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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 correlated with miR-378 in human NK cells during DENV infection. PBMCs were cultured with DENV-2 (ZS01/01 strain) at MOIs of 0, 1, and 5 for 12, 24, and 48 hours and collected for analysis of GrzB expression in NK, CD8+, and CD4+ T cells in three groups using flow cytometry. (a) Representative flow-cytometric dot plots show the percentages of GrzB+ cells in NK (CD3−CD56+), CD8+ (CD3+CD8+) T and CD4+ (CD3+CD4+) T cells between DENV2 - and Mock-infected cells. Numbers in each plot indicate the percentage of GrzB+ cells. (b) Overlaying flow-cytometric plots of the GrzB+ NK cells in DENV2 (red curve) and Mock (blue curve) groups at 24 h post-infection. Pooled data show that the MFI of GrzB in NK cells is significantly increased in DENV2 cells cultured within 24 h compared to Mock cells. (c) Pooled data show that the percentages of GrzB+ cells in CD8+ or CD4+ T cells are not significantly different between DENV2 and Mock cells within 48 h post-infection. The above representative data are from 12 samples and four independent experiments (mean ± SD; paired two-tailed Student's t-test, **p < 0.01). NK cells were also sorted from DENV-infected PBMCs at an MOI of 5 for 24 h as a non-infected control group. (d) Represented flow-cytometric plots show GrzB expression levels in sorted NK (CD3−CD56+) cells between DENV2 and Mock groups, and numbers indicate the MFI of GrzB in CD56dim and CD56bright subsets in each plot. The overlaying plot displays GrzB+ cells in CD56dim subsets in DENV2 (red curve) and Mock (blue curve) groups. (e) Pooled data show that GrzB expression levels in CD56dim or CD56bright subsets are significantly increased in DENV2 (left), but the mRNA levels in isolated NK cells between the two groups were not significantly different (right). (f) Pooled data of qPCR analysis show that in vitro DENV infection induces a down-regulation of miR-30 and miR-378, but not miR-27a*, in NK cells. (g) GrzB is negatively correlated with miR-378 in CD56dim or CD56bright NK cells during in vitro DENV infection. The decrement values of relative expression of miR-378 versus GrzB using mean fluorescence intensity (MFI) increment values were conducted for correlational statistical analyses using the Pearson correlation coefficient analysis to determine the correlation between miR-378 and GrzB expression. Decrement values of miR-378 were determined by subtracting miR-378 relative expression values in DENV2 cells from the expression values in Mock cells. Increased values of GrzB MFI were determined by subtracting GrzB MFI values in Mock cells from the values in DENV2 cells. The left panel shows the correlation between GrzB and miR-378 in CD56dim NK cells (p < 0.001), and the right panel shows the correlation between GrzB and miR-378 in CD56bright NK cells (p < 0.01). The above representative data are from eight samples and three independent experiments (mean ± SD; paired two-tailed Student's t-test, *p < 0.05, ***p < 0.001). DENV2, DENV-infected group (MOI 5); Mock, non-infected group.
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fig3: GrzB is negatively correlated with miR-378 in human NK cells during DENV infection. PBMCs were cultured with DENV-2 (ZS01/01 strain) at MOIs of 0, 1, and 5 for 12, 24, and 48 hours and collected for analysis of GrzB expression in NK, CD8+, and CD4+ T cells in three groups using flow cytometry. (a) Representative flow-cytometric dot plots show the percentages of GrzB+ cells in NK (CD3−CD56+), CD8+ (CD3+CD8+) T and CD4+ (CD3+CD4+) T cells between DENV2 - and Mock-infected cells. Numbers in each plot indicate the percentage of GrzB+ cells. (b) Overlaying flow-cytometric plots of the GrzB+ NK cells in DENV2 (red curve) and Mock (blue curve) groups at 24 h post-infection. Pooled data show that the MFI of GrzB in NK cells is significantly increased in DENV2 cells cultured within 24 h compared to Mock cells. (c) Pooled data show that the percentages of GrzB+ cells in CD8+ or CD4+ T cells are not significantly different between DENV2 and Mock cells within 48 h post-infection. The above representative data are from 12 samples and four independent experiments (mean ± SD; paired two-tailed Student's t-test, **p < 0.01). NK cells were also sorted from DENV-infected PBMCs at an MOI of 5 for 24 h as a non-infected control group. (d) Represented flow-cytometric plots show GrzB expression levels in sorted NK (CD3−CD56+) cells between DENV2 and Mock groups, and numbers indicate the MFI of GrzB in CD56dim and CD56bright subsets in each plot. The overlaying plot displays GrzB+ cells in CD56dim subsets in DENV2 (red curve) and Mock (blue curve) groups. (e) Pooled data show that GrzB expression levels in CD56dim or CD56bright subsets are significantly increased in DENV2 (left), but the mRNA levels in isolated NK cells between the two groups were not significantly different (right). (f) Pooled data of qPCR analysis show that in vitro DENV infection induces a down-regulation of miR-30 and miR-378, but not miR-27a*, in NK cells. (g) GrzB is negatively correlated with miR-378 in CD56dim or CD56bright NK cells during in vitro DENV infection. The decrement values of relative expression of miR-378 versus GrzB using mean fluorescence intensity (MFI) increment values were conducted for correlational statistical analyses using the Pearson correlation coefficient analysis to determine the correlation between miR-378 and GrzB expression. Decrement values of miR-378 were determined by subtracting miR-378 relative expression values in DENV2 cells from the expression values in Mock cells. Increased values of GrzB MFI were determined by subtracting GrzB MFI values in Mock cells from the values in DENV2 cells. The left panel shows the correlation between GrzB and miR-378 in CD56dim NK cells (p < 0.001), and the right panel shows the correlation between GrzB and miR-378 in CD56bright NK cells (p < 0.01). The above representative data are from eight samples and three independent experiments (mean ± SD; paired two-tailed Student's t-test, *p < 0.05, ***p < 0.001). DENV2, DENV-infected group (MOI 5); Mock, non-infected group.

Mentions: We determined whether the up-regulation of GrzB in CD56+ NK cells was directly mediated by the down-regulation of miR-378 or the secondary pathological or systemic effects during DENV infection in humans. PBMCs were successfully infected with DENV2 in vitro (Supplementary Figure S4a–c). DENV infection induced NK and T-cell activation (Supplementary Figure S5a–c), and GrzB expression was significantly up-regulated in CD56+ NK cells, but not CD8+ or CD4+ T cells, at 24 h post-infection (Figure 3a–c). Notably, both CD56dim and CD56bright subsets of CD56+ NK cells exhibited strong GrzB expression after DENV infection, but GrzB mRNA expression levels in isolated CD56+ NK cells were not significantly increased in the DENV-infected group (Figure 3d and e). These data suggest that miR-378 may participate in the regulation of GrzB expression at the translation level, and NK cells are a major source of GrzB expression during DENV infection.


Suppressed expression of miR-378 targeting gzmb in NK cells is required to control dengue virus infection
GrzB is negatively correlated with miR-378 in human NK cells during DENV infection. PBMCs were cultured with DENV-2 (ZS01/01 strain) at MOIs of 0, 1, and 5 for 12, 24, and 48 hours and collected for analysis of GrzB expression in NK, CD8+, and CD4+ T cells in three groups using flow cytometry. (a) Representative flow-cytometric dot plots show the percentages of GrzB+ cells in NK (CD3−CD56+), CD8+ (CD3+CD8+) T and CD4+ (CD3+CD4+) T cells between DENV2 - and Mock-infected cells. Numbers in each plot indicate the percentage of GrzB+ cells. (b) Overlaying flow-cytometric plots of the GrzB+ NK cells in DENV2 (red curve) and Mock (blue curve) groups at 24 h post-infection. Pooled data show that the MFI of GrzB in NK cells is significantly increased in DENV2 cells cultured within 24 h compared to Mock cells. (c) Pooled data show that the percentages of GrzB+ cells in CD8+ or CD4+ T cells are not significantly different between DENV2 and Mock cells within 48 h post-infection. The above representative data are from 12 samples and four independent experiments (mean ± SD; paired two-tailed Student's t-test, **p < 0.01). NK cells were also sorted from DENV-infected PBMCs at an MOI of 5 for 24 h as a non-infected control group. (d) Represented flow-cytometric plots show GrzB expression levels in sorted NK (CD3−CD56+) cells between DENV2 and Mock groups, and numbers indicate the MFI of GrzB in CD56dim and CD56bright subsets in each plot. The overlaying plot displays GrzB+ cells in CD56dim subsets in DENV2 (red curve) and Mock (blue curve) groups. (e) Pooled data show that GrzB expression levels in CD56dim or CD56bright subsets are significantly increased in DENV2 (left), but the mRNA levels in isolated NK cells between the two groups were not significantly different (right). (f) Pooled data of qPCR analysis show that in vitro DENV infection induces a down-regulation of miR-30 and miR-378, but not miR-27a*, in NK cells. (g) GrzB is negatively correlated with miR-378 in CD56dim or CD56bright NK cells during in vitro DENV infection. The decrement values of relative expression of miR-378 versus GrzB using mean fluorescence intensity (MFI) increment values were conducted for correlational statistical analyses using the Pearson correlation coefficient analysis to determine the correlation between miR-378 and GrzB expression. Decrement values of miR-378 were determined by subtracting miR-378 relative expression values in DENV2 cells from the expression values in Mock cells. Increased values of GrzB MFI were determined by subtracting GrzB MFI values in Mock cells from the values in DENV2 cells. The left panel shows the correlation between GrzB and miR-378 in CD56dim NK cells (p < 0.001), and the right panel shows the correlation between GrzB and miR-378 in CD56bright NK cells (p < 0.01). The above representative data are from eight samples and three independent experiments (mean ± SD; paired two-tailed Student's t-test, *p < 0.05, ***p < 0.001). DENV2, DENV-infected group (MOI 5); Mock, non-infected group.
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fig3: GrzB is negatively correlated with miR-378 in human NK cells during DENV infection. PBMCs were cultured with DENV-2 (ZS01/01 strain) at MOIs of 0, 1, and 5 for 12, 24, and 48 hours and collected for analysis of GrzB expression in NK, CD8+, and CD4+ T cells in three groups using flow cytometry. (a) Representative flow-cytometric dot plots show the percentages of GrzB+ cells in NK (CD3−CD56+), CD8+ (CD3+CD8+) T and CD4+ (CD3+CD4+) T cells between DENV2 - and Mock-infected cells. Numbers in each plot indicate the percentage of GrzB+ cells. (b) Overlaying flow-cytometric plots of the GrzB+ NK cells in DENV2 (red curve) and Mock (blue curve) groups at 24 h post-infection. Pooled data show that the MFI of GrzB in NK cells is significantly increased in DENV2 cells cultured within 24 h compared to Mock cells. (c) Pooled data show that the percentages of GrzB+ cells in CD8+ or CD4+ T cells are not significantly different between DENV2 and Mock cells within 48 h post-infection. The above representative data are from 12 samples and four independent experiments (mean ± SD; paired two-tailed Student's t-test, **p < 0.01). NK cells were also sorted from DENV-infected PBMCs at an MOI of 5 for 24 h as a non-infected control group. (d) Represented flow-cytometric plots show GrzB expression levels in sorted NK (CD3−CD56+) cells between DENV2 and Mock groups, and numbers indicate the MFI of GrzB in CD56dim and CD56bright subsets in each plot. The overlaying plot displays GrzB+ cells in CD56dim subsets in DENV2 (red curve) and Mock (blue curve) groups. (e) Pooled data show that GrzB expression levels in CD56dim or CD56bright subsets are significantly increased in DENV2 (left), but the mRNA levels in isolated NK cells between the two groups were not significantly different (right). (f) Pooled data of qPCR analysis show that in vitro DENV infection induces a down-regulation of miR-30 and miR-378, but not miR-27a*, in NK cells. (g) GrzB is negatively correlated with miR-378 in CD56dim or CD56bright NK cells during in vitro DENV infection. The decrement values of relative expression of miR-378 versus GrzB using mean fluorescence intensity (MFI) increment values were conducted for correlational statistical analyses using the Pearson correlation coefficient analysis to determine the correlation between miR-378 and GrzB expression. Decrement values of miR-378 were determined by subtracting miR-378 relative expression values in DENV2 cells from the expression values in Mock cells. Increased values of GrzB MFI were determined by subtracting GrzB MFI values in Mock cells from the values in DENV2 cells. The left panel shows the correlation between GrzB and miR-378 in CD56dim NK cells (p < 0.001), and the right panel shows the correlation between GrzB and miR-378 in CD56bright NK cells (p < 0.01). The above representative data are from eight samples and three independent experiments (mean ± SD; paired two-tailed Student's t-test, *p < 0.05, ***p < 0.001). DENV2, DENV-infected group (MOI 5); Mock, non-infected group.
Mentions: We determined whether the up-regulation of GrzB in CD56+ NK cells was directly mediated by the down-regulation of miR-378 or the secondary pathological or systemic effects during DENV infection in humans. PBMCs were successfully infected with DENV2 in vitro (Supplementary Figure S4a–c). DENV infection induced NK and T-cell activation (Supplementary Figure S5a–c), and GrzB expression was significantly up-regulated in CD56+ NK cells, but not CD8+ or CD4+ T cells, at 24 h post-infection (Figure 3a–c). Notably, both CD56dim and CD56bright subsets of CD56+ NK cells exhibited strong GrzB expression after DENV infection, but GrzB mRNA expression levels in isolated CD56+ NK cells were not significantly increased in the DENV-infected group (Figure 3d and e). These data suggest that miR-378 may participate in the regulation of GrzB expression at the translation level, and NK cells are a major source of GrzB expression 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