Discovery of functional elements in 12 Drosophila genomes using evolutionary signatures.

Sequencing of multiple related species followed by comparative genomics analysis constitutes a powerful approach for the systematic understanding of any genome. Here, we use the genomes of 12 Drosophila species for the de novo discovery of functional elements in the fly. Each type of functional element shows characteristic patterns of change, or 'evolutionary signatures', dictated by its precise selective constraints. Such signatures enable recognition of new protein-coding genes and exons, spurious and incorrect gene annotations, and numerous unusual gene structures, including abundant stop-codon readthrough. Similarly, we predict non-protein-coding RNA genes and structures, and new microRNA (miRNA) genes. We provide evidence of miRNA processing and functionality from both hairpin arms and both DNA strands. We identify several classes of pre- and post-transcriptional regulatory motifs, and predict individual motif instances with high confidence. We also study how discovery power scales with the divergence and number of species compared, and we provide general guidelines for comparative studies.

Earliest innate immune responses require macrophage RelA during pneumococcal pneumonia.

NF-κB regulates cytokine expression to initiate and control the innate immune response to lung infections. The NF-κB protein RelA is critical for pulmonary host defense during Streptococcus pneumoniae pneumonia, but the cell-specific roles of this transcription factor remain to be determined. We hypothesized that RelA in alveolar macrophages contributes to cytokine expression and host defense during pneumococcal pneumonia. To test this hypothesis, we compared mice lacking RelA exclusively in myeloid cells (RelA(Δ/Δ)) with littermate controls (RelA(F/F)). Alveolar macrophages from RelA(Δ/Δ) mice expressed no full-length RelA, demonstrating effective targeting. Alveolar macrophages from RelA(Δ/Δ) mice exhibited reduced, albeit detectable, proinflammatory cytokine responses to S. pneumoniae, compared with alveolar macrophages from RelA(F/F) mice. Concentrations of these cytokines in lung homogenates were diminished early after infection, indicating a significant contribution of macrophage RelA to the initial expression of cytokines in the lungs. However, the cytokine content in infected lungs was equivalent by 15 hours. Neutrophil recruitment during S. pneumoniae pneumonia reflected a delayed onset in RelA(Δ/Δ) mice, followed by similar rates of accumulation. Bacterial clearance was eventually effective in both genotypes, but began later in RelA(Δ/Δ) mice. Thus, during pneumococcal pneumonia, only the earliest induction of the cytokines measured depended on transcription by RelA in myeloid cells, and this transcriptional activity contributed to effective immunity.

Targeted deletion of tumor suppressor PTEN augments neutrophil function and enhances host defense in neutropenia-associated pneumonia.

Neutropenia and related infections are the most important dose-limiting toxicities in anticancer chemotherapy and radiotherapy. In this study, we explored a new strategy for augmenting host defense in neutropenia-related pneumonia. Phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) signaling in neutrophils was elevated by depleting PTEN, a phosphatidylinositol 3'-phosphatase that hydrolyzes PtdIns(3,4,5)P(3). In myeloid-specific PTEN knockout mice, significantly more neutrophils were recruited to the inflamed lungs during neutropenia-associated pneumonia. Using an adoptive transfer technique, we demonstrated that this enhancement could be caused directly by PTEN depletion in neutrophils. In addition, disruption of PTEN increased the recruitment of macrophages and elevated proinflammatory cytokines/chemokine levels in the inflamed lungs, which could also be responsible for the enhanced neutrophil recruitment. Depleting PTEN also significantly delayed apoptosis and enhanced the bacteria-killing capability of the recruited neutrophils. Finally, we provide direct evidence that enhancement of neutrophil function by elevating PtdIns(3,4,5)P(3) signaling can alleviate pneumonia-associated lung damage and decrease pneumonia-elicited mortality. Collectively, these results not only provide insight into the mechanism of action of PTEN and PtdIns(3,4,5)P(3) signaling pathway in modulating neutrophil function during lung infection and inflammation, but they also establish PTEN and related pathways as potential therapeutic targets for treating neutropenia-associated pneumonia.

Mechanisms of the hepatic acute-phase response during bacterial pneumonia.

The acute-phase response is characterized by increased circulating levels of acute-phase proteins (APPs) generated by the liver. During bacterial pneumonia, APPs correlate with the severity of disease, serve as biomarkers, and are functionally significant. The kinetics and regulatory mechanisms of APP induction in the liver during lung infection have yet to be defined. Here we show that APP mRNA transcription is induced in the livers of mice whose lungs are infected with either Escherichia coli or Streptococcus pneumoniae, and that in both cases this induction occurs in tandem with activation in the liver of the transcription factors signal transducer and activator of transcription 3 (STAT3) and NF-kappaB RelA. Interleukin-6 (IL-6) deficiency inhibited the activation of STAT3 and the induction of select APPs in the livers of pneumonic mice. Furthermore, liver RelA activation and APP induction were reduced for mice lacking all signaling receptors for tumor necrosis factor alpha and IL-1. In a murine hepatocyte cell line, knockdown of either STAT3 or RelA by small interfering RNA inhibited cytokine induction of the APP serum amyloid A-1, demonstrating that both transcription factors were independently essential for the expression of this gene. These data suggest that during pneumonia caused by gram-negative or gram-positive bacteria, the expression of APPs in the liver depends on STAT3 activation by IL-6 and on RelA activation by early-response cytokines. These signaling axes may be critical for integrating systemic responses to local infection, balancing antibacterial host defenses and inflammatory injury during acute bacterial pneumonia.

Alveolar epithelial STAT3, IL-6 family cytokines, and host defense during Escherichia coli pneumonia.

While signal transducer and activator of transcription (STAT) 3 signaling has been linked to multiple pathways influencing immune function and cell survival, the direct influence of this transcription factor on innate immunity and tissue homeostasis during pneumonia is unknown. Human patients with dominant-negative mutations in the Stat3 gene develop recurrent pneumonias, suggesting a role for STAT3 in pulmonary host defense. We hypothesized that alveolar epithelial STAT3 is activated by IL-6 family cytokines and is required for effective responses during gram-negative bacterial pneumonia. STAT3 phosphorylation was increased in pneumonic mouse lungs and in murine lung epithelial (MLE)-15 cells stimulated with pneumonic bronchoalveolar lavage fluid (BALF) through 48 hours of Escherichia coli pneumonia. Mice lacking active STAT3 in alveolar epithelial cells (Stat3(Delta/Delta)) had fewer alveolar neutrophils and more viable bacteria than control mice early after intratracheal E. coli. By 48 hours after E. coli infection, however, lung injury was increased in Stat3(Delta/Delta) mice. Bacteria were cleared from lungs of both genotypes, albeit more slowly in Stat3(Delta/Delta) mice. Of the IL-6 family cytokines measured in lungs from infected C57BL/6 mice, IL-6, oncostatin M, leukemia inhibitory factor (LIF), and IL-11 were significantly elevated. Neutralization studies demonstrated that LIF and IL-6 mediated BALF-induced STAT3 activation in MLE-15 cells. Together, these results indicate that during E. coli pneumonia, select IL-6 family members activate alveolar epithelial STAT3, which functions to promote neutrophil recruitment and to limit both infection and lung injury.

Phylogenetic footprinting analysis in the upstream regulatory regions of the Drosophila enhancer of split genes.

During Drosophila development Suppressor of Hairless [Su(H)]-dependent Notch activation upregulates transcription of the Enhancer of split-Complex [E(spl)-C] genes. Drosophila melanogaster E(spl) genes share common transcription regulators including binding sites for Su(H), proneural, and E(spl) basic-helix-loop-helix (bHLH) proteins. However, the expression patterns of E(spl) genes during development suggest that additional factors are involved. To better understand regulators responsible for these expression patterns, recently available sequence and annotation data for multiple Drosophila genomes were used to compare the E(spl) upstream regulatory regions from more than nine Drosophila species. The mgamma and mbeta regulatory regions are the most conserved of the bHLH genes. Fine analysis of Su(H) sites showed that high-affinity Su(H) paired sites and the Su(H) paired site plus proneural site (SPS + A) architecture are completely conserved in a subset of Drosophila E(spl) genes. The SPS + A module is also present in the upstream regulatory regions of the more ancient mosquito and honeybee E(spl) bHLH genes. Additional transcription factor binding sites were identified upstream of the E(spl) genes and compared between species of Drosophila. Conserved sites provide new understandings about E(spl) regulation during development. Conserved novel sequences found upstream of multiple E(spl) genes may play a role in the expression of these genes.

Regulation of signal transducer and activator of transcription signaling by the tyrosine phosphatase PTP-BL.

Signal Transducer and Activator of Transcription (STAT) proteins are a family of latent cytoplasmic transcription factors that are activated by tyrosine phosphorylation after cytokine stimulation. One mechanism by which STAT signaling is regulated is by dephosphorylation through the action of protein tyrosine phosphatases (PTP). We have identified PTP-Basophil like (PTP-BL) as a STAT PTP. PTP-BL dephosphorylates STAT proteins in vitro and in vivo, resulting in attenuation of STAT-mediated gene activation. In CD4(+) T cells, PTP-BL deficiency leads to increased and prolonged activation of STAT4 and STAT6, and consequently enhanced T helper 1 (Th1) and Th2 cell differentiation. Taken together, our findings demonstrate that PTP-BL is a physiologically important negative regulator of the STAT signaling pathway.

Functions and regulation of NF-kappaB RelA during pneumococcal pneumonia.

Eradication of bacteria in the lower respiratory tract depends on the coordinated expression of proinflammatory cytokines and consequent neutrophilic inflammation. To determine the roles of the NF-kappaB subunit RelA in facilitating these events, we infected RelA-deficient mice (generated on a TNFR1-deficient background) with Streptococcus pneumoniae. RelA deficiency decreased cytokine expression, alveolar neutrophil emigration, and lung bacterial killing. S. pneumoniae killing was also diminished in the lungs of mice expressing a dominant-negative form of IkappaBalpha in airway epithelial cells, implicating this cell type as an important locus of NF-kappaB activation during pneumonia. To study mechanisms of epithelial RelA activation, we stimulated a murine alveolar epithelial cell line (MLE-15) with bronchoalveolar lavage fluid (BALF) harvested from mice infected with S. pneumoniae. Pneumonic BALF, but not S. pneumoniae, induced degradation of IkappaBalpha and IkappaBbeta and rapid nuclear accumulation of RelA. Moreover, BALF-induced RelA activity was completely abolished following combined but not individual neutralization of TNF and IL-1 signaling, suggesting either cytokine is sufficient and necessary for alveolar epithelial RelA activation during pneumonia. Our results demonstrate that RelA is essential for the host defense response to pneumococcus in the lungs and that RelA in airway epithelial cells is primarily activated by TNF and IL-1.