Multi-omics biomarkers of endothelial dysregulation preceding chronic lung allograft dysfunction: A prospective...

Long-term survival of lung transplant recipients remains limited by chronic lung allograft dysfunction (CLAD). CLAD is only diagnosed following a persistent and substantial decline in lung function, after which irreversible damage to the lungs has occurred, limiting opportunities to effectively intervene at an early stage. There is a critical need for earlier detection prior to its clinical manifestation. The immunological drivers of CLAD remain unclear, limiting the development of predictive biomarkers and new therapies.

In this hypothesis-generating, prospective cohort study, we profiled the microbial, metabolic, lipidomic, and gene expression dynamics of longitudinally collected broncho-alveolar lavages (BALs) from 56 CLAD-free lung transplant recipients up to 30 months post-transplant, and compared BALs from 13 CLAD-free patients to BALs from 13 patients who developed CLAD. In CLAD-free patients, the first 6 months post-transplant were hallmarked by diminished microbial diversity and increased abundance of Staphylococcus and Candida, coupled with upregulated innate and adaptive immune responses, and elevated nitric oxide metabolism (FDR < 0.05). This was superseded by homeostatic tissue repair and by the reactivation of T-cell genes such as CD3, GZMA, IL2RB, CD28, CD40LG, and LCK, after tapering of maintenance immunosuppression (FDR < 0.05). In patients who developed CLAD, disease onset was preceded by the increased abundance of sphingolipids and the upregulation of glycocalyx and immune cell recruitment genes such as HAPLN3, HS3ST3B1, SULF2, CHST2, CSGALNACT1, CXCR1, CSF3R, SELL, CXCL2, and CEACAM1 (FDR < 0.05), suggesting increased vascular dysfunction and immune cell graft infiltration prior to CLAD onset. Scoring against a publicly available lung single-cell dataset showed our bulk gene transcriptomics signature to be expressed by monocytes, endothelial, and T cells. In contrast to CLAD-free patients, this signature persisted after 1.5 months post-transplant and increased in intensity upon the start of lung function decline. Multi-omics integration highlighted sphingolipid molecules and genes involved in immune cell recruitment and endothelial function as candidate biomarkers associated with the onset of CLAD. This study is limited by its small sample size.

We have identified immunological processes, metabolites, lipids, and genes associated with the onset of CLAD. Our findings are to be considered associative and not aimed at establishing causality. Future studies employing a targeted approach in independent validation cohorts, using, for example, quantitative polymerase chain reaction (PCR) and targeted mass-spectrometry, will be required to confirm these findings.