MIF Inhibitor ISO-1 Protects Photoreceptors and Reduces Gliosis in Experimental Retinal Detachment

Abstract

Photoreceptor death and retinal gliosis underlie the majority of vision threatening retinal diseases including retinal detachment (RD). Although the underlying pathobiology of vision limiting processes in RD is not fully understood, inflammation is known to play a critical role. We conducted an iTRAQ proteomic screen of up- and down-regulated proteins in a murine model of RD to identify potential targetable candidates. Macrophage migration inhibitory factor (MIF) was identified and evaluated for neurotoxic and pro-gliotic effects during RD. Systemic administration of the MIF inhibitor ISO-1 significantly blocked photoreceptor apoptosis, outer nuclear layer (ONL) thinning, and retinal gliosis. ISO-1 and MIF knockout (MIFKO) had greater accumulation of Müller glia pERK expression in the detached retina, suggesting that Müller survival pathways might underlie the neuroprotective response. Our data show the feasibility of the MIF-inhibitor ISO-1 to block pathological damage responses in retinal detachment and provide a rationale to explore MIF inhibition as a potential therapeutic option for RD.

Figure 1

Proteomic analysis of murine RD. (a) Heatmap of proteins with FDR < 0.05 identified on iTRAQ proteomic screen. Proteins extracted from individual murine retina plus vitreous at week 2 (n = 3) or week 4 (n = 3) post RD creation were labeled with iTRAQ probes in an 8-plex assay. Control murine retinas (n = 2) were sham-treated (label #119) or untreated (#121). Red represents strong protein expression and blue represents lack of expression. Validation of up-regulated proteins in homogenized retina plus vitreous was performed by ELISA at four timepoints post RD for MIF with n = 15 individual mice/group. MIF expression was significantly elevated in RD eyes at week 1 (b, *p = 0.0430).

Figure 2

MIF genetic depletion reduces apoptosis but not loss of ONL thickness in RD. MIFKO mice with BALB/c and C57BL/6 backgrounds were used with appropriate background controls (n = 5/group). Representative photographs shows TUNEL-positive cells in day 14 detached retina from BALB/c background controls (a) and MIFKO mice (b). Scale bar denotes 50 microns. TUNEL-positive cells were significantly reduced in the ONL of day 14 BALB/c and C57BL/6 MIFKO mice (c, **p = 0.0008 and *p = 0.0466, respectively). However, the loss of the ONL thickness in these day 14 RDs was not significantly reduced in either BALB/c or C57BL/6 MIFKO mice compared with background controls (d, p = 0.4169 and p = 0.2378, respectively). No significant changes were observed in cleaved caspase-3 positive cells (e, p = 0.5295). Cleaved PARP-positive cells were significantly reduced in day 14 MIFKO mice (f, *p = 0.0224). Confocal micrographs shows cleaved PARP positive cells in day 14 detached retina from C57BL/6 background controls (g) and MIFKO mice (h). Arrows denote positive cells. Scale bar denotes 25 microns.

Figure 3

ISO-1 treatment blocks photoreceptor apoptosis and loss of ONL thickness. Experimental design (a): ISO-1(40 mg/kg/day) or vehicle treatment was initiated 1 day prior to RD creation and continued daily until sacrifice at day 3 or 14 (n = 6/group). Representative photographs show TUNEL positive cells (red) in the ONL in day 3 detached retina from vehicle (b) and ISO-1 treated mice (c). Scale bar denotes 50 microns. TUNEL positive cells were significantly reduced in ISO-1 treated mice at day 3 when apoptosis is maximal (f, *p = 0.0036), but not different at day 14 (not shown). A second independent study confirmed the significant reduction in TUNEL by ISO-1 (35 mg/kg/day, n = 6/group, p = 0.008, not shown). ONL thickness in ISO-1 treated eyes was significantly thicker than that of vehicle treated eyes at day 14 (g, *p = 0.0278). Confocal micrographs shows activated caspase-3 positive cells (green) in the ONL of day 3 background controls (d) and ISO-1 treated (e) mice (n = 10/group). Scale bar denotes 25 microns. Caspase-3 positive cells were not significantly reduced in ISO-1 treated animals compared to controls in day 3 and 14 (h, n = 10/group, p = 0.2593 and p = 0.1367, respectively). Cleaved PARP protein expression in ISO-1 treated eyes was not significantly different from vehicle treated eyes at either timepoints (i, n = 6, p = 0.3269 at day3 and n = 5, p = 0.3163 at day14). AIF protein expression in ISO-1 treated eyes was not significantly increased from vehicle treated eyes at day 3 (j, n = 6/group, p = 0.2891).

Figure 4

Macrophage accumulation in RD is not affected by ISO-1. (a) Immunoperoxidase photomicrographs of F4/80+ cells (red) in the retina of C57BL/6 mice systemically treated with ISO-1 or vehicle demonstrates an accumulation of inflammation in detached retina compared with attached fellow-eye controls at day 3 and 14 (n = 6/group). (b) Quantification reveals the F4/80+ cell count was not affected by ISO-1 treatment compared with vehicle treatment at either timepoints (p = 0.2293, day3 and p = 0.4714, day14). Scale bar denotes 50 microns.

Figure 5

ISO-1 treatment and MIF genetic depletion both reduce retinal gliosis. Immunofluorescence staining shows GFAP accumulation (green) after RD (b,c,e,f) compared with attached controls (a,d) in vehicle or ISO-1 treated animals at day 3 or day 14 (n = 6/group). Treatment with ISO-1 displayed significant reduction in gliosis in day 14 eyes (k, *p = 0.0256) while GFAP expression in day 3 detached retina was not significantly decreased (k, p = 0.1898). Detached retinas in day 14 BALB/c wild type controls (h) and MIFKO mice (j) displayed increased gliosis by GFAP accumulation over adjacent attached retina in controls (g,i). Detached retina in MIFKO mice had significantly reduced GFAP accumulation compared with background mice (l, n = 5/group, *p = 0.0319). Scale bar denotes 50 microns.

Figure 6

Retinal pERK is up-regulated in ISO-1 and MIFKO RDs. Immunofluorescence staining shows pERK (green) accumulation in Müller glia after RD (b,c,e,f) compared with attached controls (a,d) in vehicle or ISO-1 treated animals at day 3 or day 14 (n = 6/group). Treatment with ISO-1 displayed significant elevation in pERK expression in day 14 eyes (k, *p = 0.0356) while pERK expression in day 3 detached retinas was not significantly elevated (k, p = 0.1948). Detached retinas in day 14 BALB/c wild type controls (h) and MIFKO mice (j) displayed increased pERK accumulation (red) compared to attached retina in controls (g,i). Detached retina in MIFKO mice had significantly elevated pERK accumulation compared with background mice (l, n = 5/group, *p = 0.0246). Scale bar denotes 50 microns.