Response of Human Liver Tissue to Innate Immune Stimuli

Abstract

Precision-cut human liver slice cultures (PCLS) have become an important alternative immunological platform in preclinical testing. To further evaluate the capacity of PCLS, we investigated the innate immune response to TLR3 agonist (poly-I:C) and TLR4 agonist (LPS) using normal and diseased liver tissue. Pathological liver tissue was obtained from patients with active chronic HCV infection, and patients with former chronic HCV infection cured by recent Direct-Acting Antiviral (DAA) drug therapy. We found that hepatic innate immunity in response to TLR3 and TLR4 agonists was not suppressed but enhanced in the HCV-infected tissue, compared with the healthy controls. Furthermore, despite recent HCV elimination, DAA-cured liver tissue manifested ongoing abnormalities in liver immunity: sustained abnormal immune gene expression in DAA-cured samples was identified in direct ex vivo measurements and in TLR3 and TLR4 stimulation assays. Genes that were up-regulated in chronic HCV-infected liver tissue were mostly characteristic of the non-parenchymal cell compartment. These results demonstrated the utility of PCLS in studying both liver pathology and innate immunity.

Keywords: direct-acting antiviral treatment; fibrosis; hepatitis C virus; inflammation; innate immunity; liver slice culture.

Figure 1

Innate immune response of human liver slices to poly-I:C or LPS treatment. Non-HCV-infected specimens are shown. IFN-β and IFN-λ expression peaked at 2-4 h, whereas interferon-stimulated gene expression for IFIT1, CXCL10 and MX1 peaked round 4-12 h. Each marker represents a patient time-point summary which consist of 2-3 biological replicates of liver slices. Relative abundance at the log2 scale is shown, with the median, 25% and 75% quantile values indicated with violin plots.

Figure 2

HCV and fibrosis analysis with human liver slices. (A) All eight chronic HCV samples (blue color) were confirmed with positive HCV RNA detection. None of the DAA cured subjects (red color) or the non-infected controls (black color) was positive for HCV RNA. The limit of reliable detection (LoD) was 1.2 IU/50 ng liver total RNA. (B) HCV RNA remained robustly detected in the day 7 liver slices cultured from chronically HCV-infected patients. The viral load between day 0 and day 7 liver slices was not statistically significant (P = 0.4688, Wilcoxon matched-pairs signed rank test). (C) Fibrosis analysis of the day 0 ex vivo liver specimens with trichrome staining and picrosirius red staining indicated that in DAA-treated, and now HCV-negative patients in this study there was persistent fibrosis, similar to untreated HCV and different from tissue without a history of HCV infection. The scoring system was based on the Scheuer/Batts-Ludwig method. The fibrosis score included 10 non-infected patients, 4 chronic HCV patients, and 8 DAA-treated patients. In other words, 4 out of the 7 HCV+ subjects, and 8 out of the 10 DAA-cured subjects were analyzed. Due to tissue availability, not all subjects could be analyzed in this way. The minimum and maximum data points for each subgroup are shown. Statistical significance was based on Mann-Whitney test. **P < 0.01. ††P < 0.01.

Figure 3

Examples of trichrome stain, picrosirius red stain, and H&E stain analysis of human liver slices. Arrow indicates the nodule formation in the cirrhosis livers. The Scheuer/Batts-Ludwig method was used for fibrosis scoring, with 0, No fibrosis; 1, portal fibrosis; 2, peri-portal fibrosis; 3, bridging fibrosis; and 4, cirrhosis. The DAA-4 liver tissue was collected 12 months after the completion of 24-week DAA therapy. DAA-9 liver tissue was collected 20 months after the completion of 12-week DAA therapy.

Figure 4

Immune gene changes at the day 7 baseline in chronic HCV-infected and DAA-cured livers. (A) Hierarchical clustering of immune genes with statistically significant elevation in HCV or DAA-cured subjects at Day 0 or Day 7 time points. The group-level fold changes are shown, which were calculated with the mean Ct value of each gene within each patient group. Decreased fold changes are colored in green, and increased fold changes are colored in red. (B) The up-regulated genes in HCV and DAA-cured at day 7 time points. Genes were mapped to liver cell types according to the cell type analysis with day 0 non-infected livers. Genes are grouped by expression patterns in cell compartments and in specimen type. The overlapped genes up-regulated at both day 0 and day 7 time points are bolded and underscored.

Figure 5

Delta-delta Ct analysis showing up-regulated genes post- poly-I:C or LPS stimulation in chronic HCV-infected and DAA-cured liver slices. The relative gene abundance was normalized to to the arithmetic mean of Ct values of ACTB, GAPDH and HPRT1. The relative abundance was further normalized to the day 7 time zero measurements. Statistical significance was based on two-tailed Mann-Whitney test. *, statistical significantly different between non-infected versus chronic HCV liver slices. †, statistical significantly different between non-infected versus DAA-cured liver slices. ‡, statistical significantly different between chronic HCV versus DAA-cured liver slices. Levels of statistical significance are indicated thus: *P < 0.05 **P < 0.01. The mean and standard deviation within each group are plotted.

Figure 6

Altered TLR3 and TLR4 response in chronic HCV-infected liver slices not restored in DAA-cured liver slices. (A) Hierarchical clustering of immune responsive genes with 2-fold or greater induction during LPS stimulation. The statistical significance P value with ΔCt method is shown. Gene abundance of individual liver slices was normalized to the arithmetic mean of Ct values of ACTB, GAPDH and HPRT. Two-tailed Mann-Whitney test was used. Greater significance P value is colored with darker red. (B) Genes up-regulated with P < 0.05 at one time point in either poly-I:C or LPS treatment were included as up-regulated genes. Genes boxes were colored with the enriched expression in cell compartment of day 0 information. Using the ΔΔCt method, genes that increased in expression during TLR3/TLR4 response are highlighted with a black bordering; genes shown to increase based on both ΔΔCt and ΔCt methods are highlighted with black bordering and black solid circles. Genes names are colored by in HCV- or DAA- samples or both. These genes were further grouped by whether they were differentially expressed in TLR3 or TLR4 treatment not at the baseline level, and whether poly-I: C or LPS treatment revealed differential responses.