STAT1 modulates tissue wasting or overgrowth downstream from PDGFRß.

Platelet-derived growth factor (PDGF) acts through two conserved receptor tyrosine kinases: PDGFRα and PDGFRß. Gain-of-function mutations in human PDGFRB have been linked recently to genetic diseases characterized by connective tissue wasting (Penttinen syndrome) or overgrowth (Kosaki overgrowth syndrome), but it is unclear whether PDGFRB mutations alone are responsible. Mice with constitutive PDGFRß signaling caused by a kinase domain mutation (D849V) develop lethal autoinflammation. Here we used a genetic approach to investigate the mechanism of autoinflammation in Pdgfrb+/D849V mice and test the hypothesis that signal transducer and activator of transcription 1 (STAT1) mediates this phenotype. We show that Pdgfrb+/D849V mice with Stat1 knockout (Stat1-/-Pdgfrb+/D849V ) are rescued from autoinflammation and have improved life span compared with Stat1+/-Pdgfrb+/D849V mice. Furthermore, PDGFRß-STAT1 signaling suppresses PDGFRß itself. Thus, Stat1-/-Pdgfrb+/D849V fibroblasts exhibit increased PDGFRß signaling, and mice develop progressive overgrowth, a distinct phenotype from the wasting seen in Stat1+/-Pdgfrb+/D849V mice. Deletion of interferon receptors (Ifnar1 or Ifngr1) does not rescue wasting in Pdgfrb+/D849V mice, indicating that interferons are not required for autoinflammation. These results provide functional evidence that elevated PDGFRß signaling causes tissue wasting or overgrowth reminiscent of human genetic syndromes and that the STAT1 pathway is a crucial modulator of this phenotypic spectrum.

Sex-specific effects of injury and beta-adrenergic activation on metabolic and inflammatory mediators in a murine model of post-traumatic osteoarthritis.

OBJECTIVE: Metabolic processes are intricately linked to the resolution of innate inflammation and tissue repair, two critical steps for treating post-traumatic osteoarthritis (PTOA). Based on lipolytic and immunoregulatory actions of norepinephrine, we hypothesized that intra-articular ß-adrenergic receptor (ßAR) stimulation would suppress PTOA-associated inflammation in the infrapatellar fat pad (IFP) and synovium. DESIGN: We used the ßAR agonist isoproterenol to perturb intra-articular metabolism 3.5 weeks after applying a non-invasive single-load compression injury to knees of 12-week-old male and female mice. We examined the acute effects of intra-articular isoproterenol treatment relative to saline on IFP histology, multiplex gene expression of synovium-IFP tissue, synovial fluid metabolomics, and mechanical allodynia. RESULTS: Injured knees developed PTOA pathology characterized by heterotopic ossification, articular cartilage loss, and IFP atrophy and fibrosis. Isoproterenol suppressed the upregulation of pro-fibrotic genes and downregulated the expression of adipose genes and pro-inflammatory genes (Adam17, Cd14, Icam1, Csf1r, and Casp1) in injured joints of female (but not male) mice. Analysis of published single-cell RNA-seq data identified elevated catecholamine-associated gene expression in resident-like synovial-IFP macrophages after injury. Injury substantially altered synovial fluid metabolites by increasing amino acids, peptides, sphingolipids, phospholipids, bile acids, and dicarboxylic acids, but these changes were not appreciably altered by isoproterenol. Intra-articular injection of either isoproterenol or saline increased mechanical allodynia in female mice, whereas neither substance affected male mice. CONCLUSIONS: Acute ßAR activation altered synovial-IFP transcription in a sex and injury-dependent manner, suggesting that women with PTOA may be more sensitive than men to treatments targeting sympathetic neural signaling pathways.

RELATIONSHIP BETWEEN REPRODUCTIVE AND BONE BIOMARKERS AND OSTEOARTHRITIS IN ZOO ASIAN (ELEPHAS MAXIMUS) AND AFRICAN (LOXODONTA AFRICANA) ELEPHANTS.

Osteoarthritis (OA) is common in zoo Asian (Elephas maximus) and African (Loxodonta africana) elephants. This study investigated the relationship between confirmed or suspected OA with ovarian cyclicity, gonadotropins, progestagens, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and collagen type I (CTX-I) in zoo elephants. In Asian elephants, odds of having confirmed or suspected OA decreased with cycling (OR = 0.22, P = 0.016; OR = 0.29, P = 0.020, respectively), however, not when adjusted for age (odds ratio [OR] = 0.31, P = 0.112; OR = 0.58, P = 0.369, respectively). In African elephants, none of the models between confirmed OA and cycling status were significant (P > 0.060), while the odds of having suspected OA decreased with cycling (OR = 0.12, P = 0.001), even after adjusting for age (OR = 0.15, P = 0.005). Progestagens (Asian elephants P > 0.096; African elephants P > 0.415), LH (Asian P > 0.129; African P > 0.359), and FSH (Asian P > 0.738; African P > 0.231) did not differ with confirmed or suspected OA status, unadjusted. CTX-I concentrations were not related to OA status (P > 0.655). This study concluded hormonal changes may not have a strong impact on OA, so additional investigation into other serologic biomarkers is warranted.

PFKFB3-dependent glucose metabolism regulates 3T3-L1 adipocyte development.

Proliferation and differentiation of preadipocytes, and other cell types, is accompanied by an increase in glucose uptake. Previous work showed that a pulse of high glucose was required during the first 3 days of differentiation in vitro, but was not required after that. The specific glucose metabolism pathways required for adipocyte differentiation are unknown. Herein, we used 3T3-L1 adipocytes as a model system to study glucose metabolism and expansion of the adipocyte metabolome during the first 3 days of differentiation. Our primary outcome measures were GLUT4 and adiponectin, key proteins associated with healthy adipocytes. Using complete media with 0 or 5 mM glucose, we distinguished between developmental features that were dependent on the differentiation cocktail of dexamethasone, insulin, and isobutylmethylxanthine alone or the cocktail plus glucose. Cocktail alone was sufficient to activate the capacity for 2-deoxglucose uptake and glycolysis, but was unable to support the expression of GLUT4 and adiponectin in mature adipocytes. In contrast, 5 mM glucose in the media promoted a transient increase in glucose uptake and glycolysis as well as a significant expansion of the adipocyte metabolome and proteome. Using genetic and pharmacologic approaches, we found that the positive effects of 5 mM glucose on adipocyte differentiation were specifically due to increased expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key regulator of glycolysis and the ancillary glucose metabolic pathways. Our data reveal a critical role for PFKFB3 activity in regulating the cellular metabolic remodeling required for adipocyte differentiation and maturation.

Glutathione as a mediator of cartilage oxidative stress resistance and resilience during aging and osteoarthritis.

Purpose: An underlying cause of osteoarthritis (OA) is the inability of chondrocytes to maintain homeostasis in response to changing stress conditions. The purpose of this article was to review and experimentally evaluate oxidative stress resistance and resilience concepts in cartilage using glutathione redox homeostasis as an example. This framework may help identify novel approaches for promoting chondrocyte homeostasis during aging and obesity.Materials and Methods: Changes in glutathione content and redox ratio were evaluated in three models of chondrocyte stress: (1) age- and tissue-specific changes in joint tissues of 10 and 30-month old F344BN rats, including ex vivo patella culture experiments to evaluate N-acetylcysteine dependent resistance to interleukin-1beta; (2) effect of different durations and patterns of cyclic compressive loading in bovine cartilage on glutathione stress resistance and resilience pathways; (3) time-dependent changes in GSH:GSSG in primary chondrocytes from wild-type and Sirt3 deficient mice challenged with the pro-oxidant menadione.Results: Glutathione was more abundant in cartilage than meniscus or infrapatellar fat pad, although cartilage was also more susceptible to age-related glutathione oxidation. Glutathione redox homeostasis was sensitive to the duration of compressive loading such that load-induced oxidation required unloaded periods to recover and increase total antioxidant capacity. Exposure to a pro-oxidant stress enhanced stress resistance by increasing glutathione content and GSH:GSSG ratio, especially in Sirt3 deficient cells. However, the rate of recovery, a marker of resilience, was delayed without Sirt3.Conclusions: OA-related models of cartilage stress reveal multiple mechanisms by which glutathione provides oxidative stress resistance and resilience.

Loss of mitochondrial protease ClpP protects mice from diet-induced obesity and insulin resistance.

Caseinolytic peptidase P (ClpP) is a mammalian quality control protease that is proposed to play an important role in the initiation of the mitochondrial unfolded protein response (UPRmt), a retrograde signaling response that helps to maintain mitochondrial protein homeostasis. Mitochondrial dysfunction is associated with the development of metabolic disorders, and to understand the effect of a defective UPRmt on metabolism, ClpP knockout (ClpP-/-) mice were analyzed. ClpP-/- mice fed ad libitum have reduced adiposity and paradoxically improved insulin sensitivity. Absence of ClpP increased whole-body energy expenditure and markers of mitochondrial biogenesis are selectively up-regulated in the white adipose tissue (WAT) of ClpP-/- mice. When challenged with a metabolic stress such as high-fat diet, despite similar caloric intake, ClpP-/- mice are protected from diet-induced obesity, glucose intolerance, insulin resistance, and hepatic steatosis. Our results show that absence of ClpP triggers compensatory responses in mice and suggest that ClpP might be dispensable for mammalian UPRmt initiation. Thus, we made an unexpected finding that deficiency of ClpP in mice is metabolically beneficial.

GC-MS metabolic profiling reveals fructose-2,6-bisphosphate regulates branched chain amino acid metabolism in the heart during fasting.

INTRODUCTION: As an insulin sensitive tissue, the heart decreases glucose usage during fasting. This response is mediated, in part, by decreasing phosphofructokinase-2 (PFK-2) activity and levels of its product fructose-2,6-bisphosphate. However, the importance of fructose-2,6-bisphosphate in the fasting response on other metabolic pathways has not been evaluated. OBJECTIVES: The goal of this study is to determine how sustaining cardiac fructose-2,6-bisphosphate levels during fasting affects the metabolomic profile. METHODS: Control and transgenic mice expressing a constitutively active form of PFK-2 (GlycoHi) were subjected to either 12-h fasting or regular feeding. Animals (n = 4 per group) were used for whole-heart extraction, followed by gas chromatography-mass spectrometry metabolic profiling and multivariate data analysis. RESULTS: Principal component analysis displayed differences between Control and GlycoHi groups under both fasting and fed conditions while a clear response to fasting was observed only for Control animals. However, pathway analysis revealed that these smaller changes in the GlycoHi group were significantly associated with branched-chain amino acid (BCAA) metabolism (~ 40% increase in all BCAAs). Correlation network analysis demonstrated clear differences in response to fasting between Control and GlycoHi groups amongst most parameters. Notably, fasting caused an increase in network density in the Control group from 0.12 to 0.14 while the GlycoHi group responded oppositely (0.17-0.15). CONCLUSIONS: Elevated cardiac PFK-2 activity during fasting selectively increases BCAAs levels and decreases global changes in metabolism.

Innate inflammation and synovial macrophages in osteoarthritis pathophysiology.

Although osteoarthritis (OA) was historically referred to as the non-inflammatory arthritis, it is now considered a condition involving persistent low-grade inflammation and activation of innate inflammatory pathways. Synovitis increases the risk of OA onset and progression and involves the recruitment of monocytes, lymphocytes, and other leukocytes. In particular, macrophages are important mediators of synovial inflammatory activity and pathologic cartilage and bone responses that are characteristic of OA. Advances in understanding how damage-associated molecular patterns (DAMPs) trigger monocyte/macrophage recruitment and activation in joints provide opportunities for disease-modifying therapies. However, the complexity and plasticity of macrophage phenotypes that exist in vivo have thus far prevented the successful development of macrophage-targeted treatments. Current studies show that synovial macrophages are derived from distinct cellular lineages, which correspond to unique functional roles for maintaining joint homeostasis. An improved understanding of the aetiology of synovial inflammation in specific OA-subtypes, such as with obesity or genetic risk, is a potential strategy for developing patient selection criteria for future precision therapies.

Innate Immune Responses and Osteoarthritis.

PURPOSE OF THE REVIEW: Osteoarthritis (OA) is a chronic, painful joint disease that affects approximately 40% of adults over 70 year. Age is the strongest predictor of OA, while obesity is considered the primary preventable risk factor for OA. Both conditions are associated with abnormal innate immune inflammatory responses that contribute to OA progression and are the focus of this review. RECENT FINDINGS: Recent studies have identified risk factors for OA progression including increased innate immune responses secondary to aging-associated myeloid skewing, obesity-related myeloid activation, and synovial tissue hyperplasia with activated macrophage infiltration. Toll-like receptor (TLR)4-induced catabolic responses also play a significant role in OA. The complex interplay between obesity and aging-associated macrophage activation, pro-inflammatory cytokine production from TLR-driven responses, and adipokines leads to a vicious cycle of synovial hyperplasia, macrophage activation, cartilage catabolism, infrapatellar fat pad fibrosis, and joint destruction.

Up-regulation of the Sirtuin 1 (Sirt1) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) genes in white adipose tissue of Id1 protein-deficient mice: implications in the protection against diet and age-induced glucose intolerance.

Id1, a helix-loop-helix (HLH) protein that inhibits the function of basic HLH E protein transcription factors in lymphoid cells, has been implicated in diet- and age-induced obesity by unknown mechanisms. Here we show that Id1-deficient mice are resistant to a high fat diet- and age-induced obesity, as revealed by reduced weight gain and body fat, increased lipid oxidation, attenuated hepatosteatosis, lower levels of lipid droplets in brown adipose tissue, and smaller white adipocytes after a high fat diet feeding or in aged animals. Id1 deficiency improves glucose tolerance, lowers serum insulin levels, and reduces TNFα gene expression in white adipose tissue. Id1 deficiency also increased expression of Sirtuin 1 and peroxisome proliferator-activated receptor γ coactivator 1α, regulators of mitochondrial biogenesis and energy expenditure, in the white adipose tissue. This effect was accompanied by the elevation of several genes encoding proteins involved in oxidative phosphorylation and fatty acid oxidation, such as cytochrome c, medium-chain acyl-CoA dehydrogenase, and adipocyte protein 2. Moreover, the phenotype for Id1 deficiency was similar to that of mice expressing an E protein dominant-positive construct, ET2, suggesting that the balance between Id and E proteins plays a role in regulating lipid metabolism and insulin sensitivity.

Deficiency in adipocyte chemokine receptor CXCR4 exacerbates obesity and compromises thermoregulatory responses of brown adipose tissue in a mouse model of diet-induced obesity.

The chemokine receptor CXCR4 is expressed on adipocytes and macrophages in adipose tissue, but its role in this tissue remains unknown. We evaluated whether deficiency in either adipocyte or myeloid leukocyte CXCR4 affects body weight (BW) and adiposity in a mouse model of high-fat-diet (HFD)-induced obesity. We found that ablation of adipocyte, but not myeloid leukocyte, CXCR4 exacerbated obesity. The HFD-fed adipocyte-specific CXCR4-knockout (AdCXCR4ko) mice, compared to wild-type C57BL/6 control mice, had increased BW (average: 52.0 g vs. 35.5 g), adiposity (average: 49.3 vs. 21.0% of total BW), and inflammatory leukocyte content in white adipose tissue (WAT), despite comparable food intake. As previously reported, HFD feeding increased uncoupling protein 1 (UCP1) expression (fold increase: 3.5) in brown adipose tissue (BAT) of the C57BL/6 control mice. However, no HFD-induced increase in UCP1 expression was observed in the AdCXCR4ko mice, which were cold sensitive. Thus, our study suggests that adipocyte CXCR4 limits development of obesity by preventing excessive inflammatory cell recruitment into WAT and by supporting thermogenic activity of BAT. Since CXCR4 is conserved between mouse and human, the newfound role of CXCR4 in mouse adipose tissue may parallel the role of this chemokine receptor in human adipose tissue.

Effect of biological sex and short-term high-fat diet on cellular proliferation, ribosomal biogenesis, and targeted protein abundance in murine articular cartilage.

Objective: To identify factors contributing to sex-differences in OA risk by evaluating the short-term effect of high-fat (HF) diet on sex-specific changes in cartilage cell proliferation, ribosomal biogenesis, and targeted extra-cellular and cellular protein abundance. Materials and methods: Knee cartilage was harvested to the subchondral bone from 20-week-old female and male C57BL/6J mice fed a low-fat or HF diet for 4 weeks and labeled with deuterium oxide for 1, 3, 5, 7, 15, or 21 days. Deuterium enrichment was quantified in isolated DNA and RNA to measure cell proliferation and ribosomal biogenesis, respectively. Protein concentration was measured using targeted high resolution accurate mass spectrometry. Results: HF diet increased the maximal deuterium incorporation into DNA from approximately 40 to 50%, albeit at a slower rate. These findings, which were magnified in female versus male mice, indicate a greater number of proliferating cells with longer half-lives under HF diet conditions. HF diet caused distinct sex-dependent effects on deuterium incorporation into RNA, increasing the fraction of ribosomes undergoing biogenesis in male mice and doubling the rate of ribosome biogenesis in female mice. HF diet altered cartilage protein abundance similarly in both sexes, except for matrilin-3, which was more abundant in HF versus LF conditions in female mice only. Overall, HF diet treatment had a stronger effect than sex on cartilage protein abundance, with most changes involving extracellular matrix and matrix-associated proteins. Conclusions: Short-term HF diet broadly altered cartilage matrix protein abundance, while sex-dependent effects primarily involved differences in cell proliferation and ribosomal biogenesis.

Teaming up to overcome challenges toward translation of new therapeutics for osteoarthritis.

As a leading global cause of musculoskeletal-related disability, osteoarthritis (OA) represents a public health urgency. Understanding of disease pathogenesis has advanced substantially in the past decade, yet no disease-modifying therapeutics have advanced to the clinic. To address this challenge, the CARE-AP (Cartilage Repair strategies to alleviate Arthritis Pain) collaborative research team was convened to bring together relevant multidisciplinary expertise and perspectives from across the VA research community nationwide. The first CARE-AP Annual Research Symposium took place (virtually) in February 2022 with roughly 90 participants. A number of innovative and therapeutic strategies were discussed, including siRNA approaches coupled with novel nanoparticle-based delivery systems, cellular engineering approaches to develop reparative cells that can probe the joint environment and respond to disease-specific cues, and novel biofabrication techniques to improve tissue engineering and effect "biological joint replacement." In addition, challenges and advances in rehabilitation approaches, imaging outcomes, and clinical studies were presented, which were integrated into a framework of recommendations for running "preclinical trials" to improve successful clinical translation.

Increased susceptibility of Trpv4-deficient mice to obesity and obesity-induced osteoarthritis with very high-fat diet.

OBJECTIVE: To test the hypotheses that: (1) the transient receptor potential vanilloid 4 (TRPV4) ion channel is protective in the obesity model of osteoarthritis (OA), resulting in more severe obesity-induced OA in Trpv4 knockout (Trpv4(-/-)) mice; and (2) loss of TRPV4 alters mesodermal stem cell differentiation. METHODS: Male Trpv4(-/-) and wild-type (Trpv4(+/+)) mice were fed a control or high-fat diet (10% kcal and 60% kcal from fat, respectively) for 22 weeks, at which time spontaneous cage activity and severity of knee OA were evaluated. In addition, the adipogenic, osteogenic and chondrogenic potential of bone marrow-derived (MSC) and adipose-derived (ASC) stem cells from Trpv4(-/-) and Trpv4(+/+) mice were compared. RESULTS: A high-fat diet significantly increased knee OA scores and reduced spontaneous cage activity in Trpv4(-/-) mice, while also increasing weight gain and adiposity. MSCs from Trpv4(-/-) mice had decreased adipogenic and osteogenic differentiation potential versus Trpv4(+/+) MSCs. ASCs from Trpv4(-/-) mice had increased adipogenic and osteogenic and reduced chondrogenic differentiation potential versus Trpv4(+/+) ASCs. CONCLUSIONS: Pan-Trpv4(-/-) mice develop more severe OA with high-fat feeding, potentially due to more severe diet-induced obesity. The altered differentiation potential of Trpv4(-/-) progenitor cells may reflect the importance of this ion channel in the maintenance and turnover of mesodermally-derived tissues.

Induction of osteoarthritis and metabolic inflammation by a very high-fat diet in mice: effects of short-term exercise.

OBJECTIVE: To test the hypotheses that obesity due to a very high-fat diet induces knee osteoarthritis (OA), and that short-term wheel-running exercise protects against obesity-induced knee OA by reducing systemic inflammation and metabolic dysregulation. METHODS: Male C57BL/6J mice were fed either a control diet (13.5% kcal from fat) or a very high-fat diet (60% kcal from fat) from age 12 weeks to age 24 weeks. From 20 to 24 weeks of age, half of the mice were housed with running wheels. The severity of knee OA was determined by assessing histopathologic features, and serum cytokines were measured using a multiplex bead immunoassay and enzyme-linked immunosorbent assays. Body composition was quantified by dual-energy x-ray absorptiometry, and insulin resistance was assessed by glucose tolerance testing. RESULTS: Feeding mice with a very high-fat diet increased knee OA scores and levels of serum leptin, adiponectin, KC (mouse analog of interleukin-8 [IL-8]), monokine induced by interferon-γ (CXCL9), and IL-1 receptor antagonist to an extent in proportion to the gain in body fat (3-fold increase in percent body fat compared to controls). Wheel-running exercise reduced progression of OA in the medial femur of obese mice. In addition, exercise disrupted the clustering of cytokine expression and improved glucose tolerance, without reducing body fat or cytokine levels. CONCLUSION: Obesity induced by a very high-fat diet in mice causes OA and systemic inflammation in proportion to body fat. Increased joint loading is not sufficient to explain the increased incidence of knee OA with obesity, as wheel running is protective rather than damaging. Exercise improves glucose tolerance and disrupts the coexpression of proinflammatory cytokines, suggesting that increased aerobic exercise may act independently of weight loss in promoting joint health.

Effects of Leptin and Body Weight on Inflammation and Knee Osteoarthritis Phenotypes in Female Rats.

Leptin is a proinflammatory adipokine that contributes to obesity-associated osteoarthritis (OA), especially in women. However, the extent to which leptin causes knee OA separate from the effect of increased body weight is not clear. We hypothesized that leptin is necessary to induce knee OA in obese female rats but not sufficient to induce knee OA in lean rats lacking systemic metabolic inflammation. The effect of obesity without leptin signaling was modeled by comparing female lean Zucker rats to pair fed obese Zucker rats, which possess mutant fa alleles of the leptin receptor gene. The effect of leptin without obesity was modeled in female F344BN F1 hybrid rats by systemically administering recombinant rat leptin versus saline for 23 weeks via osmotic pumps. Primary OA outcomes included cartilage histopathology and subchondral bone micro-computed tomography. Secondary outcomes included targeted cartilage proteomics, serum inflammation, and synovial fluid inflammation following an acute intra-articular challenge with interleukin-1ß (IL-1ß). Compared to lean Zucker rats, obese Zucker rats developed more severe tibial osteophytes and focal cartilage lesions in the medial tibial plateau, with modest changes in proximal tibial epiphysis trabecular bone structure. In contrast, exogenous leptin treatment, which increased plasma leptin sixfold without altering body weight, caused mild generalized cartilage fibrillation and reduced Safranin O staining compared to vehicle-treated animals. Leptin also significantly increased subchondral and trabecular bone volume and bone mineral density in the proximal tibia. Cartilage metabolic and antioxidant enzyme protein levels were substantially elevated with leptin deficiency and minimally suppressed with leptin treatment. In contrast, leptin treatment induced greater changes in systemic and local inflammatory mediators compared to leptin receptor deficiency, including reduced serum IL-6 and increased synovial fluid IL-1ß. In conclusion, rat models that separately elevate leptin or body weight develop distinct OA-associated phenotypes, revealing how obesity increases OA pathology through both leptin-dependent and independent pathways. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Age-Associated Changes in Knee Osteoarthritis, Pain-Related Behaviors, and Dorsal Root Ganglia Immunophenotyping of Male and Female Mice.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Imaging mass cytometry reveals tissue-specific cellular immune phenotypes in the mouse knee following ACL injury.

Objective: To develop an imaging mass cytometry method for identifying complex cell phenotypes, inter-cellular interactions, and population changes in the synovium and infrapatellar fat pad (IFP) of the mouse knee following a non-invasive compression injury. Design: Fifteen male C57BL/6 mice were fed a high-fat diet for 8 weeks prior to random assignment to sham, 0.88 â€‹mm, or 1.7 â€‹mm knee compression displacement at 24 weeks of age. 2-weeks after loading, limbs were prepared for histologic and imaging mass cytometry analysis, focusing on myeloid immune cell populations in the synovium and IFP. Results: 1.7 â€‹mm compression caused anterior cruciate ligament (ACL) rupture, development of post-traumatic osteoarthritis, and a 2- to 3-fold increase in cellularity of synovium and IFP tissues compared to sham or 0.88 â€‹mm compression. Imaging mass cytometry identified 11 myeloid cell subpopulations in synovium and 7 in IFP, of which approximately half were elevated 2 weeks after ACL injury in association with the vasculature. Notably, two monocyte/macrophage subpopulations and an MHC IIhi population were elevated 2-weeks post-injury in the synovium but not IFP. Vascular and immune cell interactions were particularly diverse in the synovium, incorporating 8 unique combinations of 5 myeloid cell populations, including a monocyte/macrophage population, an MHC IIhi population, and 3 different undefined F4/80+ myeloid populations. Conclusions: Developing an imaging mass cytometry method for the mouse enabled us to identify a diverse array of synovial and IFP vascular-associated myeloid cell subpopulations. These subpopulations were differentially elevated in synovial and IFP tissues 2-weeks post injury, providing new details on tissue-specific immune regulation.

In vivo mechanotransduction: Effect of acute exercise on the metabolomic profiles of mouse synovial fluid.

Objective: 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. Material and methods: 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. Results: 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. Conclusion: 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.

Insulin-like growth factor 1 receptor mediates photoreceptor neuroprotection.

Insulin-like growth factor I (IGF-1) is a neurotrophic factor and is the ligand for insulin-like growth factor 1 receptor (IGF-1R). Reduced expression of IGF-1 has been reported to cause deafness, mental retardation, postnatal growth failure, and microcephaly. IGF-1R is expressed in the retina and photoreceptor neurons; however, its functional role is not known. Global IGF-1 KO mice have age-related vision loss. We determined that conditional deletion of IGF-1R in photoreceptors and pan-retinal cells produces age-related visual function loss and retinal degeneration. Retinal pigment epithelial cell-secreted IGF-1 may be a source for IGF-1R activation in the retina. Altered retinal, fatty acid, and phosphoinositide metabolism are observed in photoreceptor and retinal cells lacking IGF-1R. Our results suggest that the IGF-1R pathway is indispensable for photoreceptor survival, and activation of IGF-1R may be an essential element of photoreceptor and retinal neuroprotection.