Kate L. Jeffrey

Suppression of inflammation by a synthetic histone mimic

Interaction of pathogens with cells of the immune system results in activation of inflammatory gene expression. This response, although vital for immune defence, is frequently deleterious to the host due to the exaggerated production of inflammatory proteins. The scope of inflammatory responses reflects the activation state of signalling proteins upstream of inflammatory genes as well as signal-induced assembly of nuclear chromatin complexes that support mRNA expression. Recognition of post-translationally modified histones by nuclear proteins that initiate mRNA transcription and support mRNA elongation is a critical step in the regulation of gene expression. Here we present a novel pharmacological approach that targets inflammatory gene expression by interfering with the recognition of acetylated histones by the bromodomain and extra terminal domain (BET) family of proteins. We describe a synthetic compound (I-BET) that by ‘mimicking’ acetylated histones disrupts chromatin complexes responsible for the expression of key inflammatory genes in activated macrophages, and confers protection against lipopolysaccharide-induced endotoxic shock and bacteria-induced sepsis. Our findings suggest that synthetic compounds specifically targeting proteins that recognize post-translationally modified histones can serve as a new generation of immunomodulatory drugs.

Microbial Stimulation Fully Differentiates Monocytes to DC-SIGN/CD209+ Dendritic Cells for Immune T Cell Areas

Dendritic cells (DCs), critical antigen-presenting cells for immune control, normally derive from bone marrow precursors distinct from monocytes. It is not yet established if the large reservoir of monocytes can develop into cells with critical features of DCs in vivo. We now show that fully differentiated monocyte-derived DCs (Mo-DCs) develop in mice and DC-SIGN/CD209a marks the cells. Mo-DCs are recruited from blood monocytes into lymph nodes by lipopolysaccharide and live or dead gram-negative bacteria. Mobilization requires TLR4 and its CD14 coreceptor and Trif. When tested for antigen-presenting function, Mo-DCs are as active as classical DCs, including cross-presentation of proteins and live gram-negative bacteria on MHC I in vivo. Fully differentiated Mo-DCs acquire DC morphology and localize to T cell areas via L-selectin and CCR7. Thus the blood monocyte reservoir becomes the dominant presenting cell in response to select microbes, yielding DC-SIGN(+) cells with critical functions of DCs.

Targeting dual-specificity phosphatases: manipulating MAP kinase signalling and immune responses

Dual-specificity phosphatases (DUSPs) are a subset of protein tyrosine phosphatases, many of which dephosphorylate threonine and tyrosine residues on mitogen-activated protein kinases (MAPKs), and hence are also referred to as MAPK phosphatases (MKPs). The regulated expression and activity of DUSP family members in different cells and tissues controls MAPK intensity and duration to determine the type of physiological response. For immune cells, DUSPs regulate responses in both positive and negative ways, and DUSP-deficient mice have been used to identify individual DUSPs as key regulators of immune responses. From a drug discovery perspective, DUSP family members are promising drug targets for manipulating MAPK-dependent immune responses in a cell-type and disease-context-dependent manner, to either boost or subdue immune responses in cancers, infectious diseases or inflammatory disorders.

Conserved vertebrate mir-451 provides a platform for Dicer-independent, Ago2-mediated microRNA biogenesis

Canonical animal microRNAs (miRNAs) are generated by sequential cleavage of precursor substrates by the Drosha and Dicer RNase III enzymes. Several variant pathways exploit other RNA metabolic activities to generate functional miRNAs. However, all of these pathways culminate in Dicer cleavage, suggesting that this is a unifying feature of miRNA biogenesis. Here, we show that maturation of miR-451, a functional miRNA that is perfectly conserved among vertebrates, is independent of Dicer. Instead, structure-function and knockdown studies indicate that Drosha generates a short pre-mir-451 hairpin that is directly cleaved by Ago2 and followed by resection of its 3′ terminus. We provide stringent evidence for this model by showing that Dicer knockout cells can generate mature miR-451 but not other miRNAs, whereas Ago2 knockout cells reconstituted with wild-type Ago2, but not Slicer-deficient Ago2, can process miR-451. Finally, we show that the mir-451 backbone is amenable to reprogramming, permitting vector-driven expression of diverse functional miRNAs in the absence of Dicer. Beyond the demonstration of an alternative strategy to direct gene silencing, these observations open the way for transgenic rescue of Dicer conditional knockouts.

Suppression of the antiviral response by an influenza histone mimic

Viral infection is commonly associated with virus-driven hijacking of host proteins. Here we describe a novel mechanism by which influenza virus affects host cells through the interaction of influenza non-structural protein 1 (NS1) with the infected cell epigenome. We show that the NS1 protein of influenza A H3N2 subtype possesses a histone-like sequence (histone mimic) that is used by the virus to target the human PAF1 transcription elongation complex (hPAF1C). We demonstrate that binding of NS1 to hPAF1C depends on the NS1 histone mimic and results in suppression of hPAF1C-mediated transcriptional elongation. Furthermore, human PAF1 has a crucial role in the antiviral response. Loss of hPAF1C binding by NS1 attenuates influenza infection, whereas hPAF1C deficiency reduces antiviral gene expression and renders cells more susceptible to viruses. We propose that the histone mimic in NS1 enables the influenza virus to affect inducible gene expression selectively, thus contributing to suppression of the antiviral response.

Induction and suppression of antiviral RNA interference by influenza A virus in mammalian cells

Influenza A virus (IAV) causes annual epidemics and occasional pandemics, and is one of the best-characterized human RNA viral pathogens1. However, a physiologically relevant role for the RNA interference (RNAi) suppressor activity of the IAV non-structural protein 1 (NS1), reported over a decade ago2, remains unknown3. Plant and insect viruses have evolved diverse virulence proteins to suppress RNAi as their hosts produce virus-derived small interfering RNAs (siRNAs) that direct specific antiviral defence4–7 by an RNAi mechanism dependent on the slicing activity of Argonaute proteins (AGOs)8,9. Recent studies have documented induction and suppression of antiviral RNAi in mouse embryonic stem cells and suckling mice10,11. However, it is still under debate whether infection by IAV or any other RNA virus that infects humans induces and/or suppresses antiviral RNAi in mature mammalian somatic cells12–21. Here, we demonstrate that mature human somatic cells produce abundant virus-derived siRNAs co-immunoprecipitated with AGOs in response to IAV infection. We show that the biogenesis of viral siRNAs from IAV double-stranded RNA (dsRNA) precursors in infected cells is mediated by wild-type human Dicer and potently suppressed by both NS1 of IAV as well as virion protein 35 (VP35) of Ebola and Marburg filoviruses. We further demonstrate that the slicing catalytic activity of AGO2 inhibits IAV and other RNA viruses in mature mammalian cells, in an interferon-independent fashion. Altogether, our work shows that IAV infection induces and suppresses antiviral RNAi in differentiated mammalian somatic cells.

Beyond receptors and signaling: epigenetic factors in the regulation of innate immunity.

The interaction of innate immune cells with pathogens leads to changes in gene expression that elicit our bodys first line of defense against infection. Although signaling pathways and transcription factors have a central role, it is becoming increasingly clear that epigenetic factors, in the form of DNA or histone modifications, as well as noncoding RNAs, are critical for generating the necessary cell lineage as well as context‐specific gene expression in diverse innate immune cell types. Much of the epigenetic landscape is set during cellular differentiation; however, pathogens and other environmental triggers also induce changes in histone modifications that can either promote tolerance or ‘train’ innate immune cells for a more robust antigen‐independent secondary response. Here we review the important contribution of epigenetic factors to the initiation, maintenance and training of innate immune responses. In addition, we explore how pathogens have hijacked these mechanisms for their benefit and the potential of small molecules targeting chromatin machinery as a way to boost or subdue the innate immune response in disease.

GEF-H1 controls microtubule-dependent sensing of nucleic acids for antiviral host defenses.

Detailed understanding of the signaling intermediates that confer the sensing of intracellular viral nucleic acids for induction of type I interferons is critical for strategies to curtail viral mechanisms that impede innate immune defenses. Here we show that the activation of the microtubule-associated guanine nucleotide exchange factor GEF-H1, encoded by Arhgef2, is essential for sensing of foreign RNA by RIG-I–like receptors. Activation of GEF-H1 controls RIG-I–dependent and Mda5-dependent phosphorylation of IRF3 and induction of IFN-β expression in macrophages. Generation of Arhgef2−/− mice revealed a pronounced signaling defect that prevented antiviral host responses to encephalomyocarditis virus and influenza A virus. Microtubule networks sequester GEF-H1 that upon activation is released to enable antiviral signaling by intracellular nucleic acid detection pathways.

A Quorum Sensing Signal Promotes Host Tolerance Training Through HDAC1-Mediated Epigenetic Reprogramming

The mechanisms by which pathogens evade elimination without affecting host fitness are not well understood. For the pathogen Pseudomonas aeruginosa, this evasion appears to be triggered by excretion of the quorum-sensing molecule 2-aminoacetophenone, which dampens host immune responses and modulates host metabolism, thereby enabling the bacteria to persist at a high burden level. Here, we examined how 2-aminoacetophenone trains host tissues to become tolerant to a high bacterial burden, without compromising host fitness. We found that 2-aminoacetophenone regulates histone deacetylase 1 expression and activity, resulting in hypo-acetylation of lysine 18 of histone H3 at pro-inflammatory cytokine loci. Specifically, 2-aminoacetophenone induced reprogramming of immune cells occurs via alterations in histone acetylation of immune cytokines in vivo and in vitro. This host epigenetic reprograming, which was maintained for up to 7 days, dampened host responses to subsequent exposure to 2-aminoacetophenone or other unrelated pathogen-associated molecules. The process was found to involve a distinct molecular mechanism of host chromatin regulation. Inhibition of histone deacetylase 1 prevented the immunomodulatory effects of 2-aminoacetophenone. These observations provide the first mechanistic example of a quorum-sensing molecule regulating a host epigenome to enable tolerance of infection. These insights have enormous potential for developing preventive treatments against bacterial infections.

Reply to ‘Questioning antiviral RNAi in mammals’

Jeffrey et al. reply — Benjamin tenOever purports to comment on our claim that “mammals elicit a small RNA-mediated response to RNA virus infection in somatic cells”. Our article1 is a follow-up of two published papers in 2013, which provided the first evidence for an antiviral function of RNA interference (RNAi) in mammals2,3. The 2013 studies demonstrated production of canonical virus-derived small interfering RNAs (siRNAs) in suckling mice and cultured mouse embryonic stem cells (mESCs) and hamster cells following infection with positive-strand RNA viruses. Production of the viral siRNAs in all three host systems was strongly inhibited by the B2 protein of Nodamura virus (NoV), known previously to suppress antiviral RNAi in insect cells and siRNA-induced RNAi in mammalian cells4–6. Notably, the suppressor activity of B2 is required for NoV infection in all three systems and deletion of Argonaute 2 (AGO2) in mESCs enhanced accumulation of the B2-deletion mutant of NoV significantly more than wild-type NoV, indicating B2 suppression of an AGO2-dependent antiviral RNAi mechanism in mammalian cells2,3. However, many questions remain to be addressed in mammalian antiviral RNAi. Our new study aimed firstly to understand why many previous deep sequencing studies were unable to detect viral siRNAs in mature human somatic cells infected with a range of RNA viruses. These unsuccessful attempts, including one by tenOever and colleagues, to deep sequence small RNAs from human A549 cells infected with wild-type A/Puerto Rico/8/1934(H1N1, PR8) strain of influenza A virus (IAV)7 have led to the idea that the conserved machinery of RNAi is unable to detect RNA virus infection in interferon (IFN)-competent mammalian somatic cells. Our study1 has shown that human HEK-293T, A549 cells and monkey Vero cells produce highly abundant viral siRNAs after infection with either the same PR8 strain of IAV or a related WSN strain, A/WASN/1933(H1N1). Two technical improvements were critical for our success. Firstly, host cells needed to be infected with a mutant IAV lacking function of the viral non-structural protein 1 (NS1), known to suppress antiviral RNAi in insect cells and siRNA-induced RNAi in mammalian cells4,8. Secondly, the small RNAs that are not specifically associated with AGOs and the RNA-induced silencing complex (RISC) needed to be removed by only including AGO co-immunoprecipitation RNAs into the construction of small RNA libraries for sequencing. Using this approach, the relative abundance of influenza viral siRNAs in the mature human somatic cells is comparable to those found in mESCs3 and Drosophila cells9. Notably, single species positiveand negative-strand influenza viral siRNAs are readily detectable by northern blot hybridization using regular RNA probes1, a key criteria used in microRNA (miRNA) annotation10. We demonstrated that the influenza viral siRNAs become undetectable by either deep sequencing or northern blotting in Dicer knockout cells, and that the defect in the viral siRNA biogenesis was restored by ectopic expression of human Dicer. Drosophila Dicer-2, but not Dicer-1, which are responsible for the biogenesis of viral siRNAs and cellular miRNAs, respectively11, could also produce influenza viral siRNAs in human cells, although the predominant size shifts from 22 nucleotides (nt), made by human Dicer, to 21 nt by Drosophila Dicer-2 (ref. 1). Moreover, the sequenced influenza viral siRNAs are highly enriched for the canonical siRNA duplexes with 2-nt 3ʹ-overhangs1, supporting the proposed model in which viral siRNAs are produced by human Dicer that uses doublestranded RNA (dsRNA) viral replicative intermediates as precursors. Consistent with the biogenesis of mammalian miRNAs by the same Dicer enzyme, Dicer-produced influenza viral siRNAs are abundantly loaded into AGOs and exhibit strong preference for uracil as the 5ʹ-terminal nucleotide1. Our findings explain why tenOever and colleagues failed to detect the influenza viral siRNAs in human cells infected with NS1expressing wild-type IAV7. Indeed, ectopic expression of NS1 can suppress production of siRNAs from either artificial long dsRNA12 or influenza viral dsRNA replicative intermediates1. We have also detected abundant virus-specific small RNAs not associated with RISC in infected human cells, which must be removed before the canonical properties of the viral siRNAs are visible1. We predict that AGO co-immunoprecipitation would improve the detection of viral siRNAs from viruses that do not have a suppressor to inhibit siRNA biogenesis. We did not develop a reporter system for the influenza viral siRNAs, frequently used to assay for the activity of a miRNA or siRNA to target a steadily transcribed messenger RNA. Instead, we used a genetic approach commonly employed to assess the impact of antiviral RNAi directly by comparing virus titres between wild-type and RNAi-defective mutant cells3,11,13–19. The primary mouse embryonic fibroblasts (MEFs) carrying a genetic mutation (Ago2D597A) that abolishes the activity of RISC programmed by siRNA to slice target RNA20 are the only available RNAi-defective mature somatic mammalian cells that are IFN-competent and exhibit no known defect in miRNA function20. Our results illustrate that three distinct RNA viruses replicate to significantly enhanced levels in the RNAi-defective mutant MEFs when compared to wild-type MEFs1. Our findings are consistent with the observation that RNAi-defective fruit flies are much more susceptible than wild-type flies to all viruses examined, including those expressing proteins that potently, but incompletely, suppress RNAi14–16. In the comments21 made on Benitez et al.’s article22 and related studies, pioneering work to engineer expression of miRNAs from viral RNA genomes23,24 have been presented and discussed to explain why these recombinant viruses remain susceptible to the artificial miRNAs despite expression of potent RNAi suppressors. Notably, abolishing the catalytic activity of AGO2 in MEFs is significantly more effective to enhance accumulation of the NS1-deletion mutant of IAV (delNS1) than wild-type IAV1, providing strong physiological evidence for both the induction and suppression of antiviral RNAi in mature mammalian somatic cells. One of the important questions that remain to be fully investigated is the relative contribution of antiviral RNAi to mammalian antiviral immunity. To this end, we have shown that induction of IFNstimulated genes (ISGs) is similar in wildtype and RNAi-defective MEFs1. Suppression of RNAi by the B2 protein of NoV also does not alter the expression of ISGs in infected mice2. Furthermore, production of abundant