SARS-CoV-2 N Protein Triggers Acute Lung Injury via Modulating Macrophage Activation and Infiltration in in vitro and in vivo

Dengming Lai; Kun Zhu; Sisi Li; Yi Xiao; Qi Xu; Yisheng Sun; Pingping Yao; Daqing Ma; Qiang Shu

doi:10.2147/JIR.S405722

SARS-CoV-2 N Protein Triggers Acute Lung Injury via Modulating Macrophage Activation and Infiltration in in vitro and in vivo

J Inflamm Res. 2023 Apr 28:16:1867-1877. doi: 10.2147/JIR.S405722. eCollection 2023.

Authors

Dengming Lai^#¹, Kun Zhu^#², Sisi Li^#³, Yi Xiao¹, Qi Xu⁴, Yisheng Sun⁵, Pingping Yao⁵, Daqing Ma^{6

7}, Qiang Shu⁷

Affiliations

¹ Department of Neonatal Surgery, Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, People's Republic of China.
² Department of Pathology, The Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, People's Republic of China.
³ School of Medicine, Hangzhou City University, Hangzhou, People's Republic of China.
⁴ School of Laboratory Medicine and Bioengineering, Hangzhou Medical College, Hangzhou, People's Republic of China.
⁵ Key Laboratory of Vaccine, Prevention and Control of Infectious Disease of Zhejiang Province, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, People's Republic of China.
⁶ Division of Anaesthetics, Pain Medicine & Intensive Care, Department of Surgery & Cancer, Faculty of Medicine, Imperial College London, Chelsea & Westminster Hospital, London, UK.
⁷ Department of Thoracic and Cardiovascular Surgery, Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, People's Republic of China.

^# Contributed equally.

Abstract

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.

Keywords: N protein; S protein; SARS-CoV-2; acute lung injury; cytokine; macrophage.

Grants and funding

Supported by Zhejiang University special scientific research fund for COVID-19 prevention and control (Q.S.); National Natural Science Foundation of China 82272191 (Q.S.), 81901989 (D.L.); the Fundamental Research Funds for the Central Universities 226-2022-00060 (Q.S.); Natural Science Foundation of Zhejiang Province LY21H150005 (D.L.); Foundation for The Top-Notch Youth Talent Cultivation Project of Independent Design Project of National Clinical Research Center for Child Health Q21B0007 (D.L.); Special Fund for the Incubation of Young Clinical Scientist, The Children’s Hospital of Zhejiang University School of Medicine CHZJU2022YS002 (D.L.); Natural Science Foundation of Zhejiang Province LQY19H190001 (Q.X.); Health Commission of Zhejiang Province 2019KY063, 2021KY133 (Q.X.), 2022485642, 2013KYA107 (S.L.); the Scientific Research Foundation of Zhejiang University City College J-202103(S.L.).