MicroRNA biomarkers and host response pathways in severe pulmonary hemorrhagic syndrome due to leptospirosis: A multi-omics study

Background

Severe pulmonary hemorrhagic syndrome (SPHS) remains a fatal complication of leptospirosis with poorly understood mechanisms and an urgent need for effective biomarkers.

Methods

A nested case-control analysis was conducted using blood specimens from two previous Thai leptospirosis cohorts. Candidate microRNAs were initially discovered through a global profiling of 798 serum microRNAs in five SPHS and seven non-SPHS patients, and then validated using real-time polymerase chain reactions in 168 patients. Pathways enriched from microRNA targets were compared to those from an integrated transcriptomic-proteomic analysis. Proteins pertaining to the key resulting pathway were measured to validate significance and reveal correlation with microRNA biomarkers.

Results

Serum microRNA profiling revealed a total of 81 significantly expressed microRNAs, of which seven were selected for further validation in the whole cohort of 168 leptospirosis patients, including 28 in SPHS and 140 nonSPHS groups. Among the selected microRNAs, miR-5010–3p and miR-147b-3p had significantly higher expression in SPHS group compared to nonSPHS group, with consistently higher expression after adjusting for age, sex, days of illness, comorbidity, smoking status or recruitment site. The two had area under the curve (AUC) values of 0.76 (95% CI: 0.67–0.85) and 0.70 (95% CI: 0.56–0.81) for discriminating SPHS, respectively. These microRNAs also exhibited consistent AUC values in patients tested before chest radiograph shadows manifested. Combination of miR-5010–3p with miR-548ai and miR-224–5p, as selected by Bayesian Model Averaging algorithm, substantially boosts the AUC value to 0.86 (95% CI: 0.77−0.94). The miRNA biomarkers also enhanced the predictive values of a previously validated clinical model, increasing AUC value from 0.87 to 0.92 with a significant reclassification net index. Multi-omics pathway analysis incorporating microRNA targets and transcriptomic-proteomic data suggested TNF signaling as among the key pathways. In validation, seven out of ten pathway proteins were significantly different between groups, with principal components correlated with severity and miR-5010–3p.

Conclusions

MiR-5010-3p and miR-147b-3p are novel biomarkers with good predictability and potential relevance with TNF signaling pathway, an important host response mechanism in leptospirosis SPHS.