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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

DENV infection induces a significant increase of GrzB expression. Freshly isolated PBMCs from 10 DENV-infected patients (DENV patients) and 10 Healthy Ctrls were directly stained with cell-surface and intracellular antibodies to analyze GrzB and CD107 expression in NK, CD8+ and CD4+ T cells using flow cytometry. (a) Representative flow-cytometric plots show the percentages of GrzB+ cells in total PBMCs, CD8+ and CD4+ T cells in Healthy Ctrls and DENV patients. (b) Pooled data show that the percentages of GrzB+ cells in total PBMCs and CD8+ T cells are significantly increased in DENV patients compared to Healthy Ctrls. (blue curve, Healthy Ctrl; red curve, DENV patients). (c) Representative flow-cytometric plots and pooled data show that the MFI of GrzB in NK (CD3−CD56+) cells is significantly increased in DENV patients. (d) Representative flow-cytometric plots and pooled data show that the percentages of CD107+GrzB+ cells in CD8+ T or CD56+ NK cells are significantly increased in DENV patients compared to Healthy Ctrls. (e) A representative two-tier flow-cytometric analysis and pooled data show that GrzB+ cells in PBMCs of DENV patients contain more CD3−CD56+ NK than CD3+CD8+ cells. Data are representative of at least three independent experiments (mean ± SD; independent samples t-test, *p < 0.05, **p < 0.01, ***p < 0.001).
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fig2: DENV infection induces a significant increase of GrzB expression. Freshly isolated PBMCs from 10 DENV-infected patients (DENV patients) and 10 Healthy Ctrls were directly stained with cell-surface and intracellular antibodies to analyze GrzB and CD107 expression in NK, CD8+ and CD4+ T cells using flow cytometry. (a) Representative flow-cytometric plots show the percentages of GrzB+ cells in total PBMCs, CD8+ and CD4+ T cells in Healthy Ctrls and DENV patients. (b) Pooled data show that the percentages of GrzB+ cells in total PBMCs and CD8+ T cells are significantly increased in DENV patients compared to Healthy Ctrls. (blue curve, Healthy Ctrl; red curve, DENV patients). (c) Representative flow-cytometric plots and pooled data show that the MFI of GrzB in NK (CD3−CD56+) cells is significantly increased in DENV patients. (d) Representative flow-cytometric plots and pooled data show that the percentages of CD107+GrzB+ cells in CD8+ T or CD56+ NK cells are significantly increased in DENV patients compared to Healthy Ctrls. (e) A representative two-tier flow-cytometric analysis and pooled data show that GrzB+ cells in PBMCs of DENV patients contain more CD3−CD56+ NK than CD3+CD8+ cells. Data are representative of at least three independent experiments (mean ± SD; independent samples t-test, *p < 0.05, **p < 0.01, ***p < 0.001).

Mentions: We detected GrzB expression in DENV-infected patients using intracellular cytokine staining (ICS) and flow-cytometric analysis. Supplementary Figure S2 shows the strategy for gating CD56+ NK cells, CD8+ and CD4+ T cells. GrzB protein was significantly up-regulated in total PBMCs, CD8+ T cells, and CD56+ NK cells of DENV-infected patients, but the mRNA level of GrzB in PBMCs of DENV-infected patients was not significantly increased compared with Healthy Ctrls (Figure 2a–c; Supplementary Figure S3). These results suggest the post-transcriptional targeting of GrzB mRNA by miR-378. Consistently, a marker for the degranulation capacity of cytotoxic lymphocytes, CD107a, which is a lysozyme-associated membrane glycoprotein,24,25 was also significantly up-regulated in CD8+ T cells and NK cells of DENV-infected patients (Figure 2d). Given that both CD56+ NK cells and CD8+ T cells produce appreciable amounts of GrzB, we therefore identified which cell sup-population was the major source of GrzB. Notably, the percentages of CD56+ NK (CD3−CD56+) cells in total GrzB+ PBMCs of DENV-infected patients were much higher than the percentages of CD3+CD8+ T cells (Figure 2e), which suggests that CD56+ NK cells are the major source of GrzB during DENV infection. Taken together, these results suggest that the down-regulation of miR-378 contributes to the up-regulation of GrzB protein expression, particularly in NK cells, but it does not influence GrzB mRNA levels in DENV-infected patients.


Suppressed expression of miR-378 targeting gzmb in NK cells is required to control dengue virus infection
DENV infection induces a significant increase of GrzB expression. Freshly isolated PBMCs from 10 DENV-infected patients (DENV patients) and 10 Healthy Ctrls were directly stained with cell-surface and intracellular antibodies to analyze GrzB and CD107 expression in NK, CD8+ and CD4+ T cells using flow cytometry. (a) Representative flow-cytometric plots show the percentages of GrzB+ cells in total PBMCs, CD8+ and CD4+ T cells in Healthy Ctrls and DENV patients. (b) Pooled data show that the percentages of GrzB+ cells in total PBMCs and CD8+ T cells are significantly increased in DENV patients compared to Healthy Ctrls. (blue curve, Healthy Ctrl; red curve, DENV patients). (c) Representative flow-cytometric plots and pooled data show that the MFI of GrzB in NK (CD3−CD56+) cells is significantly increased in DENV patients. (d) Representative flow-cytometric plots and pooled data show that the percentages of CD107+GrzB+ cells in CD8+ T or CD56+ NK cells are significantly increased in DENV patients compared to Healthy Ctrls. (e) A representative two-tier flow-cytometric analysis and pooled data show that GrzB+ cells in PBMCs of DENV patients contain more CD3−CD56+ NK than CD3+CD8+ cells. Data are representative of at least three independent experiments (mean ± SD; independent samples t-test, *p < 0.05, **p < 0.01, ***p < 0.001).
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fig2: DENV infection induces a significant increase of GrzB expression. Freshly isolated PBMCs from 10 DENV-infected patients (DENV patients) and 10 Healthy Ctrls were directly stained with cell-surface and intracellular antibodies to analyze GrzB and CD107 expression in NK, CD8+ and CD4+ T cells using flow cytometry. (a) Representative flow-cytometric plots show the percentages of GrzB+ cells in total PBMCs, CD8+ and CD4+ T cells in Healthy Ctrls and DENV patients. (b) Pooled data show that the percentages of GrzB+ cells in total PBMCs and CD8+ T cells are significantly increased in DENV patients compared to Healthy Ctrls. (blue curve, Healthy Ctrl; red curve, DENV patients). (c) Representative flow-cytometric plots and pooled data show that the MFI of GrzB in NK (CD3−CD56+) cells is significantly increased in DENV patients. (d) Representative flow-cytometric plots and pooled data show that the percentages of CD107+GrzB+ cells in CD8+ T or CD56+ NK cells are significantly increased in DENV patients compared to Healthy Ctrls. (e) A representative two-tier flow-cytometric analysis and pooled data show that GrzB+ cells in PBMCs of DENV patients contain more CD3−CD56+ NK than CD3+CD8+ cells. Data are representative of at least three independent experiments (mean ± SD; independent samples t-test, *p < 0.05, **p < 0.01, ***p < 0.001).
Mentions: We detected GrzB expression in DENV-infected patients using intracellular cytokine staining (ICS) and flow-cytometric analysis. Supplementary Figure S2 shows the strategy for gating CD56+ NK cells, CD8+ and CD4+ T cells. GrzB protein was significantly up-regulated in total PBMCs, CD8+ T cells, and CD56+ NK cells of DENV-infected patients, but the mRNA level of GrzB in PBMCs of DENV-infected patients was not significantly increased compared with Healthy Ctrls (Figure 2a–c; Supplementary Figure S3). These results suggest the post-transcriptional targeting of GrzB mRNA by miR-378. Consistently, a marker for the degranulation capacity of cytotoxic lymphocytes, CD107a, which is a lysozyme-associated membrane glycoprotein,24,25 was also significantly up-regulated in CD8+ T cells and NK cells of DENV-infected patients (Figure 2d). Given that both CD56+ NK cells and CD8+ T cells produce appreciable amounts of GrzB, we therefore identified which cell sup-population was the major source of GrzB. Notably, the percentages of CD56+ NK (CD3−CD56+) cells in total GrzB+ PBMCs of DENV-infected patients were much higher than the percentages of CD3+CD8+ T cells (Figure 2e), which suggests that CD56+ NK cells are the major source of GrzB during DENV infection. Taken together, these results suggest that the down-regulation of miR-378 contributes to the up-regulation of GrzB protein expression, particularly in NK cells, but it does not influence GrzB mRNA levels in DENV-infected patients.

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