Expression of Cxcl5 and Cxcr2 in mouse retina
Expressions of both Cxcl5 and Cxcr2 genes were detected in normal mouse retina, but their expressions showed no significant difference after ON injury (Cxcl5: P = 0.111; Cxcr2: P = 0.188) as compared to those without ON injury; however, with lens injury, the expression of both Cxcl5 (P = 0.004) and Cxcr2 (P = 0.001) increased significantly as compared to those without ON and lens injury (Fig. 1A and B). Immunofluorescence analysis revealed that Cxcl5 expressed in the GC layer (GCL), inner nuclear layer (INL), outer plexiform layer (OPL) and photoreceptor layer (PRL; Fig. 1C-H), whereas Cxcr2 expressed in GCL, some cells in the IPL, INL and outer nuclear layer (ONL, Fig. 1I-N). In addition, the infiltrating cells also expressed Cxcl5 and Cxcr2 in the retina of the mice with ON and lens injury, and the expression of Cxcr2 was co-localized with the markers for infiltrating macrophages and neutrophils (Figure S2).
Cxcl5 promoted ex vivo retina ganglion cell survival and neurite outgrowth
After 7-day treatment of recombinant Cxcl5, the number of RGCs (P < 0.001) was significantly higher than that of control group by 37.9%. In addition, the number of neurite outgrowth in Cxcl5-treated group was 2.80 times higher than that in the control group (P = 0.017), and the average axon length was 1.73 times longer than that in the control group (P = 0.01). Meanwhile, immunofluorescence analysis on CD68 showed that the number of activated retinal microglia cells in the GCL increased by 40.3% as compared to the control group (P = 0.034; Fig. 2A-H, Q and R). Our results showed that Cxcl5 could promote RGC survival and neurite regeneration in retinal explant culture.
Cxcr2 antagonist attenuated ex vivo RGC survival and microglia activation
To explore whether Cxcl5 protects RGCs through its receptor Cxcr2, the effect of Cxcr2 antagonist SB225002 on RGC survival was evaluated in the retinal explant culture. Immunofluorescence analysis showed that SB225002 caused a concentration-dependent inhibitory effect on RGC survival. There was no significant difference in the number of RGCs between 0.125 µM SB225002 and the vehicle groups (P > 0.999). Instead, the number of RGCs in the 1.25 and 12.5 µM groups decreased by 26.3% (P < 0.001) and 32.9% (P < 0.001) respectively, as compared to the vehicle group. Meanwhile, the number of activated microglia also significantly decreased by 73.0% in the 1.25 µM (P < 0.001) and 83.0% in the 12.5 µM (P < 0.001) groups, as compared to the vehicle group. There were no statistically significant differences in the number of RGCs (P > 0.999) and microglia (P = 0.817) between the vehicle group and the control group (Fig. 2I-R). Our results indicated involvement of Cxcr2 in the regulation of RGC survival in the retinal explant culture.
To further confirm the cell survival regulatory effect of SB225002 specifically through Cxcr2, we determined Cxcr2 gene expressions in 11 cell lines. We identified HaCaT as Cxcr2 expressing cells and B3 as cells not expressing Cxcr2. MTT assay showed that viability of HaCaT cells treated with 12.5 µM SB225002 was significantly lower than that of control group (P = 0.005); in contrast, there was no significant difference in B3 cell viability among different SB225002 concentrations (P = 0.41; Figure S3), suggesting that SB225002 reduces cell viability specifically through Cxcr2.
Cxcr2 antagonist and clodronate liposomes attenuated the promotion effect of Cxcl5 in retinal explant culture
To confirm the RGC protective effect of Cxcl5 acting through Cxcr2, the effect of co-treatment of recombinant Cxcl5 and SB225002 was assessed. The number of RGCs in the co-treatment of recombinant Cxcl5 and 12.5 µM SB225002 was 63.4% lesser than that in the Cxcl5 alone group (P < 0.001) and 52.3% lesser than the control group (P < 0.001). Similarly, the number of CD68+ microglia in the co-treatment of recombinant Cxcl5 and 12.5 µM SB225002 was 72.3% lesser than that in treated with Cxcl5 alone (P < 0.001) and 61.2% lesser than the control group (P = 0.001; Fig. 2C, G, Q and R).
To delineate whether microglia participate in the Cxcl5-mediated RGC protection, clodronate liposome was applied to remove microglia. The number of CD68+ microglia in the co-treatment of recombinant Cxcl5 and clodronate were 72.3% lesser than that in the Cxcl5 alone group (P < 0.001), and 61.2% lesser than the control group (P < 0.001). Meanwhile, the number of RGCs in the co-treatment of recombinant Cxcl5 and clodronate was 63.4% lesser than that in the Cxcl5 group (P < 0.001), and 52.3% lesser than the control group (P < 0.001; Fig. 2D, H, Q and R). As the majority of microglia had been eliminated by clodronate liposomes, the promoting effect of Cxcl5 on RGC survival ceased. Therefore, our results indicated that Cxcl5 promotes RGC survival may through the activation of microglia.
Cxcl5 promoted retina ganglion cell survival and axonal regeneration after optic nerve and lens injury
We evaluated the effect of Cxcl5 on RGC survival and axonal regeneration in mice with optic nerve and lens injury. At 3 and 7 days after ON injury or ON and lens injury, recombinant Cxcl5 protein was injected intravitreally, and the retinas harvested 14 days after ON injury. The number of RGCs in ON-injured mice treated with Cxcl5 was increased by 23.1% as compared to those treated with vehicle (P = 0.02; Fig. 3A, B and I). Remarkably, the number of RGCs in the ON and lens-injured mice treated with Cxcl5 increased by 21.4% as compared to those treated with vehicle (P = 0.001; Fig. 3C, D and I). For the inflammatory response, immunofluorescence analysis on CD68 showed that the number of activated microglia cells in ON-injured mice treated with Cxcl5 was 63 times higher than those with ON injury alone (P = 0.002); however, there was no significant difference on the number of infiltrating CD68+ cells in the vitreous cavity between these two groups (P = 0.924). On the contrary, the number of microglia cell activation in ON crush and lens-injured mice treated with Cxcl5 was 24 times higher than those with ON and lens injury alone (P = 0.004), and the number of infiltrating CD68+ cells in the vitreous cavity of the ON crush and lens-injured mice treated with Cxcl5 was 52% higher than those with ON and lens injury alone (P = 0.014, ; Fig. 3E-H, J and K). These suggested that Cxcl5 could promote RGC survival in mice after ON injury largely may due to microglia activation and partially through the infiltrating inflammatory cells induced by lens injury.
For axonal regeneration, the number of GAP43+ regenerating axons at 0.1 mm from the crushed site in ON-injured mice treated with Cxcl5 was significantly higher than those with ON injury alone (P = 0.025), although there were no significant differences at other distances (P > 0.05; Fig. 4A, B and G). Besides, Cxcl5 did not further promote axonal regeneration in ON-injured mice with lens injury, indicating that Cxcl5 did not enhance the inflammation-mediated axonal regeneration in RGCs after ON injury (Fig. 4C, D and G).
Cxcr2 antagonist SB225002 inhibit the promotion effect of Cxcl5 in vivo
In normal mice received intraperitoneal injection of 2 µg/g SB225002, there was no statistical difference in the number of RGCs group as compared to the normal mice without SB225002 treatment (P = 0.948; Fig. 5A, B and H), suggesting that 2 µg/g SB2250022 did not affect RGC survival in normal mice. Therefore, 2 µg/g SB225002 was chosen for further studies.
Immunofluorescence analysis on βIII-Tubulin revealed that SB225002 treatment showed no significant difference on the number of RGCs after ON injury as compared to the mice with ON injury alone (P = 0.871). Instead, SB225002 significantly attenuated the effect of lens injury on RGCs that the number of RGCs in ON and lens-injured mice treated with SB225002 was 38.7% lesser than that those treated with vehicle (P = 0.0009; Fig. 5C-H). Moreover, the number of regenerating axons in ON and lens-injured mice treated with SB225002 at 0.1 mm (P = 0.006), 0.2 mm (P = 0.012), 0.5 mm (P = 0.049) and 1 mm (P = 0.006) was significantly reduced as compared to the mice with ON and lens injury only (Fig. 4C, E, F, G). In addition, Immunofluorescence analysis on CD68 showed that macrophage infiltration was significantly inhibited by SB225002 in ON and lens-injured mice as compared to those treated with vehicle (P = 0.001, Fig. 6A-I, All cell counting results are available in supplementary tables). Collectively, these suggested that SB225002 would inhibit the promotion effect of lens injury on RGC survival and axonal regeneration.
Cxcr2 antagonist reduced the lens injury-induced Akt activation
Akt activation (phospho-Akt/total Akt) in mice with ON and lens injury was 1.99 folds higher than those with ON injury only (P = 0.026). With 7 day SB225002 treatment, the increased Akt activation was significantly reduced as compared to those of ON and lens-injured mice (P = 0.048). However, there was no statistically significant difference in Stat3 activation (phospho-Stat3/Stat3) among different treatment groups (P = 0.054, Fig. 7). These results suggested participation of Akt activation in the protection of ON injury by Cxcl5/Cxcr2.