Integrated in vitro and multi-cohort cross-omics analysis of HTLV-1-associated lung pathology reveals a RelA-dependent mechanism for monocyte recruitment and differentiation.

BACKGROUND: Human T-lymphotropic virus type 1 (HTLV-1) infects up to ten million people worldwide and is associated with inflammatory diseases, including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Individuals with HAM/TSP are prone to pulmonary complications such as bronchiectasis, characterized by sustained mononuclear cell infiltration and elevated inflammatory mediators in bronchoalveolar lavage fluid. However, the epithelial mechanisms linking HTLV-1 exposure to lung inflammation remain poorly defined. METHODS: To study epithelial signaling in response to HTLV-1 exposure, human alveolar epithelial A549 cells were co-cultured with HTLV-1-infected (MT-2 or MT-4 cells) T cells/supernatants or uninfected (Jurkat) T cells. Transcriptomic changes were assessed by RNA sequencing and pathway enrichment analyses, with key mediators validated by RT-qPCR. NF-κB dependency was evaluated using CRISPR/Cas9-mediated knockout of NF-κB RelA/p65. Functional consequences of epithelial activation were assessed using monocyte chemotaxis and differentiation assays in THP-1 cells and primary human monocytes. In vivo relevance was examined through integrative cross-omics analyses combining our own and publicly available bulk and single-cell transcriptomics, epigenomics, viral interactomics, and multi-ancestry genome-wide association studies (GWAS). RESULTS: HTLV-1 exposure induced a robust epithelial antiviral and inflammatory transcriptional program in A549 cells, predominantly regulated by NF-κB signaling. Among the most strongly upregulated genes in A549 MT-2 co-cultures were the monocyte chemoattractant MCP-1/CCL2 and the macrophage differentiation factor CSF1, as confirmed by RT-qPCR. CRISPR/Cas9-mediated knockout of NF-κB RelA/p65 demonstrated that CSF-1 induction is mechanistically dependent on NF-κB activation. Supernatants from HTLV-1-exposed epithelial cells promoted monocyte chemotaxis and macrophage differentiation in THP-1 cells and primary human monocytes. Transcriptomic data of people living with HTLV-1, HAM/TSP patients and idiopathic pulmonary fibrosis patients confirm in vivo expression of the in vitro gene signature, whereas single cell RNA-seq identified a unique myeloid subset in human lung, characterized by co-expression of CCL2/ISG15/CXCL10. Finally, GWAS analyses revealed ancestry-specific associations (CCL2 for European and CSF1 for African ancestry). CONCLUSIONS: We report an in vitro co-culture model that recapitulates HTLV-1-triggered lung inflammation through RelA/NF-kB-dependent release of pro-inflammatory cytokines and chemokines resulting in monocyte chemotaxis, activation and differentiation. This epithelial-myeloid inflammatory axis provides a relevant in vitro model that recapitulates in vivo HTLV-1-associated lung pathology.