Retinal microglia are phagocytic cells that are responsible for cleaning up apoptotic debris throughout the inner and outer plexiform layers of the retina.
8 Resting RMG can become activated by exposure to a variety of factors, including endotoxin or glycated albumin,
11,31 where they undergo a morphologic transformation to assume an ameboid shape and migrate to areas of tissue damage. As in macrophages, activated RMG upregulate extracellular signal-regulated kinase (ERK) and mitogen-activated protein kinase signaling, which leads to release of TNF-α.
11 Activated RMG have been linked to a variety of ocular disorders such as AMD,
31,41,42 light-induced retinal degeneration,
43 DR,
13 glaucoma,
44 and endotoxin-induced uveitis.
45
As phagocytic cells, macrophages and RMG have many functional similarities. Studies in macrophage cultures have demonstrated that AR inhibition or deficiency prevents endotoxin-induced nuclear factor–κB activation leading to production of pro-inflammatory.
29 Similarly, many studies have shown that RMG secret pro-inflammatory cytokines.
31,34 While AR inhibitors prevent endotoxin-induced inflammatory responses in macrophage cultures,
26 the potential role for AR in regulating the inflammatory response of RMG has not been investigated, particularly the inflammatory response induced by endotoxin or other factors that cause transition of RMG to an activated state.
In agreement with studies of Ramana and coworkers,
27,28 our previous studies demonstrated that reduction of AR activity, using either genetic or pharmacologic inhibitors, substantially suppressed the LPS-induced secretion of TNF-α by macrophage cultures. Reactive oxygen species (ROS) is a key factor in the induction of TNF-α through activation of p38 or ERK.
11,34 Our previous study with the RAW264.7 macrophage cell line showed that AR inhibition attenuates p38 or ERK activation and suppresses ROS production following LPS exposure
26 indicating that AR inhibition is capable of suppressing TNF-α secretion. In the current study, we evaluated the role of AR activity in response of RMG to activation. Using primary cultures of microglia generated from the mouse eye, we demonstrated that RMG have levels of AR that are similar to murine cell line RAW264.7 (
Fig. 1). In addition, we showed that loss of AR activity through BGG or sorbinil inhibition or via AR knockout mice attenuated LPS-induced TNF-α secretion in RMG (
Fig. 2). Taken together, these findings demonstrate the functional similarity between macrophages and RMG with regard to the involvement of AR in regulating cytokine production. It has been shown that TNF-α, as one of the major cytokines secreted by activated RMG,
31,46 may be involved in neurodegeneration
39 and uveitis.
47 Therefore, inhibition of TNF-α through pharmacologic inhibition of AR in microglia may hold promise as a potential new strategy against ocular inflammation.
In the inflamed eye, activated RMG migrate into the subretinal space and cause RPE disorganization.
31 Prevention of RMG migration might be another strategy to minimize collateral damage to cells during inflammation. We demonstrated that AR inhibition or genetic knock down suppresses LPS-induced cell migration in both macrophages and RMG (
Fig. 3). This is the first report indicating that AR mediates endotoxin-induced migration of immune cells. This result is also consistent with our previous study that AR inhibition prevents LPS-induced infiltration of inflammatory cells into the eye.
26 The mechanism linking AR activity and cell migration is not yet understood. Thus, to further characterize these mechanisms, we investigated whether AR mediates MMP-9 activation after LPS exposure. Importantly, MMP-9 is an enzyme that induces cellular morphologic changes promoting increased motility.
48,49 We confirmed that AR inhibition reduces the LPS-stimulated activation of MMP-9 (
Fig. 4), which further characterizes the effects of AR inhibition on mechanisms controlling cell migration.
The distribution of RMG throughout the retina changes with age. In younger animals, RMG are found primarily in the inner retina. However, with age or after adopting an “activated” state, RMG can be found in higher numbers in the subretinal space between photoreceptor outer segments and the RPE. Wong and coworkers
31 have shown than activated RMG disrupt the normal organization of RPE and alter the expression of key junctional proteins such as ZO-1. To determine whether AR inhibition or ablation in RMG influences the viability of RPE cells, we carried out coculture experiments with RMG and ARPE-19. Apoptosis was significantly higher when RPE cells were cocultured with activated RMG, which was significantly diminished by pretreating with ARIs. Similarly, RMG derived from AR-null mice were substantially less effective in inducing RPE apoptosis (
Fig. 5). Thus, either AR inhibition or ablation reduced the ability of activated RMG to induce apoptosis in ARPE-19 (
Fig. 5). Interestingly, it appears that diabetic mice that are null for the AR gene have markedly reduced levels of defects typically associated with DR, including retinal capillary degeneration and iNOS activation.
23,24 Further study will be required to determine whether the blockade of AR in RMG can translate into reduced markers of inflammation in other cells and tissues in the diabetic eye, including the retinal vasculature and neurons known to be damaged by chronic hyperglycemia such as in diabetes.
5
Despite discouraging results from clinical trials of ARI against DR and neuropathy, research continues on the development of newer generations of inhibitors for clinical study.
50 We recently identified β-glucogallin as a novel ARI from Indian gooseberry (
Emblica officinalis).
30,51 In addition to having ARI activity in aldo-keto reductase assays, we found that β-glucogallin was effective at reducing inflammatory cells in a murine uveitis model.
26 Results from the current study demonstrate that β-glucogallin suppresses many of the functional responses of macrophages and RMG to LPS exposure, including cytokine production, cell migration, and induction of MMP-9. Further study will be required to determine if β-glucogallin, or structurally-related derivatives,
51 are effective at downregulation of RMG activation in the diabetic retina.