Targeting STAT6-mediated synovial macrophage activation improves pain in experimental knee osteoarthritis.

BACKGROUND: Pain from osteoarthritis (OA) is one of the top causes of disability worldwide, but effective treatment is lacking. Nociceptive factors are released by activated synovial macrophages in OA, but depletion of synovial macrophages paradoxically worsens inflammation and tissue damage in previous studies. Rather than depleting macrophages, we hypothesized that inhibiting macrophage activation may improve pain without increasing tissue damage. We aimed to identify key mechanisms mediating synovial macrophage activation and test the role of STAT signaling in macrophages on pain outcomes in experimental knee OA. METHODS: We induced experimental knee OA in rats via knee destabilization surgery, and performed RNA sequencing analysis on sorted synovial tissue macrophages to identify macrophage activation mechanisms. Liposomes laden with STAT1 or STAT6 inhibitors, vehicle (control), or clodronate (depletion control) were delivered selectively to synovial macrophages via serial intra-articular injections up to 12 weeks after OA induction. Treatment effects on knee and hindpaw mechanical pain sensitivity were measured during OA development, along with synovitis, cartilage damage, and synovial macrophage infiltration using histopathology and immunofluorescence. Lastly, crosstalk between drug-treated synovial tissue and articular chondrocytes was assessed in co-culture. RESULTS: The majority of pathways identified by transcriptomic analyses in OA synovial macrophages involve STAT signaling. As expected, macrophage depletion reduced pain, but increased synovial tissue fibrosis and vascularization. In contrast, STAT6 inhibition in macrophages led to marked, sustained improvements in mechanical pain sensitivity and synovial inflammation without worsening synovial or cartilage pathology. During co-culture, STAT6 inhibitor-treated synovial tissue had minimal effects on healthy chondrocyte gene expression, whereas STAT1 inhibitor-treated synovium induced changes in numerous cartilage turnover-related genes. CONCLUSION: These results suggest that STAT signaling is a major mediator of synovial macrophage activation in experimental knee OA. STAT6 may be a key mechanism mediating the release of nociceptive factors from macrophages and the development of mechanical pain sensitivity. Whereas therapeutic depletion of macrophages paradoxically increases inflammation and fibrosis, blocking STAT6-mediated synovial macrophage activation may be a novel strategy for OA-pain management without accelerating tissue damage.

The effect of HLA-DRB1*04:01 on a mouse model of atherosclerosis.

Objectives: HLA-DRB1 is associated with an increased risk of cardiovascular disease in patients with rheumatoid arthritis (RA). This study aimed to determine the effect of HLA-DRB1 on atherosclerotic cardiovascular disease (ASCVD) using a novel mouse model. Methods: Mice transgenic for HLA-DRB1*04:01 (DR4tg) were crossed with low density lipoprotein receptor knock-out (Ldlr-/-) mice that develop atherosclerosis when fed a high fat, high cholesterol (HFHC) diet. Male and female DR4tgLdlr-/- (n = 48), Ldlr-/- (n = 24), DR4tg (n = 24), and C57Bl/6 (B6) background (n = 24) mice were fed HFHC or regular diet (RD) for 12 weeks. Blood samples were analyzed for serum lipoproteins using a colorimetric assay. C-reactive protein (CRP) and oxidized LDL (OxLDL) were measured using ELISA. Atherosclerosis in the aortas was assessed using the lipid stain, Sudan IV. The presence of citrulline in atherosclerotic plaque was determined by immunohistochemistry. Results: Sera low-density lipoprotein cholesterol (LDL-C) levels were higher in HFHC-fed Ldlr-/- versus DR4tgLdlr-/--; p = 0.0056, but the aortic plaque burden and degree of citrullination in the plaque were similar for these two strains. The ratio of pro-atherogenic OxLDL to LDL levels was higher in DR4tgLdlr-/- than Ldlr-/-mice; p = 0.0017. All mice had an increase in CRP when fed a HFHC diet, most pronounced for DR4tgLdlr-/-; p = 0.0009. There were no significant sex differences for DR4tgLdlr-/- mice; however, male Ldlr-/- mice had worse atherosclerosis. B6 and DR4tg mice did not have significant elevations in serum cholesterol levels and did not develop atherosclerosis. Conclusions: Expression of HLA-DRB1 resulted in an elevation of OxLDL and a reduction in the male bias for atherosclerosis, mimicking what is observed in RA.

Effects of risk factors on evoked pain patterns in rat models of experimental knee osteoarthritis.

Pain experiences in patients with knee osteoarthritis (OA) may be influenced differently by OA risk factors, reducing the translatability of preclinical research into the clinic. Our objective was to contrast evoked pain patterns after exposure to different OA risk factors including acute joint trauma, chronic instability, or obesity/metabolic syndrome using rat models of experimental knee OA. We tested longitudinal patterns of evoked pain behaviors (knee pressure pain threshold and hindpaw withdrawal threshold) in young male rats exposed to different OA-inducing risk factors including (1) nonsurgical joint trauma (impact-induced anterior cruciate ligament (ACL) rupture); (2) surgical joint destabilization (ACL + medial meniscotibial ligament transection); and (3) high fat/sucrose (HFS) diet-induced obesity. Histopathology for synovitis, cartilage damage, and subchondral bone morphology was performed. Pressure pain threshold was reduced (more pain) most, and earlier by joint trauma (Week 4-12) and HFS (Week 8-28) than by joint destabilization (Week 12). Hindpaw withdrawal threshold was reduced transiently after joint trauma (Week 4), with smaller and later reductions after joint destabilization (Week 12), but not with HFS. Synovial inflammation occurred at Week 4 after joint trauma and instability but only coincided with pain behaviors after joint trauma. Cartilage and bone histopathology were most severe after joint destabilization and least severe with HFS. The pattern, intensity, and timing of evoked pain behaviors varied due to OA risk factor exposure and were inconsistently associated with histopathological OA features. These findings may help to explain the challenges with translating preclinical OA pain research to multimorbid clinical OA contexts.

Impaired Efferocytosis by Synovial Macrophages in Patients With Knee Osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) exposes all joint tissues to physiologic stresses, increasing the need to clear apoptotic cells from tissues, including the synovium. We undertook this study to assess the burden of apoptotic cells in synovial tissue in patients with late-stage knee OA and to investigate whether OA impairs the macrophage-mediated clearance of apoptotic cells via efferocytosis. METHODS: Synovial tissue was collected from individuals with healthy knees and patients with late-stage knee OA during arthroplasty. Synovial apoptotic cell burden was assessed by immunofluorescence for cleaved caspase 3. Efferocytosis of apoptotic Jurkat cells by CD14+ synovial tissue macrophages and peripheral blood-derived macrophages was quantified using immunofluorescence microscopy. Effects of OA on macrophage-mediated efferocytosis were modeled by stimulating blood-derived macrophages with synovial fluid collected from individuals with healthy knees and patients with early- or late-stage knee OA. RESULTS: Patients with late-stage knee OA had more apoptotic synovial cells compared to healthy individuals. There was a marked reduction in the fraction of synovial tissue macrophages engaging in efferocytosis and the quantity of material efferocytosed by individual macrophages in OA patients. Blood-derived macrophages exposed to synovial fluid from patients with knee OA recapitulated the defective efferocytosis, with the greatest effect from patients with early-stage knee OA and higher disease activity (pain and inflammation). CONCLUSION: Apoptotic cells accumulate in the synovium of patients with late-stage knee OA. Our results suggest that OA impairs critical homeostatic functions of synovial macrophages, leading to accumulation of apoptotic cells.

The effect of sex on immune responses to a homocitrullinated peptide in the DR4-transgenic mouse model of Rheumatoid Arthritis.

Rheumatoid Arthritis (RA) is more common and severe in women compared to men. Both women and men with RA express autoantibodies to post-translationally modified antigens, including citrullinated and homocitrullinated proteins or peptides. These autoantibodies are strongly linked with the HLA-DR4 gene. The objective of this study was to determine sex differences in immune responses to homocitrullinated antigens. We used a humanized animal model of RA, DR4-transgenic mice and immunized them with a homocitrullinated peptide called HomoCitJED. Immune responses in these mice were measured for splenocyte proliferation by tritiated thymidine incorporation, serum autoantibody production by ELISA and cytokine levels by multiplex. We found that T cell and antibody responses to homocitrullinated antigens were similar in male and female mice. However, we found sex differences in serum cytokine profiles with female mice having higher ratio of IL-1α to IL-5, suggesting imbalances in immune regulation. This is the first study to report that immune responses to homocitrullinated antigens can be differentiated by sex.

Improved Aptamers for the Diagnosis and Potential Treatment of HER2-Positive Cancer.

Aptamers provide a potential source of alternative targeting molecules for existing antibody diagnostics and therapeutics. In this work, we selected novel DNA aptamers targeting the HER2 receptor by an adherent whole-cell SELEX approach. Individual aptamers were identified by next generation sequencing and bioinformatics analysis. Two aptamers, HeA2_1 and HeA2_3, were shown to bind the HER2 protein with affinities in the nanomolar range. In addition, both aptamers were able to bind with high specificity to HER2-overexpressing cells and HER2-positive tumor tissue samples. Furthermore, we demonstrated that aptamer HeA2_3 is being internalized into cancer cells and has an inhibitory effect on cancer cell growth and viability. In the end, we selected novel DNA aptamers with great potential for the diagnosis and possible treatment of HER2-positive cancer.