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Tim-3 negatively regulates IL-12 expression by monocytes in HCV infection.

Zhang Y, Ma CJ, Wang JM, Ji XJ, Wu XY, Jia ZS, Moorman JP, Yao ZQ - PLoS ONE (2011)

Bottom Line: 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/M(Ø), which is inversely associated with the diminished IL-12 expression in chronically HCV-infected individuals when compared to healthy subjects.Tim-3 blockade reduces HCV core-mediated expression of the negative immunoregulators PD-1 and SOCS-1 and increases STAT-1 phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Internal Medicine, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee, United States of America.

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/M(Ø)) 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/M(Ø), 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/M(Ø) incubated with HCV-expressing hepatocytes, as well as in primary M/M(Ø) 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/M(Ø). 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.

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Tim-3 and IL-12 expression in M/MØ is differentially regulated by hepatocytes expressing HCV.A) Immunofluoresence staining of HCV core protein in Huh-7 cells 48 h after HCV-JFH-1 (left) or mock (right) transfection. Higher magnificent imaging (×40) is inserted at upper left corner. B) Tim-3 expression on M/MØ co-cultured with HCV-transfected (red line) or mock-transfected (green line) Huh-7 cells without (left) or with (right) LPS/R848 stimulation. C) Representative Time-course of Tim-3 expression on CD14+ M/MØ co-cultured with HCV+ Huh-7 versus HCV− Huh-7 cells from two reproducible experiments. D) IL-12 expression in M/MØ co-cultured with HCV+ Huh-7 versus HCV− Huh-7 cells, shown as dot plot, histogram, and bar figure in multiple experiments.
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pone-0019664-g003: Tim-3 and IL-12 expression in M/MØ is differentially regulated by hepatocytes expressing HCV.A) Immunofluoresence staining of HCV core protein in Huh-7 cells 48 h after HCV-JFH-1 (left) or mock (right) transfection. Higher magnificent imaging (×40) is inserted at upper left corner. B) Tim-3 expression on M/MØ co-cultured with HCV-transfected (red line) or mock-transfected (green line) Huh-7 cells without (left) or with (right) LPS/R848 stimulation. C) Representative Time-course of Tim-3 expression on CD14+ M/MØ co-cultured with HCV+ Huh-7 versus HCV− Huh-7 cells from two reproducible experiments. D) IL-12 expression in M/MØ co-cultured with HCV+ Huh-7 versus HCV− Huh-7 cells, shown as dot plot, histogram, and bar figure in multiple experiments.

Mentions: The observed Tim-3 up-regulation on M/MØ during chronic HCV infection might be a result rather than a cause of IL-12 suppression. To further elucidate the role of HCV in regulation of Tim-3 and IL-12 expression and to more accurately mimic the in vivo setting of chronic HCV infection, we employed a newly established cell culture system by transfecting Huh-7 hepatocytes with the HCV-JFH-1 strain in vitro [19], [20] As shown in Fig. 3A, HCV core as well as NS5 protein (Fig.S1A) is detected in HCV-JFH-1-transfected Huh-7 hepatocytes, but not in mock-transfected controls, by immunofluorescent staining. HCV core mRNA is also detected by RT-PCR in the supernatant of the HCV-transfected Huh-7 cells but not in the culture of controls (Fig.S1B). Additionally, uninfected Huh-7 cells can be infected by the supernatant of JFH-1-transfected Huh-7 cells (Fig.S1C), suggesting that live HCV particles are secreted from the HCV mRNA-transfected hepatocytes. We then incubated purified healthy M/MØ with Huh-7 cells 48 h after HCV transfection, followed by detection of Tim-3 and IL-12 expressions in M/MØ with or without TLR stimulation for 18 h. As shown in Fig. 3B, Tim-3 expression is found to be up-regulated on purified M/MØ co-cultured with hepatocytes expressing live HCV when compared with HCV− hepatocytes, in both the un-stimulated and LPS/R848-stimulated state. As described above, Tim-3 is expressed at relatively high levels on M/MØ cultured with hepatocytes without TLR stimulation (Fig. 3B, left panel) when compared to those with LPS/R848 stimulation for 18 h (Fig. 3B, right panel); however, in either scenario, Tim-3 is up-regulated by HCV exposure. To determine the dynamics of Tim-3 up-regulation by HCV, we kinetically examined Tim-3 expression on M/MØ at various time-points after co-culture with HCV+ Huh-7 versus HCV− Huh-7 without TLR stimulation. As shown in Fig. 3C, at all time-points examined, the expression of Tim-3 on CD14+ M/MØ incubated with HCV-expressing Huh-7 cells is remarkably higher than those co-cultured with HCV− hepatocytes. We also examined IL-12 expression in M/MØ incubated with HCV+ hepatocytes or HCV− hepatocytes for 18 h, as shown in a Fig. 3D dot plot, histogram, and bar figure derived from multiple repeated experiments, HCV+ Huh-7 significantly inhibits IL-12 production by CD14+ M/MØ.


Tim-3 negatively regulates IL-12 expression by monocytes in HCV infection.

Zhang Y, Ma CJ, Wang JM, Ji XJ, Wu XY, Jia ZS, Moorman JP, Yao ZQ - PLoS ONE (2011)

Tim-3 and IL-12 expression in M/MØ is differentially regulated by hepatocytes expressing HCV.A) Immunofluoresence staining of HCV core protein in Huh-7 cells 48 h after HCV-JFH-1 (left) or mock (right) transfection. Higher magnificent imaging (×40) is inserted at upper left corner. B) Tim-3 expression on M/MØ co-cultured with HCV-transfected (red line) or mock-transfected (green line) Huh-7 cells without (left) or with (right) LPS/R848 stimulation. C) Representative Time-course of Tim-3 expression on CD14+ M/MØ co-cultured with HCV+ Huh-7 versus HCV− Huh-7 cells from two reproducible experiments. D) IL-12 expression in M/MØ co-cultured with HCV+ Huh-7 versus HCV− Huh-7 cells, shown as dot plot, histogram, and bar figure in multiple experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102652&req=5

pone-0019664-g003: Tim-3 and IL-12 expression in M/MØ is differentially regulated by hepatocytes expressing HCV.A) Immunofluoresence staining of HCV core protein in Huh-7 cells 48 h after HCV-JFH-1 (left) or mock (right) transfection. Higher magnificent imaging (×40) is inserted at upper left corner. B) Tim-3 expression on M/MØ co-cultured with HCV-transfected (red line) or mock-transfected (green line) Huh-7 cells without (left) or with (right) LPS/R848 stimulation. C) Representative Time-course of Tim-3 expression on CD14+ M/MØ co-cultured with HCV+ Huh-7 versus HCV− Huh-7 cells from two reproducible experiments. D) IL-12 expression in M/MØ co-cultured with HCV+ Huh-7 versus HCV− Huh-7 cells, shown as dot plot, histogram, and bar figure in multiple experiments.
Mentions: The observed Tim-3 up-regulation on M/MØ during chronic HCV infection might be a result rather than a cause of IL-12 suppression. To further elucidate the role of HCV in regulation of Tim-3 and IL-12 expression and to more accurately mimic the in vivo setting of chronic HCV infection, we employed a newly established cell culture system by transfecting Huh-7 hepatocytes with the HCV-JFH-1 strain in vitro [19], [20] As shown in Fig. 3A, HCV core as well as NS5 protein (Fig.S1A) is detected in HCV-JFH-1-transfected Huh-7 hepatocytes, but not in mock-transfected controls, by immunofluorescent staining. HCV core mRNA is also detected by RT-PCR in the supernatant of the HCV-transfected Huh-7 cells but not in the culture of controls (Fig.S1B). Additionally, uninfected Huh-7 cells can be infected by the supernatant of JFH-1-transfected Huh-7 cells (Fig.S1C), suggesting that live HCV particles are secreted from the HCV mRNA-transfected hepatocytes. We then incubated purified healthy M/MØ with Huh-7 cells 48 h after HCV transfection, followed by detection of Tim-3 and IL-12 expressions in M/MØ with or without TLR stimulation for 18 h. As shown in Fig. 3B, Tim-3 expression is found to be up-regulated on purified M/MØ co-cultured with hepatocytes expressing live HCV when compared with HCV− hepatocytes, in both the un-stimulated and LPS/R848-stimulated state. As described above, Tim-3 is expressed at relatively high levels on M/MØ cultured with hepatocytes without TLR stimulation (Fig. 3B, left panel) when compared to those with LPS/R848 stimulation for 18 h (Fig. 3B, right panel); however, in either scenario, Tim-3 is up-regulated by HCV exposure. To determine the dynamics of Tim-3 up-regulation by HCV, we kinetically examined Tim-3 expression on M/MØ at various time-points after co-culture with HCV+ Huh-7 versus HCV− Huh-7 without TLR stimulation. As shown in Fig. 3C, at all time-points examined, the expression of Tim-3 on CD14+ M/MØ incubated with HCV-expressing Huh-7 cells is remarkably higher than those co-cultured with HCV− hepatocytes. We also examined IL-12 expression in M/MØ incubated with HCV+ hepatocytes or HCV− hepatocytes for 18 h, as shown in a Fig. 3D dot plot, histogram, and bar figure derived from multiple repeated experiments, HCV+ Huh-7 significantly inhibits IL-12 production by CD14+ M/MØ.

Bottom Line: 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/M(Ø), which is inversely associated with the diminished IL-12 expression in chronically HCV-infected individuals when compared to healthy subjects.Tim-3 blockade reduces HCV core-mediated expression of the negative immunoregulators PD-1 and SOCS-1 and increases STAT-1 phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Internal Medicine, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee, United States of America.

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/M(Ø)) 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/M(Ø), 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/M(Ø) incubated with HCV-expressing hepatocytes, as well as in primary M/M(Ø) 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/M(Ø). 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.

Show MeSH
Related in: MedlinePlus