Related Experiment Videos
Paradoxical role of capsule in murine bronchoalveolar macrophage-mediated killing of Cryptococcus neoformans
1Evans Memorial Department of Clinical Research, University Hospital, Boston MA 02118.
Abstract:
Infections with the encapsulated fungus Cryptococcus neoformans are usually acquired via inhalation, and the presence of a capsule has been identified as a virulence factor. Therefore, we studied murine bronchoalveolar macrophage (BAM)-mediated killing and phagocytosis of encapsulated and acapsular strains of C. neoformans. After 2 h, BAM killed encapsulated strains CN52 and MP415 more readily than acapsular strains CN602 and CAP67 (54.9 and 36.2% vs 26.1 and 6.7%, respectively, p less than 0.001). Pre-incubating CN602 with purified capsular polysaccharide increased killing to 42.7% (p = 0.04). Significantly greater killing of the encapsulated strains also occurred in vivo. BAM-mediated killing of CN52 appeared to proceed by non-oxidative mechanisms, as BAM released minimal amounts of H2O2 after stimulation with CN52, and killing was not reduced by inhibitors or scavengers of the respiratory burst. The association between encapsulation and susceptibility to BAM fungicidal effects was not attributable to differences in yeast ingestion. Using the same low ratio of organisms to BAM as in the killing assay, greater than 95% of both CN52 and CN602 were phagocytosed. However, BAM phagocytosed significantly greater numbers of acapsular CN602 when incubated with a higher inoculum. Phagocytosis and killing of CN52 and CN602 required fresh serum as a source of C. Phagocytosis of CN52, but not CN602, was profoundly inhibited if BAM were plated on surfaces coated with mAb against the C3bR (CR1). mAb against the iC3b receptor (CR3) did not affect phagocytosis of either strain. These data demonstrate the innate ability of BAM to preferentially kill, by apparently non-oxidative mechanisms, an encapsulated as opposed to acapsular organism. Inasmuch as different receptors appear involved in phagocytosis of encapsulated versus acapsular C. neoformans, the disparity in killing may result from the greater ability of receptors mediating uptake of encapsulated organisms to trigger the antimicrobial armamentarium of the BAM.
Insights
Murine bronchoalveolar macrophages (BAM) effectively kill encapsulated Cryptococcus neoformans strains, unlike acapsular ones. This enhanced killing of encapsulated fungi by BAM involves non-oxidative mechanisms and specific immune receptors.
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- Cryptococcus neoformans infections are typically inhaled, with the fungal capsule being a key virulence factor.
- Bronchoalveolar macrophages (BAM) are crucial immune cells in the lungs responsible for clearing inhaled pathogens.
- Understanding macrophage-host-pathogen interactions is vital for developing effective antifungal therapies.
Purpose of the Study:
- To investigate the role of the Cryptococcus neoformans capsule in macrophage-mediated killing and phagocytosis.
- To elucidate the mechanisms underlying BAM's interaction with encapsulated versus acapsular fungal strains.
- To identify the specific immune receptors involved in BAM's recognition and clearance of C. neoformans.
Main Methods:
- Comparative analysis of murine bronchoalveolar macrophage (BAM) killing and phagocytosis assays using encapsulated (CN52, MP415) and acapsular (CN602, CAP67) C. neoformans strains.
- Assessment of non-oxidative killing mechanisms by measuring hydrogen peroxide (H2O2) release and using respiratory burst inhibitors.
- Investigation of phagocytosis pathways using monoclonal antibodies (mAbs) against complement receptors (C3bR/CR1 and iC3bR/CR3) and fresh serum as a complement source.
Main Results:
- BAM demonstrated significantly higher killing rates for encapsulated C. neoformans strains compared to acapsular strains (54.9% vs. 26.1% and 36.2% vs. 6.7%).
- Killing of acapsular strains increased when pre-incubated with purified capsular polysaccharide, suggesting a direct role for the capsule.
- BAM-mediated killing of encapsulated strains appeared to be non-oxidative, with minimal H2O2 release and no reduction by respiratory burst inhibitors.
Conclusions:
- Murine bronchoalveolar macrophages possess an innate capacity to preferentially kill encapsulated C. neoformans via non-oxidative mechanisms.
- Phagocytosis of encapsulated and acapsular C. neoformans involves distinct immune receptors, with C3bR (CR1) playing a significant role in recognizing encapsulated yeasts.
- The differential engagement of BAM receptors by encapsulated versus acapsular C. neoformans may dictate the efficacy of macrophage-mediated fungal killing.