Genetic Arg-304-His substitution in GRK5 protects against sepsis progression by alleviating NF-κB-mediated inflammation.

BACKGROUND: Previous studies have demonstrated that G protein-coupled receptor kinase 5 (GRK5) exerts a pivotal regulatory effect on the inflammation associated with sepsis. The present study aimed to investigate the clinical association of GRK5 genetic variants with sepsis and to further explore the underlying genetic mechanisms involved in regulating sepsis-induced inflammatory responses and the pathogenesis of sepsis. METHODS: This case-control study enrolled 1081 septic patients and 1147 matched controls for genotyping of GRK5 rs2230349 and rs2230345 polymorphisms. The effect of these genetic variants on GRK5-mediated inflammatory responses was analyzed in peripheral blood mononuclear cells (PBMCs) and THP-1 macrophages. A clinically relevant polymicrobial sepsis model was established by subjecting wild-type (WT) and GRK5-knockout mice to cecal ligation and puncture (CLP) to evaluate the role of GRK5 in sepsis. RESULTS: We identified significant differences in the genotype/allele distribution of rs2230349 G > A, but not rs2230345, between the sepsis subtype and septic shock subgroups (GA + AA vs. GG genotype, OR = 0.698, 95% CI = 0.547-0.893, P = 0.004; A vs. G allele, OR = 0.753, 95% CI = 0.620-0.919, P = 0.005) and between the survivor and nonsurvivor subgroups (GA + AA vs. GG genotype, OR = 0.702, 95% CI = 0.531-0.929, P = 0.015; A vs. G allele, OR = 0.753, 95% CI = 0.298-0.949, P = 0.017). PBMCs carrying the sepsis-associated protective A allele produced significantly lower levels of TNF-α and IL-1ß upon LPS stimulation. The results from the in vitro experiment showed that the Arg-304-His substitution caused by the rs2230349 G-to-A mutation in GRK5 significantly decreased the LPS-induced production of several proinflammatory cytokines, such as TNF-α, IL-6, IL-1ß and MCP-1, via the IκB-α/NF-κB signaling pathway in THP-1 macrophages. Furthermore, GRK5-knockout mice exhibited a significant decrease in IκB-α phosphorylation/degradation, the p-p65/p65 ratio, the p-p50/p50 ratio, p65 nuclear translocation and downstream cytokine (TNF-α, IL-6, IL-1ß and VCAM-1) production compared to WT mice after CLP surgery. A significant improvement in 7-day survival rate in GRK5-KO septic mice was observed in the presence of antibiotics. CONCLUSIONS: The Arg-304-His substitution caused by the rs2230349 G-to-A mutation in GRK5 might disrupt GRK5 function and alleviate IKB-α/NF-κB-mediated inflammatory responses, which ultimately conferred a genetic protective effect against susceptibility to sepsis progression and mortality. These results may, to some extent, explain the heterogeneity of the clinical prognoses of septic patients and provide novel opportunities for individualized approaches for sepsis treatment.

Brain Epitranscriptomic Analysis Revealed Altered A-to-I RNA Editing in Septic Patients.

Recent studies suggest that RNA editing is associated with impaired brain function and neurological and psychiatric disorders. However, the role of A-to-I RNA editing during sepsis-associated encephalopathy (SAE) remains unclear. In this study, we analyzed adenosine-to-inosine (A-to-I) RNA editing in postmortem brain tissues from septic patients and controls. A total of 3024 high-confidence A-to-I RNA editing sites were identified. In sepsis, there were fewer A-to-I RNA editing genes and editing sites than in controls. Among all A-to-I RNA editing sites, 42 genes showed significantly differential RNA editing, with 23 downregulated and 19 upregulated in sepsis compared to controls. Notably, more than 50% of these genes were highly expressed in the brain and potentially related to neurological diseases. Notably, cis-regulatory analysis showed that the level of RNA editing in six differentially edited genes was significantly correlated with the gene expression, including HAUS augmin-like complex subunit 2 (HAUS2), protein phosphatase 3 catalytic subunit beta (PPP3CB), hook microtubule tethering protein 3 (HOOK3), CUB and Sushi multiple domains 1 (CSMD1), methyltransferase-like 7A (METTL7A), and kinesin light chain 2 (KLC2). Furthermore, enrichment analysis showed that fewer gene functions and KEGG pathways were enriched by edited genes in sepsis compared to controls. These results revealed alteration of A-to-I RNA editing in the human brain associated with sepsis, thus providing an important basis for understanding its role in neuropathology in SAE.