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MicroRNA 21 is a homeostatic regulator of macrophage polarization and prevents prostaglandin E2-mediated M2 generation.

Wang Z, Brandt S, Medeiros A, Wang S, Wu H, Dent A, Serezani CH - PLoS ONE (2015)

Bottom Line: PGE2 and its downstream effectors PKA and Epac inhibit miR-21 expression and enhance expression of M2 genes, and this effect is more pronounced in miR-21-/- cells.Among potential targets involved in macrophage polarization, we found that STAT3 and SOCS1 were enhanced in miR-21-/- cells and further enhanced by PGE2.We found that STAT3 was a direct target of miR-21 in macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.

ABSTRACT
Macrophages dictate both initiation and resolution of inflammation. During acute inflammation classically activated macrophages (M1) predominate, and during the resolution phase alternative macrophages (M2) are dominant. The molecular mechanisms involved in macrophage polarization are understudied. MicroRNAs are differentially expressed in M1 and M2 macrophages that influence macrophage polarization. We identified a role of miR-21 in macrophage polarization, and found that cross-talk between miR-21 and the lipid mediator prostaglandin E2 (PGE2) is a determining factor in macrophage polarization. miR-21 inhibition impairs expression of M2 signature genes but not M1 genes. PGE2 and its downstream effectors PKA and Epac inhibit miR-21 expression and enhance expression of M2 genes, and this effect is more pronounced in miR-21-/- cells. Among potential targets involved in macrophage polarization, we found that STAT3 and SOCS1 were enhanced in miR-21-/- cells and further enhanced by PGE2. We found that STAT3 was a direct target of miR-21 in macrophages. Silencing the STAT3 gene abolished PGE2-mediated expression of M2 genes in miR-21-/- macrophages. These data shed light on the molecular brakes involved in homeostatic macrophage polarization and suggest new therapeutic strategies to prevent inflammatory responses.

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miR-21 targets STAT3 in macrophages.(A) Thioglycollate-elicited macrophages from WT and miR-21 deficient mice were stimulated or not with PGE2 for the indicated times, and the expression of STAT3, STAT1, SOCS1, SOCS2, SOCS3, TIRAP, NFκB p65, and beta actin was determined by immunoblotting. (B) Elicited macrophages from WT and miR-21 deficient mice were stimulated or not with PGE2 for 24 h, and the expression of t-STAT6, pSTAT6 (Tyr641), pSTAT1 (Tyr701), pSTAT3 (Tyr705) and beta actin was determined by immunoblotting. (C) Elicited macrophages from WT were transfected with 30 nM of scrambled control, miR-21, or miR-21 antagomir for 24 h, followed by determination of STAT3, MyD88, SOCS1, and beta-actin by immunoblotting. The numbers represent mean densitometric analysis of the bands shown from 3 independent experiments. The dashed line in the figure indicates lanes in the membrane that contained experimental conditions that were run under the same experimental conditions but not pertinent and were omitted. Data are mean ± SEM; *p < 0.01 versus WT scrambled control-treated cells. (D) Raw264.7 macrophages were transfected with a luciferase construct containing the 3′ UTR of STAT3 and empty vector expressing luciferase reporter plasmid followed by the microRNA mimic miR-21 (30 nM) for 24 h, and luciferase activity was determined. Inset: The predicted miR-21 seed sequence located in the 3′-UTR of STAT3. Sequence alignment of miR-21 and STAT3 is shown, and matches are indicated by a line. Data represent mean ± SEM from at least 3 individual experiments, each performed in triplicate. *p < 0.05 versus empty vector.
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pone.0115855.g004: miR-21 targets STAT3 in macrophages.(A) Thioglycollate-elicited macrophages from WT and miR-21 deficient mice were stimulated or not with PGE2 for the indicated times, and the expression of STAT3, STAT1, SOCS1, SOCS2, SOCS3, TIRAP, NFκB p65, and beta actin was determined by immunoblotting. (B) Elicited macrophages from WT and miR-21 deficient mice were stimulated or not with PGE2 for 24 h, and the expression of t-STAT6, pSTAT6 (Tyr641), pSTAT1 (Tyr701), pSTAT3 (Tyr705) and beta actin was determined by immunoblotting. (C) Elicited macrophages from WT were transfected with 30 nM of scrambled control, miR-21, or miR-21 antagomir for 24 h, followed by determination of STAT3, MyD88, SOCS1, and beta-actin by immunoblotting. The numbers represent mean densitometric analysis of the bands shown from 3 independent experiments. The dashed line in the figure indicates lanes in the membrane that contained experimental conditions that were run under the same experimental conditions but not pertinent and were omitted. Data are mean ± SEM; *p < 0.01 versus WT scrambled control-treated cells. (D) Raw264.7 macrophages were transfected with a luciferase construct containing the 3′ UTR of STAT3 and empty vector expressing luciferase reporter plasmid followed by the microRNA mimic miR-21 (30 nM) for 24 h, and luciferase activity was determined. Inset: The predicted miR-21 seed sequence located in the 3′-UTR of STAT3. Sequence alignment of miR-21 and STAT3 is shown, and matches are indicated by a line. Data represent mean ± SEM from at least 3 individual experiments, each performed in triplicate. *p < 0.05 versus empty vector.

Mentions: To understand the mechanisms involved in miR-21 inhibition of macrophage polarization, we initially determined the expression profiles of proteins and transcription factors involved in M1 and M2 polarization. We found that 24 h after PGE2 challenge the expression of STAT3 and STAT1 but not STAT6 was enhanced in WT macrophages (Fig. 4 A and B). We also observed an increase in STAT3 expression in miR-21 -/- macrophages, which PGE2 treatment enhanced further (Fig. 4 A). We did not observe changes in the expression of NFκB p65, SOCS3, MyD88, or TIRAP (Fig. 4 A). We found that PGE2 treatment enhanced SOCS-1 expression levels in both miR-21 -/- and WT macrophages (Fig. 4 A). Enhanced STAT3 expression also correlated with increased STAT3 phosphorylation, but not STAT1 and STAT6 activation.


MicroRNA 21 is a homeostatic regulator of macrophage polarization and prevents prostaglandin E2-mediated M2 generation.

Wang Z, Brandt S, Medeiros A, Wang S, Wu H, Dent A, Serezani CH - PLoS ONE (2015)

miR-21 targets STAT3 in macrophages.(A) Thioglycollate-elicited macrophages from WT and miR-21 deficient mice were stimulated or not with PGE2 for the indicated times, and the expression of STAT3, STAT1, SOCS1, SOCS2, SOCS3, TIRAP, NFκB p65, and beta actin was determined by immunoblotting. (B) Elicited macrophages from WT and miR-21 deficient mice were stimulated or not with PGE2 for 24 h, and the expression of t-STAT6, pSTAT6 (Tyr641), pSTAT1 (Tyr701), pSTAT3 (Tyr705) and beta actin was determined by immunoblotting. (C) Elicited macrophages from WT were transfected with 30 nM of scrambled control, miR-21, or miR-21 antagomir for 24 h, followed by determination of STAT3, MyD88, SOCS1, and beta-actin by immunoblotting. The numbers represent mean densitometric analysis of the bands shown from 3 independent experiments. The dashed line in the figure indicates lanes in the membrane that contained experimental conditions that were run under the same experimental conditions but not pertinent and were omitted. Data are mean ± SEM; *p < 0.01 versus WT scrambled control-treated cells. (D) Raw264.7 macrophages were transfected with a luciferase construct containing the 3′ UTR of STAT3 and empty vector expressing luciferase reporter plasmid followed by the microRNA mimic miR-21 (30 nM) for 24 h, and luciferase activity was determined. Inset: The predicted miR-21 seed sequence located in the 3′-UTR of STAT3. Sequence alignment of miR-21 and STAT3 is shown, and matches are indicated by a line. Data represent mean ± SEM from at least 3 individual experiments, each performed in triplicate. *p < 0.05 versus empty vector.
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pone.0115855.g004: miR-21 targets STAT3 in macrophages.(A) Thioglycollate-elicited macrophages from WT and miR-21 deficient mice were stimulated or not with PGE2 for the indicated times, and the expression of STAT3, STAT1, SOCS1, SOCS2, SOCS3, TIRAP, NFκB p65, and beta actin was determined by immunoblotting. (B) Elicited macrophages from WT and miR-21 deficient mice were stimulated or not with PGE2 for 24 h, and the expression of t-STAT6, pSTAT6 (Tyr641), pSTAT1 (Tyr701), pSTAT3 (Tyr705) and beta actin was determined by immunoblotting. (C) Elicited macrophages from WT were transfected with 30 nM of scrambled control, miR-21, or miR-21 antagomir for 24 h, followed by determination of STAT3, MyD88, SOCS1, and beta-actin by immunoblotting. The numbers represent mean densitometric analysis of the bands shown from 3 independent experiments. The dashed line in the figure indicates lanes in the membrane that contained experimental conditions that were run under the same experimental conditions but not pertinent and were omitted. Data are mean ± SEM; *p < 0.01 versus WT scrambled control-treated cells. (D) Raw264.7 macrophages were transfected with a luciferase construct containing the 3′ UTR of STAT3 and empty vector expressing luciferase reporter plasmid followed by the microRNA mimic miR-21 (30 nM) for 24 h, and luciferase activity was determined. Inset: The predicted miR-21 seed sequence located in the 3′-UTR of STAT3. Sequence alignment of miR-21 and STAT3 is shown, and matches are indicated by a line. Data represent mean ± SEM from at least 3 individual experiments, each performed in triplicate. *p < 0.05 versus empty vector.
Mentions: To understand the mechanisms involved in miR-21 inhibition of macrophage polarization, we initially determined the expression profiles of proteins and transcription factors involved in M1 and M2 polarization. We found that 24 h after PGE2 challenge the expression of STAT3 and STAT1 but not STAT6 was enhanced in WT macrophages (Fig. 4 A and B). We also observed an increase in STAT3 expression in miR-21 -/- macrophages, which PGE2 treatment enhanced further (Fig. 4 A). We did not observe changes in the expression of NFκB p65, SOCS3, MyD88, or TIRAP (Fig. 4 A). We found that PGE2 treatment enhanced SOCS-1 expression levels in both miR-21 -/- and WT macrophages (Fig. 4 A). Enhanced STAT3 expression also correlated with increased STAT3 phosphorylation, but not STAT1 and STAT6 activation.

Bottom Line: PGE2 and its downstream effectors PKA and Epac inhibit miR-21 expression and enhance expression of M2 genes, and this effect is more pronounced in miR-21-/- cells.Among potential targets involved in macrophage polarization, we found that STAT3 and SOCS1 were enhanced in miR-21-/- cells and further enhanced by PGE2.We found that STAT3 was a direct target of miR-21 in macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.

ABSTRACT
Macrophages dictate both initiation and resolution of inflammation. During acute inflammation classically activated macrophages (M1) predominate, and during the resolution phase alternative macrophages (M2) are dominant. The molecular mechanisms involved in macrophage polarization are understudied. MicroRNAs are differentially expressed in M1 and M2 macrophages that influence macrophage polarization. We identified a role of miR-21 in macrophage polarization, and found that cross-talk between miR-21 and the lipid mediator prostaglandin E2 (PGE2) is a determining factor in macrophage polarization. miR-21 inhibition impairs expression of M2 signature genes but not M1 genes. PGE2 and its downstream effectors PKA and Epac inhibit miR-21 expression and enhance expression of M2 genes, and this effect is more pronounced in miR-21-/- cells. Among potential targets involved in macrophage polarization, we found that STAT3 and SOCS1 were enhanced in miR-21-/- cells and further enhanced by PGE2. We found that STAT3 was a direct target of miR-21 in macrophages. Silencing the STAT3 gene abolished PGE2-mediated expression of M2 genes in miR-21-/- macrophages. These data shed light on the molecular brakes involved in homeostatic macrophage polarization and suggest new therapeutic strategies to prevent inflammatory responses.

Show MeSH
Related in: MedlinePlus