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. 2022 Sep 5:930:175169.
doi: 10.1016/j.ejphar.2022.175169. Epub 2022 Jul 31.

Mast cells promote viral entry of SARS-CoV-2 via formation of chymase/spike protein complex

Shuang Liu  1 , Yasuyuki Suzuki  2 , Erika Takemasa  3 , Ryusuke Watanabe  3 , Masaki Mogi  3
Affiliations

Mast cells promote viral entry of SARS-CoV-2 via formation of chymase/spike protein complex

Shuang Liu et al. Eur J Pharmacol. .

Abstract

The pulmonary pathological findings associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) result from the release of multiple proinflammatory cytokines, which causes the subsequential damage of the lungs. The current study was undertaken to investigate the responses of mast cells to viral inoculation and their contribution to host defenses from the point of view of viral entry. Pseudovirions, in which the spike glycoprotein of SARS-CoV-2 was incorporated, triggered activation of mast cells, and a mast cell-derived chymase, MCP2, formed a complex with spike protein, which promoted protease-dependent viral entry. According to the quantification results of viral entry, 10 μM quercetin, a mast cell stabilizer, potentially potently inhibited 41.3% of viral entry, while 100 μM chymostatin, which served as a chymase inhibitor, suppressed 52.1% of viral entry, compared to non-treated cells. Study using mast cell-deficient mice showed that the absence of mast cells may influence early viral loading in the upper respiratory tract, which consequently increases the risk of viral invasion into the lower respiratory system. Furthermore, mast cell-deficient mice exhibited ongoing infection in the late phase post-viral inoculation, while clearance of virus-positive cells was observed in wild-type mice. In conclusion, mast cells act as a multifaceted immune modulator that is equipped with both protective effects and pathogenic influences on viral entry of SARS-CoV-2. The utility of mast cell stabilizers and chymase inhibitors in the treatment of SARS-CoV-2-induced acute respiratory syndrome should be optimized regarding the infection stage and the risk of cytokine storm.

Keywords: Chymase; Inhibitor; Mast cell; SARS-CoV-2; Spike.

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Figures

Fig. 1
Fig. 1
Pseudovirion inoculation triggers mast cell activation and promotes viral entry. (a) Expression of SARS-CoV-2 spike glycoprotein in pseudovirus-inoculated RBL-2H3 cells at 0 min, 5 min, 30 min, 3 h, 24 h, and 72 h post viral inoculation. Lentivirus-infected cells were used as control at 72 h post-inoculation. (b) Normalized fluorescence signal of SARS-CoV-2 spike using Hoechst 33342. Cells were examined by fluorescence microscopy, and fluorescence intensity of a high-power field was divided by the number of cells to determine the fluorescence intensity per cell (n = 5). (c) Dose-response of hexosaminidase net release trigged by SARS-CoV-2 S inoculation at indicated MOI (n = 5). (d) Viral entry of SARS-CoV-2 S into HEK293 T cells cocultured in 0.5 μM thapsigargin (TG)-supplemented MEM, SARS-CoV-2 S pseudovirion-containing TG (0.5 μM)- supplemented MEM, SARS-CoV-2 S pseudovirion-containing non-treated RBL-2H3 cell culture supernatant, or SARS-CoV-2 S pseudovirion-containing TG (0.5 μM)-treated RBL-2H3 cell culture supernatant. The number of integrated provirus copies in cells was determined 72 h post-inoculation (n = 6). Values are presented as the scatter plot of individual values with mean ± SD.
Fig. 2
Fig. 2
Mast cell-derived MCP2 formed a complex with SARS-CoV-2 spike protein. (a) MCP2 and tryptase protein, which was co-immunoprecipitated using a specific antibody for spike glycoprotein of SARS-CoV-2, was detected using western blotting. RBL-2H3 cells were inoculated with pseudovirions of SARS-CoV-2 S, and protein levels were determined at the indicated time points. Upper: Expression of MCP2 in culture supernatant (lane 1), cell lysate (lane 2) of non-activated RBL-2H3 cells, and supernatant of virus-inoculated RBL-2H3 cells, which was co-immunoprecipitated with spike glycoprotein of SARS-CoV-2 at the indicated time points (lane 3–8). Middle: Expression of tryptase in each sample as described above. Lower: Expression of actin which served as a loading control. (b) Expression of MCP2 and tryptase protein levels was normalized against actin level in total cell lysates (a,u., arbitrary units). (c) Diagram of wild-type SARS-CoV-2 S protein (WT) and its truncations (M1-5). (d) The inserted fragments of reconstructed plasmids of pcDNA3.1-SARS2-S (WT), pcDNA3.1-SARS2-S1-NTD (M1), pcDNA3.1-SARS2-S1-RBD (M2), pcDNA3.1-SARS2-S1-S2 (M3), pcDNA3.1-SARS2-L (M4), and pcDNA3.1-SARS2-FP (M5) were confirmed by restriction enzyme digestion following by agarose separation. These plasmids were used for pseudovirions and production of mutations in the following experiments. (e) MCP2 protein in culture supernatant of virus-inoculated RBL-2H3 cells, which was co-immunoprecipitated using a specific antibody for C9-tag of spike glycoprotein of SARS-CoV-2, was detected using western blotting. Upper: MCP2 which was co-immunoprecipitated with C9-tag of SARS-CoV-2 (lane 1) and its mutations (lane 1–5). Lower: Expression of virus-derived p24 served as a loading control. (f) The number of integrated provirus copies in RBL-2H3 cells was determined 72 h post-inoculation of either SARS-CoV-2 S or its mutants (n = 5). Values are presented as the scatter plot of individual values with mean ± SD.
Fig. 3
Fig. 3
Efficacy of potential mast cell inhibitors on viral entry of SARS-CoV-2. Wild-type pseudovirions of SARS-CoV-2 S were inoculated into a HEK293 T-RBL-2H3 cell coculture system in vitro. Cells were pretreated with (a) quercetin, (b) chymostatin, or (c) Bowman-Birk protease inhibitor (BBI) before virus inoculation. The number of integrated provirus copies in RBL-2H3 cells was determined 72 h post-inoculation (n = 8). Values are presented as the scatter plot of individual values with mean ± SD.
Fig. 4
Fig. 4
Investigation of contribution of mast cells and MCP2 to viral entry in vivo. Both mast cell-deficient WBB6F1/Kit-KitW/KitW-v mice and wild control C57BL/6JJms mice were inoculated intranasally with 20 μL SARS-CoV-2 S pseudovirions (5 × 106 TCID50). The number of integrated provirus copies was determined on (a) day 3 and (b) day 7 after virus inoculation (n = 6–8). SARS-CoV-2 S pseudovirions, which were pretreated with MCP2, were also intranasally inoculated into wild-type mice. The number of integrated provirus copies was determined on (c) day 3 and (d) day 7 after virus inoculation (n = 6–8). Values are presented as the scatter plot of individual values with mean ± SD.
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