Wenhua Zhu

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.

A novel long noncoding RNA Lnc-HC binds hnRNPA2B1 to regulate expressions of Cyp7a1 and Abca1 in hepatocytic cholesterol metabolism

Cholesterol metabolism disorder in hepatocytes predicts a higher risk of metabolic syndrome (MetS). Long noncoding RNAs (lncRNAs) have emerged as critical players in cellular cholesterol metabolism, but their functions are not systematically clarified. Here, we have identified a novel lncRNA named lnc‐HC negatively regulating cholesterol metabolism within hepatocytes through physical interaction with hnRNPA2B1. By further binding to the target messenger RNA of Cyp7a1 or Abca1, the lnc‐HC‐hnRNPA2B1 complex decreases expressions of the two genes that are implicated in cellular cholesterol excretion. lnc‐HC knockdown can strongly recover the cholesterol disorder in vivo. In the upstream pathway, lnc‐HC is up‐regulated by high cholesterol by the transcription activator, CCAAT/enhancer‐binding protein beta. Conclusion: These findings suggest a subtle feed‐forward regulation of lnc‐HC in cholesterol metabolism and define a novel line of evidence by which lncRNAs modulate the metabolic system at the post‐transcriptional level. (Hepatology 2016;64:58‐72)

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 rats 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.

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.

TLR3 and TLR7 modulate IgE production in antigen induced pulmonary inflammation via influencing IL-4 expression in immune organs.

Background Toll-like receptors (TLRs) as pattern recognition receptors, participate in both innate and adaptive immune responses, and seem to play an important role in the pathogenesis of asthma. This study aimed to identify key TLRs involved in antigen induced pulmonary inflammation (AIPI), a rat model for asthma, and to explore the role of TLRs in the disease development. Methods and Findings E3 rats were sensitized with ovalbumin (OVA)/alum intraperitoneally and intranasally challenged with OVA to induce AIPI model. TLR1-9 and cytokine mRNA expression in spleen, lung and mediastinal lymph node (mLN) tissues were screened by quantitative real-time polymerase chain reaction. TLR7 expression was found to be significantly down-regulated in spleen while TLR3 and TLR8 expression was up-regulated in mLN of AIPI rats. Furthermore, imiquimod (a ligand of TLR7) and TLR3 specific short-hairpin RNA plasmid for RNA interference were administrated, respectively, in vivo to AIPI rats to observe their effects on the disease by assessing various asthmatic parameters. The numbers of total cells, eosinophils, macrophages and lymphocytes were counted according to differential morphology in bronchoalveolar lavage fluid. Serum IgE and OVA specific IgG1 concentration was detected by enzyme-linked immunosorbent assay. The results showed that both TLR7 ligand treatment and TLR3 RNAi in vivo decreased serum IgE level and interleukin-4 mRNA expression. Conclusion/Significance TLR3 in mLN and TLR7 in spleen both systemically modulate disease development in AIPI rats via altering serum IgE concentration relevant to Th2 responses. And these findings may provide an important clue for further research in the asthma pathogenesis and suggest a new remedy for asthma treatment.

Genetic analysis of SNPs in CACNA1C and ANK3 gene with schizophrenia: A comprehensive meta‐analysis

Recently, genome‐wide association studies (GWAS), meta‐analyses, and replication studies focusing on bipolar disorder (BD) have implicated the α‐1C subunit of the L‐type voltage‐dependent calcium channel (CACNA1C) and ankyrin 3 (ANK3) genes in BD. Based on the hypothesis that both schizophrenia (SZ) and BD may share some common genetic risk factors, we investigated the association of CACNA1C and ANK3 with SZ using meta‐analytic techniques, combining all published data up to April 2015. Nine teams, including four European decent samples and five Asian samples, contributed 14,141 cases and 30,679 controls for the analysis of CACNA1C rs1006737 and SZ. A significant difference was identified between patients and controls for the A‐allele of rs1006737 in combined studies (Z = 6.02, P = 1.74E‐09), in European studies (Z = 4.08, P = 4.50E‐05), and in Asian studies (Z = 4.60, P = 4.22E‐06). Meanwhile, for the T‐allele of ANK3 rs10761482 (1,794 cases versus 1,395 controls), a significant association was observed in combined samples (Z = 2.06, P = 0.04) and in Asian samples (Z = 3.10, P = 0.002). In summary, our study provides further evidence for the positive association of CACNA1C and ANK3 with SZ. These results support the hypothesis that both SZ and BD share common genetic risk factors. Further research is needed to examine the functions of CACNA1C and ANK3, and their interacting partners in the molecular, developmental, and pathophysiological processes in SZ.

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.

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.

Methotrexate ameliorates pristane-induced arthritis by decreasing IFN-γ and IL-17A expressions

ObjectiveThis study was carried out to test the effects of methotrexate (MTX) and black seed oil (BSO) on pristane-induced arthritis (PIA) in rats. Methods: Inbred dark agouti (DA) rats were induced by a single subcutaneous injection of pristane, and then treated with MTX or BSO. Arthritis severity was evaluated macroscopically and microscopically. Plasma nitric oxide (NO) concentration was determined by the Griess method and cytokine mRNA expression in the spleen was detected by the real-time reverse transcription-polymerase chain reaction (RT-PCR).ResultsThe clinical arthritis severity was decreased after MTX treatment, while the BSO groups did not show significant changes compared with the disease group. The plasma NO level of the MTX group was significantly decreased compared with the disease group, but the BSO groups showed no difference from the disease group in plasma NO levels. The interferon-γ (IFN-γ) and interleukin-17A (IL-17A) mRNA expressions in the spleens were significantly decreased in the MTX group, but only showed a declining trend in the BSO groups compared with the disease group. Neither MTX nor BSO had an effect on the mRNA expressions of IL-4, transforming growth factor β (TGF-β), and tumor necrosis factor-α (TNF-α) in the spleen.ConclusionsMTX, but not BSO, can reduce the arthritis severity and decrease the mRNA expressions of IFN-γ and IL-17A in pristane-induced arthritis of rats.

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

Objective and designAim of this study is to explore the expression stability of 12 commonly used housekeeping genes in inflammation condition.Materials and methodsTwo 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.ResultsThe 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.ConclusionWe 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.