Preclinical Models Of Rheumatoid Arthritis Research Articles

Liposomal AntagomiR-155-5p Restores Anti-Inflammatory Macrophages and Improves Arthritis in Preclinical Models of Rheumatoid Arthritis.

We previously reported an increased expression of microRNA-155 (miR-155) in the blood monocytes of patients with rheumatoid arthritis (RA) that could be responsible for impaired monocyte polarization to anti-inflammatory M2-like macrophages. In this study, we employed two preclinical models of RA, collagen-induced arthritis and K/BxN serum transfer arthritis, to examine the therapeutic potential of antagomiR-155-5p entrapped within PEGylated (polyethylene glycol [PEG]) liposomes in resolution of arthritis and repolarization of monocytes towards the anti-inflammatory M2 phenotype. AntagomiR-155-5p or antagomiR-control were encapsulated in PEG liposomes of 100 nm in size and -10 mV in zeta potential with high antagomiR loading efficiency (above 80%). Mice were injected intravenously with 1.5 nmol/100 μL PEG liposomes containing antagomiR-155-5p or control after the induction of arthritis. We demonstrated the biodistribution of fluorescently tagged PEG liposomes to inflamed joints one hour after the injection of fluorescently tagged PEG liposomes, as well as the liver's subsequent accumulation after 48 hours, indicative of hepatic clearance, in mice with arthritis. The injection of PEG liposomes containing antagomiR-155-5p decreased arthritis score and paw swelling compared with PEG liposomes containing antagomiR-control or the systemic delivery of free antagomiR-155-5p. Moreover, treatment with PEG liposomes containing antagomiR-155-5p led to the restoration of bone marrow monocyte defects in anti-inflammatory macrophage differentiation without any significant functional change in other immune cells, including splenic B and T cells. The injection of antagomiR-155-5p encapsulated in PEG liposomes allows the delivery of small RNA to monocytes and macrophages and reduces joint inflammation in murine models of RA, providing a promising strategy in human disease.

Extracellular sulfatase-2 is overexpressed in rheumatoid arthritis and mediates the TNF-α-induced inflammatory activation of synovial fibroblasts.

Extracellular sulfatase-2 (Sulf-2) influences receptor-ligand binding and subsequent signaling by chemokines and growth factors, yet Sulf-2 remains unexplored in inflammatory cytokine signaling in the context of rheumatoid arthritis (RA). In the present study, we characterized Sulf-2 expression in RA and investigated its potential role in TNF-α-induced synovial inflammation using primary human RA synovial fibroblasts (RASFs). Sulf-2 expression was significantly higher in serum and synovial tissues from patients with RA and in synovium and serum from hTNFtg mice. RNA sequencing analysis of TNF-α-stimulated RASFs showed that Sulf-2 siRNA modulated ~2500 genes compared to scrambled siRNA. Ingenuity Pathway Analysis of RNA sequencing data identified Sulf-2 as a primary target in fibroblasts and macrophages in RA. Western blot, ELISA, and qRT‒PCR analyses confirmed that Sulf-2 knockdown reduced the TNF-α-induced expression of ICAM1, VCAM1, CAD11, PDPN, CCL5, CX3CL1, CXCL10, and CXCL11. Signaling studies identified the protein kinase C-delta (PKCδ) and c-Jun N-terminal kinase (JNK) pathways as key in the TNF-α-mediated induction of proteins related to cellular adhesion and invasion. Knockdown of Sulf-2 abrogated TNF-α-induced RASF proliferation. Sulf-2 knockdown with siRNA and inhibition by OKN-007 suppressed the TNF-α-induced phosphorylation of PKCδ and JNK, thereby suppressing the nuclear translocation and DNA binding activity of the transcription factors AP-1 and NF-κBp65 in human RASFs. Interestingly, Sulf-2 expression positively correlated with the expression of TNF receptor 1, and coimmunoprecipitation assays demonstrated the binding of these two proteins, suggesting they exhibit crosstalk in TNF-α signaling. This study identified a novel role of Sulf-2 in TNF-α signaling and the activation of RA synoviocytes, providing the rationale for evaluating the therapeutic targeting of Sulf-2 in preclinical models of RA.

Azatricyclic Inverse Agonists of RORγt That Demonstrate Efficacy in Models of Rheumatoid Arthritis and Psoriasis.

Structure-activity relationship studies directed toward the replacement of the fused phenyl ring of the lead hexahydrobenzoindole RORγt inverse agonist series represented by 1 with heterocyclic moieties led to the identification of three novel aza analogs 5-7. The hexahydropyrrolo[3,2-f]quinoline series 5 (X = N, Y = Z=CH) showed potency and metabolic stability comparable to series 1 but with improved in vitro membrane permeability and serum free fraction. This structural modification was applied to the hexahydrocyclopentanaphthalene series 3, culminating in the discovery of 8e as a potent and selective RORγt inverse agonist with an excellent in vitro profile, good pharmacokinetic properties, and biologic-like in vivo efficacy in preclinical models of rheumatoid arthritis and psoriasis.

Quercetin: A Potential Candidate for the Treatment of Arthritis

Diet plays a significant role in ensuring healthy life, and the bioactive compounds present in food and medicinal plants may be developed as drugs that combat various illnesses. A bioactive flavanoid, quercetin which is a dietary component, possesses numerous health-promoting effects. In preclinical models of rheumatoid arthritis, gouty arthritis and osteoarthritis, quercetin has shown significant joint protective effects. Taking into account the significance of this compound, the present review discusses its anti-arthritic properties, demonstrating its mechanism of action for the treatment of arthritis with its therapeutic potential.

The Joint-Brain Axis: Insights From Rheumatoid Arthritis on the Crosstalk Between Chronic Peripheral Inflammation and the Brain.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by erosive polyarthritis. Beyond joint pathology, RA is associated with neuropsychiatric comorbidity including depression, anxiety, and an increased risk to develop neurodegenerative diseases in later life. Studies investigating the central nervous system (CNS) in preclinical models of RA have leveraged the understanding of the intimate crosstalk between peripheral and central immune responses. This mini review summarizes the current knowledge of CNS comorbidity in RA patients and known underlying cellular mechanisms. We focus on the differential regulation of CNS myeloid and glial cells in different mouse models of RA reflecting different patterns of peripheral immune activation. Moreover, we address CNS responses to anti-inflammatory treatment in human RA patients and mice. Finally, to illustrate the bidirectional communication between the CNS and chronic peripheral inflammation, we present the current knowledge about the impact of the CNS on arthritis. A comprehensive understanding of the crosstalk between the CNS and chronic peripheral inflammation will help to identify RA patients at risk of developing CNS comorbidity, setting the path for future therapeutic approaches in both RA and neuropsychiatric diseases.

Mesenchymal Stem/Stromal Cells for Rheumatoid Arthritis Treatment: An Update on Clinical Applications.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that affects the lining of the synovial joints leading to stiffness, pain, inflammation, loss of mobility, and erosion of joints. Its pathogenesis is related to aberrant immune responses against the synovium. Dysfunction of innate and adaptive immunity, including dysregulated cytokine networks and immune complex-mediated complement activation, are involved in the progression of RA. At present, drug treatments, including corticosteroids, antirheumatic drugs, and biological agents, are used in order to modulate the altered immune responses. Chronic use of these drugs may cause adverse effects to a significant number of RA patients. Additionally, some RA patients are resistant to these therapies. In recent years, mesenchymal stem/stromal cell (MSCs)-based therapies have been largely proposed as a novel and promising stem cell therapeutic approach in the treatment of RA. MSCs are multipotent progenitor cells that have immunomodulatory properties and can be obtained and expanded easily. Today, nearly one hundred studies in preclinical models of RA have shown promising trends for clinical application. Proof-of-concept clinical studies have demonstrated satisfactory safety profile of MSC therapy in RA patients. The present review discusses MSC-based therapy approaches with a focus on published clinical data, as well as on clinical trials, for treatment of RA that are currently underway.

Quantitative tracking of inflammatory activity at the peak and trough plasma levels of tofacitinib, a Janus kinase inhibitor, via in vivo 18 F-FDG PET.

To assess the capability of in vivo positron emission tomography (PET) using 18 F-fluorodeoxyglucose (18 F-FDG) to quantify changes in inflammatory activity in response to tofacitinib, a Janus kinase (JAK) inhibitor, over a timeframe of a few hours to few days in a preclinical model of rheumatoid arthritis (RA). Twenty-four mice with collagen-induced arthritis in the following groups were assessed: Group 1, where the changes in PET measures for the extremity joints were evaluated at the peak and trough plasma drug levels after administration of a single dose of tofacitinib (4hours apart); Group 2, where joint PET measures were assessed before treatment and after 6days of administration of a daily dose of tofacitinib; and group 3 (controls), where joint PET measures were derived from the same mice, 6days apart. At about peak plasma levels of the drug after a single tofacitinib administration, there was a reduction in PET measures compared to pretreatment values, suggesting decreased inflammatory activity. These measures were equivalent to those obtained after 6days of daily dosing by tofacitinib. However, PET measures at trough plasma levels of the drug from tofacitinib administration were significantly higher than those at peak plasma drug levels and equivalent to pretreatment measures. There were insignificant changes in PET measures for the control animals. 18 F-FDG PET can detect changes in inflammatory activity occurring in response to the JAK inhibitor tofacitinib: (a) during peak and trough plasma drug levels, that is within mere hours of treatment; and (b) over a span of days.

Efficacy and Pharmacodynamic Modeling of the BTK Inhibitor Evobrutinib in Autoimmune Disease Models.

Because of its role in mediating both B cell and Fc receptor signaling, Bruton's tyrosine kinase (BTK) is a promising target for the treatment of autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Evobrutinib is a novel, highly selective, irreversible BTK inhibitor that potently inhibits BCR- and Fc receptor-mediated signaling and, thus, subsequent activation and function of human B cells and innate immune cells such as monocytes and basophils. We evaluated evobrutinib in preclinical models of RA and SLE and characterized the relationship between BTK occupancy and inhibition of disease activity. In mouse models of RA and SLE, orally administered evobrutinib displayed robust efficacy, as demonstrated by reduction of disease severity and histological damage. In the SLE model, evobrutinib inhibited B cell activation, reduced autoantibody production and plasma cell numbers, and normalized B and T cell subsets. In the RA model, efficacy was achieved despite failure to reduce autoantibodies. Pharmacokinetic/pharmacodynamic modeling showed that mean BTK occupancy in blood cells of 80% was linked to near-complete disease inhibition in both RA and SLE mouse models. In addition, evobrutinib inhibited mast cell activation in a passive cutaneous anaphylaxis model. Thus, evobrutinib achieves efficacy by acting both on B cells and innate immune cells. Taken together, our data show that evobrutinib is a promising molecule for the chronic treatment of B cell-driven autoimmune disorders.

PET/CT Imaging of Human TNFα Using [89Zr]Certolizumab Pegol in a Transgenic Preclinical Model of Rheumatoid Arthritis.

Tumor necrosis factor alpha (TNFα) drives inflammation and bone degradation in patients with rheumatoid arthritis (RA). Some RA patients experience a rapid clinical response to TNFα inhibitors such as certolizumab pegol (CZP) while other patients show limited to no response. Current methods for imaging RA have limited sensitivity and do not assist in the selection of patients most likely to respond to immune-mediated therapy. Herein, we developed a novel positron emission tomography (PET) radiotracer for immuno-PET imaging of TNFα in transgenic human TNFα-expressing mice. CZP was modified with p-isothiocyanatobenzyl-deferoxamine (DFO) and radiolabeled with Zr-89. The biological activity of [89Zr]DFO-CZP was evaluated by HPLC and binding assay using human recombinant TNFα (hTNFα). The feasibility of specific immuno-PET imaging of human TNFα was assessed in a transgenic mouse model of RA that expresses human TNFα. This model resembles the progression of RA in humans by maintaining lower levels of circulating hTNFα and exhibits chronic arthritis in the forepaw and hind paw joints. The dosimetry of [89Zr]DFO-CZP in humans was estimated using microPET/CT imaging in Sprague Dawley rats. [89Zr]DFO-CZP was isolated with radiolabeling yields of 85 ± 6% (n = 5) and specific activities ranging from 74 to 185MBq/mg (n = 5). Following size exclusion purification, the radiochemical purity of [89Zr]DFO-CZP was greater than 97%. [89Zr]DFO-CZP retained high immunoreactivity with more than 95% of the radioactivity shifted into higher molecular weight complexes. Images showed increasing uptake of the tracer in forepaw and hind paw joints with disease progression. No uptake was observed in the model previously administered with an excess amount of unmodified CZP and in normal control mice, demonstrating in vivo specific uptake of [89Zr]DFO-CZP. The feasibility of immuno-PET imaging of human TNFα with [89Zr]DFO-CZP has been demonstrated in a preclinical model of RA.

Spontaneous burrowing behavior as an outcome measure of the global impact of chronic pain in preclinical models of arthritis

Abstract Aims Arthritis is the leading cause of chronic pain in European adults (painineurope.com). Arthritis-induced chronic pain is a major clinical problem that is frequently inadequately treated by current analgesics and results in significant reduction in the patient’s quality of life. Outcome measures used in pain research to assess injury-induced pain-like behaviours in preclinical models are often dependent on the assessment of withdrawal reflexes, typically evoked by mechanical or thermal stimuli.Advances inpre-dictive validity may be improved by assessing the global impact of pain and the reinstatement of innate behaviours suppressed by injury. Burrowing, an indicator of the global “wellbeing” of the animal can be objectively measured. We aimed to assess the impact of several preclinical modelsof arthritis onburrowing behaviour in mice. Methods The collagen antibody-induced arthritis (CAIA) pre-clinical model of rheumatoid arthritis, the complete Freund’s adjuvant (CFA), monosodium iodoacetate (MIA) and partial medial menisectomy (MMT) preclinical models of osteoarthritis were assessed in female and/or male C57/Black6J adult mice, comparing to control (salineorsham-operated) and naïve animals. Mechanical hypersensitivity using von Frey filaments and spontaneous burrowing behaviour were assessed over60days (CAIA), 14days (CFA), 28 days (MIA) and up to 200 days (MMT). Results We show the development of mechanical hypersen-sitivity in all preclinical models of arthritis, whereas spontaneous burrowing behaviour was altered differently between the preclin-ical models. A deficit in burrowing behaviour was observed in the preclinical models of osteoarthritis and a gain in burrowing behaviour noted in the preclinical model of rheumatoid arthritis, with no effect seen in the CFA model. Conclusions Burrowing behaviour appears more sensitive to detecting variation in preclinical models of arthritis when compared to mechanical hypersensitivity. Burrowing behaviour can detect both gain and deficits in the behaviour, which differs between preclinical models of rheumatoid and osteoarthritis.

Development and optimization of a high-throughput micro-computed tomography imaging method incorporating a novel analysis technique to evaluate bone mineral density of arthritic joints in a rodent model of collagen induced arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease resulting in joint inflammation, pain, and eventual bone loss. Bone loss and remodeling caused by symmetric polyarthritis, the hallmark of RA, is readily detectable by bone mineral density (BMD) measurement using micro-CT. Abnormalities in these measurements over time reflect the underlying pathophysiology of the bone. To evaluate the efficacy of anti-rheumatic agents in animal models of arthritis, we developed a high throughput knee and ankle joint imaging assay to measure BMD as a translational biomarker. A bone sample holder was custom designed for micro-CT scanning, which significantly increased assay throughput. Batch processing 3-dimensional image reconstruction, followed by automated image cropping, significantly reduced image processing time. In addition, we developed a novel, automated image analysis method to measure BMD and bone volume of knee and ankle joints. These improvements significantly increased the throughput of ex vivo bone sample analysis, reducing data turnaround from 5days to 24hours for a study with 200 rat hind limbs. Taken together, our data demonstrate that BMD, as quantified by micro-CT, is a robust efficacy biomarker with a high degree of sensitivity. Our innovative approach toward evaluation of BMD using optimized image acquisition and novel image processing techniques in preclinical models of RA enables high throughput assessment of anti-rheumatic agents offering a powerful tool for drug discovery.

Myeloid-derived suppressor cells have a proinflammatory role in the pathogenesis of autoimmune arthritis

ObjectivesAlthough myeloid-derived suppressor cells (MDSCs) have been linked to T cell tolerance, their role in autoimmune rheumatoid arthritis (RA) remains elusive. Here we investigate the potential association of MDSCs with...

OP0108 The Antibody-Based Delivery of IL4 to the Neo-Vasculature Cures Mice with Arthritis

BackgroundAntibody-cytokine fusion proteins (“immunocytokines”) are innovative biopharmaceutical agents, which are being considered for the therapy of cancer and chronic inflammatory conditions. Immunomodulatory fusion proteins capable of selective localization at the...

IDO2 Is a Critical Mediator of Autoantibody Production and Inflammatory Pathogenesis in a Mouse Model of Autoimmune Arthritis

Rheumatoid arthritis and other autoimmune disorders are associated with altered activity of the immunomodulatory enzyme IDO. However, the precise contributions of IDO function to autoimmunity remain unclear. In this article, we examine the effect of two different IDO enzymes, IDO1 and IDO2, on the development of autoimmune arthritis in the KRN preclinical model of rheumatoid arthritis. We find that IDO2, not IDO1, is critical for arthritis development, providing direct evidence of separate in vivo functions for IDO1 and IDO2. Mice null for Ido2 display decreased joint inflammation relative to wild-type mice owing to a reduction in pathogenic autoantibodies and Ab-secreting cells. Notably, IDO2 appears to specifically mediate autoreactive responses, but not normal B cell responses, as total serum Ig levels are not altered and IDO2 knockout mice are able to mount productive Ab responses to model Ags in vitro and in vivo. Reciprocal adoptive transfer studies confirm that autoantibody production and arthritis are modulated by IDO2 expression in a cell type extrinsic to the T cell. Taken together, our results, provide important insights into IDO2 function by defining its pathogenic contributions to autoantibody-mediated autoimmunity.

Novel Phosphoinositide 3-Kinaseδ,γInhibitor: Potent Anti-Inflammatory Effects and Joint Protection in Models of Rheumatoid Arthritis

Phosphoinositide 3-kinases γ and δ (PI3Kγ and PI3Kδ) are expressed in rheumatoid arthritis (RA) synovium and regulate innate and adaptive immune responses. We determined the effect of a potent PI3Kδ,γ inhibitor, IPI-145, in two preclinical models of RA. IPI-145 was administered orally in rat adjuvant-induced arthritis (AA) and intraperitoneally in mouse collagen-induced arthritis (CIA). Efficacy was assessed by paw swelling, clinical scores, histopathology and radiography, and microcomputed tomography scanning. Gene expression and Akt phosphorylation in joint tissues were determined by quantitative real-time polymerase chain reaction and Western blot analysis. Serum concentrations of anti-type II collagen (CII) IgG and IgE were measured by immunoassay. T-cell responses to CII were assayed using thymidine incorporation and immunoassay. IPI-145 significantly reduced arthritis severity in both RA models using dosing regimens initiated before onset of clinical disease. Treatment of established arthritis with IPI-145 in AA, but not CIA, significantly decreased arthritis progression. In AA, histology scores, radiographic joint damage, and matrix metalloproteinase (MMP)-13 expression were reduced in IPI-145-treated rats. In CIA, joint histology scores and expression of MMP-3 and MMP-13 mRNA were lower in the IPI-145 early treatment group than in the vehicle group. The ratio of anti-CII IgG2a to total IgG in CIA was modestly reduced. Interleukin-17 production in response to CII was decreased in the IPI-145-treated group, suggesting an inhibitory effect on T-helper cell 17 differentiation. These data show that PI3Kδ,γ inhibition suppresses inflammatory arthritis, as well as bone and cartilage damage, through effects on innate and adaptive immunity and that IPI-145 is a potential therapy for RA.

PLGA microspheres encapsulating siRNA anti-TNFalpha: Efficient RNAi-mediated treatment of arthritic joints

The aim of this study was to investigate potentialities of poly(dl-lactide–co-glycolide) (PLGA) microspheres for the delivery of small interfering RNAs (siRNAs) against tumor necrosis factor α (TNF-α) to achieve prolonged and efficient inhibition of TNF-α for the treatment of rheumatoid arthritis (RA). PLGA microspheres were prepared by a modified multiple emulsion–solvent evaporation method. The formulations were characterized in terms of morphology, mean diameter and siRNAs distribution, encapsulation efficiency, and in vitro release kinetics. The efficiency of this system was then evaluated both in vitro and in vivo using the murine monocytic cell line J774 and a pre-clinical model of RA, respectively. siRNA-encapsulating PLGA microspheres were characterized by a high encapsulation efficiency and a slow and prolonged anti-TNF-α siRNAs. Our results provide evidence that, upon intra-articular administration, PLGA microspheres slowly releasing siRNAs effectively inhibited the expression of TNF-α in arthritic joints. Our system might represent an alternative strategy for the design of novel anti-rheumatic therapies based on the use of RNA interference in RA.

Mesenchymal stem cells are conditionally therapeutic in preclinical models of rheumatoid arthritis

ObjectiveThe role of mesenchymal stem cells (MSC) in experimental arthritis is undoubtedly conflicting. This study explored the effect of bone marrow-derived MSC in previously untested and pathogenetically different models of...

RNAi-mediated gene silencing in inflammatory monocytes for efficient immuno-intervention in experimental arthritis

Background and objectivesThe inflammatory monocyte subset is essential in innate immunity but it also contributes to the pathogenesis of many inflammatory and autoimmune disorders including rheumatoid arthritis (RA). Strategies to...

Toward understanding the role of leptin and leptin receptor antagonism in preclinical models of rheumatoid arthritis

A potential link between obesity, circulating leptin levels and autoimmune disease symptoms suggests that targeting the leptin receptor (ObR) might be a viable novel strategy to combat rheumatoid arthritis (RA). However, studies in animal models and evaluation of clinical cases did not provide clear view on leptin's involvement in RA. To validate ObR as RA target, we used our peptide-based ObR agonists and antagonist in different in vitro and in vivo models of the disease. In human peripheral blood mononuclear cells, leptin and its agonist fragment, desI2-E1/Aca, moderately induced constitutive activation of a major proinflammatory transcription factor, NF-κB, while the ObR antagonist peptide Allo-aca inhibited the process. Leptin administration itself did not induce arthritis in rats, but worsened the clinical condition of mice given K/BxN serum transfer arthritis. Simultaneous administration of Allo-aca reduced leptin-dependent increase in disease severity by more than 50%, but the antagonist was ineffective when injected with a 3-day delay. In rats inflicted with mild adjuvant-induced arthritis, both leptin and Allo-aca reduced the extent of joint swelling and the number of arthritic joints. In a more aggressive disease stage, Allo-aca decreased the number of arthritic joints in a dose-dependent manner but did not affect other arthritis markers. In summary, leptin exerts diverse effects on RA depending on the experimental model. This might reflect the heterogeneous character of RA, which is differently impacted by leptin and is unmasked by ObR antagonism. Nevertheless, the results suggest that ObR antagonists might become useful therapeutics in leptin-sensitive early stages of RA.

Imidazo[4,5-d]thiazolo[5,4-b]pyridine based inhibitors of IKK2: Synthesis, SAR, PK/PD and activity in a preclinical model of rheumatoid arthritis

The synthesis, structure–activity relationships (SAR) and biological evaluation of thiazole based tricyclic inhibitors of IKK2 are described. Compound 9 was determined to be orally efficacious in a murine model of rheumatoid arthritis.