Experimental evidence for splicing of intron-containing transcripts of plant LTR retrotransposon Ogre

Ogre elements are a distinct group of plant Ty3/gypsy-like retrotransposons characterized by several specific features, one of which is a separation of the gag-pol region into two non-overlapping open reading frames: ORF2 coding for Gag-Pro, and ORF3 coding for RT/RH-INT proteins. Previous characterization of Ogre elements from several plant species revealed that part of their transcripts lacks the region between ORF2 and ORF3, carrying one uninterrupted ORF instead. In this work, we investigated a hypothesis that this region represents an intron that is spliced out from part of the Ogre transcripts as a means for preferential production of ORF2-encoded proteins over those encoded by the complete ORF2–ORF3 region. The experiments involved analysis of transcription patterns of well-defined Ogre populations in a model plant Medicago truncatula and examination of transcripts carrying dissected pea Ogre intron expressed within a coding sequence of chimeric reporter gene. Both experimental approaches proved that the region between ORF2 and ORF3 is spliced from Ogre transcripts and showed that this process is only partial, probably due to weak splice signals. This is one of very few known cases of spliced LTR retrotransposons and the only one where splicing does not involve parts of the element’s coding sequences, thus resembling intron splicing found in most cellular genes

CD200 Receptor Controls Sex-Specific TLR7 Responses to Viral Infection

Immunological checkpoints, such as the inhibitory CD200 receptor (CD200R), play a dual role in balancing the immune system during microbial infection. On the one hand these inhibitory signals prevent excessive immune mediated pathology but on the other hand they may impair clearance of the pathogen. We studied the influence of the inhibitory CD200-CD200R axis on clearance and pathology in two different virus infection models. We find that lack of CD200R signaling strongly enhances type I interferon (IFN) production and viral clearance and improves the outcome of mouse hepatitis corona virus (MHV) infection, particularly in female mice. MHV clearance is known to be dependent on Toll like receptor 7 (TLR7)-mediated type I IFN production and sex differences in TLR7 responses previously have been reported for humans. We therefore hypothesize that CD200R ligation suppresses TLR7 responses and that release of this inhibition enlarges sex differences in TLR7 signaling. This hypothesis is supported by our findings that in vivo administration of synthetic TLR7 ligand leads to enhanced type I IFN production, particularly in female Cd200−/− mice and that CD200R ligation inhibits TLR7 signaling in vitro. In influenza A virus infection we show that viral clearance is determined by sex but not by CD200R signaling. However, absence of CD200R in influenza A virus infection results in enhanced lung neutrophil influx and pathology in females. Thus, CD200-CD200R and sex are host factors that together determine the outcome of viral infection. Our data predict a sex bias in both beneficial and pathological immune responses to virus infection upon therapeutic targeting of CD200-CD200R

Type I Interferon Signaling Regulates Ly6Chi Monocytes and Neutrophils during Acute Viral Pneumonia in Mice

Type I interferon (IFN-I) plays a critical role in the homeostasis of hematopoietic stem cells and influences neutrophil influx to the site of inflammation. IFN-I receptor knockout (Ifnar1−/−) mice develop significant defects in the infiltration of Ly6Chi monocytes in the lung after influenza infection (A/PR/8/34, H1N1). Ly6Chi monocytes of wild-type (WT) mice are the main producers of MCP-1 while the alternatively generated Ly6Cint monocytes of Ifnar1−/− mice mainly produce KC for neutrophil influx. As a consequence, Ifnar1−/− mice recruit more neutrophils after influenza infection than do WT mice. Treatment of IFNAR1 blocking antibody on the WT bone marrow (BM) cells in vitro failed to differentiate into Ly6Chi monocytes. By using BM chimeric mice (WT BM into Ifnar1−/− and vice versa), we confirmed that IFN-I signaling in hematopoietic cells is required for the generation of Ly6Chi monocytes. Of note, WT BM reconstituted Ifnar1−/− chimeric mice with increased numbers of Ly6Chi monocytes survived longer than influenza-infected Ifnar1−/− mice. In contrast, WT mice that received Ifnar1−/− BM cells with alternative Ly6Cint monocytes and increased numbers of neutrophils exhibited higher mortality rates than WT mice given WT BM cells. Collectively, these data suggest that IFN-I contributes to resistance of influenza infection by control of monocytes and neutrophils in the lung

ChemR23 Dampens Lung Inflammation and Enhances Anti-viral Immunity in a Mouse Model of Acute Viral Pneumonia

Viral diseases of the respiratory tract, which include influenza pandemic, children acute bronchiolitis, and viral pneumonia of the elderly, represent major health problems. Plasmacytoid dendritic cells play an important role in anti-viral immunity, and these cells were recently shown to express ChemR23, the receptor for the chemoattractant protein chemerin, which is expressed by epithelial cells in the lung. Our aim was to determine the role played by the chemerin/ChemR23 system in the physiopathology of viral pneumonia, using the pneumonia virus of mice (PVM) as a model. Wild-type and ChemR23 knock-out mice were infected by PVM and followed for functional and inflammatory parameters. ChemR23−/− mice displayed higher mortality/morbidity, alteration of lung function, delayed viral clearance and increased neutrophilic infiltration. We demonstrated in these mice a lower recruitment of plasmacytoid dendritic cells and a reduction in type I interferon production. The role of plasmacytoid dendritic cells was further addressed by performing depletion and adoptive transfer experiments as well as by the generation of chimeric mice, demonstrating two opposite effects of the chemerin/ChemR23 system. First, the ChemR23-dependent recruitment of plasmacytoid dendritic cells contributes to adaptive immune responses and viral clearance, but also enhances the inflammatory response. Second, increased morbidity/mortality in ChemR23−/− mice is not due to defective plasmacytoid dendritic cells recruitment, but rather to the loss of an anti-inflammatory pathway involving ChemR23 expressed by non-leukocytic cells. The chemerin/ChemR23 system plays important roles in the physiopathology of viral pneumonia, and might therefore be considered as a therapeutic target for anti-viral and anti-inflammatory therapies

Microtubule Dynamics Regulate Cyclic Stretch-Induced Cell Alignment in Human Airway Smooth Muscle Cells

Microtubules are structural components of the cytoskeleton that determine cell shape, polarity, and motility in cooperation with the actin filaments. In order to determine the role of microtubules in cell alignment, human airway smooth muscle cells were exposed to cyclic uniaxial stretch. Human airway smooth muscle cells, cultured on type I collagen-coated elastic silicone membranes, were stretched uniaxially (20% in strain, 30 cycles/min) for 2 h. The population of airway smooth muscle cells which were originally oriented randomly aligned near perpendicular to the stretch axis in a time-dependent manner. However, when the cells treated with microtubule disruptors, nocodazole and colchicine, were subjected to the same cyclic uniaxial stretch, the cells failed to align. Lack of alignment was also observed for airway smooth muscle cells treated with a microtubule stabilizer, paclitaxel. To understand the intracellular mechanisms involved, we developed a computational model in which microtubule polymerization and attachment to focal adhesions were regulated by the preexisting tensile stress, pre-stress, on actin stress fibers. We demonstrate that microtubules play a central role in cell re-orientation when cells experience cyclic uniaxial stretching. Our findings further suggest that cell alignment and cytoskeletal reorganization in response to cyclic stretch results from the ability of the microtubule-stress fiber assembly to maintain a homeostatic strain on the stress fiber at focal adhesions. The mechanism of stretch-induced alignment we uncovered is likely involved in various airway functions as well as in the pathophysiology of airway remodeling in asthma

Immunization with Radiation-Attenuated Plasmodium berghei Sporozoites Induces Liver cCD8α+DC that Activate CD8+T Cells against Liver-Stage Malaria

Immunization with radiation (γ)-attenuated Plasmodia sporozoites (γ-spz) confers sterile and long-lasting immunity against malaria liver-stage infection. In the P. berghei γ-spz model, protection is linked to liver CD8+ T cells that express an effector/memory (TEM) phenotype, (CD44hiCD45RBloCD62Llo ), and produce IFN-γ. However, neither the antigen presenting cells (APC) that activate these CD8+ TEM cells nor the site of their induction have been fully investigated. Because conventional (c)CD8α+ DC (a subset of CD11c+ DC) are considered the major inducers of CD8+ T cells, in this study we focused primarily on cCD8α+ DC from livers of mice immunized with Pb γ-spz and asked whether the cCD8α+ DC might be involved in the activation of CD8+ TEM cells. We demonstrate that multiple exposures of mice to Pb γ-spz lead to a progressive and nearly concurrent accumulation in the liver but not the spleen of both the CD11c+NK1.1− DC and CD8+ TEM cells. Upon adoptive transfer, liver CD11c+NK1.1− DC from Pb γ-spz-immunized mice induced protective immunity against sporozoite challenge. Moreover, in an in vitro system, liver cCD8α+ DC induced naïve CD8+ T cells to express the CD8+ TEM phenotype and to secrete IFN-γ. The in vitro induction of functional CD8+ TEM cells by cCD8α+ DC was inhibited by anti-MHC class I and anti-IL-12 mAbs. These data suggest that liver cCD8α+ DC present liver-stage antigens to activate CD8+ TEM cells, the pre-eminent effectors against pre-erythrocytic malaria. These results provide important implications towards a design of anti-malaria vaccines

Critical Role of Constitutive Type I Interferon Response in Bronchial Epithelial Cell to Influenza Infection

Innate antiviral responses in bronchial epithelial cells (BECs) provide the first line of defense against respiratory viral infection and the effectiveness of this response is critically dependent on the type I interferons (IFNs). However the importance of the antiviral responses in BECs during influenza infection is not well understood. We profiled the innate immune response to infection with H3N2 and H5N1 virus using Calu-3 cells and primary BECs to model proximal airway cells. The susceptibility of BECs to influenza infection was not solely dependent on the sialic acid-bearing glycoprotein, and antiviral responses that occurred after viral endocytosis was more important in limiting viral replication. The early antiviral response and apoptosis correlated with the ability to limit viral replication. Both viruses reduced RIG-I associated antiviral responses and subsequent induction of IFN-β. However it was found that there was constitutive release of IFN-β by BECs and this was critical in inducing late antiviral signaling via type I IFN receptors, and was crucial in limiting viral infection. This study characterizes anti-influenza virus responses in airway epithelial cells and shows that constitutive IFN-β release plays a more important role in initiating protective late IFN-stimulated responses during human influenza infection in bronchial epithelial cells

Effect of Respiratory Syncytial Virus Infection on Plasmacytoid Dendritic Cell Regulation of Allergic Airway Inflammation.

Background: Respiratory syncytial virus (RSV) can infect myeloid dendritic cells (mDCs) and regulate their function in the development of allergy. It has been widely reported that plasmacytoid DCs (pDCs) play a critical role in antiviral innate immunity. In contrast, not much is known about the role of pDCs in the interaction between allergy and viral infection. The purpose of the present study was to investigate the effect of RSV infection on pDC function in the regulation of allergic airway inflammation in a murine model of Dermatophagoides farinae-sensitized allergic asthma. Methods: Splenic pDCs isolated from D. farinae-sensitized donor mice were infected with live RSV ex vivo. Subsequently, these pDCs were inoculated into the airways of D. farinae-sensitized recipient mice. Lung pathology, lung tissue cytokine profiles, the number of regulatory T cells (T(reg)) and mDCs as well as the effects of IL-10 neutralization in the lung tissue of recipient mice were determined. Results: Intranasal inoculation of D. farinae-sensitized pDCs significantly inhibited the development of allergic airway inflammation and both Th1 and Th2 immunity. Live RSV infection of these pDCs prior to inoculation interfered with their inhibitory effects through decreasing T(reg) and IL-10 and increasing mDCs. Conclusions: In asthmatic airways, pDCs mediate tolerance to inhaled allergens through the regulation of T(reg), IL-10 and mDCs. RSV infection of pDCs potentially inhibits their immunotolerogenic effects and thus exacerbates allergic airway inflammation