Spike proteins of coronaviruses activate mast cells for degranulation via stimulating Src/PI3K/AKT/Ca2+ intracellular signaling cascade

Abstract

Mast cells (MCs) are strategically located at the interface between host and environment. The non-allergic functions of MCs in immunosurveillance against pathogens have been recently underscored. However, the activation of MCs by pathogens may beneficially or detrimentally regulate immune inflammation to combat or promote pathogen invasion. We and others have conclusively demonstrated that MCs serve as a crucial mediator in the induction of hyperinflammation initiated by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), leading to substantial tissue damage across multiple organs in murine and nonhuman primate models. Whereas the precise mechanism underlying virus-induced MC activation and degranulation remains largely elusive, our previous findings have indicated that the binding of the Spike proteins to cellular receptors is sufficient to elicit MC activation for rapid degranulation. This study aims to corroborate the ubiquity of coronavirus-induced MC degranulation and elucidate the intracellular signaling pathways that mediate the activation of MCs upon Spike protein binding to the cellular receptors. Our transcriptome analysis revealed MC activation upon the stimulations with a range of Spike/RBD proteins and viral particles of coronavirus. Notably, the interaction between these Spike/RBD proteins and cellular receptors triggered the activation of src kinase, a member of Src Family Kinases (SFKs). This activation, in turn, stimulated the PI3K/AKT signaling pathway, resulting in an accumulation of intracellular calcium ions. These calcium ions subsequently facilitated microtubule-dependent granule transport, ultimately promoting MC degranulation. In summary, this study elucidates the mechanism underlying virus-triggered activation of MCs and has the potential to aid in the development of MC-targeted antiviral therapeutic strategies.

Importance: The activation and degranulation of mast cells (MCs), triggered by a variety of viruses, are intricately linked to viral pathogenesis. However, the precise mechanism underlying virus-induced MC degranulation remains largely unknown. In this study, we demonstrate the ubiquity of coronavirus-induced MC degranulation and investigate the intracellular signaling pathways that mediate this process. We reveal that the binding of Spike proteins and cellular receptors is sufficient to elicit MC activation for rapid degranulation. This binding triggers the activation of src kinase and the downstream PI3K/AKT cellular signaling pathway, resulting in an accumulation of intracellular calcium ions. These calcium ions subsequently facilitate microtubule-dependent granule transport, ultimately promoting MC degranulation. This study elucidates the mechanism underlying virus-triggered activation of MCs and has the potential to aid in the development of MC-targeted antiviral therapeutic strategies.

Keywords: coronavirus; degranulation; mast cell.

Fig 1

Binding of Spike/RBD proteins to receptors triggers MC degranulation. (A) Expression of ACE2, DPP4, and APN in LUVA and HMC-1 cells was detected by immunostaining with specific antibodies and analyzed with flow cytometry. (B) LUVA, LAD2, and HMC-1 cells (1 × 10⁶ cells for each) were treated with HCoV-229E and HCoV-NL63 (M.O.I = 1) or medium (mock) for 24 or 48 h. Viral replication was quantified by detecting the expression of nucleocapsid gene and normalizing with the gapdh gene. (C) HMC-1 and LUVA cells were exposed to Spike/RBD proteins (5 µg/mL) for 1 h at 4°C. The co-localization of these proteins with their respective receptors of APN, ACE2, and DPP4 was detected with confocal microscopy. Scale bar: 10 µm. (D through G) MC degranulation. MC degranulation in Spike/RBD protein-treated (D), HCoV-229E and HCoV-NL63 (M.O.I = 1) virus-infected LUVA, LAD2, or HMC-1 cells (E), Spike/RBD mutant protein-treated LUVA cells (F), and pre- and post-fusion S2 subunits from SARS-CoV-2-treated LUVA and HMC-1 cells (G) were detected by quantifying the β-hexosaminidase release. The compound 48/80 (C48/80) was used as the control. Data are presented as mean ± SD. One representative result from three independent repeats is shown. ***P < 0.001 is considered significant differences.

Fig 2

Transcriptome analysis reveals Spike/RBD proteins-induced MC activation. LAD2 cells were exposed to Spike/RBD proteins (5 µg/mL) or virions of HCoV-229E and HCoV-NL63 (MOI = 1) for 24 h. Total RNAs were extracted from cells, and the transcriptome analysis was conducted. Data from three independent repeats were summarized. (A) Volcano plot of DEGs comparing Spike/RBD proteins treated or HCoV-229E and HCoV-NL63 infected cells to that of mock-infection or medium-treatment. The symbols of top upregulated or downregulated genes are shown. (B) Summary of DEGs. The consistently upregulated and downregulated genes in LAD2 cells from these treatments with spike/RBD proteins and viral particles. (C) GO functional enrichment analysis of DEGs. The color bar indicates the minus logarithm of Q values, and bubble size indicates the absolute gene counts enriched in a GO term. (D-F) Expression of inflammatory factors. Total RNAs from LAD2 cells or LUVA cells were isolated. The expression of IL-1β, IL-6, TNF-α, or IL-8 in virus-infected LAD2 cells (D) or Spike/RBD protein-treated LUVA cells (E, F) were detected by real-time (RT-)PCR. Data are presented as mean ± SD. One representative result from three independent repeats is shown. *P < 0.05, **P < 0.01 and ***P < 0.001 are considered significant differences.

Fig 3

Spike/RBD proteins induce activation of the cellular Src/PI3K/AKT signaling pathway. (A) LUVA cells were exposed to Spike/RBD proteins (5 µg/mL) for 2 h. Src-pY416, Src, P-PI3K(P85), PI3K, P-AKT, and AKT levels were assessed by immunoblot analysis. (B) LUVA cells were exposed to Spike/RBD proteins (5 µg/mL) in the presence or not of LY294002 (100 µM) for 2 h. (C) LUVA or HMC-1 cells were treated with HCoV-229E and HCoV-NL63 (M.O.I = 1) for 2 h. MC degranulation was detected by quantifying the β-hexosaminidase release. Data are presented as mean ± SD. One representative result from three independent repeats is shown. ***P < 0.001 is considered significant differences.

Fig 4

The role of cytoplasmic Ca²⁺ and microtubule reorganization in Spike protein-triggered MC degranulation. (A) LUVA cells were exposed to Spike/RBD proteins (5 µg/mL) for 2 h, and the F03 intracellular calcium ion fluorescent probe was added during the treatments. Cells were observed with confocal microscopy. Scale bar: 100 µm. (B) LUVA and LAD2 cells were exposed to Spike/RBD proteins (5 µg/mL) in the presence or not of BAPTA-AM (20 µM) for the indicated time, and the dynamic change of intracellular Ca²⁺ was monitored. (C, D) MC degranulation. LUVA and LAD2 cells were exposed to spike/RBD proteins (5 µg/mL) in the presence of BAPTA-AM (20 µM) (C) or Nocodazole (20 µM) (D) for 2 h. MC degranulation was detected by quantifying the β-hexosaminidase release. Data are presented as mean ± SD. One representative result from four independent repeats is shown. ***P < 0.001 is considered significant differences.

Fig 5

Graphical illustration of MC degranulation triggered by Spike/RBD proteins. The interaction between spike/RBD proteins and cellular receptors initiates the activation of src kinase, which subsequently stimulates the PI3K/AKT signaling pathway. This, in turn, leads to an accumulation of intracellular calcium ions. The elevated calcium levels facilitate microtubule-dependent granule transport, ultimately promoting MC degranulation.