Figure 5.

Depletion of CSF-1R⁺ cells correlates with decreased inflammatory cytokine protein content and blunted STAT3 activation in the infected cornea. MAFIA mice were treated with daily IP injections of AP20187 dimerizer or VEH. Following this treatment regimen, mice were infected with 10³ PFU HSV-1/cornea or left UI as controls, and their corneas harvested and processed for protein assays. (A–F) Cornea protein extracts were analyzed for soluble factor content by suspension array. Bars depict the mean pg/mg protein concentration for each analyte ± SEM (n = 4–7 for UI groups and n = 9–14 for infected groups per time point, from 3–4 independent experiments), ***P < 0.001 versus the rest of the groups in (A); **P < 0.01 versus VEH + UI and #P < 0.05 versus AP20187 + HSV-1 groups in (B) by ANOVA followed by Bonferroni multiple comparison test. (G) Representative immunoblot images from cornea protein extracts show VEH-treated, infected corneas have significant phosphorylation (activation) of STAT3 (pSTAT3) relative to UI controls, while AP20187-treated, infected corneas resulted in a significant reduction of pSTAT3. At 6 days PI, cornea protein extracts from mice that were pretreated with the VEH had significant elevation in pSTAT3 levels compared to the UI controls. Such phosphorylation was reduced in the AP20187 pretreated, infected cornea protein samples. Phosphorylation of ERK (pERK) was detected in UI and infected corneas, regardless of pretreatment. (H, I) Summary densitometry analyses of pSTAT3 and pERK. STAT3 and ERK detections were used to measure total STAT3 and ERK, respectively, and β-actin detection was used as a loading control. Internal normalization for each band was conducted relative to β-actin, and the results are expressed as ratios (H) pSTAT3/STAT3 and (I) pERK/ERK (relative to UI) ± SEM (n = 5–6 per group from two independent experiments). ***P < 0.001 and **P < 0.01 by ANOVA followed by Bonferroni multiple comparison test.