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Microglia activation is essential for BMP7-mediated retinal reactive gliosis

View Article: PubMed Central - PubMed

ABSTRACT

Background: Our previous studies have shown that BMP7 is able to trigger activation of retinal macroglia. However, these studies showed the responsiveness of Müller glial cells and retinal astrocytes in vitro was attenuated in comparison to those in vivo, indicating other retinal cell types may be mediating the response of the macroglial cells to BMP7. In this study, we test the hypothesis that BMP7-mediated gliosis is the result of inflammatory signaling from retinal microglia.

Methods: Adult mice were injected intravitreally with BMP7 and eyes harvested 1, 3, or 7 days postinjection. Some mice were treated with PLX5622 (PLX) to ablate microglia and were subsequently injected with control or BMP7. Processed tissue was analyzed via immunofluorescence, RT-qPCR, or ELISA. In addition, cultures of retinal microglia were treated with vehicle, lipopolysaccharide, or BMP7 to determine the effects of BMP7-isolated cells.

Results: Mice injected with BMP7 showed regulation of various inflammatory markers at the RNA level, as well as changes in microglial morphology. Isolated retinal microglia also showed an upregulation of BMP-signaling components following treatment. In vitro treatment of retinal astrocytes with conditioned media from activated microglia upregulated RNA levels of gliosis markers. In the absence of microglia, the mouse retina showed a subdued gliosis and inflammatory response when exposed to BMP7.

Conclusions: Gliosis resulting from BMP7 is mediated through an inflammatory response from retinal microglia.

Electronic supplementary material: The online version of this article (doi:10.1186/s12974-017-0855-0) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Effect of BMP7 on gliosis in absence of microglia—localization of gliosis markers. Mouse retinal sections from eyes injected with vehicle or BMP7 were labeled for gliosis markers GFAP (A, B (a, d, g, j)), S100-β (A, B (b, e, h, k)), and NCAN (A, B (c, f, j, l)). Mice kept on the PLX diet did not show an increase in label for the gliosis markers GFAP and S100-β BMP7 or vehicle-injected retina 3 and 7 days postinjection (A, B (g, h, j, k)). NCAN label appeared to be similar in the BMP7 injected and the respective age-matched vehicle controls in mice kept on the PLX chow (A, B (i, l)). Mice kept on the control chow and injected with BMP7 clearly showed an increase in GFAP, S100-β, and NCAN levels 7 days postinjection, when compared to their respective vehicle control (B (a–f)). Three days postinjection, there is an increase in GFAP and NCAN label in BMP7-injected retinas in comparison to the respective vehicle control-injected retinas (A (a, c, d, f)). When comparing mice kept on control chow or the PLX chow, there is an increase in GFAP and S100-β label in the mice kept on control chow in comparison to the mice kept on the PLX chow, 7 days post BMP7 injection (B (d, e, j, k)). GFAP and S100-β label in mice kept on control chow and PLX chow appears to be similar in the BMP7-injected retinas, 3 days postinjection (A (d, e, j, k)). Magnification bar in (B, (c)) = 50 μm, for images A, B (a–l)
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Fig8: Effect of BMP7 on gliosis in absence of microglia—localization of gliosis markers. Mouse retinal sections from eyes injected with vehicle or BMP7 were labeled for gliosis markers GFAP (A, B (a, d, g, j)), S100-β (A, B (b, e, h, k)), and NCAN (A, B (c, f, j, l)). Mice kept on the PLX diet did not show an increase in label for the gliosis markers GFAP and S100-β BMP7 or vehicle-injected retina 3 and 7 days postinjection (A, B (g, h, j, k)). NCAN label appeared to be similar in the BMP7 injected and the respective age-matched vehicle controls in mice kept on the PLX chow (A, B (i, l)). Mice kept on the control chow and injected with BMP7 clearly showed an increase in GFAP, S100-β, and NCAN levels 7 days postinjection, when compared to their respective vehicle control (B (a–f)). Three days postinjection, there is an increase in GFAP and NCAN label in BMP7-injected retinas in comparison to the respective vehicle control-injected retinas (A (a, c, d, f)). When comparing mice kept on control chow or the PLX chow, there is an increase in GFAP and S100-β label in the mice kept on control chow in comparison to the mice kept on the PLX chow, 7 days post BMP7 injection (B (d, e, j, k)). GFAP and S100-β label in mice kept on control chow and PLX chow appears to be similar in the BMP7-injected retinas, 3 days postinjection (A (d, e, j, k)). Magnification bar in (B, (c)) = 50 μm, for images A, B (a–l)

Mentions: To determine if microglia were involved in BMP7-mediated gliosis response, mice with ablated microglia (PLX mice) were injected intravitreally with vehicle or BMP7, and mRNA levels of proinflammatory markers or gliosis-related molecules were determined by RT-qPCR 7 days postinjection. As in previous graphs, levels of mRNA are relative to levels in the respective vehicle-treated mice. Mice kept on control chow and treated with BMP7 showed an increase in levels of inflammatory markers including Gm-csf, Ifn-γ, Il-6, and Iba1 and gliosis markers including vimentin (Vim), Gfap, Egfr, Mmp9, lipocalin 2 (Lcn2), and thioredoxin interacting protein (Txnip) (Fig. 7a,b). Analysis of inflammatory markers of mice on PLX chow and treated with BMP7 via RT-qPCR also showed only modest increases in levels of Il-1β and Vegf compared to vehicle control (Fig. 7b). mRNA levels of Gm-csf, Ifn-γ, Il-6, Cd68, and Iba1 dropped drastically when microglia were not present (Fig. 7b). RT-qPCR analysis of PLX mice 7 days post-BMP7 treatment showed no increase in mRNA levels of gliosis markers compared to the PLX vehicle controls (Fig. 7a). Markers indicative of gliosis were further investigated by examining patterns of immunoreactivity for GFAP, S100-β, and neurocan (NCAN) (Fig. 8A, B). Three days following vehicle or BMP7 injection, the PLX mice showed similar levels of GFAP and S100-β label in control and PLX mice (Fig. 8A (a, b, d, e, g, h, j, k)). However, 7 days postinjection, the PLX mice showed decreased GFAP and S100-β label in BMP7-injected PLX retinas (Fig. 8B (j, k)), when compared to the control BMP7-injected retinas (Fig. 8B (d, e)). NCAN immunofluorescence label did not diminish following PLX treatment in comparison to controls at either 3 days (Fig. 8A (f, l)) or 7 days postinjection (Fig. 8B (f, l)). Moreover, levels of NCAN were increased in vehicle-injected eyes at both 3 and 7 days of PLX-treated mice in comparison to vehicle-injected eyes of control-treated mice (compare Fig. 8A (c and i) and Fig. 8B (c and i)), supporting a potential role for microglia in extracellular matrix remodeling. Negative controls showed no label (Additional file 3: Figure S3). Gliosis markers showed similar label in uninjected mice in comparison to the 3 day and 7 day vehicle injected mice (Additional file 4; Figure S4). Protein levels of gliosis markers GFAP, S100-β, and TXNIP were also quantified using western blot (Additional file 5: Figure S5).Fig. 7


Microglia activation is essential for BMP7-mediated retinal reactive gliosis
Effect of BMP7 on gliosis in absence of microglia—localization of gliosis markers. Mouse retinal sections from eyes injected with vehicle or BMP7 were labeled for gliosis markers GFAP (A, B (a, d, g, j)), S100-β (A, B (b, e, h, k)), and NCAN (A, B (c, f, j, l)). Mice kept on the PLX diet did not show an increase in label for the gliosis markers GFAP and S100-β BMP7 or vehicle-injected retina 3 and 7 days postinjection (A, B (g, h, j, k)). NCAN label appeared to be similar in the BMP7 injected and the respective age-matched vehicle controls in mice kept on the PLX chow (A, B (i, l)). Mice kept on the control chow and injected with BMP7 clearly showed an increase in GFAP, S100-β, and NCAN levels 7 days postinjection, when compared to their respective vehicle control (B (a–f)). Three days postinjection, there is an increase in GFAP and NCAN label in BMP7-injected retinas in comparison to the respective vehicle control-injected retinas (A (a, c, d, f)). When comparing mice kept on control chow or the PLX chow, there is an increase in GFAP and S100-β label in the mice kept on control chow in comparison to the mice kept on the PLX chow, 7 days post BMP7 injection (B (d, e, j, k)). GFAP and S100-β label in mice kept on control chow and PLX chow appears to be similar in the BMP7-injected retinas, 3 days postinjection (A (d, e, j, k)). Magnification bar in (B, (c)) = 50 μm, for images A, B (a–l)
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Fig8: Effect of BMP7 on gliosis in absence of microglia—localization of gliosis markers. Mouse retinal sections from eyes injected with vehicle or BMP7 were labeled for gliosis markers GFAP (A, B (a, d, g, j)), S100-β (A, B (b, e, h, k)), and NCAN (A, B (c, f, j, l)). Mice kept on the PLX diet did not show an increase in label for the gliosis markers GFAP and S100-β BMP7 or vehicle-injected retina 3 and 7 days postinjection (A, B (g, h, j, k)). NCAN label appeared to be similar in the BMP7 injected and the respective age-matched vehicle controls in mice kept on the PLX chow (A, B (i, l)). Mice kept on the control chow and injected with BMP7 clearly showed an increase in GFAP, S100-β, and NCAN levels 7 days postinjection, when compared to their respective vehicle control (B (a–f)). Three days postinjection, there is an increase in GFAP and NCAN label in BMP7-injected retinas in comparison to the respective vehicle control-injected retinas (A (a, c, d, f)). When comparing mice kept on control chow or the PLX chow, there is an increase in GFAP and S100-β label in the mice kept on control chow in comparison to the mice kept on the PLX chow, 7 days post BMP7 injection (B (d, e, j, k)). GFAP and S100-β label in mice kept on control chow and PLX chow appears to be similar in the BMP7-injected retinas, 3 days postinjection (A (d, e, j, k)). Magnification bar in (B, (c)) = 50 μm, for images A, B (a–l)
Mentions: To determine if microglia were involved in BMP7-mediated gliosis response, mice with ablated microglia (PLX mice) were injected intravitreally with vehicle or BMP7, and mRNA levels of proinflammatory markers or gliosis-related molecules were determined by RT-qPCR 7 days postinjection. As in previous graphs, levels of mRNA are relative to levels in the respective vehicle-treated mice. Mice kept on control chow and treated with BMP7 showed an increase in levels of inflammatory markers including Gm-csf, Ifn-γ, Il-6, and Iba1 and gliosis markers including vimentin (Vim), Gfap, Egfr, Mmp9, lipocalin 2 (Lcn2), and thioredoxin interacting protein (Txnip) (Fig. 7a,b). Analysis of inflammatory markers of mice on PLX chow and treated with BMP7 via RT-qPCR also showed only modest increases in levels of Il-1β and Vegf compared to vehicle control (Fig. 7b). mRNA levels of Gm-csf, Ifn-γ, Il-6, Cd68, and Iba1 dropped drastically when microglia were not present (Fig. 7b). RT-qPCR analysis of PLX mice 7 days post-BMP7 treatment showed no increase in mRNA levels of gliosis markers compared to the PLX vehicle controls (Fig. 7a). Markers indicative of gliosis were further investigated by examining patterns of immunoreactivity for GFAP, S100-β, and neurocan (NCAN) (Fig. 8A, B). Three days following vehicle or BMP7 injection, the PLX mice showed similar levels of GFAP and S100-β label in control and PLX mice (Fig. 8A (a, b, d, e, g, h, j, k)). However, 7 days postinjection, the PLX mice showed decreased GFAP and S100-β label in BMP7-injected PLX retinas (Fig. 8B (j, k)), when compared to the control BMP7-injected retinas (Fig. 8B (d, e)). NCAN immunofluorescence label did not diminish following PLX treatment in comparison to controls at either 3 days (Fig. 8A (f, l)) or 7 days postinjection (Fig. 8B (f, l)). Moreover, levels of NCAN were increased in vehicle-injected eyes at both 3 and 7 days of PLX-treated mice in comparison to vehicle-injected eyes of control-treated mice (compare Fig. 8A (c and i) and Fig. 8B (c and i)), supporting a potential role for microglia in extracellular matrix remodeling. Negative controls showed no label (Additional file 3: Figure S3). Gliosis markers showed similar label in uninjected mice in comparison to the 3 day and 7 day vehicle injected mice (Additional file 4; Figure S4). Protein levels of gliosis markers GFAP, S100-β, and TXNIP were also quantified using western blot (Additional file 5: Figure S5).Fig. 7

View Article: PubMed Central - PubMed

ABSTRACT

Background: Our previous studies have shown that BMP7 is able to trigger activation of retinal macroglia. However, these studies showed the responsiveness of Müller glial cells and retinal astrocytes in vitro was attenuated in comparison to those in vivo, indicating other retinal cell types may be mediating the response of the macroglial cells to BMP7. In this study, we test the hypothesis that BMP7-mediated gliosis is the result of inflammatory signaling from retinal microglia.

Methods: Adult mice were injected intravitreally with BMP7 and eyes harvested 1, 3, or 7 days postinjection. Some mice were treated with PLX5622 (PLX) to ablate microglia and were subsequently injected with control or BMP7. Processed tissue was analyzed via immunofluorescence, RT-qPCR, or ELISA. In addition, cultures of retinal microglia were treated with vehicle, lipopolysaccharide, or BMP7 to determine the effects of BMP7-isolated cells.

Results: Mice injected with BMP7 showed regulation of various inflammatory markers at the RNA level, as well as changes in microglial morphology. Isolated retinal microglia also showed an upregulation of BMP-signaling components following treatment. In vitro treatment of retinal astrocytes with conditioned media from activated microglia upregulated RNA levels of gliosis markers. In the absence of microglia, the mouse retina showed a subdued gliosis and inflammatory response when exposed to BMP7.

Conclusions: Gliosis resulting from BMP7 is mediated through an inflammatory response from retinal microglia.

Electronic supplementary material: The online version of this article (doi:10.1186/s12974-017-0855-0) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus