Single-cell RNA sequencing reveals the induction of novel myeloid and myeloid-associated cell populations in visceral fat with long-term obesity.

Macrophages and other immune cells are important contributors to obesity-associated inflammation; however, the cellular identities of these specific populations remain unknown. In this study, we identified individual populations of myeloid cells found in mouse epididymal/visceral adipose tissue by single-cell RNA sequencing, immunofluorescence, and flow cytometry. Multiple canonical correlation analysis identified 11 unique myeloid and myeloid-associate cell populations. In obese mice, we detected an increased percentage of monocyte-derived pro-inflammatory cells expressing Cd9 and Trem2, as well as significantly decreased percentages of multiple cell populations, including tissue-resident cells expressing Lyve1, Mafb, and Mrc1. We have identified and validated a novel myeloid/macrophage population defined by Ly6a expression, exhibiting both myeloid and mesenchymal characteristics, which increased with obesity and showed high pro-fibrotic characteristics in vitro. Our mouse adipose tissue myeloid cell atlas provides an important resource to investigate obesity-associated inflammation and fibrosis.

Transgenerational impact of maternal obesogenic diet on offspring bile acid homeostasis and nonalcoholic fatty liver disease.

Studies show maternal obesity is a risk factor for metabolic syndrome and nonalcoholic fatty liver disease (NAFLD) in offspring. Here we evaluated potential mechanisms underlying these phenotypes. Female C57Bl6 mice were fed chow or an obesogenic high-fat/high-sucrose (HF/HS) diet with subsequent mating of F1 and F2 female offspring to lean males to develop F2 and F3 generations, respectively. Offspring were fed chow or fibrogenic (high transfat, cholesterol, fructose) diets, and histopathological, metabolic changes, and bile acid (BA) homeostasis was evaluated. Chow-fed F1 offspring from maternal HF/HS lineages (HF/HS) developed periportal fibrosis and inflammation with aging, without differences in hepatic steatosis but increased BA pool size and shifts in BA composition. F1, but not F2 or F3, offspring from HF/HS showed increased steatosis on a fibrogenic diet, yet inflammation and fibrosis were paradoxically decreased in F1 offspring, a trend continued in F2 and F3 offspring. HF/HS feeding leads to increased periportal fibrosis and inflammation in chow-fed offspring without increased hepatic steatosis. By contrast, fibrogenic diet-fed F1 offspring from HF/HS dams exhibited worse hepatic steatosis but decreased inflammation and fibrosis. These findings highlight complex adaptations in NAFLD phenotypes with maternal diet.

Engineered Self-Regulating Macrophages for Targeted Anti-inflammatory Drug Delivery.

Background: Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by increased levels of inflammation that primarily manifests in the joints. Macrophages act as key drivers for the progression of RA, contributing to the perpetuation of chronic inflammation and dysregulation of pro-inflammatory cytokines such as interleukin 1 (IL-1). The goal of this study was to develop a macrophage-based cell therapy for biologic drug delivery in an autoregulated manner. Methods: For proof-of-concept, we developed "smart" macrophages to mitigate the effects of IL-1 by delivering its inhibitor, IL-1 receptor antagonist (IL-1Ra). Bone marrow-derived macrophages were lentivirally transduced with a synthetic gene circuit that uses an NF-κB inducible promoter upstream of either the Il1rn or firefly luciferase transgenes. Two types of joint like cells were utilized to examine therapeutic protection in vitro, miPSCs derived cartilage and isolated primary mouse synovial fibroblasts while the K/BxN mouse model of RA was utilized to examine in vivo therapeutic protection. Results: These engineered macrophages were able to repeatably produce therapeutic levels of IL-1Ra that could successfully mitigate inflammatory activation in co-culture with both tissue engineered cartilage constructs and synovial fibroblasts. Following injection in vivo, macrophages homed to sites of inflammation and mitigated disease severity in the K/BxN mouse model of RA. Conclusion: These findings demonstrate the successful development of engineered macrophages that possess the ability for controlled, autoregulated production of IL-1 based on inflammatory signaling such as the NF-κB pathway to mitigate the effects of this cytokine for applications in RA or other inflammatory diseases. This system provides proof of concept for applications in other immune cell types as self-regulating delivery systems for therapeutic applications in a range of diseases.

Chondrocytes from osteoarthritic cartilage of obese patients show altered adiponectin receptors expression and response to adiponectin.

Obesity and osteoarthritis (OA) are well-known comorbidities and their precise molecular interactions are still unidentified. Adiponectin, a major adipokine, known to have an anti-inflammatory effect in atherosclerosis or Type 2 Diabetes Mellitus (T2DM), has also been postulated to be pro-inflammatory in OA. This dual role of adiponectin is still not explained. The precise mechanism by which adiponectin affects cartilage and chondrocytes remains to be elucidated. In the present observational study chondrocytes from 30 patients with OA (18 females and 12 males) undergoing total knee replacement (TKR) were isolated. Expression of adiponectin receptors 1 and 2 (ADIPOR1 and ADIPOR2) was examined both at gene and protein levels in chondrocytes. The difference in adiponectin receptor expression between lean and obese patients with OA and the role of adiponectin in regulating pro-inflammatory genes (MCP-1, IL-6, and VCAM-1, MMP-1, MMP-2, and TIMP-1) has been investigated. We found that ADIPOR1 represented the most abundant adiponectin receptor in primary OA chondrocytes. ADIPOR1 and ADIPOR2 genes and ADIPOR1 protein were differently expressed in OA chondrocytes obtained from obese compared with lean patients with OA. Adiponectin induced gene expression of MCP-1, IL-6, and MMP-1 in all OA patients' chondrocytes. In contrast, VCAM-1 and MMP-2 were differently regulated by adiponectin depending on the patient's body mass index. This study suggests that adiponectin and ADIPOR1 may have important roles in the pathogenesis of cartilage degeneration in OA of obese subjects.

Immunoengineering the next generation of arthritis therapies.

Immunoengineering continues to revolutionize healthcare, generating new approaches for treating previously intractable diseases, particularly in regard to cancer immunotherapy. In joint diseases, such as osteoarthritis (OA) and rheumatoid arthritis (RA), biomaterials and anti-cytokine treatments have previously been at that forefront of therapeutic innovation. However, while many of the existing anti-cytokine treatments are successful for a subset of patients, these treatments can also pose severe risks, adverse events and off-target effects due to continuous delivery at high dosages or a lack of disease-specific targets. The inadequacy of these current treatments has motivated the development of new immunoengineering strategies that offer safer and more efficacious alternative therapies through the precise and controlled targeting of specific upstream immune responses, including direct and mechanistically-driven immunoengineering approaches. Advances in the understanding of the immunomodulatory pathways involved in musculoskeletal disease, in combination with the growing emphasis on personalized medicine, stress the need for carefully considering the delivery strategies and therapeutic targets when designing therapeutics to better treat RA and OA. Here, we focus on recent advances in biomaterial and cell-based immunomodulation, in combination with genetic engineering, for therapeutic applications in joint diseases. The application of immunoengineering principles to the study of joint disease will not only help to elucidate the mechanisms of disease pathogenesis but will also generate novel disease-specific therapeutics by harnessing cellular and biomaterial responses. STATEMENT OF SIGNIFICANCE: It is now apparent that joint diseases such as osteoarthritis and rheumatoid arthritis involve the immune system at both local (i.e., within the joint) and systemic levels. In this regard, targeting the immune system using both biomaterial-based or cellular approaches may generate new joint-specific treatment strategies that are well-controlled, safe, and efficacious. In this review, we focus on recent advances in immunoengineering that leverage biomaterials and/or genetically engineered cells for therapeutic applications in joint diseases. The application of such approaches, especially synergistic strategies that target multiple immunoregulatory pathways, has the potential to revolutionize our understanding, treatment, and prevention of joint diseases.

A genome-engineered bioartificial implant for autoregulated anticytokine drug delivery.

Biologic drug therapies are increasingly used for inflammatory diseases such as rheumatoid arthritis but may cause significant adverse effects when delivered continuously at high doses. We used CRISPR-Cas9 genome editing of iPSCs to create a synthetic gene circuit that senses changing levels of endogenous inflammatory cytokines to trigger a proportional therapeutic response. Cells were engineered into cartilaginous constructs that showed rapid activation and recovery in response to inflammation in vitro or in vivo. In the murine K/BxN model of inflammatory arthritis, bioengineered implants significantly mitigated disease severity as measured by joint pain, structural damage, and systemic and local inflammation. Therapeutic implants completely prevented increased pain sensitivity and bone erosions, a feat not achievable by current clinically available disease-modifying drugs. Combination tissue engineering and synthetic biology promises a range of potential applications for treating chronic diseases via custom-designed cells that express therapeutic transgenes in response to dynamically changing biological signals.

Is Obesity a Disease of Stem Cells?

Obesity disrupts physiological homeostasis and alters both systemic and local microenvironments that impact stem cell plasticity and impair regenerative capacity. We present growing evidence that reveals the bidirectionality of obesity-induced stem cell dysfunction and how the molecular changes in stem cells residing in obese environments may accelerate disease severity.

Gene therapy for follistatin mitigates systemic metabolic inflammation and post-traumatic arthritis in high-fat diet-induced obesity.

Obesity-associated inflammation and loss of muscle function play critical roles in the development of osteoarthritis (OA); thus, therapies that target muscle tissue may provide novel approaches to restoring metabolic and biomechanical dysfunction associated with obesity. Follistatin (FST), a protein that binds myostatin and activin, may have the potential to enhance muscle formation while inhibiting inflammation. Here, we hypothesized that adeno-associated virus 9 (AAV9) delivery of FST enhances muscle formation and mitigates metabolic inflammation and knee OA caused by a high-fat diet in mice. AAV-mediated FST delivery exhibited decreased obesity-induced inflammatory adipokines and cytokines systemically and in the joint synovial fluid. Regardless of diet, mice receiving FST gene therapy were protected from post-traumatic OA and bone remodeling induced by joint injury. Together, these findings suggest that FST gene therapy may provide a multifactorial therapeutic approach for injury-induced OA and metabolic inflammation in obesity.

Intergenerational Transmission of Diet-Induced Obesity, Metabolic Imbalance, and Osteoarthritis in Mice.

OBJECTIVE: Obesity and osteoarthritis (OA) are 2 major public health issues affecting millions of people worldwide. Whereas parental obesity affects the predisposition to diseases such as cancer or diabetes in children, transgenerational influences on musculoskeletal conditions such as OA are poorly understood. This study was undertaken to assess the intergenerational effects of a parental/grandparental high-fat diet on the metabolic and skeletal phenotype, systemic inflammation, and predisposition to OA in 2 generations of offspring in mice. METHODS: Metabolic phenotype and predisposition to OA were investigated in the first and second (F1 and F2) generations of offspring (n = 10-16 mice per sex per diet) bred from mice fed a high-fat diet (HFD) or a low-fat control diet. OA was induced by destabilizing the medial meniscus. OA, synovitis, and adipose tissue inflammation were determined histologically, while bone changes were measured using micro-computed tomography. Serum and synovial cytokines were measured by multiplex assay. RESULTS: Parental high-fat feeding showed an intergenerational effect, with inheritance of increased weight gain (up to 19% in the F1 generation and 9% in F2), metabolic imbalance, and injury-induced OA in at least 2 generations of mice, despite the fact that the offspring were fed the low-fat diet. Strikingly, both F1 and F2 female mice showed an increased predisposition to injury-induced OA (48% higher predisposition in F1 and 19% in F2 female mice fed the HFD) and developed bone microarchitectural changes that were attributable to parental and grandparental high-fat feeding. CONCLUSION: The results of this study reveal a detrimental effect of parental HFD and obesity on the musculoskeletal integrity of 2 generations of offspring, indicating the importance of further investigation of these effects. An improved understanding of the mechanisms involved in the transmissibility of diet-induced changes through multiple generations may help in the development of future therapies that would target the effects of obesity on OA and related conditions.

Physiologic and pathologic effects of dietary free fatty acids on cells of the joint.

Fatty acids (FAs) are potent organic compounds that not only can be used as an energy source during nutrient deprivation but are also involved in several essential signaling cascades in cells. Therefore, a balanced intake of different dietary FAs is critical for the maintenance of cellular functions and tissue homeostasis. A diet with an imbalanced fat composition creates a risk for developing metabolic syndrome and various musculoskeletal diseases, including osteoarthritis (OA). In this review, we summarize the current state of knowledge and mechanistic insights regarding the role of dietary FAs, such as saturated FAs, omega-6 polyunsaturated FAs (PUFAs), and omega-3 PUFAs on joint inflammation and OA pathogeneses. In particular, we review how different types of dietary FAs and their derivatives distinctly affect a variety of cells within the joint, including chondrocytes, osteoblasts, osteoclasts, and synoviocytes. Understanding the molecular mechanisms underlying the effects of FAs on metabolic behavior, anabolic, and catabolic processes, as well as the inflammatory response of joint cells, may help identify therapeutic targets for the prevention of metabolic joint diseases.

Effects of dietary fatty acid content on humeral cartilage and bone structure in a mouse model of diet-induced obesity.

Obesity is a primary risk factor for osteoarthritis (OA), and previous studies have shown that dietary content may play an important role in the pathogenesis of cartilage and bone in knee OA. Several previous studies have shown that the ratio of ω-3 polyunsaturated fatty acids (PUFAs), ω-6 PUFAs, and saturated fatty acids can significantly influence bone structure and OA progression. However, the influence of obesity or dietary fatty acid content on shoulder OA is not well understood. The goal of this study was to investigate the role of dietary fatty acid content on bone and cartilage structure in the mouse shoulder in a model of diet-induced obesity. For 24 weeks, mice were fed control or high-fat diets supplemented with ω-3 PUFAs, ω-6 PUFAs, or saturated fatty acids. The humeral heads were analyzed for bone morphometry and mineral density by microCT. Cartilage structure and joint synovitis were determined by histological grading, and microscale mechanical properties of the cartilage extracellular and pericellular matrices were quantified using atomic force microscopy. Diet-induced obesity significantly altered bone morphology and mineral density in a manner that was dependent on dietary free fatty acid content. In general, high-fat diet groups showed decreased bone quality, with the ω-3 diet being partially protective. Cartilage mechanical properties and OA scores showed no changes with obesity or diet. These findings are consistent with clinical literature showing little if any relationship between obesity and shoulder OA (unlike knee OA), but suggest that diet-induced obesity may influence other joint tissues. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Regional Differences Between Perisynovial and Infrapatellar Adipose Tissue Depots and Their Response to Class II and Class III Obesity in Patients With Osteoarthritis.

OBJECTIVE: Obesity is associated with an increased risk of developing osteoarthritis (OA), which is postulated to be secondary to adipose tissue-dependent inflammation. Periarticular adipose tissue depots are present in synovial joints, but the association of this tissue with OA has not been extensively explored. The aim of this study was to investigate differences in local adipose tissue depots in knees with OA and characterize the changes related to class II and class III obesity in patients with end-stage knee OA. METHODS: Synovium and the infrapatellar fat pad (IPFP) were collected during total knee replacement from 69 patients with end-stage OA. Histologic changes, changes in gene and protein expression of adiponectin, peroxisome proliferator-activated receptor γ (PPARγ), and Toll-like receptor 4 (TLR-4), and immune cell infiltration into the adipose tissue were investigated. RESULTS: IPFP and synovium adipose tissue depots differed significantly and were influenced by the patient's body mass index. Compared to adipocytes from the IPFP and synovium of lean patients, adipocytes from the IPFP of obese patients were significantly larger and the synovium of obese patients displayed marked fibrosis, increased macrophage infiltration, and higher levels of TLR4 gene expression. The adipose-related markers PPARγ in the IPFP and adiponectin and PPARγ in the synovium were expressed at lower levels in obese patients compared to lean patients. Furthermore, there were increased numbers of CD45+ hematopoietic cells, CD45+CD14+ total macrophages, and CD14+CD206+ M2-type macrophages in both the IPFP and synovial tissue of obese patients. CONCLUSION: These differences suggest that IPFP and synovium may contain 2 different white adipose tissue depots and support the theory of inflammation-induced OA in patients with class II or III obesity. These findings warrant further investigation as a potentially reversible, or at least suppressible, cause of OA in obese patients.