Interleukin-1 receptor antagonist prevents murine bronchopulmonary dysplasia induced by perinatal inflammation and hyperoxia

Abstract

Bronchopulmonary dysplasia (BPD) is a common lung disease of premature infants, with devastating short- and long-term consequences. The pathogenesis of BPD is multifactorial, but all triggers cause pulmonary inflammation. No therapy exists; therefore, we investigated whether the anti-inflammatory interleukin-1 receptor antagonist (IL-1Ra) prevents murine BPD. We precipitated BPD by perinatal inflammation (lipopolysaccharide injection to pregnant dams) and rearing pups in hyperoxia (65% or 85% O2). Pups were treated daily with IL-1Ra or vehicle for up to 28 d. Vehicle-injected animals in both levels of hyperoxia developed a severe BPD-like lung disease (alveolar number and gas exchange area decreased by up to 60%, alveolar size increased up to fourfold). IL-1Ra prevented this structural disintegration at 65%, but not 85% O2. Hyperoxia depleted pulmonary immune cells by 67%; however, extant macrophages and dendritic cells were hyperactivated, with CD11b and GR1 (Ly6G/C) highly expressed. IL-1Ra partially rescued the immune cell population in hyperoxia (doubling the viable cells), reduced the percentage that were activated by up to 63%, and abolished the unexpected persistence of IL-1α and IL-1β on day 28 in hyperoxia/vehicle-treated lungs. On day 3, perinatal inflammation and hyperoxia each triggered a distinct pulmonary immune response, with some proinflammatory mediators increasing up to 20-fold and some amenable to partial or complete reversal with IL-1Ra. In summary, our analysis reveals a pivotal role for IL-1α/β in murine BPD and an involvement for MIP (macrophage inflammatory protein)-1α and TREM (triggering receptor expressed on myeloid cells)-1. Because it effectively shields newborn mice from BPD, IL-1Ra emerges as a promising treatment for a currently irremediable disease that may potentially brighten the prognosis of the tiny preterm patients.

Keywords: anti-inflammatory therapy; cytokines; neonatal immunity; receptor blockade.

Fig. 1.

Lung histology in 28-d-old pups exposed to antenatal LPS and postnatal hyperoxia at 85%. Pregnant dams were injected with 150 μg/kg LPS at day 14 of gestation. After delivery, newborn mice were exposed to an FiO₂ of 0.21 (room air) or 0.85 and received daily s.c. injections of either vehicle or IL-1Ra (10 mg/kg); n = 10–27 per group. Another group of dams received vehicle instead of LPS antenatally; their pups breathed room air and were injected with vehicle (no antenatal LPS, air vehicle; n = 6). Lungs were assessed after 28 d. (A) One representative 500 μm × 500 μm slide per group is depicted. Scale bars: 100 μm. (B–D) Scans of whole lungs were analyzed for the number of alveoli per square millimeter (B), the size of the alveoli (C), and the SVR (D). Data are shown as means ± SEM. ^♦P < 0.05; ^♦♦♦P < 0.001 for no antenatal LPS air vehicle vs. hyperoxia-vehicle; ^❖P < 0.05 and ^❖❖❖P < 0.001 for room air vehicle vs. hyperoxia-vehicle; **P < 0.01, room air vehicle vs. room air IL-1Ra.

Fig. 2.

Abundance of pulmonary cytokines on day 3 after perinatal inflammation and 85% hyperoxia. Following antenatal LPS or vehicle, 3 d of 21% (room air) or 85% O₂ and daily postnatal s.c. injection with IL-1Ra or vehicle, cytokines were determined in the lungs by ELISA. n = 7–20 per group. Data are means of cytokine abundance normalized to total protein (t.p.) ± SEM. ^□P < 0.05 for no LPS room air vs. room air vehicle; *P < 0.05 for room air vehicle vs. room air IL-1Ra; ^#P < 0.05 for hyperoxia vehicle vs. hyperoxia IL-1Ra; ^♦P < 0.05 for no antenatal LPS air vehicle vs. hyperoxia vehicle.

Fig. 3.

Lung histology at day 28 of life after perinatal LPS or vehicle and 65% O₂. Animals were treated identically to those in Fig. 1 except for reduction of the FiO₂ to 0.65. Room air groups from each of the 28-d experiments were pooled, so the data are identical to those in Fig. 1. n = 5–27 per group. Lungs were collected and analyzed on day 28. (A) One representative 500 μm × 500 μm slide for each treatment group is presented. Scale bars: 100 μm. (B–D) Computerized quantification of the slides. Alveolar number per square millimeter (B), alveolar size (C), and SVR (D) are depicted. Data are means ± SEM. ^♦P < 0.05 and ^♦♦P < 0.01 for no antenatal LPS air vehicle vs. hyperoxia vehicle; ^❖P < 0.05 and ^❖❖P < 0.01 for room air vehicle vs. hyperoxia vehicle; **P < 0.01 for room air vehicle vs. room air IL-1Ra; ^#P < 0.05 and ^##P < 0.01 for hyperoxia vehicle vs. hyperoxia IL-1Ra. (E) Photograph of one representative mouse from the hyperoxia vehicle (Left) and the hyperoxia IL-1Ra groups (Right) on day 28.

Fig. 4.

Exploration of mediators of inflammation on days 3 and 28 of the murine BPD model. (A–E) Following the same experimental protocol as animals in Fig. 3, cytokine abundance was determined in the lungs obtained on day 3. Room air groups from each of the 3-d experiments were pooled and are therefore identical to those in Fig. 2. (C and E) IL-6 and MIP-2 were measured by ELISA. n = 8–20 per group. Graphs show means of cytokine abundance normalized to t.p. ± SEM. (A, B, D) Semiquantitative protein analysis of cytokines (A), other mediators (B), and chemokines (D) was performed by multiplex immunoblotting on the lungs of three animals from each group. Data are plotted as OD normalized to the positive control spots on each membrane in arbitrary units ± SEM. (A–E) ^□P < 0.05 and ^□□P < 0.01 for no antenatal LPS air vehicle vs. room air vehicle; *P < 0.05 for room air vehicle vs. room air IL-1Ra; ^♦P < 0.05, ^♦♦P < 0.01, and ^♦♦♦P < 0.001 for no antenatal LPS air vehicle vs. hyperoxia vehicle; ^❖P < 0.05 and ^❖❖P < 0.01 for room air vehicle vs. hyperoxia vehicle; ^#P < 0.05 for hyperoxia vehicle vs. hyperoxia IL-1Ra. (F) Sections of the same day 28 lungs used to generate Fig. 3 were subjected to IHC assessment of the abundance of pulmonary IL-1β. One representative image for each group is shown; n = 4–7 per group. Scale bars: 100 μm. IL-1α was also determined and is shown in Fig. S4.

Fig. 5.

Flow cytometric analysis of the lungs on day 28. The left lungs of the animals shown in Fig. 3 (perinatal LPS, 65% hyperoxia) were analyzed by flow cytometry. Among viable cells, all immune cells (A) or 20,000 CD45⁺ cells (B–G) were gated. The graphs show data from one experiment (n = 3–4 per group); a second experiment produced similar results (n = 3–4 per group). (A) The total number of cells isolated from the lungs is depicted. (B and C) Enumeration of CD4⁺ and CD8⁺ T cells, B220⁺CD11c⁻ B cells, B220⁺CD11c⁺ conventional DC, and B220⁻CD11c⁺ plasmacytoid DC is plotted as percent of CD45⁺ cells. (D and E) CD11c⁺CD11b⁺-activated monocytes are shown as percent of CD45⁺ cells. (E) One exemplary histogram per group of the CD11b stain is depicted; red and blue line, room air groups; orange line, hyperoxia vehicle; green line, hyperoxia IL-1Ra. (F and G) Assessment of CD11b and GR1, markers of macrophage and granulocyte activation, on CD45⁺ cells. (F) Analysis of one experiment (n = 4 per group); (G) exemplary dot plots of one lung from each group. *P < 0.05, **P < 0.01, and ***P < 0.001 for all comparisons.