key: cord-1051231-c6gpnz0f authors: Robinot, Rémy; Hubert, Mathieu; de Melo, Guilherme Dias; Lazarini, Françoise; Bruel, Timothée; Smith, Nikaïa; Levallois, Sylvain; Larrous, Florence; Fernandes, Julien; Gellenoncourt, Stacy; Rigaud, Stéphane; Gorgette, Olivier; Thouvenot, Catherine; Trébeau, Céline; Mallet, Adeline; Duménil, Guillaume; Gobaa, Samy; Etournay, Raphaël; Lledo, Pierre-Marie; Lecuit, Marc; Bourhy, Hervé; Duffy, Darragh; Michel, Vincent; Schwartz, Olivier; Chakrabarti, Lisa A. title: SARS-CoV-2 infection damages airway motile cilia and impairs mucociliary clearance date: 2020-10-06 journal: bioRxiv DOI: 10.1101/2020.10.06.328369 sha: 7f3edfbe601b0f0bdf0ce1899944f375bf174f31 doc_id: 1051231 cord_uid: c6gpnz0f Understanding how SARS-CoV-2 spreads within the respiratory tract is important to define the parameters controlling the severity of COVID-19. We examined the functional and structural consequences of SARS-CoV-2 infection in a reconstituted human bronchial epithelium model. SARS-CoV-2 replication caused a transient decrease in epithelial barrier function and disruption of tight junctions, though viral particle crossing remained limited. Rather, SARS-CoV-2 replication led to a rapid loss of the ciliary layer, characterized at the ultrastructural level by axoneme loss and misorientation of remaining basal bodies. The motile cilia function was compromised, as measured in a mucociliary clearance assay. Epithelial defense mechanisms, including basal cell mobilization and interferon-lambda induction, ramped up only after the initiation of cilia damage. Analysis of SARS-CoV-2 infection in Syrian hamsters further demonstrated the loss of motile cilia in vivo. This study identifies cilia damage as a pathogenic mechanism that could facilitate SARS-CoV-2 spread to the deeper lung parenchyma. The COVID-19 pandemic remains a worldwide public health emergency. The infection caused by the severe 2 acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, in December 2019 (Lu 3 et al., 2020; Zhou et al., 2020b; Zhu et al., 2020b) and spread to all continents except Antartica, affecting 4 over 31 million persons and causing over 970,000 deaths as of Sept. 24, 2020 (covid19.who.int). The respiratory syndrome COVID-19 ranges from mild upper respiratory tract infection to bilateral 7 pneumonia with acute respiratory distress syndrome (ARDS) and multiple organ failure (Guan et al., 2020; 8 Huang et al., 2020; Zhou et al., 2020a) . Pathological examination indicates that SARS-CoV-2 targets 9 primarily the airways and the lungs (Tian et al., 2020; Xu et al., 2020) . Severe cases are characterized by 10 diffuse alveolar damage and formation of hyaline membranes that limit gaseous exchanges. pneumonia is associated with inflammatory infiltrates in the alveolar space and a systemic cytokine storm, 12 suggesting that an exacerbated immune response contributes to damaged lung function (Huang et al., 13 2020; Liao et al., 2020) . Induction of interferons appears limited in the more severe clinical cases, pointing 14 to an imbalance between antiviral and inflammatory cytokine responses (Blanco-Melo et al., 2020; Hadjadj 15 et al., 2020) . 19 reported deaths (Peiris et al., 2003; Petersen et al., 2020) . Both viruses use the angiotensin converting 20 enzyme 2 (ACE2) as a receptor, and the transmembrane serine protease 2 (TMPRSS2) as a viral entry 21 cofactor (Hoffmann et al., 2020; Zhou et al., 2020b) . The pneumonia induced by the two coronaviruses 22 show similar features in severe cases. SARS-CoV-2 induces a mortality rate approximately ten times lower 23 than SARS-CoV-1, but shows much higher effective transmissibility, and thus represents a greater threat 24 to global health (Petersen et al., 2020) . Possible reasons for the high transmissibility of SARS-CoV-2 include 25 an active viral replication in upper airway epithelia at an early stage of infection. The number of SARS-CoV-26 2 genomic copies in nasopharyngeal swabs is generally high at symptom onset (≥10 6 viral RNA copies/mL) 27 and persists for about 5 days before declining (Wolfel et al., 2020; Zou et al., 2020) . This is in contrast to 28 the pattern observed for SARS-CoV-1, with viral RNA peaking about 10 days after symptom onset and 29 remaining of moderate magnitude (Peiris et al., 2003) . Therefore, analyzing how SARS-CoV-2 spreads in 30 the airways is relevant to understand its pandemic potential and potentially identify novel mitigation 31 strategies. 32 The epithelium lining the airways plays a key role in the defense against infections (Tilley et al., 2015) . It 1 comprises goblet cells that secrete a protective mucus able to trap inhaled particles, including microbes. 2 Ciliated cells, which constitute over half of epithelial cells, possess an apical layer of about 200 cilia that 3 beat rhythmically in a coordinated fashion, resulting in a movement of the overlaying mucus layer towards 4 the laryngopharynx, where it is ultimately swallowed (Spassky and Meunier, 2017) . This mechanism of 5 mucociliary clearance prevents the accumulation of particles and mucus within the lungs. The airways 6 basal cells, located close to the epithelial basement membrane, respond to injury by proliferating and 7 differentiating into other epithelial cell types. Studies of autopsy samples from COVID-19 patients and 8 experimental infection of tissue explants have documented SARS-CoV-2 replication predominantly in the 9 upper and lower airway epithelia and in the lung alveoli (Hou et al., 2020; Hui et al., 2020; Yao et al., 2020) . 10 Infection of reconstituted airway epithelia showed a preferential targeting of ciliated cells, with an 11 infection of goblet cells in some (Mulay et al., 2020; Pizzorno et al., 2020; Ravindra et al., 2020; Zhu et al., 12 2020a) but not all studies (Hou et al., 2020; Zhu et al., 2020b) . The functional consequences of SARS-CoV-13 2 infection on epithelial functions remain to be characterized. 14 15 To better understand the mechanism of SARS-CoV-2 dissemination in the respiratory tract, we analyzed 16 the ultrastructural and functional changes induced by infection in a reconstituted human bronchial 17 epithelium model. This system enabled the study of SARS-CoV-2 interactions with its primary target cells 18 in a well-differentiated pseudostratified epithelium. We also examined the impact of SARS-CoV-2 infection 19 on the airway mucosa in vivo, using the physiologically relevant Syrian hamster model. The reconstituted epithelia were infected with a viral suspension of SARS-CoV-2 at 10 6 pfu/mL deposited 1 on the apical side for 4h, restored to ALI conditions, and monitored for infection for 7 days. We observed 2 a rapid increase of extracellular viral RNA in apical culture supernatants, with concentrations reaching up 3 to 10 6 viral RNA copies/µL at 2 days post-infection (dpi), followed by stable or slowly decreasing viral RNA 4 levels until 7 dpi (Fig. 1C) . In contrast, minimal concentrations of viral RNA were detected in the basolateral 5 compartment (Fig. 1D) , indicating that SARS-CoV-2 particles were predominantly released from the apical 6 side of the epithelium. Infectious viral particle production in apical supernatants initially tracked with viral 7 RNA production, with a rapid increase at 2 dpi to reach a mean of 1.8 x 10 6 TCID50/mL, followed by a 8 plateau at 4 dpi (Fig. 1E) . A decrease to 6.1 x 10 4 TCID50/mL was then observed at 7 dpi, suggesting a 9 partial containment of infectious virus production. Immunofluorescence analysis of the reconstituted 10 epithelia revealed a patchy distribution of infected cells expressing the SARS-CoV-2 spike protein at 2 and 11 4 dpi, and minimal persistence of productively infected cells at 7 dpi, consistent with partial viral 12 containment after one week of infection (Fig. 1F ). To test barrier function, we measured the trans-epithelial electrical resistance (TEER) between electrodes 28 placed in the apical and basal compartments of reconstituted bronchial epithelia (Fig. 1G ). As expected, 29 TEER proved relatively stable in mock-treated cultures, with values remaining above 600 Ω.cm2. In CoV-2 infected cultures, there was a transient but significant 3.3x TEER decrease at 4 dpi ( Fig. 1H; P=0 .02). (Fig. 2B) . We did not detect infected cells expressing the goblet cell marker MUC5AC (Fig. S3B ). 24 However, further analyses by SEM documented viral budding from cells with multiple secretory pores that 25 may represent rare infected goblet cells (Fig. S3C) . Interestingly, viral production was also documented in 26 cells with a transitional phenotype characterized by the presence of both motile cilia and abundant 27 secretory vesicles (Fig. S3D ). Taken together, these results showed a preferential tropism of SARS-CoV-2 28 for ciliated epithelial cells, with occasional infection of transitional and secretory cells. As we had observed infected cells with weak b-tubulin IV staining, we asked whether SARS-CoV-2 infection 31 could perturb the layer of motile cilia present at the apical side of ciliated cells. To this goal, we quantified 32 the area occupied by b-tubulin IV staining (tubulin+ area) on projections of confocal images obtained 1 sequentially in the course of infection ( Fig. 2C-D) . The tubulin+ area remained unchanged at 2 dpi, but 2 decreased at 4 dpi (median values: 87.9% in mock, 61.7% in infected; P=0.0012), and showed only limited 3 recovery at 7 dpi (94.8 % in mock vs 67.9% in infected, P=0.0043). SEM imaging confirmed a marked cilia 4 loss at 4 dpi and showed that deciliated areas were not devoid of cells, but rather occupied by cells covered 5 by flattened microvilli (Fig. 2E ). images obtained at 2 dpi in each of three categories: ciliated cells from mock-infected epithelia (mock), 12 productively infected ciliated cells (spike+) and bystander uninfected ciliated cells (spike-) from SARS-CoV-13 2 exposed epithelia. For each cell, the averaged intensity profile of b-tubulin IV staining was measured 14 along the depth axis (Fig. 3A ). There was a bimodal distribution of b-tubulin in mock cells, with a distal 15 peak corresponding to cilia and a proximal peak located just below the plasma membrane, corresponding 16 to the area where basal bodies anchor cilia into the cytoplasm. Examination of average profiles for each 17 category suggested a specific decrease of the distal b-tubulin peak in spike+ cells. This was confirmed by 18 an analysis of the distal to proximal peak intensity ratio, which showed a significant decrease in spike+ 19 cells, as compared to spike-and mock cells (Fig. 3B) . Thus, the density of cilia decreased at 2 dpi, supporting 1 Mucociliary transport was altered at 7 dpi, at a time when barrier integrity was already restored (Fig. 1 ). 2 To better apprehend the process of epithelial regeneration, we analyzed the localization and morphology 3 of basal cells at this time point. Confocal images showed that basal cells expressing cytokeratin-5 were 4 typically flattened on the basement membrane of mock-treated epithelia, while they appeared raised 5 through the thickness of the pseudo-stratified epithelium in infected samples (Fig. 6A ). To quantify this 6 phenomenon, we first generated elevation maps of the inserts that support the cultures, and used these 7 to correct for local insert deformations (Fig. S5A ). This approach enabled to precisely quantify the mean 8 height of basal cells in the epithelia, which proved significantly higher in infected than non-infected 9 samples ( Fig. 6B-C) . SEM imaging confirmed that basal cells adopted a more rounded morphology in 10 infected epithelia (Fig. S5B) . Thus, basal cells were mobilized at 7 dpi, which may contribute to the 11 restoration of barrier integrity. However, they had not differentiated into ciliary cells at this stage, as l induction showed a different kinetics, with limited induction in apical supernatants at 2 dpi, but 23 persistent increase at 4 and 7 dpi, to reach relatively high concentrations in both the apical (501.3±81.8 24 pg/mL) and basal supernatants (343±106 pg/mL) (Fig. 6D, right) . Both IFN-b and IFN-l production showed 25 a degree of inter-sample variability, but were significantly induced as compared to mock-treated samples 26 at 4 dpi (Fig. 6E) . It remained striking, however, that viral replication already peaked at 2 dpi (Fig. 1E) , while 27 interferon production was minimal at this stage. Pretreatment of the reconstituted epithelia with 28 exogenous IFN-b or IFN-l prior to SARS-CoV-2 infection decreased viral RNA levels by 2 logs (Fig. 6F) , 29 pointing to the importance of the timing of IFN induction to achieve viral containment. Taken together, 30 these findings documented the induction of epithelial defense mechanisms following SARS-CoV-2 31 infection, including basal cell mobilization and type III interferon induction. However, the kinetics of these 1 responses appeared too slow in this model to prevent viral replication and functional impairment. (Shah et al., 2008; Tilley et al., 2015) . We documented by 31 immunofluorescence a thinning of the ciliary layer as early as 2 dpi, associated to a layer of viral spike 32 protein at the base of motile cilia. SARS-CoV-2 particles did not bud from cilia, but rather from deciliated 1 areas, as indicated by the minimal colocalization between the spike and b-tubulin markers. Clusters of viral 2 particles could be detected on microvilli, consistent with findings suggesting that SARS-CoV-2 could induce 3 the formation of actin-based filaments in transformed cells (Bouhaddou et al., 2020) . The absence of viral 4 particles within cilia may result from the restricted protein access imposed by the transition zone at the 5 base of motile cilia (Nachury and Mick, 2019). Access of cellular proteins into the ciliary axoneme is limited 6 to those bound by the intraflagellar transport machinery, which likely prevents the import of viral proteins. 7 Therefore, the destruction of motile cilia by SARS-CoV-2 seems mediated by an indirect mechanism, rather 8 than by direct viral production within these structures. TEM imaging revealed the presence of misoriented Vector representation of tracks from (A) and (B) showing bead speed and direction. A low bead 17 speed is observed in infected epithelia (D, left), with an enlarged view showing non E-F) Bead mean speed (E) and track straightness (F) measured in mock and infected epithelia at Mann Whitney tests)