OBJECTIVE:Osteoarthritis (OA) involves systemic inflammation, yet peripheral blood immunophenotypes and their underlying epigenetic landscapes remain poorly defined. We characterized cellular and chromatin accessibility profiles in knee OA and radiographic progressors (RPs). METHODS:We performed multimodal immunophenotyping using cytometry by time-of-flight mass spectrometry (CyTOF) on peripheral blood samples from 21 patients with knee OA and 11 healthy controls (HCs). To confirm findings, we developed DNA methylation-based imputation models and applied them to two independent validation cohorts (n = 723). Finally, single-cell Assay for Transposase-Accessible Chromatin sequencing (scATAC-seq) was performed to interrogate chromatin accessibility landscapes in a patient subset. RESULTS:CyTOF identified significant expansions of CD8+ central memory T cells, CD4+ Treg cells, and nonclassical monocytes in patients with OA versus HCs (all P ≤ 0.05). These expansions were robustly confirmed in the large-scale validation cohorts (all P < 0.0001). Conversely, RPs displayed validated reductions in circulating CD4+ and CD8+ central memory pools. scATAC-seq revealed extensive epigenetic remodeling, particularly within monocytes and Treg cells. Paradoxically, unsupervised clustering indicated that epigenetically defined proinflammatory clusters were depleted in OA blood. Pathway analysis revealed that these depleted clusters possessed activated, migratory phenotypes, whereas the expanded circulating cells among patients with OA displayed quiescent, nonmigratory epigenetic signatures. CONCLUSION:Knee OA is characterized by validated systemic expansions of specific Treg cell and monocyte subsets. Single-cell epigenetic profiling suggests epigenetically distinct subpopulations driven by age, OA, comorbid conditions, or migration from circulation to the periphery.

Vagus nerve stimulation (VNS) has been shown to improve chronic pain, including fibromyalgia, migraine headaches, and gastrointestinal pain, as well as inflammation in RA. Here, we investigated the efficacy of transcutaneous VNS (tVNS) in providing pain relief in a mouse model of post-traumatic osteoarthritis (PTOA). Destabilization of the medial meniscus (DMM) surgery was performed in 16-wk-old female and male mice. Beginning at 4 wk post-injury, mice were randomized to receive tVNS (10 min daily, 5 d per wk) delivered via the tragus of the ear or sham stimulation for 8 wk. Mechanical hyperalgesia, as measured using an algometer, and weight distribution were monitored at 3 time points: (1) pre-VNS, (2) 4-wk treatment, and (3) 8-wk treatment (end point of the study). Compared to the sham treatment, 4 wk of tVNS reduced mechanical hyperalgesia in female mice, and 8 wk of tVNS improved weight-bearing in male and female mice. tVNS altered serum pro-inflammatory cytokine and chemokine levels in a sex-dependent manner. Eight weeks of tVNS improved the lateral femur compartment of the DMM-affected joints in males. Our results reveal that tVNS improves PTOA-related pain and suppresses pro-inflammatory cytokine production, making it a promising, non-addictive intervention for chronic OA pain.

ObjectivesDietary interventions are a potentially powerful treatment option for knee osteoarthritis (OA). The objective of this study was to evaluate a well‐formulated ketogenic diet (KD) in the context of knee OA histology and pain using the destabilization of the medial meniscus (DMM) mouse model and correlate with gut microbiome and systemic cytokine levels.MethodsAdult male mice underwent unilateral DMM or sham surgery and were then fed eight weeks of KD or chow. At baseline and every two weeks, mechanical allodynia of the operated and contralateral knees was assessed via analgesiometry. Knee joints were collected for histology, gut microbiome analysis was performed on cecal material via 16S sequencing, and serum cytokines were analyzed via Bio‐Plex assay.ResultsKD mice had worse histopathologic OA after DMM (mean ± SEM Osteoarthritis Research Society International score: KD‐DMM: 4.0 ± 0.5 vs chow‐DMM: 2.7 ± 0.08; P = 0.02). KD mice had increased mechanical allodynia postsurgery (P = 0.005 in mixed‐effects model). The gut microbiome changed substantially with KD: 59 clades were altered by KD in DMM and 39 by KD in sham (36 were shared, 25 overlapped with previous murine OA studies). Several clades were correlated on an individual‐mouse level with both histology and allodynia (eg, Lactobacillus histology P = 0.004, allodynia P = 1 × 10−4). Serum analysis showed four cytokines increased with KD (interleukin [IL]‐1β, IL‐2, IL‐3, and IL‐13).ConclusionKD started immediately after OA induction via DMM is associated with worsened histologic outcomes. KD also worsens mechanical allodynia after either DMM or sham surgery. KD induces significant gut microbiome dysbiosis in clades previously associated with murine OA.

OBJECTIVE:Aging is a strong risk factor for osteoarthritis (OA). Previous studies have not found accelerated blood epigenetic aging in OA patients generally. The goal of this study was to evaluate peripheral blood epigenetic aging rates among OA patient subgroups. METHODS:Genome-wide baseline blood DNA methylation data from the Osteoarthritis Initiative (n = 554) and Johnston County Osteoarthritis Project (n = 128) were generated using Illumina MethylationEPICv1 arrays on baseline samples of OA progressors (radiographic and/or pain progression in 2-5 years) vs. non-progressors. Epigenetic age was calculated using Horvath, GrimAge, PhenoAge, Horvath-IEAA, Hannum, Horvath-SkinBlood, and DunedinPACE calculators and epigenetic age acceleration was compared. Age-associated proteins were imputed using GrimAge and generalized logistic models developed to differentiate progressor groups. RESULTS:Borderline epigenetic age acceleration was found among radiographic progressors using the SkinBlood calculator (difference in epigenetic age acceleration, ΔEAA=1.5 years, q = 0.02), dual progressors (pain+radiographic) (ΔEAA = 1.2y, q = 0.05) and any progressor (pain+radiographic+dual) (ΔEAA = 1.1y, q = 0.02), although this was less than the reported error of the calculator (2.5 years). Radiographic progressors also exhibited accelerated epigenetic aging using the PhenoAge calculator (ΔEAA = 2.1y, q = 0.02). Imputed age protein-based models showed marginal performance for radiographic progression (area under the curve (AUC) = 0.63) and poor performance for other progressor types (pain AUC=0.54, dual AUC=0.52, any AUC = 0.57). Proteins chosen most frequently during modeling had prior links to OA (i.e., GDF8, MATN3, and interleukin 18). CONCLUSIONS:No meaningful difference in epigenetic age was seen among radiographic OA using 5 of 7 aging calculators, with minimal and likely not-clinically-relevant epigenetic age acceleration using the SkinBlood and PhenoAge calculators.

Objective:The lack of biomarkers to predict knee osteoarthritis (OA) progression is a key unmet need in the OA field. The objective of this study was to evaluate epigenetic changes in baseline peripheral blood cells of healthy individuals who subsequently developed radiographic knee OA (incident OA) as a preclinical biomarker. Methods:Genome-wide DNA methylation data were generated using Illumina Methylation EPICv2 arrays on baseline blood samples from 234 incident OA patients and 234 controls matched by age±5y, sex, ethnicity, and BMI±5 units. Peripheral blood cellular composition was estimated. Lasso was used for CpG selection. Models were first trained using a 40-round Monte Carlo cross-validated generalized logistic modeling approach, then parsimonious models developed using CpG sites selected in ≥10 rounds. Results:Future incident OA cases were estimated to have increased monocyte frequency (0.089 ​± ​0.0001 vs. 0.083 ​± ​0.0001 mean ​± ​SEM, q ​= ​0.01), although cell composition models were not predictive (AUC ​= ​0.56 ​± ​0.01) Full methylation models accurately differentiated future incident OA patients from controls but suffered from overfitting (development: AUC ​= ​0.91 ​± ​0.001, validation: AUC ​= ​0.64 ​± ​0.006). Parsimonious models developed using 21 top CpG sites performed similarly with less overfitting (development: AUC ​= ​0.91 ​± ​0.001, validation: AUC ​= ​0.87 ​± ​0.003, accuracy 0.79 ​± ​0.004, sensitivity 0.78 ​± ​0.004, specificity 0.80 ​± ​0.006). Previously developed OA progression models did not predict incident OA (AUC ​= ​0.48 ​± ​0.002). Five of 21 CpG sites were located proximate to known genes, including RGS9BP, TOLLIP, UOX, L3MBTL1, and IFI27L1. Conclusions:Blood-based DNA methylation models may predict incident knee OA in healthy individuals using a small set of CpG sites. Further work should focus on evaluating the pathophysiological role of DNA methylation in incident OA.

Purpose (the aim of the study): Osteoarthritis (OA) is an incompletely understood chronic disease characterized by low level systemic inflammation among other factors. One reliable model for inducing OA in mice is surgical destabilization of the medial meniscus (DMM). The surgery primarily affects the central weight-bearing region of the medial tibial plateau and medial femoral condyle. The gut microbiome has come to light as a potential contributor to the onset and course of many diseases, including musculoskeletal disorders, in recent years.

Purpose (the aim of the study): Purpose: Osteoarthritis (OA) is a significant global health problem, characterized by cartilage degeneration and associated symptoms such as pain, stiffness, swelling, and functional disability. Limited treatment options required an exploration of novel interventions. Vagal nerve stimulation (VNS), FDA-approved for epilepsy and depression, activates the cholinergic anti-inflammatory pathway, reducing inflammation and pain in rheumatoid arthritis mouse models. We investigated transcutaneous-VNS (tVNS) effects on pain in destabilization of the medial meniscus (DMM) and tibial compression anterior cruciate ligament (ACL) rupture mouse models of OA.

Purpose (the aim of the study): Knee osteoarthritis (OA) is a heterogeneous disease characterized by a variety of clinical and molecular phenotypes. However, we do not yet have robust biomarkers to distinguish/predict these phenotypes. We previously published analyses of baseline peripheral blood cell DNA methylation patterns to predict future radiographic and pain progression in patients with early symptomatic knee OA. In the current study, we expand this previous analysis to include the prediction of future incident radiographic knee OA using baseline peripheral blood DNA methylation data from healthy individuals in the Osteoarthritis Initiative (OAI) cohort.

Purpose (the aim of the study): Knee osteoarthritis (OA) is a heterogeneous disease characterized by a variety of clinical and molecular phenotypes. We do not yet have robust biomarkers to distinguish/predict these phenotypes. Several chronic diseases exhibit a phenotype of epigenetic age acceleration, including diabetes, heart disease, and malignancy. Recent studies have not identified epigenetic age acceleration in knee OA generally. In the current study, we evaluated whether accelerated epigenetic aging was present in baseline blood samples from Osteoarthritis Initiative (OAI) and Johnston County Osteoarthritis Project (JoCoOA) participants who experienced rapid radiographic progression in the subsequent 48-96 months.

Objectives The Murphy Roths Large (MRL)/MpJ ‘superhealer’ mouse strain is protected from post-traumatic osteoarthritis (OA), although no studies have evaluated the microbiome in the context of this protection. This study characterised microbiome differences between MRL and wild-type mice, evaluated microbiome transplantation and OA and investigated microbiome-associated immunophenotypes. Methods Cecal material from mixed sex C57BL6/J (B6) or female MRL/MpJ (MRL) was transplanted into B6 and MRL mice, then OA was induced by disruption of the medial meniscus surgery (DMM). In other experiments, transplantation was performed after DMM and transplantation was performed into germ-free mice. Transplanted mice were bred through F2. OARSI, synovitis and osteophyte scores were determined blindly 8 weeks after DMM. 16S microbiome sequencing was performed and metagenomic function was imputed. Immunophenotypes were determined using mass cytometry. Results MRL-into-B6 transplant prior to DMM showed reduced OA histopathology (OARSI score 70% lower transplant vs B6 control), synovitis (60% reduction) and osteophyte scores (30% reduction) 8 weeks after DMM. When performed 48 hours after DMM, MRL-into-B6 transplant improved OA outcomes but not when performed 1–2 weeks after DMM. Protection was seen in F1 (60% reduction) and F2 progeny (30% reduction). Several cecal microbiome clades were correlated with either better (eg, Lactobacillus, R=−0.32, p=0.02) or worse (eg, Rikenellaceae , R=0.43, p=0.001) OA outcomes. Baseline immunophenotypes associated with MRL-into-B6 transplants and MRL included reduced double-negative T cells and increased CD25+CD4+ T cells. Conclusion The gut microbiome is responsible in part for OA protection in MRL mice and is transferrable by microbiome transplantation. Transplantation induces resting systemic immunophenotyping changes that correlate with OA protection.

Currently, there are no disease-modifying osteoarthritis (OA) drugs (DMOAD) to prevent OA progression and there are limitations on pain relieving therapeutics. Vagus nerve stimulation (VNS) delivered by an implantable device is FDA-approved for refractory epilepsy and severe depression. Here, we investigated the efficacy of transcutaneous VNS (tVNS) for preventing OA progression and providing pain relief in two mouse models of post-traumatic OA (PTOA): the surgical destabilized medial meniscus (DMM) and the non-surgical forced tibial compression anterior cruciate ligament rupture (ACLR). Here, we show that 2 weeks of tVNS significantly reduced histological OA scores in male and female mice after ACLR compared to sham stimulation. In female, but not male, mice, tVNS reduced hyperalgesia and mechanical allodynia. In the slower DMM model, 8 weeks of tVNS improved weight bearing in male and female mice, but only female mice had improved hyperalgesia. Male mice had lower OA histological scores. Serum proinflammatory cytokines were significantly reduced by tVNS in both models but differed by gender and model. Overall, these results provide strong pre-clinical evidence that tVNS reduces OA progression, improves pain, and suppresses pro-inflammatory cytokines, making it a promising DMOAD. ### Competing Interest Statement TM Griffin: Patent application pending, Compositions and methods for treating osteoarthritis using a CD14 inhibitor; consulting with Novo Nordisk

Cartilage microbial DNA patterns have been recently characterized in osteoarthritis (OA). The objectives of this study were to evaluate the gut origins of cartilage microbial DNA, to characterize cartilage microbial changes with age, obesity, and OA in mice, and correlate these to gut microbiome changes. We used 16S rRNA sequencing performed longitudinally on articular knee cartilage from germ-free (GF) mice following oral microbiome inoculation and cartilage and cecal samples from young and old wild-type mice with/without high-fat diet-induced obesity (HFD) and with/without OA induced by destabilization of the medial meniscus (DMM) to evaluate gut and cartilage microbiota. Microbial diversity was assessed, groups compared, and functional metagenomic profiles reconstructed. Findings were confirmed in an independent cohort by clade-specific qPCR. We found that cartilage microbial patterns developed at 48 h and later timepoints following oral microbiome inoculation of GF mice. Alpha diversity was increased in SPF mouse cartilage samples with age (P = 0.013), HFD (P = 5.6E-4), and OA (P = 0.029) but decreased in cecal samples with age (P = 0.014) and HFD (P = 1.5E-9). Numerous clades were altered with aging, HFD, and OA, including increases in Verrucomicrobia in both cartilage and cecal samples. Functional analysis suggested changes in dihydroorotase, glutamate-5-semialdehyde dehydrogenase, glutamate-5-kinase, and phosphoribosylamine-glycine ligase, in both cecum and cartilage, with aging, HFD, and OA. In conclusion, cartilage microbial DNA patterns develop rapidly after the introduction of a gut microbiome and change in concert with the gut microbiome during aging, HFD, and OA in mice. DMM-induced OA causes shifts in both cartilage and cecal microbiome patterns independent of other factors.