Ute Lehmann

Dendritic cells are early responders to retinal injury

The presence and activity of dendritic cells (DC) in retina is controversial, as these cells are difficult to identify in retina due to limited markers and sparse numbers. Transgenic mice that express green fluorescent protein (GFP) on the CD11c promoter to label DC allowed the visualization and quantification of retinal DC. Two retina injury models, the optic nerve crush (ONC) and light injury, were used to study their injury response. Many GFP(+) DC were tightly associated with retinal ganglion cell nerve fibers following ONC, while very few microglia (GFP(-)CD11b(+) cells) were found in close contact. The GFP(+) cells were greatly elevated in the outer plexiform layer following photic injury. All of the GFP(+) DC were CD11b(+), suggesting a myeloid origin. In addition, the GFP(+) DC upregulated expression of MHC class II after injury, while the GFP(-)CD11b(+) microglia did not. This study shows that DC were found in the retina and that they rapidly responded to neural injuries. We propose that they are a previously overlooked population, distinct from microglia, and may be important in the injury response.

Peripheral Induction of Tolerance by Retinal Antigen Expression

The contribution of peripheral expression of tissue-specific CNS Ags to the generation of tolerance is uncertain. To study this question, we examined mice transgenic (Tg) for expression of β-galactosidase (βgal) on the retinal photoreceptor cell arrestin promoter, in conjunction with TCR Tg mice producing CD4+ T cells specific for βgal (βgalTCR). Several strategies were used to test the hypothesis that βgal expressed in the retina supported thymus-independent tolerance and regulatory T cell development. Retinal expression generated an immunoregulatory response that depressed development of immune responses to βgal following systemic immunization with βgal. This regulation was transferable to naive mice by CD3+4+25+ T cells from naive retinal βgal+ donors. Experiments that removed the βgal+ retina by enucleation showed that subsequent development of a regulatory response was lost. Adoptive transfer of CD25− βgalTCR T cells into retinal βgal Tg mice on the Rag−/− background led to regulatory activity that limited lymphopenia-induced proliferation of βgalTCR T cells in mice with retinal expression of βgal and inhibited the ear-swelling assay for delayed type hypersensitivity. These results show that retinal expression of very small amounts of a tissue-specific Ag can generate tolerance that includes regulatory T cells.

Local Activation of Dendritic Cells Alters the Pathogenesis of Autoimmune Disease in the Retina

Interest in the identities, properties, functions, and origins of local APC in CNS tissues is growing. We recently reported that dendritic cells (DC) distinct from microglia were present in quiescent retina and rapidly responded to injured neurons. In this study, the disease-promoting and regulatory contributions of these APC in experimental autoimmune uveoretinitis (EAU) were examined. Local delivery of purified, exogenous DC or monocytes from bone marrow substantially increased the incidence and severity of EAU induced by adoptive transfer of activated, autoreactive CD4 or CD8 T cells that was limited to the manipulated eye. In vitro assays of APC activity of DC from quiescent retina showed that they promoted generation of Foxp3+ T cells and inhibited activation of naive T cells by splenic DC and Ag. Conversely, in vitro assays of DC purified from injured retina demonstrated an enhanced ability to activate T cells and reduced induction of Foxp3+ T cells. These findings were supported by the observation that in situ activation of DC before adoptive transfer of β-galactosidase–specific T cells dramatically increased severity and incidence of EAU. Recruitment of T cells into retina by local delivery of Ag in vivo showed that quiescent retina promoted development of parenchymal Foxp3+ T cells, but assays of preinjured retina did not. Together, these results demonstrated that local conditions in the retina determined APC function and affected the pathogenesis of EAU by both CD4 and CD8 T cells.

Retinal dendritic cell recruitment, but not function, was inhibited in MyD88 and TRIF deficient mice

BackgroundImmune system cells are known to affect loss of neurons due to injury or disease. Recruitment of immune cells following retinal/CNS injury has been shown to affect the health and survival of neurons in several models. We detected close, physical contact between dendritic cells and retinal ganglion cells following an optic nerve crush, and sought to understand the underlying mechanisms.MethodsCD11c-DTR/GFP mice producing a chimeric protein of diphtheria toxin receptor (DTR) and GFP from a transgenic CD11c promoter were used in conjunction with mice deficient in MyD88 and/or TRIF. Retinal ganglion cell injury was induced by an optic nerve crush, and the resulting interactions of the GFPhi cells and retinal ganglion cells were examined.ResultsRecruitment of GFPhi dendritic cells to the retina was significantly compromised in MyD88 and TRIF knockout mice. GFPhi dendritic cells played a significant role in clearing fluorescent-labeled retinal ganglion cells post-injury in the CD11c-DTR/GFP mice. In the TRIF and MyD88 deficient mice, the resting level of GFPhi dendritic cells was lower, and their influx was reduced following the optic nerve crush injury. The reduction in GFPhi dendritic cell numbers led to their replacement in the uptake of fluorescent-labeled debris by GFPlo microglia/macrophages. Depletion of GFPhi dendritic cells by treatment with diphtheria toxin also led to their displacement by GFPlo microglia/macrophages, which then assumed close contact with the injured neurons.ConclusionsThe contribution of recruited cells to the injury response was substantial, and regulated by MyD88 and TRIF. However, the presence of these adaptor proteins was not required for interaction with neurons, or the phagocytosis of debris. The data suggested a two-niche model in which resident microglia were maintained at a constant level post-optic nerve crush, while the injury-stimulated recruitment of dendritic cells and macrophages led to their transient appearance in numbers equivalent to or greater than the resident microglia.

Evidence for Extrathymic Generation of Regulatory T Cells Specific for a Retinal Antigen

Background: Thymic expression of a photoreceptor cell antigen, interphotoreceptor retinoid-binding protein, is known to generate regulatory T cells (Treg) that prevent spontaneous autoimmune disease of the retina. However, the contribution of other endogenous, tissue-specific antigens (Ags) expressed in the retina to the generation of Treg is uncertain. Methods: Transgenic mice that express β-galactosidase (β-gal) in photoreceptor cells, together with β-gal-specific T cell receptor transgenic mice, were used to study the induction of Treg in vivo. Results: Transgenic expression of β-gal on the arrestin promoter led to a spontaneous immunoregulatory response that inhibited the development of immune responses to β-gal. The regulation was transferred by CD3+4+25+ Treg. Several strategies were then used to show that β-gal expressed in the retina supported spontaneous, thymus-independent Treg development. The endogenous Treg also differed from the Treg induced by Ag inoculation into the anterior chamber of the eye. Conclusion: These results demonstrate that retinal expression of very small amounts of a tissue-specific Ag can generate Treg in the periphery.

Immunoproteasome deficiency protects in the retina after optic nerve crush

The immunoproteasome is upregulated by disease, oxidative stress, and inflammatory cytokines, suggesting an expanded role for the immunoproteasome in stress signaling that goes beyond its canonical role in generating peptides for antigen presentation. The signaling pathways that are regulated by the immunoproteasome remain elusive. However, previous studies suggest a role for the immunoproteasome in the regulation of PTEN and NF-κB signaling. One well-known pathway upstream of NF-κB and downstream of PTEN is the Akt signaling pathway, which is responsible for mediating cellular survival and is modulated after optic nerve crush (ONC). This study investigated the role of retinal immunoproteasome after injury induced by ONC, focusing on the Akt cell survival pathway. Retinas or retinal pigment epithelial (RPE) cells from wild type (WT) and knockout (KO) mice lacking either one (LMP2) or two (LMP7 and MECL-1) catalytic subunits of the immunoproteasome were utilized in this study. We show that mRNA and protein levels of the immunoproteasome subunits are significantly upregulated in WT retinas following ONC. Mice lacking the immunoproteasome subunits show either a delayed or dampened apoptotic response as well as altered Akt signaling, compared to WT mice after ONC. Treatment of the RPE cells with insulin growth factor-1 (IGF-1) to stimulate Akt signaling confirmed that the immunoproteasome modulates this pathway, and most likely modulates parallel pathways as well. This study links the inducible expression of the immunoproteasome following retinal injury to Akt signaling, which is important in many disease pathways.