Biology subjects

Appleton, C. T.

Publications and source records attributed to Appleton, C. T..

5 recordsLinked to original sources

bioRxiv2024

Metabolic Stress Accelerates Dysregulated Synovial Macrophage-Fibroblast Communication and Htra1 Overproduction in Osteoarthritis

Biomechanical and metabolic factors increase the risk for osteoarthritis (OA) by causing supraphysiological stresses on joint tissues. Chronic exposure to these stresses contributes to failure of the joint organ system, resulting in pain and loss of function for patients with OA. The synovium is vital for joint organ health but during OA, synovial inflammation and damage are associated with worse outcomes including pain. Unfortunately, the separate and combined effects of metabolic and biomechanical stresses on synovial tissues are not well understood. In this study, metabolic syndrome (MetS) was associated with worse knee pain in patients with early-stage knee OA, suggesting that metabolic stress may act on synovial tissues during early-stage OA, exacerbating outcomes. In a rat model of experimental knee OA, the combined effects of biomechanical and metabolic stresses induced worse knee pain, cartilage damage, and synovial inflammation than biomechanical stress alone. Further, single-cell RNA sequencing of synovial macrophages and fibroblasts identified earlier metabolic (glycolytic and respiratory) shifts, neurogenesis, dysregulated communication, and cell activation when metabolic and biomechanical stresses were combined. Lastly, using a direct contact co-culture system, we showed that metabolic stress alters macrophage-fibroblast communication leading to increased expression of Htra1, a pathogenic protease in OA. This study identifies novel mechanisms that may represent amenable therapeutic targets for patients experiencing MetS and OA. One-sentence summary: Metabolic stress may cause worse outcomes in OA through dysregulated synovial cell communication that activates synovial fibroblasts and increases Htra1 production.

physiology↗

bioRxiv2023

Synovial macrophage diversity and activation of M-CSF signaling in post-traumatic osteoarthritis

ObjectiveSynovium is home to immune and stromal cell types that orchestrate inflammation following a joint injury; in particular, macrophages are central protagonists in this process. We sought to define the cellular and temporal dynamics of the synovial immune niche in a mouse model of post-traumatic osteoarthritis (PTOA), and to identify stromal-immune crosstalk mechanisms that coordinate macrophage function and phenotype. DesignWe induced PTOA in mice using a non-invasive tibial compression model of anterior cruciate ligament rupture (ACLR). Single cell RNA-seq and flow cytometry were used to assess immune cell populations in healthy (Sham) and injured (7d and 28d post-ACLR) synovium. Characterization of synovial macrophage polarization states was performed, alongside computational modeling of macrophage differentiation, as well as implicated transcriptional regulators and stromal-immune communication axes. ResultsImmune cell types are broadly represented in healthy synovium, but experience drastic expansion and speciation in PTOA, most notably in the macrophage portion. We identified several polarization states of macrophages in synovium following joint injury, underpinned by distinct transcriptomic signatures, and regulated in part by stromal-derived macrophage colony-stimulating factor signaling. The transcription factors Pu.1, Cebp, Cebp{beta}, and Jun were predicted to control differentiation of systemically derived monocytes into pro-inflammatory synovial macrophages. ConclusionsWe defined different synovial macrophage subpopulations present in healthy and injured mouse synovium. Nuanced characterization of the distinct functions, origins, and disease kinetics of macrophage subtypes in PTOA will be critical for targeting these highly versatile cells for therapeutic purposes.

immunology↗

bioRxiv2023

Synovial macrophage activation mediates pain experiences in experimental knee osteoarthritis

It has been suggested that synovial macrophages mediate nociceptive signals in knee osteoarthritis (OA) but the underlying mechanisms are unknown. Our objectives were to investigate the role of synovial macrophages and their activation via signal transducer and activator of transcription (STAT) signaling in mediating OA pain experiences. We induced experimental OA in rats via knee destabilization surgery and then performed RNA sequencing analysis in sorted synovial macrophages to identify signaling pathways associated with macrophage activation. Next, we repeated intra-articular injections of liposomal clodronate to deplete macrophages, or liposomal inhibitors of STAT1 or STAT6 to block macrophage activation, and tested the effects on local and distal mechanical pain sensitivity. We also assessed synovitis, cartilage damage, and synovial macrophage infiltration with histopathology and immunofluorescence, and crosstalk between liposomal drug-treated synovium and articular chondrocytes in co-culture. Most enriched signaling pathways in activated OA macrophages involved STAT signalling. Macrophage depletion and STAT6 inhibition led to marked, sustained improvements in mechanical pain sensitivity and synovial inflammation compared to controls, but macrophage depletion caused increased synovial fibrosis and vascularization. In contrast, STAT1 and STAT6 inhibition in macrophages did not worsen synovial or cartilage pathology. In crosstalk assays, macrophage STAT1-inhibited synovium caused the greatest increases in the expression of anabolic and catabolic chondrocyte genes and sulphated glycosaminoglycan secretion in chondrocytes. Our results suggest that synovial macrophages play a key role in mediating pain experiences in experimental knee OA, and that selectively blocking STAT6 in synovial macrophages may reduce OA-related pain without accelerating joint tissue damage. (248/250) One Sentence SummarySelective drug targeting to synovial macrophages improves pain experiences in surgical joint destabilization-induced experimental rodent knee OA. (145/150)

physiology↗

bioRxiv2021

Genetic deletion of interleukin-15 is not associated with major structural changes following experimental post-traumatic knee osteoarthritis in rats

Post-traumatic Osteoarthritis (PTOA) is a degenerative joint disease, leading to articular cartilage breakdown, osteophyte formation, and synovitis, caused by an initial joint trauma. Pro-inflammatory cytokines increase catabolic activity and may perpetuate inflammation following joint trauma. Interleukin-15 (IL-15), a pro-inflammatory cytokine, is increased in OA patients, although its roles in OA pathophysiology are not well characterized. IL-15 levels appear to correlate to self-reported pain levels, and polymorphisms in the IL-15 receptor alpha gene correlate to a 1.5-fold increase in OA symptoms. This could be due to IL-15 effects on the activity of proteinases, such as matrix metalloproteinases (MMP) -1, -3, and -7. Here we utilized Il15 deficient rats to examine the role of IL-15 in PTOA pathogenesis in an injury-induced model of OA. OA was surgically induced in Il15 deficient rats and control wild-type rats to compare PTOA progression. Semi-quantitative scoring of the articular cartilage, subchondral bone, osteophyte size, and synovium was performed by two blinded observers. Analyses of articular cartilage damage, subchondral bone damage, and osteophyte formation revealed no significant difference between Il15 deficient rats and wild-type rats following PTOA-induction. Similarly, synovitis scoring across 6 parameters found no significant difference between genetic variants. Overall, IL-15 does not appear to play a key role in the development of structural changes in this surgically-induced rat model of PTOA.

genetics↗

bioRxiv2020

Identification of Proteinase Activated Receptor (PAR) cleaving enzymes in human osteoarthritis knee joint synovial fluids.

ObjectiveOsteoarthritis (OA) is the most prevalent joint disorder with incidence increasing worldwide. Mechanistic insights into OA pathophysiology are still evolving and there are currently no disease-modifying OA drugs available. It is well established that an increase in proteolytic enzyme activity is linked to progressive degradation of the cartilage in OA. Proteolytic enzymes can also trigger inflammation through activation of a family of G-protein coupled receptors (GPCRs) called the Proteinase Activated Receptors (PARs). Here we sought to characterize the PAR activating enzyme repertoire in human OA knee joint fluids. MethodsHuman knee joint synovial fluids derived from twenty-five OA patients and four healthy donors were screened for PAR cleavage activity using novel genetically encoded human PAR biosensor expressing cells. The class or type of enzymes cleaving the PARs was further characterized using enzyme-selective inhibitors and enzyme-specific fluorogenic substrates. ResultsActivity of PAR1, PAR2 and PAR4 activating enzymes were identified at substantially different levels in OA patients relative to healthy knee joint synovial fluids. Using enzyme class or type selective inhibitors and fluorogenic substrates we found that serine proteinases, including thrombin-like enzymes, trypsin-like enzymes, and matrix metalloproteinases are the major PAR activating enzymes present in the OA knee synovial fluids. ConclusionsMultiple enzymes activating PAR1, PAR2 and PAR4 are present in OA joint fluids. PAR signalling can trigger pro-inflammatory responses and targeting PARs has been proposed as a therapeutic approach in OA. Knowledge of the PAR activators present in the human knee joint will guide study of relevant signaling events and enable future development of novel PAR targeted therapies for OA and other inflammatory joint diseases.

pharmacology and toxicology↗