The Roles Of Micro-RNAs In The Effector Function Of Type Ⅰ Interferon And The Underlying Mechanisms

Type I interferons (IFN) are one kind of the most important cytokines in immunology, and have profound immune regulatory functions, including antiviral effect, antitumor effect and immune modulation. Among them, type I IFN play vital role in triggering antiviral immune responses. So far, it is believed that type I IFN render cells antiviral status by promoting cell to express some molecules that can counteract viral infection, inhibit viral replication, and at the same time up-regulating the expression of MHC I to facilitate the recognition and activation of CTL. In addition, type I IFN can potently activate immune cells. It can activate NK cells effectively, enhancing their cytotoxicity to eliminate virus-infected cells. It is believed that type I IFN are the most potent activator for NK cells, however, the underlying mechanism for how type I IFN rapidly and effectively activate NK cells remains to be completely exploited, although some reports show that type I IFN activate NK cells through IL-15 autocrine/paracrine manner. The effects of type I IFN on NK cell function are far from initially activation merely, which need to be further investigated.Type I IFN are now extensively applied as antivirus reagent in clinics to cure many virus-infected diseases. However, the therapeutic effect of type I IFN varies vigorously from individual to individual. Although function and signaling of type I IFN have been intensively studied in the past several decades, the sophisticated mechanism of type I IFN fulfilling their function on different cells is far from to be completely understood. By now, JAK/STAT cascade, the well-established cardinal pathway transmitting IFN signals, is found to be tightly regulated at several levels. Among them, some molecules, such as suppressor of cytokine signaling (SOCS) family members, can negatively regulate type I IFN signaling. Eventually, type I IFN stimulation alters expression of hundreds of genes in cells, which is called interferon stimulated genes (ISGs), which mediate the functions of type I IFN.Mocro-RNAs (miRNAs), an abundant class of highly conserved small (18-25 nt long) non-coding RNAs, suppress gene expression by binding to the 3'UTR of target mRNAs, which presents an entirely new approach to post-transcriptional regulation of gene expression. miRNAs have been shown to play key roles in the regulation of diverse biological processes, such as development, infection, immune response, inflammation, and tumorigenesis. Since the initial observation, more than 700 miRNAs have been identified in mammals. However, the biological functions of miRNAs in a large part remain elusive. Various functions of miRNAs in immune responses are now emerging, nevertheless, there are few reports about the regulation of type I IFN signaling and subsequent antiviral innate immunity by miRNAs to date, and neither is there report about the participation of miRNA in type I IFN effect on cells. As to NK cell activation, the hypothesis that type I IFN takes part in this process through a miRNA-dependent matter needs to been further investigated.Using genetic approaches, miR-155 has been demonstrated to have an indispensable role in humeral and cellular immunity. Regarding to innate immunity and inflammatory response, miR-155 expression is induced by TLR signals, including TLR2, TLR3, TLR4, and TLR9, or stimulations from cytokines, such as IL-1, TNF-a, and IFN-β, indicating the participation of miR-155 in these immune responses. Although the fact that miR-155 expression is induced by stimulation of poly (I:C) or IFN-p implies the involvement of miR-155 in antiviral responses, there is no report by now about the regulation of innate immune antiviral response by miR-155, or regulation of type I IFN signaling and subsequent type I IFN-mediated antiviral innate immunity by miR-155.On the basis of the research background above, we applied microarray technology to investigate miRNA expression profile of murine macrophages before and after VSV challenge, and used Illumina Solexa MASS to exploit the small RNA profile of human NK cells at different time points after type I IFN stimulation. We found that miR-155 is one of the most markedly upregulated miRNA after VSV challenge in murine macrophages. We demonstrate that the whole amount of miRNAs of human NK cells slightly decreases after type I IFN stimulation, dozens of which are significantly upregulated or downregulated. We went further to investigate the roles of the miRNAs in the regulation of effector function of human NK cells activated by type I IFN.PartⅠ:Inducible microRNA-155 feedback promotes type I interferon signaling in antiviral innate immunity by targeting SOCS11. VSV infection upregulates miR-155 expression through RIG-I/JNK/NF-κB but not TLR/MyD88 pathways.After discovery of up-regulation of miRNA-155 in VSV-infected macrophages, we next investigated the underlying mechanism by which miR-155 was induced. As it was reported that signals from TLR2, TLR3, TLR4, and TLR9 induced miR-155 expression through MyD88-or TRIF-dependent signaling pathways, first we excluded the possibility that activation of TLR signals contributed to VSV-induced upregulation of miR-155 expression. Peritoneal macrophages from TLR3, TLR4, TLR9, or MyD88-deficient mice showed identical miR-155 expression induced by VSV infection as compared to that in wild type macrophages. However, VSV-induced miR-155 expression was markedly impaired in RIG-Ⅰ-deficient macrophages and splenocytes, suggesting that inducible miR-155 expression was dependent on RIG-Ⅰsignaling. As control, LPS-induced miR-155 expression was impaired by TLR4 deficiency, but not significantly influenced by RIG-I deficiency.Since induction of miR-155 was reported to be JNK-dependent, we went further to investigate the effect of JNK inhibitor and NF-κB inhibitor on VSV-induced up-regulation of miR-155 expression in macrophages. We found that inhibition of JNK and NF-κB efficiently impaired VSV-induced miR-155 expression. However, inhibition of p38 or ERK had little effect on the induction of miR-155 by VSV.2. VSV infection-inducible miR-155 attenuates viral propagation mainly through enhancing type IIFN signaling in macrophagesIn order to investigate the biological significance of upregulated miR-155 during viral infection, we examined the effect of miR-155 on VSV replication in macrophages. By measuring VSV TCID50 in the supernatants of the infected macrophages, we found that inhibition of inducible miR-155 facilitated VSV replication, while overexpression of miR-155 suppressed VSV replication. Consistent with the data of VSV TCID50 assay in supernatants, intracellular VSV RNA replicates were also increased by miR-155 inhibition and decreased by miR-155 overexpression.To gain an insight into the mechanism of the phenomenon and further identify whether VSV-induced miR-155 expression could affect VSV-triggered response in macrophages, we investigated the role of miR-155 in type I IFN production following VSV challenge. We found that miR-155 overexpression or inhibition had no significant effect on type I IFN production at both protein level and mRNA level. Then we focused on the effect of miR-155 on type I IFN downstream signaling. VSV-induced phosphorylation of STAT1 in macrophages was detected and it was shown to reach the peak at 24 h after VSV challenge. And VSV-induced STAT1 phosphorylation was promoted by miR-155 overexpression while inhibited by miR-155 inhibition. We also examined expression of IFN-induced immune regulatory genes, such as ISG15 and IP 10. In macrophages treated with recombinant IFN-β, we found that inducible expression of ISG15 and IP 10 increased by miR-155 overexpression and decreased by miR-155 inhibition. So, inducible miR-155 in response to RNA virus infection promotes type I IFN signaling but does not significantly affect type I IFN production.3. miR-155 promotes type I IFN signaling by targeting SOCS1 in virus-infected macrophagesNext, we investigated which molecule was the major target of miR-155 that could modulate type I IFN signaling. We found that miR-155 was one of the broadly conserved miRNAs that putatively targets conserved sites of murine SOCS1's 3'UTR by computational prediction via TargetScan (http://www.targetscan.org). SOCS1 has been identified to negatively regulate various immune responses and signaling pathways, including type I IFN signaling. SOCS1 mRNA level sharply accumulates in no more than 24h after VSV challenge, but its protein level hardly changes or even slightly decreases over a time course of 48 h, suggesting that the expression of SOCS1 in macrophages is regulated by a post-transcriptional mechanism, presumably miRNA inhibition. Hence, we presumed that miR-155 might suppress SOCS1 translation in macrophages after VSV challenge.Then we examined whether the protein level of SOCS1 in macrophages was targeted and regulated by miR-155. As expected, SOCS1 expression was increased by miR-155 inhibition and decreased by miR-155 overexpression. To present direct evidence that inducible miR-155 during VSV infection suppressed SOCS1 protein expression, we examined kinetic level of SOCS1 protein and mRNA after VSV challenge when inducible miR-155 was inhibited. miR-155 inhibitor rescued SOCS1 mRNA translation upon VSV challenge, and SOCS1 mRNA kinetic level was not affected significantly. Furthermore, we proved that miR-155 regulated type I IFN signaling at the point of SOCS1 by examining kinetic phosphorylation of JAK1 and STATs in response to IFN-p, and obtained similar results. These results were consistent with a recent report showing that miR-155 directly targeted the potential conserved target site in SOCS1 mRNA 3'UTR through reporter gene assay. Together, the results show that endogenous SOCS1 is targeted and directly regulated by miR-155 in macrophages.To demonstrate that the role of targeting SOCS1 in the anti-viral function of miR-155, we did experiments of SOCS1 RNAi knockdown and overexpression. And we confirmed that SOCS1 siRNA effectively inhibited its expression in murine peritoneal macrophages, while SOCS1-stably overexpressed cell clone of RAW264.7 macrophages which expressed SOCS1 mRNA without its 3'UTR sequence had significantly elevated SOCS1 protein expression.In murine peritoneal macrophages, SOCS1 knockdown attenuated VSV replication just like the effect of miR-155 overexpression, and the effect of miR-155 inhibition on VSV replication was rescued by SOCSl knockdown. Also, recombinant IFN-β-induced upregulation of ISG15 expression is reduced by miR-155 inhibition and further rescued by SOCS1 knockdown. These results suggest that SOCS1 knockdown phenocopied the antiviral effect of induced miR-155 and counteracted the effect of miR-155 inhibition.Since the SOCS1-stably overexpressed RAW264.7 cell clones transcribed SOCS1 mRNA without its 3'UTR, miR-155 should no longer target SOCS1 expression in these cells to implement its antiviral function. We did not find that miR-155 overexpression could influence VSV proliferation in SOCS1-stably overexpressed RAW264.7 cells, documented by both measuring VSV TCID50 in the cultural supernatants and intracellular VSV RNA replicates. So, we concluded that inducible miR-155 under viral infection exhibits its antiviral function mainly through suppressing endogenous SOCS1 expression and subsequently promoting type IIFN signaling.PartⅡ:Expression profile and functional prediction of small RNA in human NK cells activated by type I IFNWe purified human CD56 positive NK cells from health human peripheral blood by FACS sorting, and the purity of the NK cells was over 95%. We stimulated NK cells with human recombinant IFN-α. After Oh,12h and 24h, RNA from NK cells was collected and the small RNA fragments (18-30nt) were sequenced by Illumina Solexa MASS, a new generation of deep sequencing system. The raw data was preliminarily summarized and analyzed.1. Overview of small RNA sequencing dataOf the raw sequencing reads about 35nt long, we got rid of low quality reads and pollution, and removed the adaptor sequences to obtain high quality sequencing reads, called clean reads library, on which all subsequence analyses based. From RNA length distribution for each reads library, we can see an obvious peak at 22nt position, where miRNA should be enriched, while siRNA and piRNA are about 24nt and 30nt long respectively, indicating majority of small RNA is miRNA. Sequences of about 93% reads are identical between each two libraries, indicating small RNAs do not change a lot during type I IFN-mediated NK activation. Human genome sequence alignment demonstrates 80% of sequences can map to the genome sequence. Sequence alignment to human miRNA precursor sequences in miRNA database (miRBase14.0) reveals the proportion of registered miRNA in each small RNA reads library, that is 61.8%(0h library),44.0%(12h library) and 55.1%(24h library), showing a slight decrease after type IIFN stimulation.2. Analysis of miRNA expression profilesThe abundance value of each registered miRNA was normalized using "transcripts per million (TPM)" in each reads library. We got the expression profile of miRNAs in normal human NK cells. In resting NK cells, there are 11 miRNAs with a TPM above 10000, has-miR-21, has-let-7f, has-let-7a, has-miR-140-3p, has-miR146b-5p, has-let-7g, has-142-3p, has-miR-101, has-miR-378, has-let-7b, and has-miR-103. They account for 47.3% of small RNA amount and 76.8% of total miRNAs amount, suggesting their possible important roles in maintaining human NK cell function. Ignoring miRNAs with little amount (TPM<100), we found that most miRNAs decrease after type I IFN activation,24 of which are markedly down-regulated (less than 0.5 fold), while minority of which increase, with 2 miRNAs up-regulated significantly (more than 2 fold). Then we confirmed these results in RNA samples of human NK cells from 15 healthy donors using real-time PCR.3. Targets prediction of changed miRNA and function hypothesisWe did target prediction using miRNA prediction software TargetScan (http://www.targetscan.org) and Mirenda (http://www.microrna.org/microrna/home.do) for markedly changed miRNA mentioned above and found three miRNAs with down-regulated expression after type I IFN stimulation, including miR-378, miR-30 family and miR-150, could target NK cell cytotoxic effectors, granzyme B and perforin. It has been reported that murine NK cells increase the expressions of effector molecules, granzyme B and perforin, through a posttranscriptional mechanism, after activation by cytokines including type I IFN. Here we demonstrated that human NK cells from peripheral blood up-regulate the expression of granzyme B and perforin after type I IFN activation, while mRNA levels of these effectors remain almost unchanged. So we hypothesis that type IIFN enhances cytotoxic function of NK cells through derepression of their effector proteins translation, granzyme B and perforin, by down-regulating miR378, miR-30 family and miR-150 levels. This might be a new mechanism that type I IFN activate human NK cell function.4. Identification of novel miRNA candidatesAfter the classification annotation of sequencing clean reads, we got the amounts of all sorts of small RNAs, including miRNAs, rRNA, tRNA, scRNA, snRNA, snoRNA, repeat-associated small RNAs, mRNAs-associated RNA and unannotated RNA. Besides miRNAs, the most abundant fraction, unannotated small RNA is the second most abundant part (15.7%-19.6% in each reads library), using which we did novel miRNA prediction. Using miRNA prediction software Mireap, we found hundreds of novel miRNA candidates, 621 from library of human NK cells without type I IFN stimulation,554 from library of human NK cells after type I IFN stimulation for 12 hour and 577 from library of human NK cells after type I IFN stimulation for 24 hour. Among them, there are 11 most probable novel miRNA candidates that have transcript number above 100 at least in one reads library and exist in at least two libraries, whose functions need further investigation.Conclusions:1. VSV infection upregulates miR-155 expression in macrophages through TLR/MyD88-independent but RIG-I/JNK/NF-KB-dependent pathways. VSV infection-inducible miR-155 attenuates viral propagation mainly through enhancing type I IFN signaling in macrophages. Furthermore, inducible miR-155 under viral infection exhibits its antiviral function mainly through suppressing endogenous SOCS1 expression and subsequently promoting type I IFN signaling.2. The expression profiles of small RNAs in NK cells stimulated with or without human type I IFN have been achieved by deep sequencing with Illumina Solexa MASS. The miRNAs which were regulated by type I IFN in NK cells have been identified, a panel of novel miRNA cadidites is discovered and the functions of altered miRNAs are predicted.