A guide to miRNAs in inflammation and innate immune responses.

The discovery of microRNAs (miRNAs) in the early 1990's has revolutionized our thinking of post-transcriptional gene control. miRNAs are involved in the regulation of almost every cellular and developmental process in eukaryotic organisms. In the context of infection and immunity, miRNAs play critical roles controlling processes involved in pathogen clearance, while ensuring a rapid return to homeostasis. In this review, we provide a broad overview of the miRNAs that have been implicated in innate immunity and inflammation in mammals over the past decade. We discuss how most miRNAs can have dual activities on inflammation, suggesting that modulation of their activity for therapeutic purposes may be context-dependent.

Hepatotoxicity after paracetamol overdose in a patient with cystic fibrosis despite early acetylcysteine and utility of microRNA to predict hepatotoxicity.

CASE DETAILS: A 19-year-old girl presented to the emergency department following overdose of 10 g of paracetamol on a background history of cystic fibrosis. Paracetamol concentration was below the nomogram line, but was treated with acetylcysteine seven hours post-overdose given her symptomatology. Nineteen hours following her overdose she developed hepatotoxicity, despite early initiation of acetylcysteine. She was discharged well six days post-ingestion. On presentation, delta miRNA-122-miR483 was 20 times that of control patients, however, alanine aminotransferase was normal. DISCUSSION: Patients with cystic fibrosis are more likely to have glutathione deficiency, and greater susceptibility to liver injury. Delta miRNA may be a better detector of early liver injury than hepatic aminotransferases. Empiric treatment with acetylcysteine and serial biochemical reassessment in this setting should be considered.

Modified Polyadenylation-Based RT-qPCR Increases Selectivity of Amplification of 3'-MicroRNA Isoforms.

MicroRNA (miRNA) detection by reverse transcription (RT) quantitative real-time PCR (RT-qPCR) is the most popular method currently used to measure miRNA expression. Although the majority of miRNA families are constituted of several 3'-end length variants ("isomiRs"), little attention has been paid to their differential detection by RT-qPCR. However, recent evidence indicates that 3'-end miRNA isoforms can exhibit 3'-length specific regulatory functions, underlining the need to develop strategies to differentiate 3'-isomiRs by RT-qPCR approaches. We demonstrate here that polyadenylation-based RT-qPCR strategies targeted to 20-21 nt isoforms amplify entire miRNA families, but that primers targeted to >22 nt isoforms were specific to >21 nt isoforms. Based on this observation, we developed a simple method to increase selectivity of polyadenylation-based RT-qPCR assays toward shorter isoforms, and demonstrate its capacity to help distinguish short RNAs from longer ones, using synthetic RNAs and biological samples with altered isomiR stoichiometry. Our approach can be adapted to many polyadenylation-based RT-qPCR technologies already exiting, providing a convenient way to distinguish long and short 3'-isomiRs.

miR-222 isoforms are differentially regulated by type-I interferon.

Endogenous microRNAs (miRNAs) often exist as multiple isoforms (known as "isomiRs") with predominant variation around their 3'-end. Increasing evidence suggests that different isomiRs of the same family can have diverse functional roles, as recently demonstrated with the example of miR-222-3p 3'-end variants. While isomiR levels from a same miRNA family can vary between tissues and cell types, change of templated isomiR stoichiometry to stimulation has not been reported to date. Relying on small RNA-sequencing analyses, we demonstrate here that miR-222-3p 3'-end variants >23 nt are specifically decreased upon interferon (IFN) ß stimulation of human fibroblasts, while shorter isoforms are spared. This length-dependent dynamic regulation of long miR-222-3p 3'-isoforms and >40 other miRNA families was confirmed in human monocyte-derived dendritic cells following infection with Salmonella Typhimurium, underlining the breadth of 3'-length regulation by infection, beyond the example of miR-222-3p. We further show that stem-loop miRNA Taqman RT-qPCR exhibits selectivity between 3'-isoforms, according to their length, and that this can lead to misinterpretation of results when these isoforms are differentially regulated. Collectively, and to our knowledge, this work constitutes the first demonstration that the stoichiometry of highly abundant templated 3'-isoforms of a same miRNA family can be dynamically regulated by a stimulus. Given that such 3'-isomiRs can have different functions, our study underlines the need to consider isomiRs when investigating miRNA-based regulation.

Topoisomerase 1 Inhibition Promotes Cyclic GMP-AMP Synthase-Dependent Antiviral Responses.

Inflammatory responses, while essential for pathogen clearance, can also be deleterious to the host. Chemical inhibition of topoisomerase 1 (Top1) by low-dose camptothecin (CPT) can suppress transcriptional induction of antiviral and inflammatory genes and protect animals from excessive and damaging inflammatory responses. We describe the unexpected finding that minor DNA damage from topoisomerase 1 inhibition with low-dose CPT can trigger a strong antiviral immune response through cyclic GMP-AMP synthase (cGAS) detection of cytoplasmic DNA. This argues against CPT having only anti-inflammatory activity. Furthermore, expression of the simian virus 40 (SV40) large T antigen was paramount to the proinflammatory antiviral activity of CPT, as it potentiated cytoplasmic DNA leakage and subsequent cGAS recruitment in human and mouse cell lines. This work suggests that the capacity of Top1 inhibitors to blunt inflammatory responses can be counteracted by viral oncogenes and that this should be taken into account for their therapeutic development.IMPORTANCE Recent studies suggest that low-dose DNA-damaging compounds traditionally used in cancer therapy can have opposite effects on antiviral responses, either suppressing (with the example of CPT) or potentiating (with the example of doxorubicin) them. Our work demonstrates that the minor DNA damage promoted by low-dose CPT can also trigger strong antiviral responses, dependent on the presence of viral oncogenes. Taken together, these results call for caution in the therapeutic use of low-dose chemotherapy agents to modulate antiviral responses in humans.

Activation of cGAS-dependent antiviral responses by DNA intercalating agents.

Acridine dyes, including proflavine and acriflavine, were commonly used as antiseptics before the advent of penicillins in the mid-1940s. While their mode of action on pathogens was originally attributed to their DNA intercalating activity, work in the early 1970s suggested involvement of the host immune responses, characterized by induction of interferon (IFN)-like activities through an unknown mechanism. We demonstrate here that sub-toxic concentrations of a mixture of acriflavine and proflavine instigate a cyclic-GMP-AMP (cGAMP) synthase (cGAS)-dependent type-I IFN antiviral response. This pertains to the capacity of these compounds to induce low level DNA damage and cytoplasmic DNA leakage, resulting in cGAS-dependent cGAMP-like activity. Critically, acriflavine:proflavine pre-treatment of human primary bronchial epithelial cells significantly reduced rhinovirus infection. Collectively, our findings constitute the first evidence that non-toxic DNA binding agents have the capacity to act as indirect agonists of cGAS, to exert potent antiviral effects in mammalian cells.