One MicroRNA Controls Both Angiogenesis and TLR-mediated Innate Immunity to Nucleic Acids
Authors: Geoffrey L Rogers, Roland W Herzog
Journal: Molecular Therapy (2014)

Upon in vivo administration of gene therapy vectors, the invading vector genome and other macromolecular structures are sensed by innate immune receptors, thereby putting the immune system on alert. Among these, Toll-like receptors (TLRs) are critical for the function of certain immune cells. How the effects of TLR expression, function, and downstream signaling are controlled is not well understood. However, a new study in Nature Immunology provides unexpected answers to some of these questions.1Agudo J Ruzo A Tung N Salmon H Leboeuf M Hashimoto D et al.The miR-126-VEGFR2 axis controls the innate response to pathogen-associated nucleic acids.Nat Immunol. 2013; 15: 54-62Crossref PubMed Scopus (107) Google Scholar Agudo et al. found that mice deficient in a specific microRNA (miR-126) failed to respond to oligonucleotides that are known potent agonists of the endosomal receptors TLR7 and TLR9. TLR7 is a sensor of single-stranded RNA molecules, and TLR9 senses unmethylated DNA such as that present in viral genomes or plasmids. Therefore, these new findings have broad implications for innate immune recognition of many different gene transfer vectors. Signaling through TLRs can have multiple profound consequences on gene transfer, such as limited transduction, reduced transgene expression, enhanced inflammation, and activation of antigen-specific immune responses.2Suzuki M Bertin TK Rogers GL Cela RG Zolotukhin I Palmer DJ et al.Differential type I interferon-dependent transgene silencing of helper-dependent adenoviral vs. adeno-associated viral vectors in vivo.Mol Ther. 2013; 21: 796-805Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar Although detrimental to gene replacement therapies, these outcomes are actually desirable to enhance vaccine efficacy, oncolytic virotherapy, and various other cancer immunotherapies, spurring the development of immune-modulatory nucleic acids.3Cerullo V Diaconu I Romano V Hirvinen M Ugolini M Escutenaire S et al.An oncolytic adenovirus enhanced for Toll-like receptor 9 stimulation increases antitumor immune responses and tumor clearance.Mol Ther. 2012; 20: 2076-2086Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar,4Dharmapuri S Peruzzi D Mennuni C Calvaruso F Giampaoli S Barbato G et al.Coadministration of telomerase genetic vaccine and a novel TLR9 agonist in nonhuman primates.Mol Ther. 2009; 17: 1804-1813Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar The resulting signal is relayed from the endosomal TLR to the cytosol via adapter molecules, ultimately triggering the proinflammatory nuclear factor-κB pathway and/or inducing synthesis of type I interferon (IFN-I). Surprisingly, mice deficient in miR-126 exhibited significantly reduced expression of IFN-α/β and also showed impaired production of the proinflammatory cytokine interleukin-6 upon administration of stimulatory oligonucleotides. Given that miR-126 had been described as being expressed predominantly in endothelial cells—functioning as a regulator of angiogenesis—there was no reason to suspect a role in immunity. However, the new study shows that miR-126 is also expressed in human and murine plasmacytoid dendritic cells (pDCs), a key component of the immune system. Among other functions, pDCs produce large amounts of IFN-α/β, which can drive cells of an infected tissue into an antiviral state and which also enhances lymphocyte responses and functionality. Examination of the miR-126 knockout mice indicated that miR-126 controls the survival and function of pDCs. Modulation of functionality probably occurs directly by suppressing the translation of Tsc1, a negative regulator of mammalian target of rapamycin (mTOR), and indirectly by controlling expression of the crucial innate response genes Nfkb1, Tlr7, and Tlr9 (Figure 1; modulation of innate immunity by direct binding of miRNA to TLRs, as recently described in other studies, did not seem to be a contributing factor here). Hence, upregulation of miR-126 or suppression of TSC1 may represent potential future avenues to enhance immune therapies. Conversely, inhibition of miR-126 or its effects could be exploited to prevent responses to gene therapy vectors, as in treatment of genetic disease. Interestingly, the efficacy of liver gene transfer with lentiviral vectors (LVs) is substantially reduced by IFN-α/β that is produced in response to vector administration. As a result, many more hepatocytes are transduced in mice lacking the receptor for IFN-α/β. One would expect pDCs to be key players in inhibiting LV gene transfer, as they are the principal producers of IFN-α/β (and also aid in the activation of conventional DCs in response to LV). LV triggers signaling in pDCs at multiple steps of infection, including recognition of the viral RNA genome by TLR7 and of the reverse-transcribed DNA genome. The latter also involves TLR-independent cytosolic sensing, probably by cyclic guanosine monophosphate–adenosine monophosphate synthase.5Agudo J Ruzo A Kitur K Sachidanandam R Blander JM Brown BD A TLR and non-TLR mediated innate response to lentiviruses restricts hepatocyte entry and can be ameliorated by pharmacological blockade.Mol Ther. 2012; 20: 2257-2267Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar,6Gao D Wu J Wu YT Du F Aroh C Yan N et al.Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses.Science. 2013; 341: 903-906Crossref PubMed Scopus (690) Google Scholar Given the fundamental defect in innate immune function in pDCs of mice deficient in miR-126, these animals produced significantly less IFN-α/β following LV injection, resulting in a robust increase in hepatocyte transduction. An additional unexpected finding by Agudo et al. was that miR-126 controls innate immunity in part through regulating the expression of vascular endothelial growth factor receptor 2 (VEGFR2)—another gene previously thought to be endothelial-specific but found here to be expressed by pDCs. Deficiency of miR-126 reduces VEGFR2 (Kdr) expression in pDCs, and lack of Kdr expression in pDCs causes a reduction in pDC numbers and impairs the IFN-I response in vivo. Using rapamycin, Agudo et al. demonstrate that VEGFR2 expression is regulated by mTOR signaling, a pathway controlled by the miR-126–Tsc1 interaction. Hence, the VEGF and TLR signaling pathways are connected (Figure 1). In wild-type animals, pharmacological suppression of IFN-α/β using glucocorticoids improves liver gene transfer with LVs.5Agudo J Ruzo A Kitur K Sachidanandam R Blander JM Brown BD A TLR and non-TLR mediated innate response to lentiviruses restricts hepatocyte entry and can be ameliorated by pharmacological blockade.Mol Ther. 2012; 20: 2257-2267Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar VEGFR2 blockade may be an alternative opportunity to intervene and suppress IFN-α/β production, thereby improving gene transfer. Rapamycin, which has previously been shown to inhibit TLR9 signaling, may have additional suppressive effects on innate immunity by blocking pDC survival signals through VEGF. Although not all the dots have yet been connected in these intersecting pathways, we now have a fundamentally new understanding of how TLR-mediated innate immunity in pDCs is regulated, thus providing us with novel targets for manipulating this response. Given that TLR9 is a critical component of innate responses to vectors with DNA genomes such as plasmids, adeno-associated viral and adenoviral vectors,2Suzuki M Bertin TK Rogers GL Cela RG Zolotukhin I Palmer DJ et al.Differential type I interferon-dependent transgene silencing of helper-dependent adenoviral vs. adeno-associated viral vectors in vivo.Mol Ther. 2013; 21: 796-805Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar a wide range of potential applications should emerge.