Hypoxia Inducible Factor 1α Plays a Key Role in Remote Ischemic Preconditioning Against Stroke by Modulating Inflammatory Responses in Rats.

BACKGROUND: Limb remote ischemic preconditioning (RIPC) protects against brain injury induced by stroke, but the underlying protective mechanisms remain unknown. As hypoxia inducible factor 1α (HIF-1α) is neuroprotective in stroke and mediates neuroinflammation, we tested the hypothesis that HIF-1α is a key factor of RIPC against stroke by mediating inflammation. METHODS AND RESULTS: Stroke was induced by transient middle cerebral artery occlusion in rats, and RIPC was conducted in both hind limbs. The HIF-1α mRNA was examined by quantitative reverse transcription polymerase chain reaction after RIPC. In addition, inflammatory cytokines in the peripheral blood and brain were measured using the AimPlex multiplex immunoassays. Data showed that RIPC reduced the infarct size, improved neurological functions, and increased HIF-1α mRNA levels, interleukin (IL)-4, and IL-10 protein levels in the peripheral blood. Intraperitoneal injection of the HIF activator, dimethyloxaloylglycine, reduced the infarct size and inhibited interferon-γ protein levels, while promoting IL-4 and IL-10 protein levels, while decreasing interferon-γ protein levels in both the peripheral blood and ischemic brain. In addition, injection of dimethyloxaloylglycine had a synergistic effect with RIPC on reducing infarction and improving neurological functions, as well as decreasing interferon-γ in the peripheral blood and ischemic brain. In contrast, injection of the HIF inhibitor, acriflavine hydrochloride, abolished the protective effects of RIPC on infarction, and reduced IL-4 and IL-10 protein levels in both the peripheral blood and ischemic brain. CONCLUSIONS: We conclude that HIF-1α plays a key role in RIPC, likely mediated by a systemic modulation of the inflammatory response.

Analysis of long non-coding RNA expression profiles following focal cerebral ischemia in mice.

Long noncoding RNAs (lncRNAs) have a variety of biological functions and play key roles in many diseases. However, the knowledge of lncRNA function during a stroke is limited. We analyzed the expression profiles of lncRNAs in the brain ischemic region of mice after a 45min middle cerebral artery occlusion (MCAO) with a 48h reperfusion. Gene ontology and pathway analysis were used to elucidate the potential functions of the differentially expressed mRNAs. A total of 255 lncRNAs (217 up-regulated and 38 down-regulated) and 894 mRNAs (870 up-regulated and 24 down-regulated) showed significantly altered expression in the ischemic brain compared to the sham controls (fold change ≫>2, P≪0.05). The gene ontology terms were mainly associated with neutrophil chemotaxis, positive regulation of inflammatory response, cell cycle, positive regulation of apoptotic process, and apoptotic process. The pathway analysis indicated that the mRNAs were mainly associated with inflammatory pathways. Additionally, the interactions between the differentially expressed lncRNAs and mRNAs are revealed by a dynamic lncRNA-mRNA network. Our findings provide an overview of aberrantly expressed lncRNAs in stroke and further broaden the understanding of stroke pathogenesis.

Amelioration of myocarditis by HVEM-overexpressing dendritic cells through induction of IL-10-producing cells.

AIMS: Herpes virus entry mediator (HVEM) is considered to be a molecular 'switch' for immune responses, and a role in immune modification has been reported. The aim of this study was to assess whether HVEM-mediated immune suppression could protect against experimental autoimmune myocarditis (EAM) induced by myosin. METHODS AND RESULTS: We constructed HVEM-expressing adenovirus (AdHVEM) and fusion protein HVEM-Ig and evaluated their roles in immunoregulation in vitro and in vivo. Immunoregulation of dendritic cells (DCs) infected with recombinant virus or treated with HVEM-Ig was then studied. DCs transfected with AdHVEM (DC-AdHVEM) were protected against EAM, whereas HVEM-Ig had no protective effect. Further study showed that DC-AdHVEMs produced a regulatory cytokine, IL-10, which had further effects on induction of IL-10 producing CD4(+) T cells. This subset of T cells was then responsible for the protection against EAM. CONCLUSION: Myosin-DC-AdHVEM cell gene therapy appears to be a safe and effective way of inhibiting the development of EAM. The signal induced by HVEM seems to play different roles in different cells.

Reverse signaling using an inducible costimulator to enhance immunogenic function of dendritic cells.

A costimulatory signal from an inducible costimulator (ICOS) of T cells plays a critical role in immunological homeostasis. This study shows that the interaction of ICOSIg and its ligand (ICOSL) on mouse bone marrow-derived dendritic cells (DCs) induces a p38-MAPK dependent elevation of interleukin 6 (IL-6). It also enhances phagocytosis and the antigen-presentation function of DCs in vitro, further favoring cell-mediated immunity in vivo. As seen for other types of costimulator molecules expressed in the T cells in the CD28 family, it is shown here for the first time that ICOS can also deliver reverse signals through its ligand to ICOSL-expressing cells. These reverse signals in turn transfer positive immunogenic information to bone marrow-derived DCs. Our work therefore provides new recognition of an ICOSL/ICOS signal pathway in immunity and also supplies more evidence that this ICOSL/ICOS signal pathway is a reasonable target for therapeutic drugs.

[Construction and expression of regulatable recombinant adenovirus vector for mouse PLP-Ig chimeric protein].

AIM: To construct a regulatable recombinant adenovirus vector expressing mouse proteolipid protein (PLP)-Ig. METHODS: Total RNA was extracted from WT-1 hybridoma cells to clone Ig heavy chain Fc fragment gene. PCR was carried out to link PLP(139-151) gene and signal peptide with Fc fragment gene. pTRE-shuttle vector was used to ligate PLP-Ig gene and the backbone of the replication-incompetent adenoviral vector. After confirming the desired recombinant adenovirus by PCR and restriction endonuclease digestion analysis, it was packaged and propagated in HEK 293 cells. Tetracycline was used to regulate the expression of PLP-Ig, and Western blot was used to detect the gene expression. RESULTS: The expression of PLP-Ig was confirmed by Western blot and its expression could be regulated by tetracycline. CONCLUSION: The regulatable recombinant adenovirus vector for mouse PLP-Ig was constructed and expressed successfully, which lays the foundation for further study on gene therapy of EAE and induction of immune tolerance.