OA susceptibility in mice is partially mediated by the gut microbiome, is transferrable via microbiome transplantation and is associated with immunophenotype changes.

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.

The Microbiome in Osteoarthritis: a Narrative Review of Recent Human and Animal Model Literature.

PURPOSE OF THE REVIEW: The microbiome has recently emerged as a powerful contributor to health and illness in chronic, systemic disorders. Furthermore, new microbiome niches beyond traditional gut locations are frequently being described. Over the past 5 years, numerous pivotal studies have demonstrated associations between changes in various microbiome niches and the development of osteoarthritis (OA). Herein, we review the most impactful recent literature, including microbiome associations with disease and the potential therapeutic value of microbiome manipulation. RECENT FINDINGS: The gut microbiome of human OA patients is enriched in specific bacterial clades, most notably Streptococcus, which correlates with OA pain, Firmicutes, and others. Most studies have focused on knee OA, although one publication demonstrated positive associations with 3 gut microbiome clades in hand OA. OA can be easily distinguished from RA by evaluating differences in oral microbiome composition. Most studies have also demonstrated a reduction in richness of the gut microbiome (alpha diversity) associated with OA. Several studies have identified bacterial signatures within human knee and hip cartilage, synovial fluid, and synovial tissue and have described changes in these patterns occurring with the development of OA. In animal models of OA, high-fat diet-induced obesity has been the most well-studied OA risk factor associated with changes in the microbiome, with numerous bacterial clades changed within the gut microbiome and associated with OA. Also in animal models, various oral supplementations, including dietary fiber, probiotics including Lactobacillus species, and cecal microbiome transplantation have all shown improvements in OA histopathology or cartilage healing. Microbiome changes are strongly associated with the OA disease process and with individual OA risk factors related to both the gut microbiome and the microbial DNA patterns in the joint. Microbiome-directed interventions have the potential to prevent or reduce the progression of OA. Future studies should investigate the mechanistic underpinnings of these microbiome associations and further define the therapeutic potential of microbiome augmentation.

COVID-19: A review of therapeutic strategies and vaccine candidates.

The world is engulfed by one of the most widespread and significant public health crises in decades as COVID-19 has become among the leading causes of death internationally. The novel SARS-CoV-2 coronavirus which causes COVID-19 has unified the scientific community in search of therapeutic and preventative solutions. The top priorities at the moment are twofold: first, to repurpose already-approved pharmacologic agents or develop novel therapies to reduce the morbidity and mortality associated with the ever-spreading virus. Secondly, the scientific and larger pharmaceutical community have been tasked with the development, testing, and production of a safe and effective vaccine as a longer-term solution to prevent further spread and recurrence throughout the populace. The purpose of this article is to review the most up-to-date published data regarding both the leading pharmacological therapies undergoing clinical trials and vaccine candidates in development to stem the threat of COVID-19.

The Development of Epigenetics in the Study of Disease Pathogenesis.

The study of epigenetics has its roots in the study of organism change over time and response to environmental change, although over the past several decades the definition has been formalized to include heritable alterations in gene expression that are not a result of alterations in underlying DNA sequence. In this chapter, we discuss first the history and milestones in the 100+ years of epigenetic study, including early discoveries of DNA methylation, histone posttranslational modification, and noncoding RNA. We then discuss how epigenetics has changed the way that we think of both health and disease, offering as examples studies examining the epigenetic contributions to aging, including the recent development of an epigenetic "clock", and explore how antiaging therapies may work through epigenetic modifications. We then discuss a nonpathogenic role for epigenetics in the clinic: epigenetic biomarkers. We conclude by offering two examples of modern state-of-the-art integrated multi-omics studies of epigenetics in disease pathogenesis, one which sought to capture shared mechanisms among multiple diseases, and another which used epigenetic big data to better understand the pathogenesis of a single tissue from one disease.

Epigenetic editing: How cutting-edge targeted epigenetic modification might provide novel avenues for autoimmune disease therapy.

Autoimmune diseases are enigmatic and complex, and most been associated with epigenetic changes. Epigenetics describes changes in gene expression related to environmental influences mediated by a variety of effectors that alter the three-dimensional structure of chromatin and facilitate transcription factor or repressor binding. Recent years have witnessed a dramatic change and acceleration in epigenetic editing approaches, spurred on by the discovery and later development of the CRISPR/Cas9 system as a highly modular and efficient site-specific DNA binding domain. The purpose of this article is to offer a review of epigenetic editing approaches to date, with a focus on alterations of DNA methylation, and to describe a few prominent published examples of epigenetic editing. We will also offer as an example work done by our laboratory demonstrating epigenetic editing of the FOXP3 gene in human T cells. Finally, we discuss briefly the future of epigenetic editing in autoimmune disease.

The Epigenomic Landscape in Osteoarthritis.

PURPOSE OF REVIEW: Epigenomics has emerged as a key player in our rapidly evolving understanding of osteoarthritis. Historical studies implicated epigenetic alterations, particularly DNA methylation, in OA pathogenesis; however, recent technological advances have resulted in numerous epigenome-wide studies examining in detail epigenetic modifications in OA. The purpose of this article is to introduce basic concepts in epigenetics and their recent applications to the study of osteoarthritis development and progression. RECENT FINDINGS: Epigenetics describes three major phenomena: DNA modification via methylation, histone sidechain modifications, and short noncoding RNA sequences which work in concert to regulate gene transcription in a heritable fashion. Cartilage has been the most widely studied tissue in OA, and differential methylation of genes involved in inflammation, cell cycle, TGFß, and HOX genes have been confirmed several times. Bone studies suggest similar findings, and the intriguing possibility of epigenetic changes in subchondral bone during many OA processes. Multiple studies have demonstrated the involvement of certain noncoding RNAs, particularly miR-140, in OA development via modulation of key catabolic factors. Although much work has been done, much is still unknown. Future epigenomic studies will no doubt continue to widen our understanding of extraarticular tissues and OA pathogenesis, and studies in animal models may offer glimpses into epigenome alterations in the earliest stages of OA.

Epigenetics and Bone Remodeling.

PURPOSE OF REVIEW: Bone remodeling is a diverse field of study with many direct clinical applications; past studies have implicated epigenetic alterations as key factors of both normal bone tissue development and function and diseases of pathologic bone remodeling. The purpose of this article is to review the most important recent advances that link epigenetic changes to the bone remodeling field. RECENT FINDINGS: Epigenetics describes three major phenomena: DNA modification via methylation, histone side chain modifications, and short non-coding RNA sequences which work in concert to regulate gene transcription in a heritable fashion. Recent findings include the role of DNA methylation changes of Wnt, RANK/RANKL, and other key signaling pathways, epigenetic regulation of osteoblast and osteoclast differentiation, and others. Although much work has been done, much is still unknown. Future epigenome-wide studies should focus on extending the tissue coverage, integrating multiple epigenetic analyses with transcriptome data, and working to uncover epigenetic changes linked with early events in aberrant bone remodeling.

Renal involvement in lupus is characterized by unique DNA methylation changes in naïve CD4+ T cells.

Systemic lupus erythematosus is a multi-system disease characterized by wide-spread DNA methylation changes. To identify epigenetic susceptibility loci for lupus nephritis, genome-wide DNA methylation changes in naïve CD4+ T cells were compared between two sets of lupus patients with and without a history of renal involvement. A total of 56 lupus patients (28 with renal involvement and 28 without renal involvement), and 56 age-, sex-, and ethnicity-matched healthy controls were included in our study. We identified 191 CG sites and 121 genes that were only differentially methylated in lupus patients with but not without a history of renal involvement. The tyrosine kinase gene TNK2 involved in cell trafficking and tissue invasion, and the phosphatase gene DUSP5 which dephosphorylates and inhibits the ERK signaling pathway, were among the most hypomethylated. Independent of disease activity, renal involvement is characterized by more robust demethylation in interferon regulated genes differentially methylated in both sets of lupus patients with and without renal involvement (fold change 1.4, P = 0.0014). The type-I interferon master regulator gene IRF7 is only hypomethylated in lupus patients with renal involvement. IRF-7 is an upstream transcription factor that regulates several loci demethylated only with renal involvement such as CD80, HERC5, IFI44, IRF7, ISG15, ISG20, ITGAX, and PARP12 (P = 1.78 × 10(-6)). Among the CG sites only hypomethylated with renal involvement, CG10152449 in CHST12 has a sensitivity of 85.7% and a specificity of 64.3% for stratifying lupus patients for a history of renal involvement (P = 0.0029). Our data identified novel epigenetic susceptibility loci that are differentially methylated with renal involvement in lupus. These loci will help better understand lupus nephritis, and provide a proof of principle for the potential applicability of specific methylation changes as predictors for specific organ involvement in lupus.

Sex-Linked Discrepancies in C57BL6/J Mouse Osteoarthritis are Associated With the Gut Microbiome and are Transferrable by Microbiome Transplantation.

OBJECTIVE: Females have reduced osteoarthritis (OA) in surgical models. The objective of the current study was to evaluate a sex-linked gut microbiome in the pathogenesis of OA. METHODS: We induced OA via destabilization of the medial meniscus surgery in adult male and female C57BL6/J mice with and without opposite-sex microbiome transplantation. Eight weeks later, animals were euthanized, and OA severity, synovitis, and osteophyte scores were determined. Serum lipopolysaccharide was measured chromogenically, and serum cytokines were quantified via multiplex immunoassay. Cecal microbiome profiles were generated using 16S deep sequencing. RESULTS: Males had worse OA histology (3.5x, P = 6 × 10-7 ), synovitis (2.4x, P = 5 × 10-4 ), and osteophyte scores (3.7x, P = 3 × 10-4 ) than females. Male-into-female transplantation worsened all outcomes (histology 1.8x, P = 0.02; synovitis 2.0x, P = 3 × 10-5 ; osteophyte 2.1x, P = 0.01) compared to females, whereas female-into-male transplantation improved all outcomes except for synovitis (histology 0.53x, P = 2 × 10-4 ; osteophyte 0.28x, P = 5 × 10-4 ) compared to males. In the gut microbiome analysis, 44 clades were different in at least one group comparison; 5 clades were correlated with the Osteoarthritis Research Society International score (Lactobacillus R = -0.40, Aldercreutzia R = -0.40, rc4_4 R = -0.55, Sutterella R = -0.37, and Clostridiales R = 0.36). In the cytokine analysis, 10 analytes were different in at least one group comparison; 3 were different in two groups (female and female-into-male transplants vs male comparisons, all reduced in female and female-into-male transplants), including interleukin-12 (0.66x, P = 0.02; 0.66x, P = 0.02, respectively), eotaxin (0.74x, P = 5 × 10-6 ; 0.57x, P = 0.03), and tumor necrosis factor ⍺ (0.49x, P = 0.03; 0.52x, P = 0.009). CONCLUSION: Sex-linked differences in the mouse gut microbiome are associated with OA outcomes, are reversible by opposite-sex microbiome transplantation, and are associated with serum cytokine changes.

Murine cartilage microbial DNA deposition occurs rapidly following the introduction of a gut microbiome and changes with obesity, aging, and knee osteoarthritis.

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.

Genome-wide DNA methylation study suggests epigenetic accessibility and transcriptional poising of interferon-regulated genes in naïve CD4+ T cells from lupus patients.

Systemic lupus erythematosus is an autoimmune disease characterized by multi-system involvement and autoantibody production. Abnormal T cell DNA methylation and type-I interferon play an important role in the pathogenesis of lupus. We performed a genome-wide DNA methylation study in two independent sets of lupus patients and matched healthy controls to characterize the DNA methylome in naïve CD4+ T cells in lupus. DNA methylation was quantified for over 485,000 methylation sites across the genome, and differentially methylated sites between lupus patients and controls were identified and then independently replicated. Gene expression analysis was also performed from the same cells to investigate the relationship between the DNA methylation changes observed and mRNA expression levels. We identified and replicated 86 differentially methylated CG sites between patients and controls in 47 genes, with the majority being hypomethylated. We observed significant hypomethylation in interferon-regulated genes in naïve CD4+ T cells from lupus patients, including IFIT1, IFIT3, MX1, STAT1, IFI44L, USP18, TRIM22 and BST2, suggesting epigenetic transcriptional accessibility in these genetic loci. Indeed, the majority of the hypomethylated genes (21 out of 35 hypomethylated genes) are regulated by type I interferon. The hypomethylation in interferon-regulated genes was not related to lupus disease activity. Gene expression analysis showed overexpression of these genes in total but not naïve CD4+ T cells from lupus patients. Our data suggest epigenetic "poising" of interferon-regulated genes in lupus naïve CD4+ T cells, argue for a novel pathogenic implication for abnormal T cell DNA methylation in lupus, and suggest a mechanism for type-I interferon hyper-responsiveness in lupus T cells.

Functional characterization of the MECP2/IRAK1 lupus risk haplotype in human T cells and a human MECP2 transgenic mouse.

Genetic polymorphism in MECP2/IRAK1 on chromosome Xq28 is a confirmed and replicated susceptibility locus for lupus. High linkage disequilibrium in this locus suggests that both MECP2 and IRAK1 are candidate genes for the disease. DNA methylation changes in lupus T cells play a central role in the pathogenesis of lupus, and MeCp-2 (encoded by MECP2) is a master regulator of gene expression and is also known to recruit DNA methyltransferase 1 (DNMT1) during DNA synthesis. Using human T cells from normal individuals with either the lupus risk or the lupus protective haplotype in MECP2/IRAK1, we demonstrate that polymorphism in this locus increases MECP2 isoform 2 mRNA expression in stimulated but not unstimulated T cells. By assessing DNA methylation levels across over 485,000 methylation sites across the entire genome, we further demonstrate that the lupus risk variant in this locus is associated with significant DNA methylation changes, including in the HLA-DR and HLA-DQ loci, as well as interferon-related genes such as IFI6, IRF6, and BST2. Further, using a human MECP2 transgenic mouse, we show that overexpression of MECP2 alters gene expression in stimulated T cells. This includes overexpression of Eif2c2 that regulates the expression of multiple microRNAs (such as miR-21), and the histone demethylase Jhdm1d. In addition, we show that MECP2 transgenic mice develop antinuclear antibodies. Our data suggest that the lupus-associated variant in the MECP2/IRAK1 locus has the potential to affect all 3 epigenetic mechanisms: DNA methylation, microRNA expression, and histone modification. Importantly, these data support the notion that variants within the MECP2 gene can alter DNA methylation in other genetic loci including the HLA and interferon-regulated genes, thereby providing evidence for genetic-epigenetic interaction in lupus.

Peripheral Blood DNA Methylation-Based Machine Learning Models for Prediction of Knee Osteoarthritis Progression: Biologic Specimens and Data From the Osteoarthritis Initiative and Johnston County Osteoarthritis Project.

OBJECTIVE: The lack of accurate biomarkers to predict knee osteoarthritis (OA) progression is a key unmet need in OA clinical research. The objective of this study was to develop baseline peripheral blood epigenetic biomarker models to predict knee OA progression. METHODS: Genome-wide buffy coat DNA methylation patterns from 554 individuals from the Osteoarthritis Biomarkers Consortium (OABC) were determined using Illumina Infinium MethylationEPIC 850K arrays. Data were divided into model development and validation sets, and machine learning models were trained to classify future OA progression by knee pain, radiographic imaging, knee pain plus radiographic imaging, and any progression (pain, radiographic, or both). Parsimonious models using the top 13 CpG sites most frequently selected during development were tested on independent samples from participants in the Johnston County Osteoarthritis (JoCo OA) Project (n = 128) and a previously published Osteoarthritis Initiative (OAI) data set (n = 55). RESULTS: Full models accurately classified future radiographic-only progression (mean ± SEM accuracy 87 ± 0.8%, area under the curve [AUC] 0.94 ± 0.004), pain-only progression (accuracy 89 ± 0.9%, AUC 0.97 ± 0.004), pain plus radiographic progression (accuracy 72 ± 0.7%, AUC 0.79 ± 0.006), and any progression (accuracy 78 ± 0.4%, AUC 0.86 ± 0.004). Pain-only and radiographic-only progressors were not distinguishable (mean ± SEM accuracy 58 ± 1%, AUC 0.62 ± 0.001). Parsimonious models showed similar performance and accurately classified future radiographic progressors in the OABC cohort and in both validation cohorts (mean ± SEM accuracy 80 ± 0.3%, AUC 0.88 ± 0.003 [using JoCo OA Project data], accuracy 80 ± 0.8%, AUC 0.89 ± 0.002 [using previous OAI data]). CONCLUSION: Our data suggest that pain and structural progression share similar early systemic immune epigenotypes. Further studies should focus on evaluating the pathophysiologic consequences of differential DNA methylation and peripheral blood cell epigenotypes in individuals with knee OA.

A Pilot Analysis of Genome-Wide DNA Methylation Patterns in Mouse Cartilage Reveals Overlapping Epigenetic Signatures of Aging and Osteoarthritis.

OBJECTIVE: Cartilage epigenetic changes are strongly associated with human osteoarthritis (OA). However, the influence of individual environmental OA risk factors on these epigenetic patterns has not been determined; herein we characterize cartilage DNA methylation patterns associated with aging and OA in a mouse model. METHODS: Murine knee cartilage DNA was extracted from healthy young (16-week, n = 6), old (82-week, n = 6), and young 4-week post-destabilization of the medial meniscus (DMM) OA (n = 6) C57BL6/J mice. Genome-wide DNA methylation patterns were determined via Illumina BeadChip. Gene set enrichment analysis was performed by Ingenuity Pathway Analysis. The top seven most differentially methylated positions (DMPs) were confirmed by pyrosequencing in an independent animal set. Results were compared to previously published human OA methylation data. RESULTS: Aging was associated with 20,940 DMPs, whereas OA was associated with 761 DMPs. Merging these two conditions revealed 279 shared DMPs. All demonstrated similar directionality and magnitude of change (Δß 1.0% ± 0.2%, mean methylation change ± SEM). Shared DMPs were enriched in OA-associated pathways, including RhoA signaling (P = 1.57 × 10-4 ), protein kinase A signaling (P = 3.38 × 10-4 ), and NFAT signaling (P = 6.14 × 10-4 ). Upstream regulators, including TET3 (P = 6.15 × 10-4 ), immunoglobulin (P = 6.14 × 10-4 ), and TLR7 (P = 7.53 × 10-4 ), were also enriched. Pyrosequencing confirmed six of the seven top DMPs in an independent cohort. CONCLUSION: Aging and early OA following DMM surgery induce similar DNA methylation changes within a murine OA model, suggesting that aging may induce pro-OA epigenetic "poising" within articular cartilage. Future research should focus on confirming and expanding these findings to other environmental OA risk factors, including obesity, as well as determining late OA changes in mice.

DNA methylation and noncoding RNA in OA: Recent findings and methodological advances.

Introduction: Osteoarthritis (OA) is a chronic musculoskeletal disease characterized by progressive loss of joint function. Historically, it has been characterized as a disease caused by mechanical trauma, so-called 'wear and tear'. Over the past two decades, it has come to be understood as a complex systemic disorder involving gene-environmental interactions. Epigenetic changes have been increasingly implicated. Recent improvements in microarray and next-generation sequencing (NGS) technologies have allowed for ever more complex evaluations of epigenetic aberrations associated with the development and progression of OA. Methods: A systematic review was conducted in the Pubmed database. We curated studies that presented the results of DNA methylation and noncoding RNA research in human OA and OA animal models since 1985. Results: Herein, we discuss recent findings and methodological advancements in OA epigenetics, including a discussion of DNA methylation, including microarray and NGS studies, and noncoding RNAs. Beyond cartilage, we also highlight studies in subchondral bone and peripheral blood mononuclear cells, which highlight widespread and potentially clinically important alterations in epigenetic patterns seen in OA patients. Finally, we discuss epigenetic editing approaches in the context of OA. Conclusions: Although a substantial body of literature has already been published in OA, much is still unknown. Future OA epigenetics studies will no doubt continue to broaden our understanding of underlying pathophysiology and perhaps offer novel diagnostics and/or treatments for human OA.

Ear wound healing in MRL/MpJ mice is associated with gut microbiome composition and is transferable to non-healer mice via microbiome transplantation.

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.

Identification of Cartilage Microbial DNA Signatures and Associations With Knee and Hip Osteoarthritis.

OBJECTIVE: Alterations 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). METHODS: We 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. RESULTS: Alpha 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. CONCLUSION: Our 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.

Uric acid directly promotes human T-cell activation.

BACKGROUND: Abnormally high serum uric acid levels have been associated with several disease conditions including gout and kidney stone disease. More recently, it was shown that uric acid crystals stimulate dendritic cell maturation, activate the NALP3 inflammasome, and enhance antigen-specific immune responses. We hypothesize that uric acid can also stimulate T cells directly and in the absence of antigen presentation. METHODS: Purified primary human T cells were incubated with and without uric acid at concentrations of 50, 100, 150, and 200 microg/mL. The expression of T-cell activation markers CD25 and CD70 was assessed by flow cytometry. In other experiments, Jurkat T cells were used and the expression of the costimulatory molecule CD70 was determined at the mRNA level. RESULTS: Uric acid directly activates primary human T cells in the absence of antigen presentation. Furthermore, primary human T cells and Jurkat T cells treated with uric acid overexpress the costimulatory molecule CD70, which plays an important role in T cell-B cell interaction and antibody production. CONCLUSIONS: The finding that uric acid directly promotes T-cell activation in an antigen-independent system is novel and might play a mechanistic role in the inflammatory response observed in gouty arthritis and other immune-mediated diseases.

A pilot study of peripheral blood DNA methylation models as predictors of knee osteoarthritis radiographic progression: data from the Osteoarthritis Initiative (OAI).

Knee osteoarthritis (OA) is a leading cause of chronic disability worldwide, but no diagnostic or prognostic biomarkers are available. Increasing evidence supports epigenetic dysregulation as a contributor to OA pathogenesis. In this pilot study, we investigated epigenetic patterns in peripheral blood mononuclear cells (PBMCs) as models to predict future radiographic progression in OA patients enrolled in the longitudinal Osteoarthritis Initiative (OAI) study. PBMC DNA was analyzed from baseline OAI visits in 58 future radiographic progressors (joint space narrowing at 24 months, sustained at 48 months) compared to 58 non-progressors. DNA methylation was quantified via Illumina microarrays and beta- and M-values were used to generate linear classification models. Data were randomly split into a 60% development and 40% validation subsets, models developed and tested, and cross-validated in a total of 40 cycles. M-value based models outperformed beta-value based models (ROC-AUC 0.81 ± 0.01 vs. 0.73 ± 0.02, mean ± SEM, comparison p = 0.002), with a mean classification accuracy of 73 ± 1% (mean ± SEM) for M- and 69 ± 1% for beta-based models. Adjusting for covariates did not significantly alter model performance. Our findings suggest that PBMC DNA methylation-based models may be useful as biomarkers of OA progression and warrant additional evaluation in larger patient cohorts.

Genome-Wide DNA Methylation Study Identifies Significant Epigenomic Changes in Osteoarthritic Subchondral Bone and Similarity to Overlying Cartilage.

OBJECTIVE: To perform a genome-wide DNA methylation study to identify differential DNA methylation patterns in subchondral bone underlying eroded and intact cartilage from patients with hip osteoarthritis (OA) and to compare these with DNA methylation patterns in overlying cartilage. METHODS: Genome-wide DNA methylation profiling using Illumina HumanMethylation 450 arrays was performed on eroded and intact cartilage and subchondral bone from within the same joint of 12 patients undergoing hip arthroplasty. Genes with differentially methylated CpG sites were analyzed to identify shared pathways, upstream regulators, and overrepresented gene ontologies, and these patterns were compared with those of the overlying cartilage. Histopathology was graded by modified Mankin score and assessed for correlation with DNA methylation. RESULTS: We identified 7,316 differentially methylated CpG sites in subchondral bone underlying eroded cartilage, most of which (∼75%) were hypomethylated, and 1,397 sites in overlying eroded cartilage, 126 of which were shared. Samples clustered into 3 groups with distinct histopathologic scores. We observed differential DNA methylation of genes including the RNA interference-processing gene AGO2, the growth factor TGFB3, the OA suppressor NFATC1, and the epigenetic effector HDAC4. Among known susceptibility genes in OA, 32 were differentially methylated in subchondral bone, 8 were differentially methylated in cartilage, and 5 were shared. Upstream regulator analysis using differentially methylated genes in OA subchondral bone showed a strong transforming growth factor ß1 signature (P = 1 × 10(-40) ) and a tumor necrosis factor family signature (P = 3.2 × 10(-28) ), among others. CONCLUSION: Our data suggest the presence of an epigenetic phenotype associated with eroded OA subchondral bone that is similar to that of overlying eroded OA cartilage.