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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.


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miR-378 overexpression in mice using an miR-378 agomir inhibits GrzB expression and promotes DENV replication. Mice were injected intraperitoneally with an miR-378 agomir (5 ng), Ctrl-agomir (5 ng) or an equal volume of PBS on days 0, 3, and 6. Mice were inoculated with the DENV-2 ZS01/01 strain (106 pfu/mouse) after the third treatment. Brains, spleens, and peripheral blood of mice were obtained for analyses of miR-378 expression and viral RNA levels at 24 h post-infection using qPCR. (a) Prediction of the binding sites between the mmu-miR-378 seed sequence and mmu-GrzB mRNA sequence by miRBase and TargetScanMouse. Numbers indicate the position of nucleotides of mouse GrzB mRNA 3′-UTR that are targeted by miR-378. (b) The pooled data show that miR-378 expression in spleens and peripheral blood in mice treated with miR-378 agomir are significantly higher than mice treated with the Ctrl-agomir or PBS. (c) Represented flow-cytometric plots show the percentages of GrzB+ of NK (CD3−CD56+) cells in spleens of mice treated with the miR-378 agomir, Ctrl-agomir or PBS. (d) Pooled data show that the percentages of GrzB+ of NK cells in spleens are significantly decreased in mice treated with the miR-378 agomir compared to mice treated with Ctrl-agomir or PBS. DENV2 RNA levels in spleens (e) and brains (f) of mice treated with miR-378 agomir via detection of viral M and E genes and the 5′-UTR sequence are significantly higher than mice treated with Ctrl-agomir or PBS. Representative data are at least three independent experiments with four to six mice per group (mean ± SD; independent samples t-test, *p < 0.05, **p < 0.01, ***p < 0.001).
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fig5: miR-378 overexpression in mice using an miR-378 agomir inhibits GrzB expression and promotes DENV replication. Mice were injected intraperitoneally with an miR-378 agomir (5 ng), Ctrl-agomir (5 ng) or an equal volume of PBS on days 0, 3, and 6. Mice were inoculated with the DENV-2 ZS01/01 strain (106 pfu/mouse) after the third treatment. Brains, spleens, and peripheral blood of mice were obtained for analyses of miR-378 expression and viral RNA levels at 24 h post-infection using qPCR. (a) Prediction of the binding sites between the mmu-miR-378 seed sequence and mmu-GrzB mRNA sequence by miRBase and TargetScanMouse. Numbers indicate the position of nucleotides of mouse GrzB mRNA 3′-UTR that are targeted by miR-378. (b) The pooled data show that miR-378 expression in spleens and peripheral blood in mice treated with miR-378 agomir are significantly higher than mice treated with the Ctrl-agomir or PBS. (c) Represented flow-cytometric plots show the percentages of GrzB+ of NK (CD3−CD56+) cells in spleens of mice treated with the miR-378 agomir, Ctrl-agomir or PBS. (d) Pooled data show that the percentages of GrzB+ of NK cells in spleens are significantly decreased in mice treated with the miR-378 agomir compared to mice treated with Ctrl-agomir or PBS. DENV2 RNA levels in spleens (e) and brains (f) of mice treated with miR-378 agomir via detection of viral M and E genes and the 5′-UTR sequence are significantly higher than mice treated with Ctrl-agomir or PBS. Representative data are at least three independent experiments with four to six mice per group (mean ± SD; independent samples t-test, *p < 0.05, **p < 0.01, ***p < 0.001).

Mentions: We observed that DENV infection induced an up-regulation of GrzB, but whether GrzB played a protective or immunopathological role during in vivo DENV infection was not clear. MiR-378 was a major regulator of GrzB expression in our model. Therefore, we examined whether the overexpression of miR-378 down-regulated GrzB expression and further induced a protective or immunopathological effect in DENV-infected mice. Bioinformatics analyses suggested that miR-378 targeted the 3′-UTR region of mouse GrzB mRNA (Figure 5a). Furthermore, treatment of mice with an miR-378 agomir, which induces overexpression of miR-378 in mouse spleens and peripheral blood (Figure 5b), significantly reduced the percentages of GrzB+ NK cells in spleens compared to treatment with a control agomir (Figure 5c and d). These results suggested that the overexpression of miR-378 inhibited GrzB expression in DENV-infected mice. Notably, the down-regulation of GrzB was associated with a significant up-regulation of viral RNA levels and M and E gene expression of DENV2 in spleens and brains (Figure 5e and f). The reduced expression of GrzB following miR-378 treatment was associated with increased expression of DENV M and E transcripts, which suggests that GrzB contributes to the control of DENV replication in vivo.


Suppressed expression of miR-378 targeting gzmb in NK cells is required to control dengue virus infection
miR-378 overexpression in mice using an miR-378 agomir inhibits GrzB expression and promotes DENV replication. Mice were injected intraperitoneally with an miR-378 agomir (5 ng), Ctrl-agomir (5 ng) or an equal volume of PBS on days 0, 3, and 6. Mice were inoculated with the DENV-2 ZS01/01 strain (106 pfu/mouse) after the third treatment. Brains, spleens, and peripheral blood of mice were obtained for analyses of miR-378 expression and viral RNA levels at 24 h post-infection using qPCR. (a) Prediction of the binding sites between the mmu-miR-378 seed sequence and mmu-GrzB mRNA sequence by miRBase and TargetScanMouse. Numbers indicate the position of nucleotides of mouse GrzB mRNA 3′-UTR that are targeted by miR-378. (b) The pooled data show that miR-378 expression in spleens and peripheral blood in mice treated with miR-378 agomir are significantly higher than mice treated with the Ctrl-agomir or PBS. (c) Represented flow-cytometric plots show the percentages of GrzB+ of NK (CD3−CD56+) cells in spleens of mice treated with the miR-378 agomir, Ctrl-agomir or PBS. (d) Pooled data show that the percentages of GrzB+ of NK cells in spleens are significantly decreased in mice treated with the miR-378 agomir compared to mice treated with Ctrl-agomir or PBS. DENV2 RNA levels in spleens (e) and brains (f) of mice treated with miR-378 agomir via detection of viral M and E genes and the 5′-UTR sequence are significantly higher than mice treated with Ctrl-agomir or PBS. Representative data are at least three independent experiments with four to six mice per group (mean ± SD; independent samples t-test, *p < 0.05, **p < 0.01, ***p < 0.001).
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fig5: miR-378 overexpression in mice using an miR-378 agomir inhibits GrzB expression and promotes DENV replication. Mice were injected intraperitoneally with an miR-378 agomir (5 ng), Ctrl-agomir (5 ng) or an equal volume of PBS on days 0, 3, and 6. Mice were inoculated with the DENV-2 ZS01/01 strain (106 pfu/mouse) after the third treatment. Brains, spleens, and peripheral blood of mice were obtained for analyses of miR-378 expression and viral RNA levels at 24 h post-infection using qPCR. (a) Prediction of the binding sites between the mmu-miR-378 seed sequence and mmu-GrzB mRNA sequence by miRBase and TargetScanMouse. Numbers indicate the position of nucleotides of mouse GrzB mRNA 3′-UTR that are targeted by miR-378. (b) The pooled data show that miR-378 expression in spleens and peripheral blood in mice treated with miR-378 agomir are significantly higher than mice treated with the Ctrl-agomir or PBS. (c) Represented flow-cytometric plots show the percentages of GrzB+ of NK (CD3−CD56+) cells in spleens of mice treated with the miR-378 agomir, Ctrl-agomir or PBS. (d) Pooled data show that the percentages of GrzB+ of NK cells in spleens are significantly decreased in mice treated with the miR-378 agomir compared to mice treated with Ctrl-agomir or PBS. DENV2 RNA levels in spleens (e) and brains (f) of mice treated with miR-378 agomir via detection of viral M and E genes and the 5′-UTR sequence are significantly higher than mice treated with Ctrl-agomir or PBS. Representative data are at least three independent experiments with four to six mice per group (mean ± SD; independent samples t-test, *p < 0.05, **p < 0.01, ***p < 0.001).
Mentions: We observed that DENV infection induced an up-regulation of GrzB, but whether GrzB played a protective or immunopathological role during in vivo DENV infection was not clear. MiR-378 was a major regulator of GrzB expression in our model. Therefore, we examined whether the overexpression of miR-378 down-regulated GrzB expression and further induced a protective or immunopathological effect in DENV-infected mice. Bioinformatics analyses suggested that miR-378 targeted the 3′-UTR region of mouse GrzB mRNA (Figure 5a). Furthermore, treatment of mice with an miR-378 agomir, which induces overexpression of miR-378 in mouse spleens and peripheral blood (Figure 5b), significantly reduced the percentages of GrzB+ NK cells in spleens compared to treatment with a control agomir (Figure 5c and d). These results suggested that the overexpression of miR-378 inhibited GrzB expression in DENV-infected mice. Notably, the down-regulation of GrzB was associated with a significant up-regulation of viral RNA levels and M and E gene expression of DENV2 in spleens and brains (Figure 5e and f). The reduced expression of GrzB following miR-378 treatment was associated with increased expression of DENV M and E transcripts, which suggests that GrzB contributes to the control of DENV replication in vivo.

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