ObjectiveAlterations of the gut microbiota have been implicated in many forms of arthritis, but an examination of cartilage microbial patterns has not been performed. This study was undertaken to characterize the microbial DNA profile of articular cartilage and determine changes associated with osteoarthritis (OA).MethodsWe performed 16S ribosomal RNA gene deep sequencing on eroded and intact cartilage samples from knee OA patients (n = 21 eroded and 21 intact samples) and hip OA patients (n = 34 eroded and 33 intact samples) and cadaver controls (n = 10 knee samples and 10 hip samples). Microbial DNA diversity was assessed, groups were compared, and metagenomic profiles were reconstructed. Confirmation was performed in an independent cohort by clade‐specific quantitative polymerase chain reaction. Findings in human cartilage were compared to those in cartilage from OA‐susceptible C57BL/6 (B6) mice and OA‐resistant MRL/MpJ (MRL) mice. Germ‐free B6 mouse cartilage was analyzed as a methodologic control.ResultsAlpha diversity was reduced in human OA versus control samples (P < 0.0001), and in hip versus knee samples (P < 0.0001). Numerous clades were different in human OA versus control samples, and similar findings were noted in comparisons of murine B6 versus MRL mice. Hip samples were microbiologically distinct from knee samples. OA microbial DNA demonstrated increased gram‐negative constituents (P = 0.02). Functional analysis demonstrated increases in lipopolysaccharide production (P = 9.9 × 10−3), phosphatidylinositol signaling (P = 4.2 × 10−4), and nitrogen metabolism (P = 8 × 10−3) and decreases in sphingolipid metabolism (P = 7.7 × 10−4) associated with OA.ConclusionOur study reveals a microbial DNA signature in human and mouse cartilage. Alterations in this signature, including increases in gram‐negative constituents, occur during the development and progression of human OA. Furthermore, our findings indicate that strain‐specific signatures exist within mouse cartilage that mirror human patterns. Further study of the establishment and potential pathogenic role of these DNA signatures is needed.
Objective Adult elastic cartilage has limited repair capacity. MRL/MpJ (MRL) mice, by contrast, are capable of spontaneously healing ear punctures. This study was undertaken to characterize microbiome differences between healer and non-healer mice and to evaluate whether this healing phenotype can be transferred via gut microbiome transplantation. Methods We orally transplanted C57BL/6J (B6) mice with MRL/MpJ cecal contents at weaning and as adults (n = 57) and measured ear hole closure 4 weeks after a 2.0mm punch and compared to vehicle-transplanted MRL and B6 (n = 25) and B6-transplanted MRL (n = 20) mice. Sex effects, timing of transplant relative to earpunch, and transgenerational heritability were evaluated. In a subset (n = 58), cecal microbiomes were profiled by 16S sequencing and compared to ear hole closure. Microbial metagenomes were imputed using PICRUSt. Results Transplantation of B6 mice with MRL microbiota, either in weanlings or adults, improved ear hole closure. B6-vehicle mice healed ear hole punches poorly (0.25±0.03mm, mm ear hole healing 4 weeks after a 2mm ear hole punch [2.0mm—final ear hole size], mean±SEM), whereas MRL-vehicle mice healed well (1.4±0.1mm). MRL-transplanted B6 mice healed roughly three times as well as B6-vehicle mice, and half as well as MRL-vehicle mice (0.74±0.05mm, P = 6.9E-10 vs. B6-vehicle, P = 5.2E-12 vs. MRL-vehicle). Transplantation of MRL mice with B6 cecal material did not reduce MRL healing (B6-transplanted MRL 1.3±0.1 vs. MRL-vehicle 1.4±0.1, p = 0.36). Transplantation prior to ear punch was associated with the greatest ear hole closure. Offspring of transplanted mice healed significantly better than non-transplanted control mice (offspring:0.63±0.03mm, mean±SEM vs. B6-vehicle control:0.25±0.03mm, n = 39 offspring, P = 4.6E-11). Several microbiome clades were correlated with healing, including Firmicutes (R = 0.84, P = 8.0E-7), Lactobacillales (R = 0.65, P = 1.1E-3), and Verrucomicrobia (R = -0.80, P = 9.2E-6). Females of all groups tended to heal better than males (B6-vehicle P = 0.059, MRL-transplanted B6 P = 0.096, offspring of MRL-transplanted B6 P = 0.0038, B6-transplanted MRL P = 1.6E-6, MRL-vehicle P = 0.0031). Many clades characteristic of female mouse cecal microbiota vs. males were the same as clades characteristic of MRL and MRL-transplanted B6 mice vs. B6 controls, including including increases in Clostridia and reductions in Verrucomicrobia in female mice. Conclusion In this study, we found an association between the microbiome and tissue regeneration in MRL mice and demonstrate that this trait can be transferred to non-healer mice via microbiome transplantation. We identified several microbiome clades associated with healing.
BackgroundExtensive evidence has correlated epigenetic alterations in articular tissues with both the presence and progression of human osteoarthritis, but few analyses of blood cell epigenetic patterns have been done in OA.ObjectivesWe examined the DNA methylation aging rate in peripheral blood mononuclear cells (PBMCs) at baseline from knee OA patients with rapid radiographic progression compared to well-matched nonprogressors enrolled in the Osteoarthritis Initiative (OAI).MethodsPBMC DNA was obtained from baseline blood draws of 64 OA patients enrolled in the OAI longitudinal study. All patients had baseline symptomatic and radiographic OA. 32 rapidly-progressive OA patients, defined as ≥1.0mm radiographic joint space loss within 24 month follow-up were compared to 32 non-progressive patients. There were no differences in age, sex, race, BMI, baseline K/L grade, or calculated PBMC subset composition between rapid- and non-progressors. DNA methylation was quantified with Illumina HumanMethylation 450k arrays. Preprocessing was performed in GenomeStudio and normalized to internal controls. Epigenetic age was estimated with the algorithm described by Horvath et. al., using 353 age-associated CpG sites. This epigenetic age was compared to chronological age to calculate epigenetic-chronological age discordance (ΔAge) and group differences compared with a Student t-test. ΔAge was correlated with individual CpG methylation sites of rapid progressors, and Pearson values calculated. Correlation was considered significant if Pearson's r values were ≤-0.55 or ≥0.55 (p≤0.001). Pathway analysis of correlated genes was performed with the Ingenuity Pathway Analysis (IPA) system.ResultsThe baseline DNA methylation aging rate in rapidly progressive (RP) knee OA patients was decelerated compared to nonprogressors (NP) and to chronological age (ΔAge-RP: -4.9±1.4 vs. ΔAge-NP: -0.071±1.3 mean±SEM years less than chronological age, p=0.015). 1165 CpG sites were correlated with ΔAge in rapid progressors, corresponding to 755 genes. Ontologic analysis of highly correlated genes showed association of the STAT3 pathway (p=6E-4), Notch signaling (p=1E-3), axonal guidance signaling (p=7E-3), CREB signaling (p=2E-2), NFAT signaling (p=2E-2), and autophagy (p=4E-2) among others. Associated upstream regulators included FGF2 (p=3E-5), SMAD4 (p=9E-4), SMAD5 (p=1E-3, TNF (p=4E-3), and TGFB1 (p=4E-3), among othersConclusionsOur data reveal that a decelerated peripheral blood differential DNA methylation age epigenotype is present at baseline in rapidly progressive knee OA patients, but not in nonprogressive knee OA patients. The genes correlated with this methylation age deceleration cluster in pathways previously associated with OA in articular tissues, suggesting that these pathways may systemically epigenetically dysregulated. Our data reinforce the notion that OA is a heterogeneous disease composed of distinct subgroups, and suggests that future epigenetic investigation of immune cell subsets may be beneficial in unraveling OA pathogenesis....
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