Comparative 5' IsomiRome Analysis Uncovers Dysregulated 5' IsomiRs in Trypanosoma cruzi-infected Macrophages

Trypanosoma cruzi (T. cruzi), the etiological agent of Chagas disease, remains a major global health threat with limited therapeutic options. Macrophages are central to host defense against T. cruzi, yet their failure to achieve complete parasite clearance contributes to disease progression. Although 5' isomiRs have emerged as functional regulators of gene expression, their expression alternations in T. cruzi-infected macrophages remain largely unknown. Here, we performed comparative analyses of 5' isomiR expression in T. cruzi-infected cells and identified a marked, selective increase in microRNA 5'-end heterogeneity in THP-1-derived macrophages but not in cardiomyocytes or epithelial cells. Comparative 5' isomiRome analysis revealed 68 differentially expressed 5' isomiRs in infected macrophages, most of which were specific to T. cruzi relative to other pathogens. Among them, 56 were associated with Argonaute proteins, including 3 derived from the miR-1246 precursor. Focusing on miR-1246|+1, a 5' isomiR generated by a 1-nucleotide downstream shift at the 5' end of miR-1246, we showed that its overexpression substantially down-regulated target genes involved in nuclear factor-κB (NF-κB) signaling, circadian rhythm, cytokine responses, and cell migration. Notably, miR-1246|+1 broadly suppressed NF-κB family transcription factors and their downstream effector genes, thereby inhibiting proinflammatory M1 macrophage phenotype. Intriguingly, the suppressed NF-κB regulators Nfkb1 and Rela, together with M1 macrophage signature genes Ccl8, Il1a, Il1b, and Tlr2, displayed rhythmic expression in murine peritoneal macrophages. Collectively, these findings reveal cell type- and pathogen-specific reprogramming of the 5' isomiR landscape in T. cruzi-infected macrophages and identify miR-1246|+1 as a potential posttranscriptional regulator of macrophage inflammatory polarization.