IL-1β production through the NLRP3 inflammasome by hepatic macrophages links hepatitis C virus infection with liver inflammation and disease

Abstract

Chronic hepatitis C virus (HCV) infection is a leading cause of liver disease. Liver inflammation underlies infection-induced fibrosis, cirrhosis and liver cancer but the processes that promote hepatic inflammation by HCV are not defined. We provide a systems biology analysis with multiple lines of evidence to indicate that interleukin-1β (IL-1β) production by intrahepatic macrophages confers liver inflammation through HCV-induced inflammasome signaling. Chronic hepatitis C patients exhibited elevated levels of serum IL-1β compared to healthy controls. Immunohistochemical analysis of healthy control and chronic hepatitis C liver sections revealed that Kupffer cells, resident hepatic macrophages, are the primary cellular source of hepatic IL-1β during HCV infection. Accordingly, we found that both blood monocyte-derived primary human macrophages, and Kupffer cells recovered from normal donor liver, produce IL-1β after HCV exposure. Using the THP-1 macrophage cell-culture model, we found that HCV drives a rapid but transient caspase-1 activation to stimulate IL-1β secretion. HCV can enter macrophages through non-CD81 mediated phagocytic uptake that is independent of productive infection. Viral RNA triggers MyD88-mediated TLR7 signaling to induce IL-1β mRNA expression. HCV uptake concomitantly induces a potassium efflux that activates the NLRP3 inflammasome for IL-1β processing and secretion. RNA sequencing analysis comparing THP1 cells and chronic hepatitis C patient liver demonstrates that viral engagement of the NLRP3 inflammasome stimulates IL-1β production to drive proinflammatory cytokine, chemokine, and immune-regulatory gene expression networks linked with HCV disease severity. These studies identify intrahepatic IL-1β production as a central feature of liver inflammation during HCV infection. Thus, strategies to suppress NLRP3 or IL-1β activity could offer therapeutic actions to reduce hepatic inflammation and mitigate disease.

Figure 1. IL-1β associates with hepatic disease and is produced by liver macrophages in chronic hepatitis C patients.

(A) Hierarchical clustering of differentially expressed genes as determined by RNA-seq analysis of liver specimens from control or HCV patients with mild (no fibrosis) and severe (cirrhosis) liver disease. Clustering analysis of a total of 158 differentially expressed genes (>1.5-fold change and FDR, 0.05) in the cytokine-cytokine receptor and chemokine signaling pathways is shown. The expression of group-3 genes were increased only in patients with severe liver disease; Group-3 genes and the expression key are shown at the right (for full description, see Table S1). For analysis see methods. (B) IL-1β levels from sera of chronic hepatitis C patients and healthy controls. (C) Immunohistochemical staining and confocal microscopy analysis of healthy liver and chronic hepatitis C patient liver samples. CD68 marks macrophages (Kupffer cells or infiltrating macrophages) (red), IL-1β (green), and DRAQ5 (blue) stains the nuclei. A quantification plot of CD68+IL-1β+ cells and CD68+IL-1β - of the total IL-1β+ cells is depicted from chronically infected (three patients,) and normal healthy liver samples. The area within the white box of the far right merged panel is enlarged and shown with cell frequency counts at right. **P = 0.0062 and ***p<0.0001 by student's t-test. Arrows (white) indicate hepatocytes adjacent to CD68+/IL-1β+ Kupffer cells (yellow arrows).

Figure 2. HCV stimulates IL-1β production upon uptake by macrophages.

(A) IL-1β mRNA expression (upper panel) and secreted IL-1β protein levels (lower panel) from primary monocyte-derived macrophages of healthy human donor blood. Cells were mock-treated or treated with infectious HCV supernatant (moi = 0.01 based on Huh7 focus forming units (ffu) or treated with 1 µg/ml of LPS and ATP 5 mM (LPS/ATP; positive control). (B) Intracellular cytokine staining of treated CD14+ cells recovered from saline washout of healthy donor liver. Cells were left untreated (unstim) or were cultured with conditioned media (mock, negative control), LPS (positive control) or treated with UV-inactivated HCV (moi = 0.01 based on Huh7 focus forming units (ffu)). Data are shown from one donor and are representative two experiments each of cells collected from two independent donors. In the analysis shown the frequency of IL-1β-expressing cells was: unstim, 2.7%; mock, 6.4%; LPS, 76.5%; UV-HCV. 67.6%. (C)–(I) Analysis of THP1 cells. (C) IL-1β mRNA expression post exposure to HCV. (D) IL-1β protein secretion after treatment with variable doses of HCV (moi = 0.001, 0.01 or 0.1 Huh7 ffu) or LPS/ATP at 1 µg/ml for 24 hr. (E) Immunoblot showing the kinetics of caspase-1 activation after HCV exposure. (F) Levels of secreted IL-1β over a time course after HCV exposure (moi = 0.01 based on Huh7 ffu). (G) IL-1β levels secreted 24 hr after exposure to (left to right) cell culture media, sucrose solution, sucrose-purified culture media, infectious HCV supernatant or sucrose-purified HCV virions. (H) IL-1β mRNA expression in pre-treated with DMSO (control), bafilomycin (2.5 uM) or cytochalasin D (10 µM) cells and exposed to media or infectious HCV supernatant (moi = 0.01 Huh7 ffu). (I) Levels of secreted IL-1β 24 hr post treatment with conditioned media alone (mock) or treatment with live infectious HCV (HCV, moi = 0.01) or UV-inactivated HCV (HCV-UV). *P = 0.0175 and ***P = 0.0005, by student t-test.

Figure 3. HCV stimulation of IL-1β expression occurs through MyD88-dependent viral RNA signaling by TLR7.

(A) THP1 cells stably expressing non-targeting shRNA (control) or shRNA specific to MAVS or MyD88 were mock treated with media alone, exposed to HCV (moi = 0.01) or treated with LPS (1 µg/ml) and IL-1β expression assessed by qRT-PCR, *P = 0.0341and *P = 0.013. (B) Immunoblot analysis of IL-1β in THP-1 stably expressing the indicated shRNA. Cells were exposed to media alone (M) or infectious HCV supernatant for 6 hr. (C) Immunoblot of caspase-1 in THP-1 stably expressing the indicated shRNA. (D) IL-1β (upper panel) and IFN-β (lower panel) mRNA expression in THP-1 post-treatment with transfection reagent alone (control) or transfected with full length HCV RNA or poly IC (transfected); or treated with media containing full length HCV RNA or poly IC (extracellular). (E) IL-1β mRNA levels in THP-1 transfected with HCV polyU/UC RNA (1 µg/ml), HCV X-region (1 µg/ml) or exposed to infectious HCV for 6 hrs. (F) IL-1β (upper panel) and IFN-β (lower panel) mRNA expression in THP1 harboring the indicated shRNA, *P = 0. 0115, **P = 0.0094 and ***P = 0.0009. (G) IL-1β (upper panel) and IFN-β (lower panel) mRNA expression in THP-1 cells transfected with increasing doses (0.125, 0.25, 0.5, 1 and 2 µg/ml) of HCV polyU/UC RNA. (H) IL-1β mRNA expression in THP1 cells stably expressing non-targeting or shRNA specific to TLR7, *P = 0.022, by student's t-test.

Figure 4. HCV triggers the NLRP3 inflammasome and IL-1β maturation through induction of potassium efflux after macrophage uptake.

(A) Secreted IL-β protein levels (upper panel) and immunoblot analysis of IL-1β (lower panel set) of THP-1 treated with transfection reagent or transfected with either with full length HCV RNA or polyU/UC RNA or exposed to HCV (moi = 0.01). (B) IL-β mRNA expression in THP1 stably expressing non-targeting control shRNA or shRNA specific to NLRP3 or caspase-1. (C) Immunoblot of caspase-1 and IL-1β in THP1 stably expressing non-targeting control the indicated shRNA. (D) THP-1 were pre-treated with DMSO (control) or with 6.25, 12.5, 25, 50, 100, 200 µM of potassium channel inhibitor glybenclamide (Glyben) for 2 hrs followed by mock treatment (M; control) or HCV (moi = 0.01) exposure in the presence of glyben for an additional 1 hr. (E) IL-1β p17 abundance in THP-1 cultured in normal media or in media containing NaCl (100 mM) or KCl (100 mM) for 1 hr followed by mock-treatment (-) or exposure to HCV (moi = 0.01) in the same media for an additional 1 hr.

Figure 5. Macrophage exposure to HCV induces inflammasome signaling of IL-1β and proinflammatory cytokine and chemokine expression.

(A and B) RNA-seq analysis to directly compare the transcription profile of THP1 cells after acute HCV (moi = 0.01) exposure for 6 and 16 hours and chronic hepatitis C patient liver staged by mild or severe disease. (A) The Venn diagram shows the number of differentially expressed genes (>1.5-fold change and FDR, 0.05) that were unique and common to THP-1 cells and chronic hepatitis C patient liver in the most highly represented KEGG pathways in both datasets, the cytokine-cytokine receptor signaling, and chemokine signaling pathways. (B) Hierarchical clustering analysis of differentially expressed genes common to both HCV-exposed THP-1 cells and chronic hepatitis C liver. Group-4 genes were expressed in both THP-1 cells and chronic hepatitis C liver (for full description, see Table S2). Group-4 genes and the expression key are shown at the right. See Methods for a description of bioinformatics analysis. (C) Model of hepatic inflammatory signaling during HCV infection. Sensing of HCV by hepatic macrophages triggers the induction of IL-1β and the inflammatory response. HCV-induced inflammasome activation is initiated by endosomal TLR7 engagement of viral RNA to drive the induction of IL-1β expression via MyD88. In addition, HCV triggers potassium efflux for NLRP3 activation to produce mature IL-1β. Secreted IL-1β induces the expression of a wide-range of proinflammatory mediators and stimulates an inflammatory response that confers liver disease during chronic infection.