Animal Models Of Arthritis Research Articles

Therapeutic outcomes and biodistribution of gold nanoparticles in collagen-induced arthritis animal model

The role of gold nanoparticles (AuNPs) in the treatment of autoimmune diseases remains mysterious. Therefore, we investigated the effect of AuNPs on collagen-induced arthritis (CIA) and the impact of particle size on the tissue distribution of AuNPs. CIA model was set up in Wistar rats using bovine collagen type ІІ. CIA rats were then divided into five groups: CIA model (positive control), AuNPs treated- (sphere shape with size: 5, 25, and 75 nm), and methotrexate (MTX)-treated-rats. Untreated rats were run in parallel as normal control. At the end of the experiment, the radiographic assessment was done. Histopathological changes in the joints and vital organs were investigated. Liver- [aspartate transaminase (AST) and alanine transaminase (ALT)] and kidney- (creatinine) functions were measured. The gold bio-accumulation was measured quantitatively with inductively coupled plasma mass spectrometry (ICP-MS). Histopathological results revealed that AuNPs reduced the severity of arthritic symptoms in CIA rats. No histological changes were detected in vital organs. Importantly, AuNPs were deposited in all the examined organs, and the accumulation was size-dependent, with 75 nm demonstrated the least distributed one. Our results revealed that AuNPs decreased both liver and kidney functions as compared to normal controls. In conclusion, our data shed light on the therapeutic efficacy of AuNPs in experimental rheumatoid arthritis compared with a choice size (25 nm). Further investigations to investigate the immunological changes associated with AuNPs are in process.

Bromelain Extract Exerts Antiarthritic Effects via Chondroprotection and the Suppression of TNF-α-Induced NF-κB and MAPK Signaling.

Bromelain, a mixture of proteases in pineapple rhizome, has beneficial biological properties. Following absorption, the compound remains biologically active in mammalian blood and tissues. Bromelain has multiple clinical and therapeutic applications because of its anti-arthritic activities. Anti-inflammation is one of the putative therapeutic effects of bromelain on osteoarthritis (OA) and rheumatoid arthritis (RA), but the molecular mechanisms in cartilage and synovial fibroblast has not been reported. Thus, in this study, interleukin (IL)-1β/oncostatin M-induced porcine cartilage and TNF-α–induced synovial fibroblast were used as the inflamed OA and RA models, respectively. The results demonstrated the chondroprotective effects of bromelain on cartilage degradation and the downregulation of inflammatory cytokine (tumor necrosis factor (TNF)-α, IL-1β, IL-6, IL-8) expression in TNF-α–induced synovial fibroblasts by suppressing NF-κB and MAPK signaling. The evidence from this study supported and explained the anti-inflammatory and analgesic effects of bromelain on arthritis in animal models and clinical studies.

Preparation and characterization of a high-affinity monoclonal antibody against nerve growth factor

Nerve growth factor (NGF) is produced and released in injured tissues or chronic pain tissues caused by other diseases. Studies have shown that monoclonal antibodies targeting NGF have a good efficacy in the treatment of osteoarthritis (OA), low back pain and chronic pain, which may be a promising therapy. In this study, DNA sequences of NGF-his and NGF-hFc were synthesized using eukaryotic expression system and subcloned into pTT5 expression vector. After that, NGF proteins were expressed by transient expression in HEK293E cells. We immunized mice with NGF-hFc protein and fused mouse spleen cells to prepare hybridomas. NGF-His protein was used to screen out the hybridoma supernatant that could directly bind to NGF. Antibodies were purified from hybridioma supernatant. Futhermore, via surface plasmon resonance (SPR) screening, six anti-NGF mAbs were screened to block the binding of NGF and TrkA receptor in the treatment of chronic pain. Among them, 58F10G10H showed high affinity (KD = 1.03 × 10−9 M) and even better than that of positive control antibody Tanezumab (KD = 1.53 × 10−9 M). Moreover, the specific reactivity of 58F10G10H was demonstrated by TF-1 cell proliferation activity experiments, competitive binding Enzyme-linked immunosorbent assay (ELISA) and the arthritis animal models in mice, respectively. In conclusion, in this study, a method for the preparation of high-yield NGF-HFC and NGF-His proteins was designed, and a high-affinity monoclonal antibody against NGF with potential for basic research and clinical application was prepared.

Effects of Naringenin on ExperimentallyInducedRheumatoid Arthritis in Wistar Rats.

Naringenin is one of the most important and abundant known flavonoids found in grapefruit and other citrus fruits. This experimental study aimed to assess the clinical effects and immune responses of naringenin in the animal model of rheumatoid arthritis (RA) according to various reports on its anti-inflammatory effects and modulation of the immune system. To this end, 40 Wistar rats in the weight range of 160-180g were randomly assigned to four groups (n=10) including healthy, control, naringenin, and methotrexate orally treated groups. To induce RA disease, a compound of 200 μl of Freund's adjuvant and collagen type II was injected subcutaneously into the rear footpads of rats. The severity of RA clinical signs was assessed based on a standard scoring method. The treatment lasted for three weeks (days7-28 after induction). The obtained data pointed out that the levels of C-reactive protein (CRP), myeloperoxidase, nitric oxide, IL-17, and IFN-γ cytokines significantly increased in the RA rats, while the level of their serum antioxidants significantly reduced, compared to the healthy rats. The inflammation of the paws and the level of CRP decreased similarly in both methotrexate and naringenin-treated groups. In the naringenin-treated group, a further decrease was detected in serum myeloperoxidase, nitric oxide, and the total antioxidant capacity occurred, as compared to the methotrexate-treated rats. Nonetheless, IL-17 and IFN-γ cytokines levels were further decreased in the methotrexate-treated group. Accordingly, it can be concluded that naringenin can be effectively used for the reduction of inflammatory effects and control of RA disease.

Biofilm Formation by Pseudomonas aeruginosa in a Novel Septic Arthritis Model.

BackgroundBacterial biofilms generally contribute to chronic infections and complicate effective treatment outcomes. To date, there have been no reports describing biofilm formation in animal models of septic arthritis caused by Pseudomonas aeruginosa (P. aeruginosa). P. aeruginosa is an opportunistic pathogenic bacterium which can lead to septic arthritis. The purpose of this study was to establish a rabbit model of septic arthritis caused by P. aeruginosa to determine whether it leads to biofilm formation in the knee joint cavity. In addition, we explored the role of cyclic di-GMP (c-di-GMP) concentrations in biofilm formation in rabbit models.MethodsTwenty rabbits were randomly assigned to five groups: PAO1 (n = 4), PAO1ΔwspF (n = 4), PAO1/plac-yhjH (n = 4) infection group, Luria–Bertani (LB) broth (n = 4), and magnesium tetrasilicate (talc) (n = 4) control groups. Inoculation in the rabbit knee of P. aeruginosa or with the same volume of sterile LB or talc in suspension (control group) was used to induce septic arthritis in the animal model. In the infection groups, septic arthritis was caused by PAO1, PAO1ΔwspF, and PAO1/plac-yhjH strains, respectively. Rabbits were euthanized after 7 days, and pathological examination of synovial membrane was performed. The biofilms on the surface of the synovial membrane were observed by scanning electron microscopy, while the biofilms’ fiber deposition was discriminated using peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH).ResultsA rabbit model for knee septic arthritis induced by P. aeruginosa was successfully established. Scanning electron microscopy revealed that PAO1 strains were surrounded in a self-produced extracellular matrix on the surface of synovial membrane and showed biofilm structures. The biofilms in the fibrous deposition were also observed by PNA-FISH. The PNA-FISH assay revealed that the red fluorescence size in the PAO1ΔwspF group was greater than in PAO1 and PAO1/plac-yhjH groups.ConclusionsThis is the first study to provide evidence that P. aeruginosa forms biofilms in a rabbit model for septic knee arthritis. The rabbit model can be used to investigate new approaches to treatment of biofilms in septic arthritis. Furthermore, c-di-GMP is a key signaling molecule which impacts on biofilm formation in rabbit models of knee septic arthritis.

Annexin A1 attenuates cardiac diastolic dysfunction in mice with inflammatory arthritis

Rheumatoid arthritis (RA) carries a twofold increased incidence of heart failure with preserved ejection fraction, accompanied by diastolic dysfunction, which can lead to death. The causes of diastolic dysfunction are unknown, and there are currently no well-characterized animal models for studying these mechanisms. Current medications for RA do not have marked beneficial cardio-protective effects. K/BxN F1 progeny and KRN control mice were analyzed over time for arthritis development, monitoring left ventricular diastolic and systolic function using echocardiography. Excised hearts were analyzed by flow cytometry, qPCR, and histology. In pharmacological experiments, K/BxN F1 mice were treated with human recombinant AnxA1 (hrAnxA1, 1 μg/mouse) or vehicle daily. K/BxN F1 mice exhibited fully developed arthritis with normal cardiac function at 4 wk; however, by week 8, all mice displayed left ventricular diastolic dysfunction with preserved ejection fraction. This dysfunction was associated with cardiac hypertrophy, myocardial inflammation and fibrosis, and inflammatory markers. Daily treatment of K/BxN F1 mice with hrAnxA1 from weeks 4 to 8 halted progression of the diastolic dysfunction. The treatment reduced cardiac transcripts of proinflammatory cytokines and profibrotic markers. At the cellular level, hrAnxA1 decreased activated T cells and increased MHC IIlow macrophage infiltration in K/BxN F1 hearts. Similar effects were obtained when hrAnxA1 was administered from week 8 to week 15. We describe an animal model of inflammatory arthritis that recapitulates the cardiomyopathy of RA. Treatment with hrAnxA1 after disease onset corrected the diastolic dysfunction through modulation of both fibroblast and inflammatory cell phenotype within the heart.

Frequent injections of high-dose human umbilical cord mesenchymal stem cells slightly aggravate arthritis and skeletal muscle cachexia in collagen-induced arthritic mice.

A single injection of low-dose human umbilical cord-derived mesenchymal stem cells (UC-MSCs) has been previously demonstrated to relieve synovitis and bone erosion in animal models of arthritis, but whether frequent injections of high-dose UC-MSCs relieve arthritis and inhibit loss of muscle mass has remained elusive. In the present study, DBA/1 mice were randomly divided into three groups: Normal (wild-type mice; n=11), collagen-induced arthritis (CIA; n=12) and CIA treated with UC-MSCs (n=11; 5x106 UC-MSCs per week for 3 weeks). Arthritis and skeletal muscle cachexia were evaluated until the end of the experiment on day 84. It was indicated that both the CIA and UC-MSC groups had lower body weights compared with the normal mice. Clinical arthritis scores, hind ankle diameters, synovitis and bone erosion progressively increased and were similar between the CIA and UC-MSC groups. Although there was no difference in food intake among the three groups, the normalized food intake of normal group was significantly higher than CIA group and UC-MSC group from day 42 onwards; there was no significance on day 77 but this could be neglected. Furthermore, gastrocnemius muscle weight in the UC-MSC group was significantly reduced compared with that in the CIA and normal groups. The UC-MSC group had higher levels of proinflammatory cytokines, such as TNF-α, IL-6 and IL-1β than those in the CIA group. However, the other cytokines assessed and the fibrosis indices in the CIA and UC-MSC groups were not different from those in the control group and there was no inflammatory cell infiltration. Thus, frequent injections of high-dose UC-MSCs slightly aggravated synovitis and muscle cachexia in the murine CIA model and should therefore be avoided in the treatment of arthritis.

Effects of α1-adrenoreceptor on Treg cell function in collagen-induced arthritis

Objective: An animal model of collagen-induced arthritis (CIA) was used to investigate the effects of norepinephrine (NE) and α1-adrenoreceptor (α1-AR) on Treg cells in CIA. Methods: Thirty-two male DBA/1 mice were randomly divided into control group and CIA model group. CIA was prepared by intradermal injection of collagen type II (CII, 100 μl) at the tail base of DBA/1 mice. On the 41th day following primary immunization, co-expression of CD4+T and α1-AR in mouse spleens was observed. Protein expressions of α1-AR in the ankle joints and the spleens of mice were measured by Western blot analysis. The CD4+ T cells were isolated from the mouse spleen tissues in CIA mice and treated with NE or α1-AR agonist phenylephrine. Percentage of Treg cells in mouse CD4+ T cells of CIA mice was determined by flow cytometry. Expressions of α1-AR, transforming growth factor-β (TGF-β) and IL-10 in CD4+T cells of CIA mice were assessed by Western blot. Results: Co-expression of CD4 and α1-AR was observed in spleens of both intact and CIA mice. Compared with intace mice, α1-AR expressions in the ankle joints and spleens were down-regulated in CIA mice. NE increased the function of Treg cells in CD4+ T cells of CIA mice compared with that of nothing-treated CD4+T cells of CIA mice. Moreover, the α1-AR agonist phenylephrine increased the Treg cell function in CD4+ T cells of CIA mice relative to that of nothing-treated CD4+T cells of CIA mice. Conclusion: Activating α1-AR on CD4+T cells of CIA mice enhances Treg cell function,facilitating a shift of CD4+T cells toward Treg polarization.

Anti-inflammatory and Pro-apoptotic Effects of 18beta-Glycyrrhetinic Acid In Vitro and In Vivo Models of Rheumatoid Arthritis.

18β-Glycyrrhetinic acid (18β-GA), an active component from Glycyrrhiza glabra L. root (licorice), has been demonstrated to be able to protect against inflammatory response and reduce methotrexate (MTX)-derived toxicity. This study was therefore designed to test the therapeutic possibility of 18β-GA on rheumatoid arthritis (RA) and to explore the underlying mechanism. LPS or TNF-α-induced inflammatory cell models and collagen-induced arthritis (CIA) animal models were applied in this study. Real-time quantitative PCR (RT-qPCR) was used to measure the mRNA levels of various cytokines and FOXO family members. The protein levels of molecules in the MAPK/NF-κB signaling pathway were analyzed using western blot. The cell proliferation assay and colony-forming assay were used to test the influence of 18β-GA on cell viability. The cell apoptosis assay and cell cycle assay were performed to detect the effect of 18β-GA on cell proliferative capacity by using flow cytometry. Hematoxylin and eosin (H&E) staining was performed to evaluate pathological changes after drug administration. The enzyme-linked immunosorbent assay (ELISA) was carried out for the detection of cytokines in serum. In vitro, we found that 18β-GA decreased the mRNA levels of IL-1β, IL-6, and COX-2 by inhibiting the MAPK/NF-κB signaling pathway in MH7A and RAW264.7 cell lines. Moreover, 18β-GA was able to suppress cell viability, trigger cell apoptosis, and G1 phase cell cycle arrest in our in vitro studies. 18β-GA dramatically enhanced the mRNA level of FOXO3 in both TNF-α- and LPS-induced inflammation models in vitro. Interestingly, after analyzing GEO datasets, we found that the FOXO3 gene was significantly decreased in the RA synovial tissue as compared to healthy donors in multiple microarray studies. In vivo, 18β-GA exhibited a promising therapeutic effect in a collagen-induced arthritis mouse model by alleviating joint pathological changes and declining serum levels of TNF-α, IL-1β, and IL-6. Finally, we observed that 18β-GA administration could mitigate liver damage caused by collagen or MTX. Collectively, the current study demonstrates for the first time that 18β-GA can inhibit inflammation and proliferation of synovial cells, and the underlying mechanism may be associated with its inhibition of MAPK/NF-κB signaling and promotion of FOXO3 signaling. Therefore, 18β-GA is expected to be a new drug candidate for RA therapy.

Probiotic Supplementation for Rheumatoid Arthritis: A Promising Adjuvant Therapy in the Gut Microbiome Era

Rheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease that ultimately leads to joint destruction and functional disability. Although the exact etiology of RA is not fully understood, it is well established that gut microbiota (GM) plays a vital role in the pathogenesis of RA, with accumulating evidence suggesting that gut dysbiosis induces a chronic inflammatory response that may be linked to disease development. Of interest, patients with RA have significant changes in the intestinal microbiota compared to healthy controls, and several studies have suggested the use of probiotics as a possible adjuvant therapy for RA. Benefits of probiotic supplementation were reported in animal models of arthritis and human studies, but the current evidence regarding the effect of probiotic supplementation in the management of RA remains insufficient to make definite recommendations. Several different strains of Lactobacillus and Bifidobacteria, as single species or in mixed culture, have been investigated, and some have demonstrated beneficial effects on disease activity in RA human subjects. As of now, L.casei probiotic bacteria seems to be the strongest candidate for application as adjuvant therapy for RA patients. In this review, we highlight the role of GM in the development and progression of RA and summarize the current knowledge on the use of probiotics as a potential adjuvant therapy for RA. We also review the proposed mechanisms whereby probiotics regulate inflammation. Finally, the role of fermented foods is discussed as a possible alternative to probiotic supplements since they have also been reported to have health benefits.

Role of Epoxide Hydrolases and Cytochrome P450s on Metabolism of KZR-616, a First-in-Class Selective Inhibitor of the Immunoproteasome.

KZR-616 is an irreversible tripeptide epoxyketone-based selective inhibitor of the human immunoproteasome. Inhibition of the immunoproteasome results in anti-inflammatory activity in vitro and based on promising therapeutic activity in animal models of rheumatoid arthritis and systemic lupus erythematosus KZR-616 is being developed for potential treatment of multiple autoimmune and inflammatory diseases. The presence of a ketoepoxide pharmacophore presents unique challenges in the study of drug metabolism during lead optimization and clinical candidate profiling. This study presents a thorough and systematic in vitro and cell-based enzymatic metabolism and kinetic investigation to identify the major enzymes involved in the metabolism and elimination of KZR-616. Upon exposure to liver microsomes in the absence of NADPH, KZR-616 and its analogs were converted to their inactive diol derivatives with varying degrees of stability. Diol formation was also shown to be the major metabolite in pharmacokinetic studies in monkeys and correlated with in vitro stability results for individual compounds. Further study in intact hepatocytes revealed that KZR-616 metabolism was sensitive to an inhibitor of microsomal epoxide hydrolase (mEH) but not inhibitors of cytochrome P450 (P450) or soluble epoxide hydrolase (sEH). Primary human hepatocytes were determined to be the most robust source of mEH activity for study in vitro. These findings also suggest that the exposure of KZR-616 in vivo is unlikely to be affected by coadministration of inhibitors or inducers of P450 and sEH. SIGNIFICANCE STATEMENT: This work presents a thorough and systematic investigation of metabolism and kinetics of KZR-616 and related analogs in in vitro and cell-based enzymatic systems. Information gained could be useful in assessing novel covalent proteasome inhibitors during lead compound optimization. These studies also demonstrate a robust source in vitro test system that correlated with in vivo pharmacokinetics for KZR-616 and two additional tripeptide epoxyketones.

METTL3 Promotes Activation and Inflammation of FLSs Through the NF-κB Signaling Pathway in Rheumatoid Arthritis.

Rheumatoid arthritis (RA), a common autoimmune disease, is extremely damaging to human health. Fibroblast-like synoviocytes (FLSs) have a vital role in the occurrence and development of RA. Methyltransferase-like 3 (METTL3), which is a crucial component of the N6-methyladenosine (m6A) methyltransferase complex, is involved in the progression of many diseases. In this study, we explored the role of METTL3 in the inflammatory response and proliferation, invasion, and migration of FLSs. We used human RA synovial tissues and the adjuvant-induced arthritis (AIA) animal model of RA. Experimental results revealed that METTL3 expression was significantly upregulated in human RA synovial tissues and in the rat AIA model. METTL3 knockdown suppressed interleukin (IL)-6, matrix metalloproteinase (MMP)-3, and MMP-9 levels in human RA-FLSs and rat AIA-FLSs. In contrast, they were increased by METTL3 overexpression. Additionally, we found that, in FLSs, METTL3 may activate the nuclear factor (NF)-κB signaling pathway. The experimental results showed that METTL3 may promote FLS activation and inflammatory response via the NF-κB signaling pathway.

Non-invasive and accurate readout of superoxide anion in biological systems by near-infrared light

Infectious and inflammatory diseases involve superoxide anion (O2•–) production. Real-time and non-invasive evaluation of O2•– in intact biological systems has been a significant challenge in biology and medicine. Here, I report that an advanced near-infrared chemiluminescent probe, MCLA-800, enables reliable non-invasive optical readout of O2•–ex vivo and in vivo. MCLA-800 allowed highly selective and sensitive monitoring of O2•– in undiluted human whole blood ex vivo. For the first time, the use of MCLA-800 revealed two reproducible types of O2•– production in response to stimulation by unopsonized zymosan particles of Saccharomyces cerevisiae, that is, slow response (S-type) and fast response (F-type), specific to each individual. O2•– production was synchronized with myeloperoxidase (MPO) activation in the former type but not in the latter. Moreover, as new findings, MCLA-800 chemiluminescence demonstrated that the chemiluminescence intensity–time properties of formyl-methionyl-leucyl-phenylalanine (fMLP)- or phorbol 12-myristate 13-acetate (PMA)-induced O2•– production and MPO activity were independent of S- and F-type zymosan-induced MCLA-800 chemiluminescence whole blood and that PMA-induced MPO activation synchronized with PMA-induced O2•– production in S- and F-type zymosan-induced MCLA-800 chemiluminescence whole blood, but fMLP-induced MPO activation did not synchronize with fMLP-induced O2•– production in both of S- and F-type blood. Furthermore, MCLA-800 spatiotemporally allowed non-invasive and clear in vivo imaging of O2•– in animal models of acute dermatitis and focal arthritis. Therefore, MCLA-800 could be possibly applied in various advanced diagnostic techniques.

Applicability and implementation of the collagen-induced arthritis mouse model, including protocols (Review).

Animal models of rheumatoid arthritis (RA) are essential for studying the pathogenesis of RA in vivo and determining the efficacy of anti-RA drugs. During the past decades, numerous rodent models of arthritis have been evaluated as potential models and the modeling methods are relatively well-developed. Among these models, the collagen-induced arthritis (CIA) mouse model is the first choice and the most widely used because it may be generated rapidly and inexpensively and is relatively similar in pathogenesis to human RA. To date, there have been numerous classic studies and reviews discussing related pathogeneses and modeling methods. Based on this knowledge, combined with the latest convenient and effective methods for CIA model construction, the present review aims to introduce the model to beginners and clarify important details regarding its use. Information on the origin and pathogenesis of the CIA model, the protocol for establishing it, the rate of successful arthritis induction and the methods used to evaluate the severity of arthritis are briefly summarized. With this information, it is expected that researchers who have recently entered the field or are not familiar with this information will be able to start quickly, avoid unnecessary errors and obtain reliable results.

(-)-Englerin-A Has Analgesic and Anti-Inflammatory Effects Independent of TRPC4 and 5.

Recently, we found that the deletion of TRPC5 leads to increased inflammation and pain-related behaviour in two animal models of arthritis. (-)-Englerin A (EA), an extract from the East African plant Phyllanthus engleri has been identified as a TRPC4/5 agonist. Here, we studied whether or not EA has any anti-inflammatory and analgesic properties via TRPC4/5 in the carrageenan model of inflammation. We found that EA treatment in CD1 mice inhibited thermal hyperalgesia and mechanical allodynia in a dose-dependent manner. Furthermore, EA significantly reduced the volume of carrageenan-induced paw oedema and the mass of the treated paws. Additionally, in dorsal root ganglion (DRG) neurons cultured from WT 129S1/SvIm mice, EA induced a dose-dependent cobalt uptake that was surprisingly preserved in cultured DRG neurons from 129S1/SvIm TRPC5 KO mice. Likewise, EA-induced anti-inflammatory and analgesic effects were preserved in the carrageenan model in animals lacking TRPC5 expression or in mice treated with TRPC4/5 antagonist ML204.This study demonstrates that while EA activates a sub-population of DRG neurons, it induces a novel TRPC4/5-independent analgesic and anti-inflammatory effect in vivo. Future studies are needed to elucidate the molecular and cellular mechanisms underlying EA’s anti-inflammatory and analgesic effects.

POS0394 NAD+ BOOSTERS REESTABLISH THE ALTERED NAD+ METABOLISM OF LEUKOCYTES FROM RHEUMATOID ARTHRITIS PATIENTS IMPROVING THEIR OXIDATIVE, APOPTOTIC AND INFLAMMATORY STATUS

Background:NAD+ is an important cofactor and second messenger for multiple cellular processes that exhibits antioxidant, anti-apoptotic and anti-inflammatory properties. Pre-clinical studies in animal models of Rheumatoid Arthritis (RA) have demonstrated the therapeutic potential of NAD+ boosters in the control of the disease activity. However, to date no studies have been set up to evaluate the NAD+ metabolism and the therapeutic effects of NAD+ boosters in RA patients.Objectives:1- To study the NAD+ metabolism in RA patients and its association with key clinical features. 2- To evaluate the effect of anti-TNF therapy in the NAD+ metabolism.3- To analyze the beneficial effects of NAD+ boosters in leukocytes from active RA patients.Methods:Plasma and PBMCs were purified from 100 RA patients and 50 healthy donors (HDs). Moreover, an additional cohort of 50 RA patients treated with Anti-TNF therapy was analyzed before and after 6 months of treatment. NAD+ levels were determined by using the NAD/NADH-Glo Assay. NAD+-consuming genes expression were analyzed by RT-PCR. In parallel, PBMCs from eight HDs and eight active RA patients were treated ex vivo with 1 mM of NAD+ boosters including nicotinamide (NAM), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN). After 24 hours, intracellular reactive oxygen species (ROS) levels (DFCHDA) and the percentage of apoptotic PBMCs (annnexin V/PI) were assessed by flow cytometry. Lastly, a panel of key pro-inflammatory genes were evaluated by RT-PCR.Results:NAD+ and NADH levels were significantly reduced in plasma and PBMCs of RA patients compared with HDs and directly related to disease activity (DAS28, CDAI, SDAI). Accordingly, the expression levels of genes involved in the consumption of NAD+ such as SIRT-1, CD38 and PARP-1 were found up-regulated in PBMCs from RA patients. Anti-TNF therapy for 6 months restored the altered NAD+ levels towards those showed by HDs. Furthermore, the clinical response promoted by Anti-TNF therapy (changes in DAS28) correlated with changes in NAD+ levels. The in vitro treatments of PBMCs isolated from active RA patients with NAD+ boosters significantly increased the NAD+ levels and promoted a deep reduction of intracellular ROS levels, the percentage of apoptotic cells and the expression levels of key inflammatory mediators, such as IL-6, IL-8, IL-1ß, TNF-α, CCL2, IL-23, and STAT-3.Conclusion:1. NAD+ metabolism is altered and associated with the disease activity of RA patients, involving both, reduced NAD+ levels and increased expression of NAD+-consuming genes. 2. Anti-TNF therapy restored NAD+ levels, which were directly linked to the clinical effectiveness. 3. NAD+ boosters reduced the oxidative, apoptotic and inflammatory profiles of RA leukocytes through the parallel increase of intracellular NAD+ levels. Thus, NAD+ boosters might be considered novel therapeutic tools for RA patients.Acknowledgements:Supported by PI18/00837, RIER RD16/0012/0015, RTI2018-100695-B-I00, P18-RT-4264 and CVI276, co-funded with FEDER.Disclosure of Interests:None declared

Infliximab prevents systemic bone loss and suppresses tendon inflammation in a collagen-induced arthritis rat model.

Reduced Bone Mineral Density (BMD) and tendon abnormalities, such as tenosynovitis and enthesitis, are prevalent comorbidities in patients with rheumatoid arthritis (RA). The aim of the present study was to investigate the effect of chronic treatment with infliximab on BMD and tendon inflammation in an animal model of inflammatory arthritis. Collagen-Induced Arthritis (CIA) was induced in rats, followed by long-term intraperitoneal administration of infliximab. Two additional groups of animals received methotrexate either as a monotherapy or as a co-treatment to infliximab. BMD was evaluated by Micro-Computed Tomography (Micro-CT) and bone histological examination. Tendon inflammation was assessed histologically and by quantitative ELISA analysis of pro-inflammatory cytokines in tendon tissues. Both methotrexate and infliximab treatment alleviated joint inflammation and reduced paw edema. Infliximab-treated animals exhibited an improved trabecular microarchitecture on micro-CT and histological analysis compared to both non-treated and methotrexate-treated animals. Infliximab almost reversed the pathological changes in tendons induced by CIA. Finally, we observed statistically significant declines in tendon TNF-a and IL-23 levels after infliximab treatment. Our study provides evidence that infliximab prevents arthritis-related osteoporosis and suppresses tendon inflammation in an animal model of inflammatory arthritis, in addition to controlling disease activity. These findings offer perspectives for the management of osteoporosis and enthesitis in RA.

Pharmacological modulation of T cell immunity results in long-term remission of autoimmune arthritis

Chronic inflammatory diseases like rheumatoid arthritis are characterized by a deficit in fully functional regulatory T cells. DNA-methylation inhibitors have previously been shown to promote regulatory T cell responses and, in the present study, we evaluated their potential to ameliorate chronic and acute animal models of rheumatoid arthritis. Of the drugs tested, decitabine was the most effective, producing a sustained therapeutic effect that was dependent on indoleamine 2,3-dioxygenase (IDO) and was associated with expansion of induced regulatory T cells, particularly at the site of disease activity. Treatment with decitabine also caused apoptosis of Th1 and Th17 cells in active arthritis in a highly selective manner. The molecular basis for this selectivity was shown to be ENT1, a nucleoside transporter, which facilitates intracellular entry of the drug and is up-regulated on effector T cells during active arthritis. It was further shown that short-term treatment with decitabine resulted in the generation of a population of regulatory T cells that were able to suppress arthritis upon adoptive transfer. In summary, a therapeutic approach using an approved drug is described that treats active inflammatory disease effectively and generates robust regulatory T cells with the IDO-dependent capacity to maintain remission.

Marham-Mafasel decrease joint inflammation and IL-1β gene expression in rheumatoid arthritis animal model.

BackgroundRheumatoid arthritis (RA) is a systemic chronic disease with synovial membrane, tendon and articular tissue inflammation. Current treatments of RA have many side effects and are quite expensive. Today, new treatments procedures and inexpensive herbal drugs are developed. Marham‐Mafasel is mainly made out of two traditional herbs (Arnebia euchroma and Martricaria chamomilla).ObjectiveIn this study, for the first time, the impact of Marham‐Mafasel on joint inflammation, histopathological changes and IL‐1β gene expression was evaluated in RA animal model.MethodsThe RA was induced by a single s.c. injection of 0.1 ml Freund's complete adjuvant into the left hind footpad. In continuous, 15 RA male Wistar rats were used in three groups: I: Control; II: Treatment I (Piroxicam) and III: Treatment II (Marham‐Mafasel). The volume of the hind paw was measured every day from 0 to 19 using water changed volume approach. The inflammation in the joint was evaluated using histopathology assay and gene expression of IL‐1β was evaluated with use of Real‐Time PCR.ResultsHind paw swelling of Marham‐Mafasel at days 10th and 19th was reduced compared with the control group (p < 0.05). There was no statistically difference in histological degrading and changes index in three groups (p ≥ 0.05). Relative expression of IL‐1β in Marham‐Mafasel group was significantly decreased compared with other groups.ConclusionThe co‐administration of M. Chamomile and A. euchroma, called Marham‐Mafasel, decreases IL‐1β gene expression that leads to a reduction in inflammation in rheumatoid arthritis (RA) animal model.

Countering Inflammatory Arthritis by 14-3-3zeta: A Novel Target

Abstract Inflammatory arthritis (IA) is a common inflammatory joint-destructive condition that affects millions of individuals worldwide. Increased immune dysfunction and bone loss have been observed both in IA affected humans and in animal models. Proinflammatory events responsible for RA pathogenesis have been studied in detail; however, the protective mechanism remains under-explored. Earlier, we reported that 14-3-3zeta (z) is a molecular determinant of skewing T cell polarization and interleukin-17A (IL-17A) signal transduction. Here we demonstrate that 14-3-3z is an endogenous suppressor of IA. Loss of 14-3-3z in a novel CRISPR/Cas9 edited knockout (KO) rat resulted in early-onset and severe arthritis in animal models of inflammatory arthritis. Arthritic 14-3-3z KO animals showed increased bone loss and immune cell infiltration in synovial joints. Rescue experiments to supplement the 14-3-3z by immunization during the pre-symptomatic phase resulted in significant arthritis suppression in both wild-type and 14-3-3z KO rats. Immunization generated a robust increase in 14-3-3z antibody and IL-17A, suppressed several cytokines, including IFN-g, TNF-a, and Cxcl-1, preserved tissue mineral bone density, and trabecular bone volume. Our findings, taken together with the prior report of the molecular function of 14-3-3z as an antigen, indicate that antigenic 14-3-3z is responsible for a previously unrecognized inflammatory arthritis-suppressive mechanism.