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. 2022 Jul 7:13:938691.
doi: 10.3389/fimmu.2022.938691. eCollection 2022.

In Vivo ETosis of Human Eosinophils: The Ultrastructural Signature Captured by TEM in Eosinophilic Diseases

Vitor H Neves  1 , Cinthia Palazzi  1 , Kennedy Bonjour  1   2 , Shigeharu Ueki  3 , Peter F Weller  4 , Rossana C N Melo  1   4
Affiliations

In Vivo ETosis of Human Eosinophils: The Ultrastructural Signature Captured by TEM in Eosinophilic Diseases

Vitor H Neves et al. Front Immunol. .

Abstract

Eosinophilic diseases, also termed eosinophil-associated diseases (EADs), are characterized by eosinophil-rich inflammatory infiltrates and extensive eosinophil degranulation with clinically relevant organ pathology. Recent evidence shows that eosinophil cytolytic degranulation, that is, the release of intact, membrane-delimited granules that arises from the eosinophil cytolysis, occurs mainly through ETosis, meaning death with a cytolytic profile and extrusion of nucleus-originated DNA extracellular traps (ETs). The ultrastructural features of eosinophil ETosis (EETosis) have been studied mostly in vitro after stimulation, but are still poorly understood in vivo. Here, we investigated in detail, by transmission electron microscopy (TEM), the ultrastructure of EETosis in selected human EADs affecting several tissues and organ systems. Biopsies of patients diagnosed with eosinophilic chronic rhinosinusitis/ECRS (frontal sinus), ulcerative colitis/UC (intestine), and hypereosinophilic syndrome/HES (skin) were processed for conventional TEM. First, we found that a large proportion of tissue-infiltrated eosinophils in all diseases (~45-65% of all eosinophils) were undergoing cytolysis with release of free extracellular granules (FEGs). Second, we compared the morphology of tissue inflammatory eosinophils with that shown by in vitro ETosis-stimulated eosinophils. By applying single-cell imaging analysis, we sought typical early and late EETosis events: chromatin decondensation; nuclear delobulation and rounding; expanded nuclear area; nuclear envelope alterations and disruption; and extracellular decondensed chromatin spread as ETs. We detected that 53% (ECRS), 37% (UC), and 82% (HES) of all tissue cytolytic eosinophils had ultrastructural features of ETosis in different degrees. Eosinophils in early ETosis significantly increased their nuclear area compared to non-cytolytic eosinophils due to excessive chromatin decondensation and expansion observed before nuclear envelope disruption. ETosis led not only to the deposition of intact granules, but also to the release of eosinophil sombrero vesicles (EoSVs) and Charcot-Leyden crystals (CLCs). Free intact EoSVs and CLCs were associated with FEGs and extracellular DNA nets. Interestingly, not all cytolytic eosinophils in the same microenvironment exhibited ultrastructure of ETosis, thus indicating that different populations of eosinophils might be selectively activated into this pathway. Altogether, our findings captured an ultrastructural signature of EETosis in vivo in prototypic EADs highlighting the importance of this event as a form of eosinophil degranulation and release of inflammatory markers (EoSVs and CLCs).

Keywords: Charcot-Leyden crystals; eosinophil; eosinophil extracellular traps; eosinophil-associated diseases; eosinophilic chronic rhinosinusitis; hypereosinophilic syndrome; transmission electron microscopy; ulcerative colitis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Piecemeal degranulation (PMD) and cytolysis are the main modes of eosinophil secretion in human eosinophil-associated diseases (EADs). (A–C) Representative micrographs of tissue eosinophils in the lamina propria of nasal sinuses (eosinophilic chronic rhinosinusitis/ECRS), lamina propria and submucosa of intestines (ulcerative colitis/UC), and papillary dermis (hypereosinophilic syndrome/HES), respectively. Eosinophils undergoing (PMD) (colored in pink) and cytolysis with deposition of free extracellular granules (FEGs, purple) are observed in the same field. Biopsy samples were prepared for TEM as before (Melo et al., 2005). (Ai–Ci) Quantitative analyses demonstrate predominance of PMD and cytolysis in representative EADs. A total of 335 eosinophils (99 for ECRS, 156 for UC and 80 for HES) were randomly evaluated in a total of 66,000 μm² of tissue area from two patients with ECRS (n = 2), UC (n = 16), and HES (n = 2). Gr, secretory granules; N, nucleus.
Figure 2
Figure 2
Human eosinophil ETosis observed in vitro after stimulation. (A, B) Immunofluorescence staining for isotype (A) or anti-citrullinated H3 histone (CitH3) antibodies(green). EETs and CitH3 are colocalized as seen by confocal fluorescence microscopy after staining for DNA (Hoechst, blue) and CitH3 (green). (Bi) Merged images of CitH3 + DNA-stained eosinophils in higher magnification. Differential interference contrast (DIC) images were obtained by confocal microscopy. Purified eosinophils were stimulated with PAF + IL-5 followed by fixation after 180 min. (C) Representative micrograph of an intact control eosinophil, stimulated with IL-5 alone, displaying a typical bilobed nucleus (colored in blue) with clear distinction between euchromatin and heterochromatin. (D–G) Ultrastructural alterations displayed by human eosinophils in process of ETosis after stimulation. Decondensation, delobulation, and rounding are observed. In (F, G), the nuclear envelope is partially disrupted and chromatin is being released as extracellular traps. Free extracellular granules (FEGs) are deposited in the extracellular medium. Samples were prepared for TEM. Nucleus and extracellular chromatin are colored in blue. Ab, antibody; Gr, secretory granules.
Figure 3
Figure 3
Tissue eosinophils undergoing ETosis are seen in inflammatory sites in the proximity of intact eosinophils. (A–C) Electron micrographs of biopsy samples from eosinophilic chronic rhinosinusitis (ECRS), ulcerative colitis (UC) and hypereosinophilic syndrome (HES) patients, respectively, show eosinophils with nuclear changes typical of ETosis, characterized by chromatin decondensation, delobulation, rounding and expansion. Note that, while the nuclei (N) of intact eosinophils show typical distinction between euchromatin/heterochromatin, the nuclei (arrowheads) of ETotic eosinophils show only euchromatin. In (A), the nuclear envelope was disrupted. Samples were collected from two patients with ECRS (n = 2), UC (n = 16), and HES (n = 2) and prepared for TEM. Gr, secretory granules; FEGs, free extracellular granules. LB, lipid body.
Figure 4
Figure 4
Early ultrastructural signs of ETosis in tissue inflammatory eosinophils. (A) A representative intact eosinophil displaying typical bilobed nucleus (N) with clear distinction between euchromatin (electron-lucent) and heterochromatin (electron-dense). (B–F) Decondensed nuclei (N), with homogenously distributed euchromatin, and in different stages of delobulation and rounding denote, early ETosis. While in (B, C) nuclear segmentation is still partially observed, (D–F) show nuclei in advanced process of delobulation and rounding. In (G), a tissue area with several decondensed, completely round nuclei (colored in purple). Biopsy intestinal samples from patients with ulcerative colitis (n= 16) prepared for TEM. Gr, secretory granules.
Figure 5
Figure 5
The nuclear area increases in eosinophil undergoing ETosis. (A) A cluster of tissue eosinophils infiltrated in the skin of a patient with hypereosinophilic syndrome (HES). While several intact eosinophils show nuclei with normal chromatin appearance (marginal heterochromatin and more internal euchromatin), an early ETotic eosinophil (upper right side) shows enlarged decondensed nucleus undergoing delobulation and rounding. Note that the nuclear space (*) of the nuclear envelope is dilated with separation of the external membrane (arrowheads) from the internal one. All eosinophil nuclei were colored in purple. In (B), quantitative analyses reveal significantly higher nuclear area in eosinophils in early ETosis (only euchromatin) compared to eosinophils with euchromatin/heterochromatin distinction. A total of 263 eosinophils [77 for eosinophilic chronic rhinosinusitis (ECRS), 120 for ulcerative colitis (UC), and 68 for HES] were evaluated and the nuclear area (µm²) measured. Samples were collected from patients with ECRS (n=2), UC (n=16), and HES (n=2) and prepared for TEM. Results are expressed as means ± SEM (*P < 0.05; ***P < 0.01; ***P < 0.001). Gr, secretory granules; LB, lipid bodies.
Figure 6
Figure 6
Nuclear envelope alterations associated with ETosis. (A and Ai) Typical ETotic eosinophils with decondensed, delobulated, and round nucleus (blue) showing separation of the inner and outer nuclear membranes with enlargement of the perinuclear compartment (boxed area seen in higher magnification in Ai). Arrowheads indicate the external nuclear membrane. Note that the same content (strings, arrows) within the perinuclear space is seen in the lumen of vesicles (*) derived from the nuclear envelope. (B) Size range (diameter) of nuclear envelope-originated vesicles in eosinophils undergoing ETosis during EADs. A total of 296 vesicles from ETotic eosinophils were analyzed [92 for eosinophilic chronic rhinosinusitis (ECRS), 88 for ulcerative colitis (UC), and 86 for hypereosinophilic syndrome (HES)]. In (C), a cytolytic non-ETotic eosinophil (nucleus with euchromatin/heterochromatin distinction) showing the same structural changes of the nuclear envelope as seen in (A). Representative images are from biopsy samples (nasal sinuses) from ECRS patients (n = 2) prepared for TEM.
Figure 7
Figure 7
ETosis represents a significant proportion of cytolytic eosinophils in eosinophil-associated diseases (EADs). (A, Ai) A tissue inflammatory site [intestinal ulcerative colitis (UC) biopsy] showing ETotic eosinophils with late signs of ETosis represented by decondensed chromatin spread extracellularly as ETs (colored in purple and indicated by arrowheads in Ai at higher magnification). FEGs (light purple) are in contact with ETs. Note that other non-ETotic eosinophils with nuclei (N) exhibiting heterochromatin (condensed)/euchromatin (decondensed) and a neutrophil (Neu) are seen in the same area. (B–D) Representative electron micrographs from an ECRS biopsy (nasal sinus tissue) highlighting the structural aspect of nuclei in cytolytic eosinophils. While the presence of condensed/decondensed chromatin denotes a non-ETotic eosinophil, eosinophils in early (C) and late (D) processes of ETosis show extensively decondensed chromatin. When in early ETosis (C), the nuclear envelope is observed and frequently displays structural changes (enlargement of the perinuclear space, arrows), in late ETosis (D), the nuclear envelope disappears and the chromatin spreads in the extracellular matrix. Note secretory granules (Gr) immersed in the released chromatin. (E) Quantitative TEM (total tissue area of 66,000 μm2 evaluated), reveals that ~37%-82% of all cytolytic eosinophils in EADs are in process of ETosis and that the proportion of eosinophils in early and late ETosis varies depending on the disease. A total of 151 cytolytic eosinophils with visible nuclear contents [40 for eosinophilic chronic rhinosinusitis (ECRS), 78 for ulcerative colitis (UC), and 33 for hypereosinophilic syndrome (HES)] were evaluated from biopsy samples collected from patients with ECRS (n = 2), UC (n = 16), and HES (n = 2) and prepared for TEM.
Figure 8
Figure 8
Free-extracellular granules (FEGs), free tubular carriers [eosinophil sombrero vesicles (EoSVs)], and Charcot-Leyden crystals (CLCs) are associated with late ETosis (A) An inflamed tissue site (nasal sinus) from a patient with eosinophilic chronic rhinosinusitis (ECRS) shows an ETotic eosinophil with decondensed chromatin (colored in purple) spreading in the extracellular matrix. Note intact secretory granules (light purple) and intact free EoSVs (pink) spread around and in contact with extruded chromatin. The boxed areas in (A) are shown in higher magnification in (Ai, Aii). Arrowheads point to typical EoSVs. In (Ai), an EoSVs associates with an intact granule. (B) Quantitative TEM of inflammatory tissue sites from all eosinophil-associated diseases (EADs) showed that intact EoSVs are found in direct contact with or close to (<1µm of distance) extruded chromatin, or spread (>1µm of distance from DNA strings) in the tissue area. A total of 744 free EoSVs [335 for ECRS, 181 for ulcerative colitis (UC), and 228 for hypereosinophilic syndrome (HES)] were evaluated in electron micrographs considering only late EETosis. (C) CLCs, free EoSVs (pink) and FEGs are enmeshed in tissue chromatin deposits (purple) in a tissue biopsy (nasal sinus) from an ECRS patient. Samples were prepared for TEM.
Figure 9
Figure 9
Charcot Leyden crystals (CLCs) are frequently seen in inflammatory sites from eosinophil-associated diseases (EADs) in association with EETosis. (A) CLCs displaying varied shapes and sizes are observed by TEM in a biopsy (intestine) of a patient with UC. Large and very small CLCs can be observed in the same microenvironment. Note free-extracellular granules (FEGs, colored in light purple) close to CLCs, an intact eosinophil (Eos), and decondensing nuclei (colored in purple). (Ai-Aii) CLCs within the boxed area in (A) seen individually in higher magnification to highlight their different morphologies.
Figure 10
Figure 10
In vivo ultrastructural features of eosinophil ETosis (EETosis) observed in tissues during inflammatory human eosinophil-associated diseases. EETosis is characterized by early and late signs, which compose an ultrastructural signature by TEM. Remarkable nuclear changes and initial breakdown of the plasma membrane (PM) indicate early signs of EETosis. The nuclear envelope can show structural changes (vesiculation and enlargement of the perinuclear space between the outer and inner nuclear membrane). Eosinophils at late ETosis show complete disruption of the PM, disruption of the nuclear envelope, and release of secretory granules and EoSVs together with spread chromatin observed as extracellular traps (ETs). Charcot-Leyden crystals (CLCs) are frequently associated with EETosis.
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