P045
Phagocytosis of Mycobacterium fortuitum by caprine alveolar macrophages is associated with iNOS and proinflammatory marker expression
M Blay-Benach(1,2) J Repullés(1,2) P Cuenca-Lara(1,2) B Pérez de Val(1,2)
1:Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), IRTA, Campus de la Universitat Autònoma de Barcelona, Bellaterra, 08193, Catalonia, Spain.; 2:Unitat Mixta d'Investigació IRTA-UAB en Sanitat Animal, CReSA, Campus UAB, Bellaterra, 08193, Catalonia, Spain.
Phagocytosis mediated by alveolar macrophages (AM) plays a crucial role in host defence against pathogens such as Mycobacterium tuberculosis. However, limitations of in vitro phagocytosis assays highlight the need for new methods to assess AM functionality. This study aimed to standardize and evaluate a novel fluorescence-based technique for measuring the phagocytosis of M. fortuitum by caprine AM and its association with AM activation and proinflammatory markers. AM were stimulated in vitro with LPS and heat-inactivated M. bovis (HIMB) for 24h. After stimulation, AM were exposed to fluorescently labelled M. fortuitum for 72h and fluorescence kinetics were measured using the Incucyte® SX5 Live-Cell Imaging and Analysis System. This method was compared to a culture-based assay using the MGIT BACTEC system. In addition, AM activation and polarization markers were assessed using flow cytometry, while a multiplex immunoassay was performed to quantify proinflammatory cytokines. The fluorescence intensity kinetics indicated that stimulation with LPS, but not HIMB, resulted in a greater signal reduction compared to unstimulated AM, suggesting a higher bacterial elimination. Similarly, LPS induced a higher percentage of mycobacterial reduction measured by MGIT culture between 2 and 72h after M. fortuitum challenge. Moreover, LPS stimulation increased the production of proinflammatory cytokines (IL-1b, IL-6, TNFa), elevated the proportion of cells co-expressing iNOS and MHC-II, and enhanced the expression of these markers within M1-polarized AM, suggesting that LPS induced a more activated proinflammatory phenotype. These findings indicate that enhanced mycobacterial phagocytic capacity of AM correlates with iNOS and MHC-II expression and a proinflammatory profile.