Studies On Regulation Mechanism Of MicroRNA-223, Interleukin-27 On Antiviral Innate Immune Responses In

Part Ⅰ Study on regulation mechanism of microRNA-223 on antiviral innate immune responses in macrophagesInnate immunity is the body’s first line of effective defense against virus invasion. When the body is infected, immune cells will recognize the virus components through pattern recognition receptors and activate downstream signaling pathways, resulting in expression of type Ⅰ interferon and proinflammatory cytokines. Type Ⅰ interferon (IFN) play a vital role in antiviral immune response, and its expression is under the body’s precise regulation. MicroRNA (miRNA) is a newly discovered class of highly conserved non-coding RNA of approximately 21-23 nucleotides in length, and it negatively regulate mRNA translation mainly through targeting the 3’non-coding region (3’UTR) of mRNA. MiRNA is involved in the regulation of a variety of biological processes including growth, differentiation, tumorigenesis and development. In addition, miRNA plays a key role in regulating innate immune responses, the interaction between virus and the body on miRNA levels is widely concerned. There are some articles reporting that miRNAs have been involved in the regulation of antiviral innate immune response reports, such as miRNA-146a and miRNA-155, which play a negative and positive role in antiviral innate immune response respectively. Therefore, we’d like to explore the miRNAs which are significantly changed in the infection process and further study the mechanism to seek effective ways to ward off virus infections.During eraly phase we found out some miRNAs which are highly expressed in myeloid cells through review the literature (including miR-21, miR-25, miR-34a, miR-106a, miR-125b, miR-146a and miR-223), and detected the changes of these miRNAs by quantitative PCR (QPCR) in the process of the RNA virus VSV infection of mouse peritoneal macrophages, and we observed the expression of miR-223 significantly increased. Furthermore we found that the expression of miR-223 was not regulated by the virus RNA synthesis and protein synthesis, but the virus triggered type Ⅰ IFN signaling, and if we blocked type Ⅰ IFN signaling pathways stimulated by VSV, the expression of miR-223 would no longer be raised. Then we transfected mouse macrophages with synthesized siRNA to simulate the internal miR-223 function or specifically inhibit its function to further research on miR-223’s function and QPCR, plaque assay, TCID50 assay, and flow cytometry were used to detected miR-223’s effect on RNA virus infections. We found overexpression of miR-223 increased type Ⅰ IFN production, and led to resistance to RNA virus infections; While inhibition of miR-223 downregulated type Ⅰ IFN production and promoted RNA virus infections. In the study of molecular mechanisms, we detected the effect of miR-223 on the key molecules of the RNA virus activated downstream signaling pathway, and found overexpression of miR-223 did not significantly enhance the phosphorylation of p38 and ERK, JNK, and IκBα and the expression of RIG-I, phosphorylation of IRF3 and phosphorylation of PKR had no significant differences, however it promoted the expression of IRF7. On the contrary, if we inhibited the expression of miR-223, IRF7 also reduced accordingly. As reported in the literature, transcription factor forkhead box O3 (FOXO3) can inhibit the transcriptional activity of the IRF7, so we speculated that miR-223 may promote the expression of IRF7 through inhibiting the FOXO3 and we found FOXO3 was a new target of miR-223 by miRNA targets prediction site analysis and luciferase reporter gene assay. At last, we demonstrated FOXO3 interference and overexpression experiments to further verify that miR-223 enhanced the transcriptional activity of IRF7 and promote type Ⅰ IFN production to play antiviral function through inhibiting the expression of FOXO3.To sum up, RNA virus infection in macrophages induced upregulation of the expression of miR-223 through promoting type Ⅰ IFN production, and induced miR-223 could target FOXO3 to enhance the transcriptional activity of IRF7 and promote type Ⅰ IFN production to play antiviral function. The findings added a new awareness to the regulation mechanisim on antiviral innate immune response of macrophages, and may provide new ideas for antiviral treatment.Part II Study on regulation mechanism of interleukin-27 on antiviral innate immune responses in macrophagesInterleukin 27 (IL-27) is a member of the IL-12 family cytokines, which is a heterodimeric cytokine, composed of EBI3 and P28 two subunits. IL-27 receptor is also a heterogeneous dimer, which is made up of a ligand-binding chain IL-27R a (WSX-1, TCCR) and the other signaling chain gp130. IL-27R a/gp130 signaling activates downstream molecule JAK and STAT1/STAT3. And activated STAT1/STAT3 controls the activation of different T-cell specific transcription factor, such as T-bet (Thl), GATA-3 (Th2) and ROR-yt (Th17). In addition, the IL-27 receptor signaling can also activate MAPK to recruit and activate downstream transcription factor c-Maf and API to act on proliferation and differentiation of the cells. In recent years, increasing evidence proved that IL-27 plays an important role in immune regulation in the body. Similar to most cytokines, IL-27 played a two-faced role in the regulation of inflammation, which can both promote inflammation and inhibit inflammation. Current researches about IL-27 mostly focus on its effects on T-cell differentiation and function, while the study with regard to its role in antiviral innate immune response is very limited. Therefore, our study aims to reveal the function of IL-27 on the regulation of antiviral innate immune responsesWe have found that RNA virus VSV infection of macrophages increased the IL-27-subunit mRNA expression significantly. And we found that recombinant mouse IL-27 (rmIL-27) pretreatment of macrophages in mice inhibited replication of VSV, but had no significant effect on expression of IFN-β in macrophages, indicating that the IL-27 may play antiviral activity independent of type I IFN. We also observed that rmIL-27 pretreatment of macrophages in mice could inhibit replication of H1N1 and HSV. Then we demonstrated further research to prove the antiviral function of IL-27, and found that compared with wild type (WT) mice, IL-27 receptor subunit IL-27 Rα knockout (IL-27Ra-/-) mice were more sensitive to VSV infection and H1N1 infection, while we also observed that knock out of IL-27 receptor subunit IL-27Rα did not affect the level of IFN-beta in serum significantly. In the study on molecular mechanism, we discovered rmIL-27 significantly increases interferon gamma inducible protein 47 (Ifi47) and interferon inducible GTPase 1 (Iigpl) expression through microarray, then we studied the role of Ifi47 and ligpl in the regulation of the body’s innate immune response against viruses, and we found IL-27 indeed played antival function by upregulate the expression of Ifi47 and Iigpl. Also we explored the upregulation mechanism of IL-27 on Ifi47 and Iigpl, and found JNK inhibitor inhibited the IL-27 induced Ifi47 and Iigpl increases, indicating that the IL-27 may raise Ifi47 and Iigpl in a INK signaling pathway-dependent manner.In summary, we found that viral infection of macrophages induced the production of IL-27, and macrophage secreted IL-27 or exogenous adding recombinant IL-27 activated INK signaling pathway via IL-27 receptors on the surface of macrophage, and raised the expression of Ifi47 and Iigpl to play an antiviral function. The results of the study provide a new understanding of the function of IL-27 in antiviral innate immunity, and may provide the basis for the use of IL-27 for antiviral therapy.