which we recently showed strong FD expression in adiponectin-positive barcodes using spatial transcriptomics 11 . Furthermore, while there is a lack of studies that directly investigate the role of FD on osteophyte formation in OA, we have previously reported a robust osteophyte phenotype in FD −/− mice 12 weeks post DMM 11 . Additionally, several studies have reported the absence of FD inhibits synovitis 9 , 10 . In this present study, we observe synovitis and osteophytes coincident with cartilage protection, similar to previous work in fat-free lipodystrophic mice 7 and 12-week evaluation of FD −/− mice 11 . These results highlight the potential separable nature of joint tissues in OA. Multiple, perhaps contradictory, roles for FD in response to DMM injury and the manifestation of pain require further investigation to understand its multiple key roles in OA progression and pathological joint tissue changes. Although osteophytes and synovitis have been implicated in pain in clinical and preclinical models 54 – 57 , our findings indicate that the role of FD on pain is not simply explained by semiquantitative measured changes in these outcomes by histology. Pressure-pain threshold was not significantly correlated to osteophyte or synovitis scores, suggesting that structural changes of the knee cannot fully explain increased pain sensitivity in either sex. However, these measures are crude, and many more advanced techniques and immune profiling are available. It is important to note that, by design, our work focuses on early onset OA to capture responses to injury (acute) and how FD affects the joint and pain over time (chronic). To date, most preclinical and clinical studies investigate the relationship between pain and structure during established and severe OA progression 11 , 55 . Precise and focused work is needed to confirm or reject the direct role of osteophytes and synovitis on FD −/− driven pain, which remains a very promising avenue for future work. Preliminary analysis of CGRP + and TH + nerve endings did not reveal any differences between strain, surgery, or sex, although greater sample sizes are needed to determine whether FD plays a role in differential sensory or sympathetic neurite sprouting after DMM. Altogether, these findings demonstrate that pain sensitivity is not simply explained by these structural changes in early-onset OA. Rather, this suggests that pain may be regulated elsewhere in, or potentially outside, of the joint. Twelve weeks after DMM, we observed alterations in pathways associated with neutrophil trap formation, immunodeficiency, insulin signaling, and calcium signaling in bulk RNA sequencing of the dorsal root ganglia neurons that innervate the knee of FD −/− vs WT mice 11 . Our ongoing efforts aim to profile the DRGs from 2 weeks and 8 weeks post-DMM to uncover acute and chronic changes to these nerves and how they may help explain the early and lasting pain phenotype in the present FD −/− male and female mice. Eicosanoids can be triggered by leptin in the synovium 58 and contribute to the perception of pain through cytokine and COX2 activity 28 , 29 , 31 , 58 . As such, eicosanoids are the targets in NSAID treatment, such as celecoxib, for OA pain. Interestingly, celecoxib has known sexually dimorphic responses in treatment efficacy. Male Sprague-Dawley rats demonstrate better pain relief compared to females 59 . Because NSAIDS can modulate the complement system 24 , we utilized a targeted lipidomics panel to understand how eicosanoids are mediated by FD in response to injury and OA disease entrenchment. Moderate overlap of eicosanoid profiles in serum and synovial fluid for both time points suggests that eicosanoid changes occur in both serum and synovial fluid but in a time-dependent manner. As expected, PLS-DA plots show distinct clusters at 2 weeks but more overlap at 8 weeks due to FD’s role in activating the alternative pathway in response to tissue injury 60 . Furthermore, lower levels of wound-healing factor 12-HHT were measured in FD −/− compared to WT after DMM. These findings suggest that FD likely plays a bigger role in the acute inflammatory phase following DMM. Differences were detected in the serum and synovial fluid at this early time point, highlighting both the local and systemic effects of FD after DMM and further supporting the paradigm that OA is a systemic disease that affects the rest of the body, and can be affected by tissues outside the joint 7 , 11 . Strikingly, eicosanoid profiling demonstrated that loss of FD results in the downregulation of pain suppression and upregulation of pain-driving factors that may be acting synergistically and explain the strong pain phenotype measured in these studies. The combined effects of reducing inhibitory factors and additive effects of pain driving factors are concordant with the pronounced pain phenotype in FD −/− . However, because pain levels were similar between FD −/− male and female mice, we focused on analyzing eicosanoid profiles in male mice. Our future work will investigate differences in the targeted eicosanoid profile and untargeted metabolome of female FD −/− and WT mice to better gain insight into how FD may be influencing pain in OA. 2 weeks after DMM surgery, we observed lower levels of PGJ2, an antagonist of the inflammatory mediator prostaglandin E2 (PGE2) 61 , in the synovial fluid of the surgical limb in FD −/− mice compared to WT. Intra-muscular injection of PGJ2 has been shown to target both PPARg and opioid receptors to prevent muscle hyperalgesia in rats 47 . In contrast, levels of 12-HETE, a molecule shown to amplify PGE2 signaling 49 , and 13-HODE, an endogenous TRPV1 ligand implicated in inflammatory pain 48 , 62 , were higher in the synovial fluid of FD −/− mice at 2 weeks post-DMM when compared to WT. 13-HODE is implicated in inflammatory pain, and administration of anti-13-HODE antibodies has been shown to significantly reduce inflammatory hyperalgesia 62 . At 8 weeks post-DMM, lower levels of 14,15-EET and 10,11-EpDPA were detected in FD −/ − mice compared to WT. Both eicosanoids are associated with anti-nociceptive effects in murine models and have been shown to reduce to neuropathic and inflammatory pain when injected 43 – 46 . Furthermore, lower levels of EETs have been associated with chronic joint pain in OA in both human and mouse models 45 . In comparison, while there were no differences in synovial fluid eicosanoid profiles, clear changes in pain related factors in the serum were detected at 8 weeks. Although we did not find any significant correlations between pressure-pain threshold and the eicosanoid factors identified above, this may be due to: (1) the multiple functions FD may serve in OA pathogenesis, (2) insufficient sample size to identify if a relationship exists with these eicosanoid factors and OA outcomes, and (3) that the 40 eicosanoids measured do not completely capture the eicosanoid profiles of FD and WT mice after DMM. Thus, our future work will leverage untargeted metabolomics approaches to provide insight into other mediators that play a significant role in FD-associated pain and uncover novel molecular signatures to identify novel pain biomarkers and therapeutic targets for OA. While this study provides new knowledge on the mechanisms of pain and structure mediated by FD post injury and over time in both male and female mice, it is not without limitations. First, the analysis of changes in nerve endings within the joint was preliminary. Additional samples are needed to thoroughly characterize whether FD plays a role in neurite sprouting at the joint. Second, we focused on male DMM samples for eicosanoid phenotyping. Therefore, future work will consider both sexes. Third, while mouse models are valuable in disentangling mechanisms of OA pain and structure, there are immunological differences between mice and humans, specifically in alternative complement vs classical complement signaling 63 – 66 . Future work will be needed in humanized mice to validate the role of FD driving pain as a key next step in translating these findings to the clinic and developing the eicosanoid targets. In conclusion, this study contributes to the growing body of evidence that OA is a systemic disease that can be driven by factors outside the joint and can reciprocally affect the whole body. We demonstrate that valuable mechanistic information on OA pain can be gleaned by leveraging models that exhibit structure-pain discordance in response to DMM and by profiling this discordance over time. By global deletion of FD, we are able to recapitulate clinical pain-structure discordance, which allowed us to characterize the time course of pain and cartilage damage separately. We determined that FD −/− has a chondroprotective effect and influences pressure-pain sensitivity in early onset OA in both male and female mice. Furthermore, our findings suggest that FD −/− may contribute to sexual dimorphisms in pressure-pain hyperalgesia and side-to-side limb loading in WT animals. Targeted eicosanoid profiling of 40 known metabolites 25 revealed changes in pain regulation both locally and systemically and demonstrates its potential in identifying key mechanistic insights into the relationship between fat, OA, and pain. These findings will lay the groundwork for identifying new fat-derived therapeutic targets downstream of FD that will be able to address pain and structure in OA pathogenesis. Declarations Ethics approval and consent to participate All experimental procedures were approved by the University of California, San Francisco Institutional Animal Care and Use Committee. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding The authors would like to thank our funding sources from the Arthritis National Research Foundation, NIH Director’s New Innovator Award (DP2AG093209-01), UCSF IRACDA Scholars program (K12GM081266-17), UCSF Training for Research on Aging and Chronic Disease (AG049663), and the National Institutes of Health K99/R00 Award (R00AR078949-04). This publication is solely the responsibility of the authors and does not necessarily represent the official view of NCRR, NIAMS, or NIH. Acknowledgements We would like to thank the Skeletal Biology and Biomechanics Core at UCSF Core Center for Musculoskeletal Biology and Medicine for their technical support with microCT scanning and to the UCSF Quantitative Metabolite Analysis Center (QMAC) for their support in conducting lipidomic analysis. QMAC is made possible by support from the Benioff Center for Microbiome Medicine, ImmunoX, and the Program for Breakthrough Biomedical Research (PBBR). We would like to extend additional gratitude to John Atkinson and Xiaobo Wu for the FD -/- mice used in this study and for scientific discussions. We would also like to thank Christine Pham for scientific discussions. FD -/- mice are available from J Atkinson and X. Wu from Washington University in St. Louis by MTA or reasonable request. Lastly, the authors would also like to thank Kristen W.Y. Chan and Reyna E. Villa for their histological analysis support for this project. 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Supplementary Files CFDKOPainManuscriptSupplementalInformation.docx Cite Share Download PDF Status: Published Journal Publication published 26 Nov, 2025 Read the published version in Arthritis Research & Therapy → Version 1 posted Editorial decision: Revision requested 21 Jul, 2025 Reviews received at journal 21 Jul, 2025 Reviews received at journal 19 Jul, 2025 Reviewers agreed at journal 19 Jul, 2025 Reviews received at journal 10 Jul, 2025 Reviewers agreed at journal 09 Jun, 2025 Reviewers agreed at journal 03 Jun, 2025 Reviewers invited by journal 03 Jun, 2025 Editor assigned by journal 26 May, 2025 Submission checks completed at journal 26 May, 2025 First submitted to journal 19 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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(B) Histological slides of the medial aspect of the left knee joint from the highest scoring joints. Sections were stained with Safranin-O/Fast Green and cartilage damage (yellow arrows) and osteophyte formation (pink arrows) are indicated. (C) Modified Mankin scores were greater in the WT groups compared to \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice 8 weeks post-DMM in male and female mice. Both sexes had greater osteophyte scores after DMM in both timepoints. Scale bar represents 500mm. Two-way ANOVA with Sidak’s post-hoc test was used to analyze between surgery. Different letters represent p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6702118/v1/d12bf1fc7473e7b11bf46263.png"},{"id":84203432,"identity":"e23e92e0-c82c-47a0-9163-36c072e2d2c4","added_by":"auto","created_at":"2025-06-09 08:41:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":628869,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynovitis scores increased after DMM\u003c/strong\u003e. (A) Representative images of histological slides stained with Hematoxylin and Eosin (H\u0026amp;E).\u003cstrong\u003e \u003c/strong\u003eYellow arrows indicate increased thickening, cellularity, and fibrosis of the synovial lining. (B) Although all DMM groups scored higher for synovitis compared to naïve, male \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice displayed greater scores than the WT DMM group at 2 weeks post-DMM. Scale bar represents 500mm. Two-way ANOVA with Sidak’s post-hoc test was used to analyze between surgery. Different letters represent p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6702118/v1/5466d74271bf6c6850700d46.png"},{"id":84205088,"identity":"7ff895e0-282d-4588-8df1-c6840d22ec77","added_by":"auto","created_at":"2025-06-09 08:57:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":119124,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroCT analysis revealed subchondral bone thickening 8 weeks post-DMM in male mice. \u003c/strong\u003e(A)\u003cstrong\u003e \u003c/strong\u003eRepresentative images of the posterior distal femur. (B) Subchondral bone thickness of the distal femur was greater in the posterior lateral compartment in WT mice. Scale bar represents 100mm. Two-way ANOVA with Sidak’s post-hoc test was used to analyze between surgery. Different letters represent p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6702118/v1/3ec4f9eafbe068b8003adc54.png"},{"id":84203433,"identity":"6fc5d7ec-c0d9-4f09-aa57-9df5f3c4a1e0","added_by":"auto","created_at":"2025-06-09 08:41:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":301873,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePain phenotypes appeared at 2 weeks post-DMM and persisted through 8 weeks in both strains. \u003c/strong\u003e(A) Male mice displayed clear pressure-pain hyperalgesia at 2 weeks and maintained for an 8-week observation period. Female \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice exhibited hyperalgesia at each timepoint. There were no differences between \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e and WT in either sexes. (B) Offloading of the surgical limb after DMM was present in both male and female mice. (C) Mechanical allodynia and (D) motor coordination was no different between groups. Two-way ANOVA analysis with Tukey’s post-hoc was done at each timepoint with surgery and strain as main effects. # denotes p\u0026lt;0.05 when comparing contralateral and DMM limb in WT mice, * denotes p\u0026lt;0.05 when comparing contralateral and DMM limb in \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice, + denotes p\u0026lt;0.05 when comparing WT and \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e DMM mice.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6702118/v1/8e91ae909908e8f58252356c.png"},{"id":84203434,"identity":"be307bb1-ccbd-4353-8686-5be6ca994826","added_by":"auto","created_at":"2025-06-09 08:41:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":128911,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFD\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/- \u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003emice exhibited dysregulation of lipid profiles in the serum and synovial fluid. \u003c/strong\u003e(A)\u003cstrong\u003e \u003c/strong\u003ePLS-DA plots show separation in systemic and synovial fluid lipidomic profiles of male \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003ecompared to WT at 2 weeks post-DMM. Wound-healing factor (12-HHT) is lower in\u003cem\u003e \u003c/em\u003ethe serum of FD\u003csup\u003e-/-\u003c/sup\u003e in comparison to WT. Pain suppression factors (PGJ2, 13-HODE, 12-HETE) are lower in the synovial fluid of the surgical limb in \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003emice compared to WT. (B) At 8 weeks post-DMM, more overlap is observed in serum and SF. Differences in factors related to pain (14,15-EET, 10,11-EpDPA) are found in the serum, not the synovial fluid, when comparing \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003eto WT mice. Unpaired t-test was used to analyze between strains. Different letters represent p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6702118/v1/bc897713ac504c29f66bfda8.png"},{"id":97178421,"identity":"4fbcee7d-299a-4103-bf4d-f981314d405c","added_by":"auto","created_at":"2025-12-01 16:09:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2941815,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6702118/v1/c25a1857-c1fd-4715-b4bc-83887c097075.pdf"},{"id":84203436,"identity":"5c6758ea-e76f-4430-8787-63da0f9b32b1","added_by":"auto","created_at":"2025-06-09 08:41:30","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2452605,"visible":true,"origin":"","legend":"","description":"","filename":"CFDKOPainManuscriptSupplementalInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6702118/v1/09d52a818fbae8e77768e637.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Complement factor D (adipsin) mediates pressure-pain hypersensitivity post destabilization of medial meniscus injury","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eOsteoarthritis (OA), a disease characterized by the loss of cartilage lining the joint, is the leading cause of pain and disability worldwide\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Although pain is the primary driver for patients to seek care, current pain management strategies are inadequate, highlighting the need for new drug targets. However, disentangling the mechanism of OA pain from injury and disease entrenchment is challenging. For example, pain severity is not always a result of structural damage to the joint\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Until recently, most studies that employ preclinical models to study OA pathogenesis rely on structural characterization of the joint and often omit pain and behavioral changes. To address this gap in knowledge, we and others\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e routinely perform pain phenotyping in all OA preclinical models to understand the concordant or divergent pain and structure phenotypes that manifest with OA in mice.\u003c/p\u003e \u003cp\u003eComplement factor D (FD), also known as adipsin, is a serine protease that cleaves complement factor B to activate alternative complement signaling, a key pathway in the innate immune response\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. FD is primarily secreted from adipose tissue, which our lab has demonstrated is a key driver of OA pathogenesis and pain\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Although prior studies have determined that the loss of several complement signaling factors was protective for cartilage\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, we have recently demonstrated that \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice displayed pronounced pressure-pain hyperalgesia, despite cartilage protection with DMM\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This model provides the opportunity to decode the discordance between structure and pain reported clinically and establishes FD as a key factor regulating fat-cartilage crosstalk in OA pain. In the present study, we leveraged this model of discordant pain and structural damage phenotype to investigate and better understand the time course changes and mechanism of pressure-pain hyperalgesia post-DMM.\u003c/p\u003e \u003cp\u003eClinical data indicate that there is a dimorphism in OA, such that female patients can report more severe pain for a given amount of structural damage\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, many in the field of OA believe female mice are a poor model to study OA, due to more modest structural changes in cartilage damage requiring a higher sample size to observe significant differences in histological OA scoring. Of note, several studies demonstrate that indeed female mice do develop hallmarks of OA in response to DMM\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Previously, increased synovial fluid C5 levels were associated with increased complement activation in male patients in late-stage knee OA compared to female patients\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Moreover, early complement activation has been reported to be higher in male humans and macaques\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. These findings are also corroborated in mice and are thought to be due to restricted pathway components to promote inflammation through complement C5b-9 complex in female mice\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Therefore, in this study, we leverage the \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e model to gain insight into dimorphisms in pain regulation due to DMM, as it is known that innate differences in the modulation of pain \u0026ndash; male mice may not adequately represent pain in female mice\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Therefore, a secondary aim of this study is to leverage the discordance in structural damage and pain in \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice to better understand potential sexually dimorphic pain responses due to DMM.\u003c/p\u003e \u003cp\u003eCurrent treatments for pain in early OA have been limited to non-steroidal anti-inflammatory drugs (NSAID)\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. However, the efficacy of these drugs can be ineffective in OA patients for long-term use\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. NSAIDs have been established as modulators of the complement system\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. This same targeted eicosanoid panel\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e demonstrated that alterations in the levels of several eicosanoid species played a key role in the transition from acute to chronic hypersensitivity in the K/BxN serum transfer model of rheumatoid arthritis through toll-like receptor (TLR) 4, another key regulator of innate immunity\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Previously, crosstalk between TLRs and complement signaling has been demonstrated to coordinate synergistic or exaggerated immune responses\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Studies have also shown that eicosanoids can be deployed downstream of complement signaling to assist with clearance and the inflammatory response to pathogens\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo begin to elucidate the complement-mediated mechanisms that regulate pain in DMM-induced OA immediately after injury, we used a novel targeted approach in assessing eicosanoids, lipid-derived metabolites associated with pain, that are modulated by NSAIDs\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e and can be downstream of complement signaling. As systemic soluble mediators appear to drive fat-cartilage crosstalk and OA after DMM\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, we posit that \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice may demonstrate alterations in pain-driving and pain-relieving through eicosanoid mediators that act through these signaling molecules to modulate pain. Leveraging this novel eicosanoid profiling approach will give mechanistic insights to systemic and local cellular mechanisms related to pain that could lead to the development of novel therapeutic targets.\u003c/p\u003e \u003cp\u003eThe purpose of this study was to determine the role of FD in the onset and persistence of pain in male and female mice acutely 2 weeks after DMM, and chronically after 8 weeks of DMM. We hypothesize that the absence of \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e will protect cartilage but result in pressure-pain hyperalgesia, which will be detectable 2 weeks post-DMM. We also hypothesize that hyperalgesia due to loss of FD would be more pronounced in male mice compared to female mice. As an exploratory hypothesis, we sought to test if eicosanoid dysregulation due to loss of FD is related to the pressure-pain hyperalgesia phenotype, potentially indicating novel fat- and lipid-derived targets that can be probed for the development of novel therapeutic strategies.\u003c/p\u003e"},{"header":"METHODS","content":" \u003ch2\u003eAnimal Studies\u003c/h2\u003e \u003cp\u003e\u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e/\u0026minus;\u003c/sup\u003e mice\u003csup\u003e6\u003c/sup\u003e (provided kindly by J. Atkinson and X. Wu; Washington University in St. Louis) were bred and maintained at the animal facility at the University of California, San Francisco. All experimental procedures were approved by the University of California, San Francisco Institutional Animal Care and Use Committee. Male and female \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and wild-type (WT) (Jackson Labs #005304) control mice were challenged with destabilization of the medial meniscus (DMM) on the left knee joints or remained na\u0026iuml;ve at 16 weeks old (n\u0026thinsp;=\u0026thinsp;5\u0026ndash;11/group) as a control. Mice were sacrificed at either 2 weeks or 8 weeks post-DMM, 18 weeks or 24 weeks old in total age, at which time serum, synovial fluid, knee joints, and DRGs were collected. A timeline of these studies is presented in Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea.\u003c/p\u003e \n\u003ch3\u003eKnee Joint Assessment\u003c/h3\u003e\n\u003cp\u003eKnee joints were prepared according to previous methods\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In brief, joints were fixed in 4% paraformaldehyde for 24 hours and stored in 70% ethanol at 4C\u0026deg;. After analyzing bone microstructure, joints were decalcified in 10% formalin solution (Cal-Ex II) for 10 hours before being processed and embedded in paraffin wax. Knee joint sections were cut at 5\u0026micro;m thickness in the frontal plane and stained with Safranin-O/Fast Green or Hematoxylin and Eosin (H\u0026amp;E). Histological assessment was performed using Modified Mankin score, Osteophyte score, and Synovitis score as previously described\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eBone Microstructure Analysis\u003c/h3\u003e\n\u003cp\u003eWhole knee joints were scanned by micro-computed tomography (SCANCO \u0026micro;CT50) and imaged according to the guidelines for \u0026micro;CT of rodent bone (energy\u0026thinsp;=\u0026thinsp;55 kVP, intensity\u0026thinsp;=\u0026thinsp;114 mA, 6 \u0026micro;m nominal voxel size, integration time\u0026thinsp;=\u0026thinsp;900ms)\u003csup\u003e32\u003c/sup\u003e at the Skeletal Biology and Biomechanics Core at University of California, San Francisco. Analysis of the trabecular bone in the medial and lateral tibial plateau was performed by manually contouring 2D transverse slices in the region between the growth plate and the subchondral bone. Trabecular bone volume fraction (BV/TV) and bone mineral density (BMD) were determined using the manufacturer\u0026rsquo;s analysis software. Subchondral bone was assessed similarly to exclude trabecular bone in the region between the distal surface of the femoral condyles and the growth plate. Subchondral bone thickness was determined using Fiji software as previously described\u003csup\u003e\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry to Quantify Sensory and Sympathetic Neurites\u003c/h3\u003e\n\u003cp\u003eTo determine the presence of and changes in neurites in the knee joint, immunohistochemistry of calcitonin gene-related peptide (CGRP) positive and tyrosine hydroxylase (TH) positive neurons was performed as described\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Briefly, knee joints were embedded in paraffin wax and cut in 20\u0026micro;m sections at the frontal plane. Sections were blocked in 10% donkey serum and Triton-X buffer before incubation with anti-CGRP and TH primary antibodies (Bio-Rad, Millipore Sigma) overnight at 4\u0026deg;C. Following 3 washes of TNT buffer, sections were incubated in Alexa Fluor 647\u0026reg; and Alexa Fluor\u0026reg; 594 secondary antibodies (Jackson ImmunoResearch) for three hours at room temperature. The sections were then washed three times before incubation in DAPI (Sigma Aldrich) for 5 minutes. Three final washes were done before mounting with Invitrogen Fluoromount-G Mounting Medium (Fisher Scientific). Serial tiled images were taken on 10x objective images using confocal microscopy (Leica DMi8 Inverted Microscope). The region of interest was defined as the area between the synovial lining at the medial femoral quadrant, medial tibial quadrant, lateral femoral quadrant, and lateral tibial quadrant. Images were processed in Fiji to exclude bone marrow. SNT for neuroanatomy plug-in was used to quantify positive signals by manual tracing\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Total pixels of positive CGRP and TH signal were reported.\u003c/p\u003e\n\u003ch3\u003ePain Assessments and Behavioral Testing\u003c/h3\u003e\n\u003cp\u003ePain assessments were conducted one week before DMM and 2-, 4-, 6-, and 8-weeks post-surgery in the order from least to most invasive at the same time of day. All mice were acclimatized to the behavioral suite and equipment prior to testing. Pressure-pain hyperalgesia was measured using a Small Animal Algometer\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e (SMALGO, Bioseb). Three to five trials of the surgical limb and nonsurgical limb was collected by applying a steady force to the lateral aspect of each limb until the mouse showed signs of discomfort such as squeaking, paw withdrawal, or grimacing. The average of these trials was reported and a maximum value of 450g was employed to avoid tissue damage to the joint\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSide-to-side limb loading was measured via static incapacitance (Bioseb). Mice were placed in the restrainer to acclimate for ~\u0026thinsp;2 minutes. Load bearing measurements for each limb were taken once the mouse was calm, had both feet on each of the force sensors, and had its paws placed on the ramp at the front. Incapacitance is measured as the difference between the right and left limbs. Three to five trials were measured, and the average was reported.\u003c/p\u003e \u003cp\u003eTo assess mechanical allodynia, a Von Frey assay was used as described\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Mice were placed in a box with a wire mesh bottom and left to acclimate for 20 minutes. Once acclimated, force was applied to the mid-plantar of the surgical limb paw three times using one in a series of five Von Frey filaments ranging from 0.16g to 1.4g. Paw withdrawal was noted as either \u0026ldquo;positive\u0026rdquo; or \u0026ldquo;negative response\u0026rdquo;. This was then repeated with each of the five Von Frey filaments, with one repeating filament for a total of 6. The order of filaments was random between observers. Paw withdrawal patterns were assessed as previously described to determine the average 50% paw withdrawal threshold from the three repetitions\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo assess motor coordination, mice were placed on a rotarod wheel with an initial speed of 4 rpm. Using the ramp function, the speed increased to a maximum of 40 rpm in 120 seconds. The time and the maximum speed at which the mouse fell were noted. The average of three to five trials was reported\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003ch2\u003eEicosanoid Profiling by Targeted Lipidomics\u003c/h2\u003e \u003cp\u003eSerum and synovial fluid\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e samples were collected and extracted from male DMM mice to determine systemic and local changes in eicosanoid profiles using a novel dual extraction method that separates proteins from metabolites from a single sample\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In brief, all samples were extracted with methanol, vortexed, and placed at -20\u0026deg;C for 30 minutes to promote protein precipitation. Next, the supernatant containing metabolites was collected and dried via vacuum concentration. A 5 \u0026micro;L aliquot of each sample was subjected to targeted metabolomic panel of 40 known eicosanoids, analyzed at the Quantitative Metabolite Analysis Center at the University of California, San Francisco\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eon a Shimadzu 30-AD UPLC in series with a SCIEX 7500 Triple Quadrupole Mass Spectrometer. Analytes were chromatographically separated using a Kinetex 2.6 \u0026micro;m Polar C18 100\u0026Aring;, liquid chromatography (LC) column 100 x 3.0 mm (Phenomenex, cat #00D-4759-Y0) with a mobile phase scheme of [A] water\u0026thinsp;+\u0026thinsp;0.1% formic acid and [B] methanol\u0026thinsp;+\u0026thinsp;0.1% formic acid. The LC method was set to a constant flow rate of 500 \u0026micro;L/min, and the timed linear gradient program consisted of: time\u0026thinsp;=\u0026thinsp;0 min, 0.10% B, time\u0026thinsp;=\u0026thinsp;0.1 min, 45% B, time\u0026thinsp;=\u0026thinsp;2 min, 45% B, time\u0026thinsp;=\u0026thinsp;16.5 min, 80% B, time\u0026thinsp;=\u0026thinsp;16.6 min, 98% B, time\u0026thinsp;=\u0026thinsp;18.5 min, 98% B, time\u0026thinsp;=\u0026thinsp;18.6 min, 10% B, and time\u0026thinsp;=\u0026thinsp;20.5, 10% B. Data was collected using polarity switching with the following source parameters: CUR\u0026thinsp;=\u0026thinsp;40, GS1\u0026thinsp;=\u0026thinsp;60, GS2\u0026thinsp;=\u0026thinsp;70, Temp\u0026thinsp;=\u0026thinsp;350 \u003csup\u003eo\u003c/sup\u003eC, ISVF\u0026thinsp;=\u0026thinsp;4500V (negative mode) and 5500V (positive mode). Optimized multiple-reaction monitoring (MRM) pairs detailed in (Supplementary Tables 1 and 2) were used with a total cycle time of 0.937 seconds, dwell time of 1 ms, settling time\u0026thinsp;=\u0026thinsp;15 ms, and pause time of 5.007ms. This method was based off of SCIEX\u0026rsquo;s comprehensive targeted method for lipid mediator analysis application note. Raw data was processed using a built-in SCIEX OS software package (version 2.1.6.59781) for peak picking, alignment, and quantitation.\u003c/p\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll results are reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean. Results from pain and behavior assays were analyzed with three-way analysis of variance (ANOVA) stratified by time point, surgery, and strain. A separate two-way ANOVA with Tukey\u0026rsquo;s post-hoc test was run by genotype and limb to determine differences within each time point. Nonparametric Spearman\u0026rsquo;s correlations were calculated to determine correlative relationships between pressure-pain threshold and osteophyte score, synovitis score, or significant eicosanoids as identified above. Lipidomic data was analyzed using an unpaired t-test on normalized abundances between WT and \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. All other outcomes are evaluated using two-way ANOVA with Sidak\u0026rsquo;s post hoc test with strain and surgery as main effects. Heat maps and PLS-DA plots of lipidomic data were created using MetaboAnalyst. Statistical significance is defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Statistical analyses were performed using GraphPad Prism version 10.0.0 for Mac (GraphPad Software, Boston, Massachusetts USA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.graphpad.com\u003c/span\u003e\u003cspan address=\"http://www.graphpad.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e "},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eMale and female\u003c/b\u003e \u003cb\u003eFD\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice displayed cartilage protection after DMM surgery\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs expected, \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e male and female mice were protected from DMM-induced structural damage as shown in images of the highest Mankin Scores in each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Concordant with previous analysis at 12-weeks post-DMM\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, male WT mice demonstrated significantly greater Modified Mankin scores between DMM and na\u0026iuml;ve groups at 8 weeks post-DMM (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice displayed no significant differences between DMM and na\u0026iuml;ve groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Similarly, female WT mice exhibited greater Modified Mankin scores at both 2 and 8 weeks (p\u0026thinsp;=\u0026thinsp;0.01, p\u0026thinsp;=\u0026thinsp;0.001), although there were no cartilage changes in \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. Despite cartilage protection in \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, significant osteophyte formation was present in both strains. WT and \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e DMM groups of male and female mice had greater osteophyte scores compared to the na\u0026iuml;ve controls at 2 weeks (male: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; female: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and 8 weeks (male: p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;=\u0026thinsp;0.02; female: p\u0026thinsp;=\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) post-DMM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilarly, synovitis scores were greater in all DMM groups compared to the na\u0026iuml;ve control at both 2 weeks (male: WT p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001; female: WT p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 8 weeks (male: WT p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u0026lt;0.001; female: WT p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) post-DMM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Interestingly, \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e presented worsening synovitis scores compared to WT at 2 weeks post-DMM (p\u0026thinsp;=\u0026thinsp;0.02) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of bone changes revealed subchondral bone thickening in the lateral posterior compartment in WT (p\u0026thinsp;=\u0026thinsp;0.01), but not \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e male at 8 weeks post-DMM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). There were no significant differences in subchondral thickness at 2 weeks post-DMM, or in female mice. There were no differences in BV/TV of the proximal tibial epiphysis due to surgery, strain, or sex (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePreliminary analysis of a subset of joints to quantify CGRP\u0026thinsp;+\u0026thinsp;and TH\u0026thinsp;+\u0026thinsp;neurites in the joint did not demonstrate any clear changes between surgical groups, strain, or sex in a subset of animals (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePressure-pain Hyperalgesia was increased in\u003c/b\u003e \u003cb\u003eFD\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice at 2-weeks, and persisted to 8 weeks\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePain and behavior assays were conducted one week before DMM at baseline, and 2, 4, 6, and 8 weeks after surgery. The surgical limb of WT and \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e DMM male mice displayed decreases in pressure-pain threshold as early as 2 weeks and through 8 weeks post-DMM when compared to their contralateral limb (WT: p\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;=\u0026thinsp;0.03, p\u0026thinsp;=\u0026thinsp;0.02, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e: p\u0026thinsp;\u0026lt;\u0026thinsp;0.002, p\u0026thinsp;=\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Surprisingly, female \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice showed similar pressure-pain hyperalgesia to male \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and WT mice starting from 2 weeks throughout 8 weeks post-DMM (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) but WT female mice did not display significantly decreased thresholds compared to the contralateral limb until 6 weeks post-DMM, which persisted to 8-weeks (p\u0026thinsp;=\u0026thinsp;0.03, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). There were no differences in pressure-pain thresholds between \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and WT DMM groups when evaluated within time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) in male mice. Male mice exhibited decreased loading of the DMM limb following injury in both \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and WT mice. However, while WT maintained offloading consistently through 6 weeks (WT: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e only demonstrated significantly increased DMM limb offloading at 2 weeks post-DMM compared to na\u0026iuml;ve mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In contrast, female mice displayed more modest DMM limb offloading behavior. Only female \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibited significantly different side-to-side limb loading acutely 2 weeks after DMM, and again chronically at 6 and 8 weeks post-DMM compared to na\u0026iuml;ve (p\u0026thinsp;=\u0026thinsp;0.03, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). There were no differences in mechanical allodynia (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) or motor coordination (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) between DMM and na\u0026iuml;ve groups, although male \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e DMM mice demonstrated significantly longer time on the rotarod than the WT group at later timepoints (p\u0026thinsp;=\u0026thinsp;0.02; p\u0026thinsp;=\u0026thinsp;0.02).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo understand if the increased pain phenotypes in \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were explained by changes in histology, correlation analysis was performed with pressure-pain hyperalgesia and static incapacitance with osteophyte and synovitis scores. Pressure-pain threshold was significantly correlated with osteophyte scores in WT male mice at 8 weeks post-DMM (r = -0.72; p\u0026thinsp;=\u0026thinsp;0.03) (Supplementary Fig.\u0026nbsp;3a), although there were no differences detected in sensory or sympathetic neurites in the osteophyte region in the preliminary set of images evaluated (Supplementary Fig.\u0026nbsp;2). Additionally, offloading of the surgical limb was significantly correlated with osteophyte scores in WT female mice at 2 weeks post-DMM (r = -0.66; p\u0026thinsp;=\u0026thinsp;0.04). However, no other significant correlations between pressure-pain threshold or static incapacitance and osteophytes were observed in male or female \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (Supplementary Fig.\u0026nbsp;3b). In comparison, synovitis scores were significantly correlated with pressure-pain threshold in WT females at 8 weeks post-DMM (r\u0026thinsp;=\u0026thinsp;0.775; p\u0026thinsp;=\u0026thinsp;0.05) (Supplementary Fig.\u0026nbsp;4a). However, there were no significant correlations in any other WT or \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice between pressure-pain threshold or static incapacitance with synovitis of both sexes. (Supplementary Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEarly eicosanoid changes in\u003c/b\u003e \u003cb\u003eFD\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice were detected in synovial fluid and serum\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine the role of known eicosanoids \u0026ndash; fat-secreted bioactive lipid mediators \u0026ndash; in the \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e pain phenotype, lipidomic profiles were assessed from serum and synovial fluid of the surgical limb in DMM male mice at 2 and 8 weeks post-DMM using LC-MS. PLS-DA plots demonstrate a clear separation of lipidomic profiles at 2 weeks post-DMM in the serum and synovial fluid. \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice 2 weeks post-DMM displayed significant decreases of wound healing factor 12-HHT(12-hydroxyheptadecatrienoic acid)\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in the serum, and pain suppression factors 14,15-EET (14,15-epoxyeicosatrienoic acid) and 10,11-EpDPA (10, 11-docosahexaenoic acid)\u003csup\u003e\u003cspan additionalcitationids=\"CR44 CR45\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e (p\u0026thinsp;=\u0026thinsp;0.02, p\u0026thinsp;=\u0026thinsp;0.03) in the synovial fluid when compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSystemic eicosanoid changes in\u003c/b\u003e \u003cb\u003eFD\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;/\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice were distinct at 8-weeks post DMM\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAt 8 weeks, serum and synovial fluid eicosanoid profiles showed moderate overlap as shown in the PLS-DA plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice displayed decreases in pain suppression factor (prostaglandin J2, or PGJ2)\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e (p\u0026thinsp;=\u0026thinsp;0.03) and increases in pain driving factors 12-HETE (12-hydroxyeicosatetraenoic acid) and 13-HODE (13-hydroxyoctadecadienoic acid)\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e (p\u0026thinsp;=\u0026thinsp;0.02, p\u0026thinsp;=\u0026thinsp;0.04) in the serum compared to WT. However, there were no differences in the eicosanoid profiles in the synovial fluid at 8 weeks post-DMM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo demonstrate the differential eicosanoid profiles in each group and over time, heat maps and PLS-DA plots of four-group comparisons between strain and time point are presented in Supplementary Figs.\u0026nbsp;5 and 6. Of note, there were no significant correlations between normalized eicosanoid abundance and pressure-pain threshold or static incapacitance in combined populations of \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and WT in the serum or synovial fluid at 2 or 8 weeks post-DMM (Supplementary Fig.\u0026nbsp;7).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe assumption that all pain in OA is derived from peripheral structural insults or changes is a major barrier to understanding the mechanisms of OA pain. This study leverages a novel preclinical mouse model that we have recently demonstrated recapitulates the clinical reports of discordance between pain and structural damage in knee OA\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Through constitutive knockout of \u003cem\u003eCfd\u003c/em\u003e, we establish that FD is a key mediator in driving cartilage damage in the onset of knee OA as early as 2 weeks post-DMM in male and female mice. Despite cartilage protection, \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals challenged with DMM exhibited pain phenotypes that were not simply explained by histological assessments of osteophytes or synovitis, or neurite growth in the joint. These data indicate that structural damage in the joint is not the primary driver for pain in acute and early OA in the \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e model. Analysis of pain outcomes 2 weeks post-DMM demonstrated heightened pain sensitivity due to DMM that remained consistent throughout early onset of OA in both groups of male mice. Surprisingly, we observed that with loss of FD, male and female mice demonstrated similar pressure-pain hyperalgesia and static incapacitance, which differed dimorphically in male and female WT mice. This suggests that FD may contribute to sexual dimorphisms in the development of pain with DMM and as OA becomes entrenched. Lastly, we used an innovative targeted approach to map the role of eicosanoids in synovial fluid and serum over time, which corroborates the notion that eicosanoids may be downstream mediators of FD-driven pain in OA in mice. We discovered that loss of FD in male mice resulted in increases in pain-driving (12-HETE, 13-HODE)\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e and decreases in pain-suppressive associated (PGJ2, 14,15-EET, and 10,11-EpDPA) \u003csup\u003e\u003cspan additionalcitationids=\"CR44 CR45 CR46\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e eicosanoids after DMM when compared to WT, thus these eicosanoids may be novel targets for the development of drugs to target OA pain.\u003c/p\u003e \u003cp\u003eHere, we demonstrate that FD drives cartilage changes in OA in male and female mice. Congruent with existing literature, \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were protected from cartilage damage compared to WT after DMM. Recent studies, including our own, indicate that male \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice showed significantly less cartilage damage in spontaneous and DMM models of established OA with similar levels of pressure-pain hyperalgesia 12 weeks post-DMM\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, previous studies focused on male mice, so little is known about the FD and alternative complement signaling in female mice. Because female mice have been reported to display more modest cartilage damage after DMM\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, they are often excluded from OA preclinical studies. Interestingly, the present data provide evidence to the contrary. With sufficient sample sizes, WT female mice displayed statistically significant cartilage damage that was not present in the \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e group, indicating that the chondroprotective effect of FD is not sex-dependent. These data are important for two key reasons. First, this supports the notion that female mice challenged with DMM is an intriguing model of OA pain. Second, our findings indicate that there may be sexual dimorphisms in the mechanism of FD-mediated pain-structure discordance that should be considered. WT female mice demonstrated more pain for a given amount of cartilage damage, which is concordant with reports of increased pain severity in female patients\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, and supports the notion that holistically phenotyping preclinical mouse studies with pain and behavior may provide more translationally relevant targets and mechanistic understanding.\u003c/p\u003e \u003cp\u003eOur findings illustrate that early pressure-pain hyperalgesia and static incapacitance are the result of DMM injury. Consistent with previous studies\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, pressure-pain hyperalgesia thresholds for both WT and \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice significantly decrease at two weeks in male mice and remained consistent through the 8-week observation period, congruent with our previous work\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In females, to our surprise, \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice demonstrated a similar decrease as males in pressure-pain threshold at 2 weeks that was maintained through 8 weeks post-DMM when compared to the contralateral limb. While we did not observe changes in mechanical allodynia in this study due to DMM or loss of FD, other studies have reported that allodynia thresholds were lowest at 2\u0026ndash;8 weeks post-DMM before a reversal of sensitivity after 8 weeks\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Taken together, these findings indicate comparable pain response between \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e male and female mice despite female mice exhibiting less severe cartilage damage, and as such, we reject the initial hypothesis that \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e male mice would have a more profound pain phenotype compared to female mice.\u003c/p\u003e \u003cp\u003eWhile several studies demonstrate a role for FD and alternative complement signaling in driving cartilage damage, FD\u0026rsquo;s influence on pathological changes in other joint changes after DMM is unclear. This is particularly important because it is known that complement factors are produced and activated in almost all joint tissues\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, including the infrapatellar fat pad, which we recently showed strong FD expression in adiponectin-positive barcodes using spatial transcriptomics\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Furthermore, while there is a lack of studies that directly investigate the role of FD on osteophyte formation in OA, we have previously reported a robust osteophyte phenotype in \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice 12 weeks post DMM\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Additionally, several studies have reported the absence of FD inhibits synovitis\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In this present study, we observe synovitis and osteophytes coincident with cartilage protection, similar to previous work in fat-free lipodystrophic mice\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and 12-week evaluation of \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice\u003csup\u003e11\u003c/sup\u003e. These results highlight the potential separable nature of joint tissues in OA. Multiple, perhaps contradictory, roles for FD in response to DMM injury and the manifestation of pain require further investigation to understand its multiple key roles in OA progression and pathological joint tissue changes.\u003c/p\u003e \u003cp\u003eAlthough osteophytes and synovitis have been implicated in pain in clinical and preclinical models\u003csup\u003e\u003cspan additionalcitationids=\"CR55 CR56\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, our findings indicate that the role of FD on pain is not simply explained by semiquantitative measured changes in these outcomes by histology. Pressure-pain threshold was not significantly correlated to osteophyte or synovitis scores, suggesting that structural changes of the knee cannot fully explain increased pain sensitivity in either sex. However, these measures are crude, and many more advanced techniques and immune profiling are available. It is important to note that, by design, our work focuses on early onset OA to capture responses to injury (acute) and how FD affects the joint and pain over time (chronic). To date, most preclinical and clinical studies investigate the relationship between pain and structure during established and severe OA progression\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Precise and focused work is needed to confirm or reject the direct role of osteophytes and synovitis on \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e driven pain, which remains a very promising avenue for future work. Preliminary analysis of CGRP\u0026thinsp;+\u0026thinsp;and TH\u0026thinsp;+\u0026thinsp;nerve endings did not reveal any differences between strain, surgery, or sex, although greater sample sizes are needed to determine whether FD plays a role in differential sensory or sympathetic neurite sprouting after DMM. Altogether, these findings demonstrate that pain sensitivity is not simply explained by these structural changes in early-onset OA. Rather, this suggests that pain may be regulated elsewhere in, or potentially outside, of the joint. Twelve weeks after DMM, we observed alterations in pathways associated with neutrophil trap formation, immunodeficiency, insulin signaling, and calcium signaling in bulk RNA sequencing of the dorsal root ganglia neurons that innervate the knee of \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e vs WT mice\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Our ongoing efforts aim to profile the DRGs from 2 weeks and 8 weeks post-DMM to uncover acute and chronic changes to these nerves and how they may help explain the early and lasting pain phenotype in the present \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e male and female mice.\u003c/p\u003e \u003cp\u003eEicosanoids can be triggered by leptin in the synovium\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e and contribute to the perception of pain through cytokine and COX2 activity\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. As such, eicosanoids are the targets in NSAID treatment, such as celecoxib, for OA pain. Interestingly, celecoxib has known sexually dimorphic responses in treatment efficacy. Male Sprague-Dawley rats demonstrate better pain relief compared to females\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Because NSAIDS can modulate the complement system\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, we utilized a targeted lipidomics panel to understand how eicosanoids are mediated by FD in response to injury and OA disease entrenchment. Moderate overlap of eicosanoid profiles in serum and synovial fluid for both time points suggests that eicosanoid changes occur in both serum and synovial fluid but in a time-dependent manner. As expected, PLS-DA plots show distinct clusters at 2 weeks but more overlap at 8 weeks due to FD\u0026rsquo;s role in activating the alternative pathway in response to tissue injury\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Furthermore, lower levels of wound-healing factor 12-HHT were measured in \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e compared to WT after DMM. These findings suggest that FD likely plays a bigger role in the acute inflammatory phase following DMM. Differences were detected in the serum and synovial fluid at this early time point, highlighting both the local and systemic effects of FD after DMM and further supporting the paradigm that OA is a systemic disease that affects the rest of the body, and can be affected by tissues outside the joint\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Strikingly, eicosanoid profiling demonstrated that loss of FD results in the downregulation of pain suppression and upregulation of pain-driving factors that may be acting synergistically and explain the strong pain phenotype measured in these studies. The combined effects of reducing inhibitory factors and additive effects of pain driving factors are concordant with the pronounced pain phenotype in \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e. However, because pain levels were similar between \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e male and female mice, we focused on analyzing eicosanoid profiles in male mice. Our future work will investigate differences in the targeted eicosanoid profile and untargeted metabolome of female \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and WT mice to better gain insight into how FD may be influencing pain in OA.\u003c/p\u003e \u003cp\u003e2 weeks after DMM surgery, we observed lower levels of PGJ2, an antagonist of the inflammatory mediator prostaglandin E2 (PGE2)\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, in the synovial fluid of the surgical limb in \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice compared to WT. Intra-muscular injection of PGJ2 has been shown to target both PPARg and opioid receptors to prevent muscle hyperalgesia in rats\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. In contrast, levels of 12-HETE, a molecule shown to amplify PGE2 signaling\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, and 13-HODE, an endogenous TRPV1 ligand implicated in inflammatory pain\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e, were higher in the synovial fluid of \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice at 2 weeks post-DMM when compared to WT. 13-HODE is implicated in inflammatory pain, and administration of anti-13-HODE antibodies has been shown to significantly reduce inflammatory hyperalgesia\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. At 8 weeks post-DMM, lower levels of 14,15-EET and 10,11-EpDPA were detected in \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u003c/em\u003e\u0026minus;\u003c/sup\u003e mice compared to WT. Both eicosanoids are associated with anti-nociceptive effects in murine models and have been shown to reduce to neuropathic and inflammatory pain when injected\u003csup\u003e\u003cspan additionalcitationids=\"CR44 CR45\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Furthermore, lower levels of EETs have been associated with chronic joint pain in OA in both human and mouse models\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In comparison, while there were no differences in synovial fluid eicosanoid profiles, clear changes in pain related factors in the serum were detected at 8 weeks.\u003c/p\u003e \u003cp\u003eAlthough we did not find any significant correlations between pressure-pain threshold and the eicosanoid factors identified above, this may be due to: (1) the multiple functions FD may serve in OA pathogenesis, (2) insufficient sample size to identify if a relationship exists with these eicosanoid factors and OA outcomes, and (3) that the 40 eicosanoids measured do not completely capture the eicosanoid profiles of FD and WT mice after DMM. Thus, our future work will leverage untargeted metabolomics approaches to provide insight into other mediators that play a significant role in FD-associated pain and uncover novel molecular signatures to identify novel pain biomarkers and therapeutic targets for OA.\u003c/p\u003e \u003cp\u003eWhile this study provides new knowledge on the mechanisms of pain and structure mediated by FD post injury and over time in both male and female mice, it is not without limitations. First, the analysis of changes in nerve endings within the joint was preliminary. Additional samples are needed to thoroughly characterize whether FD plays a role in neurite sprouting at the joint. Second, we focused on male DMM samples for eicosanoid phenotyping. Therefore, future work will consider both sexes. Third, while mouse models are valuable in disentangling mechanisms of OA pain and structure, there are immunological differences between mice and humans, specifically in alternative complement vs classical complement signaling\u003csup\u003e\u003cspan additionalcitationids=\"CR64 CR65\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Future work will be needed in humanized mice to validate the role of FD driving pain as a key next step in translating these findings to the clinic and developing the eicosanoid targets.\u003c/p\u003e \u003cp\u003eIn conclusion, this study contributes to the growing body of evidence that OA is a systemic disease that can be driven by factors outside the joint and can reciprocally affect the whole body. We demonstrate that valuable mechanistic information on OA pain can be gleaned by leveraging models that exhibit structure-pain discordance in response to DMM and by profiling this discordance over time. By global deletion of FD, we are able to recapitulate clinical pain-structure discordance, which allowed us to characterize the time course of pain and cartilage damage separately. We determined that \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e has a chondroprotective effect and influences pressure-pain sensitivity in early onset OA in both male and female mice. Furthermore, our findings suggest that \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e may contribute to sexual dimorphisms in pressure-pain hyperalgesia and side-to-side limb loading in WT animals. Targeted eicosanoid profiling of 40 known metabolites\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e revealed changes in pain regulation both locally and systemically and demonstrates its potential in identifying key mechanistic insights into the relationship between fat, OA, and pain. These findings will lay the groundwork for identifying new fat-derived therapeutic targets downstream of FD that will be able to address pain and structure in OA pathogenesis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were approved by the University of California, San Francisco Institutional Animal Care and Use Committee.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank our funding sources from the Arthritis National Research Foundation, NIH Director’s New Innovator Award (DP2AG093209-01), UCSF IRACDA Scholars program (K12GM081266-17), UCSF Training for Research on Aging and Chronic Disease (AG049663), and the National Institutes of Health K99/R00 Award (R00AR078949-04).\u0026nbsp;This publication is solely the responsibility of the authors and does not necessarily represent the official view of NCRR, NIAMS, or NIH.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the Skeletal Biology and Biomechanics Core at UCSF Core Center for Musculoskeletal Biology and Medicine for their technical support with microCT scanning and to the UCSF Quantitative Metabolite Analysis Center (QMAC) for their support in conducting lipidomic analysis. QMAC is made possible by support from the Benioff Center for Microbiome Medicine, ImmunoX, and the Program for Breakthrough Biomedical Research (PBBR). We would like to extend additional gratitude to John Atkinson and Xiaobo Wu for the \u003cem\u003eFD\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice used in this study and for scientific discussions. We would also like to thank Christine Pham for scientific discussions. \u003cem\u003eFD\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice are available from J Atkinson and X. Wu from Washington University in St. Louis by MTA or reasonable request. Lastly, the authors would also like to thank Kristen W.Y. Chan and Reyna E. Villa for their histological analysis support for this project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP.T. and K.C. conceived and executed the studies, analyzed data, and wrote the main manuscript. B.A. managed the animal colony and conducted pain testing in Figure 4. J.K. stained and imaged histology data in figures 1 and 2. M.R. imaged and analyzed nerve endings. S.S., T.K., and A.G. analyzed microCT data in figure 3. D.D. and H.W. conducted the lipidomic experiment in figure 5. H.W. additionally analyzed and interpreted lipidomic data. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBerenbaum F, Griffin TM, Liu-Bryan R. Metabolic Regulation of Inflammation in Osteoarthritis. \u003cem\u003eArthritis Rheumatol Hoboken NJ\u003c/em\u003e. 2017;69(1):9-21. doi:10.1002/art.39842\u003c/li\u003e\n \u003cli\u003eCollins KH, Herzog W, MacDonald GZ, et al. 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Prostaglandin 15d-PGJ2 targets PPAR\u0026gamma; and opioid receptors to prevent muscle hyperalgesia in rats. \u003cem\u003eNeuroreport\u003c/em\u003e. 2021;32(3):238-243. doi:10.1097/WNR.0000000000001575\u003c/li\u003e\n \u003cli\u003eMorici L, All\u0026eacute;mann E, Jordan O, Nikolić I. Promising LOX proteins for cartilage-targeting osteoarthritis therapy. \u003cem\u003ePharmacol Res\u003c/em\u003e. 2025;212:107627. doi:10.1016/j.phrs.2025.107627\u003c/li\u003e\n \u003cli\u003eMoreira V, Guti\u0026eacute;rrez JM, Lomonte B, et al. 12-HETE is a regulator of PGE2 production via COX-2 expression induced by a snake venom group IIA phospholipase A2 in isolated peritoneal macrophages. \u003cem\u003eChem Biol Interact\u003c/em\u003e. 2020;317:108903. doi:10.1016/j.cbi.2019.108903\u003c/li\u003e\n \u003cli\u003eMa HL, Blanchet TJ, Peluso D, Hopkins B, Morris EA, Glasson SS. Osteoarthritis severity is sex dependent in a surgical mouse model. \u003cem\u003eOsteoarthritis Cartilage\u003c/em\u003e. 2007;15(6):695-700. doi:10.1016/j.joca.2006.11.005\u003c/li\u003e\n \u003cli\u003eMiller RE, Ishihara S, Bhattacharyya B, et al. CHEMOGENETIC INHIBITION OF PAIN NEURONS IN A MOUSE MODEL OF OSTEOARTHRITIS. \u003cem\u003eArthritis Rheumatol Hoboken NJ\u003c/em\u003e. 2017;69(7):1429-1439. doi:10.1002/art.40118\u003c/li\u003e\n \u003cli\u003eZaki S, Smith MM, Little CB. Pathology-pain relationships in different osteoarthritis animal model phenotypes: it matters what you measure, when you measure, and how you got there. \u003cem\u003eOsteoarthritis Cartilage\u003c/em\u003e. 2021;29(10):1448-1461. doi:10.1016/j.joca.2021.03.023\u003c/li\u003e\n \u003cli\u003eAssirelli E, Pulsatelli L, Dolzani P, et al. Complement Expression and Activation in Osteoarthritis Joint Compartments. \u003cem\u003eFront Immunol\u003c/em\u003e. 2020;11:535010. doi:10.3389/fimmu.2020.535010\u003c/li\u003e\n \u003cli\u003eVincent TL. Peripheral pain mechanisms in osteoarthritis. \u003cem\u003ePain\u003c/em\u003e. 2020;161(1):S138-S146. doi:10.1097/j.pain.0000000000001923\u003c/li\u003e\n \u003cli\u003eSanchez-Lopez E, Coras R, Torres A, Lane NE, Guma M. Synovial inflammation in osteoarthritis progression. \u003cem\u003eNat Rev Rheumatol\u003c/em\u003e. 2022;18(5):258-275. doi:10.1038/s41584-022-00749-9\u003c/li\u003e\n \u003cli\u003eFan T, Chen S, Zeng M, et al. Osteophytes mediate the associations between cartilage morphology and changes in knee symptoms in patients with knee osteoarthritis. \u003cem\u003eArthritis Res Ther\u003c/em\u003e. 2022;24:217. doi:10.1186/s13075-022-02905-8\u003c/li\u003e\n \u003cli\u003eMcCulloch K, Huesa C, Dunning L, et al. 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Mechanisms of complement activation by dextran-coated superparamagnetic iron oxide (SPIO) nanoworms in mouse versus human serum. \u003cem\u003ePart Fibre Toxicol\u003c/em\u003e. 2014;11(1):64. doi:10.1186/s12989-014-0064-2\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"arthritis-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arrt","sideBox":"Learn more about [Arthritis Research \u0026 Therapy](http://arthritis-research.biomedcentral.com/)","snPcode":"13075","submissionUrl":"https://submission.nature.com/new-submission/13075/3","title":"Arthritis Research \u0026 Therapy","twitterHandle":"@ArthritisRes","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6702118/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6702118/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBACKGROUND:\u003c/p\u003e\n\u003cp\u003eAlthough osteoarthritis (OA) is the leading cause of pain and disability worldwide, there is a lack of models to probe the separable mechanism of OA structural damage and knee pain. We previously identified that deletion of complement factor D (FD) results in increased pressure-pain hyperalgesia despite cartilage protection after destabilization of the medial meniscus (DMM) surgery. However, how these discordant OA phenotypes manifest is not understood. We employed a novel targeted lipidomics approach to elucidate the role of eicosanoids in FD-mediated pain. We hypothesize that the absence of \u003cem\u003eCfd (FD\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e will protect cartilage but cause increased pressure-pain hyperalgesia and eicosanoid dysregulation that persists throughout OA development.\u003c/p\u003e\n\u003cp\u003eMETHODS:\u003c/p\u003e\n\u003cp\u003eMale and female \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003eand wild-type (WT) mice were challenged with DMM or remained naïve (n=5-11/group) at 16 weeks old. Pressure-pain hyperalgesia was measured bi-weekly for 8 weeks post-DMM. A second cohort was evaluated at 2 weeks post-DMM (n=6-10/group) to investigate DMM injury response. Structural damage was scored using the Modified Mankin system. To determine changes in eicosanoid profiles, serum and synovial fluid samples were analyzed via liquid chromatography-mass spectrometry (LC-MS). Statistical analysis was performed with unpaired t-test, two-way, or three-way ANOVA with Sidak’s posthoc test. Statistical significance is defined as p\u0026lt;0.05.\u003c/p\u003e\n\u003cp\u003eRESULTS:\u003c/p\u003e\n\u003cp\u003eIn contrast to WT mice, \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003eshowed no significant differences in Modified Mankin scores 8 weeks post-DMM. As expected, \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003ehyperalgesia levels persisted until 8 weeks post DMM, similar to WT. Changes in eicosanoid profiles of pain-associated factors in \u003cem\u003eFD\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e when compared to WT were found in the synovial fluid at 2 weeks and the serum at 8 weeks post-DMM.\u003c/p\u003e\n\u003cp\u003eCONCLUSION:\u003c/p\u003e\n\u003cp\u003eThe absence of \u003cem\u003eCfd\u003c/em\u003e drives knee hyperalgesia in male and female mice at 2 weeks-post DMM and persists through an 8-week observation period despite observing cartilage protection. Changes of eicosanoid profiles at both time points suggest that FD drives pain acutely, and the hyperalgesia phenotype emerges early in response to DMM injury, elucidating the role of the alternative complement in mediating OA pain and structural damage.\u003c/p\u003e","manuscriptTitle":"Complement factor D (adipsin) mediates pressure-pain hypersensitivity post destabilization of medial meniscus injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-09 08:41:26","doi":"10.21203/rs.3.rs-6702118/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-21T15:26:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-21T13:43:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-19T09:52:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"102312692747146247084658299307578116326","date":"2025-07-19T09:45:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-10T16:12:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"224397090299419111039909575723261385504","date":"2025-06-09T14:15:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"330026898151469996663335857418678706837","date":"2025-06-03T12:56:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-03T12:21:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-26T09:26:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-26T07:00:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Arthritis Research \u0026 Therapy","date":"2025-05-19T21:54:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"arthritis-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arrt","sideBox":"Learn more about [Arthritis Research \u0026 Therapy](http://arthritis-research.biomedcentral.com/)","snPcode":"13075","submissionUrl":"https://submission.nature.com/new-submission/13075/3","title":"Arthritis Research \u0026 Therapy","twitterHandle":"@ArthritisRes","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"eb931d05-2dda-49d3-adde-1232ce3924c0","owner":[],"postedDate":"June 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:02:05+00:00","versionOfRecord":{"articleIdentity":"rs-6702118","link":"https://doi.org/10.1186/s13075-025-03678-6","journal":{"identity":"arthritis-research-and-therapy","isVorOnly":false,"title":"Arthritis Research \u0026 Therapy"},"publishedOn":"2025-11-26 15:57:10","publishedOnDateReadable":"November 26th, 2025"},"versionCreatedAt":"2025-06-09 08:41:26","video":"","vorDoi":"10.1186/s13075-025-03678-6","vorDoiUrl":"https://doi.org/10.1186/s13075-025-03678-6","workflowStages":[]},"version":"v1","identity":"rs-6702118","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6702118","identity":"rs-6702118","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}