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SARS-CoV-2 N Protein Triggers Acute Lung Injury via Modulating Macrophage Activation and Infiltration in in vitro and
Dengming Lai1, Kun Zhu2, Sisi Li3
1Department of Neonatal Surgery, Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, People's Republic of China.
Background:
SARS-CoV-2-induced acute lung injury but its nucleocapsid (N) and/or Spike (S) protein involvements in the disease pathology remain elusive.
Methods:
In vitro, the cultured THP-1 macrophages were stimulated with alive SARS-CoV-2 virus at different loading dose, N protein or S protein with/without TICAM2-siRNA, TIRAP-siRNA or MyD88-siRNA. The TICAM2, TIRAP and MyD88 expression in the THP-1 cells after N protein stimulation were determined. In vivo, naïve mice or mice with depletion macrophages were injected with N protein or dead SARS-CoV-2. The macrophages in the lung were analyzed with flow cytometry, and lung sections were stained with H&E or immunohistochemistry. Culture supernatants and serum were harvested for cytokines measurements with cytometric bead array.
Results:
Alive SARS-CoV-2 virus or N protein but not S protein induced high cytokine releases from macrophages in a time or virus loading dependent manner. MyD88 and TIRAP but not TICAM2 were highly involved in macrophage activation triggered by N protein whilst both inhibited with siRNA decreased inflammatory responses. Moreover, N protein and dead SARS-CoV-2 caused systemic inflammation, macrophage accumulation and acute lung injury in mice. Macrophage depletion in mice decreased cytokines in response to N protein.
Conclusion:
SARS-CoV-2 and its N protein but not S protein induced acute lung injury and systemic inflammation, which was closely related to macrophage activation, infiltration and release cytokines.
Insights
The SARS-CoV-2 nucleocapsid (N) protein, not the Spike (S) protein, triggers acute lung injury and systemic inflammation by activating macrophages. This highlights the N protein
Area of Science:
- Virology
- Immunology
- Pathology
Background:
- The precise roles of SARS-CoV-2 nucleocapsid (N) and Spike (S) proteins in acute lung injury pathogenesis are not fully understood.
- Investigating the specific contributions of viral proteins to disease mechanisms is crucial for developing targeted therapies.
Purpose of the Study:
- To elucidate the distinct roles of SARS-CoV-2 N and S proteins in inducing macrophage activation and subsequent acute lung injury.
- To identify the key molecular pathways, including TICAM2, TIRAP, and MyD88, involved in N protein-mediated inflammation.
Main Methods:
- In vitro studies using THP-1 macrophages stimulated with live SARS-CoV-2, N protein, or S protein, with or without siRNA targeting TICAM2, TIRAP, or MyD88.
- In vivo experiments in mice involving injection of N protein or inactivated SARS-CoV-2, with analysis of lung macrophages, histology, and cytokine levels.
- Macrophage depletion models were used to assess their role in N protein-induced inflammation.
Main Results:
- Live SARS-CoV-2 and N protein, but not S protein, significantly elevated cytokine release from macrophages in a dose-dependent manner.
- MyD88 and TIRAP, but not TICAM2, were critical for N protein-induced macrophage activation; their inhibition via siRNA reduced inflammatory responses.
- N protein and inactivated SARS-CoV-2 induced systemic inflammation, macrophage infiltration in lungs, and acute lung injury in mice, effects diminished by macrophage depletion.
Conclusions:
- SARS-CoV-2 N protein, unlike S protein, is a key driver of acute lung injury and systemic inflammation.
- Macrophage activation, infiltration, and cytokine release are central mechanisms in N protein-induced pathology.
