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Suppressed expression of miR-378 targeting gzmb in NK cells is required to control dengue virus infection

View Article: PubMed Central - PubMed

ABSTRACT

Dengue virus (DENV) remains a major public health threat because no vaccine or drugs are available for the prevention and treatment of DENV infection, and the immunopathogenesis mechanisms of DENV infection are not fully understood. Cytotoxic molecules, such as granzyme B (GrzB), may be necessary to control viral infections. However, the exact role of GrzB during DENV infection and the mechanisms regulating GrzB expression during DENV infection are not clear. This study found that miR-27a*, miR-30e, and miR-378 were down-regulated in DENV-infected patients, and DENV infection in humans induced a significant up-regulation of GrzB in natural killer (NK) cells and CD8+ T cells. Further investigation indicated that NK cells, but not CD8+ T cells, were the major sources of GrzB, and miR-378, but not miR-27a* or miR-30e, suppressed GrzB expression in NK cells. Notably, we found that overexpression of miR-378 using a miR-378 agomir in DENV-infected mice inhibited GrzB expression and promoted DENV replication. These results suggest the critical importance of miR-378 in the regulation of GrzB expression and a protective role for GrzB in controlling DENV replication in vivo. Therefore, this study provides a new insight into the immunopathogenesis mechanism of DENV infection and a biological basis for the development of new therapeutic strategies to control DENV infection.

No MeSH data available.


Related in: MedlinePlus

GrzB is negatively regulated by miR-378 in human primary NK cells. Freshly sorted primary NK cells from PBMCs of healthy controls were transfected with Cy3 fluorescein-labeled miR-378 mimics (50 nM), control mimics (50 nM), miR-378 inhibitors (100 nM), or control inhibitors (100 nM). (a) Representative flow-cytometric plots show the purity of sorted NK cells. (b and c) Representative flow-cytometric plots show the transfection efficiency of miR-378 mimics and inhibitors. Numbers indicate the percentages of positive cells in each plot. (d and e) The pooled data show the levels of miR-378 (left) and GrzB protein (right) determined using RT-qPCR and flow-cytometric assays in NK cells transfected with miR-378 mimics or miR-378 inhibitors compared with transfection with Control mimics or Control inhibitors. The sorted NK cells were transduced using lentivirus with vectors of hsa-miR-378 (LV-miR-378), hsa-miR-378 inhibitor (LV-miR-378 inhibitor), or LV-miR-negative control (LV-miR-Ctrl) for seven days. Cells were collected for analyses of miR-378 and GrzB levels using RT-qPCR and flow cytometry, respectively. (f) Representative flow-cytometric plots show the rates of GFP+ NK cells after transduction of LV-miR-Ctrl at MOIs of 0, 10, and 20. (g) The pooled data show that miR-378 expression is significantly increased in NK cells transduced with LV-miR-378. (h) The MFI of GrzB is down-regulated in NK cells transduced with LV-miR-378, but it is up-regulated in NK cells transduced with the LV-miR-378 inhibitor compared to LV-miR-Ctrl. Representative data are at least three independent experiments with one donor in every experiment (mean ± SD; paired two-tailed Student's t-test, *p < 0.05, **p < 0.01, ***p < 0.001). Mock, non-transfected or non-transduced group.
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fig4: GrzB is negatively regulated by miR-378 in human primary NK cells. Freshly sorted primary NK cells from PBMCs of healthy controls were transfected with Cy3 fluorescein-labeled miR-378 mimics (50 nM), control mimics (50 nM), miR-378 inhibitors (100 nM), or control inhibitors (100 nM). (a) Representative flow-cytometric plots show the purity of sorted NK cells. (b and c) Representative flow-cytometric plots show the transfection efficiency of miR-378 mimics and inhibitors. Numbers indicate the percentages of positive cells in each plot. (d and e) The pooled data show the levels of miR-378 (left) and GrzB protein (right) determined using RT-qPCR and flow-cytometric assays in NK cells transfected with miR-378 mimics or miR-378 inhibitors compared with transfection with Control mimics or Control inhibitors. The sorted NK cells were transduced using lentivirus with vectors of hsa-miR-378 (LV-miR-378), hsa-miR-378 inhibitor (LV-miR-378 inhibitor), or LV-miR-negative control (LV-miR-Ctrl) for seven days. Cells were collected for analyses of miR-378 and GrzB levels using RT-qPCR and flow cytometry, respectively. (f) Representative flow-cytometric plots show the rates of GFP+ NK cells after transduction of LV-miR-Ctrl at MOIs of 0, 10, and 20. (g) The pooled data show that miR-378 expression is significantly increased in NK cells transduced with LV-miR-378. (h) The MFI of GrzB is down-regulated in NK cells transduced with LV-miR-378, but it is up-regulated in NK cells transduced with the LV-miR-378 inhibitor compared to LV-miR-Ctrl. Representative data are at least three independent experiments with one donor in every experiment (mean ± SD; paired two-tailed Student's t-test, *p < 0.05, **p < 0.01, ***p < 0.001). Mock, non-transfected or non-transduced group.

Mentions: We determined whether miR-378 specifically regulated GrzB expression in NK cells. Primary NK cells were isolated from PBMCs of healthy donors, and the purity and sorting efficiency of isolated NK cells was >90%, as measured by flow cytometry (Figure 4a). NK cells were transfected with Cy3 fluorescein-labeled synthetic miR-378 mimics, inhibitors, control mimics, or control inhibitors. Fluorescence microscopy and flow-cytometric analyses confirmed that miR-378 mimics and inhibitors were transfected in NK cells (Figure 4b and c). qPCR analysis revealed that miR-378 expression was significantly increased in NK cells after transfection of miR-378 mimics compared with control mimics (Figure 4d). ICS/flow-cytometric analyses of GrzB showed that transfection of miR-378 mimics induced a significant decrease in the MFI of GrzB in NK cells compared with control mimics (Figure 4e). Conversely, transfection of miR-378 inhibitors induced a significant increase in the MFI of GrzB in NK cells compared to control inhibitors (Figure 4e). Furthermore, we used a lentiviral transduction method to stably overexpress miR-378 or inhibit intrinsic miR-378 in human primary NK cells (Figure 4f and g). Similar decreases in the MFI of GrzB after transduction of a lentiviral vector overexpressing miR-378 and increases in the MFI of GrzB in NK cells after transduction of a lentiviral vector that inhibited intrinsic miR-378 compared to controls were observed (Figure 4h). These results collectively suggest that miR-378 regulates GrzB expression in human NK cells during DENV infection.


Suppressed expression of miR-378 targeting gzmb in NK cells is required to control dengue virus infection
GrzB is negatively regulated by miR-378 in human primary NK cells. Freshly sorted primary NK cells from PBMCs of healthy controls were transfected with Cy3 fluorescein-labeled miR-378 mimics (50 nM), control mimics (50 nM), miR-378 inhibitors (100 nM), or control inhibitors (100 nM). (a) Representative flow-cytometric plots show the purity of sorted NK cells. (b and c) Representative flow-cytometric plots show the transfection efficiency of miR-378 mimics and inhibitors. Numbers indicate the percentages of positive cells in each plot. (d and e) The pooled data show the levels of miR-378 (left) and GrzB protein (right) determined using RT-qPCR and flow-cytometric assays in NK cells transfected with miR-378 mimics or miR-378 inhibitors compared with transfection with Control mimics or Control inhibitors. The sorted NK cells were transduced using lentivirus with vectors of hsa-miR-378 (LV-miR-378), hsa-miR-378 inhibitor (LV-miR-378 inhibitor), or LV-miR-negative control (LV-miR-Ctrl) for seven days. Cells were collected for analyses of miR-378 and GrzB levels using RT-qPCR and flow cytometry, respectively. (f) Representative flow-cytometric plots show the rates of GFP+ NK cells after transduction of LV-miR-Ctrl at MOIs of 0, 10, and 20. (g) The pooled data show that miR-378 expression is significantly increased in NK cells transduced with LV-miR-378. (h) The MFI of GrzB is down-regulated in NK cells transduced with LV-miR-378, but it is up-regulated in NK cells transduced with the LV-miR-378 inhibitor compared to LV-miR-Ctrl. Representative data are at least three independent experiments with one donor in every experiment (mean ± SD; paired two-tailed Student's t-test, *p < 0.05, **p < 0.01, ***p < 0.001). Mock, non-transfected or non-transduced group.
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fig4: GrzB is negatively regulated by miR-378 in human primary NK cells. Freshly sorted primary NK cells from PBMCs of healthy controls were transfected with Cy3 fluorescein-labeled miR-378 mimics (50 nM), control mimics (50 nM), miR-378 inhibitors (100 nM), or control inhibitors (100 nM). (a) Representative flow-cytometric plots show the purity of sorted NK cells. (b and c) Representative flow-cytometric plots show the transfection efficiency of miR-378 mimics and inhibitors. Numbers indicate the percentages of positive cells in each plot. (d and e) The pooled data show the levels of miR-378 (left) and GrzB protein (right) determined using RT-qPCR and flow-cytometric assays in NK cells transfected with miR-378 mimics or miR-378 inhibitors compared with transfection with Control mimics or Control inhibitors. The sorted NK cells were transduced using lentivirus with vectors of hsa-miR-378 (LV-miR-378), hsa-miR-378 inhibitor (LV-miR-378 inhibitor), or LV-miR-negative control (LV-miR-Ctrl) for seven days. Cells were collected for analyses of miR-378 and GrzB levels using RT-qPCR and flow cytometry, respectively. (f) Representative flow-cytometric plots show the rates of GFP+ NK cells after transduction of LV-miR-Ctrl at MOIs of 0, 10, and 20. (g) The pooled data show that miR-378 expression is significantly increased in NK cells transduced with LV-miR-378. (h) The MFI of GrzB is down-regulated in NK cells transduced with LV-miR-378, but it is up-regulated in NK cells transduced with the LV-miR-378 inhibitor compared to LV-miR-Ctrl. Representative data are at least three independent experiments with one donor in every experiment (mean ± SD; paired two-tailed Student's t-test, *p < 0.05, **p < 0.01, ***p < 0.001). Mock, non-transfected or non-transduced group.
Mentions: We determined whether miR-378 specifically regulated GrzB expression in NK cells. Primary NK cells were isolated from PBMCs of healthy donors, and the purity and sorting efficiency of isolated NK cells was >90%, as measured by flow cytometry (Figure 4a). NK cells were transfected with Cy3 fluorescein-labeled synthetic miR-378 mimics, inhibitors, control mimics, or control inhibitors. Fluorescence microscopy and flow-cytometric analyses confirmed that miR-378 mimics and inhibitors were transfected in NK cells (Figure 4b and c). qPCR analysis revealed that miR-378 expression was significantly increased in NK cells after transfection of miR-378 mimics compared with control mimics (Figure 4d). ICS/flow-cytometric analyses of GrzB showed that transfection of miR-378 mimics induced a significant decrease in the MFI of GrzB in NK cells compared with control mimics (Figure 4e). Conversely, transfection of miR-378 inhibitors induced a significant increase in the MFI of GrzB in NK cells compared to control inhibitors (Figure 4e). Furthermore, we used a lentiviral transduction method to stably overexpress miR-378 or inhibit intrinsic miR-378 in human primary NK cells (Figure 4f and g). Similar decreases in the MFI of GrzB after transduction of a lentiviral vector overexpressing miR-378 and increases in the MFI of GrzB in NK cells after transduction of a lentiviral vector that inhibited intrinsic miR-378 compared to controls were observed (Figure 4h). These results collectively suggest that miR-378 regulates GrzB expression in human NK cells during DENV infection.

View Article: PubMed Central - PubMed

ABSTRACT

Dengue virus (DENV) remains a major public health threat because no vaccine or drugs are available for the prevention and treatment of DENV infection, and the immunopathogenesis mechanisms of DENV infection are not fully understood. Cytotoxic molecules, such as granzyme B (GrzB), may be necessary to control viral infections. However, the exact role of GrzB during DENV infection and the mechanisms regulating GrzB expression during DENV infection are not clear. This study found that miR-27a*, miR-30e, and miR-378 were down-regulated in DENV-infected patients, and DENV infection in humans induced a significant up-regulation of GrzB in natural killer (NK) cells and CD8+ T cells. Further investigation indicated that NK cells, but not CD8+ T cells, were the major sources of GrzB, and miR-378, but not miR-27a* or miR-30e, suppressed GrzB expression in NK cells. Notably, we found that overexpression of miR-378 using a miR-378 agomir in DENV-infected mice inhibited GrzB expression and promoted DENV replication. These results suggest the critical importance of miR-378 in the regulation of GrzB expression and a protective role for GrzB in controlling DENV replication in vivo. Therefore, this study provides a new insight into the immunopathogenesis mechanism of DENV infection and a biological basis for the development of new therapeutic strategies to control DENV infection.

No MeSH data available.


Related in: MedlinePlus