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

Abstract

Background: Immune 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.

Methods: CD11c-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.

Results: Recruitment 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.

Conclusions: The 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.

Figure 1

CDG and B6 retinal ganglion cells (RGC) survive similarly after an optic nerve crush (ONC). RGC survive in greater numbers using DSAEK forceps for the ONC. *All post-ONC counts of RGC differed from normal controls, as well as the contralateral RGC, P < 0.05. Normal control RGC counts represent the RGC counts of both retinas after unilateral injection into the superior colliculus. Ipsilateral - manipulated side; contralateral - opposite, unmanipulated side. Counts are average RGC numbers/retinal field where each retina count is the average of eight fields/retina, ± SD. N = number of mice. For example, an N of 4 represents 32 fields. Field size = 0.190 mm².

Figure 2

Retinal GFP ^hi cells are CD11b ⁺ and express F4/80. The GFP^hi cells in the retina of CDG mice are found in the CD45^med region associated with microglia (MG), and in the CD45^hi populations. (A), Analysis of F4/80 and GFP expression in retinal CD45^med cells from naive B6, naive CDG, and CDG mice post-ONC. (B), Expression of GFP and F4/80 in CD45^hi cells from the same retinas shown in (A). The quadrants are labeled with the percent of cells contained in each quadrant.

Figure 3

Recruitment, redistribution and interaction of retinal GFP ^hi DC with retinal ganglion cells (RGC) and their axons following an optic nerve crush (ONC) injury. (A), GFP^hi DC in naive retina, and at 1, 3, 5, 7, and 11 days post-ONC. GFP^hi DC, green; isolectin B4-stained blood vessels, red; anti-β3 tubulin stained RGC somata (blue/white arrows), nerve fibers (large blue arrows), and dendrites (small blue arrows). (B), GFP^hi DC surround and engulf RGC somata ten days post-ONC. DAPI, blue; GFP^hi DC, green.

Figure 4

Detection of retinal ganglion cell (RGC) phagocytosis by GFP ^hi and GFP ^lo CD11b ⁺ cells in CDG retina post-ONC. (A) Depiction of CD45^hi and CD45^med gating, elimination of 1A8⁺ PMN, and selection for CD11b⁺ cells that are GFP^lo or GFP^hi. (B) Sequential confocal sections of GFP^hi DC engulfing DiI-labeled RGC seven days post-ONC. DiI-labeled RGC, red; GFP^hi DC, green; DAPI-stained nuclei, blue. (C) Detection and quantitation of DiI-labeled CD45^medCD11b⁺GFP^hi DC and GFP^lo cells by flow cytometry. Ipsilateral retinas were labeled by injection of DiI into the superior colliculus. After seven days, mice were given an ONC; retinas were harvested six days later.

Figure 5

Phagocytosis of retinal ganglion cells (RGC) following an optic nerve injury (ONC). (A) Time course of RGC apoptosis following an ONC. (B) Analysis of DiI uptake in GFP^hi DC and GFP^lo MG/macrophages following an ONC. Retinas were labeled by injection of DiI into the superior colliculus. After 7 days the mice were given an ONC; retinas were harvested 7, 10, 13, and 17 days later. CD45⁺ cells were examined by flow cytometry as shown in Figure 4. All cells in (B) are CD11b⁺.

Figure 6

Recruitment of CD45 ^med GFP ^hi cells to retina in Tko, Mko, or MTdko mice on the CDG background. Retinas were harvested ten days after an ONC. Numbers in the quadrants are percents, and are the averages of at least four samples. P-values for differences in the populations are shown.

Figure 7

DiI uptake by CD45 ^hi cells. (A) Flow cytometric analysis of DiI uptake by GFP^hi and GFP^lo cells in the CD45^hi population of retina from the indicated strains harvested after retrograde labeling of RGC with DiI. The retina was DiI labeled by injection of DiI into the superior colliculus seven days prior to the ONC. Retina was harvested ten days post-ONC where indicated. (B) Summary of cell counts and P-values in mice receiving DiI and an ONC.

Figure 8

DiI uptake by CD45 ^med cells. (A) Flow cytometric analysis of the uptake of DiI by GFP^hi and GFP^lo cells in the CD45^med population of retina from the indicated strains harvested after retrograde labeling of RGC with DiI via injection of DiI into the superior colliculus seven days prior to the ONC. Retina was harvested ten days post-ONC. Application of the DiI label, and performance of the ONC were as indicated on the axis. (B) Summary of cell counts and P-values.

Figure 9

Systemic administration of DTx to the CD11c-DTR/GFP mice leads to depletion of the GFP ^hi DC. (A-C) Serial fundus photographs of the same retina following an ONC and DTx treatment via ip injection. (A) GFP^hi DC at 12 days post-ONC. (B) Fundus photograph of retina one day after ip injection of 200 ng DTx. (C) Photograph of retina two days post-ip DTx injection. (D) Flow cytometry of retina following ip injection of DTx under two different protocols; six daily injections of 10 ng into a naive mouse, and 2 injections of 100 ng into a mouse at days 11 and 12 post-ONC. (E) Daily serial injections of 10 ng DTx in naive mice. (F-K) Confocal microscopy of retinal flatmounts in ONC-injured retinas. (F-H) DTx (200 ng) was injected ip 48 hours prior to harvest, at 15 days post-ONC. (F) Cells and nerve fibers in the NFL post-DTx. (G-H) Cells at two levels in the IPL. All images in each vertical column are from the same confocal field at different depths into the retina. (I-K) Saline, 1 μl, was injected ip 48 hours prior to harvest, at 15 days post-ONC. (I) Cells and nerve fibers in the NFL. (J-K) Cells at two levels in the IPL. GFP^loCD11b⁺ cells stained red; GFP^hiCD11b⁺ cells stained yellow-green. NFL, nerve fiber layer; IPL, inner plexiform layer at two depths is shown, immediately below the retinal ganglion cell layer (RGC) soma, and adjacent to the INL.

Figure 10

A two-niche model for resident and recruited cells in retina post-injury. The sizes of the niches, N1 (MG/macrophage niche) and N2 (GFP^hi DC niche), as well as the arrows denoting pathways, were drawn to suggest relative sizes, and changes due to injury. VEC, vascular endothelial cells.