Interrupting pro-inflammatory pulmonary-vascular cell interactions to reduce pulmonary capillary leak in critically ill children with bronchiolitis

Project Summary Viral infection of the lower respiratory tract in children less than two years old, known as bronchiolitis, is the #1 reason children seek unplanned medical care. Severe disease is also prevalent; more than 4,000 children per year in the US will require mechanical ventilation for hypoxemic respiratory failure. Thankfully, mortality is low, but morbidity remains too high. Viruses infect airway epithelial cells, initiating an innate immune response directed in part by tightly regulated interleukin-1 (IL-1) signaling. Beyond this immune response, IL-1 induces pulmonary endothelial cell (EC) dysfunction. Dysfunctional ECs no longer maintain alveolar barrier integrity. Leak of fluid, solutes, and activated immune cells worsens lung compliance and blood and air flow matching (V/Q) and increases distance for gas diffusion, producing hypoxemic respiratory failure. Our overarching hypothesis is that hypoxic respiratory failure in children with bronchiolitis is due to inappropriately intense IL-1 signaling from alveolar cells to pulmonary ECs, causing dysfunction. Our long-term goal is to understand the regulation of IL-1 signaling in bronchiolitis to establish markers of disease severity and therapies that reduce the effects of pulmonary capillary leak and, ultimately, reduce the severity of hypoxic respiratory failure. Our proposal is based on prior human and animal studies and our single-cell RNA-sequencing and proteomic analysis of tracheo- bronchioalveolar lavage (tBAL) samples identifying differential regulation of IL-1 signaling molecules, such as IL-1RN, IL-1R2, TSG-6 and pirin in critically ill children with bronchiolitis. We add vascular function assays with these samples to demonstrate infection induces EC dysfunction. The overall objective of this application is to establish specific molecules responsible for the resolution of hypoxic respiratory failure and demonstrate that these molecules mitigate breakdown of pulmonary EC barrier. We will perform a prospective observational cohort study of children with confirmed bronchiolitis requiring invasive mechanical ventilation. We will collect tBAL samples from patients on intubation and throughout their disease. Each sample will be immediately processed for single-cell RNA sequencing, proteomics, and metabolomics. Aim 1 will use causal inference to establish which cellular processes and molecules are responsible for hypoxemic respiratory failure. In Aim 2, we will test how these molecules regulate IL-1 mediated paracellular leak using cultured human pulmonary microvascular ECs and pediatric precision cut human lung slices and test inhibitors using trans-endothelial electric resistance assays, transcriptional profiling, confocal microscopy, and immunoblotting. In Aim 3, we will establish a blockade of IL-1 signaling and treat hypoxemic respiratory failure using mouse models of viral lung injury with and without FDA-approved inhibitors of IL-1 signaling. We will show IL-1 inhibitors block signaling via high-parameter microscopy and transcriptomics and improve gas exchange, protein leak, lung weight, and compliance. This proposal is significant because it will advance our understanding of how pulmonary capillary dysfunction drives hypoxemic respiratory failure and set the stage for repurposing existing therapies to treat bronchiolitis.