A106-20 A Novel Genetic Cause of Interstitial Lung Disease Reveals Critical Role for EML4 in Normal Lung Homeostasis

Abstract Rationale Alveolar macrophages (AMs) play a critical role in maintaining lung homeostasis. Variants in genes such as CSF2RA, CSF2RB and GATA2 can disrupt AM development and function and lead to interstitial lung disease (ILD). Identifying new genetic causes of AM dysfunction can provide critical mechanistic insights into lung homeostasis in health and disease. We report two siblings, born to consanguineous parents who presented with short stature, failure to thrive (FTT), atopic disease (eosinophilic esophagitis, severe eczema, and food allergies) and ILD. Lung biopsy and bronchoalveolar lavage revealed features of lipoid pneumonia and pulmonary alveolar proteinosis. Whole genome sequencing revealed a homozygous loss of a ∼120kb region in both siblings which included the transcription start site and first exon of the EML4 gene, which encodes for the Echinoderm microtubule-associated protein-like 4 (EML4) protein. The function of EML4 is poorly characterized and its potential role in the pathogenesis of ILD is unknown. Methods Mice with an orthologous genomic deletion in Eml4 were generated by CRISPR/Cas9 editing. The lungs of the mice were examined by flow cytometry, single cell RNA sequencing (scRNA-seq) and histopathology. To determine the effect of exposure to diverse microbial flora and pathogens, EML4-deficient mice were co-housed with ‘dirty’ mice. Results EML4-deficient mice were significantly smaller than wild-type (WT) littermates, consistent with FTT in the patients. They also had decreased AMs compared to WT mice, but no significant changes in other immune cell populations in the lung. The AM-focused impact of EML4-deficiency was confirmed by scRNA-seq which showed that the highest number of differentially expressed genes between WT and EML4-deficient mice were in AMs. Further, EML4-deficient AMs accumulated increased lipid droplets reminiscent of the foamy macrophages observed in the bronchioalveolar lavage fluid and lung biopsy of the patients. Histopathological analysis of lung sections from EML4-deficient mice housed under specific pathogen free (SPF) conditions did not show any lung infiltration or disease. However, EML4-deficient mice co-housed with ‘dirty’ mice that carried diverse mycobacteria and pathogens including Mycoplasma pulmonis developed florid immune infiltration and fibrotic lung damage compared to co-housed WT mice. Conclusions EML4-deficiency causes defects in AM number and function. Upon microbial exposure, EML4 deficiency can trigger inappropriate inflammatory responses and subsequent lung fibrosis, indicating the EML4 plays a critical role in maintaining lung homeostasis. Thus, loss of EML4 function is the likely cause of ILD in these 2 patients. This abstract is funded by: National Health and Medical Research Council