Proteomic Analysis of Bronchoalveolar Lavage Fluid from Children with Bronchiolitis Obliterans Syndrome Identifies Potential Treatment Strategies

Background Bronchiolitis obliterans syndrome (BOS) is a severe complication of HSCT, commonly occurring in conjunction with other chronic GVHD. It is unclear why some patients with chronic GVHD develop BOS, while most do not. We hypothesized that proteomic analysis of bronchoalveolar lavage fluid (BALF) from children with and without BOS could identify novel opportunities for BOS prophylaxis and treatment. Methods BALF from 5 children with and 5 without BOS was submitted for aptamer-based proteomic analysis using the 7,000 protein SomaScan platform (Somalogic). Two patients with BOS were male and three female. Two controls were female and 3 male. Median age of cases was 9 years and controls 8.2 years. Differentially expressed proteins were analyzed using Enrichr (MSigDB Hallmark 2020) and Ingenuity Pathway Analyses (IPA; Qiagen). We explored biological effects of IL17 by incubating primary pulmonary small airway epithelial cells and pulmonary microvascular endothelial cells with IL17 ligand and performed qPCR to measure gene expression. Results Proteomic analysis identified 305 differentially expressed proteins. The most significant differentially regulated protein was reduction in IL17 receptor in BOS cases (p= 6 × 10e-5); IL17 ligand was increased in BOS cases compared with controls (p=0.01). MSigDB Hallmark 2020 Pathway analysis identified differential regulation of potential targetable pathways, including complement, interferon gamma and TNF alpha (Figure 1). IPA analysis (Figure 2) identified differential regulation of a number of pathways dysregulated in BOS after lung transplantation, such as IL17 and IL13 signaling but also identified differential regulation of BEX2 signaling, ID1 signaling and CHUK (a regulator of NF-kb) pathways which have not been previously associated with BOS. We incubated pulmonary epithelial and endothelial cells with IL17 and saw downregulation of IL17ra, in agreement with the proteomic data. In addition, we saw a very different response to IL17 exposure between small airway epithelial and endothelial cells, illustrating the complex physiological changes happening in BOS (Figure 3A). Network analysis (Cytoscape stringApp) of proteins significantly different in BOS vs control samples shows protein-protein interactions between dysregulated in BOS including IL-17 receptor A (Figure 3B). Potentially novel BOS pathways are being validated using ELISA. Conclusions Our model identified multiple dysregulated pathways in BOS after HSCT, some similar and some dissimilar to those reported in BOS after lung transplant. We identified novel, targetable pathways that are being further studied as potential treatments for BOS after HSCT.