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Autophagy facilitates antibody-enhanced dengue virus infection in human pre-basophil/mast cells.

Fang YT, Wan SW, Lu YT, Yao JH, Lin CF, Hsu LJ, Brown MG, Marshall JS, Anderson R, Lin YS - PLoS ONE (2014)

Bottom Line: Dengue virus (DENV) infection can cause severe hemorrhagic disease in humans.Our results demonstrate a significant induction of autophagy in antibody-enhanced DENV infection of pre-basophil-like KU812 and immature mast cell-like HMC-1 cells.Given the importance of ADE and FcR-bearing cells such as monocytes, macrophages and basophil/mast cells in dengue disease, the results provide insights into dengue pathogenesis and therapeutic means of control.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, National Cheng Kung University Medical College, Tainan, Taiwan.

ABSTRACT

Background: Dengue virus (DENV) infection can cause severe hemorrhagic disease in humans. Although the pathogenic mechanisms underlying severe DENV disease remain unclear, one of the possible contributing factors is antibody-dependent enhancement (ADE) which occurs when sub-neutralizing antibodies derived from a previous DENV infection enhance viral infection through interaction between virus-antibody complexes and FcR-bearing cells, such as macrophages and basophil/mast cells. Although recent reports showed that DENV induces autophagy, the relationship between antibody-enhanced DENV infection and autophagy is not clear.

Methodology/principal findings: We showed that sub-neutralizing antibodies derived from dengue patient sera enhanced DENV infection and autophagy in the KU812 pre-basophil-like cell line as well as the HMC-1 immature mast cell line. Antibody-enhanced DENV infection of KU812 cells increased the number of autophagosome vesicles, LC3 punctation, LC3-II accumulation, and p62 degradation over that seen in cells infected with DENV alone. The percentages of DENV envelope (E) protein-positive cells and LC3 puncta following antibody-enhanced DENV infection of KU812 cells were reduced by the autophagy inhibitor 3-MA. Antibody-enhanced DENV infection of HMC-1 cells showed co-localization of DENV E protein and dsRNA with autophagosomes, which was inhibited by 3-MA treatment. Furthermore, DENV infection and replication were reduced when KU812 cells were transfected with the autophagy-inhibiting Atg4BC74A mutant.

Conclusions/significance: Our results demonstrate a significant induction of autophagy in antibody-enhanced DENV infection of pre-basophil-like KU812 and immature mast cell-like HMC-1 cells. Also, autophagy plays an important role in DENV infection and replication in these cells. Given the importance of ADE and FcR-bearing cells such as monocytes, macrophages and basophil/mast cells in dengue disease, the results provide insights into dengue pathogenesis and therapeutic means of control.

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Co-localization of DENV E protein and LC3 punctation in antibody-enhanced DENV infection of KU812 cells.(A) KU812 cells were incubated with medium alone (Mock), with DENV alone, with DENV in the presence of sub-neutralizing dengue patient sera, sub-neutralizing dengue patient sera alone, with UV-inactivated DENV (iDENV) alone, or iDENV in the presence of sub-neutralizing dengue patient sera. After infection, cells were fixed, permeabilized, and stained with anti-DENV E protein (red), anti-LC3 (green), and DAPI (blue). Cells were then mounted and observed by confocal microscopy. The square areas are zoomed-in images and shown in the right panels (merge, zoom). Bar: 10 µm. The imaging data were repeated three times and one set of representative results is shown. (B) The quantification of E-positive cells (A, filled arrowheads) is shown. The means ± SD of three independent experiments are shown. ***P<0.005. (C) The quantification of LC3 punctation cells (A, empty arrowheads) is shown. The means ± SD of three independent experiments are shown. ***P<0.005. (D) The percentages of cells with E-positive and LC3 punctation (A, arrows) are shown. The means ± SD of three independent experiments are shown. ***P<0.005. (E) After 24 h post-infection, the protein levels of LC3, p62, and NS4B from total cell lysates were detected by Western blotting. β-actin served as internal control. NC: negative control (nutrient-rich medium); PC: positive control (starvation; Hank's balanced salt solution). (F and G) KU812 cells were pre-treated with or without 5 mM 3-MA for 1 h before incubation with medium alone (Mock), DENV alone, or DENV with sub-neutralizing dengue patient sera. 3-MA was maintained in the medium during DENV infection. After 24 h post-infection, the expression of DENV E protein (F) and NS4B protein (G) was detected by flow cytometry. The means ± SD of three independent experiments are shown. **P<0.01, ***P<0.005.
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pone-0110655-g002: Co-localization of DENV E protein and LC3 punctation in antibody-enhanced DENV infection of KU812 cells.(A) KU812 cells were incubated with medium alone (Mock), with DENV alone, with DENV in the presence of sub-neutralizing dengue patient sera, sub-neutralizing dengue patient sera alone, with UV-inactivated DENV (iDENV) alone, or iDENV in the presence of sub-neutralizing dengue patient sera. After infection, cells were fixed, permeabilized, and stained with anti-DENV E protein (red), anti-LC3 (green), and DAPI (blue). Cells were then mounted and observed by confocal microscopy. The square areas are zoomed-in images and shown in the right panels (merge, zoom). Bar: 10 µm. The imaging data were repeated three times and one set of representative results is shown. (B) The quantification of E-positive cells (A, filled arrowheads) is shown. The means ± SD of three independent experiments are shown. ***P<0.005. (C) The quantification of LC3 punctation cells (A, empty arrowheads) is shown. The means ± SD of three independent experiments are shown. ***P<0.005. (D) The percentages of cells with E-positive and LC3 punctation (A, arrows) are shown. The means ± SD of three independent experiments are shown. ***P<0.005. (E) After 24 h post-infection, the protein levels of LC3, p62, and NS4B from total cell lysates were detected by Western blotting. β-actin served as internal control. NC: negative control (nutrient-rich medium); PC: positive control (starvation; Hank's balanced salt solution). (F and G) KU812 cells were pre-treated with or without 5 mM 3-MA for 1 h before incubation with medium alone (Mock), DENV alone, or DENV with sub-neutralizing dengue patient sera. 3-MA was maintained in the medium during DENV infection. After 24 h post-infection, the expression of DENV E protein (F) and NS4B protein (G) was detected by flow cytometry. The means ± SD of three independent experiments are shown. **P<0.01, ***P<0.005.

Mentions: We further confirmed autophagy in DENV-infected KU812 cells using confocal microscopy to analyze the expression of DENV E protein and LC3 punctation, as a marker for autophagy. In addition to increased DENV E protein expression (Figure 2A, filled arrowhead), antibody-enhanced DENV infection also increased LC3 punctation (Figure 2A, empty arrowhead). Furthermore, KU812 cells were infected with iDENV in the presence and absence of subneutralizing dengue patient sera. The LC3 punctation also apparently increased in the iDENV infection of KUB12 cells with subneutralizing dengue patient sera (Figure 2A). The co-localization of E-protein and LC3 punctation is also shown (merge, zoom, yellow). The quantified results of E protein expression and LC3 punctation from Figure 2A are shown in Figure 2B and Figure 2C, respectively. Not every cell expressing E protein showed LC3 punctation and vice versa. We therefore further analyzed the percentages of cells with both E protein expression and LC3 punctation (Figure 2A, merge, arrow, and Figure 2D). Cell lysates collected from cells infected with DENV alone or antibody-enhanced DENV were analyzed by Western blotting to confirm autophagy induction and viral infection. LC3-II accumulation and p62 degradation as autophagy indicators as well as DENV NS4B expression were increased in the antibody-enhanced DENV-infected cells (Figure 2E). In addition, nutrient-rich medium incubation served as a negative control and stimulation of autophagy by starvation served as a positive control of autophagy (Figure 2E). It has been previously demonstrated that dsRNA, as an indicator of DENV replication, can co-localize with LC3 punctation structures in hepatocytes [28]. Here, we found that the number of dsRNA-positive KU812 cells (Figure S1, filled arrowhead) as well as LC3 puncta (Figure S1, empty arrowhead) were increased in antibody-enhanced, compared to DENV alone, infection. Also, numbers of dsRNA/LC3 punctation-positive KU812 cells were increased in antibody-enhanced DENV infection (Figure S1, merge, arrow). The co-localization of dsRNA and LC3 punctation is also shown (Figure S1, zoom, yellow). It is noteworthy that co-localization of dsRNA and LC3 punctation occurs sporadically, indicating either a transient interaction or an interaction involving only a subset of the total dsRNA. Furthermore, the autophagy inhibitor 3-MA was used to assess if modulation of autophagy may alter DENV infection. KU812 cells were infected with DENV alone or in combination with enhancing antibody and with or without 3-MA. Results showed that treatment with 3-MA inhibited DENV E and NS4B protein expression in KU812 cells whether they were inoculated with DENV alone or with enhancing antibodies (Figure 2F and 2G). A representative histogram of each group is shown in Figure S2.


Autophagy facilitates antibody-enhanced dengue virus infection in human pre-basophil/mast cells.

Fang YT, Wan SW, Lu YT, Yao JH, Lin CF, Hsu LJ, Brown MG, Marshall JS, Anderson R, Lin YS - PLoS ONE (2014)

Co-localization of DENV E protein and LC3 punctation in antibody-enhanced DENV infection of KU812 cells.(A) KU812 cells were incubated with medium alone (Mock), with DENV alone, with DENV in the presence of sub-neutralizing dengue patient sera, sub-neutralizing dengue patient sera alone, with UV-inactivated DENV (iDENV) alone, or iDENV in the presence of sub-neutralizing dengue patient sera. After infection, cells were fixed, permeabilized, and stained with anti-DENV E protein (red), anti-LC3 (green), and DAPI (blue). Cells were then mounted and observed by confocal microscopy. The square areas are zoomed-in images and shown in the right panels (merge, zoom). Bar: 10 µm. The imaging data were repeated three times and one set of representative results is shown. (B) The quantification of E-positive cells (A, filled arrowheads) is shown. The means ± SD of three independent experiments are shown. ***P<0.005. (C) The quantification of LC3 punctation cells (A, empty arrowheads) is shown. The means ± SD of three independent experiments are shown. ***P<0.005. (D) The percentages of cells with E-positive and LC3 punctation (A, arrows) are shown. The means ± SD of three independent experiments are shown. ***P<0.005. (E) After 24 h post-infection, the protein levels of LC3, p62, and NS4B from total cell lysates were detected by Western blotting. β-actin served as internal control. NC: negative control (nutrient-rich medium); PC: positive control (starvation; Hank's balanced salt solution). (F and G) KU812 cells were pre-treated with or without 5 mM 3-MA for 1 h before incubation with medium alone (Mock), DENV alone, or DENV with sub-neutralizing dengue patient sera. 3-MA was maintained in the medium during DENV infection. After 24 h post-infection, the expression of DENV E protein (F) and NS4B protein (G) was detected by flow cytometry. The means ± SD of three independent experiments are shown. **P<0.01, ***P<0.005.
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pone-0110655-g002: Co-localization of DENV E protein and LC3 punctation in antibody-enhanced DENV infection of KU812 cells.(A) KU812 cells were incubated with medium alone (Mock), with DENV alone, with DENV in the presence of sub-neutralizing dengue patient sera, sub-neutralizing dengue patient sera alone, with UV-inactivated DENV (iDENV) alone, or iDENV in the presence of sub-neutralizing dengue patient sera. After infection, cells were fixed, permeabilized, and stained with anti-DENV E protein (red), anti-LC3 (green), and DAPI (blue). Cells were then mounted and observed by confocal microscopy. The square areas are zoomed-in images and shown in the right panels (merge, zoom). Bar: 10 µm. The imaging data were repeated three times and one set of representative results is shown. (B) The quantification of E-positive cells (A, filled arrowheads) is shown. The means ± SD of three independent experiments are shown. ***P<0.005. (C) The quantification of LC3 punctation cells (A, empty arrowheads) is shown. The means ± SD of three independent experiments are shown. ***P<0.005. (D) The percentages of cells with E-positive and LC3 punctation (A, arrows) are shown. The means ± SD of three independent experiments are shown. ***P<0.005. (E) After 24 h post-infection, the protein levels of LC3, p62, and NS4B from total cell lysates were detected by Western blotting. β-actin served as internal control. NC: negative control (nutrient-rich medium); PC: positive control (starvation; Hank's balanced salt solution). (F and G) KU812 cells were pre-treated with or without 5 mM 3-MA for 1 h before incubation with medium alone (Mock), DENV alone, or DENV with sub-neutralizing dengue patient sera. 3-MA was maintained in the medium during DENV infection. After 24 h post-infection, the expression of DENV E protein (F) and NS4B protein (G) was detected by flow cytometry. The means ± SD of three independent experiments are shown. **P<0.01, ***P<0.005.
Mentions: We further confirmed autophagy in DENV-infected KU812 cells using confocal microscopy to analyze the expression of DENV E protein and LC3 punctation, as a marker for autophagy. In addition to increased DENV E protein expression (Figure 2A, filled arrowhead), antibody-enhanced DENV infection also increased LC3 punctation (Figure 2A, empty arrowhead). Furthermore, KU812 cells were infected with iDENV in the presence and absence of subneutralizing dengue patient sera. The LC3 punctation also apparently increased in the iDENV infection of KUB12 cells with subneutralizing dengue patient sera (Figure 2A). The co-localization of E-protein and LC3 punctation is also shown (merge, zoom, yellow). The quantified results of E protein expression and LC3 punctation from Figure 2A are shown in Figure 2B and Figure 2C, respectively. Not every cell expressing E protein showed LC3 punctation and vice versa. We therefore further analyzed the percentages of cells with both E protein expression and LC3 punctation (Figure 2A, merge, arrow, and Figure 2D). Cell lysates collected from cells infected with DENV alone or antibody-enhanced DENV were analyzed by Western blotting to confirm autophagy induction and viral infection. LC3-II accumulation and p62 degradation as autophagy indicators as well as DENV NS4B expression were increased in the antibody-enhanced DENV-infected cells (Figure 2E). In addition, nutrient-rich medium incubation served as a negative control and stimulation of autophagy by starvation served as a positive control of autophagy (Figure 2E). It has been previously demonstrated that dsRNA, as an indicator of DENV replication, can co-localize with LC3 punctation structures in hepatocytes [28]. Here, we found that the number of dsRNA-positive KU812 cells (Figure S1, filled arrowhead) as well as LC3 puncta (Figure S1, empty arrowhead) were increased in antibody-enhanced, compared to DENV alone, infection. Also, numbers of dsRNA/LC3 punctation-positive KU812 cells were increased in antibody-enhanced DENV infection (Figure S1, merge, arrow). The co-localization of dsRNA and LC3 punctation is also shown (Figure S1, zoom, yellow). It is noteworthy that co-localization of dsRNA and LC3 punctation occurs sporadically, indicating either a transient interaction or an interaction involving only a subset of the total dsRNA. Furthermore, the autophagy inhibitor 3-MA was used to assess if modulation of autophagy may alter DENV infection. KU812 cells were infected with DENV alone or in combination with enhancing antibody and with or without 3-MA. Results showed that treatment with 3-MA inhibited DENV E and NS4B protein expression in KU812 cells whether they were inoculated with DENV alone or with enhancing antibodies (Figure 2F and 2G). A representative histogram of each group is shown in Figure S2.

Bottom Line: Dengue virus (DENV) infection can cause severe hemorrhagic disease in humans.Our results demonstrate a significant induction of autophagy in antibody-enhanced DENV infection of pre-basophil-like KU812 and immature mast cell-like HMC-1 cells.Given the importance of ADE and FcR-bearing cells such as monocytes, macrophages and basophil/mast cells in dengue disease, the results provide insights into dengue pathogenesis and therapeutic means of control.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, National Cheng Kung University Medical College, Tainan, Taiwan.

ABSTRACT

Background: Dengue virus (DENV) infection can cause severe hemorrhagic disease in humans. Although the pathogenic mechanisms underlying severe DENV disease remain unclear, one of the possible contributing factors is antibody-dependent enhancement (ADE) which occurs when sub-neutralizing antibodies derived from a previous DENV infection enhance viral infection through interaction between virus-antibody complexes and FcR-bearing cells, such as macrophages and basophil/mast cells. Although recent reports showed that DENV induces autophagy, the relationship between antibody-enhanced DENV infection and autophagy is not clear.

Methodology/principal findings: We showed that sub-neutralizing antibodies derived from dengue patient sera enhanced DENV infection and autophagy in the KU812 pre-basophil-like cell line as well as the HMC-1 immature mast cell line. Antibody-enhanced DENV infection of KU812 cells increased the number of autophagosome vesicles, LC3 punctation, LC3-II accumulation, and p62 degradation over that seen in cells infected with DENV alone. The percentages of DENV envelope (E) protein-positive cells and LC3 puncta following antibody-enhanced DENV infection of KU812 cells were reduced by the autophagy inhibitor 3-MA. Antibody-enhanced DENV infection of HMC-1 cells showed co-localization of DENV E protein and dsRNA with autophagosomes, which was inhibited by 3-MA treatment. Furthermore, DENV infection and replication were reduced when KU812 cells were transfected with the autophagy-inhibiting Atg4BC74A mutant.

Conclusions/significance: Our results demonstrate a significant induction of autophagy in antibody-enhanced DENV infection of pre-basophil-like KU812 and immature mast cell-like HMC-1 cells. Also, autophagy plays an important role in DENV infection and replication in these cells. Given the importance of ADE and FcR-bearing cells such as monocytes, macrophages and basophil/mast cells in dengue disease, the results provide insights into dengue pathogenesis and therapeutic means of control.

Show MeSH
Related in: MedlinePlus