Tnfrsf1b Protects Zebrafish Against Klebsiella pneumoniae Infection

Yanfei Jing,1,* Ze Xu,2,* Feng Ju,3,* Mingyong Wang,4 Fan Tu,5 Xiaohong Rui,5 Futao Cao,6 Jun Liu5 1Department of Function, Affiliated Wuxi Fifth Hospital of Jiangnan University, Wuxi, 214005, People’s Republic of China; 2Department of Laboratory Medicine, The Affiliated Wuxi Center for Disease Control and Prevention of Nanjing Medical University, Wuxi, 214023, People’s Republic of China; 3Department of Digestive, Wuxi Fifth People’s Hospital Affiliated Jiangnan University, Wuxi, 214005, People’s Republic of China; 4Murui Biological Technology Co., Ltd., Suzhou Industrial Park, Suzhou, People’s Republic of China; 5Department of Laboratory Medicine, Wuxi Fifth People’s Hospital Affiliated Jiangnan University, Wuxi, 214005, People’s Republic of China; 6Department of Emergency, Jiangnan University Medical Center, Wuxi, 214005, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jun Liu, Department of Laboratory Medicine, Wuxi Fifth People’s Hospital Affiliated Jiangnan University, Wuxi, 214005, People’s Republic of China, Email [email protected] Futao Cao, Department of Emergency, Jiangnan University Medical Center, Wuxi, 214005, People’s Republic of China, Email [email protected]: Klebsiella (K). pneumoniae is an opportunistic pathogen that causes severe infections in the lungs, urinary tract, and liver, yet its pathogenesis remains unclear. Tnfrsf1b, a member of the tumor necrosis factor receptor superfamily, is associated with inflammation and tissue damage. The objective of this study was to investigate the functional role of tnfrsf1b in zebrafish during K. pneumoniae infection and tnfrsf1b should be considered a candidate target for further investigation.Methods: A tnfrsf1b-knockout zebrafish line was generated using CRISPR/Cas9 technology. Both wild-type and mutant zebrafish were infected with the hypervirulent K. pneumoniae strain NTUH-K2044. Phenotypic changes, immune cell recruitment, and cytokine production were assessed using Alcian blue staining, Sudan Black B staining, neutral red staining, qRT-PCR, and ELISA. Rescue experiments were performed by injecting capped tnfrsf1b mRNA into mutants and wild-type zebrafish embryos. Pharmacological inhibition was tested using the TNFR inhibitor R7050.Results: Compared with wild-type zebrafish, tnfrsf1b-knockout mutants exhibited significantly higher malformation and mortality rates, increased recruitment of macrophages and neutrophils, and elevated levels of pro-inflammatory cytokines (TNF-α, IL-1β). Similar phenotypes were observed in R7050-treated zebrafish. K. pneumoniae infection further exacerbated these immune dysfunctions in tnfrsf1b mutants. Injection of capped tnfrsf1b mRNA into mutants partially rescued developmental and immune defects, while overexpression in wild-type zebrafish conferred protection against K. pneumoniae-induced damage.Conclusion: tnfrsf1b plays a critical role in regulating host immune responses and protecting zebrafish from K. pneumoniae infection. Targeting the tnfrsf1b signaling pathway may represent a promising therapeutic approach for managing infections caused by hypervirulent K. pneumoniae.Keywords: tnfrsf1b, Klebsiella pneumoniae, zebrafish, inflammation, infection susceptibility