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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 is over-expressed and IL-12 expression suppressed on CD14+ M/MØ in chronically HCV-infected individuals.PBMC from chronically HCV-infected subjects (n = 21) and healthy subjects (n = 12) were stimulated with or without TLR ligands LPS and R848 for 18 h, followed by immunostaining with antibodies against Tim-3, IL-12 and CD14. Tim-3 and IL-12 expressions in un-stimulated and LPS/R848-stimulated CD14+ M/MØ were examined by flow cytometry. A) Representative flow cytometric dot plots measuring Tim-3 expression and IL-12 production in CD14+ M/MØ. B) Time-course of Tim-3 expression in primary healthy M/MØ. PBMC stimulated with LPS and R848 for the indicated times. Tim-3 expression on CD14+ M/MØ was assessed by flow cytometry; corresponding changes in % gated and MFI are shown with isotype control. C) Summary data of the percentage of Tim-3+ and D) IL-12+ cells in CD14+ M/MØ of healthy subjects and chronically HCV-infected individuals, in naïve and TLR-activated state, are shown. Each symbol represents an individual subject, and the horizontal bars represent median values. The p value (**<0.01; ***<0.001) is denoted above the group of study subjects.
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pone-0019664-g001: Tim-3 is over-expressed and IL-12 expression suppressed on CD14+ M/MØ in chronically HCV-infected individuals.PBMC from chronically HCV-infected subjects (n = 21) and healthy subjects (n = 12) were stimulated with or without TLR ligands LPS and R848 for 18 h, followed by immunostaining with antibodies against Tim-3, IL-12 and CD14. Tim-3 and IL-12 expressions in un-stimulated and LPS/R848-stimulated CD14+ M/MØ were examined by flow cytometry. A) Representative flow cytometric dot plots measuring Tim-3 expression and IL-12 production in CD14+ M/MØ. B) Time-course of Tim-3 expression in primary healthy M/MØ. PBMC stimulated with LPS and R848 for the indicated times. Tim-3 expression on CD14+ M/MØ was assessed by flow cytometry; corresponding changes in % gated and MFI are shown with isotype control. C) Summary data of the percentage of Tim-3+ and D) IL-12+ cells in CD14+ M/MØ of healthy subjects and chronically HCV-infected individuals, in naïve and TLR-activated state, are shown. Each symbol represents an individual subject, and the horizontal bars represent median values. The p value (**<0.01; ***<0.001) is denoted above the group of study subjects.

Mentions: Tim-3 has been shown to be over-expressed on and involved in inhibiting viral specific T cell responses during HCV infection [9]. The expression and role of Tim-3 in regulation of M/MØ function during HCV infection remains undefined. To address this, Tim-3 expression, along with intracellular IL-12 production, in resting and TLR-stimulated M/MØ of chronically HCV-infected patients and healthy subjects, were examined by flow cytometry. As shown in the representative dot plots, time-course, and summary data of Tim-3 expression on CD14+ M/MØ of healthy subjects (n = 12, multiple assays, p<0.001) and HCV-infected individuals (n = 21, p<0.001) in Fig. 1A through D, Tim-3 is constitutively expressed on resting M/MØ; its expression significantly decreases upon cell activation, as early as 6 h, after stimulation with Toll-like receptor 4 (TLR4) ligand - lipopolysaccharide (LPS) and TLR7/8 ligand - R848, which can synergistically activate primary M/MØ to produce IL-12 [17], [18]. Notably, chronically HCV-infected individual exhibits significantly elevated Tim-3 expression on CD14+ M/MØ, in both the un-stimulated and TLR-stimulated states, when compared to healthy subjects. In contrast to the elevated Tim-3 expression, IL-12 expression is barely detectable in resting M/MØ; its expression is significantly increased in CD14+ M/MØ following TLR stimulation, but to a lesser extent and leading to an impaired IL-12 expression in the setting of chronic HCV infection compared to healthy subjects.


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 is over-expressed and IL-12 expression suppressed on CD14+ M/MØ in chronically HCV-infected individuals.PBMC from chronically HCV-infected subjects (n = 21) and healthy subjects (n = 12) were stimulated with or without TLR ligands LPS and R848 for 18 h, followed by immunostaining with antibodies against Tim-3, IL-12 and CD14. Tim-3 and IL-12 expressions in un-stimulated and LPS/R848-stimulated CD14+ M/MØ were examined by flow cytometry. A) Representative flow cytometric dot plots measuring Tim-3 expression and IL-12 production in CD14+ M/MØ. B) Time-course of Tim-3 expression in primary healthy M/MØ. PBMC stimulated with LPS and R848 for the indicated times. Tim-3 expression on CD14+ M/MØ was assessed by flow cytometry; corresponding changes in % gated and MFI are shown with isotype control. C) Summary data of the percentage of Tim-3+ and D) IL-12+ cells in CD14+ M/MØ of healthy subjects and chronically HCV-infected individuals, in naïve and TLR-activated state, are shown. Each symbol represents an individual subject, and the horizontal bars represent median values. The p value (**<0.01; ***<0.001) is denoted above the group of study subjects.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102652&req=5

pone-0019664-g001: Tim-3 is over-expressed and IL-12 expression suppressed on CD14+ M/MØ in chronically HCV-infected individuals.PBMC from chronically HCV-infected subjects (n = 21) and healthy subjects (n = 12) were stimulated with or without TLR ligands LPS and R848 for 18 h, followed by immunostaining with antibodies against Tim-3, IL-12 and CD14. Tim-3 and IL-12 expressions in un-stimulated and LPS/R848-stimulated CD14+ M/MØ were examined by flow cytometry. A) Representative flow cytometric dot plots measuring Tim-3 expression and IL-12 production in CD14+ M/MØ. B) Time-course of Tim-3 expression in primary healthy M/MØ. PBMC stimulated with LPS and R848 for the indicated times. Tim-3 expression on CD14+ M/MØ was assessed by flow cytometry; corresponding changes in % gated and MFI are shown with isotype control. C) Summary data of the percentage of Tim-3+ and D) IL-12+ cells in CD14+ M/MØ of healthy subjects and chronically HCV-infected individuals, in naïve and TLR-activated state, are shown. Each symbol represents an individual subject, and the horizontal bars represent median values. The p value (**<0.01; ***<0.001) is denoted above the group of study subjects.
Mentions: Tim-3 has been shown to be over-expressed on and involved in inhibiting viral specific T cell responses during HCV infection [9]. The expression and role of Tim-3 in regulation of M/MØ function during HCV infection remains undefined. To address this, Tim-3 expression, along with intracellular IL-12 production, in resting and TLR-stimulated M/MØ of chronically HCV-infected patients and healthy subjects, were examined by flow cytometry. As shown in the representative dot plots, time-course, and summary data of Tim-3 expression on CD14+ M/MØ of healthy subjects (n = 12, multiple assays, p<0.001) and HCV-infected individuals (n = 21, p<0.001) in Fig. 1A through D, Tim-3 is constitutively expressed on resting M/MØ; its expression significantly decreases upon cell activation, as early as 6 h, after stimulation with Toll-like receptor 4 (TLR4) ligand - lipopolysaccharide (LPS) and TLR7/8 ligand - R848, which can synergistically activate primary M/MØ to produce IL-12 [17], [18]. Notably, chronically HCV-infected individual exhibits significantly elevated Tim-3 expression on CD14+ M/MØ, in both the un-stimulated and TLR-stimulated states, when compared to healthy subjects. In contrast to the elevated Tim-3 expression, IL-12 expression is barely detectable in resting M/MØ; its expression is significantly increased in CD14+ M/MØ following TLR stimulation, but to a lesser extent and leading to an impaired IL-12 expression in the setting of chronic HCV infection compared to healthy subjects.

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