Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo

Abstract

Neutrophil extracellular traps (NETs) are released as neutrophils die in vitro in a process requiring hours, leaving a temporal gap that invasive microbes may exploit. Neutrophils capable of migration and phagocytosis while undergoing NETosis have not been documented. During Gram-positive skin infections, we directly visualized live polymorphonuclear cells (PMNs) in vivo rapidly releasing NETs, which prevented systemic bacterial dissemination. NETosis occurred during crawling, thereby casting large areas of NETs. NET-releasing PMNs developed diffuse decondensed nuclei, ultimately becoming devoid of DNA. Cells with abnormal nuclei showed unusual crawling behavior highlighted by erratic pseudopods and hyperpolarization consistent with the nucleus being a fulcrum for crawling. A requirement for both Toll-like receptor 2 and complement-mediated opsonization tightly regulated NET release. Additionally, live human PMNs injected into mouse skin developed decondensed nuclei and formed NETS in vivo, and intact anuclear neutrophils were abundant in Gram-positive human abscesses. Therefore early in infection NETosis involves neutrophils that do not undergo lysis and retain the ability to multitask.

Figure 1

Rapid in vivo NETosis during acute Gram-positive bacterial infections is directly visualized in vivo. (a) Method 1: NET release was visualized as extracellular DNA (SYTOX Orange, white arrows) in vivo during infection (S. aureus, Xen29), but not during sterile inflammation (MIP-2 superfusion) (neutrophils are green, while NETs are red)(6 mice). (b) Method 2: NET quantification using fluorochrome conjugated histone or neutrophil elastase specific antibodies following live S. aureus, S. pyogenes or killed bacteria (S. aureus Xen8.1). Control animals received fluorochrome conjugated IgG isotype control (n = 4 for each group, n = 3 for dead bacteria). (c) Temporal NET tissue accumulation with PMN images removed for clarity of NETs. (d) Method 3: In vivo PMN nuclei pre-stained with the cell-permeable DNA dye (SYTO 60, blue) during sterile inflammation (left) or during infection with GFP-S. aureus (GFP-USA300)(right). A PMN with a normal nucleus is circled in blue, while a PMN with a diffuse nucleus is circled in green. NETs are demonstrated during infection 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^−/− animals (n = 3 each group). (f) Impaired DNA release in wild type, Tlr2^−/− and C3^−/− mice (n = 3 for all groups). NET area was determined using Volocity imaging software (* = P < 0.05 for treatment versus control, or treatment versus sterile inflammation, or knockout versus wild type, # = P < 0.05 for treatment versus C3^−/−).

Figure 2

PMN are viable and functional during nuclear breakdown and chromatin decondensation. PMN and nuclei were evaluated in vivo following 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 towards a GFP-bacterium (green arrow) while releasing DNA. Beneath each 2D spinning disk image is a panel of confocal 3D reconstruction using various degrees of transparency to distinguish the DNA in relation to the PMN outer membrane. The NETosing PMN in this image (middle panels) 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 S. aureus (GFP-bacteria, green). (b) Three nuclear phenotypes were visualized; normal, diffuse, or absent. Top panels show extracellular membrane and nuclear staining, while the lower panels 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-staphylococcus by neutrophils was quantified in wildtype, Tlr2^−/− or C3^−/− mice. The ability for neutrophils with decondensed or absent nuclei to phagocytose was quantified in (e) wildtype, Tlr2^−/− or C3^−/− animals. (n = 3 experiments per group). (** = P < 0.01 and *** = P < 0.001 for phagocytosis versus no phagocytosis)

Figure 3

NET-forming PMN display a novel crawling phenotype in vivo related to nuclear structure. (a) 2D images of in vivo PMN with a 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 Tlr2^−/− and C3^−/− animals. (b) Cell polarities in relation to nuclear morphology were quantified. (c) The relationship of pseudopod formation of crawling PMN 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 < 0.001, ** = P < 0.01, * = P < 0.05 compared to sterile inflammation and ### = P < 0.001, # = P < 0.05 compared to wild type).

Figure 4

NETs are essential to limit acute S. aureus dissemination. (a) Photon imaging of live luminescent S. aureus (Xen8.1, 1 × 10⁸ CFU per 100 μl saline injection) within the mouse skin at 1 h and 4 h. (b) Live luminescent S. aureus were quantified within the skin of mice pretreated with either DNase (1,000U i.p.) or saline (i.p.) at 1 h, 4 h and 8 h post-infection (6 mice). (c) Bacterial dissemination from the skin to the blood at 4 h post-infection (Xen8.1) (6 mice). (d) CFUs grown from a 3.5 mm skin biopsy of the injection site at 24 h (6 mice). (* = P < 0.05).

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 (PMN), red blood cells (RBCs), activated neutrophils with vesicles in the cytoplasm (PMNv), as well as numerous anuclear neutrophils with cytoplasmic granules and nuclear vesicles (arrowheads). (b) A typical anuclear neutrophil is demonstrated undergoing NET formation (arrowhead) and a second cell is shown, enlarged in (c), following 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 EM. (d) An anuclear neutrophil with nuclear envelope breakdown 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 PE-conjugated CD66b specific antibody, a PerCP-conjugated anti-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 pre-labeled with SYTO 60. NETs were visualized and quantified using the NET ratio. Exogenous DNase decreased visible NETs and decreased the NET ratio. (c) Normal PMN were stained with PE-conjugated CD16 specific antibody and SYTO 60. PMN were injected into mouse skin alone or with GFP-S. aureus. A NET is being released from the PMN stimulated with bacteria and the GFP-staphylococcus can be seen attached to the NET. NET release by in vivo human PMN is quantified as NET ratio (** P < 0.01 for untreated versus treated). (d) PMN nuclei were quantified in vivo from uninfected normal human PMN, human PMN infected with S. aureus and in human abscess PMN.