Mouse Model Of Arthritis Research Articles

NFIC attenuates rheumatoid arthritis-induced inflammatory response in mice by regulating PTEN/SENP8 transcription

ObjectiveTo explore whether nuclear factor I C (NFIC) alleviated inflammatory response of synovial fibroblasts (SFs) caused by rheumatoid arthritis (RA) by regulating transcription levels of phosphatase and tension homolog deleted on chromosome 10 (PTEN) and sentrin-specific protease 8 (SENP8). MethodsNFIC, PTEN, and SENP8 levels in RASFs and normal SFs (NSFs) were measured by qRT-PCR and western blotting. The levels of Bax, Bcl-2, MMP-3, and MMP-13, as well as the content of superoxide dismutase (SOD) and malondialdehyde (MDA) were determined in RASFs and NSFs using western blotting and ELISA. The binding of NFIC to promoter sequences of PTEN and SENP8 was predicted and verified. A mouse model of collagen-induced arthritis (CIA) was established and evaluated according to the degree of joint swelling and arthritis index. ResultsNFIC, PTEN, and SENP8 were downregulated in RASFs. RASFs had increased viability and MDA levels as well as decreased cell apoptosis and SOD content. NFIC was demonstrated to modulate the transcription of PTEN and SENP8 as their transcription factor. NFIC ameliorated the inflammatory response induced by RA in vivo by promoting the transcription of PTEN and SENP8. ConclusionNFIC acted as a transcription factor to facilitate the transcription of PTEN and SENP8, thereby inducing apoptosis of RASFs and effectively attenuating inflammatory response in CIA mice.

Bifidobacterium pseudocatenulatum-Mediated Bile Acid Metabolism to Prevent Rheumatoid Arthritis via the Gut–Joint Axis

Early intervention in rheumatoid arthritis (RA) is critical for optimal treatment, but initiation of pharmacotherapy to prevent damage remains unsatisfactory currently. Manipulation of the gut microbiome and microbial metabolites can be effective in protecting against RA. Thus, probiotics can be utilized to explore new strategies for preventing joint damage. The aim of this study was to explore the metabolites and mechanisms by which Bifidobacterium pseudocatenulatum affects RA. Based on 16S rRNA sequencing and UPLC-MS/MS assays, we focused on bile acid (BA) metabolism. In a collagen-induced arthritis (CIA) mouse model, B. pseudocatenulatum prevented joint damage by protecting the intestinal barrier and reshaped gut microbial composition, thereby elevating bile salt hydrolase (BSH) enzyme activity and increasing the levels of unconjugated secondary BAs to suppress aberrant T-helper 1/17-type immune responses; however, these benefits were eliminated by the Takeda G protein-coupled receptor 5 (TGR5) antagonist SBI-115. The results suggested that a single bacterium, B. pseudocatenulatum, can prevent RA, indicating that prophylactic administration of probiotics may be an effective therapy.

Injectable Decorin/Gellan Gum Hydrogel Encapsulating Adipose-Derived Stem Cells Enhances Anti-Inflammatory Effect in Cartilage Injury via Autophagy Signaling

Adipose-derived stem cells (ADSCs) are employed as a promising alternative in treating cartilage injury. Regulating the inflammatory “fingerprint” of ADSCs to improve their anti-inflammatory properties could enhance therapy efficiency. Herein, a novel injectable decorin/gellan gum hydrogel combined with ADSCs encapsulation for arthritis cartilage treatment is proposed. Decorin/gellan gum hydrogel was prepared according to the previous manufacturing protocol. The liquid–solid form transition of gellan gum hydrogel is perfectly suitable for intra-articular injection. Decorin-enriched matrix showing an immunomodulatory ability to enhance ADSCs anti-inflammatory phenotype under inflammation microenvironment by regulating autophagy signaling. This decorin/gellan gum/ADSCs hydrogel efficiently reverses interleukin-1β-induced cellular injury in chondrocytes. Through a mono-iodoacetate-induced arthritis mice model, the synergistic therapeutic effect of this ADSCs-loaded hydrogel, including inflammation attenuation and cartilage protection, is demonstrated. These results make the decorin/gellan gum hydrogel laden with ADSCs an ideal candidate for treating inflammatory joint disorders.

Therapeutic Potential of Catechin as an IKK-β Inhibitor for the Management of Arthritis: In vitro and In vivo Approach.

Rheumatoid arthritis (RA) is associated with increased levels of cytokines, for instance, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and interleukin-1 (IL-1), which exhibit potent pro-inflammatory effects and are contributing factors to disease progression. A range of cytokines, cell adhesion molecules, and enzymes that are implicated in the debilitating effects of RA are transcribed by nuclear factor kappa. The purpose of this research was to characterize the efficacy of "catechin" as an IkappaB kinase-beta (IKK-β) inhibitor in collagen-induced arthritis (CIA) model in mice, as IKK-β is crucial in the transmission of signal-inducible NF-κβ activation. Arthritis was brought on in Bagg and Albino, but it is written BALB/c (BALB/c) male mice through subcutaneous immunization with bovine type II collagen on days 0 and 21. Catechin is given orally every day after the onset of the disease. Clinical evaluation of the prevalence and severity of the condition was done throughout the trial, and biochemical testing was done at the end (day 42). In vitro findings of the study demonstrated catechin as a potent inhibitor of IKK-β with Half maximal Inhibitory Concentration (IC50) values of 2.90 μM and 4.358 μM in IKK-β and NF-κβ transactivation activity assay, respectively. Furthermore, catechin (dose range of 10-100 mg/kg, p.o.) was effective in reducing disease incidence and clinical signs in a dose-dependent manner, with an Effective Dose for 50% of the population (ED50) value of 79.579 mg/kg. The findings of this study demonstrate dose-dependent efficacy in terms of both disease severity (clinical scoring) and inflammatory markers (biochemical evaluation of the serum and joints). IKK inhibitors are a prospective target for the creation of new therapeutics for arthritis and other inflammatory diseases because it has been suggested that this enzyme is crucial in the pathophysiology of RA. The finding of this study suggests that "catechin" represents a novel inhibitor of IKK-β with promising anti-inflammatory activity.

Intra-articular delivery of AAV vectors encoding PD-L1 attenuates joint inflammation and tissue damage in a mouse model of rheumatoid arthritis

Intra-articular delivery of AAV vectors encoding PD-L1 attenuates joint inflammation and tissue damage in a mouse model of rheumatoid arthritis

Tea polyphenol carrier-enhanced dexamethasone nanomedicines for inflammation-targeted treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by synovial inflammation, cartilage damage and bone erosion. In the progression of RA, the inflammatory mediators including ROS, NO, TNF-α, and IL-6 play important roles in the aggravation of inflammation. Hence, reducing the generation and release of inflammatory mediators is of great importance. However, the high dose and frequent administration of clinical anti-inflammatory drugs such as glucocorticoids (GCs) usually lead to severe side effects. The development of nanotechnology provides a promising strategy to overcome these issues. Here, polyphenol-based nanoparticles with inherent anti-oxidative and anti-inflammatory activities were developed and used as a kind of nanocarrier to deliver dexamethasone (Dex). The in vitro experiments confirmed that the nanoparticles and drugs could act synergistically for suppressing inflammatory mediators in the LPS/INF-γ-induced inflammatory cell model. After intravenous administration, the Dex-loaded nanoparticles with good biosafety showed effective accumulation in inflamed joints and improved therapeutic efficacy by inducing anesis of synovial inflammation and cartilage destruction over free Dex in a collagen-induced arthritis (CIA) mouse model. The results demonstrated that polyphenol-based nanoparticles with therapeutic functions may serve as an innovative platform to synergize with chemotherapeutic agents for enhanced treatment of inflammatory diseases.

Bone Marrow Mesenchymal Stem Cells Release miR-378a-5p-Carried Extracellular Vesicles to Alleviate Rheumatoid Arthritis

This study investigates whether bone marrow mesenchymal stem cell (BMSC)-derived extracellular vesicles (EVs) can affect rheumatoid arthritis (RA) by delivering microRNA (miR)-378a-5p to regulate the interferon regulatory factor 1/signal transducer and transcription 1 (IRF1/STAT1) axis. We identified RA-associated miRNAs using the GEO microarray dataset GSE121894. We found the most important miRNAs in RA synovial tissues using RT-qPCR. BMSC-derived EVs were ultracentrifuged and cocultured with human synovial microvascular endothelial cells (HSMECs) in vitro. Dual-luciferase and RNA immunoprecipitation studies examined miR-378a-5p’s specific binding to IRF1. We also measured angiogenesis, migration, and proliferation using CCK-8, Transwell, and tube formation assays. Collagen-induced arthritis (CIA) mice models were created by inducing arthritis and scoring it. RA synovial tissues had low miR-378a-5p expression, whereas BMSC-derived EVs had high levels. The transfer of miR-378a-5p by BMSC-derived EVs to HSMECs boosted proliferation, migration, and angiogenesis. miR-378a-5p inhibited IRF1. MiR-378a-5p-containing BMSC-derived EVs decreased STAT1 phosphorylation and HSMEC IRF1 expression. EVs with miR-378a-5p mimic promoted HSMEC proliferation, migration, and angiogenesis, whereas dexmedetomidine inhibited STAT1 phosphorylation. In CIA mice, BMSC-derived EVs containing miR-378a-5p enhanced synovial vascular remodeling and histopathology. Thus, miR-378a-5p from BMSC-derived EVs promotes HSMEC proliferation, migration, and angiogenesis, inactivating the IRF1/STAT1 axis and preventing RA.

Construction and evaluation of a clinically relevant model of septic arthritis.

Despite the creation of several experimental animal models for the study of septic arthritis, a protocol detailing the development of a reliable and easily reproducible animal model has not yet been reported. The experimental protocol described herein for the development of a clinically relevant mouse model of septic arthritis includes two main study stages: the first stage consisting of the preparation of the mice and of the methicillin-resistant Staphylococcus aureus (MRSA) cultures, followed by direct inoculation of MRSA into the knee joints of C57BL/6J mice (25-40 min); and a second study stage consisting of multiple sample collection and data analysis (1-3 days). This protocol may be carried out by researchers skilled in mouse care and trained to work with biosafety-level-2 agents such as MRSA. The model of septic arthritis described here has demonstrated clinical relevance in developing intra-articular inflammation and cartilage destruction akin to that of human patients. Moreover, we describe methods for serum, synovial fluid and knee joint tissue analysis that were used to confirm the development of septic arthritis in this model, and to test potential treatments. This protocol confers the advantages of enabling granular evaluation of the pathophysiology of MRSA infection and of the efficacy of therapeutic medications; it may also be employed to study a range of native joint diseases beyond inflammatory pathologies alone.

Ninjin'yoeito suppressed the onset of arthritis, pain, and muscle atrophy in rheumatoid arthritis model mice.

Rheumatoid arthritis is one of the most common diseases in orthopedic surgery. The main symptoms are joint pain and systemic symptoms. In recent years, rheumatoid arthritis is known to cause sarcopenia. Ninjin'yoeito (NYT), a traditional Japanese medicine, has been prescribed for patients with post-illness or post-operative weakness, fatigue, loss of appetite, rash, cold limbs, and anemia. In addition to its traditional use, NYT has been prescribed for treating frailty in gastrointestinal, respiratory, and urinary functions. Further, NYT is known to be effective in suppressing muscle atrophy in the prior literature. The present study aimed to investigate whether NYT suppresses various symptoms of the Collagen-induced arthritis (CIA) model. Long-term administration of NYT inhibited the increases in arthritis scores, decreases pain threshold, and muscle atrophy in the CIA model. In addition, NYT inhibited the elevation of the plasma IL-6 level. These results suggest that NYT may have therapeutic effects on symptoms, muscle atrophy and increase in plasma IL-6 level caused by rheumatoid arthritis.

Activation of angiotensin II type 2 receptor attenuates lung injury of collagen-induced arthritis by alleviating endothelial cell injury and promoting Ly6Clo monocyte transition

As one of the most frequent extra-articular manifestations of rheumatoid arthritis (RA), interstitial lung disease (ILD) is still challenging due to unrevealed pathophysiological mechanism. To address this question, in the present study, we used the classical collagen-induced arthritis (CIA) mouse model to determine the related-immune mechanism of lung injury and possible pharmacological treatment for RA-ILD. At the peak of arthritis, we found CIA mice developed apparent lung injury, characterized by interstitial thickening, inflammatory cell infiltration, and lymphocyte follicle formation. Additionally, the endothelial injury occurred as the number of endothelial cells (ECs) and their CD31 expression decreased. Along with those, monocytes, predominantly Ly6Chi monocytes with pro-inflammatory phenotype, were also increased. While in the remission period of arthritis, ECs gradually increased with retrieved CD31 expression, leading to decreased infiltrating monocytes, but boosted Ly6Clo population. Ly6Clo monocytes were prone to locate around damaged ECs, promoted ECs proliferation and vascular tube formation, and lessened the expression of adhesion molecules. In addition, we evaluated angiotensin II type 2 receptor (Agtr2), which has been demonstrated to be protective against lung injury, could be beneficial in RA-ILD. We found elevated Agtr2 in CIA lung tissue, and activation of Agtr2, within its specific agonist C21, alleviated the pulmonary inflammation in vivo, reduced ECs injury, and promoted monocytes conversion from Ly6Chi to Ly6Clo monocytes in vitro. Our data reveal a potential pathological mechanism of RA-ILD that involves ECs damage and inflammatory monocytes infiltration and provide a potential drug target, Agtr2, for RA-ILD treatment.

Chemically modified small interfering RNA targeting Hedgehog signaling pathway for rheumatoid arthritis therapy

Rheumatoid arthritis (RA) is an inflammatory disease that leads to disability; however, existing therapies are still unsatisfactory. Activated fibroblast-like synoviocytes (FLSs) play an essential role in synovitis formation and joint destruction in RA. The Hedgehog signaling pathway is aberrantly activated and contributes to the aggressive phenotype of RA-FLSs. However, it remains uncertain whether inhibiting Smoothened (SMO), a critical component of the Hedgehog signaling pathway, is an effective treatment for RA. Here, we design a series of small interfering RNAs (siRNAs) that specifically target the SMO gene. With precise chemical modifications, siRNAs' efficacy and stability are significantly improved, and the off-target effects are minimized. The optimized chemically modified siRNA (si-S1A3-Chol) decreases RA-FLS proliferation and invasiveness without the transfection reagent. Furthermore, si-S1A3-Chol injected intra-articularly effectively alleviates joint destruction and improves motor function in collagen-induced arthritis mouse models. Consequently, our results demonstrate that chemically modified siRNA targeting the Hedgehog signaling pathway may be a potential therapy for RA.

Metabolomic Profiling of Red Blood Cells to Identify Molecular Markers of Methotrexate Response in the Collagen Induced Arthritis Mouse Model

Although methotrexate (MTX) is the first line disease-modifying therapy used in the treatment of autoimmune arthritis, it is limited by its unpredictable and variable response profile and lack of therapeutic biomarkers to predict or monitor therapeutic response. The purpose of this work is to evaluate the utility of red blood cell (RBC) metabolite profiles to screen for molecular biomarkers associated with MTX response. Methods: Utilizing the collagen-induced arthritis mouse model, DBA/1J mice were treated with subcutaneous MTX (20 mg/kg/week) and RBC samples were collected and analyzed by semi-targeted global metabolomic profiling and analyzed by univariate analysis. Results: MTX treatment normalized the following RBC metabolite levels that were found to be altered by disease induction: N-methylisoleucine, nudifloramide, phenylacetylglycine, 1-methyl-L-histidine, PC 42:1, PE 36:4e, PC 42:3, PE 36:4e (16:0e/20:4), and SM d34:0. Changes in the RBC metabolome weakly but significantly correlated with changes in the plasma metabolome following MTX treatment (ρ = 0.24, p = 1.1 × 10−13). The RBC metabolome resulted in the detection of nine significant discriminatory biomarkers, whereas the plasma metabolome resulted in two. Overall, the RBC metabolome yielded more highly sensitive and specific biomarkers of MTX response compared to the plasma metabolome. N-methylisoleucine was found to be highly discriminatory in both plasma and RBCs. Conclusions: Our results suggest that RBCs represent a promising biological matrix for metabolomics and future studies should consider the RBC metabolome in their biomarker discovery strategy.

Dysbiotic but nonpathogenic shift in the fecal mycobiota of patients with rheumatoid arthritis

ABSTRACT Rheumatoid arthritis (RA) is closely associated with the oral and gut microbiomes. Fungal cell wall components initiate inflammatory arthritis in mouse models. However, little is known regarding the role of the fungal community in the pathogenesis of RA. To evaluate the association between RA and the gut microbiome, investigations of bacterial and fungal communities in patients with RA are necessary. Therefore, we investigated the compositions and associations of fecal bacterial and fungal communities in 30 healthy controls and 99 patients with RA. The relative abundances of Bifidobacterium and Blautia decreased, whereas the relative abundance of Streptococcus increased, in patients with RA. The relative abundance of Candida in the fecal fungal community was higher in patients with RA than in healthy controls, while the relative abundance of Aspergillus was higher in healthy controls than in patients with RA. Candida species-specific gene amplification showed that C. albicans was the most abundant species of Candida. Ordination analysis and random forest classification models supported the findings of structural changes in bacterial and fungal communities. Aspergillus was the core fecal fungal genus in healthy controls, although Saccharomyces spp. are typically predominant in Western cohorts. In addition, bacterial–fungal association analyses showed that the hub node had shifted from fungi to bacteria in patients with RA. The finding of fungal dysbiosis in patients with RA suggests that fungi play critical roles in the fecal microbial communities and pathogenesis of RA.

Inhibitory effects of Ganoderma lucidum spore oil on rheumatoid arthritis in a collagen-induced arthritis mouse model

Holistic healthcare practitioners have now started to focus on specific traditional medicinal mushrooms to treat rheumatoid arthritis (RA). Ganoderma lucidum (GL) is one of the oldest mushrooms that have been used in ancient Chinese medicine to treat inflammatory ailments, including autoimmune diseases such as RA. Spores from this mushroom have specific effects on immunomodulation, aging, and cancer. However, the effect of G. lucidum spores (GLS) on arthritis remains unclear. Therefore, we investigated the effects of GLS oil in a collagen-induced rheumatoid arthritis (CIA) model. Metabolomics analysis revealed that GLS oil contains ten acids, of which oleic acid (52.12%) and linoleic acid (16.77%) predominated. The GLS oil-treated CIA mice had a significantly lower clinical score (p = 0.0384) for RA than the control CIA mice. Moreover, GLS oil reduced CIA-induced cartilage degeneration and synovial membrane inflammation in the knee. The GLS oil group showed significantly reduced knee eosinophilia (p = 0.0056). Immunostaining of neutrophils revealed that neutrophils infiltrated the CIA group; however, infiltrated neutrophils were significantly reduced in the GLS oil group in both the knees (p = 0.0006) and ankles (p = 0.0023). GLS oil treatment substantially suppressed LPS- or TNF-α–induced IL-6 mRNA expression in primary cultured chondrocytes. IL-6 immunohistochemistry results showed that the protein levels of IL-6 were attenuated in the GLS oil group compared to the CIA group. These findings suggest that GLS oil may be useful for the development of RA drugs. Further clinical research is required to identify significant improvements.

Artemisitene suppresses rheumatoid arthritis progression via modulating METTL3-mediated N6-methyladenosine modification of ICAM2mRNA in fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is a chronic autoimmune disease. We previously revealed that the natural compound artemisitene (ATT) exhibits excellent broad anticancer activities without toxicity on normal tissues. Nevertheless, the effect of ATT on RA is undiscovered. Herein, we aim to study the effect and potential mechanism of ATT on RA management. A collagen-induced arthritis (CIA) mouse model was employed to confirm the anti-RA potential of ATT. Cell Counting Kit-8(CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays, cell cycle and apoptosis analysis, immunofluorescence, migration and invasion assays, quantitative real-time PCR (RT-qPCR), Western blot, RNA-sequencing (RNA-seq) analysis, plasmid construction and lentivirus infection, and methylated RNA immunoprecipitation and chromatin immunoprecipitation assays, were carried out to confirm the effect and potential mechanism of ATT on RA management. ATT relieved CIA in mice. ATT inhibited proliferation and induced apoptosis of RA-fibroblast-like synoviocytes (FLSs). ATT restrained RA-FLSs migration and invasion via suppressing epithelial-mesenchymal transition. RNA-sequencing analysis and bioinformatics analysis identified intercellular adhesion molecule 2 (ICAM2) as a promoter of RA progression in RA-FLSs. ATT inhibits RA progression by suppressing ICAM2/phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/p300 pathway in RA-FLSs. Moreover, ATT inhibited methyltransferase-like 3 (METTL3)-mediated N6-methyladenosine methylation of ICAM2 mRNA in RA-FLSs. Interestingly, p300 directly facilitated METTL3 transcription, which could be restrained by ATT in RA-FLSs. Importantly, METTL3, ICAM2 and p300 expressions in synovium tissues of RA patients were related to clinical characteristics and therapy response. We provided strong evidence that ATT has therapeutic potential for RA management by suppressing proliferation, migration and invasion, in addition to inducing apoptosis of RA-FLSs through modulating METTL3/ICAM2/PI3K/AKT/p300 feedback loop, supplying the fundamental basis for the clinical application of ATT in RA therapy. Moreover, METTL3, ICAM2 and p300 might serve as biomarkers for the therapy response of RA patients.

Pro-resolving and anti-arthritic properties of the MC1 selective agonist PL8177.

Melanocortins are peptides endowed with anti-inflammatory and pro-resolving activities. Many of these effects are mediated by the Melanocortin receptor 1 (MC1) as reported in several experimental settings. As such, MC1 can be a viable target for the development of new therapies that mimic endogenous pro-resolving mediators. The aim of this study was to assess the immunopharmacology of a selective MC1 agonist (PL8177) in vitro and in a mouse model of inflammatory arthritis. PL8177 and the natural agonist αMSH were tested for activation of mouse and human Melanocortin receptors (MC1,3,4,5), monitoring cAMP accumulation and ERK1/2 phosphorylation, using transiently transfected HEK293A cells. The anti-inflammatory and pro-resolving effects of PL8177 and αMSH were evaluated using mouse peritoneal Macrophages. Finally, a model of K/BxN serum transfer induced arthritis was used to determine the in vivo potential of PL8177. PL8177 activates mouse and human MC1 with apparent EC50 values of 0.01 and 1.49 nM, respectively, using the cAMP accumulation assay. Similar profiles were observed for the induction of ERK phosphorylation (EC50: 0.05 and 1.39 nM). PL8177 displays pro-resolving activity (enhanced Macrophage efferocytosis) and counteracts the inflammatory profile of zymosan-stimulated macrophages, reducing the release of IL-1β, IL-6, TNF-α and CCL-2. In the context of joint inflammation, PL8177 (3mg/kg i.p.) reduces clinical score, paw swelling and incidence of severe disease as well as the recruitment of immune cells into the arthritic joint. These results demonstrate that the MC1 agonism with PL8177 affords therapeutic effects in inflammatory conditions including arthritis. Drugs targeting the Melanocortin system have emerged as promising therapeutics for several conditions including inflammation or obesity. Multiple candidates are under clinical development, and some have already reached approval. Here we present the characterization of a novel drug candidate, PL8177, selective for the Melanocortin 1 receptor (MC1), demonstrating its selectivity profile on cAMP and ERK1/2 phosphorylation signaling pathways, of relevance as selective drugs will translate into lesser off-target effect. PL8177 also demonstrated, not only anti-inflammatory activity, but pro-resolving actions due to its ability to enhance efferocytosis (i.e. the phagocytosis of apoptotic cells), endowing this molecule with therapeutic advantages compared to classical anti-inflammatory drugs. Using a mouse model of inflammatory arthritis, the compound demonstrated in vivo efficacy by reducing clinical score, paw swelling and overall disease severity. Taken together, these results present Melanocortin-based therapies, and specifically targeting MC1 receptor, as a promising strategy to manage chronic inflammatory diseases.

Suppression of Autoimmune Rheumatoid Arthritis with Hybrid Nanoparticles That Induce B and T Cell Tolerance to Self-Antigen.

Autoimmune diseases affect over 4% of the world's population. Treatments are generally palliative or use broad spectrum immunosuppressants to reduce symptoms and disease progression. In some diseases, antibodies generated to a single autoantigen are the major cause of pathogenic inflammation, suggesting that treatments to induce tolerance to the autoantigen could be therapeutic. Here we report the development of hybrid nanoparticles (NPs) that induce tolerance in both T cells and B cells. The NPs comprise a lipid monolayer encapsulating a PLGA core loaded with rapamycin that promotes development of regulatory T cells (Tregs). The lipid monolayer displays the protein antigen and a ligand of the B cell inhibitory co-receptor CD22 (CD22L) that act together to suppress activation of B cells recognizing the antigen. We demonstrate that the hybrid NPs decorated with ovalbumin (OVA) elicit tolerance to OVA in naı̈ve mice, as judged by low OVA-specific antibody titers after the challenge. In the K/BxN mouse model of rheumatoid arthritis caused by B and T cell-dependent responses to the self-antigen glucose-6-phosphate-isomerase (GPI), we show that GPI hybrid NPs delay development of disease, with some treated mice remaining arthritis-free for 300 days. We provide evidence that the mechanism of rheumatoid arthritis suppression involves induction of B cell tolerance, as measured by low anti-GPI antibodies and decreased plasma cell populations, and T cell tolerance, as measured by increased Tregs. The results show the potential of this versatile NP platform for inducing immune tolerance to a self-antigen and suppressing autoimmune disease.

The Therapeutic Effect of Phosphopeptide P140 Attenuates Inflammation Induced by Uric Acid Crystals in Gout Arthritis Mouse Model

Gout is a painful form of inflammatory arthritis characterized by the deposition of monosodium urate (MSU) crystals in the joints. The aim of this study was to investigate the effect of peptide P140 on the inflammatory responses in crystal-induced mouse models of gout and cell models including MSU-treated human cells. Injection of MSU crystals into the knee joint of mice induced neutrophil influx and inflammatory hypernociception. Injection of MSU crystals subcutaneously into the hind paw induced edema and increased pro-inflammatory cytokines levels. Treatment with P140 effectively reduced hypernociception, the neutrophil influx, and pro-inflammatory cytokine levels in these experimental models. Furthermore, P140 modulated neutrophils chemotaxis in vitro and increased apoptosis pathways through augmented caspase 3 activity and reduced NFκB phosphorylation. Moreover, P140 increased the production of the pro-resolving mediator annexin A1 and decreased the expression of the autophagy-related ATG5-ATG12 complex and HSPA8 chaperone protein. Overall, these findings suggest that P140 exerts a significant beneficial effect in a neutrophilic inflammation observed in the model of gout that can be of special interest in the design of new therapeutic strategies.

Discovery of ABBV-3373, an Anti-TNF Glucocorticoid Receptor Modulator Immunology Antibody Drug Conjugate.

Using a convergent synthetic route to enable multiple points of diversity, a series of glucocorticoid receptor modulators (GRM) were profiled for potency, selectivity, and drug-like properties in vitro. Despite covering a large range of diversity, profiling the nonconjugated small molecule was suboptimal and they were conjugated to a mouse antitumor necrosis factor (TNF) antibody using the MP-Ala-Ala linker. Screening of the resulting antibody drug conjugates (ADCs) provided a better assessment of efficacy and physical properties, reinforcing the need to conduct structure-activity relationship studies on the complete ADC. DAR4 ADCs were screened in an acute mouse contact hypersensitivity model measuring biomarkers to ensure a sufficient therapeutic window. In a chronic mouse arthritis model, mouse anti-TNF GRM ADCs were efficacious after a single dose of 10 mg/kg i.p. for over 30 days. Data on the unconjugated payloads and mouse surrogate anti-TNF ADCs identified payload 17 which was conjugated to a human anti-TNF antibody and advanced to the clinic as ABBV-3373.

Contribution of MicroRNA-27b-3p to Synovial Fibrotic Responses in Knee Osteoarthritis.

Synovial fibrosis contributes to osteoarthritis (OA) pathology, but the underlying mechanisms remain unknown. We have observed increased microRNA-27b-3p (miR-27b-3p) levels in synovial fluid of patients with late-stage radiographic knee OA. Here, we investigated the contribution of miR-27b-3p to synovial fibrosis in patients with severe knee OA and in a mouse model of knee OA. We stained synovium sections obtained from patients with radiographic knee OA scored according to the Kellgren/Lawrence scale and mice that underwent destabilization of the medial meniscus (DMM) for miR-27b-3p using in situ hybridization. We examined the effects of intraarticular injection of miR-27b-3p mimic into naive mouse knee joints and intraarticular injection of a miR-27b-3p inhibitor into mouse knee joints after DMM. We performed transfection with miR-27b-3p mimic and miR-27b-3p inhibitor in human OA fibroblast-like synoviocytes (FLS) using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) array, RNA sequencing, RT-qPCR, Western blotting, immunofluorescence, and migration assays. We observed increased miR-27b-3p expression in the synovium from patients with knee OA and in mice with DMM-induced arthritis. Injection of the miR-27b-3p mimic in mouse knee joints induced a synovial fibrosis-like phenotype, increased synovitis scores, and increased COL1A1 and α-smooth muscle actin (α-SMA) expression. In the mouse model of DMM-induced arthritis, injection of the miR-27b-3p inhibitor decreased α-SMA but did not change COL1A1 expression levels or synovitis scores. Transfection with the miR-27b-3p mimic in human OA FLS induced profibrotic responses, including increased migration and expression of key extracellular matrix (ECM) genes, but transfection with the miR-27b-3p inhibitor had the opposite effects. RNA sequencing identified a PPARG/ADAMTS8 signaling axis regulated by miR-27b-3p in OA FLS. Human OA FLS transfected with miR-27b-3p mimic and then treated with the PPARG agonist rosiglitazone or with ADAMTS8 small interfering RNA exhibited altered expression of select ECM genes. Our findings demonstrate that miR-27b-3p has a key role in ECM regulation associated with synovial fibrosis during OA.