RNAi Induces Innate Immunity through Multiple Cellular Signaling Pathways

<div><p>Background & Aims</p><p>Our previous results showed that the knockdown of woodchuck hepatitis virus (WHV) by RNA interference (RNAi) led to upregulation of interferon stimulated genes (ISGs) in primary hepatocytes. In the present study, we tested the hypothesis that the cellular signaling pathways recognizing RNA molecules may be involved the ISG stimulation by RNAi.</p><p>Methods</p><p>Primary murine hepatocytes (PMHs) from wild type mice and WHV transgenic (Tg) mice were prepared and treated with defined siRNAs. The mRNA levels of target genes and ISGs were detected by real-time RT-PCR. The involvement of the signaling pathways including RIG-I/MDA5, PKR, and TLR3/7/8/9 was examined by specific inhibition and the analysis of their activation by Western blotting.</p><p>Results</p><p>In PMHs from WHV Tg mice, specific siRNAs targeting WHV, mouse β-actin, and GAPDH reduced the levels of targeted mRNAs and increased the mRNA expression of IFN-β, MxA, and IP-10. The enhanced ISG expression by siRNA transfection were abolished by siRNA-specific 2′-O-methyl antisense RNA and the inhibitors 2-AP and chloroquine blocking PKR and other TLR-mediated signaling pathways. Furthermore, Western blotting revealed that RNAi results in an increase in PKR phosphorylation and nuclear translocation of IRF3 and NF-êB, indicating the possible role of IRF3 in the RNAi-directed induction of ISGs. In contrast, silencing of RIG-I and MDA5 failed to block RNAi-mediated MxA induction.</p><p>Conclusions</p><p>RNAi is capable of enhancing innate immune responses through the PKR- and TLR-dependent signaling pathways in primary hepatocytes. The immune stimulation by RNAi may contribute to the antiviral activity of siRNAs in vivo.</p></div

Data_Sheet_1_Prognosis prediction performs better in patients with non-cirrhosis hepatitis B virus-related acute-on-chronic liver failure than those with cirrhosis.docx

BackgroundThe accurate prediction of the outcome of hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) is impeded by population heterogeneity. The study aimed to assess the impact of underlying cirrhosis on the performance of clinical prediction models (CPMs).MethodsUsing data from two multicenter, prospective cohorts of patients with HBV-ACLF, the discrimination, calibration, and clinical benefit were assessed for CPMs predicting 28-day and 90-day outcomes in patients with cirrhosis and those without, respectively.ResultsA total of 919 patients with HBV-ACLF were identified by Chinese Group on the Study of Severe Hepatitis B (COSSH) criteria, including 675 with cirrhosis and 244 without. COSSH-ACLF IIs, COSSH-ACLFs, Chronic Liver Failure-Consortium Acute-on-Chronic Liver Failure score (CLIF-C ACLFs), Tongji Prognostic Predictor Model score (TPPMs), Model for End-Stage Liver Disease score (MELDs), and MELD-Sodium score (MELD-Nas) were all strong predictors of short-term mortality in patients with HBV-ACLF. In contrast to a high model discriminative capacity in ACLF without cirrhosis, each prognostic model represents a marked decline of C-index, net reclassification index (NRI), and integrated discrimination improvement (IDI) in predicting either 28-day or 90-day prognosis of patients with cirrhosis. The hazard analysis identified largely overlapping risk factors of poor outcomes in both subgroups, while serum bilirubin was specifically associated with short-term mortality in patients with cirrhosis and blood urea nitrogen in patients without cirrhosis. A subgroup analysis in patients with cirrhosis showed a decline of discrimination of CPMS in those with ascites or infections compared to that in those without.ConclusionPredicting the short-term outcome of HBV-ACLF by CPMs is optimal in patients without cirrhosis but limited in those with cirrhosis, at least partially due to the complicated ascites or infections.</p