Interaction of pathogens with cells of the immune system results in activation of inflammatory gene expression. This response, while 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 expression1–4. 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 expression5–10. 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 LPS-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.

SUMMARY 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 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.

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.

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 Slicerdeficient 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.Slicer | gene suppression | miRNA reprogramming

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.

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Mitogen-activated protein kinases facilitate many cellular processes and are essential for immune cell function. Their activity is controlled by kinases and dual-specificity phosphatases. A comprehensive microarray analysis of human leukocytes identified DUSP2 (encoding the phosphatase PAC-1) as one of the most highly induced transcripts in activated immune cells. We generated Dusp2(-/-) mice and found considerably reduced inflammatory responses in the 'K/BxN' model of rheumatoid arthritis. PAC-1 deficiency led to increased activity of Jun kinase (Jnk) but unexpected impairment of the activity of extracellular signal-regulated kinase (Erk) and the kinase p38, reduced activity of the transcription factor Elk1 and a complex of mobilized transcription factor NFAT and the AP-1 transcription factor and decreased effector immune cell function. Thus, PAC-1 is a key positive regulator of inflammatory cell signaling and effector functions, mediated through Jnk and Erk mitogen-activated protein kinase crosstalk.

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.