Infection of lung megakaryocytes and platelets by SARS-CoV-2 anticipate fatal COVID-19

Abstract

SARS-CoV-2, although not being a circulatory virus, spread from the respiratory tract resulting in multiorgan failures and thrombotic complications, the hallmarks of fatal COVID-19. A convergent contributor could be platelets that beyond hemostatic functions can carry infectious viruses. Here, we profiled 52 patients with severe COVID-19 and demonstrated that circulating platelets of 19 out 20 non-survivor patients contain SARS-CoV-2 in robust correlation with fatal outcome. Platelets containing SARS-CoV-2 might originate from bone marrow and lung megakaryocytes (MKs), the platelet precursors, which were found infected by SARS-CoV-2 in COVID-19 autopsies. Accordingly, MKs undergoing shortened differentiation and expressing anti-viral IFITM1 and IFITM3 RNA as a sign of viral sensing were enriched in the circulation of deadly COVID-19. Infected MKs reach the lung concomitant with a specific MK-related cytokine storm rich in VEGF, PDGF and inflammatory molecules, anticipating fatal outcome. Lung macrophages capture SARS-CoV-2-containing platelets in vivo. The virus contained by platelets is infectious as capture of platelets carrying SARS-CoV-2 propagates infection to macrophages in vitro, in a process blocked by an anti-GPIIbIIIa drug. Altogether, platelets containing infectious SARS-CoV-2 alter COVID-19 pathogenesis and provide a powerful fatality marker. Clinical targeting of platelets might prevent viral spread, thrombus formation and exacerbated inflammation at once and increase survival in COVID-19.

Keywords: COVID-19; Lung; Macrophages; Megakaryocytes; Platelets; SARS-CoV-2.

Fig. 1

Platelets from non-survivor patients with COVID-19 harbor SARS-CoV-2. A Copies of SARS-CoV-2 ORF1 (blue), Spike (S, magenta) and Nucleocapsid (N, orange) RNA per million platelets detected by RT-qPCR, from COVID-19 survivors, COVID-19 non-survivors and healthy donor samples. Asterisk indicates statistical significance in the comparison between survivors and non-survivors per detected gene target (Kruskal–Wallis between the three groups). LOD = limit of detection. B Combined detection of SARS-CoV-2 spike protein and SARS-CoV-2 RNA by flow cytometry (FISH-flow). On the left, SARS-CoV-2 spike⁺/RNA⁺ detection gate (red) showing an example of healthy donor, COVID-19 survivor, and COVID-19 non-survivor. On the right, the percentage of SARS-CoV-2 spike⁺/RNA⁺ platelets among platelets from COVID-19 survivors and non-survivors, normalized by detected events in healthy donor samples. Samples were classified as negative (gray) or positive (orange) for the presence of SARS-CoV-2 in platelets. Asterisk indicates statistical significance in the comparison between survivors and non-survivors (Mann-Whitney between the two groups). C Number of COVID-19 survivors (blue) and non-survivors (magenta) among individuals tested for the presence of SARS-CoV-2 in platelets (negative or positive). OR: odds ratio. D-E Representative confocal microscopy images after CD41 (green) immunolabeling and SARS-CoV-2 RNA in situ hybridization (red) for SARS-CoV-2 (+) RNA (D) or SARS-CoV-2 (-) RNA (E) in platelet samples from a COVID-19 non-survivor. Images show low magnification (upper, bar = 10 μm), three-dimensional projections (xy, xz and yz, lower left, bar = 2 μm) and three-dimensional rendering (lower right, bar = 1 μm). Arrowheads indicate SARS-CoV-2 RNA, showing definite intracellular localization of the virus within the platelets. F Representative electron microscopy images of platelets with spherical crowned SARS-CoV-2 particles of 50-80 nm in diameter (arrowheads) located in the lumen of OCS in platelets of non-survivors (bars = 200 nm). Dotted line indicates area magnified as shown in the insets (bar = 100 nm). G Representative immunogold labeling with a polyclonal anti-spike antibody of non-survivor platelets otherwise tested positive for the presence of SARS-CoV-2 by FISH-Flow techniques (two examples, upper and lower images). Dotted squares point magnified regions where spike proteins are immunolocalized (red arrowhead). No spike immunolabeling was observed on platelet surface. Bar = 100, 200 or 500 nm

Fig. 2

In COVID-19 non-survivors, MKs are infected and express viral sensing genes. A Frequency of MKs detected among PBMC from healthy donors and COVID-19 survivors and non-survivors as quantified by flow cytometry. Asterisks indicate statistical significance (Kruskal–Wallis test). B–F Transcriptional identity of MKs in COVID-19 patients by single-cell RNA sequencing reveals distinct phenotypes in non-survivor patients. B UMAP of single-cell transcriptomic data of MKs detected among PBMC from non-COVID-19 healthy donors (n = 30), COVID-19 survivors (n = 140) and COVID-19 non-survivors (n = 13). Unsupervised clustering detected 9 different clusters (0 to 8) of all cells analyzed. MK singlets are indicated by blue region. C Proportion of each cluster in healthy donors, COVID-19 survivors and non-survivors. D Scored gene signature expression of classical (upper) and non-classical (bottom) MK differentiation. E Fraction of cells from cluster 3 in comparison to all other clusters in individual patient samples categorized as healthy donors, COVID-19 survivors and non-survivors. F Heatmap of the genes that significantly change along pseudotime trajectory of MK development (p < 0.05 and Morans I score > 0.25). G Hematoxylin/eosin histology of bone marrow tissue obtained after COVID-19 non-survivor autopsy (low magnification (bar = 50 μm)) in which some MKs surrounded by blue circles are shown in high magnification insets (bar = 20 μm). Arrowheads indicate MKs. H Representative confocal microscopy images after CD41 (green) immunolabeling and replicative SARS-CoV-2 (-) RNA strand in situ hybridization (red) in bone marrow samples obtained from tissue autopsies of three different COVID-19 non-survivors (bar = 10 μm). Arrowheads indicate SARS-CoV-2 (-) RNA inside MKs

Fig. 3

SARS-CoV-2 in platelets and MKs in lung from non-survivor COVID-19 patients. A Quantification of different cytokines/chemokines in bronchoalveolar lavage samples from COVID-19 survivors (blue) and non-survivors (magenta). Asterisk indicates statistical significance in the comparison between survivors and non-survivors (Mann–Whitney). B Representative confocal microscopy images after SARS-CoV-2 RNA in situ hybridization (red) for positive (+) RNA strand and immunolabeling of either CD41 (upper row) or vWF (lower row), both in green, in bronchoalveolar lavage samples from two different COVID-19 non-survivors (bar = 10 μm or 5 μm for the inset in upper image). Arrowheads indicate SARS-CoV-2 RNA inside MKs. C Frequency of MKs and platelets among the cell population detected in bronchoalveolar lavage samples from COVID-19 survivors (blue) and non-survivors (magenta) detected by flow cytometry. D Frequency of SARS-CoV-2⁺ MKs and platelets among the population of MKs and platelets detected in bronchoalveolar lavage samples from COVID-19 survivors (blue) and non-survivors (magenta) as quantified by flow cytometry. Asterisk indicates statistical significance in the comparison between survivors and non-survivors (Mann–Whitney). E Hematoxylin/eosin/saffron histology and vWF immunohistochemistry (lower inset) of lung tissue from COVID-19 autopsy, showing low (bar = 50 μm) and high magnification (bar = 20 and 25 μm) images. Blue arrowheads indicate MKs. F Hematoxylin/eosin histology of lung tissue in which some MKs indicated by dotted square region (left), resided to reside inside alveolar space (right) (bar = 25 μm). Orange arrowheads indicate MKs. G Representative confocal microscopy images after CD41 (green) immunolabeling and replicative SARS-CoV-2 (-) RNA strand in situ hybridization (red) in lung samples obtained from tissue autopsies of  five different COVID-19 non-survivors (bar = 10 μm). Arrowheads indicate SARS-CoV-2 (-) RNA inside MKs

Fig. 4

SARS-CoV-2 sheltered by platelets from non-survivor patients with COVID-19 is infectious to macrophages. A Upper row: Hematoxylin/eosin/saffron stain histology of representative lung tissue autopsy of COVID-19 patients showing a macrophage, indicated by dotted square region (left) in the process of phagocytosing a red blood cell as shown in higher magnification (right, blue arrowhead). Bar = 10 μm. Lower panel: Immunohistochemistry for vWF of alveoli from non-survivor lung autopsy (indicated by blue dotted square at low magnification, bar = 150 μm) where a macrophage (indicated by blue dotted square at middle magnification, bar = 30 μm) contained hemophagocytosed vWF⁺ platelets (blue arrowheads in high magnification image, bar = 5 μm). B Representative confocal microscopy images after vWF (green) and CD68 (purple) immunolabeling and SARS-CoV-2 RNA in situ hybridization (red) for positive (+) RNA strand in BAL samples from COVID-19 non-survivors. Images show three-dimensional projections (xy, xz and yz, bar = 3 μm). Arrowheads indicate SARS-CoV-2 RNA inside platelets engulfed by macrophages (representative of n = 3 different individuals). C Frequency of macrophage-platelet conjugates among macrophages in bronchoalveolar lavage samples from COVID-19 survivors (blue) and non-survivors (magenta) detected by flow cytometry. Asterisk indicates statistical significance in the comparison between survivors and non-survivors (Mann–Whitney). D Paired comparison of percentages of SARS-CoV-2 RNA⁺/dsRNA⁺ Vero cells treated with releasate from platelets treated or not with TRAP, from 3 different non-survivors. The detection threshold (dotted red line) was established with healthy donor platelets treated equally. The percentages were converted in PFU per million platelets using the standard curve we established. Mann–Whitney test. The estimated mean PFU per million platelets is shown in blue, with 95% confidence intervals. E Scheme of the experiments evaluating platelet-mediated SARS-CoV-2 transfer of infection to macrophages in vitro. SARS-CoV-2 -containing platelets from non-survivors interacted with macrophages in the presence or absence of abciximab (anti-GpIIbIIIa) for 2 h (pulse) followed by 24-h chase. At these time-points, macrophages harboring (+) and (-) SARS-CoV-2 RNA were enumerated by in situ hybridization and macrophage supernatants were collected and further evaluated for infectious virus content in reporter Vero cells. SARS-CoV-2 RNA⁺/dsRNA⁺ Vero cells were detected by in situ hybridization and quantified by FISH-flow. F Confocal microscopy images of SARS-CoV-2 RNA in situ hybridization (red) for positive (+) and negative (-) strand RNA in macrophages that interacted in vitro with platelets samples from COVID-19 non-survivors. Images were acquired after 2 h (pulse, left) or 24 h (chase, right) of interaction with platelets. Images show three-dimensional projections (xy, xz and yz, bar = 10 μm). Arrowheads indicate SARS-CoV-2 RNA. Macrophage nuclei are stained with DAPI (blue). G Outgrowth in Vero reporter cells of SARS-CoV-2 produced by macrophages after platelet-mediated infection. On the left, confocal microscopy image of double-strand RNA (dsRNA, red) in Vero cells cultivated with macrophage supernatants for 24 h. Arrowheads indicate infected Vero cells (bar = 30 μm), Vero cells nuclei are stained with DAPI (blue). On the middle graph, infected Vero cells were quantified by FISH-flow and expressed as % of SARS-CoV-2 RNA⁺/dsRNA⁺ Vero cells, comparing negative controls (medium, gray = non-infected Vero), positive controls (virus, green = primary SARS-CoV-2 obtained from patient bronchoalveolar lavage) and COVID-19 non-survivors platelets after pulse (2 h interaction with macrophages, light red) and chase (24 h after interaction with macrophages, red). Asterisk indicates statistical significance in the comparison between pulse and chase (Mann–Whitney). The right graph shows the outgrowth of SARS-CoV-2 in Vero cells incubated with macrophages that interacted with COVID-19 non-survivors’ platelets in the presence or not of abciximab (10 μg/ml) also quantified by FISH-Flow. Results are expressed as % of SARS-CoV-2 RNA⁺/dsRNA⁺ Vero cells in the two conditions. The % inhibition of Vero cell infection in the presence of abciximab is indicated, with asterisk corresponding to statistical significance in the comparison between the two groups (Student T-test)

Fig. 5

Scheme: Platelets harboring SARS-CoV-2 offer a convergent therapeutical target in severe COVID-19 with multiple manifestations. 1- SARS-CoV-2 favors emergency inflammatory megakaryopoiesis. SARS-CoV-2 infected MKs in the bone marrow (containing both viruses and replicating SARS-CoV-2 (-) RNA) migrate to the lungs where they contribute to thrombopoiesis and produce SARS-CoV-2-containing platelets in the pulmonary circulation. 2- These infectious platelets will then spread the virus and contribute to the systemic inflammatory component of severe COVID-19. As platelets sheltering SARS-CoV-2 are coated with von Willebrand Factor, indicating their highly activated status, they will also contribute to thrombus formation typical of COVID-19 complications. 3- Increase in lung VEGF-A and PDGF-BB participates to alveolar endothelial destruction and effraction allowing platelets carrying SARS-CoV-2 to reach and infect alveolar macrophages. 4- Increased lung PF4/CXCL4 released by platelets and S100A8 likely contribute to the maintenance of a highly inflammatory environment, macrophage activation, and cytokine storm. These four platelet-mediated components of severe COVID-19 suggest that targeting platelets, with the use of anti-platelet drugs like anti-GPIIbIIIa, might be an efficient strategy to block viral spread, thrombus formation and exacerbated inflammation at once, increasing the chance of survival