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Role of JAK-STAT signaling in maturation of phagosomes containing Staphylococcus aureus.

Zhu F, Zhou Y, Jiang C, Zhang X - Sci Rep (2015)

Bottom Line: Phagocytosis is a required mechanism for the defense against pathogens.However, the mechanism of phagocytosis against S. aureus has not been intensively investigated.This finding presented that the PG-activated JAK-STAT pathway was required for phagosome maturation.

View Article: PubMed Central - PubMed

Affiliation: Collaborative Innovation Center of Deep Sea Biology, Key Laboratory of Animal Virology of Ministry of Agriculture and College of Life Sciences, Zhejiang University, Hangzhou 310058, China.

ABSTRACT
Phagocytosis is a required mechanism for the defense against pathogens. Staphylococcus aureus, an important bacterial pathogen, can promptly escape from phagosomes and proliferate within the cytoplasm of host. However, the mechanism of phagocytosis against S. aureus has not been intensively investigated. In this study, the S. aureus was engulfed by macrophages (RAW264.7 cells) but not digested by the cells, suggesting that the phagosomes did not maturate in macrophages. Further investigation revealed that peptidoglycan (PG) induced the phagosome maturation of macrophages, resulting in the eradication of S. aureus. Genome-wide analysis and quantitative real-time PCR indicated that the JAK-STAT pathway was activated by PG during the phagosome maturation of macrophages against S. aureus. This finding presented that the PG-activated JAK-STAT pathway was required for phagosome maturation. Therefore, our study contributed evidence that revealed a novel aspect of PG-triggered JAK-STAT pathway in the phagosome maturation of macrophages.

No MeSH data available.


Related in: MedlinePlus

The role of JAK-STAT pathway in phagosome maturation.(A) Confocal microscopy of phagocytosis of inactivated S. aureus by RAW264.7 cells. RAW264.7 cells were inoculated with the JAK2 inhibitor, followed by the incubation with the FITC-labeled inactivated S. aureus (up) or the pHrodo-labeled S. aureus (down) in the presence or absence of PG. One hour later, the cells were examined with confocal microscopy. The cells without the JAK2 inhibitor were used as controls. Scale bar, 10 μm. (B) Localization of LAMP1 protein expressed in RAW 264.7 cells. The green points indicated the FITC-labeled S. aureus. Scale bar, 10 μm. (C) The evaluation of the total S. aureus with a pH-independent strategy. The RAW 264.7 cells were treated with PG, the JAK2 inhibitor and S. aureus. At 2 h after treatments, the total S. aureus in cells were detected with PCR using the S. aureus nuc gene-specific primers. M indicates the DNA marker. (D) The percentage of phagocytosed pHrodo-labeled inactivated S. aureus in RAW264.7 cells. The number of cells phagocytosing the pHrodo-labeled inactivated S. aureus was quantified using flow cytometry at 1 h after inoculation of S. aureus. The cells without the JAK2 inhibitor were used as controls. Statistically significant differences between treatments were indicated with asterisks (**P < 0.01). (E) The measurement of pH values. The phagosome pH was measured in RAW 264.7 cells using the dual-labeled S. aureus ratiometric assay. (F) Effects of the inhibition of JAK2 activity on expressions of genes of JAK-STAT pathway. RAW264.7 cells were treated with the JAK2 inhibitor. Six hours later, the expressions of STAT3 and STAT5a genes were evaluated using real-time PCR. Statistically significant differences between treatments were indicated with asterisks (*P < 0.05). (G) The influence of the inhibition of JAK activity on the STAT3 phosphorylation. The JAK2-inhibitor-treated RAW264.7 cells were analyzed with Western blot using phosphorylated STAT3-specific antibody. Actin was used as a control. (H) The model of PG-triggered JAK-STAT signaling pathway in the phagosome maturation.
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f3: The role of JAK-STAT pathway in phagosome maturation.(A) Confocal microscopy of phagocytosis of inactivated S. aureus by RAW264.7 cells. RAW264.7 cells were inoculated with the JAK2 inhibitor, followed by the incubation with the FITC-labeled inactivated S. aureus (up) or the pHrodo-labeled S. aureus (down) in the presence or absence of PG. One hour later, the cells were examined with confocal microscopy. The cells without the JAK2 inhibitor were used as controls. Scale bar, 10 μm. (B) Localization of LAMP1 protein expressed in RAW 264.7 cells. The green points indicated the FITC-labeled S. aureus. Scale bar, 10 μm. (C) The evaluation of the total S. aureus with a pH-independent strategy. The RAW 264.7 cells were treated with PG, the JAK2 inhibitor and S. aureus. At 2 h after treatments, the total S. aureus in cells were detected with PCR using the S. aureus nuc gene-specific primers. M indicates the DNA marker. (D) The percentage of phagocytosed pHrodo-labeled inactivated S. aureus in RAW264.7 cells. The number of cells phagocytosing the pHrodo-labeled inactivated S. aureus was quantified using flow cytometry at 1 h after inoculation of S. aureus. The cells without the JAK2 inhibitor were used as controls. Statistically significant differences between treatments were indicated with asterisks (**P < 0.01). (E) The measurement of pH values. The phagosome pH was measured in RAW 264.7 cells using the dual-labeled S. aureus ratiometric assay. (F) Effects of the inhibition of JAK2 activity on expressions of genes of JAK-STAT pathway. RAW264.7 cells were treated with the JAK2 inhibitor. Six hours later, the expressions of STAT3 and STAT5a genes were evaluated using real-time PCR. Statistically significant differences between treatments were indicated with asterisks (*P < 0.05). (G) The influence of the inhibition of JAK activity on the STAT3 phosphorylation. The JAK2-inhibitor-treated RAW264.7 cells were analyzed with Western blot using phosphorylated STAT3-specific antibody. Actin was used as a control. (H) The model of PG-triggered JAK-STAT signaling pathway in the phagosome maturation.

Mentions: To determine the role of JAK-STAT pathway in the maturation of phagosomes, RAW264.7 cells were treated with the JAK2 inhibitor, followed by the phagocytosis assays using pHrodo-labeled inactivated S. aureus. Phagosome maturation was evident after the inoculation of PG and inactivated S. aureus but not in the control (inactivated S. aureus alone) (Fig. 3A). After the addition of the JAK2 inhibitor to cells inoculated with inactivated S. aureus and PG, phagosome maturation was abolished (Fig. 3A), indicating that the PG-triggered JAK-STAT signaling pathway was essential for the phagosome maturation during phagocytosis. For colocalization of phagosome and S. aureus, we labeled LAMP1 (Lysosomal-associated membrane protein 1) with PE-modified rat antibody which shows the red fluorescence. As expected, the LAMP1 proteins localized to most lysosomes in cells treated with PG and inactivated S. aureus but not in cells treated with inactivated S. aureus alone (Fig. 3B). After the addition of the JAK2 inhibitor, the LAMP1 did not significantly localize with FITC-labeled inactivated S. aureus (Fig. 3B). We also determined the presence of S. aureus by PCR. S. aureus was detected in the RAW264.7 cells treated with the JAK2 inhibitor (Fig. 3C), which further confirmed our previous findings. The percentage of phagocytosed pHrodo-labeled inactivated S. aureus in PG-induced RAW264.7 cells was significantly higher (P < 0.01) than in PG-induced cells treated with the JAK2 inhibitor (Fig. 3D). The pH detection of phagosomes also showed that phagosomes acidified to pH < 5.0 by 30min in PG-treated cells (Fig. 3E). These findings demonstrate that the JAK-STAT pathway was required for phagosome maturation.


Role of JAK-STAT signaling in maturation of phagosomes containing Staphylococcus aureus.

Zhu F, Zhou Y, Jiang C, Zhang X - Sci Rep (2015)

The role of JAK-STAT pathway in phagosome maturation.(A) Confocal microscopy of phagocytosis of inactivated S. aureus by RAW264.7 cells. RAW264.7 cells were inoculated with the JAK2 inhibitor, followed by the incubation with the FITC-labeled inactivated S. aureus (up) or the pHrodo-labeled S. aureus (down) in the presence or absence of PG. One hour later, the cells were examined with confocal microscopy. The cells without the JAK2 inhibitor were used as controls. Scale bar, 10 μm. (B) Localization of LAMP1 protein expressed in RAW 264.7 cells. The green points indicated the FITC-labeled S. aureus. Scale bar, 10 μm. (C) The evaluation of the total S. aureus with a pH-independent strategy. The RAW 264.7 cells were treated with PG, the JAK2 inhibitor and S. aureus. At 2 h after treatments, the total S. aureus in cells were detected with PCR using the S. aureus nuc gene-specific primers. M indicates the DNA marker. (D) The percentage of phagocytosed pHrodo-labeled inactivated S. aureus in RAW264.7 cells. The number of cells phagocytosing the pHrodo-labeled inactivated S. aureus was quantified using flow cytometry at 1 h after inoculation of S. aureus. The cells without the JAK2 inhibitor were used as controls. Statistically significant differences between treatments were indicated with asterisks (**P < 0.01). (E) The measurement of pH values. The phagosome pH was measured in RAW 264.7 cells using the dual-labeled S. aureus ratiometric assay. (F) Effects of the inhibition of JAK2 activity on expressions of genes of JAK-STAT pathway. RAW264.7 cells were treated with the JAK2 inhibitor. Six hours later, the expressions of STAT3 and STAT5a genes were evaluated using real-time PCR. Statistically significant differences between treatments were indicated with asterisks (*P < 0.05). (G) The influence of the inhibition of JAK activity on the STAT3 phosphorylation. The JAK2-inhibitor-treated RAW264.7 cells were analyzed with Western blot using phosphorylated STAT3-specific antibody. Actin was used as a control. (H) The model of PG-triggered JAK-STAT signaling pathway in the phagosome maturation.
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f3: The role of JAK-STAT pathway in phagosome maturation.(A) Confocal microscopy of phagocytosis of inactivated S. aureus by RAW264.7 cells. RAW264.7 cells were inoculated with the JAK2 inhibitor, followed by the incubation with the FITC-labeled inactivated S. aureus (up) or the pHrodo-labeled S. aureus (down) in the presence or absence of PG. One hour later, the cells were examined with confocal microscopy. The cells without the JAK2 inhibitor were used as controls. Scale bar, 10 μm. (B) Localization of LAMP1 protein expressed in RAW 264.7 cells. The green points indicated the FITC-labeled S. aureus. Scale bar, 10 μm. (C) The evaluation of the total S. aureus with a pH-independent strategy. The RAW 264.7 cells were treated with PG, the JAK2 inhibitor and S. aureus. At 2 h after treatments, the total S. aureus in cells were detected with PCR using the S. aureus nuc gene-specific primers. M indicates the DNA marker. (D) The percentage of phagocytosed pHrodo-labeled inactivated S. aureus in RAW264.7 cells. The number of cells phagocytosing the pHrodo-labeled inactivated S. aureus was quantified using flow cytometry at 1 h after inoculation of S. aureus. The cells without the JAK2 inhibitor were used as controls. Statistically significant differences between treatments were indicated with asterisks (**P < 0.01). (E) The measurement of pH values. The phagosome pH was measured in RAW 264.7 cells using the dual-labeled S. aureus ratiometric assay. (F) Effects of the inhibition of JAK2 activity on expressions of genes of JAK-STAT pathway. RAW264.7 cells were treated with the JAK2 inhibitor. Six hours later, the expressions of STAT3 and STAT5a genes were evaluated using real-time PCR. Statistically significant differences between treatments were indicated with asterisks (*P < 0.05). (G) The influence of the inhibition of JAK activity on the STAT3 phosphorylation. The JAK2-inhibitor-treated RAW264.7 cells were analyzed with Western blot using phosphorylated STAT3-specific antibody. Actin was used as a control. (H) The model of PG-triggered JAK-STAT signaling pathway in the phagosome maturation.
Mentions: To determine the role of JAK-STAT pathway in the maturation of phagosomes, RAW264.7 cells were treated with the JAK2 inhibitor, followed by the phagocytosis assays using pHrodo-labeled inactivated S. aureus. Phagosome maturation was evident after the inoculation of PG and inactivated S. aureus but not in the control (inactivated S. aureus alone) (Fig. 3A). After the addition of the JAK2 inhibitor to cells inoculated with inactivated S. aureus and PG, phagosome maturation was abolished (Fig. 3A), indicating that the PG-triggered JAK-STAT signaling pathway was essential for the phagosome maturation during phagocytosis. For colocalization of phagosome and S. aureus, we labeled LAMP1 (Lysosomal-associated membrane protein 1) with PE-modified rat antibody which shows the red fluorescence. As expected, the LAMP1 proteins localized to most lysosomes in cells treated with PG and inactivated S. aureus but not in cells treated with inactivated S. aureus alone (Fig. 3B). After the addition of the JAK2 inhibitor, the LAMP1 did not significantly localize with FITC-labeled inactivated S. aureus (Fig. 3B). We also determined the presence of S. aureus by PCR. S. aureus was detected in the RAW264.7 cells treated with the JAK2 inhibitor (Fig. 3C), which further confirmed our previous findings. The percentage of phagocytosed pHrodo-labeled inactivated S. aureus in PG-induced RAW264.7 cells was significantly higher (P < 0.01) than in PG-induced cells treated with the JAK2 inhibitor (Fig. 3D). The pH detection of phagosomes also showed that phagosomes acidified to pH < 5.0 by 30min in PG-treated cells (Fig. 3E). These findings demonstrate that the JAK-STAT pathway was required for phagosome maturation.

Bottom Line: Phagocytosis is a required mechanism for the defense against pathogens.However, the mechanism of phagocytosis against S. aureus has not been intensively investigated.This finding presented that the PG-activated JAK-STAT pathway was required for phagosome maturation.

View Article: PubMed Central - PubMed

Affiliation: Collaborative Innovation Center of Deep Sea Biology, Key Laboratory of Animal Virology of Ministry of Agriculture and College of Life Sciences, Zhejiang University, Hangzhou 310058, China.

ABSTRACT
Phagocytosis is a required mechanism for the defense against pathogens. Staphylococcus aureus, an important bacterial pathogen, can promptly escape from phagosomes and proliferate within the cytoplasm of host. However, the mechanism of phagocytosis against S. aureus has not been intensively investigated. In this study, the S. aureus was engulfed by macrophages (RAW264.7 cells) but not digested by the cells, suggesting that the phagosomes did not maturate in macrophages. Further investigation revealed that peptidoglycan (PG) induced the phagosome maturation of macrophages, resulting in the eradication of S. aureus. Genome-wide analysis and quantitative real-time PCR indicated that the JAK-STAT pathway was activated by PG during the phagosome maturation of macrophages against S. aureus. This finding presented that the PG-activated JAK-STAT pathway was required for phagosome maturation. Therefore, our study contributed evidence that revealed a novel aspect of PG-triggered JAK-STAT pathway in the phagosome maturation of macrophages.

No MeSH data available.


Related in: MedlinePlus