Cellular and Molecular Gastroenterology and Hepatology最新文献

Background & aims: Mechanisms of hepatitis C virus (HCV) adaptation and escape from broadly neutralizing antibodies (bNAbs) have been primarily studied in vitro. Here, we used a previously developed in vivo adapted J6/JFH1_A876P virus and the highly bNAb sensitive hypervariable region 1 (HVR1) deleted variant, J6/JFH1_A876P,ΔHVR1, to study adaptation and bNAb AR5A escape in the HCV-permissive human-liver chimeric mouse model.

Methods: In vitro identified AR5A escape substitution, L665S, was introduced into J6/JFH1_A876P with or without HVR1. The infection of human liver chimeric mice with these recombinants revealed adaptive mutations, and the potential mechanism of adaptation was extensively characterized in vitro. Finally, we tested the barrier to resistance of AR5A in vivo by challenging passively immunized animals with HVR1-deleted viruses, either with or without the AR5A escape substitution, L665S.

Results: L665S was found to be an escape substitution in vivo. Furthermore, sequence analysis showed that the escape substitution L665S arose as early as 2 weeks post infection. At week 8, we also identified antibody escape substitutions as well as several potential in vivo adaptive substitutions in E2. For J6/JFH1_A876P, S449P and M702L increased cell-free particle infection and broadly affected antibody sensitivity for virus with HVR1. For J6/JFH1_A876P,ΔHVR1, N430D and M702L substitutions increased both cell-free particle mediated infection and cell-to-cell spread, whereas N430D also increased thermal stability at 37°C.

Conclusions: We show that L665S is an AR5A escape mutation in vivo, supporting the use of cost-effective vaccine escape studies in vitro. We also identify novel in vivo adaptive mutations and characterize their mechanism of action, thus facilitating interpretation of future HCV in vivo studies.

Background & aims: Localised acidification from immune cell infiltration and heightened glycolysis contributes to colitis pathology by activating acid-sensing receptors such as G protein-coupled receptor 68 (GPR68), a proton-sensing G protein-coupled receptor (GPCR) expressed on immune and stromal cells. Single-cell RNA sequencing (RNA-seq) analysis revealed GPR68 is also expressed in colonic sensory neurons, prompting us to investigate its role in acid-induced colonic nociception.

Methods: Expression of GPR68 in colonic nociceptors and tissue from people with colitis was confirmed by in silico analysis of our RNA-seq databases. Its contribution to disease activity was assessed using the acute dextran sulphate sodium (DSS) model of colitis. Acid-evoked sensory signalling was evaluated via colonic afferent recordings and Ca²⁺ imaging in DRG neurons from wild-type and GPR68^-/- mice, supported by pharmacological studies using Ogerin (a GPR68 positive allosteric modulator) and Ogremorphin (a GPR68 antagonist).

Results: RNA-seq analysis showed GPR68 is robustly expressed in Trpv1⁺ colonic nociceptors and upregulated in tissue from people with inflammatory bowel disease, consistent with reduced disease activity in DSS-treated GPR68^-/- mice. Genetic deletion of GPR68 abolished colonic afferent responses to acid, which were also attenuated by Ogremorphin and enhanced by Ogerin. In Ca²⁺-free buffer, dorsal root ganglion neurons from GPR68^-/- mice or those pretreated with Ogremorphin showed significantly reduced acid-evoked intracellular Ca²⁺ responses. By contrast, the colonic afferent and dorsal root ganglion Ca²⁺ response (in Ca²⁺-containing buffer) to capsaicin was comparable between tissue from wild-type and GPR68^-/- mice highlighting the involvement of divergent proton-dependent cellular signaling cascades.

Conclusions: These findings identify GPR68 as a key mediator of acid-induced colonic nociception and highlight its potential as a therapeutic target for the treatment of pain in colitis.