Identification of novel amino acid variants in the Han Chinese population that impair Toll-like Receptor 4 signaling and confer hyporesponsiveness to lipopolysaccharide

Bin Zhou^1,2Zhaoying Lv¹Ruijuan Zu¹Jianbo Liu¹Yuting Yang¹Zong-Guang Zhou^1*Yuan Li^1*

• ¹Sichuan University, Chengdu, China
• ²Laboratory of Digestive Surgery, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China school of medicine, Sichuan University., Chengdu, China

Severe acute pancreatitis (SAP) is a life-threatening condition characterized by profound pancreatic inflammation that can trigger multiorgan failure, leading to significant morbidity and mortality. Toll-like receptor 4 (TLR4), a critical sensor for lipopolysaccharide (LPS) from Gram-negative bacteria, plays a central role in SAP pathogenesis. Through sangon sequencing in a Han Chinese cohort with acute pancreatitis, we identified five novel missense variants in the TLR4 gene. In silico analyses predicted subtle alterations to the protein's secondary structure for all five variants, with the p.(Arg763Cys) variant notably poised to affect the transmembrane domain and theoretical isoelectric point. Co-immunoprecipitation assays demonstrated that the p.(Gly715Ser), p.(Arg763Cys), and p.(Arg804Trp) variants failed to interact with MyD88. Functional characterization in human embryonic kidney (HEK) 293T cells using luciferase reporter assays and interleukin 8 (IL-8) expression tests confirmed that these three variants suppressed nuclear factor kappa B (NF-κB) activation and downstream inflammatory signaling. To validate the clinical relevance of the p.(Arg804Trp) variant, we generated knock-in mice and established models of SAP. Homozygous mutant mice exhibited significantly lower histological scores, reduced apoptosis, decreased neutrophil and macrophage infiltration, and lower serum levels of amylase, lipase, IL-6, and IL-10 compared to wild-type controls. In conclusion, we identified novel TLR4 variants that impair its signaling capacity, leading to LPS hyporesponsiveness and milder SAP. Our findings highlight the influence of genetic factors on acute pancreatitis severity and suggest that targeting TLR4 could be a promising therapeutic strategy to mitigate SAP.