key: cord-1011524-kwox3910
authors: Tomris, Ilhan; Bouwman, Kim M.; Adolfs, Youri; Noack, Danny; van der Woude, Roosmarijn; Herfst, Sander; Boons, Geert-Jan; Haagmans, Bart L.; Pasterkamp, R. Jeroen; Rockx, Barry; de Vries, Robert P.
title: 3D visualization of SARS-CoV-2 infection and receptor distribution in Syrian hamster lung lobes display distinct spatial arrangements
date: 2021-03-24
journal: bioRxiv
DOI: 10.1101/2021.03.24.435771
sha: 65f6279bcab5758eac1460f8115c407920ff2102
doc_id: 1011524
cord_uid: kwox3910

SARS-CoV-2 attaches to angiotensin-converting enzyme 2 (ACE2) to gain entry into cells after which the spike protein is cleaved by the transmembrane serine protease 2 (TMPRRS2) to facilitate viral-host membrane fusion. ACE2 and TMPRRS2 expression profiles have been analyzed at the genomic, transcriptomic, and single-cell RNAseq level, however, biologically relevant protein receptor organization in whole tissues is still poorly understood. To describe the organ-level architecture of receptor expression, related to the ability of ACE2 and TMPRRS2 to mediate infectivity, we performed a volumetric analysis of whole Syrian hamster lung lobes. Lung tissue of infected and control animals were stained using antibodies against ACE2 and TMPRRS2, combined with fluorescent spike protein and SARS-CoV-2 nucleoprotein staining. This was followed by light-sheet microscopy imaging to visualize expression patterns. The data demonstrates that infection is restricted to sites with both ACE2 and TMPRRS2, the latter is expressed in the primary and secondary bronchi whereas ACE2 is predominantly observed in the terminal bronchioles and alveoli. Conversely, infection completely overlaps at these sites where ACE2 and TMPRSS2 co-localize.

SARS-CoV-2 has sparked a pandemic and additional means to understand infection dynamics of this virus will facilitate counter-measures. SARS coronaviruses carry a single protruding envelope protein, called spike, that is essential for binding to and 55 subsequent infection of host cells. The trimeric spike protein binds to angiotensinconverting enzyme 2 (ACE2), which functions as an entry receptor for SARS-CoV 1,2 .

After binding and internalization, TMPRSS2 induces the spike protein into its fusogenic form allowing fusion of the viral and target membrane. Several other attachment factors and/or receptors have been reported 3-6 , but it is generally accepted 60 that ACE2 and TMPRSS2 are essential.

ACE2 and TMPRSS2 are expressed in a wide variety of tissues and have been analyzed using several different genetic techniques [7] [8] [9] . Suprisingly a common denominator in these studies is the high expression of these proteins in extrapulmonary tissues, whereas 65 the virus mainly infects the respiratory tract. A drawback of these genetic studies is that they do not determine biochemical expression and provide limited spatial information.

High-resolution mapping of three-dimensional (3D) structures in intact tissues are indispensable in many biological studies, yet hardly employed to study viral receptors in their host organs concerning viral infection. The conventional method of histological 70 sectioning followed by the imaging of individual sections is commonly used and rather valuable, but this process does not provide spatial information. Recent developments in whole organ clearing, imaging, and analysis of these large data sets do now allow for the characterization of whole organs 10-12 . 75 Different animal models have been employed to recapitulate SARS-CoV-2 infection in humans, these are instrumental and indispensable to develop vaccines and therapeutics 13 . Several reviews are available that succinctly compare the advantages and disadvantages of different animal models 14, 15 , and the Syrian hamster is now widely accepted as an extremely suitable small animal model [16] [17] [18] . 80

Here we aimed to elucidate receptor distribution in the lungs of Syrian hamsters and correlate these patterns with the location of infection. To do so we stained whole lung lobes of SARS-CoV-2 infected Syrian hamsters and control animals, using a variety of antibodies against ACE2, TMPRRS2, and the viral nucleoprotein to detect the accepted 85 functional receptor, restriction factor, and location of infection. The results clearly show that ACE2 and TMPRSS2 are unequally distributed in the lung and that only overlapping regions are infected by SARS-CoV-2.

We initially started our studies into receptor binding of SARS-CoV trimeric receptor binding domain (RBD) proteins and the detection of ACE2 in serial tissue slices of Syrian hamster lungs 19 . We now extended these studies by detecting TMPRSS2 to 95 determine where ACE2 and TMPRRS overlap and if infection would occur in those portions of the lung. SARS-CoV-2 RBDs bind to the apical side of the bronchioles and alveoli of Syrian hamster tissue slides ( Fig. 1 A1-3) . In Syrian hamster lungs 4-days post-infection (dpi, SARS-CoV-2 infected) SARS-CoV-2 RBD failed to bind as previously shown (Fig. 1 A4-6 ), whereas significant ACE2 expression is observed in 100 the alveoli and bronchioles of mock and 4-dpi Syrian hamster lungs ( Fig. 1 B1-6 ). Even though we observe alveolar staining of ACE2 in infected Syrian hamster lungs bronchiolar staining is predominant ( Fig. 1 B4-6) , whilst in non-infected Syrian hamster, the signal is accumulated in both the bronchioles and alveoli ( Fig. 1 B1-3) .

We next characterized TMPRSS2 expression; bronchiolar and alveolar staining was 105 observed in non-infected Syrian hamster lungs, for infected Syrian hamster lungs staining is predominantly visualized in the bronchioles with minor alveolar staining ( Fig. 1 C1-6 ). For infected Syrian hamster lungs an overall reduced signal intensity is observed, in line with previous reports that demonstrate a possible downregulation of receptors and restriction factors 19, 20 . Further staining was performed with an anti-110 keratin antibody, as keratin 8 (K8) and keratin 18 (K18) are expressed in human alveolar and bronchial epithelial cells 21, 22 . We used this antibody to assess bronchiolar and alveolar staining of TMPRSS2, ACE2, and trimeric SARS-CoV-2 RBDs (Fig. 1 D1-6). In non-infected tissue slides K8/K18 is present in the alveoli and bronchioles ( Fig. 1 D1-3) , in infected Syrian hamsters a distinct reduction of K8/K18 is observed, 115 probably due to pulmonary damage ( Fig. D4-6 ). Thus, we can detect SARS-CoV receptor binding related to ACE2 and TMPRSS2 specifically in different parts of the bronchiolar and alveolar system. However, the use of tissue slides does not provide spatial information and prompted us to explore volumetric reconstructions instead.

factors in a distinct spatial morphological manner.

Volumetric analysis was performed on Syrian hamster lung lobes to assess spatial ACE2 and TMPRSS2 expression in organ samples. The workflow of volumetric imaging and data analysis is described in Figure 2A . To determine SARS-CoV-2 receptor distribution through the Syrian hamster lung, we started with our fluorescent 130 trimeric RBD proteins 19 . The trimeric SARS-CoV-2 RBD bound throughout the lung lobes but does not bind the vascular system. We observe distinct signal in the tertiary bronchus, all the way to the alveolar sacs ( Fig. 2 B1-4) . In the 3D render and orthoslice of trimeric SARS-CoV-2 RBD stained samples ( Fig. 2 B1 and B3 ), we observe a lack of signal in the primary/secondary bronchus relative to the tertiary bronchus. ACE2 135 receptor expression was highly similar to the SARS-CoV-2 RBD staining ( indicating a lack of a-specific binding while tissue penetration occurs when using primary antibodies (Fig. 2 F1-4) . In supplementary Fig 1, the light-sheet data is presented without the autofluorescence channel. Conclusively we observe different spatial expression patterns, which encouraged us to expand our approach by analyzing lung lobes for ACE2 and TMPRRS2 simultaneously. 155 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) Binding of recombinant trimeric SARS-CoV-2 throughout the lung, fluorescence signal is detected in tertiary bronchi and alveoli, without significant signal in the primary/secondary bronchi. (C) ACE2 is similarly distributed over the lung as trimeric SARS-CoV-2, with nonuniform signal in the tertiary bronchi, bronchioles and alveoli. (D) Significant TMPRSS2 expression in primary, secondary and tertiary bronchi with minor alveolar presence. (E) Intense K8/K18 staining in the primary and secondary bronchi, with occasional signal in tertiary bronchi, bronchioles and alveoli. (F) Autofluorescence in the 647 channel and minor staining of the outer regions of the lung for the secondary antibody control, pattern does not overlap with previous stains.

To assess where potential viral binding and membrane fusion can occur, co-localization of ACE2 and TMPRSS2 was determined in Syrian hamster lung lobes. Substantial staining with anti-TMPRSS2 antibody in the secondary and several tertiary bronchi is observed ( Fig. 3 A1 We wondered if our assessment is statistically significant. Indeed, after quantification of TMPRSS2 and ACE2 signal in various regions in the lungs, we observe a top-tobottom gradient for TMPRSS2 and a bottom-to-top gradient for ACE2 expression (Fig.  3C ). TMPRSS2 signal is predominantly present in the larger branches, with decreasing signal intensity in the alveoli (Fig. 3D ). ACE2 signal is relatively lower in the larger 185 branches in comparison to the alveoli (Fig. 3E ). 

To determine in which specific lung regions SARS-CoV-2 infection takes place, we 190 used hamster lung lobes isolated at four days post-infection, and stained these with anti-NP antibodies. High signal intensity is observed in the bronchi and bronchioles with several tertiary bronchi remaining unstained (Fig. 4 A1 and A2) , with minor fluorescence signal in the alveoli (Fig. 4 A3) . Additionally, the staining pattern Thus an adequate analysis of commercial antibodies cannot be overstated, as we have seen differential results with anti-ACE2 antibodies previously 19 . The antibody control 205 (secondary antibodies) for the infected hamster lung samples displays minor background staining without any similarity to the NP stain ( Fig. 3 C1-3) . Conclusively, we can confidently detect infected regions within the whole lung lobes of infected Syrian hamsters. Now with the ability to detect ACE2, TMPRSS2, and infected cells in a 3D format, we determined the overlap of ACE2 and SARS-CoV-2 infection to assess if infection was completely restricted to regions with ACE2 expression (Fig. 5 A and B) . We observed NP staining in several regions of the secondary bronchi extending to tertiary bronchi 220 and bronchioles (Fig. 5 A1 and supplementary Fig. 5 A) . The primary bronchi and the other secondary bronchi in the upper portion of the lung lobe appear to be completely clear of infection. NP staining is observed in the alveoli, albeit at a relatively low signal intensity (Fig. 5 B1 and supplementary Fig. 5 B) , additionally over the entire lung a large number of foci are present (Fig. 5 B2 and supplementary Fig. 5 B) . ACE2 225 expression is detected again mainly in the tertiary bronchi in the lower parts of the lung lobe (Fig. 5 A2 and supplementary Fig. 5 A) . No significant signal is detected in the primary bronchi and the other secondary bronchi also appear to express low levels of ACE2. ACE2 patterns were strikingly similar to the NP patterns, including a major overlap (Fig. 5 A3/B3, supplementary Fig. 5 A/B, and supplementary video 3/4) . NP 230 staining appears to overlap significantly with ACE2 staining in the tertiary bronchi, bronchioles, and alveoli (foci). However, ACE2 staining does not completely overlap with NP staining, in the lower regions of infected Syrian hamster lung lobes there appears to be a lack of NP staining whilst significant ACE2 staining is observed. Thus, SARS-CoV-2 infection is restricted to the middle portion of the lung lobe, with 235 receptors in the lower parts not utilized, perhaps due to lower levels of TMPRSS2.

Next, we analyzed the spatial distribution of TMPRSS2 concerning ACE2 in infected Syrian hamster lungs (Fig. 5 C and D) . A similar expression profile is observed compared to the duplicate experiment in non-infected lungs (Fig. 3) TMPRSS2 and ACE2 expression gradients in infected Syrian hamster lungs appeared to be near identical to non-infected hamster lungs. Whereas TMPRSS2 expression is predominantly restricted to the bronchi and upper regions of the lung, ACE2 appears to 255 be prominently present lower in the lung lobe. We observed these gradients in multiple different lung lobes with different staining approaches. As for the non-infected lung lobe, after quantification, we observe a similar pattern. We observe a higher TMPRSS2 signal in the upper branches and a significantly lower signal when reaching the "Bottom alveoli" (Fig. 3 C/D/E). This is also the case for ACE2 but then inverted, with decreased 260 ACE2 signal in the "Upper branch" and increased signal towards the "Bottom alveoli".

We also quantified the alveolar signal in other portions of this infected and co-stained lung the alveolar TMPRSS2 signal is dependent on the location and that alveoli in higher regions of the lung lobe may have a higher apparent fluorescence signal (Fig. 5 E and F). In this example the pattern for ACE2 is similar to the pattern observed before, 265 an elevated signal in the alveoli and reduced fluorescence signal in the larger branches Fig. 6 A and B) . TMPRSS2 in infected hamster lobes provides a 270 comparable impression by displaying immense fluorescence signal in the "Upper branch" and noticeably decreased signal in the "Bottom-lower branch" (Supplementary Fig. 6 C and D) . We, therefore, hypothesize that infection occurs where ACE2 and TMPRSS2 are both present in sufficient density.

In this study, we demonstrate distinct spatial distribution, or gradient, of ACE2 and be of interest to investigate extrapulmonary infection patterns but we also envision that these approaches can be utilized for other pathogens 10, 31, 32 . 300

An intriguing observation is that in previous normal lung cohorts hardly any ACE2 protein expression was observed in the human lung and bronchus 33 . Indeed it is known that ACE2 primarily resides in the alveoli, and thus in line with our light-sheet microscopy observations. On the other hand, TMPRSS2 protein expression distribution 305 is hardly known. Transcriptomic data suggests at least low expression levels of ACE2 in respiratory cells and indicates that the co-factor TMPRSS2 is highly expressed with broader distribution 8 . We demonstrate that the TMPRSS2 co-factor indeed appears to be present with a relatively higher abundance compared to ACE2 (Fig. 4 and 6) , albeit being predominantly restricted to the major lung branches in Syrian hamsters. The fact 310

that several studies using anti-ACE2 antibodies appeared not to correlate with infection patterns 33, 34 , and our observation of broad SARS-CoV-2 trimeric RBD binding, indicates that infection is a process with multiple complexities. Finally, it might be possible to extend our data into the increased ACE2 receptor 315 binding affinities of circulating variant viruses [35] [36] [37] . A hypothesis would be that these viruses can infect the upper respiratory tract, in which ACE2 is scarce, more efficiently leading to increased transmission, a highly similar model as observed for human influenza A viruses 38, 39 . The protein gradient of ACE2 and TMPRSS2 can also be discussed to this extend, whereas for a2-6 linked sialic acids, the receptor for human 320 influenza viruses, also displays a top to bottom gradient 40 . However, the biological function of these gradients remains to be elucidated.

Animals were handled in a BSL3 biocontainment laboratory. Animals were housed in groups of 2 animals in filter top cages (T3, Techniplast), in Class III isolators allowing social interactions, under controlled conditions of humidity, temperature, and light (12hour light/12-hour dark cycles). Food and water were available ad libitum. Animals 330 were cared for and monitored (pre-and post-infection) by qualified personnel. The animals were sedated/anesthetized for all invasive procedures.

Female Syrian golden hamsters (Mesocricetus auratus; 6-week-old hamsters from 335 Janvier, France) were anesthetized by chamber induction (5 liters 100% O2/min and 3 to 5% isoflurane). Animals were inoculated with 10 5 TCID50 of SARS-CoV-2 or PBS (mock controls) in a 100 μl volume via the intranasal route. Animals were monitored for general health status and behavior daily and were weighed regularly for the duration of the study (up to 22 days post-inoculation; d.p.i.). Animals were euthanized on day 4 340 after inoculation, and lung samples were removed and stored in 10% formalin for histopathology. formalin, for longer storage hamster lungs were kept in PBS and 0.01% sodium azide.

Dehydration of the lungs was performed by washing with PBS for 1.5 hours, followed by 50% methanol for 1.5 hours, 80% methanol for 1.5 hours, and as of last 100% methanol for 1.5 hours on a tilting laboratory shaker. Bleaching was performed 375 overnight at 4 °C in 90% methanol (100% v/v) and 10% H2O2 (30% v/v). Tissues were rehydrated with 100% methanol for 1 hour, followed by 100% methanol for 1 hour, 80% methanol, 50% methanol, and 1x PBS for 1 hour on a tilting shaker. Syrian hamster lungs were subsequently blocked for 24 hours at room temperature (22 °C) in 1x PBS with 0.2% gelatin, 0.5% triton-x-100, and 0.01% sodium azide (PBSGT) on a 380 tilting shaker. Hamster lungs were stained with trimeric SARS-CoV-2 RBD protein and/or with primary/secondary antibodies possessing an alexa647 or alexa750 dye.

After blocking samples were incubated for 120 hours with primary antibody or trimeric RBD protein in PBSGT with 0.1% saponin (S2149, Sigma-Aldrich) on a shaking incubator at 200 rpm, following 120 hours incubation, hamster lungs were washed with 385 PBSGT six times for 1 hour on a tilting shaker. Post-PBSGT wash, samples stained with primary antibodies were incubated with secondary antibodies that possess alexa647 or alexa750 dyes, the antibodies were diluted in PBSGT with 0.1% saponin and filtered with 0.45 µm filter for potential antibody aggregates. Hereafter samples were incubated for 120 hours at room temperature on a shaking incubator at 200 rpm. 390

Samples stained with trimeric SARS-CoV-2 RBD were incubated with primary antibody strepmabHRP (2-1509-001, IBA lifesciences) with specificity towards the TwinStrep-tag at the C-terminus of the trimeric protein for 120 hours in PBSGT with 0.1% saponin. Following secondary antibody staining and tissues treated with trimeric RBD proteins, the hamster lungs were washed with PBSGT six times for 1 hour on a 395 tilting shaker. Secondary antibody staining was also performed for trimeric RBD stained sample either using antibodies with alexa647 or alexa750 dyes with an incubation period of 120 hours, after staining washing was performed with PBSGT six times for 1 hour on a tilting shaker. Immunostained samples were subsequently treated with 50% methanol for 24 hours, 80% methanol for 24 hours, 100% methanol for 24 400 hours followed by 100% methanol for 24 hours on a tilting shaker for dehydration of the lungs. Hereafter lipid solubilization was performed with dichloromethane stacks were analyzed with ImageJ v1.54f and Imaris 9.6. Stills of image slices (orthoslices) were generated in ImageJ, whilst 3D renders were generated in Imaris.

TIFF files generated by the light-sheet microscope were imported to ImageJ with "File 425 -> Import -> Bio-formats", stack is viewed as "Hyperstack" color mode was set to "Composite" and "Display metadata" with "Display OME-XML metadata" was enabled to obtain voxel information. Brightness adjustments were performed for each channel with setting "Image -> Adjust -> Brightness/Contrast". The 488 channel color was set to hex code #FFFFFF, the 647 channel was set to hex code #FF0000 and the 430 730 channel was set to hex code #8080FF. Snapshots of the Z-stack were saved as JPEGs and the orthoslice animations with "Save As -> Avi -> Compression JPEG and Frame Rate 30 FPS". For the 3D renders obtained imaging data was imported into Imaris, display adjustments were made for each channel. Snapshots and animations were made with 3D renders (3D View) within Imaris. Volume mode was set to 435 maximum intensity projection (MIP) and the rendering quality was set to highest.

Frame settings were used for the outer bounding box of the 3D render, "Box", "Tickmarks", and "Axis labels" were enabled. The spacing of the tickmarks was set to µm 500 for the X, Y, and Z-axis. The 488 channel color was set to hex code #FFFFFF, the 647 channel was set to hex code #FF0000 and the 730 channel was set to hex code 440 #8080FF. The 3D animations were made with 1920 x 1080 resolution (16:9) and 360 frames, 360° horizontal turn.

For TMPRSS2 and ACE2 quantification surfaces and masks were generated in Imaris 445 with the "Surfaces Creation Wizard" (according to the reference manual). In this surface/mask regions of interest were selected, i.e. the branches and alveoli in the hamster lung. Regions of interest were selected in the surface tool with the corresponding source channel, whereby the 647 channel with TMPRSS2 signal was used for TMPRSS2 and ACE2 double-stained samples and 730 source channel for 450 ACE2 and anti-NP double-stained samples. For the TMPRSS2 single stained sample, the 647 channel was used as the source channel. Thresholding (Absolute Intensity) was performed arbitrarily to obtain complete coverage of the alveoli and branches. The automatically provided value for "Sphere Diameter" was used. For the separation of two or more objects that are identified as one, the "Split touching Objects (Region 455 Growing)" setting was utilized. The "Seed Points Diameter" value was set to 35 µm.

The generated surfaces were subsequently used to mask the TMPRSS2 and ACE2 channels, after which statistical values were obtained and exported to GraphPad Prism 9. 

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