感染症：感染によって誘導されるNETosisは複数の役割を同時に果たす好中球が関与するin vivoでの動的過程である

Abstract Abstract• References• Author information• Supplementary information 好中球細胞外トラップ（neutrophil extracellular trap：NET）は、 in vitro では好中球が死ぬ際に数時間を必要とする過程を介して放出されるため、微生物が侵入できる時間的ギャップが生じる。遊走やファゴサイトーシスが可能な好中球でNETosis状態にあるものは、まだ報告されていない。我々は、グラム陽性菌による皮膚感染の際に、生きている多形核細胞（PMN）が in vivo で迅速にNETを放出する様子を直接可視化した。この放出によって、細菌の全身への播種が防止された。NETosisは、好中球が這い回っている間に起こったので、NETの大部分が投げ広げられた。NET放出中の好中球は、分散して脱凝縮した核を持ち、最終的にはDNAを持たなくなった。異常な核を持つ好中球は、不規則な形の偽足と過剰な極性化を特徴とする独特の這い回り動態を示し、こうした動態は核が這い回りの支点となることと一致していた。Toll様受容体2と補体介在性オプソニン化が共に必要とされることで、NET放出は厳密に調節されていた。さらに、ヒトの生きているPMNをマウス皮膚に注入すると in vivo で脱凝縮した核が生じてNETが形成され、グラム陽性ヒト膿瘍には無傷の無核好中球が多数存在していた。したがって、感染初期のNETosisには、細胞溶解を起こしておらず、複数の役割を同時に果たす好中球が関与している。 View full text At a glance Figures View all figures Figure 1: Rapid in vivo NETosis during acute Gram-positive bacterial infections is directly visualized in vivo. (a) Method 1: NET release from emigrated PMNs visualized as extracellular DNA (SYTOX Orange, white arrows) in vivo during S. aureus skin infection (Xen29), but not during chemokine (MIP-2) induced sterile inflammation (neutrophils are green; NETs are red) (six mice). (b) Method 2: NET quantification using fluorochrome-conjugated histone-specific antibody or neutrophil elastase–specific antibody after injection of live S. aureus, S. pyogenes or killed bacteria (S. aureus Xen8.1) into the skin. Control mice received fluorochrome-conjugated IgG isotype control (n = 4 for each group, n = 3 for dead bacteria). (c) Temporal NET tissue accumulation with PMN images digitally removed for clarity of NETs. (d) Method 3: In vivo PMN nuclei prestained with the cell-permeable DNA dye (SYTO 60, blue) during chemokine (MIP-2) induced sterile inflammation (left) or during infection with GFP-expressing S. aureus (right). A PMN with a normal nucleus is circled in blue, and a PMN with a diffuse nucleus is circled in green. NETs during infection are indicated with a white arrow. NET release is quantified by a ratio of extracellular DNA to intracellular DNA. (e) Impaired histone release in Tlr2−/− and C3−/− mice (n = 3 each group). (f) Impaired DNA release in wild-type, Tlr2−/− and C3−/− mice (n = 3 for all groups). NETosis is restored in C3−/− mice treated with normal mouse serum (n = 3 mice). NET area was determined using Volocity imaging software. *P < 0.05 for treatment versus control, treatment versus sterile inflammation, or knockout versus wild type, #P < 0.05 for treatment versus C3−/−. Data are expressed as means ± s.e.m. Figure 2: PMNs are viable and functional during nuclear breakdown and chromatin decondensation. PMNs and nuclei were evaluated in vivo after incubation with live S. aureus (GFP-USA300). (a) A normal PMN that has captured a live bacterium (green) is shown by 2D imaging (top left) and contrasted by a NET-forming PMN (top middle) that is chemotaxing toward a GFP-expressing S. aureus (green arrow) while releasing DNA. Beneath each 2D spinning-disk image is a panel of confocal 3D reconstructions using various degrees of transparency to distinguish the DNA in relation to the PMN outer membrane. The NETosing PMN in this image (middle) can be observed crawling toward the live bacteria in Supplementary Video 3. Four-color spinning-disk confocal and 3D reconstruction reveals a PMN (yellow, Alexa 750–conjugated GR-1–specific antibody) with a diffuse nucleus (cell-permeable SYTO 60, blue) releasing an extracellular NET (cell-impermeable SYTOX Orange, red) while retaining live GFP-expressing S. aureus (green). (b) Three nuclear phenotypes were visualized: normal, diffuse or absent. Top images show extracellular membrane and nuclear staining; bottom images show the nucleus alone. (c) 3D reconstruction reveals the morphology of each nuclei group. The 3D image rendering results in an artificial white reflection on the cell surface that does not represent an authentic fluorescent stain or nuclei. (d) Phagocytosis of live GFP-expressing S. aureus by neutrophils was quantified in wild-type, Tlr2−/− or C3−/− mice. (e) Quantification of the ability of neutrophils with decondensed or absent nuclei to phagocytose in wild-type, Tlr2−/− or C3−/− mice. A zero indicates no cells were observed (n = 3 experiments per group). **P < 0.01 and ***P < 0.001 for phagocytosis versus no phagocytosis. Data are expressed as means ± s.e.m. Figure 3: NET-forming PMNs show a unique crawling phenotype in vivo related to nuclear structure. (a) 2D images of in vivo PMNs with normal nuclei, NET-forming cells with diffuse nuclei and an in vitro–generated anuclear human PMN (cytoplast). Contouring analysis of each cell is shown beneath the image. The PMN outer membrane is traced every 30 s (PMN in the middle image with white arrow is traced). Typical crawling phenotypes are shown for PMNs from Tlr2−/− and C3−/− mice. (b) Quantification of cell polarities in relation to nuclear morphology. (c) The relationship of pseudopod formation of crawling PMNs compared to their nuclear architecture or between wild-type, Tlr2−/− and C3−/− mice. (d) PMN velocity and meandering index during live staphylococcus infection. (e) Cellular tracking during live staphylococcus infection. *P < 0.05, **P < 0.01, ***P < 0.001 compared to sterile inflammation and #P < 0.05, ###P < 0.001 compared to wild type. Figure 4: NETs are essential for limiting acute S. aureus dissemination. (a) Photon imaging of live luminescent S. aureus (Xen8.1, 1 × 108 CFUs per 100 μl saline injection) within the mouse skin at 1 h and 4 h. (b) Live luminescent S. aureus quantified within the skin of mice pretreated with either DNase (1,000 U intraperitoneally (i.p.)) or saline (i.p.) at 1 h, 4 h and 8 h after infection (six mice). (c) Bacterial dissemination from the skin to the blood at 4 h after infection (Xen8.1) (six mice). (d) CFUs grown from a 3.5-mm skin biopsy of the injection site at 24 h (six mice). *P < 0.05. Data are expressed as means ± s.e.m. Figure 5: NETosis occurs in human abscesses due to Gram-positive bacterial infections. Five human patients presenting with Gram-positive abscesses were evaluated. Transmission electron microscopy was performed on freshly obtained clinical samples. (a) The abscesses contained intact neutrophils (PMNs), red blood cells (RBCs), activated neutrophils with vesicles in the cytoplasm (PMN(v)), as well as numerous anuclear neutrophils with cytoplasmic granules and nuclear vesicles (arrowheads). (b,c) A typical anuclear neutrophil is shown undergoing NET formation (arrowhead), and a second cell is shown, enlarged in c, after nuclear envelope breakdown with dispersed chromatin and nuclear vesicles. The remnants of the nuclear envelope are highlighted (arrowhead). NETs are identified by the prototypical 'beads on a string' appearance on electron microscopy. (d) An anuclear neutrophil with nuclear envelope breakdown and decondensed and dispersed chromatin (arrowhead), with vesicles and granules fusing with the outer plasma membrane (arrowhead). (e) Late-stage neutrophils that have released chromatin and granules into the extracellular space. Figure 6: Immunofluorescence imaging of NETosis during human abscess formation. (a) Fresh abscess aspirates were stained with a phycoerythrin (PE)-conjugated CD66b-specific antibody, a PerCP-conjugated CD45-specific antibody and SYTOX Green. The white arrow highlights an anuclear PMN adjacent to a NET that is outlined by a dotted line. The yellow arrow highlights a normal PMN with multilobar condensed nuclei. (b) Fresh live abscess aspirates stained with PE-conjugated CD16-specific antibody were directly injected into mouse skin to mimic our in vivo experiments, and nuclei were prelabeled with SYTO 60. NETs were visualized and quantified using the NET ratio. (c) Normal PMNs were stained with PE-conjugated CD16-specific antibody and SYTO 60. PMNs were injected into mouse skin alone or with GFP-expressing S. aureus. A NET is being released from the PMN stimulated with bacteria, and the GFP-expressing S. aureus can be seen attached to the NET. NET release by in vivo human PMNs is quantified as NET ratio (**P < 0.01 for untreated versus treated). (d) PMN nuclei quantified in vivo from uninfected normal human PMNs, human PMNs infected with S. aureus and in human abscess PMNs. Zero normal human PMNs were observed in the absent nuclei category without an infectious stimuli. Data are expressed as means ± s.e.m.