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Cited by 169Immune cells in cancerMicroRNA in disease regulationMacrophage Migration Inhibitory Factor

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MicroRNA 21 Is a Homeostatic Regulator of Macrophage Polarization and Prevents Prostaglandin E2-Mediated M2 Generation

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Wang, Zhuo, Brandt, Stephanie, Medeiros, Alexandra, Wang, Soujuan, Wu, Hao, Dent, Alexander, Serezani, C. Henrique·2015·PLOS ONE
DOI 10.1371/journal.pone.0115855PMC PMC4338261
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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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Figures

Fig 1 miR-21 deficiency impairs M1 and favors M2 macrophage polarization. (A) Thioglycollate-elicited macrophages from WT and miR-21 deficient mice were isolated, and the expression of Tnfa, Il12p40, Il1b, Arg1, Il10, IL-6, Retnla, Chi3l3, and Actin mRNA was determined by real time PCR. (B) Elicited macrophages were transfected with the miR-21mimic or the scrambled control (30 nM each), and the expression of Retnla, Arg1,Tnfa, Il1b and Actin was determined by real time PCR. Inset: miR-21 mRNA expression of macrophages transfected as in B. Data represent mean ± SEM from 3–5 individual experiments, each performed in triplicate. *p < 0.05 versus WT cells or mimic control.
Fig 1 miR-21 deficiency impairs M1 and favors M2 macrophage polarization. (A) Thioglycollate-elicited macrophages from WT and miR-21 deficient mice were isolated, and the expression of Tnfa, Il12p40, Il1b, Arg1, Il10, IL-6, Retnla, Chi3l3, and Actin mRNA was determined by real time PCR. (B) Elicited macrophages were transfected with the miR-21mimic or the scrambled control (30 nM each), and the expression of Retnla, Arg1,Tnfa, Il1b and Actin was determined by real time PCR. Inset: miR-21 mRNA expression of macrophages transfected as in B. Data represent mean ± SEM from 3–5 individual experiments, each performed in triplicate. *p < 0.05 versus WT cells or mimic control.CC BY · PubMed Central
Fig 2 PGE2/PKA/Epac axis inhibits miR-21 expression in macrophages. Elicited macrophages were treated or not with 1 μM PGE2 (A) or 500 μM of PKA-specific cAMP analog 6-Bnz-cAMP or Epac-specific cAMP analog 8-pCPT-2-O-Me-cAMP each (B) for the indicated times, and the expression of miR-21 was determined by real time PCR. Data represent mean ± SEM from 3–5 individual experiments, each performed in triplicate. *p < 0.05 versus unstimulated cells; # p<0.05 versus cells stimulated with PKA.
Fig 2 PGE2/PKA/Epac axis inhibits miR-21 expression in macrophages. Elicited macrophages were treated or not with 1 μM PGE2 (A) or 500 μM of PKA-specific cAMP analog 6-Bnz-cAMP or Epac-specific cAMP analog 8-pCPT-2-O-Me-cAMP each (B) for the indicated times, and the expression of miR-21 was determined by real time PCR. Data represent mean ± SEM from 3–5 individual experiments, each performed in triplicate. *p < 0.05 versus unstimulated cells; # p<0.05 versus cells stimulated with PKA.CC BY · PubMed Central
Fig 3 Absence of miR-21 amplifies PGE2-mediated M2 polarization. Thioglycollate-elicited macrophages from WT and miR-21 deficient mice were stimulated or not with PGE2 for 4 or 24 h, and the expression of Tnfa, Nos2, Arg1, Mmp2, and Chi3l3 mRNA was determined by real time PCR. Data represent mean ± SEM from 3–5 individual experiments, each performed in triplicate. *p < 0.05 versus unstimulated cells and p<0.05 versus WT macrophages; # p < 0.05 versus WT cells stimulated with PGE2 for 24h.
Fig 3 Absence of miR-21 amplifies PGE2-mediated M2 polarization. Thioglycollate-elicited macrophages from WT and miR-21 deficient mice were stimulated or not with PGE2 for 4 or 24 h, and the expression of Tnfa, Nos2, Arg1, Mmp2, and Chi3l3 mRNA was determined by real time PCR. Data represent mean ± SEM from 3–5 individual experiments, each performed in triplicate. *p < 0.05 versus unstimulated cells and p<0.05 versus WT macrophages; # p < 0.05 versus WT cells stimulated with PGE2 for 24h.CC BY · PubMed Central
Fig 4 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.
Fig 4 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.CC BY · PubMed Central
Fig 5 PGE2 utilizes STAT3 signaling to enhance M2 macrophages in miR-21 deficient macrophages. WT and miR-21 deficient macrophages were treated with 30 nM STAT3 siRNA or siRNA control for 24 h and stimulated or not with PGE2 for another 24 h. Expression of (A) Stat3, (B) Chi3l3, (C) Retnla, and (D) Il6, (E) Il10 mRNA were determined by real time PCR. (F) Proposed model of miR-21 and PGE2-mediated M2 macrophage generation. Data represent mean ± SEM from at least 3 individual experiments, each performed in triplicate. *p < 0.05 versus WT siControl; #p<0.05 versus miR-21 -/- cells and &p<0.05 versus STAT3 siRNA control.
Fig 5 PGE2 utilizes STAT3 signaling to enhance M2 macrophages in miR-21 deficient macrophages. WT and miR-21 deficient macrophages were treated with 30 nM STAT3 siRNA or siRNA control for 24 h and stimulated or not with PGE2 for another 24 h. Expression of (A) Stat3, (B) Chi3l3, (C) Retnla, and (D) Il6, (E) Il10 mRNA were determined by real time PCR. (F) Proposed model of miR-21 and PGE2-mediated M2 macrophage generation. Data represent mean ± SEM from at least 3 individual experiments, each performed in triplicate. *p < 0.05 versus WT siControl; #p<0.05 versus miR-21 -/- cells and &p<0.05 versus STAT3 siRNA control.CC BY · PubMed Central

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Metadata

Authors
Wang, Z., Brandt, S., Medeiros, A., Wang, S., Wu, H., Dent, A., & Serezani, C. H.
Affiliations
Indiana University School of Medicine (US)
Corresponding author
Serezani, C. Henrique
ORCID
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2015-02-23
Volume
10
Issue
2
Pages
e0115855
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0115855
PMC
PMC4338261
OpenAlex
W2015054805
Cited by
169
References
58
Citations by year
2026: 7 · 2025: 9 · 2024: 12 · 2023: 6 · 2022: 22 · 2021: 23 · 2020: 18 · 2019: 17 · 2018: 14 · 2017: 15 · 2016: 9 · 2015: 3
FWCI
6.74
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Acervo Digital da Universidade Estadual Paulista (Universidade Estadual Paulista) (submittedVersion, other-oa)
  • Americanae (AECID Library) (submittedVersion, other-oa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • LA Referencia (Red Federada de Repositorios Institucionales de Publicaciones Científicas) (submittedVersion, other-oa)
  • IUScholarWorks (Indiana University) (submittedVersion)
  • PMC
Funders
  • Ralph W. and Grace M. Showalter Research Trust Fund
  • National Institutes of Health (HL-103777-01, HL-124159-01, HL-103777)
  • National Heart, Lung, and Blood Institute (HL-124159-01, HL10377701, K99HL103777, R01HL124159, R00HL103777)
  • National Institute of Allergy and Infectious Diseases (R21AI079349, T32AI060519)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Immune cells in cancer
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Life Sciences › Immunology and Microbiology › Immunology › Macrophage Migration Inhibitory Factor
Keywords
miR-21, macrophage polarization, M2 macrophages, prostaglandin E2, STAT3, SOCS1, protein kinase A, Epac, inflammation resolution, microRNA regulation, M1 macrophages
MeSH terms
Suppressor of Cytokine Signaling 1 Protein, Acetylcysteine, Animals, Erythromycin, Female, Homeostasis, Macrophage Activation, Macrophages, Dinoprostone, Cell Polarity, Cyclic AMP-Dependent Protein Kinases, Mice, Knockout, Gene Silencing, MicroRNAs, STAT3 Transcription Factor, Suppressor of Cytokine Signaling Proteins, Mice

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