Mechanosensitive extrusion of Enterovirus A71-infected cells from colonic organoids

Figures

• 

  Fig. 1. Susceptibility of apical-out colon organoids to EV-A71 virus. a, Apical-out, differentiated colon epithelial organoids express the EV-A71 receptor SCARB2 (red), localizing to intracellular membranes. The integrity of the apical actin (white) microvillus brush border is visible. b, Colon organoids were infected with EV-A71. Viral titres were monitored over time by plaque assay. Data from four independent experiments using two different colonoid donors are shown. c, EV-A71-infected cells were observed by immunofluorescence assay following staining for double-stranded vRNA after 48 h of infection. Right: increased magnification of infected cell denoted by yellow arrowhead in the left panel. Scale bars, 10 μm. Source data

• 

  Fig. 2. Specific extrusion of EV-A71-infected cells from colonoids. a–c, Infected colonoids were fixed at 48 h post infection and stained for double-stranded vRNA. d, Infected cells were extruded from colonoids with higher frequency than uninfected cells. The percentages of infected or uninfected cells extruding from an individual organoid are shown as small circles. The proportion of cells extruding across all organoids in each experiment are shown as triangles of the same colour as the individual colonoids. At least ten organoids were quantified per experiment: **P < 0.01; paired, two-tailed t-test, N = 3 experiments. e, Percentages of infected extruding and non-extruding cells were measured similarly. Of the cells that were extruding, a higher percentage was infected. **P < 0.01; repeated measures one-way ANOVA with Tukey’s multiple comparisons test, N = 3 experiments. In d and e, raw cell numbers represented in the graphs are displayed in Supplementary Table 1. f, EV-A71-infected colonoids were stained for Muc2 expression, a marker for goblet cells. Arrowhead indicates infected cell. Arrow indicates goblet cell shown in g. g, Muc2-expressing goblet cell. h,i, Villin expression…

• 

  Fig. 3. EV-A71-infected cell extrusion is not driven by apoptosis. Infected colonoids were visualized by immunofluorescence confocal microscopy after 48 h of infection. Caspase 3 and 7 activity was visualized using a fluorogenic substrate. a, Individual infected organoids with extruding cells. b, Increased magnification of single uninfected, apoptotic extruding cell in a. c, Increased magnification of two infected, non-apoptotic extruding cells in a. d, Individual infected organoids with extruding cells. e, Magnification of two extruding cells in d. f, Proportions of infected extruding and uninfected extruding cells that were apoptotic were quantified, with overall values for each experiment shown as triangles. Each colour represents an independent experiment, with measurements for each individual organoid shown as small circles. *P < 0.05; paired, two-tailed t-test, N = 3; horizontal and error bars represent mean ± s.d. Raw cell numbers represented in this graph are displayed in Supplementary Table 3. Scale bars, 10 µm. Source data

• 

  Fig. 4. Mechanosensitive signalling promotes infected cell extrusion. EV-A71-infected organoids were exposed to compounds capable of inhibiting cellular factors implicated in different mechanisms of extrusion. a–d, Infected organoids were exposed to 0.5% DMSO vehicle control (a), 50 μM para-nitro-blebbistatin (b), 100 μM Z-VAD-FMK (c) or 20 μM GsMTx4 (d). Infected cells in organoids were visually inspected by confocal microscopy. Yellow arrowheads indicate infected cells in representative organoids. Scale bars, 10 μm. e, Blebbistatin inhibits both apoptotic and mechanosensitive extrusions, Z-VAD-FMK inhibits only apoptotic extrusion and GsMTx4 inhibits only mechanosensitive extrusion. f, The percentage of infected cells undergoing extrusion after 7 h of infection was enumerated. Each colour shows an independent experiment. Overall proportion of infected cells extruding per experiment shown as triangles, with measurements for each organoid shown as small circles. **P < 0.01; NS, not significant; repeated measures one-way ANOVA with Dunnett’s multiple comparisons test, N = 3. g, Viral titres quantified at 7 h post infection from infected suspension organoid cultures show no significant…

• 

  Fig. 5. Extruded cells can infect cultured cells and organoids. a, EV-A71-infected colonoid cultures were collected at 8 h post infection, and components were collected by differential sedimentation. b, To assess effectiveness of washing extruded cells, 106 PFU exogenous free virus was spiked-in to Cells + Media samples. Fractions including Whole Well (blank bars), Cells + Media (small dotted bars) and Cells (large-dotted bars) were subjected to freeze–thaw and plaque assay. Repeated-measures one-way ANOVA with the Holm–Šídák multiple comparisons test. NS, not significant. c, Distribution of virus in Cells and in Media. Ratio paired, two-tailed t-test. d, Stability of virus from Cells and Media fractions. Collected fractions were incubated at 37 °C for the times indicated, subjected to repetitive freeze–thaw and subsequent plaque assay. Amounts of virus are normalized to values from c before incubation. Two-way ANOVA with Geisser–Greenhouse correction. e, Intact extruded Cells fractions and free virus from the final wash of Cells were used to infect RD cell monolayers. Viral titres in the infected RD cells were measured 1 h and 16 h after initiating secondary infection. Ratio paired…

• 

  Fig. 6. Model for implications of infected cell extrusion on EV-A71 spread. EV-A71-infected cells extruded from the colon into the gastrointestinal lumen may play an integral role in faecal–oral transmission. When cells carrying infectious virus are extruded, progeny virions are expected to transit through the gut and be excreted in stool within live extruded cells. Within cells, virions may be protected from intestinal contents such as mucosal antibodies. Within-cell bundling of virions might additionally facilitate en bloc transmission, allowing for viral genomic diversity to be maintained. The potential for host benefits of infected cell extrusion in vivo such as quicker viral clearance or improved tolerance to infection remain intriguing open questions for further study.

• 

  Extended Data Fig. 1. Poliovirus-infected cells extrude from infected colon organoids. Cells infected with poliovirus Type 1 (Mahoney) were observed extruding from organoids by immunofluorescence. (a-b) Colon organoids infected with an MOI = 1 PFU/cell for 42 h. MOI was determined by viral titer on HeLa cells. Scale bars equal 10 μm.

• 

  Extended Data Fig. 2. Apical extrusion of EV-A71 infected cells occurs regardless of organoid polarity. Differentiated colonoids with basolateral-out organoid polarity were infected with EV-A71 and visualized by immunofluorescence at 8 hours post-infection. (a) Strong F-actin staining of the apical microvillus brush border was observed on the interior lumen of infected colonoids. Virus infected cells can be observed both within the epithelial layer, as well as within the apical lumen. (b, c) EV-A71 infected cells were apically extruded into the interior lumen of basolateral-out colonoids. Scale bars equal 10 μm.

• 

  Extended Data Fig. 3. Infected cells accumulate in organoids after 7 h infection and diminish after 9 h. (a) Infected organoids were fixed at timepoints over the first cycle of infection and the number of infected and uninfected cells were enumerated using fluorescence microscopy. Each dot represents the proportion of infected cells within an individual organoid; triangles represent values averaged across all organoids for each timepoint. One experiment with N = 10 organoids quantified per timepoint. A peak in the proportion of infected cells within organoids emerged at 7 hpi and was reduced after 9 hpi. Raw cell numbers represented in this graph are displayed in Supplementary Table 2. (b–d) Representative infected cells from each timepoint. Extruding infected cells contain similarly abundant vRNA signal regardless of timepoint visualized. Scale bars equal 10 μm. Source data

• 

  Extended Data Fig. 4. Extruded cells undergo anoikis by 48 hpi. Cells that had fully extruded from infected colonoids in the presence of the fluorogenic caspase 3/7 substrate CellEvent were collected at 48 hpi, fixed, and examined by fluorescence microscopy at 20x magnification. (a) 3D renderings of imaged cells were visualized using Volocity image analysis software. Infected cells are surrounded by yellow boxes. (b) Individual cells were identified from 20 unique fields of view as in A using computational measurement tools in Volocity. Data were collected from N = 743 individual cells. Cells with pixel intensities greater than 1.5 per cubic micron in the vRNA channel were identified as infected; N = 39 infected cells, 5.2% of total. (c) The caspase 3/7 activity of cells defined in B were compared. Caspase activity in infected cells was comparable to that of uninfected cells; unpaired, two-sided t-test; data presented as mean ± SD; N = 39 infected cells; N = 704 uninfected cells. Source data

• 

  Extended Data Fig. 5. Examination of the Organoid fraction of suspension organoid cultures. Following fractionation of organoid cultures, all fractions were visually examined on a tissue culture microscope to assess collection of target populations. (a) Visual examination of the Organoid fraction at 40x magnification confirms that organoids are collected with gravity pellet (1 x g). (b) Region highlighted in A. Single cells were also present at low abundance in the organoid fraction (arrows indicate examples), potentially a result of ongoing cell extrusion from organoids during fractionation (arrowheads).

• 

  Extended Data Fig. 6. Potential mechanisms of mechanosensitive ion channel activation in infected cells. We provide two hypotheses for potential means of activation of mechanosensitive ion channels in infected cells that may drive infected cell extrusion. Mechanosensitive ion channels such as Piezo1 can be activated by (a) external compression forces that induce membrane deformation, as well as (b) cell-intrinsic mechanical forces transmitted through tethered cytoskeletal filaments. (c, d) We present conceptual models for how viral infection may induce channel activation via both outside-in or cell-intrinsic forces. (c) In uninfected cells, cytoskeletal filaments provide structural support beneath the plasma membrane, reducing membrane deformation from cell-extrinsic forces. In infected cells wherein actin is rearranged, this structural support may be missing, resulting in ‘softer’ membranes more easily deformed by external compression forces. (d) In the force-through-filament model, cell-intrinsic mechanical forces are transmitted to Piezo channels via tethering to cytoskeletal filaments. In infected cells, cytoskeletal rearrangements may transmit forces directly to tethered Piezo channels, inducing…