Long Non-Coding RNA NEAT1 Regulation of Innate Immune Signaling in Mycobacterium tuberculosis-Infected Macrophages: Molecular Mechanism and Therapeutic Target Validation

Long non-coding RNAs (lncRNAs) are emerging as critical regulators of innate immune signaling pathways, yet their roles in M. tuberculosis pathogenesis within macrophage contexts remain incompletely defined, particularly in the context of African clinical strain diversity. NEAT1, a nuclear paraspeckle organizer lncRNA, has been reported to modulate NF-kB and inflammasome activity, but its mechanistic role in TB-infected macrophages has not been functionally characterized using clinical strains from Nigerian patients. This dissertation investigates the molecular mechanism of NEAT1-mediated innate immune regulation in M. tuberculosis-infected human macrophages, using clinical strains isolated from pulmonary TB patients in Kaduna State, Northwest Nigeria, and validates NEAT1 as a host-directed therapy target. Clinical M. tuberculosis isolates spanning three major lineages (L4 Euro-American, L5 West African 1, L6 West African 2) were used to infect THP-1-derived macrophages and primary human alveolar macrophages. NEAT1 expression was quantified by strand-specific RT-qPCR. siRNA-mediated knockdown and CRISPRi repression of NEAT1 were performed, followed by transcriptomic profiling by RNA-seq and proteomic analysis by tandem mass tag (TMT) mass spectrometry. NEAT1 knockdown resulted in significantly enhanced NF-kB nuclear translocation, increased pro-inflammatory cytokine secretion (TNF-alpha, IL-6, IL-1beta), and reduced intracellular mycobacterial survival across all three clinical lineages. Mechanistically, NEAT1 was found to sequester the p65 NF-kB subunit co-factor HNRNPA1 within nuclear paraspeckles, impeding its cytoplasmic transport and dampening NF-kB activation. Clinical significance was supported by significantly lower NEAT1 expression in macrophages from HIV-TB coinfected patients compared to TB-only patients, correlating with higher inflammatory cytokine burden. NEAT1 represents a novel host-directed therapy target whose pharmacological inhibition could enhance macrophage bactericidal activity across diverse M. tuberculosis lineages in Nigerian patients. Keywords: NEAT1, lncRNA, tuberculosis, innate immunity, host-directed therapy.