Abrogation of CC chemokine receptor 9 ameliorates collagen-induced arthritis of mice

Introduction

Biological drugs are effective in patients with rheumatoid arthritis (RA), but increase severe infections. The CC chemokine receptor (CCR) 9 antagonist was effective for Crohn’s disease without critical adverse effects including infections in clinical trials. The present study was carried out to explore the pathogenic roles of chemokine (C-C motif) ligand (CCL) 25 and its receptor, CCR9, in autoimmune arthritis and to study if the CCR9 antagonist could be a new treatment for RA.

Methods

CCL25 and CCR9 expression was examined with immunohistochemistry and Western blotting. Concentration of interleukin (IL)-6, matrix metalloproteinase (MMP)-3 and tumor necrosis factor (TNF)-α was measured with enzyme-linked immunosorbent assays. Effects of abrogating CCR9 on collagen-induced arthritis (CIA) was evaluated using CCR9-deficient mice or the CCR9 antagonist, CCX8037. Fluorescence labeled-CD11b⁺ splenocytes from CIA mice were transferred to recipient CIA mice and those infiltrating into the synovial tissues of the recipient mice were counted.

Results

CCL25 and CCR9 proteins were found in the RA synovial tissues. CCR9 was expressed on macrophages, fibroblast-like synoviocytes (FLS) and dendritic cells in the synovial tissues. Stimulation with CCL25 increased IL-6 and MMP-3 production from RA FLS, and IL-6 and TNF-α production from peripheral blood monocytes. CIA was suppressed in CCR9-deficient mice. CCX8037 also inhibited CIA and the migration of transferred CD11b⁺ splenocytes into the synovial tissues.

Conclusions

The interaction between CCL25 and CCR9 may play important roles in cell infiltration into the RA synovial tissues and inflammatory mediator production. Blocking CCL25 or CCR9 may represent a novel safe therapy for RA.     

miR‐124a inhibits the proliferation and inflammation in rheumatoid arthritis fibroblast‐like synoviocytes via targeting PIK3/NF‐κB pathway

Abnormal hyperplasia of fibroblast‐like synoviocytes (FLS) leads to the progression of rheumatoid arthritis (RA). This study aimed to investigate the role of miR‐124a in the pathogenesis of RA. The viability and cell cycle of FLS in rheumatoid arthritis (RAFLS) were evaluated by Cell Counting Kit 8 and flow cytometry assay. The expression of PIK3CA, Akt, and NF‐κB in RAFLS was examined by real‐time PCR and Western blot analysis. The production of tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 was detected by ELISA. The joint swelling and inflammation in collagen‐induced arthritis (CIA) mice were examined by histological and immunohistochemical analysis. We found that miR‐124a suppressed the viability and proliferation of RAFLS and increased the percentage of cells in the G1 phase. miR‐124a suppressed PIK3CA 3'UTR luciferase reporter activity and decreased the expression of PIK3CA at mRNA and protein levels. Furthermore, miR‐124a inhibited the expression of the key components of the PIK3/Akt/NF‐κB signal pathway and inhibited the expression of pro‐inflammatory factors TNF‐α and IL‐6. Local overexpression of miR‐124a in the joints of CIA mice inhibited inflammation and promoted apoptosis in FLS by decreasing PIK3CA expression. In conclusion, miR‐124a inhibits the proliferation and inflammation in RAFLS via targeting PIK3/NF‐κB pathway. miR‐124a is a promising therapeutic target for RA.     

IL-23 Dependent and Independent Stages of Experimental Arthritis: No Clinical Effect of Therapeutic IL-23p19 Inhibition in Collagen-induced Arthritis

IL-23p19 deficient mice have revealed a critical role of IL-23 in the development of experimental autoimmune diseases, such as collagen-induced arthritis (CIA). Neutralizing IL-23 after onset of CIA in rats has been shown to reduce paw volume, but the effect on synovial inflammation and the immunological autoimmune response is not clear. In this study, we examined the role of IL-23 at different stages of CIA and during T cell memory mediated flare-up arthritis with focus on changes in B cell activity and Th1/Th17 modulation. Anti-IL-23p19 antibody (anti-IL23p19) treatment, starting 15 days after the type II collagen (CII)-immunization but before clinical signs of disease onset, significantly suppressed the severity of CIA. This was accompanied with significantly lower CII-specific IgG1 levels and lower IgG2a levels in the anti-IL-23p19 treated mice compared to the control group. Importantly, neutralizing IL-23 after the first signs of CIA did not ameliorate the disease. This was in contrast to arthritic mice that underwent an arthritis flare-up since a significantly lower disease score was observed in the IL-23p19 treated mice compared to the control group, accompanied by lower synovial IL-17A and IL-22 expression in the knee joints of these mice. These data show IL-23-dependent and IL-23-independent stages during autoimmune CIA. Furthermore, the memory T cell mediated flare-up arthritis is IL-23-mediated. These data suggest that specific neutralization of IL-23p19 after onset of autoimmune arthritis may not be beneficial as a therapeutic therapy for patients with rheumatoid arthritis (RA). However, T cell mediated arthritis relapses in patients with RA might be controlled by anti-IL-23p19 treatment.     

Relationship between angiogenesis and inflammation in experimental arthritis

Background. Angiogenesis is involved in rheumatoid arthritis (RA) leading to leucocyte recruitment and inflammation in the synovium. Furthermore, synovial inflammation itself further potentiates endothelial proliferation and angiogenesis. In this study, we aimed at evaluating the reciprocical relationship between synovial inflammation and angiogenesis in a RA model, namely collagen-induced arthritis (CIA). Methods. CIA was induced by immunization of DBA/1 mice with collagen type II in adjuvant. Endothelial cells were detected using a GSL-1 lectin-specific immunohistochemical staining on knee joint sections. Angiogenesis, clinical scores and histological signs of arthritis were evaluated from the induction of CIA until the end of the experiment. Angiogenesis was quantified by counting both the isolated endothelial cells and vessels stained on each section. To evaluate the effect of increased angiogenesis on CIA, VEGF gene transfer was performed using an adeno-associated virus encoding VEGF (AAV-VEGF), by intra-muscular or intra-articular injection in mice with CIA. Results. We showed an increase in synovial angiogenesis from day 6 to day 55 after CIA induction, and, moreover, joint vascularization and clinical scores of arthritis were correlated (p < 0.0001, r = 0.61). Vascularization and histological scores were also correlated (p = 0.0006, r = 0.51). Systemic VEGF overexpression in mice with CIA was followed by an aggravation of arthritis as compared to AAV-lacZ control group (p < 0.0001). In contrast, there was no difference in clinical scores between control mice and mice injected within the knee with AAV-VEGF, even if joint vascularization was higher in this group than in all other groups (p = 0,05 versus non-injected group). Intra-articular AAV-VEGF injections induced more severe signs of histological inflammation and bone destruction than AAV-Lac Z or no injection. Conclusion. Angiogenesis and joint inflammation evolve in parallel during collagen-induced arthritis. Furthermore, this work shows that exogenous VEGF can aggravate CIA. It is direct evidence that the increase in joint vascularization leads to an exacerbation of arthritis. Taken together, these results emphasize the role of angiogenesis in inflammatory arthritis. It also suggests an early involvement of angiogenesis in joint inflammation.     

Vitamin K and rheumatoid arthritis

Vitamin K2 [menaquinone-4 (MK-4)] has been reported to induce apoptosis in hepatocellular carcinoma, leukemia, and MDS cell lines. The effects of MK-4 on the development of arthritis have never been addressed so far. In this study, we investigated the effect of MK-4 upon the proliferation of rheumatoid synovial cells and the development of arthritis in collagen-induced arthritis (CIA). We analyzed the effect of MK-4 on the proliferation of fibroblast-like synoviocytes (FLSs) using the 3-(4,5-demethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The proapoptotic effect of MK-4 upon FLS was investigated with annexin V staining and DNA fragmentation and caspase 3/7 assays. Moreover, we analyzed the effect of MK-4 on the development of CIA in female dark agouti rats. Our results indicated that MK-4 inhibited the proliferation of FLS and the development of CIA in a dose-dependent manner. We concluded that MK-4 may represent a new agent for the treatment of RA in the setting of combination therapy with other disease-modifying antirheumatic drugs.     

Evaluation of the cholesterol influence in type II collagen-induced arthritis in DBA/1J mice: an autoradiographic study

In order to verify the cholesterol influence in RA severity in DBA/1J mice, we quantified the cholesterol present in the knee joints of normal (N) and with collagen II induced arthritis (CIA). Forty male DBA/1J mice, were divided in normal (n=20) and CIA group (n=20). Mice in CIA group were injected with 100 μg of collagen II emulsified in Freund's complete adjuvant. Sixteen DBA/1J (8 N and 8 CIA) received an injection of 2.96 × 10⁶ Bq of 3H-cholesterol and were anesthetized and sacrificed. Semi-fine sections were covered with LM-1 emulsion, exposed for six weeks and developed. Collagen induced edema, erythema and dysfunction of knee joints in CIA group. Radioactive cholesterol was located more on the synovial membrane, where we found the greatest density of silver grains, significantly ( P <0.0001) higher in group CIA vs. controls (61±2.3 × 18±0.7). We conclude that the cholesterol deposits on the synovial membrane is related to CIA severity.     

Inhibition of TNF-induced IL-6 by the TWEAK-Fn14 interaction in rheumatoid arthritis fibroblast like synoviocytes

ObjectivesTNF-like weak inducer of apoptosis (TWEAK), a member of the TNF superfamily, has been shown to increase cytokine production by rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). In this study, we determined the effect of interaction between TWEAK and its receptor fibroblast growth factor-inducible-14 (Fn14) on cytokine expression in RAFLS.MethodsRAFLS were obtained from surgical synovial specimens and used at passage 5–10. Cytokine protein and mRNA expression were measured with ELISA and real time-PCR, respectively. Apoptotic cells were detected by TUNEL assay. RelB activation was detected by Western blot analysis.ResultsTWEAK inhibited IL-6 production from total synovial cells from RA. TWEAK weakly induced FLS IL-6 and IL-8, but in contrast TWEAK dose-dependently inhibited IL-6 and IL-8 production by TNFα-activated FLS. TWEAK did not induce apoptosis in FLS but inhibited proliferation of TNFα-activated FLS. TWEAK induced RelB activation and suppressed IL-6 mRNA expression in TNFα-activated FLS and both of these phenomenon were abolished by inhibition of new protein synthesis with cycloheximide.ConclusionsTWEAK has a previously unsuspected inhibitory effect on cytokine production by TNFα-activated RAFLS. This observation suggests that the effects of TWEAK on cytokine expression varies with the pro-inflammatory context, and that in TNFα-activated states such as RA TWEAK may have a net inhibitory effect.     

小鼠胶原诱导性关节炎模型的建立及其免疫学变化研究

目的 胶原诱导性关节炎模型（collagen induced arthritis,CIA）是研究类风湿性关节炎发病机制和治疗药物筛选的理想模型,也是目前国际上公认的关节炎模型.但是,目前鲜见Ⅱ型胶原诱导CIA模型的系统免疫学变化的报道.因此,本研究采用DBA/1小鼠诱导了CIA模型,并对其免疫学改变进行了系统研究.方法 将牛Ⅱ型胶原与完全弗氏佐剂混和并充分乳化,于DBA/1小鼠尾根部皮内注射进行初次免疫,20 d后同样方法进行再次免疫.应用千分尺测量CIA模型小鼠的左右两侧足掌厚度,并进行关节炎评分.酶联免疫吸附试验测定小鼠血清Ⅱ型胶原特异性抗体,Luminex技术和αLISA技术测定血清及培养上清中的细胞因子水平.结果CIA小鼠于造模后23 d开始,陆续出现前肢、后肢的红肿、功能障碍,发病率高达100％,且随着时间的延长其关节肿胀程度呈进行性加重,关节炎评分增高.CIA小鼠脾脏指数较正常组明显升高,且Ⅱ型胶原刺激的特异性T细胞增殖明显增强.细胞因子检测结果表明,脾细胞培养上清中IFN-γ和1L-4含量及IFN-γ/IL-4比值明显升高,TNF-α和IL-1β水平亦显著升高.此外,CIA小鼠血清中存在高水平的Ⅱ型胶原特异性抗体.结论 Ⅱ型胶原诱导CIA模型发病率高,免疫学改变以Th1细胞因子升高为主,兼有细胞免疫功能及体液免疫功能损伤.     

Methionine aminopeptidase-2 blockade reduces chronic collagen-induced arthritis: potential role for angiogenesis inhibition

The enzyme methionine aminopeptidase-2 (MetAP-2) is thought to play an important function in human endothelial cell proliferation, and as such provides a valuable target in both inflammation and cancer. Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with increased synovial vascularity, and hence is a potential therapeutic target for angiogenesis inhibitors. We examined the use of PPI-2458, a selective non-reversible inhibitor of MetAP-2, in disease models of RA, namely acute and chronic collagen-induced arthritis (CIA) in mice. Whilst acute CIA is a monophasic disease, CIA induced with murine collagen type II manifests as a chronic relapsing arthritis and mimics more closely the disease course of RA. Our study showed PPI-2458 was able to reduce clinical signs of arthritis in both acute and chronic CIA models. This reduction in arthritis was paralleled by decreased joint inflammation and destruction. Detailed mechanism of action studies demonstrated that PPI-2458 inhibited human endothelial cell proliferation and angiogenesis in vitro, without affecting production of inflammatory cytokines. Furthermore, we also investigated release of inflammatory cytokines and chemokines from human RA synovial cell cultures, and observed no effect of PPI-2458 on spontaneous expression of cytokines and chemokines, or indeed on the angiogenic molecule vascular endothelial growth factor (VEGF). These results highlight MetAP-2 as a good candidate for therapeutic intervention in RA.     

Involvement of TNF-like weak inducer of apoptosis in the pathogenesis of collagen-induced arthritis

TNF-like weak inducer of apoptosis (TWEAK) is a type II membrane protein belonging to the TNF family that regulates apoptotic cell death, cellular proliferation, angiogenesis, and inflammation. However, the role of TWEAK in the pathogenesis of rheumatoid arthritis (RA) remains unclear. In this study, we have investigated the effect of neutralizing anti-TWEAK mAb on the development of collagen-induced arthritis (CIA), a well-established murine model of RA. Administration of anti-TWEAK mAb significantly ameliorated paw swelling, synovial hyperplasia, and infiltration of inflammatory cells. The levels of proinflammatory chemokines such as MCP-1 and MIP-2 in serum and knee joints were reduced by this treatment. Consistently, recombinant TWEAK enhanced the proliferation of MCP-1 and MIP-2 production by synovial cells from CIA mice in vitro. Histological examination also revealed that the treatment with anti-TWEAK mAb suppressed the development of small vessels in synovial tissues. These results indicated anti-inflammatory and antiangiogenic effects of the TWEAK blockade in CIA, which may be also beneficial for the treatment of RA.     

Fibroblast-like synoviocytes from rheumatoid arthritis patients express functional IL-15 receptor complex: endogenous IL-15 in autocrine fashion enhances cell proliferation and expression of Bcl-x(L) and Bcl-2

The hallmarks of rheumatoid arthritis (RA) are leukocytic infiltration of the synovium and expansiveness of fibroblast-like synoviocytes (FLS). The abnormal proliferation of FLS and their resistance to apoptosis is mediated, at least in part, by present in RA joints proinflammatory cytokines and growth factors. Because IL-15 exerts properties of antiapoptotic and growth factors, and is produced by RA FLS, we hypothesized that IL-15 participates in RA FLS activation. To test this hypothesis, we first examined whether RA FLS express chains required for high affinity functional IL-15R. Indeed, RA FLS express IL-15Ralpha at mRNA and protein levels. Moreover, we confirmed the presence of IL-2Rbeta and common gamma-chains. Interestingly, TNF-alpha or IL-1beta triggered significant elevation of IL-15Ralpha chain at mRNA and protein levels. Next, we investigated the effects of exogenous or endogenous IL-15 on Bcl-2 and Bcl-x(L) expression, FLS proliferation, and apoptosis. Exogenous IL-15 enhanced RA FLS proliferation and increased the level of mRNA-encoding Bcl-x(L). To test the role of endogenous IL-15 in the activation of RA FLS, an IL-15 mutant/Fcgamma2a protein exerting properties of specific antagonist to the IL-15Ralpha chain was used. We found that blocking IL-15 biological activities using this protein substantially reduced endogenous expression of Bcl-2 and Bcl-x(L), and RA FLS proliferation that was reflected by increased apoptosis. Thus, we have demonstrated that a distinctive phenotype of RA FLS, i.e., persistent activation, proliferation, and resistance to apoptosis, is related to the autocrine activation of IL-15Rs by FLS-derived IL-15.     

Local treatment with the selective IkappaB kinase beta inhibitor NEMO-binding domain peptide ameliorates synovial inflammation

Nuclear factor (NF)-kappaB is a key regulator of synovial inflammation. We investigated the effect of local NF-kappaB inhibition in rat adjuvant arthritis (AA), using the specific IkappaB kinase (IKK)-beta blocking NF-kappaB essential modulator-binding domain (NBD) peptide. The effects of the NBD peptide on human fibroblast-like synoviocytes (FLS) and macrophages, as well as rheumatoid arthritis (RA) whole-tissue biopsies, were also evaluated. First, we investigated the effects of the NBD peptide on RA FLS in vitro. Subsequently, NBD peptides were administered intra-articularly into the right ankle joint of rats at the onset of disease. The severity of arthritis was monitored over time, rats were sacrificed on day 20, and tissue specimens were collected for routine histology and x-rays of the ankle joints. Human macrophages or RA synovial tissues were cultured ex vivo in the presence or absence of NBD peptides, and cytokine production was measured in the supernatant by enzyme-linked immunosorbent assay. The NBD peptide blocked interleukin (IL)-1-beta-induced IkappaB alpha phosphorylation and IL-6 production in RA FLS. Intra-articular injection of the NBD peptide led to significantly reduced severity of arthritis (p < 0.0001) and reduced radiological damage (p = 0.04). This was associated with decreased synovial cellularity and reduced expression of tumor necrosis factor (TNF)-alpha and IL-1-beta in the synovium. Incubation of human macrophages with NBD peptides resulted in 50% inhibition of IL-1-beta-induced TNF-alpha production in the supernatant (p < 0.01). In addition, the NBD peptide decreased TNF-alpha-induced IL-6 production by human RA synovial tissue biopsies by approximately 42% (p < 0.01). Specific NF-kappaB blockade using a small peptide inhibitor of IKK-beta has anti-inflammatory effects in AA and human RA synovial tissue as well as in two important cell types in the pathogenesis of RA: macrophages and FLS. These results indicate that IKK-beta-targeted NF-kappaB blockade using the NBD peptide could offer a new approach for the local treatment of arthritis.     

Geldanamycin induces apoptosis and inhibits inflammation in fibroblast-like synoviocytes isolated from rheumatoid arthritis patients

Rheumatoid arthritis (RA) is an autoimmune disease of the joints characterized by synovial hyperplasia and chronic inflammation. Fibroblasts-like synoviocytes (FLS), major cells in the synovium, together with infiltrated leukocytes, contribute greatly to RA progression. In our study, we hypothesized that geldanamycin (GA), a cancer drug might be able to inhibit RA FLS growth. To test the idea, RA FLS were isolated and cultured for cancer drug test. The results showed that GA can specifically inhibit the growth of RA FLS compared with normal FLS. Essentially, GA was found to promote reactive oxygen species production in RA FLS and induce programmed cell death. The annexinV/propidium iodide and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining confirmed that GA can directly induce apoptosis and subsequently inhibit the growth of RA FLS, which was also confirmed by Western blot assay. In addition, our data demonstrated that inflammation was inhibited by suppressed nuclear factor κB signaling pathway. The therapeutic effect of GA was explored in collagen-induced arthritis mice. In short, GA was a promising drug for the treatment of RA by specifically inhibiting the proliferation and inflammation of RA FLS.     

Suppression of immune induction of collagen-induced arthritis in IL-17-deficient mice

Interleukin-17 is a T cell-derived proinflammatory cytokine. This cytokine is suspected to be involved in the development of rheumatoid arthritis (RA) because this cytokine expression is augmented in synovial tissues of RA patients. The pathogenic roles of IL-17 in the development of RA, however, still remain to be elucidated. In this study, effects of IL-17 deficiency on collagen-induced arthritis (CIA) model were examined using IL-17-deficient mice (IL-17(-/-) mice). We found that CIA was markedly suppressed in IL-17(-/-) mice. IL-17 was responsible for the priming of collagen-specific T cells and collagen-specific IgG2a production. Thus, these observations suggest that IL-17 plays a crucial role in the development of CIA by activating autoantigen-specific cellular and humoral immune responses.     

Arginine-rich anti-vascular endothelial growth factor (anti-VEGF) hexapeptide inhibits collagen-induced arthritis and VEGF-stimulated productions of TNF-alpha and IL-6 by human monocytes

Vascular endothelial growth factor (VEGF) has been suggested to play a critical role in the pathogenesis of rheumatoid arthritis (RA). We previously identified a novel RRKRRR hexapeptide that blocked the interaction between VEGF and its receptor through the screening of peptide libraries. In this study, we investigated whether anti-VEGF peptide RRKRRR (dRK6) could suppress collagen-induced arthritis (CIA) and regulate the activation of mononuclear cells of RA patients. A s.c. injection of dRK6 resulted in a dose-dependent decrease in the severity and incidence of CIA and suppressed synovial infiltration of inflammatory cells in DBA/1 mice. In these mice, the T cell responses to type II collagen (CII) in lymph node cells and circulating IgG Abs to CII were also dose-dependently inhibited by the peptides. In addition, VEGF directly increased the production of TNF-alpha and IL-6 from human PBMC. Synovial fluid mononuclear cells of RA patients showed a greater response to VEGF stimulation than the PBMC of healthy controls. The major cell types responding to VEGF were monocytes. Moreover, anti-VEGF dRK6 inhibited the VEGF-induced production of TNF-alpha and IL-6 from synovial fluid mononuclear cells of RA patients and decreased serum IL-6 levels in CIA mice. In summary, we observed first that dRK6 suppressed the ongoing paw inflammation in mice and blocked the VEGF-induced production of proinflammatory cytokines. These data suggest that dRK6 may be an effective strategy in the treatment of RA, and could be applied to modulate various chronic VEGF-dependent inflammatory diseases.     

NK4 inhibits the proliferation and induces apoptosis of human rheumatoid arthritis synovial cells

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by proliferation and insufficient apoptosis of synovial cells. NK4 is a hepatocyte growth factor antagonist and is implicated in cell proliferation, viability, and apoptosis of many tumour cells. This study aimed to investigate the role of NK4 in the regulation of human RA synovial cell proliferation and apoptosis. Fibroblast‐like synoviocytes (FLSs) isolated from RA patients and MH7A synovial cells were subjected to MTT, flow cytometry, and Western blot analysis. We found that NK4 suppressed cell proliferation through cell cycle arrest at the G0/G1 phase and induced apoptosis in RA synovial cells. Furthermore, NK4 altered the expression of cell cycle and apoptosis‐related proteins such as cyclin D1, cyclin B1, PCNA, p21, p53, Bcl‐2, Bax, cleaved caspase‐9, and cleaved caspase‐3. Additionally, NK4 reduced the phosphorylation level of NF‐κB p65 and upregulated the expression of sirt1, but did not change the levels of p38 and p‐p38 in RA‐FLS and MH7A cells. In conclusion, NK4 inhibits the proliferation and induces apoptosis of human RA synovial cells. NK4 is a promising therapeutic target for RA. We demonstrated that NK4 inhibited cell proliferation by inducing apoptosis and arresting cell cycle in RA‐FLS and MH7A cells. The apoptotic effects of NK4 may be mediated in part by decreasing Bcl‐2 protein level, increasing Bax and caspase 3 protein levels, and inhibiting NF‐κB signalling in RA‐FLS and MH7A cells. These findings reveal potential mechanism underlying the role of NK4 in RA synovial cells and suggest that NK4 is a promising agent for RA treatment.     

Reactivity of rat bone marrow-derived macrophages to neurotransmitter stimulation in the context of collagen II-induced arthritis

IntroductionNumerous observations indicate that rheumatoid arthritis (RA) has a bone marrow component. In parallel, local synovial changes depend on neuronal components of the peripheral sympathetic nervous system. Here, we wanted to analyze whether collagen II-induced arthritis (CIA) has an impact on number, adhesion, apoptosis, and proliferation of the macrophage subset of bone marrow cells and how alterations in neurotransmitter microenvironment affect these properties.MethodsBone marrow-derived macrophages (BMMs) were isolated from Dark Agouti rats at different stages of CIA, and number, adhesion, caspase 3/7 activity, and proliferation were analyzed in the presence of acetylcholine (ACh), noradrenaline (NA), and vasoactive intestinal peptide (VIP).ResultsOpposed to enhanced CD11b⁺ (cluster of differentiation 11b-positive) and EMR1⁺ (epidermal growth factor-like module-containing mucin-like hormone receptor-like 1-positive) cells, characterizing the macrophage subset, in native bone marrow of rats with acute inflammatory arthritis, we found decreased numbers of CIA macrophages after enrichment and culture in comparison with healthy (control) animals. Adhesion studies revealed significantly reduced attachment to plastic in acute arthritis and collagen type I and fibronectin in chronic arthritis. Additionally, we found a strong reduction in proliferation of BMMs at CIA onset and in the chronic phase of CIA. Apoptosis remained unaffected. Neurotransmitter stimulation profoundly affected proliferation, adhesion, and apoptosis of BMMs from CIA and control rats, depending on disease time point. Cultured BMMs from CIA and control animals expressed neurotransmitter receptors for ACh, VIP and NA, but the expression profile seemed not to be affected by CIA.ConclusionsInduction of CIA distinctly inhibits proliferation of BMMs in low- and non-inflammatory phases and reduces attachment to plastic at the acute inflammatory arthritis stage and adhesion to collagen I and fibronectin at the chronic stage. Influence of neurotransmitter stimulation on adhesion, apoptosis, and proliferation is altered by CIA depending on disease stage. We suggest an altered reactivity of BMMs to neurotransmitter stimulation caused by CIA and maybe also by aging.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0684-4) contains supplementary material, which is available to authorized users.     

MiRNA-126 expression inhibits IL-23R mediated TNF-α or IFN-γ production in fibroblast-like synoviocytes in a mice model of collagen-induced rheumatoid arthritis

Both miR-126 and IL-23R affect rheumatoid arthritis (RA) procession. This study aimed to investigate the association of miR-126 and IL-23R and the possible modulation of miR-126 to RA pathogenesis. Serum, synovial tissue and synovial fluid were collected from patients with RA, and expression of miR-126, IL-23R, TNF-α and IFN-γ were detected. Fibroblast-like synoviocytes (FLS) was established using a collagen-induced arthritis mice model. The expression of miR-126 was manual intervened using pro-miR-126 and anti-miR-126 encoding lentivirus plasmids, or miR-126 agonists and corresponding negative controls. MiR-126 expression was inhibited in RA patients when compared with controls (P?<?0.05). TNF-α and IFN-γ production and IL-23R expression were significantly upregulated in RA patients when compared to controls (P?<?0.05). In pro-miR-126 treated FLS cells, the administration of pro-miR-126 plasmids upregulated miR-126, but inhibited IL-23R, TNF-α and IFN-γ expression or production. Moreover, the miR-126 agonist reversed the effects of the anti-miR-126 plasmid on FLS. These results revealed that miR-126 negative regulated the expression of IL-23R, TNF-α and IFN-γ. These results suggest the key impact of miR-126 on RA procession. Moreover, pro-miR-126 might be explored to be a potential therapy for RA.