Alpha/beta interferon immune defenses are essential for resistance to viruses and can be triggered through the actions of the cytoplasmic helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiationassociated gene 5 (MDA5). Signaling by each is initiated by the recognition of viral products such as RNA and occurs through downstream interaction with the IPS-1 adaptor protein. We directly compared the innate immune signaling requirements of representative viruses of the Flaviviridae, Orthomyxoviridae, Paramyxoviridae, and Reoviridae for RIG-I, MDA5, and interferon promoter-stimulating factor 1 (IPS-1). In cultured fibroblasts, IPS-1 was essential for innate immune signaling of downstream interferon regulatory factor 3 activation and interferon-stimulated gene expression, but the requirements for RIG-I and MDA5 were variable. Each was individually dispensable for signaling triggered by reovirus and dengue virus, whereas RIG-I was essential for signaling by influenza A virus, influenza B virus, and human respiratory syncytial virus. Functional genomics analyses identified cellular genes triggered during influenza A virus infection whose expression was strictly dependent on RIG-I and which are involved in processes of innate or adaptive immunity, apoptosis, cytokine signaling, and inflammation associated with the host response to contemporary and pandemic strains of influenza virus. These results define IPS-1-dependent signaling as an essential feature of host immunity to RNA virus infection. Our observations further demonstrate differential and redundant roles for RIG-I and MDA5 in pathogen recognition and innate immune signaling that may reflect unique and shared biologic properties of RNA viruses whose differential triggering and control of gene expression may impact pathogenesis and infection.

Plasmacytoid dendritic cells (pDCs) play essential roles in directing immune responses. These cells may be particularly important in determining the nature of immune responses to viral infections in patients with allergic asthma as well those with other atopic diseases. The purposes of this study were 1) to compare the functional capacity of pDCs in patients with one type of allergic disorder, allergic asthma, and controls; 2) to determine whether IgE cross-linking affects antiviral responses of influenza-exposed pDCs; and 3) to determine whether evidence of counterregulation of FcεRIα and IFN-α pathways exists in these cells. pDC function was assessed in a subset of asthma patients and in controls by measuring IFN-α production after exposure of purified pDCs to influenza viruses. FcεRIα expression on pDCs was determined by flow cytometry in blood samples from patients with allergic asthma and controls. pDCs from patients with asthma secreted significantly less IFN-α upon exposure to influenza A (572 versus 2815; p = 0.03), and secretion was inversely correlated with serum IgE levels. Moreover, IgE cross-linking prior to viral challenge resulted in 1) abrogation of the influenza-induced pDC IFN-α response; 2) diminished influenza and gardiquimod-induced TLR-7 upregulation in pDCs; and 3) interruption of influenza-induced upregulation of pDC maturation/costimulatory molecules. In addition, exposure to influenza and gardiquimod resulted in upregulation of TLR-7, with concomitant downregulation of FcεRIα expression in pDCs. These data suggest that counterregulation of FcεRI and TLR-7 pathways exists in pDCs, and that IgE cross-linking impairs pDC antiviral responses.

Genetic modification is critically enabling for studies addressing specification and maintenance of cell fate, however methods for engineering modifications are inefficient. We demonstrate a rapid and efficient recombination system in which an inducible, floxed cre allele replaces itself with an incoming transgene. We target this inducible cassette exchange (ICE) allele to the HPRT locus, and demonstrate recombination in murine embryonic stem (ES) cells and primary cells from derivative ICE mice. Using lentivectors, we demonstrate recombination at a randomly integrated ICE locus in human ES cells. To illustrate the utility of this system, we insert the myogenic regulator, Myf5, into the ICE locus in each platform. This enables efficient directed differentiation of mouse and human ES cells into skeletal muscle, and conditional myogenic transdetermination of primary cells cultured in vitro. This versatile tool is thus well suited to gain-of-function studies probing gene function in the specification and reprogramming of cell fate.

Plasmacytoid dendritic cells (pDCs) are vital to antiviral defense, directing immune responses via secretion of huge concentrations of IFN-α. These cells are critical in protecting the lung against clinically relevant respiratory viruses, particularly influenza (Flu), a virus responsible for substantial worldwide morbidity and mortality. How pDC responses to such viral pathogens are regulated, however, is poorly understood in humans. Using an unbiased approach of gene chip analysis, we discovered that Flu significantly impacts metabolism in primary human pDCs. We demonstrate that Flu and RV, another common respiratory virus, induce glycolysis in pDCs and that this metabolic pathway regulates pDC antiviral functions including IFN-α production and phenotypic maturation. Intranasal vaccination of human volunteers with live influenza virus also increases glycolysis in circulating pDCs, highlighting a previously unrecognized potential role for metabolism in regulating pDC immune responses to viral infections in humans.