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Cited by 159Neuroinflammation and Neurodegeneration MechanismsTGF-β signaling in diseasesImmune cells in cancer

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TGFβ signalling plays an important role in IL4-induced alternative activation of microglia

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Zhou, Xiaolai, Spittau, Björn, Krieglstein, Kerstin·2012·Journal of Neuroinflammation
DOI 10.1186/1742-2094-9-210PMC PMC3488564
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Abstract

BACKGROUND: Microglia are the resident immune cells of the central nervous system and are accepted to be involved in a variety of neurodegenerative diseases. Several studies have demonstrated that microglia, like peripheral macrophages, exhibit two entirely different functional activation states, referred to as classical (M1) and alternative (M2) activation. TGFβ is one of the most important anti-inflammatory cytokines and its effect on inhibiting microglia or macrophage classical activation has been extensively studied. However, the role of TGFβ during alternative activation of microglia has not been described yet. METHODS: To investigate the role of TGFβ in IL4-induced microglia alternative activation, both, BV2 as well as primary microglia from new born C57BL/6 mice were used. Quantitative RT-PCR and western blots were performed to detect mRNA and protein levels of the alternative activation markers Arginase1 (Arg1) and Chitinase 3-like 3 (Ym1) after treatment with IL4, TGFβ or both. Endogenous TGFβ release after IL4 treatment was evaluated using the mink lung epithelial cell (MLEC) assay and a direct TGFβ2 ELISA. TGFβ receptor type I inhibitor and MAPK inhibitor were applied to address the involvement of TGFβ signalling and MAPK signalling in IL4-induced alternative activation of microglia. RESULTS: TGFβ enhances IL4-induced microglia alternative activation by strongly increasing the expression of Arg1 and Ym1. This synergistic effect on Arg1 induction is almost completely blocked by the application of the MAPK inhibitor, PD98059. Further, treatment of primary microglia with IL4 increased the expression and secretion of TGFβ2, suggesting an involvement of endogenous TGFβ in IL4-mediated microglia activation process. Moreover, IL4-mediated induction of Arg1 and Ym1 is impaired after blocking the TGFβ receptor I indicating that IL4-induced microglia alternative activation is dependent on active TGFβ signalling. Interestingly, treatment of primary microglia with TGFβ alone results in up regulation of the IL4 receptor alpha, indicating that TGFβ increases the sensitivity of microglia for IL4 signals. CONCLUSIONS: Taken together, our data reveal a new role for TGFβ during IL4-induced alternative activation of microglia and consolidate the essential functions of TGFβ as an anti-inflammatory molecule and immunoregulatory factor for microglia.

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Figure 1 TGFβ1 enhances IL4-induced alternative activation of microglia. (A) Primary cultured microglial cells changed their morphology from round-shaped into ramified after treated with IL4 (10 ng/ml) and TGFβ1 (1 ng/ml) for 24 hours. This cellular morphological change was enhanced when microglia were co-treated with IL4 and TGFβ1. (B) Immunofluorescence staining for Arg1 demonstrated increased staining intensity for Arg1 after treatment with IL4 or TGFβ alone. Combined treatment with IL4 and TGFβ1 strongly enhanced Arg1 immunoreactivity. Scale bars indicate 100 μm. Quantitative RT-PCR showed increased Arg1 (C) and Ym1 (D) mRNA levels in primary microglia after IL4 treatment. Co-treatment with IL4 and TGFβ1 for 24 hours significantly increased Arg1 and Ym1 mRNA levels in primary microglia. (E) Western blotting revealed increased Arg1 and Ym1 protein levels in primary microglia after treatment with IL4. Again, co-treatment with IL4 and TGFβ1 increased the protein levels of Arg1 and Ym1. Representative western blot results from at least three independent experiments are shown. GAPDH was used as control for equal protein loading. (F) Densitometric evaluation of Arg1 and Ym1 band intensities and statistical analysis. Data are given as means ± standard error from three independent experiments: *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance).
Figure 1 TGFβ1 enhances IL4-induced alternative activation of microglia. (A) Primary cultured microglial cells changed their morphology from round-shaped into ramified after treated with IL4 (10 ng/ml) and TGFβ1 (1 ng/ml) for 24 hours. This cellular morphological change was enhanced when microglia were co-treated with IL4 and TGFβ1. (B) Immunofluorescence staining for Arg1 demonstrated increased staining intensity for Arg1 after treatment with IL4 or TGFβ alone. Combined treatment with IL4 and TGFβ1 strongly enhanced Arg1 immunoreactivity. Scale bars indicate 100 μm. Quantitative RT-PCR showed increased Arg1 (C) and Ym1 (D) mRNA levels in primary microglia after IL4 treatment. Co-treatment with IL4 and TGFβ1 for 24 hours significantly increased Arg1 and Ym1 mRNA levels in primary microglia. (E) Western blotting revealed increased Arg1 and Ym1 protein levels in primary microglia after treatment with IL4. Again, co-treatment with IL4 and TGFβ1 increased the protein levels of Arg1 and Ym1. Representative western blot results from at least three independent experiments are shown. GAPDH was used as control for equal protein loading. (F) Densitometric evaluation of Arg1 and Ym1 band intensities and statistical analysis. Data are given as means ± standard error from three independent experiments: *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance).CC BY · PubMed Central
Figure 2 Arg1 and Ym1 expression induced by IL4 was blocked in the presence of a TGFβ receptor type I inhibitor. Primary microglia were treated with IL4 (10 ng/ml) combined either with or without TGFβ receptor type I kinase inhibitor IV (TβKI, 2 μM) for 24 hours. RNA and proteins were isolated for quantitative RT-PCR and western blotting, respectively. Quantitative RT-PCR shows that IL4 treatment significantly increased Arg1 (A) and Ym1 (B) mRNA levels (P < 0.001) which was partially blocked by co-treatment with TβKI (P < 0.01). Western blotting (C) shows Arg1 and Ym1 protein levels in primary microglia after different treatments, which were quantified by densitometric analysis, and normalized to Gapdh (D). IL4 treatment significantly increased Arg1 and Ym1 protein levels in primary microglia (P < 0.01), which was significantly blocked by TβKI in microglia (P < 0.05). Data are presented as mean ± standard error from three independent experiments: *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance).
Figure 2 Arg1 and Ym1 expression induced by IL4 was blocked in the presence of a TGFβ receptor type I inhibitor. Primary microglia were treated with IL4 (10 ng/ml) combined either with or without TGFβ receptor type I kinase inhibitor IV (TβKI, 2 μM) for 24 hours. RNA and proteins were isolated for quantitative RT-PCR and western blotting, respectively. Quantitative RT-PCR shows that IL4 treatment significantly increased Arg1 (A) and Ym1 (B) mRNA levels (P < 0.001) which was partially blocked by co-treatment with TβKI (P < 0.01). Western blotting (C) shows Arg1 and Ym1 protein levels in primary microglia after different treatments, which were quantified by densitometric analysis, and normalized to Gapdh (D). IL4 treatment significantly increased Arg1 and Ym1 protein levels in primary microglia (P < 0.01), which was significantly blocked by TβKI in microglia (P < 0.05). Data are presented as mean ± standard error from three independent experiments: *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance).CC BY · PubMed Central
Figure 3 Treatment of microglia with IL4 increased TGFβ2 expression and secretion. Primary microglia were treated with or without IL4 (10 ng/ml) for 24 hours. Total mRNA and proteins were isolated from the cells for RT-PCR and western blotting, respectively. Conditioned medium from IL4-treated microglia (MCM-IL4) as well as non-treated microglia (MCM) was collected and the mink lung epithelial cell (MLEC) assay and enzyme-linked immunosorbent assay (ELISA) were performed. Quantitative RT-PCR for TGFβ1 (A), TGFβ2 (B) and TGFβ3 (C) revealed increased TGFβ2 expression after IL4 treatment. Intracellular TGFβ2 protein levels were significantly increased (P < 0.05) in primary microglia after treatment with IL4 (D). MLEC assay (E) shows that primary microglia secreted a certain amount of inactive TGFβ, which was significantly increased by IL4 treatment (P < 0.01). Direct TGFβ2 ELISA (F) showed that TGFβ2 secretion was significantly increased after IL4 treatment (P < 0.05). All experiments were repeated at least three times. Data are presented as mean ± standard error: *P < 0.05, **P < 0.01, ***P < 0.001(Student’s t-test).
Figure 3 Treatment of microglia with IL4 increased TGFβ2 expression and secretion. Primary microglia were treated with or without IL4 (10 ng/ml) for 24 hours. Total mRNA and proteins were isolated from the cells for RT-PCR and western blotting, respectively. Conditioned medium from IL4-treated microglia (MCM-IL4) as well as non-treated microglia (MCM) was collected and the mink lung epithelial cell (MLEC) assay and enzyme-linked immunosorbent assay (ELISA) were performed. Quantitative RT-PCR for TGFβ1 (A), TGFβ2 (B) and TGFβ3 (C) revealed increased TGFβ2 expression after IL4 treatment. Intracellular TGFβ2 protein levels were significantly increased (P < 0.05) in primary microglia after treatment with IL4 (D). MLEC assay (E) shows that primary microglia secreted a certain amount of inactive TGFβ, which was significantly increased by IL4 treatment (P < 0.01). Direct TGFβ2 ELISA (F) showed that TGFβ2 secretion was significantly increased after IL4 treatment (P < 0.05). All experiments were repeated at least three times. Data are presented as mean ± standard error: *P < 0.05, **P < 0.01, ***P < 0.001(Student’s t-test).CC BY · PubMed Central
Figure 4 TGFβ1 up regulates the IL4Rα. Primary microglia were treated with TGFβ1 (1 ng/ml) for different time points and the cells were either harvested for analysing IL4Rα mRNA and protein levels, or fixed with 4% paraformaldehyde (PFA) for IL4Rα immunostaining. Quantitative RT-PCR showed that treatment with TGFβ1 increased IL4Rα mRNA levels starting after 1 hour and peaking at around 2 hours. Afterwards mRNA levels decreased again and returned to basal levels at 24 hours (A). Western blotting showed IL4Rα protein expression starting to increase after treatment with TGFβ1 for 1 hour and peaking at around 6 to 12 hours after treatment (B). After treatment with TGFβ1 for 6 and 12 hours, IL4Rα immunoreactivity (red) was increased and cell morphology changed towards a ramified phenotype compared to control cells. After 24 hours, the IL4Rα immunoreactivity (red) was decreased but the cells still presented a ramified shape with long processes (C). Scale represents 20 μm. Treatment of primary microglia with TGFβ1 (n = 5) had no effect on IL4 mRNA levels (D). Analysis of TGFβ1-mediated changes in microglial cytokine release (n = 2) demonstrated no differences in IL4 levels after TGFβ1 treatment (E). Data (A,B, D) are presented as means ± standard error: **P < 0.01, ***P < 0.001 (one-way analysis of variance).
Figure 4 TGFβ1 up regulates the IL4Rα. Primary microglia were treated with TGFβ1 (1 ng/ml) for different time points and the cells were either harvested for analysing IL4Rα mRNA and protein levels, or fixed with 4% paraformaldehyde (PFA) for IL4Rα immunostaining. Quantitative RT-PCR showed that treatment with TGFβ1 increased IL4Rα mRNA levels starting after 1 hour and peaking at around 2 hours. Afterwards mRNA levels decreased again and returned to basal levels at 24 hours (A). Western blotting showed IL4Rα protein expression starting to increase after treatment with TGFβ1 for 1 hour and peaking at around 6 to 12 hours after treatment (B). After treatment with TGFβ1 for 6 and 12 hours, IL4Rα immunoreactivity (red) was increased and cell morphology changed towards a ramified phenotype compared to control cells. After 24 hours, the IL4Rα immunoreactivity (red) was decreased but the cells still presented a ramified shape with long processes (C). Scale represents 20 μm. Treatment of primary microglia with TGFβ1 (n = 5) had no effect on IL4 mRNA levels (D). Analysis of TGFβ1-mediated changes in microglial cytokine release (n = 2) demonstrated no differences in IL4 levels after TGFβ1 treatment (E). Data (A,B, D) are presented as means ± standard error: **P < 0.01, ***P < 0.001 (one-way analysis of variance).CC BY · PubMed Central
Figure 5 Direct interactions were not observed between the TGFβ/Smad signalling pathway and the IL4/Stat6 signalling pathways. Primary microglial cells were treated with either with IL4 (10 ng/ml), TGFβ1 (1 ng/ml) alone or together for 1 and 2 hours. The cells were either fixed with 4% paraformaldehyde (PFA) for pSmad2/3 immunostaining or harvested for testing pStat6 and pSmad2 levels. The treatment of TGFβ1 alone or TGFβ1 combining IL4-induced pSmad2/3 nuclear translocation both at 1 and 2 hours while IL4 treatment alone was not able to induced pSmad2/3 nuclear translocation (A). Western blotting showed that IL4 treatment alone exclusively induced Stat6 phosphorylation but not Smad2 phosphorylation after 1 and 2 hours, while TGFβ1 treatment alone or TGFβ1 in combination with IL4 only, induced Smad2 phosphorylation but not Stat6 phosphorylation (B).
Figure 5 Direct interactions were not observed between the TGFβ/Smad signalling pathway and the IL4/Stat6 signalling pathways. Primary microglial cells were treated with either with IL4 (10 ng/ml), TGFβ1 (1 ng/ml) alone or together for 1 and 2 hours. The cells were either fixed with 4% paraformaldehyde (PFA) for pSmad2/3 immunostaining or harvested for testing pStat6 and pSmad2 levels. The treatment of TGFβ1 alone or TGFβ1 combining IL4-induced pSmad2/3 nuclear translocation both at 1 and 2 hours while IL4 treatment alone was not able to induced pSmad2/3 nuclear translocation (A). Western blotting showed that IL4 treatment alone exclusively induced Stat6 phosphorylation but not Smad2 phosphorylation after 1 and 2 hours, while TGFβ1 treatment alone or TGFβ1 in combination with IL4 only, induced Smad2 phosphorylation but not Stat6 phosphorylation (B).CC BY · PubMed Central
Figure 6 TGFβ1-mediated enhancement of IL4-induced Arg1 expression is dependent on the mitogen-activated protein (MAP) kinase pathway. BV2 cells and primary microglia were treated with IL4 (10 ng/ml), TGFβ1 (1 ng/ml) and IL4/TGFβ1 in the presence or absence of the MAP kinase inhibitor PD98059 (10 μM) for 24 hours. Total proteins were isolated and used for electrophoresis and western blotting. Arg1 protein levels were analysed by densitometric evaluation and normalised to GAPDH. The expression levels of Arg1 after co-treatment with IL4 and TGFβ1 were reduced in the presence of PD98059 in both BV2 (A) and primary microglia (B). Quantification of Arg1 expression levels in primary microglia after different treatments (C). Data are presented as means ± standard error from three independent experiments: **P < 0.01, ***P < 0.001 (two-way analysis of variance).
Figure 6 TGFβ1-mediated enhancement of IL4-induced Arg1 expression is dependent on the mitogen-activated protein (MAP) kinase pathway. BV2 cells and primary microglia were treated with IL4 (10 ng/ml), TGFβ1 (1 ng/ml) and IL4/TGFβ1 in the presence or absence of the MAP kinase inhibitor PD98059 (10 μM) for 24 hours. Total proteins were isolated and used for electrophoresis and western blotting. Arg1 protein levels were analysed by densitometric evaluation and normalised to GAPDH. The expression levels of Arg1 after co-treatment with IL4 and TGFβ1 were reduced in the presence of PD98059 in both BV2 (A) and primary microglia (B). Quantification of Arg1 expression levels in primary microglia after different treatments (C). Data are presented as means ± standard error from three independent experiments: **P < 0.01, ***P < 0.001 (two-way analysis of variance).CC BY · PubMed Central
Figure 7 Proposed model for the role of TGFβ in IL4-induced alternative microglia activation. IL4 induces expression of the alternative activation markers Ym1 and Arg1 via the IL4Rα-Stat6 pathway. TGFβ binds to its receptors TGFβ type I and type II, which form a heteromeric complex and initiate Smad-dependent and Smad-independent pathways. Exogenous TGFβ1 enhances IL4-induced Ym1 and Arg1 expression either by a direct effect on Ym1/Arg1 promoter activity or indirectly by up regulating the IL4Rα through activation of the MAP kinase (Smad-independent) pathway. Furthermore, IL4 treatment alone increased endogenous TGFβ2 expression and secretion. Autocrine TGFβ2 in turn might be able to enhance IL4-induced Arg1 expression by using similar signalling mechanisms to exogenous TGFβ1.
Figure 7 Proposed model for the role of TGFβ in IL4-induced alternative microglia activation. IL4 induces expression of the alternative activation markers Ym1 and Arg1 via the IL4Rα-Stat6 pathway. TGFβ binds to its receptors TGFβ type I and type II, which form a heteromeric complex and initiate Smad-dependent and Smad-independent pathways. Exogenous TGFβ1 enhances IL4-induced Ym1 and Arg1 expression either by a direct effect on Ym1/Arg1 promoter activity or indirectly by up regulating the IL4Rα through activation of the MAP kinase (Smad-independent) pathway. Furthermore, IL4 treatment alone increased endogenous TGFβ2 expression and secretion. Autocrine TGFβ2 in turn might be able to enhance IL4-induced Arg1 expression by using similar signalling mechanisms to exogenous TGFβ1.CC BY · PubMed Central

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Metadata

Authors
Zhou, X., Spittau, B., & Krieglstein, K.
Affiliations
University of Freiburg (DE)
Corresponding author
  • Spittau, Björn
  • Krieglstein, Kerstin
ORCID
Venue
Journal of Neuroinflammation
Publisher
Springer Science and Business Media LLC
Published
2012-09-04
Volume
9
Issue
1
Pages
210-210
ISSN
1742-2094 (ISSN-L)
DOI
10.1186/1742-2094-9-210
PMC
PMC3488564
OpenAlex
W2128215483
Cited by
159
References
60
Citations by year
2026: 3 · 2025: 12 · 2024: 16 · 2023: 13 · 2022: 11 · 2021: 14 · 2020: 10 · 2019: 10 · 2018: 13 · 2017: 6 · 2016: 16 · 2015: 14 · 2014: 12 · 2013: 9
FWCI
5.63
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Neuroinflammation (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • FreiDok plus (Universitätsbibliothek Freiburg) (submittedVersion)
Funders
Deutsche Forschungsgemeinschaft (Kr1477/10-3)
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › TGF-β signaling in diseases
  • Life Sciences › Immunology and Microbiology › Immunology › Immune cells in cancer
Keywords
microglia, alternative activation, M2 microglia, IL-4, Ym1, MAPK signaling, anti-inflammatory cytokines, neuroinflammation, neurodegenerative diseases
MeSH terms
Analysis of Variance, Animals, Animals, Newborn, Arginase, beta-N-Acetylhexosaminidases, Brain, Cells, Cultured, Enzyme Inhibitors, Enzyme-Linked Immunosorbent Assay, Mice, Inbred C57BL, Receptors, Cell Surface, RNA, Messenger, Signal Transduction, Interleukin-4, Up-Regulation, Cytokines, Transforming Growth Factor beta, Microglia, Mitogen-Activated Protein Kinases, Lectins, Mice, Interleukin-4 Receptor alpha Subunit

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