Role of estrogen signaling in fibroblastic reticular cells for innate and adaptive immune responses in antigen-induced arthritis.

Women are more prone to develop rheumatoid arthritis, with peak incidence occurring around menopause. Estrogen has major effects on the immune system and is protective against arthritis. We have previously shown that treatment with estrogen inhibits inflammation and joint destruction in murine models of arthritis, although the mechanisms involved remain unclear. Fibroblastic reticular cells (FRCs) are specialized stromal cells that generate the three-dimensional structure of lymph nodes (LNs). FRCs are vital for coordinating immune responses from within LNs and are characterized by the expression of the chemokine CCL19, which attracts immune cells. The aim of this study was to determine whether the influence of estrogen on innate and adaptive immune cells in arthritis is mediated by estrogen signaling in FRCs. Conditional knockout mice lacking estrogen receptor α (ERα) in CCL19-expressing cells (Ccl19-CreERαfl/fl) were generated and tested. Ccl19-CreERαfl/fl mice and littermate controls were ovariectomized, treated with vehicle or estradiol and subjected to the 28-day-long antigen-induced arthritis model to enable analyses of differentiated T- and B-cell populations and innate cells in LNs by flow cytometry. The results reveal that while the response to estradiol treatment in numbers of FRCs per LN is significantly reduced in mice lacking ERα in FRCs, estrogen does not inhibit joint inflammation or markedly affect immune responses in this arthritis model. Thus, this study validates the Ccl19-CreERαfl/fl strain for studying estrogen signaling in FRCs within inflammatory diseases, although the chosen arthritis model is deemed unsuitable for addressing this question.

Local Immune Activation and Age Impact on Humoral Immunity in Mice, with a Focus on IgG Sialylation.

Age alters the host's susceptibility to immune induction. Humoral immunity with circulating antibodies, particularly immunoglobulin G (IgG), plays an essential role in immune response. IgG glycosylation in the fragment crystallizable (Fc) region, including sialylation, is important in regulating the effector function by interacting with Fc gamma receptors (FcγRs). Glycosylation is fundamentally changed with age and inflammatory responses. We aimed to explore the regulation of humoral immunity by comparing responses to antigen-induced immune challenges in young and adult mice using a local antigen-induced arthritis mouse model. This study examines the differences in immune response between healthy and immune-challenged states across these groups. Our initial assessment of the arthritis model indicated that adult mice presented more severe knee swelling than their younger counterparts. In contrast, we found that neither histological assessment, bone mineral density, nor the number of osteoclasts differs. Our data revealed an age-associated but not immune challenge increase in total IgG; the only subtype affected by immune challenge was IgG1 and partially IgG3. Interestingly, the sialylation of IgG2b and IgG3 is affected by age and immune challenges but not stimulated further by immune challenges in adult mice. This suggests a shift in IgG towards a pro-inflammatory and potentially pathogenic state with age and inflammation.

Non-invasive management of rheumatoid arthritis using hollow microneedles as a tool for transdermal delivery of teriflunomide loaded solid lipid nanoparticles.

Conventional RA treatments required prolonged therapy courses that have been accompanied with numerous side effects impairing the patient's quality of life. Therefore, microneedles combined with nanotechnology emerged as a promising alternative non-invasive, effective and self-administrating treatment option. Hence, the main aim of this study is to reduce the side effects associated with systemic teriflunomide administration through its encapsulation in solid lipid nanoparticles (TER-SLNs) and their administration through transdermal route using AdminPen™ hollow microneedles array in the affected joint area directly. In vitro characterization studies were conducted including particle size, zeta potential, encapsulation efficiency and in vitro drug release. Also, ex vivo insertion properties of AdminPen™ hollow microneedles array was carried out. Besides, in vivo evaluation in rats with antigen induced arthritis model were also conducted by assessment of joint diameter, histopathological examination of the dissected joints and testing the levels of TNF-α, IL1B, IL7, MDA, MMP 3, and NRF2 at the end of the experiment. The selected TER-SLNs formulation was about 155.3 nm with negative surface charge and 96.45 % entrapment efficiency. TER-SLNs had a spherical shape and provided sustained release for nearly 96 h. In vivo results demonstrated that nanoencapsulation along with the use of hollow microneedles had a significant influence in improving TER anti-arthritic effects compared with TER suspension with no significant difference from the negative control group.

A COX-2 Inhibitor Does Not Interfere With the Bone-Protective Effects of Loading in Male Mice With Arthritis.

Mechanical loading enhances bone strength and counteracts arthritis-induced inflammation-mediated bone loss in female mice. It is unknown whether nonsteroidal anti-inflammatory drugs (NSAIDs; eg, COX-2 inhibitors) can reduce inflammation without affecting the loading-associated bone formation in male mice. The aim of this study was to investigate if loading combined with a COX-2 inhibitor (NS-398) could prevent arthritis-induced bone loss and inflammation in male mice. Four-month-old male C57BL/6J mice were subjected to axial tibial mechanical loading three times/week for 2 weeks. Local mono-arthritis was induced with a systemic injection of methylated bovine serum albumin on the first day of loading, followed by a local injection in one knee 1 week later. The arthritis induction, knee swelling, bone architecture, and osteoclast number were evaluated in the hind limbs. C-terminal cross-links as a marker for osteoclast activity was measured in serum. Compared with loading and arthritis alone, loading of the arthritic joint enhanced swelling that was partly counteracted by NS-398. Loading of the arthritic joint enhanced synovitis and articular cartilage damage compared with loading alone. Loading increased cortical bone and counteracted the arthritis-induced decrease in epiphyseal bone. NS-398 did not alter the bone-protective effects of loading. C-terminal cross-links, a bone resorption marker, was increased by arthritis but not loading. In conclusion, loading prevented arthritis-induced epiphyseal and metaphyseal bone loss, and NS-398 reduced knee swelling without affecting the bone-protective effects of loading. If our results can be extrapolated to the human situation, specific COX-2 inhibitors could be used in combination with loading exercise to prevent pain and swelling of the joint without influencing the bone-protective effects of loading. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Pathological pathway analysis in an experimental rheumatoid arthritis model and the tissue repair effect of acupuncture at ST36.

Rheumatoid arthritis (RA) is an autoimmune disease that generally affects the joints. In the face of inflammation-induced cartilage and bone damage, RA treatment remains insufficient. While research evidence indicates that acupuncture can exert anti-inflammatory and analgesic effects, improve the joint function of RA patients, and delay the disease, data on whether it can promote RA repair are lacking. Findings from the present work demonstrated that both the antigen-induced arthritis (AIA) and collagen-induced arthritis (CIA) models can simulate joint swelling of RA. The AIA model was more stable than the CIA model, with a higher incidence of successful arthritis modeling. Moreover, the AIA mice model could simulate the signal molecules and related pathological processes of the autoimmune response in RA, as well as major pathways related to RA and antigen immune response mechanisms. Manual acupuncture (MA) at Zusanli (ST36) significantly improved paw redness and swelling, pain, and inflammatory cell infiltration in the joints in AIA mice. The therapeutic effect of MA on AIA is achieved primarily through the regulation of steroid hormone biosynthesis, cell metabolism, and tissue repair processes. MA at ST36 can increase the gene contents of tissue repair growth factors, including PEG3, GADD45A, GDF5, FGF5, SOX2, and ATP6V1C2 in the inflammatory side joints of AIA mice, as well as the gene expression of the anti-inflammatory cytokine IL-10. In conclusion, acupuncture may alleviate RA in the joints via modulating the tissue healing process.

Fasciola hepatica extract suppresses fibroblast-like synoviocytes in vitro and alleviates experimental arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial inflammation, fibroblast-like synoviocytes (FLS) activation and joint destruction. Fasciola hepatica is a platyhelminth that releases excretory-secretory immunomodulatory products capable of suppressing the Th1 immune response. Despite the effectiveness of available treatments for inducing disease remission, current options are not successful in all patients and may cause side effects. Thus, we evaluated the therapeutic potential of F. hepatica extract on FLS from RA patients and arthritis models. METHODS: FLS were isolated from synovial fluid of RA patients, cultured, and exposed to F. hepatica extract (60, 80, and 100 µg/ml) for different time points to assess cell viability, adherence, migration and invasion. For in vivo experiments, mice with antigen (AIA) and collagen (CIA) induced arthritis received a 200 µg/dose of F. hepatica extract daily. Statistical analysis was performed by ANOVA and Student's t-test using GraphPad Prism 6.0. RESULTS: In vitro assays showed that extract decreased FLS cell viability at concentration of 100 µg/ml (83.8% ± 5.0 extract vs. 100.0% ± 0.0 control; p < 0.05), adherence in 20% (92.0 cells ± 5.8 extract vs. 116.3 cells ± 7.9 control; p < 0.05), migratory potential (69.5% ± 17.6 extract vs. 100.0% control; p < 0.05), and cell invasiveness potential through the matrigel (76.0% ± 8.4 extract vs. 100.0% control; p < 0.01). The extract reduced leukocyte migration by 56% (40 × 104 leukocytes/knee ± 19.00) compared to control (90.90 × 104 leukocytes/knee ± 12.90) (p < 0.01) and nociception (6.37 g ± 0.99 extract vs. 3.81 g ± 1.44 control; p < 0.001) in AIA and delayed clinical onset of CIA (11.75 ± 2.96 extract vs. 14.00 ± 2.56 control; p = 0.126). CONCLUSION: Our results point out a potential immunomodulatory effect of F. hepatica extract in RA models. Therefore, the characterization of promising new immunomodulatory molecules should be pursued, as they can promote the development of new therapies. Trial registration Collection of synovial liquid and in vitro procedures were approved by the Ethics Committee with Certificate of Presentation of Ethical Appreciation in Plataforma Brasil (CAAE: 89044918.8.0000.5327; date of registration: 26/07/2018).

The Effects of Intra-articular Platelet-Rich Plasma Injections in Rheumatoid Arthritis: A Narrative Review.

Platelet-rich plasma injections have been a therapeutic option with exponential growth in several pathologies in the last decades, particularly musculoskeletal for their effect on improving pain and functionality. Rheumatoid arthritis is a chronic joint disease, which involves inflammation of the synovial membrane with cartilage and juxta-articular bone destruction. Conventional and biological disease-modifying anti-rheumatoid drugs are the cornerstone of the treatment of this disease. However, the use of intra-articular glucocorticoids is often necessary and the role of platelet-rich plasma injections in these patients remains uncertain. A literature review was carried out through the PubMed database, Cochrane and Google Scholar for the search terms "rheumatoid arthritis" and "platelet-rich plasma". Eleven studies have been included in this review: two of these are in vitro studies, five are animal studies, one case report, two case series and one randomized controlled trial. Most of the studies demonstrated a decrease in pain and inflammatory mediators and improvement of functional outcomes, with no severe adverse effects reported. However, the quantity and quality of literature about the effects and safety of plasma-rich plasma injections in rheumatoid arthritis patients are still scarce. It is essential that well-designed randomized controlled trials are made on this topic to understand if platelet-rich plasma may be useful as a coadjuvant therapy in rheumatoid arthritis.

Folate Receptor Beta for Macrophage Imaging in Rheumatoid Arthritis.

Non-invasive imaging modalities constitute an increasingly important tool in diagnostic and therapy response monitoring of patients with autoimmune diseases, including rheumatoid arthritis (RA). In particular, macrophage imaging with positron emission tomography (PET) using novel radiotracers based on differential expression of plasma membrane proteins and functioning of cellular processes may be suited for this. Over the past decade, selective expression of folate receptor ß (FRß), a glycosylphosphatidylinositol-anchored plasma membrane protein, on myeloid cells has emerged as an attractive target for macrophage imaging by exploiting the high binding affinity of folate-based PET tracers. This work discusses molecular, biochemical and functional properties of FRß, describes the preclinical development of a folate-PET tracer and the evaluation of this tracer in a translational model of arthritis for diagnostics and therapy-response monitoring, and finally the first clinical application of the folate-PET tracer in RA patients with active disease. Consequently, folate-based PET tracers hold great promise for macrophage imaging in a variety of (chronic) inflammatory (autoimmune) diseases beyond RA.

Protective effect of methyl gallate on murine antigen-induced arthritis by inhibiting inflammatory process and bone erosion.

Methyl gallate (MG) is a plant-derived phenolic compound known to present remarkable anti-inflammatory effect in different experimental models, such as paw oedema, pleurisy, zymosan-induced arthritis and colitis. Herein we investigated the effect of MG in the mice model of antigen-induced arthritis (AIA), a model with complex inflammatory response, driven primally by immune process and that cause bone and cartilage erosion similarly found in rheumatoid arthritis. Arthritis was induced by intra-articular injection of albumin methylated from bovine serum (mBSA) in C57BL/6 male mice previously immunized. The dose-response analysis of MG (0.7-70 mg/kg; p.o) showed that maximum inhibition was reached with the dose of 7 mg/kg on paw oedema and cell infiltration induced by AIA at 7 h. Treatment with MG (7 mg/kg; p.o) or with the positive control, dexamethasone (Dexa, 10 mg/kg, ip) reduced AIA oedema formation, leukocyte infiltration, release of extracellular DNA and cytokine production 7 and 24 h (acute response). Mice treated daily with MG for 7 days showed no significant weight loss or liver and kidney toxicity contrary to dexamethasone that induced some degree of toxicity. Prolonged treatment with MG inhibited the late inflammatory response (28 days) reducing oedema formation, cell infiltration, synovial hyperplasia, pannus formation and cartilage degradation as observed in histopathological analyses. Ultimately, MG reduced bone resorption as evidenced by a decrease in tartrate-resistant acid phosphate (TRAP)-positive cells number in femur histology. Altogether, we demonstrate that MG ameliorates the inflammatory reaction driven primarily by the immune process, suggesting a potential therapeutic application in arthritis treatment.

Role of Suppressor of cytokine signaling 2 during the development and resolution of an experimental arthritis.

Rheumatoid arthritis(RA) is a debilitating chronic inflammatory disease. Suppressors of Cytokine Signaling(SOCS) proteins regulate homeostasis and pathogenesis in several diseases. The intersection between RA pathophysiology and SOCS2 is unclear. Herein, we investigated the roles of SOCS2 during the development of an experimental antigen-induced arthritis(AIA). In wild type mice, joint SOCS2 expression was reduced during AIA development. At the peak of inflammation, SOCS2-/- mice presented with reduced numbers of infiltrated cells in their joints. At the late phase of AIA, however, exhibited increased adhesion/infiltration of neutrophils, macrophages, CD4+-T cells, CD4+CD8+-T cells, and CD4-CD8--T cells associated with elevated IL-17 and IFN-γ levels, joint damage, proteoglycan loss, and nociception. SOCS2 deficiency resulted in lower numbers of apoptotic neutrophils and reduced efferocytosis. The present study demonstrated the vital role of SOCS2 during the development and resolution of an experimental RA model. Hence, this protein may be a novel therapeutic target for this disorder.

Toxic mechanisms of cigarette smoke and heat-not-burn tobacco vapor inhalation on rheumatoid arthritis.

Tobacco combustion exposure worsens rheumatoid arthritis (RA). Non-combustible tobacco devices, as heat-not-burn tobacco (HNBT), are emerging as harm reduction to smokers by releasing nicotine and lower combustible tobacco products. Nevertheless, HNBT toxicity remains unclear. Hence, here we investigated the impacts of the tobacco combustible product (cigarette smoke; CS) or HNBT vapor exposures on antigen-induced arthritis (AIA) in C57BL/6 mice. Animals were exposed to airflow, HNBT vapor, or CS during 1 h/twice a day, under the Health Canada Intense (HCI) smoking regime, between days 14 to 20 after the first immunization. At day 21, 16 h after the last exposures, mice were i.a. challenged and the AIA effects were evaluated 24 h later. CS- or HNBT-exposed mice presented equivalent blood nicotine levels. CS exposure worsened articular symptoms, pulmonary inflammation, and expression of lung metallothioneins. Nevertheless, CS or HNBT exposures reduced lymphoid organs' cellularity, splenocyte proliferation and IL-2 secretion. Additional in vitro CS or HNBT exposures confirmed the harmful effects on splenocytes, which were partially mediated by the activation of nicotine/α7nAchR pathway. Associated, data demonstrate the toxic mechanisms of CS or HNBT inhalation at HCI regime on RA, and highlight that further investigations are fundamental to assure the toxicity of emerging tobacco products on the immune system during specific challenges.

Evaluation of the therapeutic efficacy of human bone marrow mesenchymal stem cells with COX-2 silence and TGF-ß3 overexpression in rabbits with antigen-induced arthritis.

OBJECTIVE: Mesenchymal stem cells (MSCs), especially genetically modified MSCs, have become a promising therapeutic approach for the treatment of rheumatoid arthritis (RA) through modulating immune responses. However, most MSCs used in the treatment of RA are modified based on a single gene. In this study, we evaluated the therapeutic effects of human BMSCs (hBMSCs) with COX-2 silence and TGF-ß3 overexpression in the treatment of RA in a rabbit model. MATERIALS AND METHODS: hBMSCs were cotransfected with shCOX-2 and TGF-ß3 through lentiviral vector delivery. After SPIO-Molday ION Rhodamine-B™ (MIRB) labeling, lenti-shCOX2-TGF-ß3 hBMSCs, lenti-shCOX2 hBMSCs, lenti-TGF-ß3 hBMSCs, hBMSCs without genetic modification, or phosphate-buffered saline (PBS) were injected into the knee joint of rabbits with antigen-induced arthritis (AIA). The diameter of the knee joint and soft-tissue swelling score (STS) were recorded, and the levels of inflammatory mediators, including interleukin-1ß (IL-1ß), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) were evaluated by ELISA. Clinical 3.0T MR imaging (MRI) was used to track the distribution and dynamic migration of hBMSCs in the joint. Histopathological and immunohistochemical assays were conducted to localize labeled hBMSCs and assess the alteration of synovial hyperplasia, inflammatory cell infiltration, and cartilage damage. RESULTS: COX-2 silencing and TGF-ß3 overexpression in hBMSCs were confirmed through real-time PCR and Western blot analyses. Reduced joint diameter, soft-tissue swelling (STS) score, and PGE2, IL-1ß, and TNF-α expression were detected 4 weeks after injection of MIRB-labeled lenti-shCOX2-TGF-ß3 hBMSCs into the joint in rabbits with AIA. Eight weeks after hBMSC injection, reduced inflammatory cell infiltration, improved hyperplasia of the synovial lining, recovered cartilage damage, and increased matrix staining were observed in joints injected with lenti-shCOX2-TGF-ß3 hBMSCs and lenti-shCOX2 hBMSCs. Slight synovial hyperplasia, no surface fibrillation, and strong positive expression of collagen II staining in chondrocytes and cartilage matrix were detected in the joints 12 weeks after injection of lenti-shCOX2-TGF-ß3 hBMSCs. In addition, hBMSCs were detected by MRI imaging throughout the process of hBMSC treatment. CONCLUSION: Intra-articular injection of hBMSCs with COX-2 silence and TGFß3 overexpression not only significantly inhibited joint inflammation and synovium hyperplasia, but also protected articular cartilage at the early stage. In addition, intra-articular injection of hBMSCs with COX-2 silence and TGFß3 overexpression promoted chondrocyte and matrix proliferation. This study provides an alternative therapeutic strategy for the treatment of RA using genetically modified hBMSCs.

Panax notoginseng saponins alleviate osteoporosis and joint destruction in rabbits with antigen-induced arthritis.

Although a number of anti-rheumatic drugs and biologics may be used to alleviate the symptoms of rheumatoid arthritis (RA), these compounds have been associated with bone loss and joint destruction; thus, alternative treatment approaches are required. In the present study, various plant extracts were evaluated for their capacity to inhibit joint destruction, and Panax notoginseng saponins (PNS), obtained from the Traditional Chinese Medicine Panax notoginseng, was identified as such a compound. Therefore, a rabbit antigen-induced arthritis (AIA) model was generated by immunization with ovalbumin in Freund's complete adjuvant, followed by treatment with PNS for 3 months. The morphology of the quadriceps femoris muscle, cartilage chondrocytes and skeletal elements was histologically observed by transmission electron microscopy (TEM), as well as micro-computed tomography. The results revealed that PNS significantly reduced the histopathological alterations associated with arthritic muscular atrophy and inflammation. In addition, TEM demonstrated that PNS protected chondrocytes from RA-associated damage. Furthermore, the bone density and microarchitecture in rabbits treated with PNS were markedly improved compared with those of the model group. Collectively, these data indicated that treatment with PNS may relieve osteoporosis and prevent joint and bone destruction in AIA.

Hydroquinone Exposure Worsens Rheumatoid Arthritis through the Activation of the Aryl Hydrocarbon Receptor and Interleukin-17 Pathways.

Rheumatoid arthritis (RA) development is strongly associated with cigarette smoke exposure, which activates the aryl hydrocarbon receptor (AhR) as a trigger for Th17 inflammatory pathways. We previously demonstrated that the exposure to hydroquinone (HQ), one of the major compounds of cigarette tar, aggravates the arthritis symptomatology in rats. However, the mechanisms related to the HQ-related RA still remain elusive. Cell viability, cytokine secretion, and gene expression were measured in RA human fibroblast-like synoviocytes (RAHFLS) treated with HQ and stimulated or not with TNF-α. Antigen-induced arthritis (AIA) was also elicited in wild type (WT), AhR -/- or IL-17R -/- C57BL/6 mice upon daily exposure to nebulized HQ (25ppm) between days 15 to 21. At day 21, mice were challenged with mBSA and inflammatory parameters were assessed. The in vitro HQ treatment up-regulated TNFR1, TNFR2 expression, and increased ROS production. The co-treatment of HQ and TNF-α enhanced the IL-6 and IL-8 secretion. However, the pre-incubation of RAHFLS with an AhR antagonist inhibited the HQ-mediated cell proliferation and gene expression profile. About the in vivo approach, the HQ exposure worsened the AIA symptoms (edema, pain, cytokines secretion and NETs formation) in WT mice. These AIA effects were abolished in HQ-exposed AhR -/- and IL-17R -/- animals though. Our data demonstrated the harmful HQ influence over the onset of arthritis through the activation and proliferation of synoviocytes. The HQ-related RA severity was also associated with the activation of AhR and IL-17 pathways, highlighting how cigarette smoke compounds can contribute to the RA progression.

PLA-coated Imwitor® 900 K-based herbal colloidal carriers as novel candidates for the intra-articular treatment of arthritis.

Although there are several treatments for rheumatoid arthritis (RA), outcomes are unsatisfactory and often associated with many side effects. We attempted to improve RA therapeutic outcomes by intra-articular administration of dual drug-loaded poly(lactic) acid (PLA)-coated herbal colloidal carriers (HCCs). Curcumin (CU) and resveratrol (RES) were loaded into HCCs because of their safety and significant anti-inflammatory activity. HCCs were prepared using a high-pressure, hot homogenization technique and evaluated in vitro and in vivo using a complete Freund's adjuvant-induced arthritis model. Transmission electron microscope (TEM) evaluated coating selected formulations with PLA, which increased particle sizes from 52 to 89.14 nm. The entrapment efficiency of both formulations was approximately 76%. HCCs significantly increased the amount of RES and CU released compared with the drug suspensions alone. The in vivo treated groups showed a significant improvement in joint healing. PLA-coated HCCs, followed by uncoated HCCs, yielded the highest reductions in knee diameter, myeloperoxidase (MPO) levels, and tumor necrosis factor-alpha (TNFα) levels. Histological examination of the dissected joints revealed that PLA-coated HCCs followed by uncoated HCCs exhibited the most significant joint healing effects. Our results demonstrate the superiority of intra-articularly administered HCCs to suppress RA progression compared with RES or CU suspensions alone.

The potential anti-inflammatory and anti-nociceptive effects of rat hemopressin (PVNFKFLSH) in experimental arthritis.

Inflammatory arthritis, such as rheumatoid arthritis (RA), stands out as one of the main sources of pain and impairment to the quality of life. The use of hemopressin (PVNFKFLSH; Hp), an inverse agonist of type 1 cannabinoid receptor, has proven to be effective in producing analgesia in pain models, but its effect on neuro-inflammatory aspects of RA is limited. In this study, antigen-induced arthritis (AIA) was evoked by the intraarticular (i.art.) injection of methylated bovine serum albumin (mBSA) in male Sprague Dawley rats. Phosphate buffered saline (PBS)-injected ipsilateral knee joints or AIA contralateral were used as control. Nociceptive and inflammatory parameters such as knee joint oedema and leukocyte influx and histopathological changes were carried out in addition to the local measurement of interleukins (IL) IL-6, IL-1ß, tumor necrosis factor-α and the immunoreactivity of the neuropeptides substance P (SP) and calcitonin gene related peptide (CGRP) in the spinal cord (lumbar L3-5 segments) of AIA rats. For 4 days, AIA rats were treated daily with a single administration of saline, Hp injected (10 or 20 µg/day, i.art.), Hp given orally (20 µg/Kg, p.o.) or indomethacin (Indo; 5 mg/Kg, i.p.). In comparison to the PBS control group, the induction of AIA produced a significant and progressive mono-arthritis condition. The degree of AIA severity progressively compromised the normal walking pattern and impaired mobility over the next four days in relation to PBS-injected rats or contralateral knee joints. In AIA rats, the reduction of the distance between footprints and disturbances of gait evidenced signs of nociception. This response worsened at day 4, and a loss of footprint from the ipsilateral hind paw was evident. Daily treatment of the animals with Hp either i.art. (10 and 20 µg/knee) or p.o. (20 µg/Kg) as well as Indo (5 mg/Kg, i.p.) ameliorated the impaired mobility in a time-dependent manner (P < 0.05). In parallel, the AIA-injected ipsilateral knee joints reach a peak of swelling 24 h after AIA induction, which persisted over the next four days in relation to PBS-injected rats or contralateral knee joints. There was a significant but not dose-dependent inhibitory effect produced by all dosages and routes of Hp treatments on AIA-induced knee joint swelling (P < 0.05). In addition, the increased synovial levels of MPO activity, total leukocytes number and IL-6, but not IL-1ß, were significantly reduced by the lower i.art. dose of Hp. In conclusion, these results successfully demonstrate that Hp may represent a novel therapeutic strategy to treat RA, an effect which is unrelated to the proinflammatory actions of the neuropeptides CGRP and SP.

Hydroquinone exposure worsens rheumatoid arthritis through the activation of the aryl hydrocarbon receptor and interleukin-17 pathways

Rheumatoid arthritis (RA) development is strongly associated with cigarette smoke exposure, which activates the aryl hydrocarbon receptor (AhR) as a trigger for Th17 inflammatory pathways. We previously demonstrated that the exposure to hydroquinone (HQ), one of the major compounds of cigarette tar, aggravates the arthritis symptomatology in rats. However, the mechanisms related to the HQ-related RA still remain elusive. Cell viability, cytokine secretion, and gene expression were measured in RA human fibroblast-like synoviocytes (RAHFLS) treated with HQ and stimulated or not with TNF-α. Antigen-induced arthritis (AIA) was also elicited in wild type (WT), AhR −/− or IL-17R −/− C57BL/6 mice upon daily exposure to nebulized HQ (25ppm) between days 15 to 21. At day 21, mice were challenged with mBSA and inflammatory parameters were assessed. The in vitro HQ treatment up-regulated TNFR1, TNFR2 expression, and increased ROS production. The co-treatment of HQ and TNF-α enhanced the IL-6 and IL-8 secretion. However, the pre-incubation of RAHFLS with an AhR antagonist inhibited the HQ-mediated cell proliferation and gene expression profile. About the in vivo approach, the HQ exposure worsened the AIA symptoms (edema, pain, cytokines secretion and NETs formation) in WT mice. These AIA effects were abolished in HQ-exposed AhR −/− and IL-17R −/− animals though. Our data demonstrated the harmful HQ influence over the onset of arthritis through the activation and proliferation of synoviocytes. The HQ-related RA severity was also associated with the activation of AhR and IL-17 pathways, highlighting how cigarette smoke compounds can contribute to the RA progression.

RNA sequencing data from osteochondroprogenitor populations in synovial joints of mice during murine model of rheumatoid arthritis.

The aim of this study was to analyze the transcriptome of TER119-CD31-CD45-CD51+CD200+CD105- population (further, CD200+), potential early osteocondroprogenitors, whose frequency is reduced in the joints of mice with antigen-induced arthritis (AIA) [1]. A population defined by similar surface markers has been previously identified as murine skeletal stem cells in bone [2]. In order to confirm their identity this population was compared to TER119-CD31-CD45-CD51+CD200-CD105+ (further, CD105+) cells, which possibly represent committed progenitors, or other non-progenitor population such as synovial fibroblasts. In order to asses changes in CD200+ population in inflammatory setting, it was also compared to the same population from healthy mice. AIA was induced by immunization of mice with methylated bovine serum albumin (mBSA) and subsequent intra-articular injection of mBSA, while non-immunized mice were injected with phosphate-buffered saline at all timepoints. Ten days after intra-articular injection, knee joints were harvested and synovial cells were released by collagenase digestion. Using fluorescence-activated cell sorting, 200-500 cells from selected populations were sorted directly into cell lysis buffer, RNA was reversely transcribed, and first strand cDNA product was amplified. cDNA amplicons were used for library preparation. Bioinformatics analysis was performed using cutadapt [3], HISAT2 [4], Samtools [5] and StringTie [6] tools, and egdeR [7], limma [8], and ClusterProfiler [9] Bioconductor packages. In addition to access to raw data at the NCBI Gene Expression Omnibus repository, this article also provides sample similarity analysis, tables of differentially expressed genes, graphic visualisations of differential expression and gene set enrichment analysis performed on publicly available GO terms. Interpretation of osteochondroprogenitor phenotype of CD200+ population based on analysis of presented data is provided in the article "What do we know about bone morphogenetic proteins and osteochondroprogenitors in inflammatory conditions?" [10]. Reuse of this data may help researchers elucidate alterations of synovial stromal and osteochondroprogenitor populations in inflammatory settings and define their role in structural damage in rheumatoid arthritis.

Targeting interleukin-4 to the arthritic joint.

Anti-inflammatory cytokines are a promising class of therapeutics for treatment of rheumatoid arthritis (RA), but their use is currently limited by a rapid clearance and systemic toxicity. Interleukin-4 is a small cytokine with potential for RA therapy. To increase its pharmacokinetic features, we engineered a murine IL4 conjugate by incorporating an unnatural amino acid through genetic code expansion to which PEG-folate, as a targeting moiety and PEG alone as control, were site-specifically bound. Both IL4 conjugates retained bioactivity and induced primary murine macrophage polarization into an alternatively activated (M2) related phenotype. The PEGylated conjugates had a terminal half-life of about four hours in healthy mice compared to unPEGylated IL4 (0.76 h). We showed that both conjugates successfully accumulated into arthritic joints in an antigen-induced arthritis (AIA) mouse model, as assessed by non-invasive fluorescence imaging. The modular nature of the IL4 conjugate chemistry presented herein facilitates easy adaption of PEG chain length and targeting moieties for further improvement of half-life and targeting function for future efficacy studies.

Differential patterns of pathology in and interaction between joint tissues in long-term osteoarthritis with different initiating causes: phenotype matters.

OBJECTIVE: To determine if osteoarthritis (OA) progression and joint tissue-pathology associations link specific animal models to different human OA phenotypes. DESIGN: Male 11-week-old C57BL6 mice had unilateral medial-meniscal-destabilization (DMM) or antigen-induced-arthritis (AIA). Joint tissue histopathology was scored day-3 to week-16. Tissue-pathology associations (corrected for time and at week-16) were determined by partial correlation coefficients, and odds ratios (OR) calculated for likelihood of cartilage damage and joint inflammation by ordinal-logistic-regression. RESULTS: Despite distinct temporal patterns of progression, by week-16 joint-wide OA pathology in DMM and AIA was equivalent. Significant pathology associations common to both models included: osteophyte size and maturity (r > 0.4); subchondral bone (SCB) sclerosis and osteophyte maturity (r > 0.25); cartilage erosion and chondrocyte hypertrophy/apoptosis (r > 0.4), SCB sclerosis (r > 0.26), osteophyte size (r > 0.3), and maturity (r > 0.32). DMM-specific associations were between cartilage proteoglycan loss and structural damage (r = 0.56), osteophyte maturity (r = 0.49), size (r = 0.45), and SCB sclerosis (r = 0.28). AIA-specific associations were between SCB sclerosis and chondrocyte hypertrophy/apoptosis (r = 0.40) and osteophyte size (r = 0.37); and synovitis with cartilage structural damage (r = 0.18). No tissue-pathology associations were common to both models at week-16. Increased likelihood of cartilage structural damage was associated with: chondrocyte hypertrophy/apoptosis (OR>1.7), and osteophyte size (OR>2.3) in both models; SCB sclerosis (OR = 2.0) and proteoglycan loss (OR = 2.4) in DMM; and synovitis (OR = 1.2) in AIA. Joint inflammation was associated positively with cartilage proteoglycan loss (OR = 1.4) and inversely with osteophyte size (OR = 0.21) in AIA only. CONCLUSION: This study highlights the importance of defining OA-models by initiating mechanisms and progression, not just end-stage joint-tissue pathology.