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. 2022 May 4;23(9):5124.
doi: 10.3390/ijms23095124.

SARS-CoV-2 Infection Dysregulates Cilia and Basal Cell Homeostasis in the Respiratory Epithelium of Hamsters

Tom Schreiner  1   2 , Lisa Allnoch  1   2 , Georg Beythien  1 , Katarzyna Marek  1   2 , Kathrin Becker  1 , Dirk Schaudien  3 , Stephanie Stanelle-Bertram  4 , Berfin Schaumburg  4 , Nancy Mounogou Kouassi  4 , Sebastian Beck  4 , Martin Zickler  4 , Gülsah Gabriel  4 , Wolfgang Baumgärtner  1   2 , Federico Armando  1 , Malgorzata Ciurkiewicz  1
Affiliations

SARS-CoV-2 Infection Dysregulates Cilia and Basal Cell Homeostasis in the Respiratory Epithelium of Hamsters

Tom Schreiner et al. Int J Mol Sci. .

Abstract

Similar to many other respiratory viruses, SARS-CoV-2 targets the ciliated cells of the respiratory epithelium and compromises mucociliary clearance, thereby facilitating spread to the lungs and paving the way for secondary infections. A detailed understanding of mechanism involved in ciliary loss and subsequent regeneration is crucial to assess the possible long-term consequences of COVID-19. The aim of this study was to characterize the sequence of histological and ultrastructural changes observed in the ciliated epithelium during and after SARS-CoV-2 infection in the golden Syrian hamster model. We show that acute infection induces a severe, transient loss of cilia, which is, at least in part, caused by cilia internalization. Internalized cilia colocalize with membrane invaginations, facilitating virus entry into the cell. Infection also results in a progressive decline in cells expressing the regulator of ciliogenesis FOXJ1, which persists beyond virus clearance and the termination of inflammatory changes. Ciliary loss triggers the mobilization of p73+ and CK14+ basal cells, which ceases after regeneration of the cilia. Although ciliation is restored after two weeks despite the lack of FOXJ1, an increased frequency of cilia with ultrastructural alterations indicative of secondary ciliary dyskinesia is observed. In summary, the work provides new insights into SARS-CoV-2 pathogenesis and expands our understanding of virally induced damage to defense mechanisms in the conducting airways.

Keywords: COVID-19; SARS-CoV-2; cilia; golden Syrian hamster; histology; immunohistochemistry; respiratory epithelium; scanning electron microscopy; trachea; transmission electron microscopy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Characterization of inflammatory alterations and virus load in the trachea of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected hamsters. Representative images of tracheal sections from SARS-CoV-2-infected and control animals, immunolabeled with a SARS-CoV-2 nucleoprotein (NP) antibody (A–E) or stained with hematoxylin and eosin (H&E) (G–K) at 1, 3, 6, and 14 days post infection (dpi), respectively. (A) Control animals showed no positive staining for SARS-CoV-2 NP. (B) A high amount of the NP+ signal was detected at 1 dpi in the cytoplasm and nucleus of ciliated (arrowhead in (B)) and nonciliated cells (arrows in (B)). (C) Only single cells showed the NP+ signal (arrow in (C)) in SARS-CoV-2-infected animals at 3 dpi. (D,E) A positive signal was only rudimentarily observed in SARS-CoV-2-infected animals at 6 and 14 dpi. (F) Quantification of SARS-CoV-2 NP+ cells showed the highest amount of NP+ cells at 1 dpi and a significantly higher amount of NP+ cells at 3 dpi compared with control animals. (G) H&E stained tracheal sections of control animals showed an intact, columnar, pseudostratified, ciliated epithelium. (H) At 1 dpi, the tracheal epithelium of SARS-CoV-2-infected animals was composed of moderate to severe, subepithelial, heterophilic, and lymphohistiocytic inflammation (asterisk in (H)) with rare apoptotic cells (arrowheads in (H)) and mild to moderate exocytosis of heterophils (arrow in (H)). (I) At 3 dpi, SARS-CoV-2-infected animals showed moderate, subepithelial, heterophilic, and lymphohistiocytic inflammation (asterisk in (I)) with low numbers of apoptotic figures (arrowheads in (I)) and mild to moderate exocytosis of heterophils (arrow in (I)). (J,K) Epithelial hyperplasia could be observed at 6 and 14 dpi (arrowhead in (J,K)) in the trachea of SARS-CoV-2-infected animals. (L) Inflammation in the trachea was assessed using a semiquantitative scoring system, including the extent and severity of inflammation (for details, see material and methods: 4.2 Histology). The semiquantitative evaluation revealed significantly higher scores in SARS-CoV-2-infected animals at 1, 3, and 6 dpi. (F,L): Box and whisker plots show the medians, quartiles and ranges. Asterisks indicate significant differences * p < 0.05, ** p < 0.01, *** p < 0.001, Mann–Whitney-U test. (A–E,G–K): Bars: 50 µm.
Figure 2
Figure 2
SARS-CoV-2 causes transient ciliary loss followed by cilia regeneration within two weeks, and this is accompanied by the prolonged reduction of FOXJ1+ cells. (A) Scanning electron microscopic (SEM) findings in the tracheal luminal epithelium. Representative SEM images of the tracheal epithelium of control and SARS-CoV-2-infected animals euthanized at 1, 3, 6, and 14 days post-infection (dpi). Cilia are colored in red, nonciliated areas are in brown, and cellular debris, mucus and unidentifiable material are in blue. Bars: 10 µm. In control animals, the epithelial surface showed ciliated cells with long cilia covering approximately 50% of the surface. In SARS-CoV-2-infected animals, there was mild reduction of ciliated cells at 1 dpi and a marked reduction at 3 dpi. At 6 dpi, ciliated cells were partly restored, but cilia appeared shortened (arrows). At 14, no differences were observed between control and SARS-CoV-2-infected animals. (B) Quantification of ciliated cells. At 3 and 6 dpi, SARS-CoV-2-infected animals showed significantly fewer ciliated cells compared with controls. (C) Quantification of cCAS3+ cells showed a mildly elevated percentage of apoptotic cells at 3 dpi. (D) Quantification of FOXJ1 showed a significant decrease in the percentage of FOXJ1+ cells in SARS-CoV-2-infected animals compared with controls at 1, 3, 6, and 14 dpi. B, C, D: Box and whisker plots with medians, quartiles, and ranges. Asterisks indicate significant differences: * p < 0.05, ** p < 0.01, *** p < 0.001, Mann–Whitney-U-test.
Figure 3
Figure 3
Cilia retraction and virus entry and spread in the tracheal epithelium of SARS-CoV-infected hamsters at 1 day post infection (dpi). (A–F) Representative transmission electron micrographs showing different stages of ciliary retraction and a schematic illustration of the proposed sequence of cilia internalization and SARS-CoV-2 entry into the cell. (A) Numerous virions are attached to the cilia and the microvilli surface. (B) Retraction of an entire cilium into the cytoplasm. Virus particles are in close contact with the retracted axoneme. (C) Cross-section of a completely internalized ciliary axoneme surrounded by virus particles and an infolding of the plasma membrane. (D) Internalized cilium with an associated virus particle located in a vacuole (arrow in (D,F)). (E) Numerous separated axonemes (white arrows in (E,F)) and basal bodies (black arrowheads in (E,F)) within the cytoplasm. Magnifications are 12,500× in (A,B), 31,500× in (C,D), and 10,000× in (E). (F) Schematic illustration of proposed sequence of cilia internalization and SARS-CoV-2 entry into the cell. (G–I) Virus spread between adjacent cells in the trachea of a SARS-CoV-2-infected hamster at 1 dpi. (G) SARS-CoV-2-infected epithelial cell with virus particles aligned along the basal cell pole (white arrows). (H) Higher magnification of G showing the basal cell pole. Virus particles lined up at the intercellular space and in the cytoplasm of the underlying cell. (I) Virus spreading from the intercellular space into a neighboring cell (arrow). Magnifications: 6300× in (G), 16,000× in (H), and 12,500× in (I).
Figure 4
Figure 4
Ciliary regeneration in the trachea of a hamster 6 days after SARS-CoV-2 infection. (A–G) Representative transmission electron micrographs showing different stages of ciliary regeneration and a schematic illustration of the sequence of ciliary regeneration. (A) The centriole starts to produce a primary cilium by forming vesicles at the distal end (white arrows in (A,G)). (B) A ciliary bud develops at the distal end of the centriole invaginating the ciliary vesicle (white arrowheads in (B,G)). (C) The ciliary bud migrates to the apical pole of the cell and bulges the cell membrane. (D) The basal body develops a lateral rootlet (rt in (D,G)). (E) The organelle elongates apically and forms the axoneme (black arrows in (E,G)). (F) Mature cilia with regularly formed axoneme (ax in (F,G)) and basal body (bb in (F,G)). Magnifications are 31,500× in (A), 40,000× in (B), 10,000× in (C,D) and 12,500× in (E,F).
Figure 5
Figure 5
Ultrastructural abnormalities of cilia from SARS-CoV-2-infected hamsters in the tracheal epithelium before and after ciliary regeneration. (A,B) Examples of ciliary alterations observed by transmission electron microscopy of tracheal samples. The most common ultrastructural changes included compound cilia of the bulging type (A) and adhesive type (B). Magnifications are 16,000× in (A), 20,000× in (B). (C) The quantification of compound cilia of both types showed a significant increase in SARS-CoV-2-infected animals at 1 and 14 days post-infection (dpi). Box and whisker plots show medians, quartiles, and ranges. Asterisks indicate significant differences: * p < 0.05; Mann–Whitney-U-test.
Figure 6
Figure 6
Histologic quantification of CK+ and p73+ immunolabelled cells in tracheal cross-sections. (A) Immunolabeling of CK14+ basal cells in SARS-CoV-2-infected animals revealed significant increases at 3, 6, and 14 days post-infection (dpi) in positive cells compared with controls. (B) Immunolabeling of p73+ basal cells in SARS-CoV-2-infected animals revealed significant increases in positive cells at 1 and 3 dpi and a significant decrease in positive cells at 14 dpi compared with controls. Box and whisker plots show medians, quartiles, and ranges. Asterisks indicate significant differences: * p < 0.05, ** p < 0.01, Mann–Whitney-U-test.
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