Fig 1.
Volumetric analyzes of ACE2 and TMPRSS2 in non-infected Syrian hamster lung lobes delineate expression patterns.
(A) Lung lobes from Syrian hamsters are extracted and fixated in 10% formalin. Tissue preparation is performed with bleaching, followed by blocking, immunostaining, dehydration, lipid solubilization, and refractive index matching. Refractive index matching for increased light penetration (pre-clearing vs. post-clearing on 1 mm paper), after which imaging is performed and data is analyzed. Orthogonal slices are generated with ImageJ and 3D renders with Imaris. Autofluorescence in grey (488 channel) and staining in red (647 channel). (B) ACE2 is distributed over the lung with nonuniform signals in the tertiary bronchi, bronchioles, and alveoli. (C) Significant TMPRSS2 expression in primary, secondary, and tertiary bronchi with minor alveolar presence. (D) Intense K8/K18 staining in the primary and secondary bronchi, with occasional signal in tertiary bronchi, bronchioles, and alveoli. (E) Autofluorescence in the 647 channel and minor staining of the outer regions of the lung for the secondary antibody control, the pattern does not overlap with previous stains.
Fig 2.
ACE2 and TMPRSS2 partially overlap.
Autofluorescence in grey (488 channel), co-staining in red (647 channel), and purple (730 channel), with overlapping regions in pink (colorblind-proof). (A) TMPRSS2 expression is seen in the primary/secondary bronchi and several tertiary bronchi, with signal spreading to the bronchioles. ACE2 is predominantly located in the tertiary bronchi with relatively lower signals in the primary and secondary bronchi. With consistent expression for TMPRSS2 and bottom-to-top expression for ACE2. Overlap in several regions can be observed. 0.63 zoom, voxel resolution X,Y,Z: 4.79 μm, 4.79 μm, 5 μm. (B) Minor alveolar TMPRSS2 expression and mostly present at the transitioning point of the bronchioles to the alveolar sacs, a similar pattern is also observed for ACE2 expression. The expression profiles of TMPRSS2 and ACE2 overlap in the bronchioles and alveoli. 6.3 zoom, voxel resolution X,Y,Z: 0.48 μm, 0.48 μm, 2 μm. MP4 files of 3D render and orthoslice merge are provided.
Fig 3.
Detecting infected cells within a whole Syrian hamster lung lobe.
Autofluorescence in grey (488 channel) and staining in red (647 channel). (A) Significant NP staining in the primary/secondary bronchi and bronchioles, with occasional tertiary bronchi and alveolar staining (light-sheet and confocal imaging data). (B) Non-specific binding in non-infected when using the anti-NP antibody in the bronchi and outer regions of the lung, although with a lower apparent signal intensity relative to infected lungs (light-sheet and confocal imaging data). (C) Minor autofluorescence and a-specific staining with secondary antibodies, the observed pattern does not overlap with previous stains (light-sheet and confocal imaging data).
Fig 4.
Visualizing tropism of SARS-CoV-2 infection with ACE2 and TMPRSS2 expression patterns at 4-dpi.
Autofluorescence in grey (488 channel), co-staining in red (647 channel), and purple (730 channel), with overlapping regions in pink (colorblind-proof). (A) In several regions of the lung, NP staining is observed from the secondary bronchi extending to the tertiary bronchi, a top-to-bottom gradient. ACE2 is again a bottom-to-top gradient, with signals in the alveoli and various tertiary bronchi. There is a major overlap of NP with ACE2 in the tertiary bronchi, bronchioles, and alveoli (foci). In the lower portion of the lung, there is no apparent overlap of NP and ACE2. 0.63 zoom, voxel resolution X,Y,Z: 4.79 μm, 4.79 μm, 5 μm. (B) In the alveoli there is a significant overlap of NP and ACE2, the previously observed foci appear to be alveolar. Several NP-stained foci do not superimpose with ACE2 stained foci. 6.3 zoom, voxel resolution X,Y,Z: 0.48 μm, 0.48 μm, 2 μm. (C) The expression profile of TMPRSS2 in infected Syrian hamster lungs is highly similar to the expression in non-infected lungs. The fluorescence signal is spreading towards the bronchioles. Likewise, ACE2 expression is also highly similar to mock lungs, with protein staining in tertiary bronchi and bronchioles, 0.63 zoom, voxel resolution X,Y,Z: 4.79 μm, 4.79 μm, 5 μm. (D) At the transitioning point of bronchioles and alveoli TMPRSS2 and ACE2 superimpose, whilst in the alveoli only ACE2 expression is present. 6.3 zoom, voxel resolution X,Y,Z: 0.48 μm, 0.48 μm, 2 μm. MP4 files of 3D render and orthoslice merges are provided.
Fig 5.
SARS-CoV-2 infection necessitates sufficient amounts of ACE2 and TMPRSS2 expression at 4-dpi.
TMPRSS2 was imaged in the 561 channel, NP in the 647 channel, and ACE2 in the 730 channel. With fluorescence signal for the single channels shown in red (ACE2), green (NP), blue (TMPRSS2), and background in grey. (A/B) ACE2 and TMPRSS2 signals in the nasal cavity towards the nasopharynx overlaps with SARS-CoV-2 infection seen in both the 3D render and orthoslices. 0.63 zoom, voxel resolution X,Y,Z: 4.79 μm, 4.79 μm, 5 μm. (C/D) In the left lobe TMPRSS2 signal is seen in the larger branches and upper portions of the lung lobe spreading towards the alveoli. ACE2 signal is predominantly present in the alveoli, bronchioles, and tertiary branches lower in the lung lobe. SARS-CoV-2 infection is transposing in regions where both ACE2 and TMPRSS2 signal is present. (E/F) In the accessory lobe fluorescence signal of TMPRSS2, NP, and ACE2 is mostly visible in the larger branch, with no apparent signal in the alveoli. (G/H) The right upper/mid lobe contains TMPRSS2 signal in the larger branches with spread towards the alveoli in higher in the lung, whilst for ACE2 the signal is distributed mostly in the alveoli and lower portions of the lobe. Regions with SARS-CoV-2 infection, whereby high TMPRSS2 and low ACE2 are detected, also regions with low TMPRSS2 and high ACE2 signal.
Fig 6.
ACE2 is differentially expressed in (non-)infected Syrian hamster lung lobes.
ACE2 signal shown in purple with autofluorescence (488 channel) in grey. The selected regions “upper” in red and “bottom” in blue. (A) Signals of “upper” and “bottom” were quantified, with significant differences in intensity between these regions (P = 0.033). (B) In panel A-C 1 3D render of ACE2 channel shown, hereafter a mask for the “upper” was generated, shown with and without the 488 channel (gray) in panel panel A-C 2–3. Masks for the “bottom” regions was also generated with and without the 488 channel in panel A-C 4–5. Selected regions were generated with the “Seed Points Diameter” tool in Imaris 9.7, after which all values were averaged for grouped analysis to assess the statistical significance.
Fig 7.
ACE2 and TMPRSS2 expression in the ferret animal model.
TMPRSS2 fluorescence signal in red and ACE2 signal in purple. (A) In the ferret trachea, TMPRSS2 expression is observed with occasional ACE2 signal. Ferret trachea was cut into multiple pieces due to dimensional limitations of the microscope and after imaging and analysis the ferret trachea imaging data was placed in the order the ferret trachea was cut in, with panels 1–4 comprosing these cut trachea parts. Panel 1, part of the trachea that attaches to the ferret head with panel 4 being the part that is attached to the left cranial lobe. Panel 2 and 3 are parts of the trachea that are attached to the parts in panel 1 and 4. (B) In the left cranial lobe, minimal ACE2 expression is observed with no apparent TMPRSS2 signal. The left cranial lobe was cut into seven pieces, shown in panel 1–7. With panel 1 being the upper portion and panel 6/7 being the lower portion of the lobe, panel 2–5 are the pieces that connect panel 1 and 6/7 to form the entire cranial lung lobe.