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PI3K/Akt signaling pathway modulates influenza virus induced mouse alveolar macrophage polarization to M1/M2b.

Zhao X, Dai J, Xiao X, Wu L, Zeng J, Sheng J, Su J, Chen X, Wang G, Li K - PLoS ONE (2014)

Bottom Line: Protein expression assay showed similar results as the gene expression analysis for phenotype verification.Immunofluorescence showed that iNOS expression was up-regulated but not Arg1 expression.In conclusion, our results demonstrate the dynamic polarization of AM induced by influenza viruses, and suggested that PI3K/Akt signaling pathway modulates AM polarization to M1/M2b.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Shantou University Medical College, College, Shantou, Guangdong, China.

ABSTRACT
Macrophages polarized to M1 (pro-inflammation) or M2 (anti-inflammation) phenotypes in response to environmental signals. In this study, we examined the polarization of alveolar macrophage (AM), following induction by different influenza virus strains (ST169 (H1N1), ST602 (H3N2) and HKG9 (H9N2)). Macrophages from other tissues or cell line exert alternative responding pattern, and AM is necessary for investigating the respiratory system. AM polarized toward the M1 phenotype after 4 hours of infection by all three virus strains, and AM to presented M2b phenotype after 8 hours induction, and immunosuppressive phenotype after 24 hours of induction. Protein expression assay showed similar results as the gene expression analysis for phenotype verification. The ELISA assay showed that TNF-α secretion was up-regulated after 4 and 8 hours of infection by influenza viruses, and it returned to basal levels after 24 hours of infection. IL-10 expression was elevated after 8 and 24 hours of infection. Immunofluorescence showed that iNOS expression was up-regulated but not Arg1 expression. Influenza virus notably increased phospho-Akt but not phospho-Erk1/2 or phospho-p38, and the AM polarization pattern have been changed by LY294002 (PI3K inhibitor). In conclusion, our results demonstrate the dynamic polarization of AM induced by influenza viruses, and suggested that PI3K/Akt signaling pathway modulates AM polarization to M1/M2b.

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Related in: MedlinePlus

The Quantity and Purity of AM.(A) showed that AM was detected by flow cytometry, result (a) showed that AM (R1, framed region) is 51.1% of BAL cells. (b) and (c) showed AM is framed as Alexa Fluor 488 labeled F4/80 positive cells (d) showed AM was purified by adhere to the plate and the purity was detected as 95.2% by flow cytometry. (B) showed AM was purified by adhere to the coverslip, Alexa Fluor 488 labeled rabbit anti mouse F4/80 was used as the macrophage membrane marker, and the nuclear was stained by Hoechst, negative cells were red arrow headed; negative (MDCK cells) and positive (Raw264.7 cells) control was processed follow the same condition. (C) showed the purity from five independent microscope captures (of picture B) was detected as 96.8%.
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pone-0104506-g001: The Quantity and Purity of AM.(A) showed that AM was detected by flow cytometry, result (a) showed that AM (R1, framed region) is 51.1% of BAL cells. (b) and (c) showed AM is framed as Alexa Fluor 488 labeled F4/80 positive cells (d) showed AM was purified by adhere to the plate and the purity was detected as 95.2% by flow cytometry. (B) showed AM was purified by adhere to the coverslip, Alexa Fluor 488 labeled rabbit anti mouse F4/80 was used as the macrophage membrane marker, and the nuclear was stained by Hoechst, negative cells were red arrow headed; negative (MDCK cells) and positive (Raw264.7 cells) control was processed follow the same condition. (C) showed the purity from five independent microscope captures (of picture B) was detected as 96.8%.

Mentions: Converging studies have shown that M1 and M2 macrophages are functionally polarized in response to microorganisms and host mediators. Here, we freshly purified AM to detect the polarization pattern induced by different influenza subtypes and to demonstrate the function of AM in acute lung injury induced by influenza virus. Flow cytometry showed that the F4/80 positive cells were AM (Figure 1A.a.b.c), and the total number of AM obtained from 36 mice is approximately 1.5×107 cells. Then, AM was purified by adherence to a culture dish, and the purity was determined through two experiments. Immunofluorescence showed AM have a purity to be 96.8% (Figure 1B and C), red arrow headed the negative cells (Figure 1B); Figure 1A.d showed flow cytometer analysis result, in which AM has a purity of 95.2%, which is similar to the result of Figure 1B and C.


PI3K/Akt signaling pathway modulates influenza virus induced mouse alveolar macrophage polarization to M1/M2b.

Zhao X, Dai J, Xiao X, Wu L, Zeng J, Sheng J, Su J, Chen X, Wang G, Li K - PLoS ONE (2014)

The Quantity and Purity of AM.(A) showed that AM was detected by flow cytometry, result (a) showed that AM (R1, framed region) is 51.1% of BAL cells. (b) and (c) showed AM is framed as Alexa Fluor 488 labeled F4/80 positive cells (d) showed AM was purified by adhere to the plate and the purity was detected as 95.2% by flow cytometry. (B) showed AM was purified by adhere to the coverslip, Alexa Fluor 488 labeled rabbit anti mouse F4/80 was used as the macrophage membrane marker, and the nuclear was stained by Hoechst, negative cells were red arrow headed; negative (MDCK cells) and positive (Raw264.7 cells) control was processed follow the same condition. (C) showed the purity from five independent microscope captures (of picture B) was detected as 96.8%.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126709&req=5

pone-0104506-g001: The Quantity and Purity of AM.(A) showed that AM was detected by flow cytometry, result (a) showed that AM (R1, framed region) is 51.1% of BAL cells. (b) and (c) showed AM is framed as Alexa Fluor 488 labeled F4/80 positive cells (d) showed AM was purified by adhere to the plate and the purity was detected as 95.2% by flow cytometry. (B) showed AM was purified by adhere to the coverslip, Alexa Fluor 488 labeled rabbit anti mouse F4/80 was used as the macrophage membrane marker, and the nuclear was stained by Hoechst, negative cells were red arrow headed; negative (MDCK cells) and positive (Raw264.7 cells) control was processed follow the same condition. (C) showed the purity from five independent microscope captures (of picture B) was detected as 96.8%.
Mentions: Converging studies have shown that M1 and M2 macrophages are functionally polarized in response to microorganisms and host mediators. Here, we freshly purified AM to detect the polarization pattern induced by different influenza subtypes and to demonstrate the function of AM in acute lung injury induced by influenza virus. Flow cytometry showed that the F4/80 positive cells were AM (Figure 1A.a.b.c), and the total number of AM obtained from 36 mice is approximately 1.5×107 cells. Then, AM was purified by adherence to a culture dish, and the purity was determined through two experiments. Immunofluorescence showed AM have a purity to be 96.8% (Figure 1B and C), red arrow headed the negative cells (Figure 1B); Figure 1A.d showed flow cytometer analysis result, in which AM has a purity of 95.2%, which is similar to the result of Figure 1B and C.

Bottom Line: Protein expression assay showed similar results as the gene expression analysis for phenotype verification.Immunofluorescence showed that iNOS expression was up-regulated but not Arg1 expression.In conclusion, our results demonstrate the dynamic polarization of AM induced by influenza viruses, and suggested that PI3K/Akt signaling pathway modulates AM polarization to M1/M2b.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Shantou University Medical College, College, Shantou, Guangdong, China.

ABSTRACT
Macrophages polarized to M1 (pro-inflammation) or M2 (anti-inflammation) phenotypes in response to environmental signals. In this study, we examined the polarization of alveolar macrophage (AM), following induction by different influenza virus strains (ST169 (H1N1), ST602 (H3N2) and HKG9 (H9N2)). Macrophages from other tissues or cell line exert alternative responding pattern, and AM is necessary for investigating the respiratory system. AM polarized toward the M1 phenotype after 4 hours of infection by all three virus strains, and AM to presented M2b phenotype after 8 hours induction, and immunosuppressive phenotype after 24 hours of induction. Protein expression assay showed similar results as the gene expression analysis for phenotype verification. The ELISA assay showed that TNF-α secretion was up-regulated after 4 and 8 hours of infection by influenza viruses, and it returned to basal levels after 24 hours of infection. IL-10 expression was elevated after 8 and 24 hours of infection. Immunofluorescence showed that iNOS expression was up-regulated but not Arg1 expression. Influenza virus notably increased phospho-Akt but not phospho-Erk1/2 or phospho-p38, and the AM polarization pattern have been changed by LY294002 (PI3K inhibitor). In conclusion, our results demonstrate the dynamic polarization of AM induced by influenza viruses, and suggested that PI3K/Akt signaling pathway modulates AM polarization to M1/M2b.

Show MeSH
Related in: MedlinePlus