Tim-3 Negatively Regulates IL-12 Expression by Monocytes in HCV Infection

Abstract

T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is a newly identified negative immunomodulator that is up-regulated on dysfunctional T cells during viral infections. The expression and function of Tim-3 on human innate immune responses during HCV infection, however, remains poorly characterized. In this study, we report that Tim-3 is constitutively expressed on human resting CD14+ monocyte/macrophages (M/MØ) and functions as a cap to block IL-12, a key pro-inflammatory cytokine linking innate and adaptive immune responses. Tim-3 expression is significantly reduced and IL-12 expression increased upon stimulation with Toll-like receptor 4 (TLR4) ligand - lipopolysaccharide (LPS) and TLR7/8 ligand - R848. Notably, Tim-3 is over-expressed on un-stimulated as well as TLR-stimulated M/MØ, which is inversely associated with the diminished IL-12 expression in chronically HCV-infected individuals when compared to healthy subjects. Up-regulation of Tim-3 and inhibition of IL-12 are also observed in M/MØ incubated with HCV-expressing hepatocytes, as well as in primary M/MØ or monocytic THP-1 cells incubated with HCV core protein, an effect that mimics the function of complement C1q and is reversible by blocking the HCV core/gC1qR interaction. Importantly, blockade of Tim-3 signaling significantly rescues HCV-mediated inhibition of IL-12, which is primarily expressed by Tim-3 negative M/MØ. Tim-3 blockade reduces HCV core-mediated expression of the negative immunoregulators PD-1 and SOCS-1 and increases STAT-1 phosphorylation. Conversely, blocking PD-1 or silencing SOCS-1 gene expression also decreases Tim-3 expression and enhances IL-12 secretion and STAT-1 phosphorylation. These findings suggest that Tim-3 plays a crucial role in negative regulation of innate immune responses, through crosstalk with PD-1 and SOCS-1 and limiting STAT-1 phosphorylation, and may be a novel target for immunotherapy to HCV infection.

Role of Tim-3 in hepatitis B virus infection: An overview

“…Concordantly, expression is increased on monocytes from patients with HCV infection and is positively correlated with IL-17 levels in CD4 + T cells, thus promoting Th17 cell accumulation. However, blocking Tim-3 on monocytes restores the balance of IL-12, IL-23 and IL-17 signaling via the STAT3 pathway [50,51] .…”

KLRG1 Negatively Regulates Natural Killer Cell Functions through the Akt Pathway in Individuals with Chronic Hepatitis C Virus Infection

“…Flow cytometry. Procedures for detection of cell surface markers and intracellular cytokine staining were performed essentially as described previously (42,43). Briefly, PBMCs (0.2 ϫ 10 6 per well in a 96-well plate) were stimulated with 10 ng/ml recombinant human interleukin-12 (rhIL-12; eBioscience, San Diego, CA) for 18 h, followed by 1 g/ml Brefeldin A (BioLegend, San Diego, CA) 4 h prior to harvesting the cells, thus forbidding cytokine secretion.…”

“…Transfection of Huh-7 hepatocytes (kindly provided by T. J. Liang, Liver Section, NIH NIDDK) with HCV JFH-1 strain (kindly provided by T. Wakita) was carried out as described previously (42,43). Prior to the coculture experiment, HCV-transfected or untransfected Huh-7 hepatocytes were serum starved for 18 h, then activated with rhIFN-␥ (0.1 g/ml; R&D Systems) for 48 h. Activated hepatocytes were removed from plates with 0.05% trypsin-EDTA and then plated at 5 ϫ 10 5 cells/well in a 12-well plate.…”

“…Additionally, the driving force for KLRG1 upregulation during HCV infection remains to be determined. To further elucidate the role of HCV in regulation of KLRG1 expression, we employed a newly established cell culture system by transfecting Huh-7 hepatocytes with the HCV JFH-1 strain in vitro to mimic the in vivo setting of early HCV infection (42,43). To this end, PBMCs or purified NKs from HS were cocultured with HCV-transfected or untransfected Huh-7 hepatocytes for 48 h, followed by flow cytometric analysis for KLRG1 expression and NK functions.…”

The Role of Monocytes/Macrophages in HBV and HCV Infection

“…The possible mechanism is that the STAT-1 phosphorylation is enhanced during the treatment [52]. Tim-3, acting as a negative regulator, inhibits monocytes'/macrophages' function in HCV infection in some researches [53]. Myeloid-derived suppressor cell (MDSC) is a kind of inhibitory cell that originated in the bone marrow and was irst identiied as natural suppressor cell in tumour-bearing mice in the mid-1960s [54].…”