The findings of the current study confirm a key role for Müller cells and Ccl2 in the retinal neuroinflammatory response in the light-damage model of retinal degeneration. Firstly, using both in situ hybridization and immunohistochemistry, we confirmed the efficacy of siRNA transfection in targeted suppression of Ccl2 expression in Müller glia following damage. Second, we found that suppression of Ccl2 mRNA in Müller cells inhibited the recruitment of both ED1-positive and IBA1-positive monocytes/microglia to the injured retina after BCL exposure. Third, our data showed that photoreceptor death was reduced after BCL when Ccl2 expression was inhibited by Ccl2 siRNA.
Previous investigators have theorized that Müller cells or retinal pigment epithelial cells (RPE) may be the source of chemokines that mediate neuroinflammation following light-induced degeneration ,and several studies have shown that RPE cells in vitro express Ccl2 in response to stimulatory cytokines in the extracellular environment [68–72]. The present study is the first, to our knowledge, to directly confirm that Müller cells guide monocyte/microglia recruitment in the retina through the expression of Ccl2 mRNA, and that such expression exacerbates photoreceptor death following the initial damaging-light stimulus. This is consistent with our previous investigation, which found that Müller cells express Ccl2 in spatiotemporal correlation with the recruitment of ED1-positive monocytes and photoreceptor death following BCL exposure .
Our data point to a crucial role for chemokines in the propagation of local neuroinflammatory responses driven by the neural retina. Ccl2 is a strong chemoattractant and activator of monocytes  and microglia in vitro, and is induced in the CNS in a range of pathologies (reviewed in ). Our data indicate that Ccl2 upregulation by Müller cells promotes the recruitment of two monocyte/microglia populations immunoreactive for the markers ED1 and IBA1 in the retina following BCL exposure . First are parenchymal microglia immunoreactive for IBA1, which infiltrate the OPL and ONL after BCL [63, 65]. Second, there is modulation of ED1+/IBA1+ nuclei recruited from the retinal and choroidal blood supplies, which is consistent with the markers, morphology, and distribution of bone-marrow-derived ‘hematogenus’ monocytes [65, 66, 73]. These findings are supported by a previous study in the CNS using Ccr2-knockout mice subjected to partial sciatic nerve ligation, which showed that the Ccr2 chemokine receptor, of which Ccl2 is a known ligand , mediates the recruitment of both hematogenous and resident microglia/monocytes immunoreactive for IBA1 .
Because both bone-marrow and resident microglia/monocytes are implicated in the clearance of debris and dead photoreceptors after injury , the expression of Ccl2 by Müller cells may promote homeostasis and recovery through efficient recruitment and activation of phagocytes to sites of photoreceptor degeneration. However, given that we found a decrease in photoreceptor apoptosis and expression of AP-1 following suppression of Ccl2 by siRNA, the secretion of Ccl2 by Müller cells may be a maladapted process, which is prone to eliciting exaggerated and damaging microglial responses. A number of studies have shown that microglial activation and aggregation exacerbates photoreceptor degeneration in the light-damage model [32, 33], whereas activated microglia induce apoptosis of cultured photoreceptors through the secretion of cytotoxic factors in vitro. Moreover, the introduction of synthetic Ccl2 to cultured microglial cells or monocytes has been shown to promote their activation and cytotoxicity toward co-cultured photoreceptors and RPE cells [47, 76]. The signaling events that govern the synthesis of Ccl2 by Müller cells are unknown, although upregulation of Ccl2 may be stimulated as a result of local photoreceptor death, because increased levels of Ccl2 in Müller cells correlates spatially with the localization of light-induced photoreceptor apoptosis, as shown in our previous investigation . Alternatively, or perhaps concurrently, Ccl2 synthesis may be stimulated by the presence of cytokines in the extracellular environment, such as IL-1β, IL-7, and TNF-α [68, 71, 77], following BCL exposure.
Our findings are consistent with other studies that have characterized Ccl2 as a non-redundant factor in the guidance of microglia/monocytes in a variety of degenerative models. In the retina, an investigation in experimental retinal detachment using Ccl2−/− mice and Ccl2-specific antibody neutralization noted a substantial decrease in the recruitment of parenchymal microglia to the ONL following detachment, in conjunction with reduced photoreceptor death . Deficiencies in monocyte recruitment have also been reported after Ccl2 inhibition in other models such as skin inflammation , thioglycollate challenge , experimental autoimmune encephalomyelitis , pulmonary granuloma , and peripheral endotoxin insult . Despite this, a previous investigation did not observe modulation in a population of F4/80-positive macrophages in the subretinal space following light-induced damage to Ccl2−/− mice . As discussed in our previous investigation, however , the authors in that investigation did not quantify those cells, nor did they assess the distribution of other microglial markers such as ED1 and IBA1.
Relevance to human retinal dystrophies
Exposure to bright continuous light in rats has been used to model retinal degeneration for over 40 years [83, 84]. Several lines of evidence also indicate that light damage is a useful model of AMD [56, 85–87]. This model, like the established laser-induced model of neovascular AMD, uses an acute damaging stimulus to evoke site-specific AMD-like retinal degeneration. Although the rat retina lacks a macula and fovea centralis, it includes an homologous feature, the area centralis, in superiotemporal retina [88–90]. Previous studies have identified the focal degeneration of photoreceptors and RPE cells and associated changes to the blood–retinal barrier as being localized to the area centralis, thus mimicking many of the histopathological aspects of advanced ‘dry’ AMD [56, 85–87].
Recruitment of monocytes/microglia has been associated with the progression and severity of AMD pathology for many years [2, 12–15], while several investigations have shown that microglial attenuation reduces lesion size in the laser-induced model of neovascular ‘wet’ AMD [91–93]. Retinas from human donors show increased expression for Ccl2 in all forms of AMD , while increased levels of Ccl2 protein have been detected in aqueous humor samples taken from patients in advanced stages of ‘wet’ and ‘dry’ AMD [95, 96]. Increased Cc2 expression has also been described in the retinas of aged (20-month-old) mice, compared with young (3-month-old) mice . Moreover, studies in experimental laser-induced choroidal neovascularization (CNV) have shown that ablation of either Ccl2 or the receptor Ccr2 inhibits the infiltration of monocytes/microglia and reduces lesion size following CNV [98, 99]. Conversely, it has been previously suggested that aging Ccl2−/− Ccr2−/− mice develop AMD-like retinal degeneration [100, 101], indicating that a degree of Ccl2 signaling is also required for homeostasis, although the AMD-like phenotype in the knockout has been questioned .
siRNA-mediated gene therapy is considered to have therapeutic potential in knocking down deleterious genes in various human pathologies (reviewed in [102, 103]). Our investigation is the first to show that monocyte recruitment, and in turn photoreceptor death, may be modified in the retina by siRNA-mediated suppression of Ccl2 in vivo in the CNS. Previous studies in AMD have shown that intravitreally injected siRNA targeting vascular endothelia growth factor ameliorates retinal degeneration in experimental CNV [104, 105], and has also been the basis for several clinical trials . However, unlike the current investigation, these early studies used ‘naked’ unmodified siRNA molecules, which are now known to produce non-specific effects via Toll-like receptor 3 signaling in the retina . Nevertheless, modulation of Ccl2 expression using appropriately targeted RNAi may provide a powerful means to control excessive microglial recruitment and activation in retinal dystrophies such as AMD.