Journal: Immunity

Article Title: Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation

doi: 10.1016/j.immuni.2021.07.012

Figure Lengend Snippet: The confocal IVM movie captures the reperfusion phase of a cremaster muscle post-capillary venular segment following 40 min of ischemia of the tissue from a chimeric Atg5 ΔEC mouse exhibiting GFP + neutrophils (green) and tdTomato - (white; WT) or tdTomato + (magenta; Atg5 −/− ) ECs. EC junctions were immunostained in vivo with an AF-647-anti-PECAM-1 mAb (blue). The movie tracks 4 luminal neutrophils migrating through the same Atg5 −/− junction (formed between two Atg5 −/− ECs) at different time points of the reperfusion period. Each individual neutrophil TEM event is associated with the formation of a transient pore within the PECAM-1 labeled EC junction. After completion of TEM, neutrophils crawl along the abluminal side of the EC surface prior to entering the interstitial space. The image sequence captures the first 2 hours of the reperfusion phase. For enhanced clarity, tracked neutrophils undergoing TEM were isolated from the inflammatory response by creating an isosurface using Imaris software. Scale bar, 10 μm.

Article Snippet: Anti-human Atg5 (clone D5F5U) , Cell Signaling Technology , Cat#12994; RRID: AB_2630393.

Techniques:

Journal: Immunity

Article Title: Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation

doi: 10.1016/j.immuni.2021.07.012

Figure Lengend Snippet: The confocal IVM movie captures the reperfusion phase of a cremaster muscle post-capillary venular segment following 40 min of ischemia of the tissue from a chimeric Atg5 ΔEC mouse exhibiting GFP + neutrophils (green) and tdTomato - (white; WT) or tdTomato + (magenta; Atg5 −/− ) ECs. EC junctions were labeled in vivo with an AF-647-anti-PECAM-1 mAb (blue). The video tracks up to 8 neutrophils migrating through the same PECAM-1 junctional pore formed between a WT and an adjacent Atg5 −/− EC over a period of 2 hours. Of note, the PECAM-1 pore remains open during the entire image acquisition period. For enhanced clarity, tracked neutrophils undergoing TEM were isolated from the inflammatory response by creating an isosurface using Imaris software. Scale bar, 5 μm.

Article Snippet: Anti-human Atg5 (clone D5F5U) , Cell Signaling Technology , Cat#12994; RRID: AB_2630393.

Techniques:

Journal: Immunity

Article Title: Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation

doi: 10.1016/j.immuni.2021.07.012

Figure Lengend Snippet: Video illustrating the workflow developed to target a region of interest (ROI) by confocal microscopy for subsequent imaging by serial block face scanning electron microscopy (SBF SEM) in the IR-stimulated microcirculation of an Atg5 ΔEC mouse. The designated ROI shows a combination of WT (tdTomato - ) and Atg5 −/− (tdTomato + ) ECs. The position of the ROI selected by confocal microscopy was identified in the cremaster tissue by X-ray micro-computed tomography (micro-CT) guided by tissue shape and venules as landmarks allowing the tissue to be trimmed around the ROI. The tdTomato signal of the confocal 63 × Z stack image was aligned to the SBF SEM data based on nuclei positions and overlaid to the EM data.

Article Snippet: Anti-human Atg5 (clone D5F5U) , Cell Signaling Technology , Cat#12994; RRID: AB_2630393.

Techniques:

Journal: Immunity

Article Title: Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation

doi: 10.1016/j.immuni.2021.07.012

Figure Lengend Snippet: Serial block face scanning electron microscopy imaging of the ROI targeted as shown in Video S3 in an IR-stimulated Atg5 ΔEC venule. The lower panel illustrates the selection and segmentation of ECs through the generation of AreaLists using the TrakEM2 Fiji tool in the region of thickened PECAM-1 + junctional sites depicted by confocal microscopy. BM; Basement membrane. Scale bar, 5 μm. Still images corresponding to this video are shown in Figures 4I, S4E, and S4F.

Article Snippet: Anti-human Atg5 (clone D5F5U) , Cell Signaling Technology , Cat#12994; RRID: AB_2630393.

Techniques:

Journal: Immunity

Article Title: Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation

doi: 10.1016/j.immuni.2021.07.012

Figure Lengend Snippet: 3D reconstruction of the segmented ECs imaged by SBF-SEM in the ROI selected by confocal microscopy within an IR-stimulated Atg5 ΔEC venule. Individual ECs are depicted using different colors and cell-cell contacts (closely apposed plasma membranes) are shown between Atg5 −/− - Atg5 −/− and WT- Atg5 −/− (white) or WT-WT (cyan) ECs. Arrows indicate enlarged contacts and membrane flaps between Atg5 −/− ECs. Scale bar, 5 μm. Still images corresponding to this video are shown in Figures 4K–4N and S4G.

Article Snippet: Anti-human Atg5 (clone D5F5U) , Cell Signaling Technology , Cat#12994; RRID: AB_2630393.

Techniques:

Journal: Immunity

Article Title: Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation

doi: 10.1016/j.immuni.2021.07.012

Figure Lengend Snippet: Modulation of EC autophagy controls neutrophil extravasation and cellular tissue damage (A and B) Neutrophil extravasation in chimeric Atg5 fl/fl and Atg5 ΔEC mice (A) treated i.s. with PBS or LPS and (B) subjected to local IR injury (n = 3–8 mice/group). (C) Representative (n = 8) confocal images of IR-stimulated cremasteric PCVs (PECAM-1) immunostained for neutrophils (MRP14) (scale bar, 30 μm). (D) Neutrophil extravasation in Atg5 fl/fl and Atg5 iΔEC mice subjected to local IR injury (n = 6 mice/group). (E) Propidium iodide (PI) + cells in Atg5 fl/fl and Atg5 ΔEC mice subjected to IR injury, as quantified by confocal IVM (n = 3–5 mice/group). (F and G) GFP-LC3 puncta or endogenous LC3 puncta per venular EC area in cremasteric PCVs of (F) GFP-Map1lc3 TG/+ mice and (G) Atg5 fl/fl and Atg5 ΔEC mice treated i.s. with scrambled or Tat-Beclin 1 peptide (n = 3–5 mice/group). (H and I) Neutrophil extravasation in (H) WT mice and (I) Atg5 fl/fl and Atg5 iΔEC mice subjected to local IR injury and treated i.s. with scrambled or Tat-Beclin 1 (n = 4–6 mice/group). (J and K) Intravascular neutrophils in WT mice subjected to local IR injury and treated i.s. with scrambled or Tat-Beclin 1 (n = 3–4 mice/group) (J), and (K) representative confocal images (n = 3–6) of cremasteric PCVs (PECAM-1) immunostained for neutrophils (MRP14) (scale bar, 30 μm). Means ± SEMs. Statistically significant difference from controls or between indicated groups is shown by ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; ns, not significant. See also Figure S2 .

Article Snippet: Anti-human Atg5 (clone D5F5U) , Cell Signaling Technology , Cat#12994; RRID: AB_2630393.

Techniques:

Journal: Immunity

Article Title: Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation

doi: 10.1016/j.immuni.2021.07.012

Figure Lengend Snippet: EC ATG5 deficiency promotes increased and faster neutrophil TEM (A–C) Chimeric Atg5 fl/fl and Atg5 ΔEC mice were subjected to local IR injury and neutrophil responses in cremasteric PCVs analyzed by confocal real-time IVM. Neutrophil (A) adhesion, (B) TEM, and (C) duration of TEM. (D) Representative confocal images (n = 7) of cremasteric PCVs (PECAM-1) immunostained for neutrophils (MRP14), highlighting mosaic distribution of ECs, with arrows indicating Atg5 −/− and WT junctions (scale bars, 15 μm). (E–G) Neutrophil (E) paracellular and (F) transcellular TEM and (G) TEM through hotspots in WT and Atg5 −/− junctions of chimeric Atg5 ΔEC mice. (H and I) Pore (H) opening duration and (I) number within chimeric Atg5 ΔEC mice (n = 5–7 mice/group). (J) Representative (n = 5) confocal images illustrating an Atg5 −/− junction exhibiting multiple PECAM-1 pores (arrows; scale bars, 15 μm). Dashed boxes delineate magnified areas. Means ± SEMs. Statistically significant difference from controls or between indicated groups is shown by ∗ p < 0.05 and ∗∗ p < 0.01; ns, not significant. See also Figure S3 .

Article Snippet: Anti-human Atg5 (clone D5F5U) , Cell Signaling Technology , Cat#12994; RRID: AB_2630393.

Techniques:

Journal: Immunity

Article Title: Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation

doi: 10.1016/j.immuni.2021.07.012

Figure Lengend Snippet: ATG5-dependent autophagy regulates the architecture and molecular composition of EC contacts (A–F) Atg5 fl/fl and Atg5 ΔEC mice were subjected to local IR injury. (A and B) Representative confocal images (n = 3–4) of cremasteric PCVs (PECAM-1 and VE-cadherin) showing aberrant, thickened junctional structures (arrowheads) (A) and (B) frequency of thickened junctions (n = 3–4 mice/group). (C–F) Quantification of PECAM-1 and VE-cadherin (C and D) junctional width and (E and F) junctional enrichment (n = 3 mice/group). (G and H) Cell surface proteins under (G) basal and (H) endotoxemia conditions in WT and ATG5-deficient lung ECs from Atg5 ΔEC mice (n = 3–8 mice/group). (I–N) Correlative light electron microscopy (CLEM) analysis of a venular segment in an IR-stimulated Atg5 ΔEC mouse (n = 1). (I) Serial-block face scanning electron microscopy (SBF-SEM) micrograph of the region of interest (ROI) (targeted as shown in ), illustrating segmentation of ECs. (J) Confocal image showing WT and Atg5 −/− ECs within the ROI, with the latter exhibiting thickened PECAM-1 junctional structures (arrowhead and asterisk). (K) 3D reconstruction of segmented ECs and cell-cell contacts of the venular area depicted in (J). (L–N) Enlargements of the 3D model illustrating (L) WT-WT and (M and N) Atg5 −/− - Atg5 −/− cell contacts showing areas of enlarged contacts (arrowhead) and membrane flaps (asterisk, scale bars, 1 μm). (O) Neutrophil extravasation in Atg5 fl/fl and Atg5 ΔEC mice subjected to IR injury and treated with an isotype control or anti-PECAM-1-blocking mAb (n = 3–4 mice/group). Dashed boxes delineate magnified areas. Means ± SEMs. Statistically significant difference from controls or between indicated groups is shown by ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Scale bars, 5 μm, unless otherwise specified. See also Figure S4 .

Article Snippet: Anti-human Atg5 (clone D5F5U) , Cell Signaling Technology , Cat#12994; RRID: AB_2630393.

Techniques: Electron Microscopy, Blocking Assay, Membrane, Control

Journal: Immunity

Article Title: Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation

doi: 10.1016/j.immuni.2021.07.012

Figure Lengend Snippet: EC autophagy machinery regulates cell-surface PECAM-1 intracellular trafficking and degradation (A–C) Schematic illustrating cell-surface biotinylation method. Control and ATG5 siRNA-silenced HUVECs were stimulated with LPS (A) and (B) immunoblotted for total and biotinylated PECAM-1, ATG5, and β-actin at the indicated times postbiotin incubation and (C) analyzed for fold change in cell surface-derived PECAM-1 (n = 7). (D–G) GFP-LC3 transfected HUVECs were stimulated with LPS before antibody feeding using a nonblocking anti-PECAM-1 mAb. (D) Number of GFP-LC3 + / PECAM-1 + vesicles at the indicated times after incubation with anti-PECAM-1 mAb (n = 3–5; 40–100 cells analyzed per condition). (E) Time-lapse confocal images showing the formation of GFP-LC3 + /PECAM-1 + vesicles (scale bars, 10 μm). (F and G) Representative (n = 4) confocal images of GFP-LC3 transfected HUVECs immunostained for PECAM-1 and WIPI2 (scale bars, 10 μm and enlargements, 3 μm) (F) and (G) quantification of the number of GFP-LC3 + / PECAM-1 + vesicles WIPI2 -/+ (n = 4; >100 cells analyzed per condition). Means ± SEMs. Statistically significant difference from controls is shown by ∗ p < 0.05 and ∗∗ p < 0.01. See also Figure S5 .

Article Snippet: Anti-human Atg5 (clone D5F5U) , Cell Signaling Technology , Cat#12994; RRID: AB_2630393.

Techniques: Control, Incubation, Derivative Assay, Transfection

Journal: Immunity

Article Title: Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation

doi: 10.1016/j.immuni.2021.07.012

Figure Lengend Snippet: Non-canonical autophagy operates in microvascular ECs and regulates PECAM-1 distribution in response to IR injury (A and B) GFP-Map1lc3 TG/+ mice were subjected to local IR injury. (A and B) Representative (n = 3) confocal images of cremasteric PCVs (PECAM-1) immunostained for WIPI2, showing GFP-LC3 + and WIPI2 + puncta (scale bar, 5 μm) (A) and (B) number of GFP-LC3 + /WIPI2 -/+ puncta per venular EC area (n = 3 mice/group). (C and D) Atg5 ΔEC , WT, and Atg16L1 E230 mice were subjected to local IR injury. (C) Representative (n = 3–5) confocal images of cremasteric PCVs (PECAM-1) immunostained for endogenous LC3, with arrows indicating localization of LC3 puncta (scale bar, 5 μm). (D) Number of LC3 puncta per venular EC area (n = 3–5 mice/group). (E–G) WT and Atg16L1 E230 mice were subjected to local IR injury. (E) Representative (n = 3) confocal images of cremasteric PCVs (PECAM-1, VE-cadherin) (scale bars, 5 and 3 μm for enlargements) and associated quantification of PECAM-1. (F and G) Junctional width (F) and (G) junctional enrichment (n = 3 mice/group). Means ± SEMs. Statistically significant difference from controls is shown by ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001. See also Figure S6 .

Article Snippet: Anti-human Atg5 (clone D5F5U) , Cell Signaling Technology , Cat#12994; RRID: AB_2630393.

Techniques:

Journal: Immunity

Article Title: Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation

doi: 10.1016/j.immuni.2021.07.012

Figure Lengend Snippet: Genetic ablation of vascular autophagy (canonical and non-canonical) promotes exaggerated and more rapid leukocyte trafficking (A and B) Chimeric Atg5 fl/fl and Atg5 ΔEC mice were subjected to (A) LPS-induced peritonitis (n = 6–12 mice/group) or (B) skin inflammation (n = 5–6 mice/group), and neutrophil infiltration was quantified by flow cytometry or myeloperoxidase (MPO) activity, respectively. (C) Atg5 fl/fl and Atg5 iΔEC mice were subjected to LPS-induced peritonitis, and neutrophil infiltration was quantified by flow cytometry (n = 4–9 mice/group). (D) WT and Atg16L1 E230 mice were subjected to MDP-induced peritonitis, and neutrophil infiltration was quantified by flow cytometry (n = 4–5 mice/group). (E and F) Non-chimeric (E) and (F) chimeric WT and Atg16L1 E230 mice were subjected to local IR injury, and neutrophil extravasation was assessed by confocal microscopy (n = 6–8 mice/group). (G–I) Chimeric Atg5 fl/fl and Atg5 ΔEC mice were subjected to zymosan-induced peritonitis, and infiltration of (G) neutrophils, (H) monocytes, and (I) eosinophils at the indicated times was quantified by flow cytometry (n = 3–9 mice/group). Means ± SEMs. Statistically significant difference from controls or between indicated groups is shown by ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Anti-human Atg5 (clone D5F5U) , Cell Signaling Technology , Cat#12994; RRID: AB_2630393.

Techniques: Flow Cytometry, Activity Assay, Confocal Microscopy

Journal: Immunity

Article Title: Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation

doi: 10.1016/j.immuni.2021.07.012

Figure Lengend Snippet:

Article Snippet: Anti-human Atg5 (clone D5F5U) , Cell Signaling Technology , Cat#12994; RRID: AB_2630393.

Techniques: Blocking Assay, Control, Generated, Recombinant, Antibody Labeling, cDNA Synthesis, SYBR Green Assay, Luciferase, Real-time Polymerase Chain Reaction, Plasmid Preparation, Software