NF-κB translocation prevents host cell death after low-dose challenge by Legionella pneumophila

Legionella pneumophila, the causative agent of Legionnaires' disease, grows within macrophages and manipulates target cell signaling. Formation of a Legionella-containing replication vacuole requires the function of the bacterial type IV secretion system (Dot/Icm), which transfers protein substrates into the host cell cytoplasm. A global microarray analysis was used to examine the response of human macrophage-like U937 cells to low-dose infections with L. pneumophila. The most striking change in expression was the Dot/Icm-dependent up-regulation of antiapoptotic genes positively controlled by the transcriptional regulator nuclear factor κB (NF-κB). Consistent with this finding, L. pneumophila triggered nuclear localization of NF-κB in human and mouse macrophages in a Dot/Icm-dependent manner. The mechanism of activation at low-dose infections involved a signaling pathway that occurred independently of the Toll-like receptor adaptor MyD88 and the cytoplasmic sensor Nod1. In contrast, high multiplicity of infection conditions caused a host cell response that masked the unique Dot/Icm-dependent activation of NF-κB. Inhibition of NF-κB translocation into the nucleus resulted in premature host cell death and termination of bacterial replication. In the absence of one antiapoptotic protein, plasminogen activator inhibitor–2, host cell death increased in response to L. pneumophila infection, indicating that induction of antiapoptotic genes is critical for host cell survival

Binucleation ramps up gene expression meeting the physiological demands of an organism.

In this issue of PLOS Biology, van Rijnberk and colleagues show how polyploidy, via binucleation, enables Caenorhabditis elegans intestinal cells to ramp up gene expression supplying the oocytes with the necessary lipids for optimal organismal growth and reproductive fitness

Wound-Induced Polyploidization: Regulation by Hippo and JNK Signaling and Conservation in Mammals.

Tissue integrity and homeostasis often rely on the proliferation of stem cells or differentiated cells to replace lost, aged, or damaged cells. Recently, we described an alternative source of cell replacement- the expansion of resident, non-dividing diploid cells by wound-induced polyploidization (WIP). Here we show that the magnitude of WIP is proportional to the extent of cell loss using a new semi-automated assay with single cell resolution. Hippo and JNK signaling regulate WIP; unexpectedly however, JNK signaling through AP-1 limits rather than stimulates the level of Yki activation and polyploidization in the Drosophila epidermis. We found that polyploidization also quantitatively compensates for cell loss in a mammalian tissue, mouse corneal endothelium, where increased cell death occurs with age in a mouse model of Fuchs Endothelial Corneal Dystrophy (FECD). Our results suggest that WIP is an evolutionarily conserved homeostatic mechanism that maintains the size and synthetic capacity of adult tissues

Polyploidization and Cell Fusion Contribute to Wound Healing in the Adult Drosophila Epithelium

SummaryBackgroundReestablishing epithelial integrity and biosynthetic capacity is critically important following tissue damage. The adult Drosophila abdominal epithelium provides an attractive new system to address how postmitotic diploid cells contribute to repair.ResultsPuncture wounds to the adult Drosophila epidermis close initially by forming a melanized scab. We found that epithelial cells near the wound site fuse to form a giant syncytium, which sends lamellae under the scab to re-epithelialize the damaged site. Other large cells arise more peripherally by initiating endocycles and becoming polyploid, or by cell fusion. Rac GTPase activity is needed for syncytium formation, while the Hippo signaling effector Yorkie modulates both polyploidization and cell fusion. Large cell formation is functionally important because when both polyploidization and fusion are blocked, wounds do not re-epithelialize.ConclusionsOur observations indicate that cell mass lost upon wounding can be replaced by polyploidization instead of mitotic proliferation. We propose that large cells generated by polyploidization or cell fusion are essential because they are better able than diploid cells to mechanically stabilize wounds, especially those containing permanent acellular structures, such as scar tissue

Mouse cornea endothelial cells increase ploidy to compensate for cell loss in Fuchs dystrophy mouse model.

<p>(A and B) Spatial distribution and ploidy values (as indicated by color code) of mouse endothelial nuclei from WT or <i>Col8a2</i>^{<i>Q455K/Q455K</i>} mice at 7 months old. Nuclei not determined (ND, gray). Scale bar, 50μm. (C) Ploidy (%) distribution in indicate mouse genotypes and ages. (D) Endothelial nuclear number significantly declines in <i>Col8a2</i>^{<i>Q455K/Q455K</i>} compared to WT mice by 7 months. (E) Total endothelial ploidy is not significantly different between WT and <i>Col8a2</i>^{<i>Q455K/Q455K</i>} mice. (C-E) Quantified from 3 representative endothelial (150,000μm²) regions within corneas from age matched male mice as represented in A and B. Error bars represent standard deviation where *<i>p</i><0.05 and n.s., not significant (<i>p</i>>0.05) are based on two-tailed Student's <i>t</i> test.</p

Wound-induced polyploidization compensates for cell loss in adult Drosophila epidermis.

<p>Schematic of the semi-automated approach to identify and measure DNA content within fly epidermis. Fly epidermal nuclei were identified by epidermal specific expression of nuclear GFP. Nuclei were outlined (red) using Fiji’s trace function and transferred to the corresponding DAPI image. Nuclear traces were color-coded based on the normalized ploidy value as indicated. Nuclei overlapping with non-epidermal nuclei were not determined (ND, gray). Epidermal cells polyploidize in response to injury: (A) Uninjured (-), (B) 3d post injury (+), and (C) % of epidermal nuclei in the indicated color-coded ploidy ranges. (D) Average epidermal ploidy (black or gray lines for injured (+) or uninjured (-), respectively) and nuclear density (brown line) versus distance from the wound center or image center in ctrl. (E) Epidermal nuclear number is reduced 3d post injury. (F) Total epidermal ploidy is not significantly different (n.s.) at 3d post injury. All analysis was preformed with 2 uninjured and 5 injured flies. Error bars represent standard deviation where *<i>p</i><0.05 is based on two-tailed Student's <i>t</i> test.</p

Hippo pathway controls the extent of endoreplication post injury.

<p>EdU marks endoreplicating cells around the wound scar (W, white dashed line) at 2d post injury. Immunofluorescent images of EdU staining in (A) ctrl or (B) epidermal specific Yki overexpression (<i>yki</i>^<i>OE</i>). The boundary of EdU+ area is outlined (red dashed line). (C) Diagram of core Hippo signaling pathway regulating entry into the endocycle. (D-F) Quantification of the effect of core Hippo genes on EdU response where (D) represents the average number EdU+ nuclei, (E) the average area containing EdU+ cells, (F) average EdU intensity per nucleus, and (G) the wound scar size at 2d post injury. All constructs were expressed with epidermal specific-Gal4 driver and examined at 2d post injury. At least 3 flies were scored for each condition. Error bars represent standard deviation where *<i>p</i><0.05, **<i>p</i><0.01, and n.s., not significant (<i>p</i>>0.05) are based on two-tailed Student's <i>t</i> test.</p

Yki tunes the extent of polyploization post injury.

<p>Spatial distrubution and ploidy values (as indicated by color code) of fly abdominal epidermal nuclei expressing <i>yki</i>^<i>OE</i> or <i>yki</i>^<i>RNAi</i> (A and C) Uninjured and (B and D) 3d post injury, respectively. Nuclei not determined (ND, gray). Scale bar, 50μm. (E) Ploidy (%) distribution in uninjured (-) and 3d post injury (+) in indicate fly genotypes. (F) Average epidermal ploidy and nuclear density versus distance from the wound center or image center in ctrl in the indicated conditions. (G) Epidermal nuclear number significantly declines 3 days post injury, except when <i>yki</i> is overexpressed in fly epidermis. (H) Total tissue ploidy is altered by <i>yki</i> expression. (E-H) All analysis was preformed with at least 2 uninjured and 3 injured flies for each condition. Error bars represent standard deviation where **<i>p</i><0.01 and n.s., not significant (p>0.05) are based on two-tailed Student's <i>t</i> test.</p

AP-1 regulates wound-induced polyploidy by affecting Yki.

<p>(A) Yki (<i>dIAP-lacZ</i>) and JNK (<i>puc</i>-Gal4, UAS-GFP) reporters are upregulated by injury and co-localize around wound site at 2d. (B) Spatial distribution and ploidy values (as indicated by color code) of fly abdominal epidermal nuclei expressing <i>jun</i>^<i>RNAi</i>. Nuclei not determined (ND, gray). (C) Ploidy (%) distribution in uninjured (-) and 3d post injury (+) in indicated fly genotypes. Analysis was performed with 2 uninjured and 5 injured flies. (D-G) Immunofluorescent images of EdU stained fly abdomens at 2d post injury expressing indicated transgenes. (H and I) AP-1 transcription factors, <i>jun</i> and <i>fos</i>, affect endoreplication as measured by EdU incorportation. (I) is the average EdU intensity per nucleus. All transgenes were expressed with epidermal specific-Gal4 driver and examined at 2d post injury. At least 3 flies were scored for each condition. Error bars represent standard deviation where *<i>p</i><0.05, **<i>p</i><0.01, and n.s., not significant (p>0.05) are based on two-tailed Student's <i>t</i> test.</p