P1.159: Targeting Apoptosis-Associated Speck-Like Protein Containing a Caspase Recruitment Domain to Alleviate Oxygen-Induced Lung And Brain Injury in Mice

Background: The activation of inflammasome cascade is implicated in the development of bronchopulmonary dysplasia (BPD) and hyperoxia-related brain injury. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly. IC100 is a humanized IgG4 monoclonal antibody that disrupts ASC oligomerization and speck formation.
This study is designed to evaluate:
1. if hyperoxia induces ASC speck formation in lungs,
2. the efficacy of IC100, directed against ASC, in the treatment of BPD in hyperoxia-exposed neonatal mice,
3. the efficacy of IC100 in preventing hyperoxia induced brain injury.

Methods: 1. To test ASC speck formation in lung: ASC-citrine reporter mice expressing ASC fusion protein with a C-terminal Citrine (fluorescent GFP isoform) were exposed to 85% oxygen (O2) from postnatal day (P)1 to P14 that can induce BPD-like pathology. The lungs were dissected on P14 to examine ASC specks.
2. To assess the effects of IC100 in treatment of BPD: Newborn mice (C57BL/6J) were randomized to following groups from P1 to P14: room air (RA) with placebo (PBS) (RA-PBS), RA + IC100 intraperitoneal injection (RA-IC100), 85% O2 with PBS (O2-PBS), 85% O2 + IC100 intraperitoneal injection (O2-IC100). Lungs dissected for histological evaluation on P14 and structures assessed by radial alveolar count (RAC), inflammation assessed by macrophage infiltration.
3. To assess the efficacy of IC100 in preventing hyperoxia induced brain injury: Newborn mice randomized to 3 groups from P1 to P14: room air (RA), 85% O2 (O2), 85% O2 + IC100 intraperitoneal injection (O2-IC100). Brain was dissected for evaluation on P14 and hippocampus was assessed for cell proliferation with Ki67 staining and apoptosis with TUNEL staining.

Data were presented as mean ± SD and analyzed by ANOVA.

Results: As per Figure 1: hyperoxia exposure induced ASC specks in alveolar infiltrated cells, suggesting ASC activation.
As per Figure 2: O2-PBS group had lower RAC than RA-PBS group. However, O2-IC100 group had higher RAC than O2-PBS group, suggesting IC100 improves alveolarization under hyperoxia. O2-PBS group also had higher infiltrated cells in alveolar airspaces than RA-PBS group. While O2-IC100 group had fewer infiltrated cells in alveolar airspaces than O2-PBS group, indicating IC100 reduce hyperoxia-induced lung inflammation.
As per Figure 3: O2-IC100 group had improved cell proliferation similar to RA group and lower apoptosis similar to RA group suggesting IC100’s ability to reduce hyperoxia -induced brain injury and return growth to baseline.

We are currently working on various other markers of brain injury and recovery and hope to have additional results prior to presentation.

Conclusion: These data demonstrate the crucial role of ASC in pathogenesis of hyperoxia induced organ injury and efficacy of a humanized therapeutic anti-ASC antibody in treating these hyperoxia related injury in mice.