Osteoarthritis (OA) is a leading cause of chronic pain, yet OA pain management remains poor. Age is the strongest predictor of OA development, and mechanisms driving OA pain are unclear. We undertook ...this study to characterize age-associated changes in knee OA, pain-related behaviors, and dorsal root ganglion (DRG) molecular phenotypes in mice of both sexes.
Male or female C57BL/6 mice 6 or 20 months of age were evaluated for histopathologic knee OA, pain-related behaviors, and L3-L5 DRG immune characterization via flow cytometry. DRG gene expression in older mice and humans was also examined.
Male mice at 20 months of age had worse cartilage degeneration than 6-month-old mice. Older female mouse knees showed increased cartilage degeneration but to a lesser degree than those of male mice. Older mice of both sexes had worse mechanical allodynia, knee hyperalgesia, and grip strength compared to younger mice. For both sexes, DRGs from older mice showed decreased CD45+ cells and a significant increase in F4/80+ macrophages and CD11c+ dendritic cells. Older male mouse DRGs showed increased expression of Ccl2 and Ccl5, and older female mouse DRGs showed increased Cxcr4 and Ccl3 expression compared to 6-month-old mouse DRGs, among other differentially expressed genes. Human DRG analysis from 6 individuals >80 years of age revealed elevated CCL2 in men compared to women, whereas CCL3 was higher in DRGs from women.
We found that aging in male and female mice is accompanied by mild knee OA, mechanical sensitization, and changes to immune cell populations in the DRG, suggesting novel avenues for development of OA therapies.
•The timescale of obesity-related knee osteoarthritis pathogenesis is slower than systemic metabolic and functional changes.•A high-fat diet and voluntary wheel running altered osteophyte formation, ...subchondral tibial bone mineral density, and relative tibial trabecular bone volume prior to diet or activity-induced changes in cartilage damage.•“Pre-osteoarthritis” high-fat diet and exercise-dependent phenotypes were explored using correlation network analyses to examine associations between systemic factors (i.e., metabolic, inflammatory, and biomechanical outcomes) and local knee structural changes during early-stage osteoarthritis.•Systemic and local knee structural outcomes in diet-induced obese mice were more strongly associated with circadian-related running patterns rather than running distance.•Sedentary and exercise networks were nearly completely distinct, suggesting that the biological relationships among systemic and local osteoarthritis-related variables are substantially altered the level of physical activity.
Obesity increases knee osteoarthritis (OA) risk through metabolic, inflammatory, and biomechanical factors, but how these systemic and local mediators interact to drive OA pathology is not well understood. We tested the effect of voluntary running exercise after chronic diet-induced obesity on knee OA-related cartilage and bone pathology in mice. We then used a correlation-based network analysis to identify systemic and local factors associated with early-stage knee OA phenotypes among the different diet and exercise groups.
Male C57BL/6J mice were fed a defined control (10% kcal fat) or high fat (HF) (60% kcal fat) diet from 6 to 37 weeks of age. At 25 weeks, one-half of the mice from each diet group were housed in cages with running wheels for the remainder of the study. Histology, micro computed tomography, and magnetic resonance imaging were used to evaluate changes in joint tissue structure and OA pathology. These local variables were then compared to systemic metabolic (body mass, body fat, and glucose tolerance), inflammatory (serum adipokines and inflammatory mediators), and functional (mechanical tactile sensitivity and grip strength) outcomes using a correlation-based network analysis. Diet and exercise effects were evaluated by two-way analysis of variance.
An HF diet increased the infrapatellar fat pad size and posterior joint osteophytes, and wheel running primarily altered the subchondral cortical and trabecular bone. Neither HF diet nor exercise altered average knee cartilage OA scores compared to control groups. However, the coefficient of variation was ≥25% for many outcomes, and some mice in both diet groups developed moderate OA (≥33% maximum score). This supported using correlation-based network analyses to identify systemic and local factors associated with early-stage knee OA phenotypes. In wheel-running cohorts, an HF diet reduced the network size compared to the control diet group despite similar running distances, suggesting that diet-induced obesity dampens the effects of exercise on systemic and local OA-related factors. Each of the 4 diet and activity groups showed mostly unique networks of local and systemic factors correlated with early-stage knee OA.
Despite minimal group-level effects of chronic diet-induced obesity and voluntary wheel running on knee OA pathology under the current test durations, diet and exercise substantially altered the relationships among systemic and local variables associated with early-stage knee OA. These results suggest that distinct pre-OA phenotypes may exist prior to the development of disease.
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Obesity is one of the most significant risk factors for knee osteoarthritis. However, therapeutic strategies to prevent or treat obesity-associated osteoarthritis are limited because of uncertainty ...about the etiology of disease, particularly with regard to metabolic factors. High-fat-diet-induced obese mice have become a widely used model for testing hypotheses about how obesity increases the risk of osteoarthritis, but progress has been limited by variation in disease severity, with some reports concluding that dietary treatment alone is insufficient to induce osteoarthritis in mice. We hypothesized that increased sucrose content of typical low-fat control diets contributes to osteoarthritis pathology and thus alters outcomes when evaluating the effects of a high-fat diet. We tested this hypothesis in male C57BL/6J mice by comparing the effects of purified diets that independently varied sucrose or fat content from 6 to 26 weeks of age. Outcomes included osteoarthritis pathology, serum metabolites, and cartilage gene and protein changes associated with cellular metabolism and stress-response pathways. We found that the relative content of sucrose versus cornstarch in low-fat iso-caloric purified diets caused substantial differences in serum metabolites, joint pathology, and cartilage metabolic and stress-response pathways, despite no differences in body mass or body fat. We also found that higher dietary fat increased fatty acid metabolic enzymes in cartilage. The findings indicate that the choice of control diets should be carefully considered in mouse osteoarthritis studies. Our study also indicates that altered cartilage metabolism might be a contributing factor to how diet and obesity increase the risk of osteoarthritis.
Aging and female sex are the strongest risk factors for nontraumatic osteoarthritis (OA); whereas obesity is a modifiable risk factor accelerating OA. Prior studies indicate that the innate immune ...receptor toll-like receptor 4 (TLR4) mediates obesity-induced metabolic inflammation and cartilage catabolism via recognition of damage-associated molecular patterns and is increased with aging in OA joints. TLR4 responses are limited by innate immunoreceptor adapter protein DNAX-activating protein of 12kDA (DAP12). We undertook this study to test the hypothesis that TLR4 promotes, whereas DAP12 limits, obesity-accelerated OA in aged female mice. We fed 13- to 15-month-old female WT, TLR4 KO, and DAP12 KO mice a high-fat diet (HFD) or a control diet for 12 weeks, and changes in body composition, glucose tolerance, serum cytokines, and insulin levels were compared. Knee OA was evaluated by histopathology and μCT. Infrapatellar fat pads (IFPs) were analyzed by histomorphometry and F4/80+ crown-like structures were quantified. IFPs and synovium gene expression were analyzed using a targeted insulin resistance and inflammation array. All HFD-treated mice became obese, but only WT and TLR4 KO mice developed glucose intolerance. HFD induced cartilage catabolism in WT and DAP12 KO female mice, but not in TLR4 KO mice. Gene-expression analysis of IFPs and synovium showed significant differences in insulin signaling, adipokines, and inflammation between genotypes and diets. Unlike young mice, systemic inflammation was not induced by HFD in the older female mice independent of genotype. Our findings support the conclusion that TLR4 promotes and DAP12 limits HFD-induced cartilage catabolism in middle-aged female mice.
Immune Contributions to Osteoarthritis Lopes, Erika Barboza Prado; Filiberti, Adrian; Husain, Syed Ali ...
Current osteoporosis reports,
12/2017, Letnik:
15, Številka:
6
Journal Article
Recenzirano
Purpose of the Review
Mounting evidence supports a role of low-grade inflammation in the pathophysiology of osteoarthritis (OA). We review and discuss the role of synovitis, complement activation, ...cytokines, and immune cell population in OA.
Recent Findings
Using newer imaging modalities, synovitis is found in the majority of knees with OA. Complement activation and pro-inflammatory cytokines play a significant role in the development of cartilage destruction and synovitis. Immune cell infiltration of OA synovial tissue by sub-populations of T cells and activated macrophages correlates with OA disease progression and pain.
Summary
The innate and acquired immune system plays a key role in the low-grade inflammation found associated with OA. Targets of these pathways my hold promise for future disease-modifying osteoarthritis drugs (DMOADs).
To explore how systemic factors that modify knee osteoarthritis risk are connected to ‘whole-joint’ structural changes by evaluating the effects of high-fat diet and wheel running exercise on ...synovial fluid (SF) metabolomics.
Male mice were fed a defined control or high-fat (60% kcal fat) diet from 6 to 52 weeks of age, and half the animals were housed with running wheels from 26 to 52 weeks of age (n = 9–13 per group). Joint tissue structure and osteoarthritis pathology were evaluated by histology and micro-computed tomography. Systemic metabolic and inflammatory changes were evaluated by body composition, glucose tolerance testing, and serum biomarkers. SF metabolites were analyzed by high performance-liquid chromatography mass spectrometry. We built correlation-based network models to evaluate the connectivity between systemic and local metabolic biomarkers and osteoarthritis structural pathology within each experimental group.
High-fat diet caused moderate osteoarthritis, including cartilage pathology, synovitis and increased subchondral bone density. In contrast, voluntary exercise had a negligible effect on these joint structure components. 1,412 SF metabolite features were detected, with high-fat sedentary mice being the most distinct. Diet and activity uniquely altered SF metabolites attributed to amino acids, lipids, and steroids. Notably, high-fat diet increased network connections to systemic biomarkers such as interleukin-1β and glucose intolerance. In contrast, exercise increased local joint-level network connections, especially among subchondral bone features and SF metabolites.
Network mapping showed that obesity strengthened SF metabolite links to blood glucose and inflammation, whereas exercise strengthened SF metabolite links to subchondral bone structure.
Exercise is known to induce beneficial effects in synovial joints. However, the mechanisms underlying these are unclear. Synovial joints experience repeated mechanical loading during exercise. These ...mechanical stimuli are transduced into biological responses through cellular mechanotransduction. Mechanotransduction in synovial joints is typically studied in tissues. However, synovial fluid directly contacts all components of the joint, and thus may produce a whole-joint picture of the mechanotransduction response to loading. The objective of this study was to determine if metabolic phenotypes are present in the synovial fluid after acute exercise as a first step to understanding the beneficial effects of exercise on the joint.
Mice underwent a single night of voluntary wheel running or standard housing and synovial fluid was harvested for global metabolomic profiling by LC-MS. Hierarchical unsupervised clustering, partial least squares discriminant, and pathway analysis provided insight into exercise-induced mechanotransduction.
Acute exercise produced a distinct metabolic phenotype in synovial fluid. Mechanosensitive metabolites included coenzyme A derivatives, prostaglandin derivatives, phospholipid species, tryptophan, methionine, vitamin D3, fatty acids, and thiocholesterol. Enrichment analysis identified several pathways previously linked to exercise including amino acid metabolism, inflammatory pathways, citrulline-nitric oxide cycle, catecholamine biosynthesis, ubiquinol biosynthesis, and phospholipid metabolism.
To our knowledge, this is the first study to investigate metabolomic profiles of synovial fluid during in vivo mechanotransduction. These profiles indicate that exercise induced stress-response processes including both pro- and anti-inflammatory pathways. Further research will expand these results and define the relationship between the synovial fluid and the serum.
Abstract
The reliance on flow cytometry for phenotypical and functional analyses of immune components in both research and clinical laboratories has increased the need flow cytometric assay ...standardization. Dried-down reagent cocktails pre-optimized with regards to fluorophore choice for each specificity as well as concentration of each conjugate can improve standardization. Use of a pre-optimized dried-down cocktail provides an efficient assay workflow which can minimize operator error in addition to offering enhanced reagent stability and reduced variability across instruments and sites.
We developed a dried-down reagent cocktail for the identification and characterization of CD4+ and CD8+ T-cell memory and naïve subsets, including stem cell memory populations. The BD Horizon™ Dri Memory T cell panel contains optimized antibody-fluorophore conjugates against CD3, CD4, CD8, CD45RA, CD197, CD27 and CD95. Additional markers can be accommodated as drop-ins for a more comprehensive analysis.
This cocktail was optimized for stain index in all channels of a red, blue, violet laser flow cytometer (BD FACSLyric™) in liquid format, then converted to dry format. The performance of the dried-down cocktail, with regards to MFI and % positivity of T-cell subsets, is comparable to that of its optimized liquid equivalents. Additionally, results are consistent across FACSLyric instruments and similarly when acquired on a BD LSRFortessa™.
The comparable performance of the dried-down cocktail tube, along with the advantages offered by using a dried reagent cocktail, and the convenience of adding liquid drop-in reagents, reveal the BD Horizon Dri Memory T cell panel tube to be a valuable option for memory T-cell flow cytometry analysis.
Abstract only
BACKGROUND
Aged skeletal muscle presents with an accumulation of extracellular matrix (ECM) proteins, including collagen, that are stiffer and fibrotic (i.e., unresolvable and resistant ...to turnover) which alters the cellular microenvironment leading to reduced muscle function. A limitation of current assessments of protein turnover using a single timepoint is that they do not account for how much of a protein pool is actively turning over. In addition, defining the proportion of the protein pool actively turning over also informs on how much of the same pool is fibrotic. Thus, determining aging muscle changes in both collagen protein synthesis and the collagen protein pool actively turning over is vital for a more comprehensive understanding of fibrosis in aged skeletal muscle ECM.
PURPOSE
To test the hypothesis that with age the fraction of collagen proteins actively turning over would decrease, indicating an increase in fibrotic collagen, and that the fraction of the protein pool turning over would be inverse to the amount of collagen cross‐linking and overall protein oxidative damage.
METHODS
Young adult (6 mo.) and old (23 mo.) female C57BL/6 mice were labeled with the stable isotope deuterium oxide (D
2
O) for 4, 15, 30, 45, or 60 days (n=3–5/timepoint). Gastrocnemius muscles were analyzed for collagen protein synthesis (
k
1/day) and the plateau (
P
) value for fraction new as a novel indicator of the protein pool capable of turning over (with 1.0 equal to 100% of the total pool turning over). Collagen cross‐linking was assessed using a pepsin digestion collagen solubility and hydroxyproline assay and Western Blotting for lysyl oxidase (LOX, catalyzes collagen cross‐links), while muscle protein carbonylation was assessed as a marker of oxidative damage.
RESULTS
Collagen protein synthesis and collagen plateau fraction new were significantly (p < 0.05) lower in old compared to young (0.011±0.002 vs 0.027±0.003
k
1/day and 0.141±0.015 vs 0.280±0.051
P
, respectively). The lower collagen turnover in old compared to young muscle was paralleled by higher total LOX expression (20183±600 vs 16333±435 arbitrary density units, respectively), lower pepsin soluble collagen (15.1±1.28 vs 37.4±5.96 ng/mg muscle, respectively), higher pepsin insoluble (285±17.5 vs 201±16.4 ng/mg muscle, respectively) and total collagen (303±19.8 vs 237±18.4 ng/mg muscle, respectively), and unchanged protein carbonylation (0.083±0.002 vs 0.083±0.008 arbitrary density units, respectively).
CONCLUSIONS
The pool of collagen proteins actively turning over was significantly reduced in aged compared to young muscle, indicating that a greater proportion of the collagen proteins in aged muscle were fibrotic. The reduced proportion of collagen turning over was associated with increased markers of collagen cross‐linking, but not overall protein oxidative damage. Further research is warranted to identify which individual ECM proteins become fibrotic with aging to develop therapeutics to rescue muscle function.
Support or Funding Information
This research was supported by NIA Training Grant T32AG052363.
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