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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

BMP7 injection triggers inflammatory changes in the mouse retina. Expression levels of a panel of proinflammatory markers were analyzed by RT-qPCR in RNA samples from mouse retina injected with vehicle or BMP7, harvested 3 and 7 days postinjection (a). At 3 days post-BMP7 injection, about a 2-fold increase in RNA levels, relative to the vehicle controls, was observed in levels of Ifn-γ, Tnf-α, and Il-1β. Seven days post-BMP7 injection, 2-fold increase in levels was observed in Csf, Vegf, Thbs1, and Thbs2, and greater than 3-fold increase in Gm-csf, Ifn-γ, Il6, and CD68 RNA levels relative to the vehicle-injected control. Mouse retinal microglial cells treated with BMP7 for 3, 6, 12, and 24 h were also analyzed for changes in RNA levels of inflammatory markers (b), with LPS treatment used as a positive control (c). In vitro treatments showed a significant increase in Ifn-γ levels at the 3-h time point. At 6 h post-BMP7 treatment, mRNA levels of Gm-csf, Ifn-γ, Csf, Tnfα, Il-6, and Cd68 were increased to 1.5-fold or greater. By 12 h, we observed no significant differences between BMP7 and vehicle-treated samples. At the 24-h time point, however, we observed significant increases in the levels of Ifn-γ and Thbs. The LPS-treated microglia showed a relative increase in most of the markers, with significant increases observed in levels of Gm-csf, Ifn-γ, Il-6, and Thbs2 (c). Protein levels of IFN-γ was also determined via ELISA (d). We observed a 2-fold increase in levels in medium from microglial cells incubated with BMP7 for 24 h and in protein from whole retinal tissue from mice injected with BMP7 for 3 days, when compared to their respective vehicle control. Protein from 7 days BMP7-injected retina showed a 5-fold increase in protein levels compared to the vehicle control. Data shown in graphs represent relative expression levels of RNA or protein of BMP7 or LPS-treated samples to their respective vehicle control. Bars above a level of 1.0 (solid black line) represent an increase in mRNA levels while bars below the level of 1.0 represent a decrease in mRNA levels relative to the corresponding vehicle control. Statistical analysis was performed by unpaired Student’s t test. *p value <0.05. Abbreviations: CD cluster of differentiation, Csf colony stimulating factor, Gm-csf granulocyte macrophage colony stimulating factor, Ifn interferon, Il interleukin, Tnf-α tumor necrosis factor alpha, Thbs thrombospondin, Vegf vascular endothelial growth factor
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Fig2: BMP7 injection triggers inflammatory changes in the mouse retina. Expression levels of a panel of proinflammatory markers were analyzed by RT-qPCR in RNA samples from mouse retina injected with vehicle or BMP7, harvested 3 and 7 days postinjection (a). At 3 days post-BMP7 injection, about a 2-fold increase in RNA levels, relative to the vehicle controls, was observed in levels of Ifn-γ, Tnf-α, and Il-1β. Seven days post-BMP7 injection, 2-fold increase in levels was observed in Csf, Vegf, Thbs1, and Thbs2, and greater than 3-fold increase in Gm-csf, Ifn-γ, Il6, and CD68 RNA levels relative to the vehicle-injected control. Mouse retinal microglial cells treated with BMP7 for 3, 6, 12, and 24 h were also analyzed for changes in RNA levels of inflammatory markers (b), with LPS treatment used as a positive control (c). In vitro treatments showed a significant increase in Ifn-γ levels at the 3-h time point. At 6 h post-BMP7 treatment, mRNA levels of Gm-csf, Ifn-γ, Csf, Tnfα, Il-6, and Cd68 were increased to 1.5-fold or greater. By 12 h, we observed no significant differences between BMP7 and vehicle-treated samples. At the 24-h time point, however, we observed significant increases in the levels of Ifn-γ and Thbs. The LPS-treated microglia showed a relative increase in most of the markers, with significant increases observed in levels of Gm-csf, Ifn-γ, Il-6, and Thbs2 (c). Protein levels of IFN-γ was also determined via ELISA (d). We observed a 2-fold increase in levels in medium from microglial cells incubated with BMP7 for 24 h and in protein from whole retinal tissue from mice injected with BMP7 for 3 days, when compared to their respective vehicle control. Protein from 7 days BMP7-injected retina showed a 5-fold increase in protein levels compared to the vehicle control. Data shown in graphs represent relative expression levels of RNA or protein of BMP7 or LPS-treated samples to their respective vehicle control. Bars above a level of 1.0 (solid black line) represent an increase in mRNA levels while bars below the level of 1.0 represent a decrease in mRNA levels relative to the corresponding vehicle control. Statistical analysis was performed by unpaired Student’s t test. *p value <0.05. Abbreviations: CD cluster of differentiation, Csf colony stimulating factor, Gm-csf granulocyte macrophage colony stimulating factor, Ifn interferon, Il interleukin, Tnf-α tumor necrosis factor alpha, Thbs thrombospondin, Vegf vascular endothelial growth factor

Mentions: To determine whether BMP7 regulated inflammatory signals that could then either trigger or enhance the gliosis response, BMP7-treated retinas were analyzed for messenger RNA (mRNA) levels of proinflammatory markers (Fig. 2a). For the analyses of mRNA levels, values plotted in graphs were all relative to control levels which were set to a value of 1.0; hence, increases in mRNA levels in comparison to controls are bars above a level of 1.0, while a decrease is represented by bars below the level of 1.0. Three days postinjection, increases of 1.5-fold or more in mRNA levels of Tnf-α, Il-1β, and Ifn-γ were present. However, larger increases were evident in multiple factors 7 days postinjection, including granulocyte macrophage colony stimulating factor (Gm-Csf), colony stimulating factor (Csf), Ifn-α, Ifn-γ, Il-6, Vegf, thrombospondins-1 and-2 (Thbs1 & Thbs2), and Cd68. We also observed more than a 2-fold increase in microglial marker Iba1 and Irf8, markers for activated microglia.Fig. 2


Microglia activation is essential for BMP7-mediated retinal reactive gliosis
BMP7 injection triggers inflammatory changes in the mouse retina. Expression levels of a panel of proinflammatory markers were analyzed by RT-qPCR in RNA samples from mouse retina injected with vehicle or BMP7, harvested 3 and 7 days postinjection (a). At 3 days post-BMP7 injection, about a 2-fold increase in RNA levels, relative to the vehicle controls, was observed in levels of Ifn-γ, Tnf-α, and Il-1β. Seven days post-BMP7 injection, 2-fold increase in levels was observed in Csf, Vegf, Thbs1, and Thbs2, and greater than 3-fold increase in Gm-csf, Ifn-γ, Il6, and CD68 RNA levels relative to the vehicle-injected control. Mouse retinal microglial cells treated with BMP7 for 3, 6, 12, and 24 h were also analyzed for changes in RNA levels of inflammatory markers (b), with LPS treatment used as a positive control (c). In vitro treatments showed a significant increase in Ifn-γ levels at the 3-h time point. At 6 h post-BMP7 treatment, mRNA levels of Gm-csf, Ifn-γ, Csf, Tnfα, Il-6, and Cd68 were increased to 1.5-fold or greater. By 12 h, we observed no significant differences between BMP7 and vehicle-treated samples. At the 24-h time point, however, we observed significant increases in the levels of Ifn-γ and Thbs. The LPS-treated microglia showed a relative increase in most of the markers, with significant increases observed in levels of Gm-csf, Ifn-γ, Il-6, and Thbs2 (c). Protein levels of IFN-γ was also determined via ELISA (d). We observed a 2-fold increase in levels in medium from microglial cells incubated with BMP7 for 24 h and in protein from whole retinal tissue from mice injected with BMP7 for 3 days, when compared to their respective vehicle control. Protein from 7 days BMP7-injected retina showed a 5-fold increase in protein levels compared to the vehicle control. Data shown in graphs represent relative expression levels of RNA or protein of BMP7 or LPS-treated samples to their respective vehicle control. Bars above a level of 1.0 (solid black line) represent an increase in mRNA levels while bars below the level of 1.0 represent a decrease in mRNA levels relative to the corresponding vehicle control. Statistical analysis was performed by unpaired Student’s t test. *p value <0.05. Abbreviations: CD cluster of differentiation, Csf colony stimulating factor, Gm-csf granulocyte macrophage colony stimulating factor, Ifn interferon, Il interleukin, Tnf-α tumor necrosis factor alpha, Thbs thrombospondin, Vegf vascular endothelial growth factor
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Fig2: BMP7 injection triggers inflammatory changes in the mouse retina. Expression levels of a panel of proinflammatory markers were analyzed by RT-qPCR in RNA samples from mouse retina injected with vehicle or BMP7, harvested 3 and 7 days postinjection (a). At 3 days post-BMP7 injection, about a 2-fold increase in RNA levels, relative to the vehicle controls, was observed in levels of Ifn-γ, Tnf-α, and Il-1β. Seven days post-BMP7 injection, 2-fold increase in levels was observed in Csf, Vegf, Thbs1, and Thbs2, and greater than 3-fold increase in Gm-csf, Ifn-γ, Il6, and CD68 RNA levels relative to the vehicle-injected control. Mouse retinal microglial cells treated with BMP7 for 3, 6, 12, and 24 h were also analyzed for changes in RNA levels of inflammatory markers (b), with LPS treatment used as a positive control (c). In vitro treatments showed a significant increase in Ifn-γ levels at the 3-h time point. At 6 h post-BMP7 treatment, mRNA levels of Gm-csf, Ifn-γ, Csf, Tnfα, Il-6, and Cd68 were increased to 1.5-fold or greater. By 12 h, we observed no significant differences between BMP7 and vehicle-treated samples. At the 24-h time point, however, we observed significant increases in the levels of Ifn-γ and Thbs. The LPS-treated microglia showed a relative increase in most of the markers, with significant increases observed in levels of Gm-csf, Ifn-γ, Il-6, and Thbs2 (c). Protein levels of IFN-γ was also determined via ELISA (d). We observed a 2-fold increase in levels in medium from microglial cells incubated with BMP7 for 24 h and in protein from whole retinal tissue from mice injected with BMP7 for 3 days, when compared to their respective vehicle control. Protein from 7 days BMP7-injected retina showed a 5-fold increase in protein levels compared to the vehicle control. Data shown in graphs represent relative expression levels of RNA or protein of BMP7 or LPS-treated samples to their respective vehicle control. Bars above a level of 1.0 (solid black line) represent an increase in mRNA levels while bars below the level of 1.0 represent a decrease in mRNA levels relative to the corresponding vehicle control. Statistical analysis was performed by unpaired Student’s t test. *p value <0.05. Abbreviations: CD cluster of differentiation, Csf colony stimulating factor, Gm-csf granulocyte macrophage colony stimulating factor, Ifn interferon, Il interleukin, Tnf-α tumor necrosis factor alpha, Thbs thrombospondin, Vegf vascular endothelial growth factor
Mentions: To determine whether BMP7 regulated inflammatory signals that could then either trigger or enhance the gliosis response, BMP7-treated retinas were analyzed for messenger RNA (mRNA) levels of proinflammatory markers (Fig. 2a). For the analyses of mRNA levels, values plotted in graphs were all relative to control levels which were set to a value of 1.0; hence, increases in mRNA levels in comparison to controls are bars above a level of 1.0, while a decrease is represented by bars below the level of 1.0. Three days postinjection, increases of 1.5-fold or more in mRNA levels of Tnf-α, Il-1β, and Ifn-γ were present. However, larger increases were evident in multiple factors 7 days postinjection, including granulocyte macrophage colony stimulating factor (Gm-Csf), colony stimulating factor (Csf), Ifn-α, Ifn-γ, Il-6, Vegf, thrombospondins-1 and-2 (Thbs1 & Thbs2), and Cd68. We also observed more than a 2-fold increase in microglial marker Iba1 and Irf8, markers for activated microglia.Fig. 2

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 M&uuml;ller 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&nbsp;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