Basal epithelial stem cells cross an alarmin checkpoint for postviral lung disease

Epithelial cells are charged with protection at barrier sites, but whether this normally beneficial response might sometimes become dysfunctional still needs definition. Here, we recognized a pattern of imbalance marked by basal epithelial cell growth and differentiation that replaced normal airspaces in a mouse model of progressive postviral lung disease due to the Sendai virus. Single-cell and lineage-tracing technologies identified a distinct subset of basal epithelial stem cells (basal ESCs) that extended into gas-exchange tissue to form long-term bronchiolar-alveolar remodeling regions. Moreover, this cell subset was selectively expanded by crossing a cell-growth and survival checkpoint linked to the nuclear-localized alarmin IL-33 that was independent of IL-33 receptor signaling and instead connected to autocrine chromatin accessibility. This mechanism creates an activated stem-progenitor cell lineage with potential for physiological or pathological function. Thus, conditional loss of Il33 gene function in basal epithelial cells disrupted the homeostasis of the epithelial barrier at skin and gut sites but also markedly attenuated postviral disease in the lung based on the downregulation of remodeling and inflammation. Thus, we define a basal ESC strategy to deploy innate immune machinery that appears to overshoot the primordial goal of self-defense. Our findings reveal new targets to stratify and correct chronic and often deadly postviral disease

Long-term IL-33-producing epithelial progenitor cells in chronic obstructive lung disease

Chronic obstructive lung disease is characterized by persistent abnormalities in epithelial and immune cell function that are driven, at least in part, by infection. Analysis of parainfluenza virus infection in mice revealed an unexpected role for innate immune cells in IL-13–dependent chronic lung disease, but the upstream driver for the immune axis in this model and in humans with similar disease was undefined. We demonstrate here that lung levels of IL-33 are selectively increased in postviral mice with chronic obstructive lung disease and in humans with very severe chronic obstructive pulmonary disease (COPD). In the mouse model, IL-33/IL-33 receptor signaling was required for Il13 and mucin gene expression, and Il33 gene expression was localized to a virus-induced subset of airway serous cells and a constitutive subset of alveolar type 2 cells that are both linked conventionally to progenitor function. In humans with COPD, IL33 gene expression was also associated with IL13 and mucin gene expression, and IL33 induction was traceable to a subset of airway basal cells with increased capacities for pluripotency and ATP-regulated release of IL-33. Together, these findings provide a paradigm for the role of the innate immune system in chronic disease based on the influence of long-term epithelial progenitor cells programmed for excess IL-33 production

Template-dependent polymerization across discontinuous templates by the heterodimeric primase from the hyperthermophilic archaeon Sulfolobus solfataricus

The eukaryotic-like primase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsoPriSL) exhibits a range of activities including template-dependent de novo primer synthesis, primer extension and template-independent terminal nucleotidyl transfer using either rNTPs or dNTPs. Remarkably, the enzyme is able to synthesize products far longer than templates in vitro. Here we show that the long products resulted from template-dependent polymerization across discontinuous templates (PADT) by SsoPriSL. PADT was initiated through either primer synthesis or terminal transfer, and occurred efficiently on templates containing contiguous dCs. Template switching took place when the 3′-end of a growing strand synthesized on one template annealed to another template directly or following the terminal addition of nucleotides, and was subsequently extended on the new template. The key to PADT was the ability of SsoPriSL to promote strand annealing. SsoPriSL catalyzed PADT with either dNTPs or rNTPs as the substrates but preferred the latter. The enzyme remained active in PADT but became inefficient in primer synthesis in vitro when temperature was raised from 55°C to 70°C. Our results suggest that SsoPriSL is capable of bridging noncomplementary DNA ends and, therefore, may serve a role in double-strand DNA break repair in Archaea

Improved Control of Tuberculosis and Activation of Macrophages in Mice Lacking Protein Kinase R

Host factors that microbial pathogens exploit for their propagation are potential targets for therapeuic countermeasures. No host enzyme has been identified whose genetic absence benefits the intact mammalian host in vivo during infection with Mycobacterium tuberculosis (Mtb), the leading cause of death from bacterial infection. Here, we report that the dsRNA-dependent protein kinase (PKR) is such an enzyme. PKR-deficient mice contained fewer viable Mtb and showed less pulmonary pathology than wild type mice. We identified two potential mechanisms for the protective effect of PKR deficiency: increased apoptosis of macrophages in response to Mtb and enhanced activation of macrophages in response to IFN-gamma. The restraining effect of PKR on macrophage activation was explained by its mediation of a previously unrecognized ability of IFN-gamma to induce low levels of the macrophage deactivating factor interleukin 10 (IL10). These observations suggest that PKR inhibitors may prove useful as an adjunctive treatment for tuberculosis

Correction: Improved Control of Tuberculosis and Activation of Macrophages in Mice Lacking Protein Kinase R.

[This corrects the article DOI: 10.1371/journal.pone.0030512.]

Characteristics and Comparative Analysis of the Special-Structure (Non-Single-Circle) Mitochondrial Genome of <i>Capsicum pubescens</i> Ruiz & Pav

Chilean peppers, cultivated from Capsicum pubescens, are globally renowned as popular vegetable and spice crops. C. pubescens belongs to the Capsicum L. (pepper) family and is one of the five pepper cultivars grown in China. In this study, we assembled and annotated the complete mt genome of C. pubescens. We investigated several aspects of its genome, including characteristics, codon usage, RNA editing sites, repeat sequences, selective pressure, gene clusters, and phylogenetic relationships. Furthermore, we compared it with other plant mt genomes. The data we obtained will provide valuable information for studying evolutionary processes in the Capsicum genus and will assist in the functional analysis of Capsicum mitogenomes

Course of Mtb infection in PKR-deficient and wild type mice.

<p>(A) Time course of burden of colony-forming units (CFU) in lung, liver and spleen over 24 weeks following infection by inhalation of an average of 30 CFU by wild type mice and 37 CFU by PKR-deficient mice. Results are means ± SD for 5 mice per time point. Asterisks mark time points at which the differences had p values≤0.01 by Student's t test. (B) Mtb burdens (mean CFU ± SEM for 4–6 mice per strain) in lungs of mice from 5 independent experiments at the latest time point post-infection evaluated in each experiment. Red bars: PKR^−/− mice. Blue bars: C57LB/6 wild type controls. Green bar: 129S1/SvImJ wild type controls. The initial bacterial burdens were comparable between mouse strains within each experiment as assessed by the following CFU counts 24 hours post infection (pairs are means for wild type mice followed by PKR-deficient mice in individual experiments in the order depicted in the figure): 30, 37; 15, 20; 24, 22; 488, 468; 40, 45. The unusually low CFU seen in the first experiment are depicted with reference to an expanded Y-axis. (C and D) Histopathology (C) and acid-fast staining of Mtb (D) in lungs from Mtb-infected wild type and PKR^−/− mice at day 168 from a representative experiment. (E and F) Comparable uptake and control of Mtb by PKR-deficient and wild type macrophages in vitro. (E) Uptake of Mtb 4 hours after addition at the indicated MOI, with and without exposure of macrophages to IFN-gamma (10 ng/mL) overnight in advance of infection. (F) Intracellular growth of Mtb (MOI = 3) with and without exposure of macrophages to IFN-gamma (10 ng/mL) overnight in advance of infection. With the other MOIs tested (1 and 10), there was likewise no significant difference in the numbers of CFU in PKR-deficient and wild type macrophages over the 3 days studied.</p

Activation of PKR by IFN-gamma.

<p>(A) Autophosphorylation of PKR in macrophages at the indicated times after addition of IFN-gamma (10 ng/mL). A duplicate gel western blotted with antibody to beta-tubulin served as a loading control. One of four similar experiments. (B) Immunofluorescent localization of PKR in wild type macrophages with and without Mtb infection and/or IFN-gamma treatment. Macrophages from wild type and PKR^−/− mice were incubated with or without IFN-gamma (10 ng/mL) at 37°C overnight and then infected with Mtb at MOI 10 for 24 h. Macrophages were fixed with 4% <i>p</i>-formaldehyde for 2 h and immunofluorescent staining for PKR (green) and DNA (DAPI; blue) was conducted. Images were recorded by confocal microscopy.</p

Enhanced expression of iNOS in PKR-deficient mice and macrophages in response to Mtb and IFN-gamma.

<p>(A) Nitrite plus nitrate in sera from Mtb-infected wild type and PKR-deficient mice. Nitrite and nitrate in serum from Mtb-infected mice at day 168 were measured by the Griess reaction after nitrate was reduced to nitrite as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030512#pone.0030512-MacMicking2" target="_blank">[30]</a>. Means ± SD of 4–5 mice in one experiment representative of two. ***, p<0.0005 (unpaired <i>t</i> test). (B) Secretion of nitrite by macropahges in vitro. Macrophages (5×10⁵) were treated with Mtb (MOI 3), IFN-gamma (10 ng/mL), or pre-treated with IFN-gamma (10 ng/mL) overnight followed by infection with Mtb (MOI 3) for 24 h. Nitrite in the supernatant was determined by the Griess reaction. Means ± SD of triplicates. (C and D) Mature and nascent iNOS transcripts. Macrophages were incubated as in (B) for 3 h. qRT-PCR signals for iNOS were normalized to GAPDH. Means ± SD of triplicates. In (B), (C) and (D), results are from one of two similar experiments. Results were similar in additional experiments at MOI's of 0.3, 1 and 3 and IFN-gamma concentrations of 1, 10 and 100 ng/mL. (E) Western blot (left) and its densitometric assessment (right) for iNOS in macrophages incubated with the indicated concentrations of IFN-gamma for 24 h. Immunoblot for beta-tubulin served as a loading control. (F) Impact of PKR deficiency on stability of iNOS mRNA. Macrophages were treated with IFN-gamma (10 ng/mL) followed 3 h later by actinomycin D (10 micrograms/mL). RNA was extracted at the indicated times for RT-PCR and normalized to GAPDH.</p