In vitro priming with Mycobacterium fortuitum induces trained immunity-associated phenotypes in caprine alveolar macrophages and enhances phagosomal maturation and antimicrobial functions upon homologous re-stimulation

Background Trained immunity refers to the stimuli-induced epigenetic and metabolic reprogramming of innate immune cells, enabling enhanced non-specific responses upon secondary stimulation. Alveolar macrophages (AMs) constitute the primary immune barrier in the lung and represent a key target in enhancing protection against pulmonary pathogens. Methods In this study, an in vitro priming-resting-re-stimulation protocol was used to characterise the ability of Mycobacterium fortuitum (MF) to induce functional features of trained immunity in caprine AMs in vitro . Pro-inflammatory cytokines (TNFα, IL-1β, IL-6) and nitric oxide synthase (iNOS)-positive cells were quantified by multiplex assay and flow cytometry, respectively. Phagosomal acidification was detected by flow cytometry and live-cell fluorescent microscopy after MF-challenge using pH-sensitive labelled bacteria. Results MF-trained AMs displayed significantly elevated TNFα and IL-1β production and a higher frequency of inducible iNOS-producing cells compared to non-stimulated controls, consistent with a trained immunity-associated pro-inflammatory and antimicrobial phenotype. Phagosomal acidification was enhanced and sustained in MF-trained AMs following homologous challenge with MF, suggesting enhanced phagocytic activity against mycobacteria. Conclusion These findings provide evidence that M. fortuitum induces trained immunity-associated functional responses in caprine AMs and support further trained immunity studies in the caprine lung mucosa against pulmonary infections.