ABSTRACT
Interleukin-6 (IL-6) is a major pro-inflammatory cytokine for which the levels in plasma demonstrate a robust correlation with age and body mass index (BMI) as part of the senescence-associated secretory phenotype. IL-6 cytokines also play a crucial role in metabolic homeostasis and regenerative processes, primarily via the canonical STAT3 pathway. Thus, selective modulation of IL-6 signaling may offer a unique opportunity for therapeutic interventions. Recently, we discovered that a non-canonical signaling pathway downstream of tyrosine (Y) 814 within the intracellular domain of gp130, the IL-6 co-receptor, is responsible for the recruitment and activation of SRC family of kinases (SFK). Mice with constitutive genetic inactivation of gp130 Y814 (F814 mice) show accelerated resolution of inflammatory response and superior regenerative outcomes in skin wound healing and posttraumatic models of osteoarthritis. The current study was designed to explore if selective genetic or pharmacological inhibition of the non-canonical gp130-Y814/SFK signaling reduces systemic chronic inflammation and multimorbidity in a high-fat diet (HFD)-induced model of accelerated aging. F814 mice showed significantly reduced inflammatory response to HFD in adipose and liver tissue, with significantly reduced levels of systemic inflammation compared to wild type mice. F814 mice were also protected from HFD-induced bone loss and cartilage degeneration. Pharmacological inhibition of gp130-Y814/SFK in mice on HFD mirrored the effects observed in F814 mice on HFD; furthermore, this pharmacological treatment also demonstrated a marked increase in physical activity levels and protective effects against inflammation-associated suppression of neurogenesis in the brain tissue compared to the control group. These findings suggest that selective inhibition of SFK signaling downstream of gp130 receptor represents a promising strategy to alleviate systemic chronic inflammation. Increased degenerative changes and tissue senescence are inevitable in obese and aged organisms, but we demonstrated that the systemic response and inflammation-associated multi-morbidity can be therapeutically mitigated.
ABSTRACT
Adult mammals are incapable of multitissue regeneration, and augmentation of this potential may shift current therapeutic paradigms. We found that a common co-receptor of interleukin 6 (IL-6) cytokines, glycoprotein 130 (gp130), serves as a major nexus integrating various context-specific signaling inputs to either promote regenerative outcomes or aggravate disease progression. Via genetic and pharmacological experiments in vitro and in vivo, we demonstrated that a signaling tyrosine 814 (Y814) within gp130 serves as a major cellular stress sensor. Mice with constitutively inactivated Y814 (F814) were resistant to surgically induced osteoarthritis as reflected by reduced loss of proteoglycans, reduced synovitis, and synovial fibrosis. The F814 mice also exhibited enhanced regenerative, not reparative, responses after wounding in the skin. In addition, pharmacological modulation of gp130 Y814 upstream of the SRC and MAPK circuit by a small molecule, R805, elicited a protective effect on tissues after injury. Topical administration of R805 on mouse skin wounds resulted in enhanced hair follicle neogenesis and dermal regeneration. Intra-articular administration of R805 to rats after medial meniscal tear and to canines after arthroscopic meniscal release markedly mitigated the appearance of osteoarthritis. Single-cell sequencing data demonstrated that genetic and pharmacological modulation of Y814 resulted in attenuation of inflammatory gene signature as visualized by the anti-inflammatory macrophage and nonpathological fibroblast subpopulations in the skin and joint tissue after injury. Together, our study characterized a molecular mechanism that, if manipulated, enhances the intrinsic regenerative capacity of tissues through suppression of a proinflammatory milieu and prevents pathological outcomes in injury and disease.
Subject(s)
Cytokines , Osteoarthritis , Mice , Rats , Animals , Dogs , Cytokine Receptor gp130 , Interleukin-6 , Proteoglycans , MammalsABSTRACT
Epigenetic mechanisms guiding articular cartilage regeneration and age-related disease such as osteoarthritis (OA) are poorly understood. STAT3 is a critical age-patterned transcription factor highly active in fetal and OA chondrocytes, but the context-specific role of STAT3 in regulating the epigenome of cartilage cells remain elusive. In this study, DNA methylation profiling was performed across human chondrocyte ontogeny to build an epigenetic clock and establish an association between CpG methylation and human chondrocyte age. Exposure of adult chondrocytes to a small molecule STAT3 agonist decreased DNA methylation, while genetic ablation of STAT3 in fetal chondrocytes induced global hypermethylation. CUT&RUN assay and subsequent transcriptional validation revealed DNA methyltransferase 3 beta (DNMT3B) as one of the putative STAT3 targets in chondrocyte development and OA. Functional assessment of human OA chondrocytes showed the acquisition of progenitor-like immature phenotype by a significant subset of cells. Finally, conditional deletion of Stat3 in cartilage cells increased DNMT3B expression in articular chondrocytes in the knee joint in vivo and resulted in a more prominent OA progression in a post-traumatic OA (PTOA) mouse model induced by destabilization of the medial meniscus (DMM). Taken together these data reveal a novel role for STAT3 in regulating DNA methylation in cartilage development and disease. Our findings also suggest that elevated levels of active STAT3 in OA chondrocytes may indicate an intrinsic attempt of the tissue to regenerate by promoting a progenitor-like phenotype. However, it is likely that chronic activation of this pathway, induced by IL-6 cytokines, is detrimental and leads to tissue degeneration.
Subject(s)
Cartilage, Articular , Osteoarthritis , Mice , Animals , Humans , Chondrocytes/metabolism , Cells, Cultured , Osteoarthritis/genetics , Osteoarthritis/metabolism , Cartilage, Articular/metabolism , Epigenesis, Genetic , DNA Methylation/genetics , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolismABSTRACT
Human adipose-derived mesenchymal stem cells (ASCs) transduced with a lentiviral vector system to express bone morphogenetic protein 2 (LV-BMP-2) have been shown to reliably heal bone defects in animal models. However, the influence of donor characteristics such as age, sex, race, and medical co-morbidities on ASC yield, growth and bone regenerative capacity, while critical to the successful clinical translation of stem cell-based therapies, are not well understood. Human ASCs isolated from the infrapatellar fat pads in 122 ASC donors were evaluated for cell growth characteristics; 44 underwent additional analyses to evaluate in vitro osteogenic potential, with and without LV-BMP-2 transduction. We found that while female donors demonstrated significantly higher cell yield and ASC growth rates, age, race, and the presence of co-morbid conditions were not associated with differences in proliferation. Donor demographics or the presence of comorbidities were not associated with differences in in vitro osteogenic potential or stem cell differentiation, except that transduced ASCs from healthy donors produced more BMP-2 at day 2. Overall, donor age, sex, race, and the presence of co-morbid conditions had a limited influence on cell yield, proliferation, self-renewal capacity, and osteogenic potential for non-transduced and transduced (LV-BMP-2) ASCs. These results suggest that ASCs are a promising resource for both autologous and allogeneic cell-based gene therapy applications.
Subject(s)
Adipose Tissue , Mesenchymal Stem Cells , Animals , Humans , Female , Adipose Tissue/metabolism , Osteogenesis , Cell Differentiation/genetics , Mesenchymal Stem Cells/metabolism , Bone RegenerationABSTRACT
BACKGROUND: Autologous fat grafting is commonly used for soft-tissue repair (approximately 90,000 cases per year in the United States), but outcomes are limited by volume loss (20% to 80%) over time. Human allograft adipose matrix (AAM) stimulates de novo adipogenesis in vivo, but retention requires optimization. The extracellular matrix derived from superficial fascia, interstitial within the adipose layer, is typically removed during AAM processing. Thus, fascia, which contains numerous important proteins, might cooperate with AAM to stimulate de novo adipogenesis, improving long-term retention compared to AAM alone. METHODS: Human AAM and fascia matrix proteins (back and upper leg regions) were identified by mass spectrometry and annotated by gene ontology. A three-dimensional in vitro angiogenesis assay was performed. Finally, AAM and/or fascia (1 mL) was implanted into 6- to 8-week-old male Fischer rats. After 8 weeks, the authors assessed graft retention by gas pycnometry and angiogenesis (CD31) and adipocyte counts (hematoxylin and eosin) histologically. RESULTS: Gene ontology annotation revealed an angiogenic enrichment pattern unique to the fascia, including lactadherin, collagen alpha-3(V) chain, and tenascin-C. In vitro, AAM stimulated 1.0 ± 0.17 angiogenic sprouts per bead. The addition of fascia matrix increased sprouting by 88% (2.0 ± 0.12; P < 0.001). A similar angiogenic response (CD31) was observed in vivo. Graft retention volume was 25% (0.25 ± 0.13) for AAM, significantly increasing to 60% (0.60 ± 0.14) for AAM/fascia ( P < 0.05). De novo adipogenesis was 12% (12.4 ± 7.4) for AAM, significantly increasing to 51% (51.2 ± 8.0) for AAM/fascia ( P < 0.001) by means of adipocyte quantification. CONCLUSIONS: Combining fascia matrix with AAM improves angiogenesis and adipogenesis compared to AAM alone in rats. These preliminary in vitro and pilot animal studies should be further validated before definitive clinical adoption. CLINICAL RELEVANCE STATEMENT: When producing an off-the-shelf adipose inducing product by adding a connective tissue fascial component (that is normally discarded) to the mix of adipose matrix, vasculogenesis is increased and, thus, adipogenesis and graft survival is improved. This is a significant advance in this line of product.
Subject(s)
Adipose Tissue , Rodentia , Rats , Male , Humans , Animals , Adipose Tissue/transplantation , Adipogenesis/physiology , Obesity , Fascia/transplantation , AllograftsABSTRACT
Growth of long bones and vertebrae is maintained postnatally by a long-lasting pool of progenitor cells. Little is known about the molecular mechanisms that regulate the output and maintenance of the cells that give rise to mature cartilage. Here we demonstrate that postnatal chondrocyte-specific deletion of a transcription factor Stat3 results in severely reduced proliferation coupled with increased hypertrophy, growth plate fusion, stunting and signs of progressive dysfunction of the articular cartilage. This effect is dimorphic, with females more strongly affected than males. Chondrocyte-specific deletion of the IL-6 family cytokine receptor gp130, which activates Stat3, phenocopied Stat3-deletion; deletion of Lifr, one of many co-receptors that signals through gp130, resulted in a milder phenotype. These data define a molecular circuit that regulates chondrogenic cell maintenance and output and reveals a pivotal positive function of IL-6 family cytokines in the skeletal system with direct implications for skeletal development and regeneration.
Subject(s)
Chondrocytes/metabolism , Cytokine Receptor gp130/genetics , Growth Plate/metabolism , Mice/genetics , STAT3 Transcription Factor/genetics , Animals , Cell Proliferation/genetics , Cytokine Receptor gp130/metabolism , Homeostasis/genetics , Mice/growth & development , STAT3 Transcription Factor/metabolismABSTRACT
Osteoarthritis (OA) impacts hundreds of millions of people worldwide, with those affected incurring significant physical and financial burdens. Injuries such as focal defects to the articular surface are a major contributing risk factor for the development of OA. Current cartilage repair strategies are moderately effective at reducing pain but often replace damaged tissue with biomechanically inferior fibrocartilage. Here we describe the development, transcriptomic ontogenetic characterization and quality assessment at the single cell level, as well as the scaled manufacturing of an allogeneic human pluripotent stem cell-derived articular chondrocyte formulation that exhibits long-term functional repair of porcine articular cartilage. These results define a new potential clinical paradigm for articular cartilage repair and mitigation of the associated risk of OA.
ABSTRACT
Cartilage tissue is comprised of extracellular matrix and chondrocytes, a cell type with very low cellular turnover in adults, providing limited capacity for regeneration. However, in development a significant number of chondrocytes actively proliferate and remodel the surrounding matrix. Uncoupling the microenvironmental influences that determine the balance between clonogenic potential and terminal differentiation of these cells is essential for the development of novel approaches for cartilage regeneration. Unfortunately, most of the existing methods are not applicable for the analysis of functional properties of chondrocytes at a single cell resolution. Here we demonstrate that a novel 3D culture method provides a long-term and permissive in vitro niche that selects for highly clonogenic, colony-forming chondrocytes which maintain cartilage-specific matrix production, thus recapitulating the in vivo niche. As a proof of concept, clonogenicity of Sox9 IRES-EGFP mouse chondrocytes is almost exclusively found in the highest GFP+ fraction known to be enriched for chondrocyte progenitor cells. Although clonogenic chondrocytes are very rare in adult cartilage, we have optimized this system to support large, single cell-derived chondrogenic organoids with complex zonal architecture and robust chondrogenic phenotype from adult pig and human articular chondrocytes. Moreover, we have demonstrated that growth trajectory and matrix biosynthesis in these organoids respond to a pro-inflammatory environment. This culture method offers a robust, defined and controllable system that can be further used to interrogate the effects of various microenvironmental signals on chondrocytes, providing a high throughput platform to assess genetic and environmental factors in development and disease.