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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: 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.Therefore, our study contributed evidence that revealed a novel aspect of PG-triggered JAK-STAT pathway in the phagosome maturation of macrophages.

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 effect of PG-induced JAK-STAT pathway on phagocytosis against live S. aureus.(A) RAW264.7 cells were activated by PG, followed by incubation with live S. aureus. One hour or one day later, the cells were examined with transmission electron microscope. The black arrows indicate the intracellular S. aureus. Lane headings indicated the treatments. The box indicated the enlarged image. Scale bar, 2 μm (up) or 1 μm (down). (B) Localization of LAMP1 expressed in RAW 264.7 cells. The green points showed the FITC-labeled S. aureus. Scale bar, 10 μm. (C) The expressions of genes from the JAK-STAT pathway in RAW264.7 cells in response to PG challenge. At 1 h after challenge with live S. aureus or live S. aureus + PG or PG alone, the expression profiles of Jak2, Stat3, Stat5a, Cish, Csf1, IL-6, Socs3 and Nfkb1, TNF-α and IFN-γ genes in RAW264.7 cells were examined with real-time PCR. The GAPDH gene was used as a control. Statistically significant differences between treatments were indicated with asterisks (**P < 0.01). Control, RAW264.7 cells only. (D) RAW264.7 cells were inoculated with the JAK2 inhibitor, followed by incubation with the FITC-labeled 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 micrscopy. The cells without the JAK2 inhibitor were used as controls. Scale bar, 10 μm. (E) The pH values in macrophages. The phagosome pH was measured in RAW 264.7 cells using the dual-labeled S. aureus ratiometric assay. (F) The examination of the total S. aureus in RAW 264.7 cells. 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. (G) The percentage of phagocytosed pHrodo-labeled S. aureus in RAW264.7 cells. The number of cells phagocytosed the pHrodo-labeled 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).
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f4: The effect of PG-induced JAK-STAT pathway on phagocytosis against live S. aureus.(A) RAW264.7 cells were activated by PG, followed by incubation with live S. aureus. One hour or one day later, the cells were examined with transmission electron microscope. The black arrows indicate the intracellular S. aureus. Lane headings indicated the treatments. The box indicated the enlarged image. Scale bar, 2 μm (up) or 1 μm (down). (B) Localization of LAMP1 expressed in RAW 264.7 cells. The green points showed the FITC-labeled S. aureus. Scale bar, 10 μm. (C) The expressions of genes from the JAK-STAT pathway in RAW264.7 cells in response to PG challenge. At 1 h after challenge with live S. aureus or live S. aureus + PG or PG alone, the expression profiles of Jak2, Stat3, Stat5a, Cish, Csf1, IL-6, Socs3 and Nfkb1, TNF-α and IFN-γ genes in RAW264.7 cells were examined with real-time PCR. The GAPDH gene was used as a control. Statistically significant differences between treatments were indicated with asterisks (**P < 0.01). Control, RAW264.7 cells only. (D) RAW264.7 cells were inoculated with the JAK2 inhibitor, followed by incubation with the FITC-labeled 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 micrscopy. The cells without the JAK2 inhibitor were used as controls. Scale bar, 10 μm. (E) The pH values in macrophages. The phagosome pH was measured in RAW 264.7 cells using the dual-labeled S. aureus ratiometric assay. (F) The examination of the total S. aureus in RAW 264.7 cells. 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. (G) The percentage of phagocytosed pHrodo-labeled S. aureus in RAW264.7 cells. The number of cells phagocytosed the pHrodo-labeled 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).

Mentions: We next determined the effects of PG on phagosome maturation after RAW264.7 cells were inoculated with live S. aureus. Using transmission electron microscopy, we observed no bacteria in the cells at 1 hour after inoculation (Fig. 4A). At one day after inoculation, the cells treated with live S. aureus alone were dead and many bacteria were present (Fig. 4A). However, the cells treated with live S. aureus and PG grew normally and no bacteria were present (Fig. 4A). The LAMP1 proteins were colocalized with many lysosomes in cells treated with PG and S.aureus but not in cells treated with S. aureus alone (Fig. 4B). These data suggested that PG could activate phagosome maturation in order to phagocytose bacteria.


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

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

The effect of PG-induced JAK-STAT pathway on phagocytosis against live S. aureus.(A) RAW264.7 cells were activated by PG, followed by incubation with live S. aureus. One hour or one day later, the cells were examined with transmission electron microscope. The black arrows indicate the intracellular S. aureus. Lane headings indicated the treatments. The box indicated the enlarged image. Scale bar, 2 μm (up) or 1 μm (down). (B) Localization of LAMP1 expressed in RAW 264.7 cells. The green points showed the FITC-labeled S. aureus. Scale bar, 10 μm. (C) The expressions of genes from the JAK-STAT pathway in RAW264.7 cells in response to PG challenge. At 1 h after challenge with live S. aureus or live S. aureus + PG or PG alone, the expression profiles of Jak2, Stat3, Stat5a, Cish, Csf1, IL-6, Socs3 and Nfkb1, TNF-α and IFN-γ genes in RAW264.7 cells were examined with real-time PCR. The GAPDH gene was used as a control. Statistically significant differences between treatments were indicated with asterisks (**P < 0.01). Control, RAW264.7 cells only. (D) RAW264.7 cells were inoculated with the JAK2 inhibitor, followed by incubation with the FITC-labeled 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 micrscopy. The cells without the JAK2 inhibitor were used as controls. Scale bar, 10 μm. (E) The pH values in macrophages. The phagosome pH was measured in RAW 264.7 cells using the dual-labeled S. aureus ratiometric assay. (F) The examination of the total S. aureus in RAW 264.7 cells. 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. (G) The percentage of phagocytosed pHrodo-labeled S. aureus in RAW264.7 cells. The number of cells phagocytosed the pHrodo-labeled 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).
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f4: The effect of PG-induced JAK-STAT pathway on phagocytosis against live S. aureus.(A) RAW264.7 cells were activated by PG, followed by incubation with live S. aureus. One hour or one day later, the cells were examined with transmission electron microscope. The black arrows indicate the intracellular S. aureus. Lane headings indicated the treatments. The box indicated the enlarged image. Scale bar, 2 μm (up) or 1 μm (down). (B) Localization of LAMP1 expressed in RAW 264.7 cells. The green points showed the FITC-labeled S. aureus. Scale bar, 10 μm. (C) The expressions of genes from the JAK-STAT pathway in RAW264.7 cells in response to PG challenge. At 1 h after challenge with live S. aureus or live S. aureus + PG or PG alone, the expression profiles of Jak2, Stat3, Stat5a, Cish, Csf1, IL-6, Socs3 and Nfkb1, TNF-α and IFN-γ genes in RAW264.7 cells were examined with real-time PCR. The GAPDH gene was used as a control. Statistically significant differences between treatments were indicated with asterisks (**P < 0.01). Control, RAW264.7 cells only. (D) RAW264.7 cells were inoculated with the JAK2 inhibitor, followed by incubation with the FITC-labeled 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 micrscopy. The cells without the JAK2 inhibitor were used as controls. Scale bar, 10 μm. (E) The pH values in macrophages. The phagosome pH was measured in RAW 264.7 cells using the dual-labeled S. aureus ratiometric assay. (F) The examination of the total S. aureus in RAW 264.7 cells. 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. (G) The percentage of phagocytosed pHrodo-labeled S. aureus in RAW264.7 cells. The number of cells phagocytosed the pHrodo-labeled 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).
Mentions: We next determined the effects of PG on phagosome maturation after RAW264.7 cells were inoculated with live S. aureus. Using transmission electron microscopy, we observed no bacteria in the cells at 1 hour after inoculation (Fig. 4A). At one day after inoculation, the cells treated with live S. aureus alone were dead and many bacteria were present (Fig. 4A). However, the cells treated with live S. aureus and PG grew normally and no bacteria were present (Fig. 4A). The LAMP1 proteins were colocalized with many lysosomes in cells treated with PG and S.aureus but not in cells treated with S. aureus alone (Fig. 4B). These data suggested that PG could activate phagosome maturation in order to phagocytose bacteria.

Bottom Line: 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.Therefore, our study contributed evidence that revealed a novel aspect of PG-triggered JAK-STAT pathway in the phagosome maturation of macrophages.

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