Dual human lung models reveal compartment-specific activity of anti-tuberculosis drugs and host-directed therapies

ABSTRACT Tuberculosis (TB) remains a major global health challenge that requires new therapeutic strategies to improve drug efficacy, shorten treatment duration, prevent drug resistance, and limit Mycobacterium tuberculosis (Mtb) persistence. Here, we established complementary in vitro human lung models integrating alveolar macrophage-like (AML) cells and airway air–liquid interface (ALI) cultures to evaluate standard-of-care antibiotics, host-directed therapies, and virulence-targeting agents. AMLs recapitulated key morphological, transcriptional, and functional features of primary alveolar macrophages, including a CD16 + immunoregulatory phenotype highly permissive to Mtb infection. In parallel, ALI cultures maintained epithelial barrier integrity and secretory functions, allowing apical Mtb infection, drug penetration analysis, and inflammatory profiling. Benchmarking of standard-of-care antibiotics revealed compartment-specific activity: isoniazid, rifampicin, and moxifloxacin were effective in both systems, while pyrazinamide was active only in AMLs. Anti-inflammatory host-directed therapies, such as ibuprofen and doramapimod, selectively reduced cytokine production without affecting bacterial load. Together, this dual-platform system offers a physiologically relevant and scalable model to assess antimicrobial efficacy and host modulation across distinct pulmonary niches, bridging the gap between conventional macrophage assays and the complex human lung. IMPORTANCE Tuberculosis remains one of the world's deadliest infectious diseases. The development of new therapies is limited by the absence of human-relevant models that reproduce the distinct lung niches encountered by Mycobacterium tuberculosis . Current macrophage or epithelial monocultures fail to predict how drugs act in the alveolar versus airway compartments, where intracellular and extracellular bacteria coexist and trigger different immune responses. Here, we introduce a dual human lung platform integrating alveolar macrophage-like cells and air–liquid interface airway epithelium. These models recapitulate key physiological features, including macrophage immunoregulatory programming, epithelial barrier function, mucociliary activity, and compartment-specific drug penetration. Benchmarking standard antibiotics, host-directed therapies, and antivirulence strategies revealed striking niche-dependent differences in antimicrobial and immunomodulatory activities. This system provides a powerful and accessible preclinical tool to evaluate antimicrobial and host-directed interventions in relevant human lung environments, helping bridge the gap between simplified in vitro assays and the complex biology of human tuberculosis.