A crucial role for Kupffer cell-derived galectin-9 in regulation of T cell immunity in hepatitis C infection

Abstract

Approximately 200 million people throughout the world are infected with hepatitis C virus (HCV). One of the most striking features of HCV infection is its high propensity to establish persistence (approximately 70-80%) and progressive liver injury. Galectins are evolutionarily conserved glycan-binding proteins with diverse roles in innate and adaptive immune responses. Here, we demonstrate that galectin-9, the natural ligand for the T cell immunoglobulin domain and mucin domain protein 3 (Tim-3), circulates at very high levels in the serum and its hepatic expression (particularly on Kupffer cells) is significantly increased in patients with chronic HCV as compared to normal controls. Galectin-9 production from monocytes and macrophages is induced by IFN-gamma, which has been shown to be elevated in chronic HCV infection. In turn, galectin-9 induces pro-inflammatory cytokines in liver-derived and peripheral mononuclear cells; galectin-9 also induces anti-inflammatory cytokines from peripheral but not hepatic mononuclear cells. Galectin-9 results in expansion of CD4(+)CD25(+)FoxP3(+)CD127(low) regulatory T cells, contraction of CD4(+) effector T cells, and apoptosis of HCV-specific CTLs. In conclusion, galectin-9 production by Kupffer cells links the innate and adaptive immune response, providing a potential novel immunotherapeutic target in this common viral infection.

Figure 1. Galectin-9 is elevated in the plasma of patients with chronic HCV and higher levels are seen in hepatocellular carcinoma (HCC).

Galectin-9 levels were analyzed in the plasma of 10 normal controls, 22 patients with HCV and 11 patients with non-viral causes of liver disease using a sandwich ELISA . Seven patients with HCV and HCC are denoted by open squares; they had significantly higher galectin-9 levels compared to HCV patients without HCC, p = 0.0334. Plasma from 11 patients with non-viral liver disease was analyzed. Three patients had alcoholic liver disease, 3 patients had Primary Biliary Cirrhosis, 3 patients had autoimmune hepatitis and 2 patients had non-alcoholic steatohepatitis. P- values were calculated using the two tailed Mann-Whitney test. NS denotes non-significant, p values>0.05.

Figure 2. HCV patients have increased Galectin-9 in Kupffer cells and periportal areas.

Immunohistochemistry of paraffin embedded samples (see Methods for details) was used to analyze 39 samples from HCV-infected patients and 15 normal subjects for galectin-9 staining. Staining for CD68 was used to identify Kupffer cells. The analyzing pathologist (M.S.) was blinded to patient identity and medical history. (A) Negative control with secondary antibody only, 20× magnification. (B) Galectin-9 staining in normal liver (brown) 20×; (C) Galectin-9 staining in a HCV patient 20×, (D) Periportal galectin-9 staining in HCV patient, 200×. (E) Co-localization of galectin-9 (brown) and CD68 (red) 600×. (F–H) Intensity of galectin-9 was scored from 0 to 3, and frequency was scored 0 (<10%), 1 (10–40%), 2 (40–70%), 3 (>70%). Distribution of scores by category expressed as a percent of the total patients comparing normal patients to patients with HCV. P-values calculated by the Chi-square test for trend.

Figure 3. Interferon gamma increases galectin-9 levels in macrophages.

CD14 positive cells from five normal controls and five HCV patients were bead selected from PBMCs and cultured for 48 hours with 25 ng/ml of MCSF to differentiate them into macrophages. Cells were stimulated for 48 hours with the stimuli shown at 2×10⁶/ml. IFN-γ was used at 25 ng/ml, HCV core and β-gal were used at 10 ug/ml, LPS was used at 100 ng/ml, and IL-1β was used at 10 ng/ml. Cell lysates were prepared and analyzed by Western Blot. (A) Western blot from one representative normal patient and one representative HCV patient to galectin-9 and β-Actin. (B) Fold induction was determined by densitometry using β-Actin as a loading control (n = 4 normal, and 3 HCV-positive patients).

Figure 4. Galectin-9 induces cytokine secretion from liver-derived and peripheral mononuclear cells.

Hepatic (n = 8) or peripheral (n = 8) mononuclear cells (2×10⁶/ml) were cultured in 96-well plates (200 ul/well) for 48 hours in media alone (RPMI+10% human serum) ± galectin-9 (5 ug/ml). After the culture period supernatants were collected and cytokine levels were measured using cytokine multiplex (Luminex™) technology as described in materials and methods. P-values were calculated using the Wilcoxan matched-pairs signed rank test.

Figure 5. Galectin-9 induces regulatory T cells (T_regs) via TGF-β.

Peripheral mononuclear cells (2×10⁶/ml) isolated from 5 chronic HCV patients and 6 normal controls were cultured for 5 days in media alone (RPMI+10% human serum) ± galectin-9 (5 ug/ml). After the culture period Treg levels were estimated using flow cytometry. Tregs were defined as CD4⁺CD25⁺FoxP3⁺CD127⁻ T cells. Culture in the presence of galectin-9 induced Tregs (A) and a concomitant reduction in the CD4⁺CD25⁻FoxP3⁻ effector population was observed (B). (C) CD14⁺ monocytes were isolated from 3 chronic HCV patients and cultured in for 48 hours in media alone (RPMI+10% human serum) ± Galectin-9 (5 ug/ml). Galectin-9 increased the relative expression of TGF-β1 mRNA as analyzed by real time PCR. (D) Depletion of CD14⁺ cells from PBMC (8 normal patients) cultures treated with Galectin-9 attenuated the induction of Tregs at 5 days. (E) The addition of anti-TGF-β antibody at day 0 and day 2 of culture with PBMC and galectin-9 blocked induction of the Treg population. Representative flow plots are shown; the bar graphs are derived from three separate experiments. (F) THP-1 monocyte cell line constitutively expresses Galectin-9 which is upregulated by IFN-γ (25 ng/ml for 48 hours). Both the western blot and the densitometry analysis are shown, samples analyzed in triplicate. (G) Galectin-9 treatment induces TGF-β production from THP-1 cells. THP-1 cells were cultured with galectin-9 at 2.5 ug/ml for 24 hours then incubated with the latency associated peptide (LAP) then anti-LAP-PE to detect TGF-β. (H) IFN-γ treated THP-1 cells induce Treg from CD4⁺ T cells. Cells were cultured at a 1∶1 ratio for 5 days. Tregs were defined as CD4⁺CD25⁺FoxP3⁺CD127⁻ T cells and graphed as a % of total CD4⁺ cells. P-values were calculated using the Wilcoxan matched-pairs signed rank test.

Figure 6. Galectin-9 induces apoptosis of HCV-specific CTLs through caspase-8 activation.

CD8⁺ T cells clones specific for NS3∶1406, NS3∶1436 and NS5∶2594 were incubated for 6 hours in media alone (A) or with 5 ug/ml of galectin-9 (B) followed by staining with Annexin V and 7-AAD. Representative histograms showing the percent of HCV-specific T cells staining with Annexin V following the indicated treatment. (C) Combined data for 5 CTL clones from different patients demonstrated an increase in Annexin V/7-AAD with galectin-9 treatment. P-values were calculated using the Wilcoxan matched-pairs signed rank test. (D) Plot showing NS3∶1436-specific CD8⁺ T cells by pentamer staining ex vivo. (E) Annexin V staining of NS3∶1436- specific CD8⁺ T cells from two HCV patients cultured for 6 hours with media (grey shading) or galectin-9 (solid line, no shading). (F). Galectin-9 induces T cell apoptosis through caspase-8 activation. Shown are PBMC from a HCV-positive patient treated for 1 hour with 5 ug/ml galectin-9 or media alone. A FITC-conjugated caspase-8 inhibitor that binds specifically to activated caspase-8 was added for the last hour of culture. Cells were then stained with anti-CD8 and 1436-pentamer and the percentage of HCV-specific T cells with activated caspase-8 was determined by flow cytometric analysis.

Figure 7. Paradigm for central role of galectin-9 in regulation of hepatitis C immunity.

Liver sinusoids are lined by a fenestrated layer of sinusoidal endothelial cells. The unique architecture of the liver allows interaction of antigen-presenting cells with T cells. a) Chronic HCV infection is characterized by hepatic infiltration of Th1, Tc1, NK and NKT cells that secrete IFN-γ which stimulates KCs to produce galectin-9. b) Galectin-9 (large brown arrows) expands CD4⁺CD25⁺FoxP3⁺CD127^low regulatory T cells via TGF-β dependent mechanisms. c) HCV-specific CTLs expressing high levels of the Tim-3 receptor are engaged and become apoptotic. d) Galectin-9 also induces production of pro-inflammatory cytokines (TNF-α, IL-1β, IFN-γ) and pro-fibrotic cytokines (TGF-β) that can act on hepatocytes and hepatic stellate cells (HSC). The space of Disse contains the HSC. Hepatocytes (HC), liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC), Th1 (CD4⁺ T cells), Tc1 (CD8⁺ T cells), natural killer (NK) and natural killer T (NKT) cells, hepatic stellate cells (HSC). Inset, the strategic location of KCs and the slow blood flow through the sinusoids allows contact with infiltrating lymphocytes (structural relationship of hepatic cells adapted from [18], [45]).