Rapid reversal of innate immune dysregulation in blood of patients and livers of humanized mice with HCV following DAA therapy

<div><p>Chronic hepatitis C virus (HCV) infection results in sustained immune activation in both the periphery and hepatic tissue. HCV infection induces innate immune signaling that is responsible for recognition of dsRNA, leading to activation of transcription factors and production of Type I and III IFNs, as well as pro-inflammatory cytokines and chemokines. Continued activation of host-immune mediated inflammation is thought to contribute to pathologic changes that result in progressive hepatic fibrosis. The current standard treatment for chronic HCV infection is directly-acting antivirals (DAAs), which have provided the unique opportunity to determine whether successful, rapid treatment-induced eradication of viral RNA normalizes the dysregulated antiviral innate immune response in patients chronically infected with HCV.</p><p>Results</p><p>First, in patients receiving two different combinations of DAAs, we found that DAAs induced not only rapid viral clearance, but also a re-setting of antiviral immune responses in the peripheral blood. Specifically, we see a rapid decline in the expression of genes associated with chronic IFN stimulation (IFIT3, USP18, IFIT1) as well as a rapid decline in genes associated with inflammation (IL1β, CXCL10, CXCL11) in the peripheral blood that precedes the complete removal of virus from the blood. Interestingly, this rapid reversal of innate immune activation was not seen in patients who successfully clear chronic HCV infection using IFN-based therapy. Next, using a novel humanized mouse model <i>(Fah</i>^<i>-/-</i><i>RAG2</i>^<i>-/-</i><i>IL2rg</i>^<i>null</i>—FRG), we assessed the changes that occur in the hepatic tissue following DAA treatment. DAA-mediated rapid HCV clearance resulted in blunting of the expression of proinflammatory responses while functionally restoring the RIG-I/MAVS axis in the liver of humanized mice.</p><p>Conclusions</p><p>Collectively, our data demonstrate that the rapid viral clearance following treatment with DAAs results in the rebalancing of innate antiviral response in both the peripheral blood and the liver as well as enhanced antiviral signaling within previously infected hepatocytes.</p></div

Differential transcriptional changes are associated with IFN-free DAA therapy and IFN/Ribavirin therapy.

<p><b>(A)</b> Representation of the shared and different transcriptional changes in DAA therapy (EOT) compared to IFNα/Ribavirin therapy (wk 10) (19). The top 1000 genes statistically significantly changed from pretreatment in both data sets were compared to the expression of the given gene in the other treatment cohort. <b>(B)</b> List of the 32 genes that were statistically changed from post treatment to pretreatment in both data sets. <b>(C)</b> Quantitative RT-PCR comparing fold change from pretreatment at twelve weeks following DAA therapy (Black Bars) and twenty-four weeks following IFNα/Ribavirin therapy (White bars). P value represents comparison between both treatments. P<0.05, ***P<0.001, NS = Not significant. N = 8-11patients per group.</p

DAA therapy results in a reduction in innate immune activation in the peripheral blood.

<p><b>(A)</b> Graphical representation of the measured or predicted activation state (IPA) of innate immune signaling molecules at the end of DAA therapy compared to pretreatment. Green is inhibited whereas red represents increased expression. <b>(B)</b> Semi-quantitative RT-PCR of select genes associated with HCV-induced inflammation from PBMCs treated with two different regimens of DAA at end of treatment compared to pretreatment in DAA cohort 1 (n = 18, top) or DAA cohort 2 (n = 11, bottom). <b>(C)</b> Western blot analysis of the levels of pS-NFκB, and NFκB. Top: representative images of 6 patient samples prior to (Pre) and twelve weeks following completion of DAA therapy (Post) in DAA cohort 2. <b>(D)</b> Quantification of the ratio of pS-NFκB to NFκB (right) prior to treatment (circles) and at twelve weeks following the end of treatment (squares). Lines represent individual patient samples. *P = 0.03, Wilcoxon matched-pairs signed rank test. N = 11 patients.</p

DAA therapy induces rapid suppression of some but not all antiviral signaling molecules.

<p>Kinetic analysis of transcriptional levels of CXCL10 (black), CXCL11 (purple), RIG-I (yellow), IRF3 (blue), IFIT1(ISG56) (red), IFITM1 (grey), USP18 (green), IL1β (black circles, dotted line) and plasma viral load (black crosses with black dashed line) from PBMCs in DAA patient cohort 2 (n = 11).</p

Top transcriptional changes in the peripheral blood of patients receiving DAA therapy.

<p>Fold changes and p values are a paired comparison between end of treatment and pretreatment.</p

Restoration of HCV-suppressed antiviral signaling in the liver of humanized mice treated with DAAs.

<p><b>(A)</b> Serum levels of HCV in mice injected with 1.4 x 10⁶ IU HCV intravenously. <b>(B)</b> HCV viral loads after treatment with Sofosbuvir (2.0mg daily), Daclatasvir (0.3mg daily), Asunaprevir (0.5mg twice daily) or DMSO vehicle control via oral gavage. <b>(C)</b> Protein expression of NS3, MAVS, IFITM1, and IRF3 following 14 days of DMSO treatment or DAA treatment in whole liver tissue via immunofluorescence staining. N = 3 mice per group. ****P<0.0001, NS = Not significant.</p

Predicted changes in activation status of upstream regulators (IPA) in the peripheral blood of patients receiving DAA therapy.

<p>Predicted changes in activation status of upstream regulators (IPA) in the peripheral blood of patients receiving DAA therapy.</p