In situ analysis of lung antigen-presenting cells during murine pulmonary infection with virulent Mycobacterium tuberculosis

Scarce information exists about the role of lung antigen-presenting cells (APCs) in vivo during pulmonary tuberculosis. As APCs activate cellular immunity, following intratracheal inoculation with virulent Mycobacterium tuberculosis, we assessed in situ lung APC recruitment, distribution, granuloma involvement, morphology and mycobacterial burden by using MHC-CII, CD14, scavenger receptor class A (SRA), the murine dendritic cell (DC)-restricted marker CD11c and Ziehl-Neelsen staining. CD11c⁺ DC and CD14 ⁺ cell recruitment into lungs appeared by day 14, continuing until day 60. MHC-CII⁺ cells increased since day 7, persisting until day 60. Thus, virulent mycobacteria delays (14-21 days) lung APC recruitment compared to model antigens and nonvirulent bacilli (24-48 h). Regarding granuloma constitution, highly bacillary CD14⁺ and SRA⁺ cells were centrally located. MHC-CII⁺ cells were more peripheral, with less mycobacteria. CD11c⁺ cells were heterogeneously distributed within granulomas, with scarce bacilli. When labelling lung suspensions for MHC-CII and classifying cells as macrophages or DC, then staining for Ziehl-Neelsen, a remarkable segregation was found regarding bacillary burden. Most macrophage-like cells contained numerous bacilli, while DC had no or scarce mycobacteria. This implies differential APC contributions in situ during pulmonary tuberculosis regarding mycobacterial uptake, granuloma involvement and perhaps bacillary growth.