Diabetic retinopathy (DR) is the most common complication of diabetes and a leading
cause of vision loss worldwide (1). Unfortunately, there are no treatments targeting
early stages of the disease prior to the onset of sight-threatening vascular defects
such as macular edema or neovascularization. A better understanding of the etiology
of DR is needed to identify therapeutic targets to halt early disease progression.
To this end, numerous studies demonstrated that a low-grade inflammation occurs in
retinas of diabetic animal models and suggest that inflammation contributes a role
in DR progression. Various mechanisms leading to retinal inflammation in DR have been
described, with the majority of studies implicating retinal Müller glial cells and
microglia as the initiators of retinal inflammation (for review, see ref. 2). However,
seldom does a study make a strong connection between these two cell types. In this
issue of Diabetes, Portillo et al. (3) describe a mechanism in which retinal inflammation
in diabetic mice is dependent upon expression of the cluster of differentiation gene
40 (CD40) receptor by Müller cells (Fig. 1). The study suggests that CD40 activation
induces Müller cells to release ATP, leading to activation of P2X7 purinergic receptors
on retinal microglia and their subsequent expression of inflammatory cytokines. Importantly,
the requirement of Müller cell–specific CD40 expression to recapitulate the appearance
of acellular capillaries in diabetic retinas also suggests that inflammation is necessary
for the loss of vascular cells associated with DR pathology.
Figure 1
Proposed role of CD40 and P2X7 receptors in diabetic retinal neuroinflammation. The
CD40 receptor on Müller cells (yellow) is activated in retinas of diabetic mice, presumably
by binding to CD40L. CD40 activation triggers phospholipase Cγ1 (PLC) activation,
leading to an increase in intracellular calcium (Ca2+) resulting in release of ATP.
CD40 activation also causes Müller cells to express the chemokine CCL2. Extracellular
ATP activates P2X7 receptors on a subset of retinal microglia (green), which is necessary
for their expression of the cytokines TNF-α and IL-1β. Thus, CD40 activation on Müller
cells links macroglial and microglial inflammatory responses in DR.
CD40 is mainly known as an immune costimulatory molecule, and interactions between
CD40 and its major ligand (CD40L) play key roles in immunological licensing of antigen-presenting
cells by CD4+ T cells and for B-cell activation, proliferation, class switching, and
immunoglobin production (4). In a previous study Portillo et al. (5) found that germline
deletion of the CD40 gene blocked intracellular adhesion molecule 1 expression, leukostasis,
and the appearance of acellular capillaries in the retinas of diabetic mice. These
authors also found that in the retina Müller glial cells express CD40, as do endothelial
cells, microglia, and retinal ganglion cells (RGC) (6). Since CD40 was deleted in
all cells of the germline knockout mice, including circulating immune cells, the mechanism
and cell type in which CD40 contributed to DR pathology were unclear. In the current
study CD40 was “added back” to the knockout mice in such a way that it was expressed
exclusively by Müller cells, which was sufficient to restore the diabetes-induced
inflammation and vascular pathology.
Müller cell–targeted transgenic add-back of CD40 represents an elegant means of testing
the hypothesis that CD40 expression by this cell type is sufficient for low-grade
retinal inflammation in this diabetic model. However, the choice of targeting these
glial cells is not obvious. Müller cells span radially across retina layers providing
structural, metabolic, and neurotrophic support necessary for homeostasis (7). CD40
is an immune costimulatory molecule, and a role for CD40–CD40L interaction in Müller
cell pathophysiology is unprecedented. Furthermore, one might have expected leukostasis
and capillary dropout in the diabetic retina to be dependent on CD40 expression on
endothelial cells, since the interaction of luminal CD40 with CD40L on activated platelets
causes endothelial activation and adhesion molecule upregulation leading to leukocyte
adherence (8,9).
Importantly, Portillo et al. (3) provide compelling evidence that Müller cells initiate
retinal inflammation in the diabetic retina and signal to microglia to elicit their
participation. The study concludes that diabetes triggers retinal neuroinflammation
directly through CD40 stimulation on Müller cells and indirectly through ATP release
by Müller cells, leading to stimulation of P2X7 purinergic receptors on microglia/macrophages.
One might have expected inflammatory responses to be dependent on direct CD40 stimulation
on microglia. Microglia represent the major resident innate immune cells of the retina
and other neuronal tissues and are uniquely equipped to mount inflammatory responses
to infection and tissue damage. In the retina, the conventional view is that microglia
are the first responders, initiating an inflammatory response that leads to Müller
cell reactive gliosis (10). Furthermore, in the brain, CD40 is mainly found on microglia,
where CD40 deficiency or neutralization of CD40L inhibits microglial activation, alleviates
brain pathology, and improves cognitive performance in mouse models of Alzheimer disease
(11).
The ATP-mediated mechanism described by Portillo et al. (3) may have implications
for other aspects of DR pathology as well. Several purinergic receptors, including
P2X7, play key roles in retinal physiology and pathophysiology, including modulation
of retinal neurotransmission, control of vascular tone, and Müller cell swelling and
gliosis, as well as RGC apoptosis (12). Diabetes was found to increase the susceptibility
of retinal microvessels to transmembrane pore formation in response to P2X7 activation,
suggesting that extracellular ATP may cause mural cell loss in DR (13). In addition,
P2X7 and P2X4 receptor antagonists inhibited the induction of endothelial cell inflammation
and permeability by high glucose (14). Release of ATP by Müller cells could also contribute
to the death of RGC observed in DR, as evidence suggests that the release of ATP by
gliotic Müller cells induces RGC apoptosis through P2X7 activation (15,16).
Finally, the current study suggests the possibility that CD40–CD40L and ATP–P2X7 interactions
represent promising new therapeutic targets for prevention of DR progression. Although
the study did not establish the source of CD40L in diabetic retinas, plasma levels
of soluble CD40L were significantly increased in the diabetic mice, suggesting a systemic
influence (platelet activation, perhaps) on DR pathology in this model. Plasma levels
of soluble CD40L are also elevated in patients with type 1 and type 2 diabetes (17,18).
Several systemic treatments targeting the CD40–CD40L system have been developed to
treat cancer, inflammation, and autoimmune disease (4). Although clinical trials of
monoclonal antibodies blocking CD40L failed due to occurrence of thromboembolisms
(19), such a complication is unlikely with intravitreal applications of these biologics.
Also, development of blood-brain-barrier–penetrant P2X7 antagonists for treatment
of neuroinflammation has been under way for some time (20). Although the role for
this receptor in normal retinal function suggests caution, it is possible that inhibition
of P2X7 on retinal microglia could modulate their function to prevent neuroinflammation
from progressing toward sight-threatening DR.