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Ubiquitous Over-Expression of Chromatin Remodeling Factor SRG3 Ameliorates the T Cell-Mediated Exacerbation of EAE by Modulating the Phenotypes of both Dendritic Cells and Macrophages.

Lee SW, Park HJ, Jeon SH, Lee C, Seong RH, Park SH, Hong S - PLoS ONE (2015)

Bottom Line: We found that the outcome of EAE development was completely different depending on the expression pattern of SRG3.SRG3 over-expression not only reduced pro-inflammatory cytokine production by DCs but also shifted macrophages from the inducible nitric oxide synthase (iNOS)-expressing M1 phenotype to the arginase-1-expressing M2 phenotype during EAE.Taken together, our results provide the first evidence supporting the development of a new therapeutic strategy for EAE involving the modulation of SRG3 expression to induce M2 and Th2 polarization, thereby inhibiting inflammatory immune responses.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Bioscience and Biotechnology, Institute of Bioscience, Sejong University, Seoul 143-747, Korea; School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea.

ABSTRACT
Although SWI3-related gene (SRG3), a chromatin remodeling factor, is critical for various biological processes including early embryogenesis and thymocyte development, it is unclear whether SRG3 is involved in the differentiation of CD4+ T cells, the key mediator of adaptive immune responses. Because it is known that experimental autoimmune encephalomyelitis (EAE) development is determined by the activation of CD4+ T helper cells, here, we investigated the role of SRG3 in EAE development using SRG3 transgenic mouse models exhibiting two distinct SRG3 expression patterns: SRG3 expression driven by either the CD2 or β-actin promoter. We found that the outcome of EAE development was completely different depending on the expression pattern of SRG3. The specific over-expression of SRG3 using the CD2 promoter facilitated EAE via the induction of Th1 and Th17 cells, whereas the ubiquitous over-expression of SRG3 using the β-actin promoter inhibited EAE by promoting Th2 differentiation and suppressing Th1 and Th17 differentiation. In addition, the ubiquitous over-expression of SRG3 polarized CD4+ T cell differentiation towards the Th2 phenotype by converting dendritic cells (DCs) or macrophages to Th2 types. SRG3 over-expression not only reduced pro-inflammatory cytokine production by DCs but also shifted macrophages from the inducible nitric oxide synthase (iNOS)-expressing M1 phenotype to the arginase-1-expressing M2 phenotype during EAE. In addition, Th2 differentiation in β-actin-SRG3 Tg mice during EAE was associated with an increase in the basophil and mast cell populations and in IL4 production. Furthermore, the increased frequency of Treg cells in the spinal cord of β-actin-SRG3 Tg mice might induce the suppression of and accelerate the recovery from EAE symptoms. Taken together, our results provide the first evidence supporting the development of a new therapeutic strategy for EAE involving the modulation of SRG3 expression to induce M2 and Th2 polarization, thereby inhibiting inflammatory immune responses.

No MeSH data available.


Related in: MedlinePlus

SRG3 over-expression driven by the β-actin promoter promotes the phenotypic shift of macrophages from M1 to M2 in response to LPS.(A-B) Peritoneal macrophages isolated from both WT and β-actin-SRG3 Tg B6 mice were primed for 5 hrs with either IFNγ (20 ng/ml) or IL4 (20 ng/ml). (A) IFNγ-primed macrophages were stimulated with either vehicle or LPS (40, 200, or 1000 ng/ml), and 12 hrs later, IL12p40 expression was analyzed via flow cytometry. Representative data from three independent experiments are shown. (B) IL4-primed macrophages were stimulated with either vehicle or LPS (40, 200, or 1000 ng/ml) for 12 hrs, and subsequently, intracellular IL10 production was analyzed via flow cytometry. Representative data are shown (n = 3). (C-D) WT, β-actin-SRG3 Tg and CD2-SRG3 Tg B6 mice were i.p. injected with LPS (2 μg) or vehicle. (C) Sixteen hrs later, intracellular TNFα, IL12p40, and iNOS production in splenic macrophages (CD11c-CD11b+F4/80+) was assessed via flow cytometry. The means ± SD are shown in the graphs (n = 4; *P<0.05, **P<0.01). (D) After 16 hrs of stimulation, intracellular arginase-1 and IL10 production and the surface expression of Dectin-1 and MR1 in macrophages (CD11c-CD11b+F4/80+) were assessed via flow cytometry. The means ± SD are shown in the graphs (n = 4; *P<0.05, **P<0.01).
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pone.0132329.g002: SRG3 over-expression driven by the β-actin promoter promotes the phenotypic shift of macrophages from M1 to M2 in response to LPS.(A-B) Peritoneal macrophages isolated from both WT and β-actin-SRG3 Tg B6 mice were primed for 5 hrs with either IFNγ (20 ng/ml) or IL4 (20 ng/ml). (A) IFNγ-primed macrophages were stimulated with either vehicle or LPS (40, 200, or 1000 ng/ml), and 12 hrs later, IL12p40 expression was analyzed via flow cytometry. Representative data from three independent experiments are shown. (B) IL4-primed macrophages were stimulated with either vehicle or LPS (40, 200, or 1000 ng/ml) for 12 hrs, and subsequently, intracellular IL10 production was analyzed via flow cytometry. Representative data are shown (n = 3). (C-D) WT, β-actin-SRG3 Tg and CD2-SRG3 Tg B6 mice were i.p. injected with LPS (2 μg) or vehicle. (C) Sixteen hrs later, intracellular TNFα, IL12p40, and iNOS production in splenic macrophages (CD11c-CD11b+F4/80+) was assessed via flow cytometry. The means ± SD are shown in the graphs (n = 4; *P<0.05, **P<0.01). (D) After 16 hrs of stimulation, intracellular arginase-1 and IL10 production and the surface expression of Dectin-1 and MR1 in macrophages (CD11c-CD11b+F4/80+) were assessed via flow cytometry. The means ± SD are shown in the graphs (n = 4; *P<0.05, **P<0.01).

Mentions: Emerging evidence has demonstrated that macrophages are involved in many physiological regulatory functions, such as thermogenesis and wound healing, in addition to classical immune functions such as phagocytosis; the observation that macrophages perform multiple functions implies the existence of subsets of macrophages that perform distinct functions. In fact, macrophages are classified into two groups according to their cytokine profile; type 1 macrophages (M1) primarily produce IL12 and IL23, whereas type 2 macrophages (M2) produce IL10 and TGFβ [11]. Recently, it has been demonstrated that M2 macrophages modulate the development of autoimmune diseases such as EAE [26]. To investigate whether SRG3 expression in macrophages affects the phenotypes of these two distinct macrophage subsets, peritoneal macrophages from β-actin-SRG3 Tg and WT B6 mice were isolated and subsequently stimulated with IFNγ or IL4 in vitro to induce differentiation into M1 or M2 macrophages, respectively. We found that peritoneal macrophages from β-actin-SRG3 Tg mice produced IL12p40 at levels comparable to those of the control non-Tg mice. This result suggests that SRG3 over-expression does not affect the differentiation of IFNγ-stimulated peritoneal macrophages into the M1 phenotype (Fig 2A; Fig A in S2 Fig). However, we discovered that SRG3 expression significantly increased IL10-producing M2 macrophages, which might help CD4+ T cells to differentiate into Th2 types (Fig 2B; Fig B in S2 Fig). Whereas SRG3 over-expression in DCs down-regulated IL12p40 production, as shown in Fig 1A, SRG3 over-expression induced macrophages to differentiate into the IL10-producing M2 type. Taken together, these results strongly indicated that SRG3 over-expression in DCs or macrophages alters their capability to produce cytokines in a somewhat distinct manner. In addition, we examined whether the effect of SRG3 over-expression on M1/M2 polarization upon LPS-mediated activation was detectable in vivo. For this purpose, CD2-SRG3 Tg, β-actin-SRG3 Tg and WT B6 mice were i.p. injected with LPS (2 μg) or vehicle, and 16 hrs later, splenic macrophages were analyzed for the expression of M1 markers (TNFα, IL12p40, and iNOS) and M2 markers (arginase-1, Dectin-1, MR1, and IL10) via flow cytometry. Strikingly, SRG3 over-expression driven by the β-actin promoter suppressed the expression of M1 markers (TNFα, IL12p40, and iNOS) but enhanced the expression of M2 markers (arginase-1, Dectin-1, MR1, and IL10) in macrophages under both basal and LPS-activated conditions, although CD2 promoter-driven SRG3 over-expression did not affect the expression of M1 or M2 markers in macrophages. These results indicated that macrophages in CD2-SRG3 Tg mice do not overexpress SRG3 due to the lack of CD2 expression in macrophages (Fig 2C and 2D; Fig C in S2 Fig). Under LPS-stimulated conditions, the β-actin-SRG3 Tg mice displayed the down-regulation of most M2 markers (i.e., arginase-1, Dectin-1, and MR-1) except for IL10, whose production was increased in macrophages compared to the vehicle-treated conditions (Fig 2D; Fig C in S2 Fig). Thus, these results demonstrated that preferential polarization toward the M2 phenotype by β-actin promoter-driven SRG3 over-expression in macrophages was closely associated with the inhibition of M1-type gene expression, implying reciprocal regulation between the M1 and M2 subtypes.


Ubiquitous Over-Expression of Chromatin Remodeling Factor SRG3 Ameliorates the T Cell-Mediated Exacerbation of EAE by Modulating the Phenotypes of both Dendritic Cells and Macrophages.

Lee SW, Park HJ, Jeon SH, Lee C, Seong RH, Park SH, Hong S - PLoS ONE (2015)

SRG3 over-expression driven by the β-actin promoter promotes the phenotypic shift of macrophages from M1 to M2 in response to LPS.(A-B) Peritoneal macrophages isolated from both WT and β-actin-SRG3 Tg B6 mice were primed for 5 hrs with either IFNγ (20 ng/ml) or IL4 (20 ng/ml). (A) IFNγ-primed macrophages were stimulated with either vehicle or LPS (40, 200, or 1000 ng/ml), and 12 hrs later, IL12p40 expression was analyzed via flow cytometry. Representative data from three independent experiments are shown. (B) IL4-primed macrophages were stimulated with either vehicle or LPS (40, 200, or 1000 ng/ml) for 12 hrs, and subsequently, intracellular IL10 production was analyzed via flow cytometry. Representative data are shown (n = 3). (C-D) WT, β-actin-SRG3 Tg and CD2-SRG3 Tg B6 mice were i.p. injected with LPS (2 μg) or vehicle. (C) Sixteen hrs later, intracellular TNFα, IL12p40, and iNOS production in splenic macrophages (CD11c-CD11b+F4/80+) was assessed via flow cytometry. The means ± SD are shown in the graphs (n = 4; *P<0.05, **P<0.01). (D) After 16 hrs of stimulation, intracellular arginase-1 and IL10 production and the surface expression of Dectin-1 and MR1 in macrophages (CD11c-CD11b+F4/80+) were assessed via flow cytometry. The means ± SD are shown in the graphs (n = 4; *P<0.05, **P<0.01).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4492541&req=5

pone.0132329.g002: SRG3 over-expression driven by the β-actin promoter promotes the phenotypic shift of macrophages from M1 to M2 in response to LPS.(A-B) Peritoneal macrophages isolated from both WT and β-actin-SRG3 Tg B6 mice were primed for 5 hrs with either IFNγ (20 ng/ml) or IL4 (20 ng/ml). (A) IFNγ-primed macrophages were stimulated with either vehicle or LPS (40, 200, or 1000 ng/ml), and 12 hrs later, IL12p40 expression was analyzed via flow cytometry. Representative data from three independent experiments are shown. (B) IL4-primed macrophages were stimulated with either vehicle or LPS (40, 200, or 1000 ng/ml) for 12 hrs, and subsequently, intracellular IL10 production was analyzed via flow cytometry. Representative data are shown (n = 3). (C-D) WT, β-actin-SRG3 Tg and CD2-SRG3 Tg B6 mice were i.p. injected with LPS (2 μg) or vehicle. (C) Sixteen hrs later, intracellular TNFα, IL12p40, and iNOS production in splenic macrophages (CD11c-CD11b+F4/80+) was assessed via flow cytometry. The means ± SD are shown in the graphs (n = 4; *P<0.05, **P<0.01). (D) After 16 hrs of stimulation, intracellular arginase-1 and IL10 production and the surface expression of Dectin-1 and MR1 in macrophages (CD11c-CD11b+F4/80+) were assessed via flow cytometry. The means ± SD are shown in the graphs (n = 4; *P<0.05, **P<0.01).
Mentions: Emerging evidence has demonstrated that macrophages are involved in many physiological regulatory functions, such as thermogenesis and wound healing, in addition to classical immune functions such as phagocytosis; the observation that macrophages perform multiple functions implies the existence of subsets of macrophages that perform distinct functions. In fact, macrophages are classified into two groups according to their cytokine profile; type 1 macrophages (M1) primarily produce IL12 and IL23, whereas type 2 macrophages (M2) produce IL10 and TGFβ [11]. Recently, it has been demonstrated that M2 macrophages modulate the development of autoimmune diseases such as EAE [26]. To investigate whether SRG3 expression in macrophages affects the phenotypes of these two distinct macrophage subsets, peritoneal macrophages from β-actin-SRG3 Tg and WT B6 mice were isolated and subsequently stimulated with IFNγ or IL4 in vitro to induce differentiation into M1 or M2 macrophages, respectively. We found that peritoneal macrophages from β-actin-SRG3 Tg mice produced IL12p40 at levels comparable to those of the control non-Tg mice. This result suggests that SRG3 over-expression does not affect the differentiation of IFNγ-stimulated peritoneal macrophages into the M1 phenotype (Fig 2A; Fig A in S2 Fig). However, we discovered that SRG3 expression significantly increased IL10-producing M2 macrophages, which might help CD4+ T cells to differentiate into Th2 types (Fig 2B; Fig B in S2 Fig). Whereas SRG3 over-expression in DCs down-regulated IL12p40 production, as shown in Fig 1A, SRG3 over-expression induced macrophages to differentiate into the IL10-producing M2 type. Taken together, these results strongly indicated that SRG3 over-expression in DCs or macrophages alters their capability to produce cytokines in a somewhat distinct manner. In addition, we examined whether the effect of SRG3 over-expression on M1/M2 polarization upon LPS-mediated activation was detectable in vivo. For this purpose, CD2-SRG3 Tg, β-actin-SRG3 Tg and WT B6 mice were i.p. injected with LPS (2 μg) or vehicle, and 16 hrs later, splenic macrophages were analyzed for the expression of M1 markers (TNFα, IL12p40, and iNOS) and M2 markers (arginase-1, Dectin-1, MR1, and IL10) via flow cytometry. Strikingly, SRG3 over-expression driven by the β-actin promoter suppressed the expression of M1 markers (TNFα, IL12p40, and iNOS) but enhanced the expression of M2 markers (arginase-1, Dectin-1, MR1, and IL10) in macrophages under both basal and LPS-activated conditions, although CD2 promoter-driven SRG3 over-expression did not affect the expression of M1 or M2 markers in macrophages. These results indicated that macrophages in CD2-SRG3 Tg mice do not overexpress SRG3 due to the lack of CD2 expression in macrophages (Fig 2C and 2D; Fig C in S2 Fig). Under LPS-stimulated conditions, the β-actin-SRG3 Tg mice displayed the down-regulation of most M2 markers (i.e., arginase-1, Dectin-1, and MR-1) except for IL10, whose production was increased in macrophages compared to the vehicle-treated conditions (Fig 2D; Fig C in S2 Fig). Thus, these results demonstrated that preferential polarization toward the M2 phenotype by β-actin promoter-driven SRG3 over-expression in macrophages was closely associated with the inhibition of M1-type gene expression, implying reciprocal regulation between the M1 and M2 subtypes.

Bottom Line: We found that the outcome of EAE development was completely different depending on the expression pattern of SRG3.SRG3 over-expression not only reduced pro-inflammatory cytokine production by DCs but also shifted macrophages from the inducible nitric oxide synthase (iNOS)-expressing M1 phenotype to the arginase-1-expressing M2 phenotype during EAE.Taken together, our results provide the first evidence supporting the development of a new therapeutic strategy for EAE involving the modulation of SRG3 expression to induce M2 and Th2 polarization, thereby inhibiting inflammatory immune responses.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Bioscience and Biotechnology, Institute of Bioscience, Sejong University, Seoul 143-747, Korea; School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea.

ABSTRACT
Although SWI3-related gene (SRG3), a chromatin remodeling factor, is critical for various biological processes including early embryogenesis and thymocyte development, it is unclear whether SRG3 is involved in the differentiation of CD4+ T cells, the key mediator of adaptive immune responses. Because it is known that experimental autoimmune encephalomyelitis (EAE) development is determined by the activation of CD4+ T helper cells, here, we investigated the role of SRG3 in EAE development using SRG3 transgenic mouse models exhibiting two distinct SRG3 expression patterns: SRG3 expression driven by either the CD2 or β-actin promoter. We found that the outcome of EAE development was completely different depending on the expression pattern of SRG3. The specific over-expression of SRG3 using the CD2 promoter facilitated EAE via the induction of Th1 and Th17 cells, whereas the ubiquitous over-expression of SRG3 using the β-actin promoter inhibited EAE by promoting Th2 differentiation and suppressing Th1 and Th17 differentiation. In addition, the ubiquitous over-expression of SRG3 polarized CD4+ T cell differentiation towards the Th2 phenotype by converting dendritic cells (DCs) or macrophages to Th2 types. SRG3 over-expression not only reduced pro-inflammatory cytokine production by DCs but also shifted macrophages from the inducible nitric oxide synthase (iNOS)-expressing M1 phenotype to the arginase-1-expressing M2 phenotype during EAE. In addition, Th2 differentiation in β-actin-SRG3 Tg mice during EAE was associated with an increase in the basophil and mast cell populations and in IL4 production. Furthermore, the increased frequency of Treg cells in the spinal cord of β-actin-SRG3 Tg mice might induce the suppression of and accelerate the recovery from EAE symptoms. Taken together, our results provide the first evidence supporting the development of a new therapeutic strategy for EAE involving the modulation of SRG3 expression to induce M2 and Th2 polarization, thereby inhibiting inflammatory immune responses.

No MeSH data available.


Related in: MedlinePlus