In vitro differentiation of human macrophages with enhanced antimycobacterial activity
Guillaume Vogt, Carl Nathan
Guillaume Vogt, Carl Nathan
View: Text | PDF
Technical Advance Infectious disease

In vitro differentiation of human macrophages with enhanced antimycobacterial activity

Abstract

Mycobacterium tuberculosis causes widespread, persistent infection, often residing in macrophages that neither sterilize the bacilli nor allow them to cause disease. How macrophages restrict growth of pathogens is one of many aspects of human phagocyte biology whose study relies largely on macrophages differentiated from monocytes in vitro. However, such cells fail to recapitulate the phenotype of tissue macrophages in key respects, including that they support early, extensive replication of M. tuberculosis and die in several days. Here we found that human macrophages could survive infection, kill Mycobacterium bovis BCG, and severely limit the replication of M. tuberculosis for several weeks if differentiated in 40% human plasma under 5%–10% (physiologic) oxygen in the presence of GM-CSF and/or TNF-α followed by IFN-γ. Control was lost with fetal bovine serum, 20% oxygen, M-CSF, higher concentrations of cytokines, or premature exposure to IFN-γ. We believe that the new culture method will enable inquiries into the antimicrobial mechanisms of human macrophages.

Authors

Guillaume Vogt, Carl Nathan

×

Figure 7

Reproducibility of control of M. tuberculosis replication by MDMs differentiated with GM-CSF and TNF-α in combination and activated with various cytokines in 5% O2.

Options: View larger image (or click on image) Download as PowerPoint
Reproducibility of control of M. tuberculosis replication by MDMs differ...
(A) Reproducible control of M. tuberculosis with appropriately differentiated MDMs that were activated with IFN-γ at a physiologic tissue O2 tension. MDMs from 24 donors were differentiated in 5% O2 with no cytokines or with GM-CSF plus TNF-α (0.5 ng/ml each) for 14 days, exposed or not to IFN-γ (3 ng/ml), infected with M. tuberculosis on day 16 (MOI of 0.17), and lysed 2 weeks later for determination of CFU. Results from all donors were pooled to calculate averages. (B) Comparison of IFN-γ and other cytokines during the activation period. In the same experiments illustrated in A, MDMs were also activated with the indicated cytokines (each at 50 ng/ml, except for IFN-γ as indicated at 3 or 0.5 ng/ml). CFU counts from MDMs lysed 2 weeks infection are presented as percentage of those for the same donor’s MDMs given PBS instead of exogenous cytokines during the activation period.