Background
The mammalian retina consists of at least two distinct glial populations: the macroglia, which includes Müller glia and retinal astrocytes, and the microglia. The Müller glia are the primary glial cells found in the retina, having their nucleus in the inner nuclear layer (INL) with processes extending from the inner limiting membrane at the vitreal border to the outer limiting membrane at the base of the photoreceptor inner segments [
1]. Retinal astrocytes migrate into the retina from the optic nerve and reside in the nerve fiber layer [
2]. The microglia are the resident macrophages found scattered through all the retinal layers [
3]. The retina of some species also contain oligodendrocytes and another glial-like cell type, known as the non-astrocytic inner retinal glia-like (NIRG) cells, that reside in the INL of the chick retina [
4,
5].
Müller glial cells and retinal astrocytes are essential for maintaining retinal homeostasis. Any injury or disease leading to retinal damage or disruption of the homeostasis triggers the glial cells to become active, a response termed reactive gliosis. Reactive gliosis has been observed in all retinal disease and injury models including glaucoma, age-related macular degeneration, and diabetic retinopathy [
6‐
9]. Reactive gliosis is characterized by hypertrophy, altered function brought about by changes in expression of proteins such as glutamine synthetase (GS), S100-β, extracellular matrix proteins, chondroitin sulfate proteoglycans (CSPG), matrix metalloproteinases (MMP), and an increase in growth factors such as ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), and vascular endothelial growth factor (VEGF) [
10,
11]. Multiple factors can trigger gliosis, including the bone morphogenetic proteins (BMPs) [
12‐
14]. Recent evidence from the Belecky-Adams laboratory showed that BMP7 triggered gliosis in both the Müller glia and astrocytes of the mouse retina; however, the mechanism by which BMP7 triggers gliosis is unknown [
11].
The BMPs are growth factors that belong to the transforming growth factor beta (TGF-β) superfamily. BMP signaling is initiated following the binding of the ligand to serine threonine kinase receptors. This leads to the activation of the receptors and the subsequent phosphorylation and activation of downstream signaling components. In the canonical pathway, the BMP signals by phosphorylation and activation of downstream receptor SMADs (RSMADs). The RSMADs form a dimer with the co-SMAD (SMAD4) and are shuttled to the nucleus to regulate transcription. BMP can also mediate the activation of a non-canonical pathway referred to as the BMP mitogen-activated protein kinase pathway (BMP-MAPK). In the BMP-MAPK pathway, the receptors recruit the X-linked inhibitor of apoptosis (XIAP) to a complex containing TAB1 and TAK1, thereby activating TAK1. TAK1 then activates downstream kinases, eventually activating NF-κB, p38, and JNK MAPKs [
15,
16]. In the CNS, BMP regulation has been observed in various diseases and injury models, such as spinal cord injuries, axonal damage, and ischemia [
14,
17,
18]. In the retina, upregulation of BMPs and their signaling components are observed in the photo-damaged retina injury model and in diabetic retinopathy [
19‐
21].
Microglia are the innate immune cells of the retina. In their resting state, the microglia act as sentinels, extending their processes throughout the retina. In the mouse retina, the microglia are initially found in the ganglion cell layer, entering the retina from the ciliary marginal zone and vitreous. By postnatal day 7, the microglia spread to the rest of the retinal layers, finally resting in the plexiform layers [
22]. Upon receiving signals from injured or dying cells, the microglial cells become activated: they retract their processes, undergo an increase in cellular area, become amoeboid in shape, and migrate to the area of injury or disease to phagocytize cellular debris and metabolic products [
23,
24]. Stimuli such as neuronal loss or damage, inflammation, and nerve degeneration activate the microglia into a motile effector cell with altered morphological characteristics [
25,
26].
Microglial activation has been observed in all retinal diseases, including diabetic retinopathy, age-related macular degeneration, glaucoma, and models of retinal pathologies. In addition to the morphological changes following activation, microglia also induce a change in production of various cytokines such as interleukin 1 beta (IL-1β), IL-6, and interferon gamma (IFN-γ), chemokines such as RANTES, MCP1, growth factors such as colony stimulating factor (CSF) and VEGF, and various scavenger receptors and antigen-presenting molecules such as the scavenger receptor A (SR-A) and major histocompatibility complex (MHC) [
3,
27]. Furthermore, research has revealed that activated microglia can be further classified into the following phenotypes: the M1 or proinflammatory phenotype and the M2 or the anti-inflammatory phenotype [
28,
29]. Polarization to the M1 phenotype, following exposure to factors such as lipopolysaccharide (LPS) and IFN-γ, the microglia upregulate proinflammatory factors such as IL-1β, tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS), SRs, and MHC-II [
30,
31]. The M2 phenotype plays a role in the resolution of the inflammation and tissue remodeling. This phenotype is induced by factors such as IL-4 and IL-10 or through the maturation of the M1 cells. This phenotype was characterized by an upregulation of markers such as arginase-1 (
Arg-1) and mannose receptor (
Mr), cytokines such as IL-10 and IL-13, and growth factors such as TGF-β and VEGF [
30,
32].
Signals from neurons and macroglia, such as fractalkine, neurotransmitters, and neurotrophins help keep the glial population in the quiescent state [
6,
33]. Activation of the glial cells has been found to be mediated by similar stimuli in vitro and in retinal disease models in vivo [
6,
25,
34‐
36]. Cytokines and other inflammatory markers such as TNF-α, iNOS, CNTF, and LIF are not only regulated during gliosis but are also factors known to act on the glial cells and regulate gliosis [
20,
37,
38]. Activated microglia are known to regulate Müller cell activity directly, regulating cell morphology, proliferation, and gene expression [
26,
39]. Activated microglia can also regulate the generation of Müller glia-derived progenitors [
40]. Here, we provide evidence that supports the hypothesis that BMP7 indirectly triggers gliosis by activating the proinflammatory state of retinal microglia.
Discussion
Our lab previously showed that BMP7 is able to trigger reactive gliosis in the retina. Here, we show that the Müller cell gliosis triggered by BMP7 is an indirect effect resulting from microglial activation to a proinflammatory state. Following exposure to BMP7, microglia upregulated at least two molecules, IFN-γ and IL-6, both of which have been shown in previous studies to trigger gliosis [
50‐
54]. The CSFR1 inhibitor PLX was used to specifically target and ablate retinal microglia without affecting numbers of other retinal cells, in order to show that the BMP7 triggers gliosis through microglial activation. We observed that BMP7 injection into retinas lacking microglia produced an abated inflammatory response and a complete loss of gliosis, suggesting an important role for the microglia in mediating the gliosis response.
BMP pathway in retinal disease
BMPs have been previously shown to be regulated in injury and disease models of the CNS and retina [
21,
55‐
57]. The BMP receptors type 1A and 1B regulate hypertrophic and scarring responses of astrocytes following spinal cord injury [
12]. In the retina, BMP signaling components, phospho SMAD 1/5/8, have also shown to be upregulated following NMDA-induced injury and promote retinal ganglion cell’s survival [
8]. We observed an increase in pTAK1 label in IBA-labeled cells in the retina, as well as in other cells of the inner nuclear layer. Increases in expression of pTAK1 in neurons have been previously reported in the brain following cerebral ischemia and is known to be expressed in axonal arbors of sensory neurons [
58,
59]. BMPs have also been shown to be important in retinal cell proliferation and regeneration in the chick retina [
60]. Ueki and Reh [
61] showed that SMAD upregulation was essential in mediating EGF dependent Müller glial cell proliferation in the mouse. The presence of BMPs in disease states is consistent with a potential role for them playing a role in retinal gliosis.
Activated microglia drive retinal gliosis
We had previously reported that BMP7 was able to trigger gliosis in retinal glia in vitro and in vivo. However, we observed a higher response in the in vivo model, which suggested there may be other cells involved in this response. Microglia are the resident macrophages in the retina. Similar to the macroglia, these cells also undergo activation. Their activation has been observed in various disease and injury models, such as retinitis pigmentosa, diabetic retinopathy, retinal detachment, and glaucoma [
62‐
66]. Activated microglia change morphology from a ramified cell to an amoeboid cell, along with changes in expression of cell surface markers, such as the cluster of differentiation molecule 11b (CD11b), CD68, major histocompatibility complexes (MHC), scavenger receptors, and TLR and secreted actors such as RANTES, interferon, interleukins, and TNFα. These changes serve to enhance the phagocytic effect of the microglial cells as well as the cytotoxic effect on injured cells and foreign pathogen [
23,
67]. Müller glia also undergo activation following disruption of retinal homeostasis. The reactive Müller glia hypertrophy and upregulated expression of various growth factors, reactive oxygen species scavengers, protect neurons from excitotoxicity and, in some organisms, can regenerate retinal neurons. These changes serve to protect the damaged retina. However, gliosis can also have detrimental effects by remodeling the extra cellular matrix and due to loss of normal glial functions which are necessary for normal neuronal activity [
6,
7].
The retinal astrocytes and Müller glial cells exhibit similar responses to injury, such as hypertrophy, upregulation of GFAP, vimentin, and GS, as observed in rat models of retinal detachment and retinitis pigmentosa [
68‐
70]. However, research has also revealed that there are differences in the response of the two cell types. GFAP upregulation was observed in Müller glia and not in the astrocytes in rats subjected to episcleral vein cauterization [
71]. Similarly, upregulation of GFAP was observed in the Müller glial cells in the retina subjected to laser-induced ocular hypertension, while the astrocytes of the contralateral control eyes also exhibited an increase in GFAP and a change in the area covered by the astrocytes [
72,
73]. The differences observed may suggest distinct functional roles for the astrocytes and Müller glia, which cooperate to restore retinal homeostasis.
Here, we observed a decreased gliosis response in the retina following BMP7 treatment in mice lacking microglia. We used a novel CSF1R inhibitor (PLX) to selectively ablate microglia. Following microglial ablation, mice were treated with BMP7 to assess gliosis in the retina. The inclusion of the inhibitor in the chow allowed its continual application over a longer period of time, enabling the maintenance of a microglia-free environment in the retina in which we could test the role of the microglia in BMP7-mediated gliosis. Without continual application of the inhibitor, microglia could repopulate the retina from one of two sources; bone marrow-derived stem cells can penetrate the blood-brain barrier and differentiate into microglia or residential microglia can proliferate and replace lost cells [
74,
75]. The two sources of microglia are not equivalent; residential microglia primarily give rise to microglia that display an M1 inflammatory phenotype, whereas the bone marrow-derived cells give rise to microglia with an M2 anti-inflammatory phenotype [
75]. At any rate, in order for us to test the role of BMP7 in indirectly triggering gliosis, we had to maintain a microglial-free environment for the duration of the experiments.
BMP and inflammation
Activation of microglia and macroglia have been studied in various models. While there are differences in the responses of the two glial populations, they do exhibit similarities. These include regulation of inflammatory markers, antigen presentation complexes, and various factors such as IFN, TNFα, and TLR [
3,
6,
36]. While several different factors have been shown to regulate macrophage and microglia activation, the effect of BMPs is still not completely characterized [
39,
40,
76,
77]. BMP6 regulates expression of inflammatory markers such as IL-6, IL-1β, and nitric oxide synthase in macrophages [
78‐
80]. In addition, more recent studies indicate that BMP exposure particularly leads to the M2 or anti-inflammatory phenotype of the macrophages promoting tissue repair [
81‐
84]. Microglia are descendants of immature macrophages and are thought to act as macrophages in disease and injury states [
85]. In our studies, BMP7 increased the proinflammatory state of the microglia. Further studies are necessary to determine if all microglia respond to BMP7 by increasing proinflammatory markers or if this is a response unique to certain populations of microglia.
In this study, we observed that microglia showed an upregulation of inflammatory markers in response to BMP7 treatment, indicative of activation. Furthermore, in the PLX treated mice, the gliosis response was subdued in comparison to control BMP7 treated retinas, suggesting that microglia are an essential mediator of retinal gliosis. These results support our hypothesis that microglia are activated by BMP7, which in turn regulate factors causing Müller cell gliosis.
In the PLX-treated mice (both vehicle and BMP7-injected), we also observe an increase in neurocan levels in the retina. Müller glia secrete MMPs that regulate neurocan levels in the extracellular matrix. In addition, microglia also secrete these enzymes [
86,
87]. Their upregulation has been observed in the CNS during inflammation in various injury models. Furthermore, microglia-derived factors such as TNF-α have also shown to regulate MMP expression by the Müller glia [
88]. Thus, we propose that the lack of microglia in the retina contributes to the increase in neurocan by regulating MMP levels either directly or indirectly by regulating Müller glia.
Comparing the mRNA and protein levels in the control and PLX-injected retinas, we observed a difference in expression patterns (Fig.
7, Additional file
5: Figure S5). Although the mRNA levels of S100 and TXNIP was reduced in the BMP7-injected PLX mice, we did not observe a similar change at the protein level. Non-correlation between mRNA and protein levels has been noted in other studies [
89‐
92]. mRNA translation and protein stability in the cell is regulated by multiple systems including micro RNAs (miRNAs), mRNA localization translational repression, and protein stability [
92,
93]. miRNAs have been previously reported to be regulated in neural tissue under conditions of stress [
94‐
96]. Furthermore, BMPs can regulate translation by regulating cytoplasmic polyadenylation element binding protein (CPEB) via the TAK pathway [
97,
98]. Further studies will be required to determine what pathway(s) mediate this non-correlation between the mRNA and protein levels.
Microglia release inflammatory factors prior to formation of gliosis
We observed a decrease in expression of GFAP and S100-β in mice kept on the PLX diet and treated with BMP7. BMP7 treatment also revealed decreased RNA levels of gliosis and inflammatory markers in PLX mice when compared to the mice kept on the normal diet. Previously, it has been reported that microglia respond early to changes in microenvironment and become activated. Bosco et al. showed that microglia become activated early in the retina, prior to any increases in IOP in the DBA/2J mice [
25]. Similarly, early activation of microglia has also been observed and implicated in progression of Parkinson’s disease [
99]. Furthermore, in the ocular hypertension mouse model studied in Gallego et al., the authors suggest that upregulation of MHC-II in microglia in the controlateral eye regulated the morphological changes of retinal astrocytes [
73]. Thus, we propose that microglia respond to the BMP7 first and become activated. These activated microglia upregulate factors, which in turn can trigger Müller cell gliosis. Consistent with this notion, our findings indicate the
Ifn-γ and other inflammatory factors were upregulated as early as 3 h following incubation of microglial cells with BMP7 in vitro, and these levels were further increased 6, 12, and 24 h postincubation with BMP7. In contrast, factors associated with gliosis do not begin to increase until 3 days in vivo, with most markers increasing after 7 days.
Potential factors regulating microglia-mediated activation of Müller glia
Previous studies looking into microglia and macroglia interactions have revealed several secreted as well as membrane bound factors which could activate the macroglia, such as IL-1β, IL-18, TGF-β, and TNF-α [
23,
100]. Morphological changes and increases in RNA levels of inflammatory markers in the microglia following BMP7 treatment indicate activation of the microglia. We observed in our analysis that RNA levels of
Ifn-γ,
Il-6,
Vegf, and
Thbs1 to be greater following Müller glia activation. Previously, Cotinet et.al and Goureau showed that IFN-γ can trigger Müller glia to regulate TNF-α and nitric oxide (NO) [
101,
102]. Similarly, IL-6 has been shown to induce Müller glia-derived progenitor cells in the injured zebrafish and chick retina [
40,
103]. We propose that BMP7 causes activation of microglia, which leads to upregulation of factors such as IFN-γ and IL-6, which in turn trigger Müller cell gliosis.
Our findings indicated an important role for microglia in Müller cell gliosis in the murine retina. However, the mechanism and potential factors that play a role in microglia and Müller glia interactions are not known. Future studies will aim to identify the potential role of IFN-γ and IL-6, upregulated by BMP7 in the retina, in microglia function, and gliosis.
Acknowledgements
The authors would like to thank Plexxikon Inc. for chow laced with PLX5622.