Mediator responses of alveolar macrophages and kinetics of mononuclear phagocyte subset recruitment during acute primary and secondary mycobacterial infections in the lungs of mice

Abstract

SummaryLittle is known about the activation programme induced in alveolar macrophages upon phagocytosis of mycobacteria and the concomitant mononuclear phagocyte migratory responses that shape the acute phase of mycobacterial infection. Using high-speed cell sorting in conjunction with real-time RT-PCR analysis, we show that sorted alveolar macrophages of transgenic CX3CR1 +/GFP mice infected with red fluorescent-labelled Mycobacterium bovis BCG exhibited weak transcriptional changes of lysosomal cathepsins B, L, D, K and S, whereas antimicrobial cathepsin G was strongly induced upon infection. Moreover, mRNA levels of pattern recognition receptors TLR2, TLR4 and NOD2 were downregulated as were neutrophil chemoattractants KC, MIP-2 and IP-10, whereas highly upregulated mRNA levels of the monocyte chemoattractant CCL2 were observed. M. bovis BCG infection of the mice elicited the alveolar accumulation of highly CX 3CR1-positive alveolar dendritic cells but not neutrophils within the alveolar compartment, whereas no increased accumulation of CX3CR1 high lung parenchymal dendritic cells (lung DC) or CX3CR1 neg lung macrophages compared with controls was noted. In contrast, CX3CR1 +/GFP mice previously immunized with M. bovis BCG rapidly responded with increased accumulations of both CX 3CR1 high alveolar and lung parenchymal DC and CX3CR1 neg lung macrophages upon intratracheal M. bovis BCG challenge. Moreover, alveolar and lung macrophages and lung DC were found to contribute to early uptake of M. bovis BCG. Together, acute mycobacterial infection triggers both rapid changes in gene expression profiles in infected alveolar macrophages and a compartment-specific accumulation of mononuclear phagocyte subsets contributing to M. bovis BCG uptake in vivo.

A Caspase-Independent Pathway Mediates Macrophage Cell Death in Response toMycobacterium tuberculosisInfection

“…However, further experiments with more specific inhibitors are needed to confirm these findings. Interestingly, the expression of cathepsin G, a highly cationic serine protease with antimicrobial activity, is upregulated in murine macrophages by M. tuberculosis infection (45). We are currently investigating the possibility that one or more of these serine proteases is involved in M. tuberculosis-mediated cell death.…”

Mice That Overexpress CC Chemokine Ligand 2 in Their Lungs Show Increased Protective Immunity to Infection withMycobacterium bovisBacille Calmette‐Guérin

“…M. bovis BCG (strain Pasteur) was cultured in Middlebrook 7H9 medium until mid-log phase [3]. Thereafter, the culture was harvested and the suspension was frozen in 1-mL aliquots at Ϫ80°C until use.…”

Functional Impairment of Murine Dendritic Cell Subsets following Infection with Infective Larval Stage 3 of Brugia malayi

“…Thereafter, the cells were washed, centrifuged, and incubated with CD11c beads (1 l beads/10 6 cells) for 20 min at 4°C. After subsequent washes, the cells were passed over a MACS MS column that was finally flushed with MACS buffer to obtain CD11c-positive cells, as described recently (67). Aliquots of CD11c-positive cells were assessed for purity using a BD FACS Aria flow cytometer and were consistently found to contain ϳ90% CD11c-positive cells.…”

“…Briefly, single-cell suspensions of spleens from at least 6 to 8 identically treated mice were pooled, and CD11c-positive cells were enriched using CD11c beads, as described above. Sorting was carried out using a high-speed FACS Aria flow cytometer (BD Biosciences) fitted with a 70-m nozzle, as described recently (11,67). After sorting, the sorted cells were subjected to analysis to ascertain their purity, and a small fraction was used to prepare cytospins (Fig.…”