Pristane-induced Arthritis Rats Research Articles

Pristane promotes anaerobic glycolysis to facilitate proinflammatory activation of macrophages and development of arthritis

Pristane-induced arthritis (PIA) could be adoptively transferred by splenic T cells in rats, and innate immunity should play critical roles in T cell activation. However, in pre-clinical stage, the activation mechanism of innate cells like macrophages remains unclear. Here we found that PIA was dependent on macrophages since cell depletion alleviated disease severity. Splenic macrophages of PIA rats showed M1 phenotypic shifting. The quantitative proteomics analysis suggested that macrophages initiated metabolic reprogramming with the conversion of aerobic oxidation to glycolysis in response to pristane in vivo. Notably, macrophages treated with pristane showed mitochondrial dysregulation and increased glycolysis flux and enzyme activity. Additionally, TNFα production, strongly associating with the glycolysis enzyme Ldha/Ldhb, could be reduced as glycolysis was inhibited or be enhanced as citrate cycle was blocked. This work provides detailed insights into the molecular mechanisms of pristane-mediated metabolic reprogramming in macrophages and suggests a new therapeutic strategy for arthritic disorders.

RTD-1 therapeutically normalizes synovial gene signatures in rat autoimmune arthritis and suppresses proinflammatory mediators in RA synovial fibroblasts.

Rhesus theta defensin-1 (RTD-1), a macrocyclic immunomodulatory host defense peptide from Old World monkeys, is therapeutic in pristane-induced arthritis (PIA) in rats, a model of rheumatoid arthritis (RA). RNA-sequence (RNA-Seq) analysis was used to interrogate the changes in gene expression in PIA rats, which identified 617 differentially expressed genes (DEGs) in PIA synovial tissue of diseased rats. Upstream regulator analysis showed upregulation of gene expression pathways regulated by TNF, IL1B, IL6, proinflammatory cytokines, and matrix metalloproteases (MMPs) involved in RA. In contrast, ligand-dependent nuclear receptors like the liver X-receptors NR1H2 and NR1H3 and peroxisome proliferator-activated receptor gamma (PPARG) were downregulated in arthritic synovia. Daily RTD-1 treatment of PIA rats for 1-5 days following disease presentation modulated 340 of the 617 disease genes, and synovial gene expression in PIA rats treated 5 days with RTD-1 closely resembled the gene signature of naive synovium. Systemic RTD-1 inhibited proinflammatory upstream regulators such as TNF, IL1, and IL6 and activated antiarthritic ligand-dependent nuclear receptor pathways, including PPARG, NR1H2, and NR1H3, that were suppressed in untreated PIA rats. RTD-1 also inhibited proinflammatory responses in IL-1β-stimulated human RA fibroblast-like synoviocytes (FLS) in vitro and diminished expression of human orthologs of disease genes that are induced in rat PIA synovium. Thus, the antiarthritic mechanisms of systemic RTD-1 include homeostatic regulation of arthritogenic gene networks in a manner that correlates temporally with clinical resolution of rat PIA.

Pristane-induced arthritis in dark Agouti rat is a relevant model for mimicking vascular dysfunction and lipid paradox in rheumatoid arthritis.

To understand the pathophysiology of cardiovascular (CV) dysfunction in rheumatoid arthritis (RA) is crucial, but limited by the paucity of animal models able to mimic CV impairments. We wanted to determine if the rat model of Pristane-Induced Arthritis (PIA) reproduced cardiometabolic impairments of RA. Dark Agouti rats received an injection of pristane or saline (controls) at day 0. Reactivity to vasoconstrictors and vasodilators was studied in aortic rings and mesenteric arteries at day 28 (acute) and day 120 post-induction (chronic phase). Circulating markers of inflammation, lipid and glucose levels, arthritis and radiographic scores were assessed. In aortic rings, PIA induced a reduced vasoconstriction to phenylephrine and serotonin in both phases of the model. The relaxant effect of acetylcholine was decreased in PIA in acute (P < 0.05) but not in chronic phase. In mesenteric arteries, only the acetylcholine-induced vasorelaxation was impaired in PIA rats in the chronic phase (P < 0.001). Serum interleukin-6 levels were higher, total cholesterol and triglycerides levels were lower in PIA in both phases (P < 0.001) whereas myeloperoxidase activity and blood glucose were unchanged. Adiponectine levels were lower in PIA in acute (P < 0.001) but not in chronic phase. Endothelial function correlated with interleukin-6, total cholesterol levels and arthritis score in aorta but not in mesenteric arteries. As new information, PIA induces endothelial dysfunction in micro-/macro-vascular beds and low lipid levels, like in RA. This model of chronic arthritis might be useful to study CV pathophysiology and to screen new therapeutic options for reducing CV risk in RA.

The CD25+/CD4+ T cell ratio and levels of CII, CIX and CXI antibodies in serum may serve as biomarkers of pristane-induced arthritis in rats and Rheumatoid Arthritis in humans

ObjectiveCollagen antibodies in serum are involved in the pathogenesis of Rheumatoid Arthritis (RA). The objective of this study was to identify the subtype of collagen antibodies and T cell subtype distribution in pristane-induced arthritis (PIA) and to clarify their roles in the initiation and maintenance of arthritis. MethodsArthritis was induced in Dark Agouti (DA) rats by injection of pristane. The severity was evaluated by macroscopic and microscopic score systems. The alteration of CD25+/CD4+ T cell ratio in rats was detected by flow cytometry. Collagen type II (CII), CIX, or CXI antibody in serum was determined by ELISA. The levels of Nitric oxide (NO) and tartrate-resistant acid phosphatase (TRAP) were measured by kits. ResultsThe serum levels of CII, CIX, CXI antibodies were significantly increased in RA patients while slightly increased in PIA rats. The ratio of CD25+/CD4+ T cells was significantly higher in RA rats than that in the control group. The serum levels of NO and TRAP in PIA rats and RA patients were higher than that in the control groups, which suggested that the activity of osteoclast was increased in RA. ConclusionThe ratio of CD25+/CD4+ T cells plays a pivotal role in the development of PIA. The serum levels of NO and TRAP are inflammatory and osteoclast activity indicators. The serum levels of CII, CIX and CXI antibodies may serve as the clinical diagnostic indicators. These findings are important to our understanding of the pathogenesis of RA, and may provide biomarkers of RA diagnosis and therapeutic targets for the treatment of RA.

Increased expression of Th17 cytokines and interleukin-22 correlates with disease activity in pristane-induced arthritis in rats.

The objective of this study was to identify the key changed subtype of T helper cells (Th cells) and their cytokines in pristane-induced arthritis (PIA) in rats. The severity of arthritis was evaluated by body weight, clinical score, the perimeter of ankle and mid-paw and histological assessment of ankle joints. Cytokines of Th1, Th2 and Th17 were determined in the spleen and inguinal lymph nodes at 28 days after pristane injection by real-time qPCR. The mRNA levels of IL-22 receptors, IL-22R1 and IL-22BP, in the spleen were quantified by real-time qPCR. Additionally, IL-22 expression in synovial membrane was detected by Western blotting, and serum IL-22 concentration was determined by ELISA. Correlation between IL-22 concentration and clinical score was analyzed. By screening the cytokines of Th1, Th2 and Th17 expression profile, we found that the mRNA levels of Th17 cytokines were significantly increased in PIA rats. Particularly, a significant increase in the protein expression of IL-22 was determined in synovial membrane and serum from PIA rats, and correlated with clinical score. We conclude that IL-22 expression level was increased and correlated with disease severity, which indicated that IL-22 may play an important role in development of PIA, and was helpful to explorer the pathogenesis of rheumatoid arthritis.

IL‑22 expression is increased variedly in the initial phase, onset and chronic phase of a pristane‑induced arthritis rat model.

The aim of the present study was to investigate the expression pattern of T helper (Th) 17 and Th22 cell-related factors in a pristane-induced arthritis (PIA) rat model. PIA rats were divided into the initial phase group [day (D) 6 post-pristane injection], the onset of clinical arthritis group (D12), the acute arthritis group (D26) and the chronic arthritis group (D70). Rats injected with saline alone were used as the control group. The mRNA expression levels of interleukin (IL)-17A, IL-17F, interferon (IFN)-γ, IL-22, IL-22 receptor (R) 1, IL-22 binding protein (BP) and RAR-related orphan receptor α were examined in the spleen and/or synovium of the various phases of PIA rats by reverse transcription-quantitative polymerase chain reaction analysis. The results demonstrated that, in the spleen, IL-22 exhibited an increasing trend in both the initial phase and the onset of disease, while the ratio of IL-22R1/IL-22BP increased in both phases, compared with the control group. During the acute arthritis phase, IL-17F and IFN-γ were significantly increased and IL-17A exhibited an increasing tendency in the synovium, compared with the control group. In the chronic phase, IL-22, IL-22R1 and IFN-γ were increased in the spleen, while IL-22 exhibited an increasing trend in the synovium. In addition, immunohistochemistry analysis was used to evaluate the expression of IL-17A, IL-21, IL-22 and IL-22R1 in the ankle joints of D26 PIA rats. IL-17A was mainly expressed in infiltrated inflammatory cells in the synovium. IL-21 and IL-22 were both expressed in the inflammatory cells and in the articular chondrocyte of the proliferative zone. IL-22R1 was expressed in proliferating synovial cells. In conclusion, Th17 and Th22-related factor expression varied in different disease progression phases and in different tissues in PIA rats. IL-22 expression exhibited an increasing trend in the initial phase and the onset phase of arthritis and increased significantly with progression to chronic arthritis in the PIA rat model. It is thought that IL-22 may serve an important role in the pathological process of PIA, particularly in the chronic fluctuation phase. Therefore, it may be a candidate molecule for the treatment of rheumatoid arthritis.

Pristane primed rat T cells enhance TLR3 expression of fibroblast-like synoviocytes via TNF-α initiated p38 MAPK and NF-κB pathways

Based on pristane-induced arthritis (PIA), we found that T cells mediate TLR3 overexpression in fibroblast-like synoviocytes (FLS). The aim of this study is to determine key factors by which T cells induce TLR3 expression. Rat FLS were co-cultured with pristane primed T cell conditioned medium (PPT medium), and TLR3 expression of FLS was significantly induced. TNF-α, IFN-γ and IL-17 were dominantly expressed in PIA T cells. The overexpression of TLR3 and its related genes in FLS co-cultured with PPT medium could be reduced through blocking TNF-α pathway. CD4+ T cells from spleen of PIA rats showed increase of TNF-α secretion. P38 MAPK and NF-κB were activated in FLS by PPT medium, and their inhibitors decreased TLR3 upregulation significantly. Finally, TNF-α induced TLR3 expression was confirmed in human synovial cells. Summarily, TNF-α derived from pristane primed T cells induced TLR3 expression of FLS through activating p38 MAPK and NF-κB pathways.

MicroRNA-26a negatively regulates toll-like receptor 3 expression of rat macrophages and ameliorates pristane induced arthritis in rats

IntroductionAbnormal toll-like receptor (TLR)3 signaling plays an indispensable role in pathogenesis of both experimental and human rheumatoid arthritis, and microRNAs (miRNAs) might orchestrate this signaling pathway. This study was performed to determine the relationship between miR-26a and TLR3 in rat macrophages and to observe effects of miR-26a mimic on pristane induced arthritis (PIA) in rats.MethodsDual luciferase reporter assay was used to validate the direct interaction between miR-26a (a candidate miRNA to target tlr3 mRNA) and tlr3 3′UTR. MiR-26a regulation on TLR3 gene expression was determined using RT-qPCR and Western blotting after miR-26a mimics and inhibitors were transfected into rat macrophage line NR8383 cells. Poly I:C (TLR3 ligand) was used to trigger TLR3 activation, and mRNA expression of its downstream cytokines interferon (ifn)-β and tumor necrosis factor (tnf)-α was accordingly detected to determine the regulation of TLR3 signaling. Expressions of TLR3 and miR-26a were detected during rat bone marrow derived macrophage (BMDM) induction, in pristane stimulated NR8383 cells and spleens from methotrexate (MTX) treated PIA rats. A miR-26a mimic was administrated intraperitoneally to PIA rats, and arthritis severity was evaluated by macroscopic or microscopic observations.ResultsDirect target relationship between miR-26a and tlr3 mRNA in rats was confirmed. Modifications of miR-26a function by transfection of miR-26a mimics and inhibitors exhibited corresponding repression and augmentation of TLR3 and its signaling downstream cytokine expressions in NR8383 cells. The alteration of miR-26a expression was negatively related with TLR3 expression during BMDM induction, in pristane-primed NR8383 cells and PIA rat spleens. Moreover, both abnormal expressions were rescued in MTX treated arthritis rat spleens. The miR-26a mimic treatment displayed the depression of TLR3 expression and ameliorated the disease severity in the rats with pristane induced arthritis.ConclusionsMiR-26a negatively regulates TLR3 signaling via targeting of TLR3 itself in rat macrophages, and this finding provides a novel insight into abnormal TLR3 overexpression during experimental arthritis.

FRI0024 Antagonizing endosomal toll-like receptors diminishes inflammatory arthritis

BackgroundRheumatoid arthritis (RA), the most common chronic inflammatory joint disease, has a definite autoimmune background. There is evidence that release of endogenous nucleic acids triggers autoimmune reactions important for the...

Overexpression of Toll‐Like Receptor 3 in Spleen is Associated with Experimental Arthritis in Rats

This study is to investigate the regulation of Toll-like receptor (TLR) expression in systemic immune reactions in different arthritis rat models, which will provide evidence to understand the mechanisms of rheumatoid arthritis further. Arthritis-susceptible DA rats were used to induce arthritis by pristane or collagen type II, and TLR2, 3, 4 and 7 expression levels in spleen were detected by real-time quantitative polymerase chain reaction. TLR3 mRNA expression in spleen of both collagen-induced arthritis and pristane-induced arthritis (PIA) rats was increased significantly at 26 and 70 days after arthritis induction. The overexpression of TLR3 was confirmed by Western blotting. Methotrexate was administrated peritoneally to PIA rats, and phytol was applied subcutaneously to PIA rats. Both methotrexate and phytol treatment could alleviate arthritis severity and block TLR3 induction. However, in arthritis-resistant E3 rats injected with pristane, TLR3 expression of spleen was unaltered. PIA in MHC congenic DA.1U rats had mild symptoms, whereas TLR3 mRNA expression in spleen of DA.1U rats showed an impaired induction at D26. So we conclude that overexpression of splenic TLR3 is strongly associated with arthritis in rats, which suggests that TLR3 should be a most vital TLR in spleen to regulate the initiation and development of experimental arthritis and may be as an intriguing therapeutic opportunity for human rheumatoid arthritis.

Induction of toll-like receptor 2 positive antigen-presenting cells in spleen of pristane-induced arthritis in rats

Toll-like receptors (TLRs) have been found to contribute to the pathogenesis of rheumatoid arthritis (RA). The aim of this study is to investigate the regulation and potential role of TLR2 in spleen of pristane-induced arthritis (PIA) rat, which can be used to further understand the mechanisms of RA. Arthritis in DA rats was induced by pristane. TLR2 expression in spleen was detected by real-time quantitative PCR and western blotting, and TLR2 expression at both mRNA and protein levels was upregulated in PIA rats. Peptidoglycan (PGN) was systemically administrated to PIA rats, and arthritis severity was evaluated macroscopically and microscopically. Results showed that systemic administration of PGN to PIA rats obviously deteriorated arthritis severity. TLR2 expression on splenocytes and different types of immune cells was measured by flow cytometry. And it was found that TLR2 was mainly expressed on antigen-presenting cells (APCs) of spleen, and the proportion of TLR2(+) dendritic cells and macrophages in spleen of PIA rats was increased remarkably. Thus, we conclude that the induction of TLR2(+) APCs in spleen may participate in the maintenance of PIA.

Arthritis is associated with T-cell-induced upregulation of Toll-like receptor 3 on synovial fibroblasts

IntroductionToll-like receptors (TLRs) are likely to play crucial roles in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to determine the key TLRs in synovium and explore their roles in the activation of fibroblast-like synoviocytes (FLSs) mediated by T cells in arthritis.MethodsPristane-induced arthritis (PIA) was established by subcutaneous injection with pristane at the base of the rat's tail. TLR expression in synovium from PIA rats was detected at different time points by performing real-time PCR. Polyinosinic:polycytidylic acid (poly(I:C)) was intra-articularly administrated to PIA rats, and arthritis was monitored macroscopically and microscopically. Synovial TLR3 was detected by immunohistochemical staining. Rat FLSs were stimulated with pristane-primed T cells or pristane-primed, T-cell conditioned medium. The intervention of TLR3 in FLSs was achieved by specific short-hairpin RNA (shRNA) or an antibody. The migration ability of FLSs was measured by using the scratch test, and gene expression was detected by using real-time PCR. FLSs from RA patients were stimulated with various cytokines and TLR ligands, and TLR3 expression was detected by performing real-time PCR. In addition, with different concentrations of poly(I:C) stimulation, TLR3 expression of FLSs from RA patients and patients with osteoarthritis (OA) was compared.ResultsSynovium TLR3 displayed early and persistent overexpression in PIA rats. TLR3 was expressed in FLSs, and local treatment with poly(I:C) synergistically aggravated the arthritis. Rat FLSs co-cultured with pristane-primed T cells showed strengthened migration ability and significant upregulation of TLR3, IFN-β, IL-6 and matrix metalloproteinase 3 (MMP3) expression, which could also be induced by pristane-primed, T-cell conditioned medium. The upregulation of cytokines and MMPs was blocked by shRNA or TLR3 antibodies. In RA FLSs with cytokine or TLR ligand stimulation, TLR3 expression exhibited remarkable upregulation. Furthermore, RA FLSs showed higher reactivity than OA FLSs to poly(I:C).ConclusionsTLR3 in the synovium of PIA rats was overexpressed, and activation of the TLR3 signaling pathway could aggravate this arthritis. The induction of TLR3 in FLSs resulted from T cell-derived inflammatory stimulation and could further mediate FLS activation in arthritis. We conclude that TLR3 upregulation of FLSs activated by T cells results in articular inflammation.

Toll-like receptor 3 upregulation in macrophages participates in the initiation and maintenance of pristane-induced arthritis in rats

IntroductionToll-like receptors (TLRs) are involved in both innate and adaptive immune responses and are likely to play a complex role in the pathogenesis of human rheumatoid arthritis (RA) and experimental arthritis. The objective of this study was to identify the key TLR in pristane-induced arthritis (PIA), a rat model for RA, and to clarify its roles in the initiation and maintenance of arthritis.MethodsArthritis in DA rats was induced by pristane and the severity was evaluated by macroscopic and microscopic score systems. Spleen TLR and cytokine expression was detected at different time points by real-time polymerase chain reaction (PCR) and flow cytometry. Polyinosine-polycytidylic acid (polyI:C, a ligand of TLR3) or TLR3 specific short-hairpin RNA plasmid for RNA interference was administrated to PIA rats in vivo. Serum nitrogen oxide concentration was determined by Griess method, and tumor necrosis factor alpha (TNF-α) was determined by L929 biotest. In splenic macrophages, TLR3 expression was measured by flow cytometry. A rat macrophage cell line (NR8383) was stimulated by pristane, and anti-TLR3 antibody were used to block TLR3 pathway. TLR3 and cytokine expression in NR8383 were detected by real-time PCR.ResultsBy screening the TLR expression profile in spleen of DA rats after pristane injection, we found that TLR3 was the most early and prominently upregulated TLR. Both TLR3 mRNA and protein expression of spleen were upregulated at 6 and 26 days after pristane injection. Furthermore, administration of polyI:C exacerbated, whereas RNA interference targeting TLR3 ameliorated, the arthritis. Particularly, TLR3 expression was induced in splenic macrophages of PIA rats, and also in the NR8383 cell line after pristane stimulation in a dose- and time- dependent manner. Upregulation of interferon beta (IFN-β) and TNF-α by pristane stimulation was blocked by anti-TLR3 antibody in NR8383.ConclusionsTLR3 plays a pivotal role in the initiation and development of PIA which may dependent on macrophage. These findings are useful to understand the pathogenesis of RA and may provide an intriguing therapeutic opportunity for RA.

Housekeeping gene stability in pristane-induced arthritis and antigen-induced pulmonary inflammation of rats

Aim of this study is to explore the expression stability of 12 commonly used housekeeping genes in inflammation condition. Two rat models, pristane-induced arthritis (PIA) and antigen-induced pulmonary inflammation (AIPI) were established and reverse transcription-PCR was performed from rat spleens and inguinal lymph nodes. The reference gene expression fluctuated in the immune-associated tissues and 18S rRNA expression from the various inflammatory conditions was the most stably in all examined gene expression. Besides, peptidylprolyl isomerase A and beta-glucuronidase in PIA spleens, beta-actin and GAPDH in PIA inguinal lymph nodes, and peptidylprolyl isomerase A and beta-actin in AIPI spleens, expressed more stably than others. These results were further confirmed by expression study of TNF-alpha in PIA rat inguinal lymph nodes. We suggest that the stable gene mentioned above or geometric mean of three best references in each experimental condition should be used as the reference and the variable ones should be avoided in similar study designs.