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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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SOCS-1 silencing regulates HCV core-mediated Tim-3 expression, IL-12 production, and STAT-1 phosphorylation in THP-1 Cells.A) and B) THP-1 cells were transfected with SOCS-1 siRNA or control siRNA, followed by HCV core and LPS/R848 stimulation for another 48 and 72 h, then subjected for the expression of Tim-3 and IL-12 analysis by flow cytometry. C) The same cells were also subject to Western blot for detection of STAT-1 phosphorylation. The figures show one representative blot and summary densitometry data of 3 repeated experiments.
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pone-0019664-g008: SOCS-1 silencing regulates HCV core-mediated Tim-3 expression, IL-12 production, and STAT-1 phosphorylation in THP-1 Cells.A) and B) THP-1 cells were transfected with SOCS-1 siRNA or control siRNA, followed by HCV core and LPS/R848 stimulation for another 48 and 72 h, then subjected for the expression of Tim-3 and IL-12 analysis by flow cytometry. C) The same cells were also subject to Western blot for detection of STAT-1 phosphorylation. The figures show one representative blot and summary densitometry data of 3 repeated experiments.

Mentions: To further characterize the relationship between Tim-3 and SOCS-1 in negative regulation of TLR/core-mediated IL-12 regulation, we silenced SOCS-1 gene expression in THP-1 cells by transfection of a small interfering RNA (siRNA) specific for SOCS-1, followed by the treatment of HCV core and LPS/R848 for 48 h and 72 h. Compared with the control siRNA, THP-1 cells transfected with SOCS-1 siRNA exhibit significantly inhibited expression of SOCS-1 protein at both 48 and 72 h after transfection [34]. We measured Tim-3 expression and IL-12 production in THP-1 cells after SOCS-1 siRNA transfection and LPS/R848/core treatment for 48 h and 72 h. As shown in Fig. 8A and B, compared with control siRNA, Tim-3 expression is significantly suppressed on SOCS-1 siRNA-transfected THP-1 cells at both time points, but more significantly at 72 h after transfection. Functionally, transfection of SOCS-1 siRNA improved HCV core-suppressed IL-12 expression, especially at 72 h after transfection of SOCS-1 siRNA versus control siRNA. Similar to the reactivation of HCV core-mediated inhibition of STAT-1 phosphorylation by Tim-3 blocking (Fig. 7B), silencing SOCS-1 also rescued HCV core-induced STAT-1 dephosphorylation (Fig. 8C). Collectively, these data suggest that Tim-3 negatively regulates M/MØ IL-12 expression by crosstalk with other inhibitory molecules, including PD-1 and SOCS-1, through limiting STAT-1 phosphorylation.


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)

SOCS-1 silencing regulates HCV core-mediated Tim-3 expression, IL-12 production, and STAT-1 phosphorylation in THP-1 Cells.A) and B) THP-1 cells were transfected with SOCS-1 siRNA or control siRNA, followed by HCV core and LPS/R848 stimulation for another 48 and 72 h, then subjected for the expression of Tim-3 and IL-12 analysis by flow cytometry. C) The same cells were also subject to Western blot for detection of STAT-1 phosphorylation. The figures show one representative blot and summary densitometry data of 3 repeated experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102652&req=5

pone-0019664-g008: SOCS-1 silencing regulates HCV core-mediated Tim-3 expression, IL-12 production, and STAT-1 phosphorylation in THP-1 Cells.A) and B) THP-1 cells were transfected with SOCS-1 siRNA or control siRNA, followed by HCV core and LPS/R848 stimulation for another 48 and 72 h, then subjected for the expression of Tim-3 and IL-12 analysis by flow cytometry. C) The same cells were also subject to Western blot for detection of STAT-1 phosphorylation. The figures show one representative blot and summary densitometry data of 3 repeated experiments.
Mentions: To further characterize the relationship between Tim-3 and SOCS-1 in negative regulation of TLR/core-mediated IL-12 regulation, we silenced SOCS-1 gene expression in THP-1 cells by transfection of a small interfering RNA (siRNA) specific for SOCS-1, followed by the treatment of HCV core and LPS/R848 for 48 h and 72 h. Compared with the control siRNA, THP-1 cells transfected with SOCS-1 siRNA exhibit significantly inhibited expression of SOCS-1 protein at both 48 and 72 h after transfection [34]. We measured Tim-3 expression and IL-12 production in THP-1 cells after SOCS-1 siRNA transfection and LPS/R848/core treatment for 48 h and 72 h. As shown in Fig. 8A and B, compared with control siRNA, Tim-3 expression is significantly suppressed on SOCS-1 siRNA-transfected THP-1 cells at both time points, but more significantly at 72 h after transfection. Functionally, transfection of SOCS-1 siRNA improved HCV core-suppressed IL-12 expression, especially at 72 h after transfection of SOCS-1 siRNA versus control siRNA. Similar to the reactivation of HCV core-mediated inhibition of STAT-1 phosphorylation by Tim-3 blocking (Fig. 7B), silencing SOCS-1 also rescued HCV core-induced STAT-1 dephosphorylation (Fig. 8C). Collectively, these data suggest that Tim-3 negatively regulates M/MØ IL-12 expression by crosstalk with other inhibitory molecules, including PD-1 and SOCS-1, through limiting STAT-1 phosphorylation.

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