647 - Blocking IL-17a Signaling Decreases Lung Inflammation and Improves Alveolarization in Experimental Bronchopulmonary Dysplasia

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease of preterm infants that is associated with life-long morbidities. Inflammatory insults contribute to BPD pathogenesis. While the pro-inflammatory cytokine, IL-17a, plays a role in various neonatal inflammatory disorders, its role in BPD pathogenesis is unclear.

Objective:

To test the hypothesis that blocking IL-17a signaling decreases lipopolysaccharide (LPS)-mediated experimental BPD in neonatal mice.

Design/Methods:

Wild-type (WT) mice were injected intraperitoneally (i.p.) with PBS or LPS on post-natal days (P) 3, 4, and 5 (N=11). On P6, the IL-17a expression was determined in whole lung cells by enzyme-linked immunosorbent assay (ELISA) and by flow cytometry (FC) after stimulating lung cells with PMA and ionomycin in vitro. Immune cells known to produce IL-22 and IL-17a were also analyzed via FC. Statistical significance between the groups was determined by t-test. To assess the role of IL-17a in LPS-induced lung injury, we injected WT-mice i.p. with isotype antibody (Ab) or IL-17a neutralizing Ab on P2 and P4, while they were treated with PBS or LPS from P3 to P5, resulting in four study groups [PBS + Isotype Ab (n=3), PBS + IL-17a Ab (n=3), LPS + Isotype Ab (n=3), LPS + IL-17a Ab (N=3)]. We quantified inflammatory markers by western blotting on P6 and alveolarization by estimating the mean linear intercept (MLI) on P14. Statistical significance between the four groups was determined by ANOVA.

Results:

LPS-injected mice had higher IL-17a protein levels (p-value 0.002) and IL-17a+ and IL-22+ cells (p-values 0.02 and 0.01, respectively) than PBS-injected mice. More than 96% of CD45+IL-17+ cells were CD4- and >91% were CD127+ suggesting that non-T lymphoid cells are the primary producers of IL-17a in this model. In the neutralizing IL-17a Ab experiments, LPS treatment increased STAT1, STAT3, and STAT6 levels to a greater extent in mice treated with isotype Ab than those treated with IL-17a Ab (p-values < 0.001, < 0.01, and < 0.05, respectively). Further, LPS-mediated alveolar simplification was significantly greater in mice treated with isotype Ab than those treated with IL-17a Ab (p-value < 0.01).

Conclusion(s):

IL-17a protein levels and IL-17a+ and IL-22+ cells are increased in LPS-induced murine experimental BPD, and non-T lymphoid cells appear to be the primary producers of IL-17a in this model. Further, blocking IL-17a signaling decreases LPS-mediated experimental BPD in neonatal mice. Our results suggest that IL-17a could potentially be a therapeutic target for BPD infants with a predominant inflammatory phenotype.