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.The sequencing of the human genome and the genomes of dozens of other metazoan species has intensified the need for systematic methods to extract biological information directly from DNA sequence. Comparative genomics has emerged as a powerful methodology for this endeavour 1,2 . Comparison of few (two-four) closely related genomes has proven successful for the discovery of protein-coding genes 3-5 , RNA genes 6,7 , miRNA genes 8-11 and catalogues of regulatory elements 3,4,12-14 . The resolution and discovery power of these studies should increase with the number of genomes [15][16][17][18][19][20] , in principle enabling the systematic discovery of all conserved functional elements.The fruitfly Drosophila melanogaster is an ideal system for developing and evaluating comparative genomics methodologies. Over the past century, Drosophila has been a pioneering model in which many of the basic principles governing animal development and population biology were established 21 . In the past decade, the genome sequence of D. melanogaster provided one of the first systematic views *These authors contributed equally to this work. {Lists of participants and affiliations appear at the end of the paper.

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 D/D ) 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 D/D mice. Bacteria were cleared from lungs of both genotypes, albeit more slowly in Stat3 D/D 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.Keywords: lung; neutrophils; STAT3; pneumonia; cytokines Lung infections account for a tremendous burden of disease worldwide and are a leading cause of acute lung injury (1, 2). While Streptococcus pneumoniae is the most common agent in patients with community-acquired pneumonia (3), gram-negative rods such as Escherichia coli are a frequent cause of nosocomial pneumonia (4). Elimination of these and other pathogens from the lower respiratory tract is made possible by an effective innate immune response (5), which is necessary yet potentially dangerous to the infected host. For this reason, cytokine networks, neutrophil emigration, plasma extravasation, and other characteristics of acute inflammation must be precisely regulated to maintain tissue homeostasis.The STAT3 transcription factor influences both immunity and inflammatory injury, but the importance of STAT3 signaling during pneumonia is unknown. STAT3 activity has been attributed both inflammatory (6-9) and anti-inflammatory (10-12) roles. Likewise, the cytokine interleukin (IL)-6, which largely signals through STAT3 (13, 14), has also been described as both pro-(15-19) and anti-inflammatory (16, 20-22), depending on the biological context. During E. coli pneumonia, neutrophil recruitment and bacterial clearance are impaired in IL-6-deficient mice (15). While the mechanisms through which IL-6 functions during this i...

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-κB 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 IκBα in airway epithelial cells, implicating this cell type as an important locus of NF-κB 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 IκBα and IκBβ 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.

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.

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 myeloidspecific PTEN knockout mice, significantly more neutrophils were recruited to the inflamed lungs during neutropeniaassociated 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. (Blood. 2009; 113:4930-4941)