Inhibition of SARS-CoV-2-mediated thromboinflammation by CLEC2.Fc

Abstract

Thromboinflammation is the major cause of morbidity and mortality in COVID-19 patients, and post-mortem examination demonstrates the presence of platelet-rich thrombi and microangiopathy in visceral organs. Moreover, persistent microclots were detected in both acute COVID-19 and long COVID plasma samples. However, the molecular mechanism of SARS-CoV-2-induced thromboinflammation is still unclear. We found that the spleen tyrosine kinase (Syk)-coupled C-type lectin member 2 (CLEC2), which was highly expressed in platelets and alveolar macrophages, interacted with the receptor-binding domain (RBD) of SARS-CoV-2 spike protein (SARS-CoV-2 RBD) directly. Unlike the thread-like NETs, SARS-CoV-2-induced aggregated NET formation in the presence of wild-type (WT), but not CLEC2-deficient platelets. Furthermore, SARS-CoV-2 spike pseudotyped lentivirus was able to induce NET formation via CLEC2, indicating SARS-CoV-2 RBD engaged CLEC2 to activate platelets to enhance NET formation. Administration of CLEC2.Fc inhibited SARS-CoV-2-induced NET formation and thromboinflammation in AAV-ACE2-infected mice. Thus, CLEC2 is a novel pattern recognition receptor for SARS-CoV-2, and CLEC2.Fc and may become a promising therapeutic agent to inhibit SARS-CoV-2-induced thromboinflammation and reduced the risk of post-acute sequelae of COVID-19 (PASC) in the future.

Keywords: CLEC2; NET; SARS-CoV-2; platelet; thromboinflammation.

Figure 1. Human CLEC2 specifically binds to the RBD of SARS‐CoV‐2 variants

1. A, B

   Interaction between several human C‐type lectins (50 μg/ml) and recombinant SARS‐CoV‐2 RBD (50 μl/well) was determined by ELISA (A), and the interactions of hCLEC2.Fc with SARS‐CoV‐2 RBDs from various strains (B).

2. C

   The binding affinity of SARS‐CoV‐2‐spike RBD and human CLEC2.Fc fusion protein was measured by biolayer interferometry. K_a: Rate constants for association. K_d: Rate constants for dissociation. K_D: Equilibrium dissociation constant.

Data information: Data in (A) is presented as mean ± SEM, each experiment was repeated independently at least four times. *P < 0.05, ***P < 0.001 (Student's t‐test). Source data are available online for this figure.

Figure 2. Sequence alignment of SARS‐CoV‐2 RBD strains and SARS‐CoV RBD

The RBD sequences of SARS‐CoV‐2 (Taiwan strain, WT, alpha, beta, gamma, delta, omicron BA.1, omicron BA.2, omicron BA.3, omicron BA.4 & 5) and of SARS‐CoV were aligned with Clustal program under MacVector software (version 17.5.6). The star sign denotes the amino acid residues (K417, G446, Y449, Y453 E484, F486, N487, Y489, G496, Q498, T500, and G502) critical for SARS‐CoV‐2 binding to ACE2 (Hoffmann et al, 2020). The dot sign in black denotes the distinct amino acid residues in RBDs among SARS‐CoV‐2 strains. WT: wild type SARS‐CoV‐2 RBD.

Figure 3. SARS‐CoV‐2 activates platelet CLEC2 to induce NET formation via spike RBD

Human neutrophils (PMNs, 4 × 10⁵/ml) were incubated with SARS‐CoV‐2 (MOI = 0.1 and 1) with or without autologous platelets (PLTs, 4 × 10⁶/ml) for 5 and 20 h at 37°C.

1. A

   The image of NETs (arrowheads: aggregated NETs).

2. B

   Quantitation of NET area by citrullinated Histone H3 (Cit‐H3) area.

3. C

   Human platelets were pretreated with anti‐CLEC2 mAb (AYP1, 2.5 μg/ml) or isotype control for 15 min at room temperature before incubation with neutrophils and SARS‐CoV‐2 stimulation.

4. D

   Neutrophils (4 × 10⁵/ml) from wild type (WT) mice were incubated with SARS‐CoV‐2 (MOI = 1) in the presence of WT platelets (4 × 10⁶/ml) or clec2 ^−/− platelets (4 × 10⁶/ml) for 5 h at 37°C.

5. E, F

   Human neutrophils were stimulated with SARS‐CoV‐2 spike RBD pseudotyped virus (pseudotyped virus; MOI = 0.1) in presence of autologous platelets at 37°C for 3 h (E). Platelets were incubated with anti‐CLEC2 mAb (2.5 μg/ml) at room temperature for 15 min before incubation with neutrophils and pseudotyped virus stimulation at 37°C for 3 h (F). The level of NET formation was determined using Cit‐H3 area (μm²).

Data information: Data are mean ± SD from at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student's t‐test). #: The less NETs attached to the plate at 20 h (in the presence of platelets) are due to the robust aggregated NETs which were detached from the culture plate. Source data are available online for this figure.

Figure 4. The prophylactic and therapeutic effects of CLEC2.Fc in SARS‐CoV‐2‐challenged AAV‐hACE2 mice

1. A, B

   Human neutrophils were stimulated with SARS‐CoV‐2 (MOI = 0.1 and 1) at 37°C in the presence of isotype control (hIgG1, 3 μg/ml) or human CLEC2.Fc (3 μg/ml) and with or without autologous platelets for 5 h (A) or with or without anti‐SARS‐CoV‐2 spike RBD antibody (3 μg/ml) for 20 h (B). Data are means ± SD from at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student's t‐test).

2. C–E

   C57BL/6 mice were inoculated with AAV‐hACE2 at day 14 before the SARS‐CoV‐2 challenge. For the prophylactic use of hCLEC2.Fc, mice were intravenously (i.v.) injected with CLEC2.Fc (100 μg/mouse) then intratracheally injected with SARS‐CoV‐2 (8 × 10⁴ PFU). For the therapeutic use of hCLEC2.Fc, mice were intratracheally inoculated with SARS‐CoV‐2 (8 × 10⁴ PFU) and intraperitoneally injected with CLEC2.Fc (100 μg/per mice). Lung tissues were collected at day 3 day and day 5 post‐infection. (C) The expression levels of viral copy number (D), cytokines and chemokines in lung were measured by qPCR (E).

Data information: Data are mean ± SEM from two independent experiments (saline: n = 3 for 3 d.p.i., n = 4 for 5 d.p.i.; isotype: n = 3 for 3 d.p.i., n = 4 for 5 d.p.i.; prophylactic treatment of CLEC2.Fc treatment: n = 5 for 3 d.p.i. and 5 d.p.i.; therapeutic treatment of CLEC2.Fc: n = 3 for 3 d.p.i. and 5 d.p.i.). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two‐way ANOVA). ‘d.p.i.’ stands for ‘day post‐infection’. Source data are available online for this figure.

Figure 5. CLEC2.Fc inhibits SARS‐CoV‐2‐induced immunothrombosis in lung

1. A

   AAV‐hACE2 mice were treated with saline and challenged with SARS‐CoV‐2 (8 × 10⁴ PFU) for 5 days. Lung tissue sections were stained with DAPI (blue), anti‐MPO antibody (green), anti‐citrullinated histone H3 (Cit‐H3) antibody (red), anti‐CD42b antibody (yellow) and anti‐CD31 antibody (gray). yellow arrow: NETs (DNA⁺Gr‐1⁺MPO⁺Cit‐H3⁺); red arrow: immunothrombosis (NETs + thrombus (CD42b⁺)), Scale bar is 100 μm.

2. B, C

   For prophylactic treatment, CLEC2.Fc (or vehicle) was given at 1 h before virus challenge; for therapeutic treatment, CLEC2.Fc was given at 8 h post‐infection. The area of NET (colocalization area of MPO and Cit‐H3) (B) and immunothrombosis (colocalization area of Cit‐H3 and CD42b) (C) were measured using MetaMorph software.

Data information: Data are mean ± SEM from two independent experiments (saline: n = 3 for 3 d.p.i., n = 4 for 5 d.p.i.; isotype: n = 3 for 3 d.p.i., n = 4 for 5 d.p.i.; prophylactic treatment of CLEC2.Fc treatment: n = 5 for 3 d.p.i. and 5 d.p.i.; therapeutic treatment of CLEC2.Fc: n = 3 for 3 d.p.i. and 5 d.p.i.). *P < 0.05, **P < 0.01 (two‐way ANOVA). Source data are available online for this figure.

Figure 6. CLEC2.Fc inhibits SARS‐CoV‐2‐induced collagen deposition in lung

1. A, B

   AAV‐hACE2 mice were treated with saline and challenged with SARS‐CoV‐2 (8 × 10⁴ PFU) for 5 days. Samples were collected at day 5 post‐infection, and the collagen was stained with Picro Sirius red (A), while collagen deposition was quantified using MetaMorph software and presented as area (μm²) (B). The images were captured by light microscopy (upper panel) and polarized light microscopy (lower panel).

Data information: Data are mean ± SEM from two independent experiments (saline: n = 3 for 3 d.p.i., n = 4 for 5 d.p.i.; isotype: n = 3 for 3 d.p.i., n = 4 for 5 d.p.i.; prophylactic treatment of CLEC2.Fc treatment: n = 5 for 3 d.p.i. and 5 d.p.i.; therapeutic treatment of CLEC2.Fc: n = 3 for 3 d.p.i. and 5 d.p.i.). ***P < 0.001, ****P < 0.0001 (two‐way ANOVA). Source data are available online for this figure.