Macrophage Immunoregulatory Galectin-9 Production is increased by Contact with Hepatitis C Virus-infected Hepatocytes and Differentiation
Noah M. Harwood, Lucy Golden-Mason, Hugo R. Rosen, John A. Mengshol
Gastroenterology/Hepatology' Universify of Colorado SOM, Denver VA Medical Center, Denver, CO
Hepatitis C Virus (HCV) infects 3. 2 million people in the United States and leads to cirrhosis in 20% over 20 years. Although therapy has dramatically improved, difficult to treat populations remain. The immunoregulatory protein Galectin-9 (Gal-9) may be partially responsible for the development and maintenance of persistent infection. In HCV patients, Gal-9 is elevated in the sera and liver, and localizes to Kupffer cells. Gal-9 induces apoptosis of HCV antigen-specific T cells and increases inhibitory regulatory T-cells via the receptor Tim-3 (PLoS ONE 2010 5(3): e9504). Our current study focuses on elucidating the signals that increase Gal-9 in Kupffer cells. Methods: しsing quantitative real-time PCR, we analyzed Gal-9 mRNA in co-cultures of the Huh7. 5 cell line infected with JFH-1 HCV and either the THP-1 monocyte cell line or human monocytes from healthy donors. Additionally, we examined Gal-9 levels upon phorbol 12-myristate 13-acetate (PMA)-induced maturation of THP-1 cells to macrophages, and in human monocytes matured to proinflammatory macrophages (M1) with GM-CSF, or alternative (M2) with M-CSF. Flow cytometry confirmed protein expression. Results: THP-1 cells upregulate Gal-9 three to five-fold (p<0.001) when exposed to HCV-infected Huh7. 5s. Induction is likely dependent on contact or proximity, as Gal-9 mRNA levels remain constant when THP-1s and infected Huh7. 5s are cultured on opposite sides of a permeable membrane. Furthermore, medium from infected hepatocytes fails to increase Gal-9 in THP-1s. Gal-9 induction by infected Huh7. 5s is interferon-y independent since a neutralizing antibody has no effect on Gal-9 levels. Gal-9 mRNA is elevated up to 20-fold in co-cultures of infected Huh7. 5s and human monocytes after four days (P=0.02), and increases further with time. To test whether Gal9 levels change with differentiation, we induced macrophage maturation in THP-1 cells with PMA. Basal Gal-9 increases fourfold in mature THP-1 macrophages compared to THP-1 monocytes, with a further Gal-9 increase in THP-1 macrophages exposed to HCV-infected hepatocytes or IFN-y. Human monocytes differentiated into M1s produced 22-fold more Gal-9 than those differentiated into M2s (p<0.02). Conclusions: Gal-9 is produced by monocytes after exposure to HCV-infected Huh7. 5s, and is further heightened during monocyte to macrophage differentiation. HCV-infected cells directly stimulate Gal-9, possibly via contact and uptake by monocytes or macrophages. We are currently investigating whether this occurs via endosomal toll-like receptors. Therapies targeting Gal-9 might provide an immune boost to help eradicate virus in HCV patients.
The following people have nothing to disclose: Noah M. Harwood, Lucy GoldenMason, Hugo R. Rosen, John A. Mengshol