Antenatal IL-1-dependent inflammation persists postnatally and causes retinal and sub-retinal vasculopathy in progeny

Abstract

Antenatal inflammation as seen with chorioamnionitis is harmful to foetal/neonatal organ development including to eyes. Although the major pro-inflammatory cytokine IL-1β participates in retinopathy induced by hyperoxia (a predisposing factor to retinopathy of prematurity), the specific role of antenatal IL-1β associated with preterm birth (PTB) in retinal vasculopathy (independent of hyperoxia) is unknown. Using a murine model of PTB induced with IL-1β injection in utero, we studied consequent retinal and choroidal vascular development; in this process we evaluated the efficacy of IL-1R antagonists. Eyes of foetuses exposed only to IL-1β displayed high levels of pro-inflammatory genes, and a persistent postnatal infiltration of inflammatory cells. This prolonged inflammatory response was associated with: (1) a marked delay in retinal vessel growth; (2) long-lasting thinning of the choroid; and (3) long-term morphological and functional alterations of the retina. Antenatal administration of IL-1R antagonists - 101.10 (a modulator of IL-1R) more so than Kineret (competitive IL-1R antagonist) - prevented all deleterious effects of inflammation. This study unveils a key role for IL-1β, a major mediator of chorioamnionitis, in causing sustained ocular inflammation and perinatal vascular eye injury, and highlights the efficacy of antenatal 101.10 to suppress deleterious inflammation.

Figure 1

Murine model of inflammation-induced PTB employed for the study. (A) IL-1β (1 µg) was administered intrauterine at G16.5, whereas 101.10 (1 mg/Kg/12 h), Kineret (4 mg/Kg/12 h) or vehicle were administered subcutaneously for 2 consecutive days, with the first dose administered 30 min prior to IL-1β. (B) Intrauterine injection of IL-1β was made between two foetal membranes, without penetrating the amniotic cavity.

Figure 2

Inflammatory response in the retina and sub-retina of the foetus and newborn. (A–C) Foetal eyes were collected at G17 (A), G17.5 (B) and G18 (C) after in utero exposure to IL-1β to perform quantitative PCR. Results are relative to 18 S and plotted as fold change vs. the control groups. n = 3–8 dams/group; 4 foetal eyes per sample. (D–F) Cytokine levels in eyes of newborns exposed to the indicated treatments in utero (Fig. 1). n = 4 dams/group; 4 eyes per sample. (G–H) Quantitative PCR of IL-1, IL-6 and IL-8 performed on isolated retina (G) and choroid (H) collected on foetuses at G17.5; n = 3 dams/group; 10 retinas or sub-retinas per sample. (I) Iba-1-stained cryosections of retina from foetuses at G17.5 exposed to the indicated treatments in utero; n = 3 foetuses/group. Kin refers to Kineret. Scale bar, 300 μm. Values are presented as mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Dunnett’s post-analysis.

Figure 3

Infiltration of immune cells in eyes during development of the pups. (A–C) Quantification of iba-1⁺ cells observed on retinal and choroidal flat mounts at Pt 1 (A), Pt 15 (B) and Pt 30 (C). n = 3–6 pup/group for each time point. Values are presented as mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Dunnett’s post-analysis.

Figure 4

Delay of retinal vessel growth in progeny. (A–C) Lectin-stained flat-mounts of retinas from pups at Pt 1 (A), Pt 4 (B) and Pt 8 (C) previously exposed in utero to the indicated treatments (Fig. 1). Images are representative of 3 to 5 separate pups per treatment group. Dotted lines depict the vascular front. Scale bar for A, 1500 μm; scale bar for B, 2500 μm; scale bar for C, 3000 μm. Right panels show quantification of the vascular area, n = 3–5 dams/group. (D) Magnification of lectin-stained flat-mounts of retinas from pups at Pt15 showing vascular density. Images are representative of 5 to 8 separate pups per treatment group. Scale bar, 150 μm. Right panel shows quantification of the vascular area at Pt8 and Pt15, n = 5–8 dams/group. Values are presented as mean ± S.E.M. *p < 0.05, ***p < 0.001 by one-way ANOVA with Dunnett’s post-analysis.

Figure 5

Choroidal thinning precedes retinal degeneration and thinning of the inner nuclear layer. (A–B) Representative images (top panels) and quantification (bottom panels) of lectin-stained cross-sections of choroids from pups at Pt 1 (A) and Pt 21 (B) previously exposed to the indicated treatments in utero (Fig. 1). Vertical bars represent the average choroidal thickness. Scale bar for A, 30 μm; scale bar for B, 50 μm. n = 3–4 pups/group. (C,D) Representative images (top panels) and quantification (bottom panels) of DAPI-stained cross-sections of retinas from pups at Pt1 (C) and Pt21 (D) previously exposed to the indicated treatments in utero (Fig. 1); n = 3–4 pups/group. Vertical bars represent the average retinal thickness. Scale bar for C, 250 μm; scale bar for B, 500 μm. (E,F) Quantification of DAPI-stained cross-sections of the ONL (E) and INL (F) from the same retinas measured in D. Values are presented as mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Dunnett’s post-analysis. ONBL, outer neuroblastic layer; INBL, inner neuroblastic layer; NFL, nerve fibre layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

Figure 6

Electroretinogram readings after completion of ocular development. (A) Representative ERG response of pups at Pt 30 previously exposed to the indicated treatments in utero (Fig. 1). (B,C) a-wave amplitude (B) and latency (C) of scotopic ERGs of corresponding animals. (D,E) b-wave amplitude (D) and latency (E) of scotopic ERGs of corresponding animals. n = 8–22 pups/group. Values are presented as mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Dunnett’s post-analysis.