The stress-responsive cytotoxic effect of diesel exhaust particles on lymphatic endothelial cells

Abstract Diesel exhaust particles (DEPs) are very small (typically < 0.2 μm) fragments that have become major air pollutants. DEPs are comprised of a carbonaceous core surrounded by organic compounds such as polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs. Inhaled DEPs reach the deepest sites in the respiratory system where they could induce respiratory/cardiovascular dysfunction. Additionally, a previous study has revealed that a portion of inhaled DEPs often activate immune cells and subsequently induce somatic inflammation. Moreover, DEPs are known to localize in lymph nodes. Therefore, in this study we explored the effect of DEPs on the lymphatic endothelial cells (LECs) that are a constituent of the walls of lymph nodes. DEP exposure induced cell death in a reactive oxygen species (ROS)-dependent manner. Following exposure to DEPs, next-generation sequence (NGS) analysis identified an upregulation of the integrated stress response (ISR) pathway and cell death cascades. Both the soluble and insoluble components of DEPs generated intracellular ROS. Three-dimensional Raman imaging revealed that DEPs are taken up by LECs, which suggests internalized DEP cores produce ROS, as well as soluble DEP components. However, significant cell death pathways such as apoptosis, necroptosis, ferroptosis, pyroptosis, and parthanatos seem unlikely to be involved in DEP-induced cell death in LECs. This study clarifies how DEPs invading the body might affect the lymphatic system through the induction of cell death in LECs

Testicular germ cell-specific elimination of Inv::GFP protein in Inv-KD mice.

<p>Testicular sections prepared from adult (a–f and i–p) or postnatal (3 day) (g and h) Inv-KD mice (b, d, f, h, j, l, n and p) or control Inv::GFP-rescue mice (a, c, e, g, i, k, m and o) were stained with anti-GFP antibodies (g, h, m and n, red) or double-stained with anti-GFP antibodies (c, d, i–l, o and p, red) and testicular germ cell-specific anti-calmegin antibodies (e, f, k and l, green) or vascular endothelial cell-specific PECAM-1 antibodies (o and p, green). Hoechst-stained images are shown to identify the cell types of the testis (a, b, g–i and m–p, blue). Inv::GFP protein was mainly observed in the spermatid cells of testicular germ cells (a, c, e, i and k, arrowhead) and somatic cells, such as connective tissue cells and Leydig cells (m, double arrows and arrow) and vascular endothelial cells (o, arrow) in Inv::GFP-rescue mice. Inv::GFP protein was specifically eliminated in the germ cells of Inv-KD mice (b, d, f, j, and l), and was down-regulated in connective tissue and Leydig cells (n, double arrow and arrow) and vascular endothelial cells (p, arrow). In Inv-KD mice, Inv::GFP protein was eliminated in anti-calmegin antibody-positive germ cells in adult testis, and in spermatogonia cells in postnatal mice (day 3) testis (h, arrowhead) but not in Inv::GFP-rescue mice (g, arrowhead). Scale bars: 200 µm (a and b); 50 µm (g−j and m–p).</p

shRNA-based gene silencing of the Inv::GFP fusion gene.

<p>a: Construct of the Inv::GFP fusion gene introduced into Inv::GFP-rescue mice. EGFP coding cDNA was fused to the C terminus of Inv coding cDNA and inserted downstream of the EF1αpromoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089652#pone.0089652-Watanabe1" target="_blank">[28]</a>. b: Construct of the pH1/siRNAEGFP-CAG/HcRed1 (pGtoR) transgene. Two oligonucleotides containing sense and antisense 21 nt sequences from the EGFP coding region and a spacer sequence that provided a loop structure were inserted downstream of the H1 promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089652#pone.0089652-Hasuwa1" target="_blank">[6]</a>. HcRed1 coding cDNA was inserted into the vector downstream of the CAG promoter. c: Western blot analysis of Inv::GFP protein of embryonic day 18.5 (e18.5) embryos and adult testis. The lysates of e18.5 embryos (#1 line) and adult testis (#1 and #4 line) of pGtoR-integrated Inv-KD mice (<i>inv/inv</i>, <i>Inv::GFP</i>, <i>pGtoR</i>) and control litter mate Inv::GFP-rescue mice (<i>inv/inv</i>, <i>inv::GFP</i>) were analyzed by western blotting with anti-GFP antibodies. A band of approximately 140 kDa of Inv::GFP fusion protein was detected in Inv::GFP-rescue mice, but was specifically down-regulated in the pGtoR integrated Inv-KD mice embryos and testis. Accuracy of sample loading is indicated by the Coomassie-stained gel shown below. Bright field (d, e and j) and Hc-Red (f, g and k) or GFP (h, i and l) fluorescent observation of e8.0 embryos (d–i, arrowheads) and new bone (j–l) of Inv-KD mice (d, f, h and j–l, left) and control Inv::GFP-rescue mice (e, g, i and j–l, right) are shown. Hc-Red fluorescent was specifically expressed in Inv-KD mice but not control Inv::GFP-rescue mice (f, g and k). Inv::GFP fluorescent is specifically down-regulated in Inv-KD mice compared with Inv::GFP-rescue mice (h, i and l).</p

Down-regulation of Inv::GFP protein in Inv-KD mouse kidneys.

<p>Cross sections prepared from adult (12 month-old) kidneys of Inv-KD mice (g–l) and control Inv::GFP-rescue mice (a–f) were double-stained with anti-GFP antibodies (a–c, red) and PNA-FITC (b, green) or anti-GFP (d, g and j, red) and anti-acetylated tubulin antibodies (e, h and k, green). Nuclei were counterstained with Hoechst dye (a and c). High magnification image of the boxed region (a and b) are shown in panel (c). Merged and high magnification image of (de, gh and jk) are shown in panel (f, i and l) respectively. Inv::GFP protein was detected in proximal (PNA-negative) and distal (PNA-positive) tubules of Inv::GFP-rescue mice (a and b). Localization of Inv::GFP protein was detected in the cilia of both proximal and distal tubules (c, arrowhead).Inv::GFP protein was specifically down-regulated in the renal tubules of Inv-KD mice (g and j) compared with Inv::GFP-rescue mice (a and d). Inv::GFP fusion proteins were localized in the proximal region of anti-acetylated tubulin positive cilia of the renal tubules of Inv::GFP-rescue mice (d–f, arrowheads), but were down-regulated in Inv-KD mice (g–i, arrowheads). Inv::GFP protein was significantly down-regulated in the cystic tubules (j–l) compared with the non-cystic tubules (g–i). Images were taken at 10×40 (a and b) and 10×60 (d, e, g, h, j and k) magnification. Scale bars: 50 µm (a, d, g and j); 5 µm (c, f, i and l).</p

Multiple renal cyst development in Inv-KD mice.

<p>Cross sections prepared from adult (12 months old) Inv-KD mice (b) and control litter mate Inv::GFP-rescue mouse kidneys (a). High magnification image of the boxed region of (b) is shown in panel (c). Multiple renal cysts were observed in Inv-KD mice. The average number of cysts per single kidney cross section at 1–1.5 months old (lanes 1–4) or 12 months old (lanes 5–8) of kidneys in Inv-KD mice (lanes 2, 4, 6 and 8) and control litter mate Inv::GFP-rescue mice (lanes 1, 3, 5 and 7), diameter 400–800 µm (lanes 1, 2, 5 and 6) and over 800 µm (lanes 3, 4, 7 and 8) are shown (d). Average percentage of cyst area per single kidney cross section of adult (12 months old) Inv::GFP-rescue and Inv-KD mice are shown (e). Error bars represent S.E.M. (*) and (**) indicate Student’s t-test values <0.05 and <0.001, respectively. Scale bars: 1 mm.</p

Anatomical analysis of adult Inv-KD mice.

<p>Anatomical analysis of pGtoR integrated adult (18 months old) Inv-KD mice (b and c) and control litter mate Inv::GFP-rescue mice (a). High magnification images of the kidney of (a–c) are shown in panels (d–f) and (g–i). Inv-KD mice developed fibrotic kidney with multiple cysts, but this was not observed in Inv::GFP-rescue mice. Highly developed multiple cysts were observed on the surface of Inv-KD mouse kidneys (h and i, arrowheads). Enlarged spleens were observed in adult Inv-KD mice (b and c, arrows).</p